Prof. dr. ing Marcel Christelle Popa

Delaite

Ing Dipl Corina-Lenuța

OF CHEMICAL ENGINEERING AND ENVIRONMENTAL PROTECTION "CRISTOFOR SIMIONESCU"

à base de polysaccharides modifiés, micro / nanoparticules et sous forme de film, pour la libération contrôlée de principes actifs)

Chapter 6. Polyglobalide-based porous networks containing poly(ethylene glycol)

structures prepared by photoinitiated thiol-ene coupling

Introduction

Classical thiol-ene chemistry, i.e., the radical addition of thiols to nonactivated carbon-carbon double bonds, is a powerful tool that is efficiently employed to synthesize and functionalize different types of polymeric materials important for biological applications, such as hydrogels, nanogels, micelles, and nanoparticles, and for surface grafting and film patterning. Discovered in 1839 by Goodyear, thiol-ene chemistry was used extensively in the 20th century as a method to synthesize crosslinked networks. Over the years, increased attention has been paid to Diels-Alder reactions, metal-free dipolar cycloadditions, and a series of thiol-based reactions, including the thiol-ene, thiol-yne, thiol-isocyanate, and thiol-Michael addition processes 1,2 . The addition of thiol groups to double bonds may occur through either radical or nucleophilic mechanisms. The radical thiol-ene reaction occurs through a chain reaction mechanism similar to the chain transfer polymerization mechanism 3 . The ideal thiol-ene radical reaction consists of the alternation between thiyl (RS .

) radical propagation across the "ene" functional group and a chain-transfer reaction that involves abstraction of a hydrogen radical from the thiol by the carbon-centered radical 4, 5 . Regarding the initiation part of the reaction, radical generation can be achieved thermally or photochemically. Both photochemical and thermal radical conditions were tested to explore the efficiency of the thiol-ene reaction, and it was found that using photochemical conditions leads to a faster and, in many cases, quantitative reaction 6 .

In recent years, many reports on successful polymer conjugation with a low-molecular-weight compound (either a thiol or an alkene) by thiol-ene radical chemistry have been performed and extensively reviewed, e.g., in the work of Lowe 7 . In many cases, it has been shown that thiol-ene chemistry works reasonably well for the conjugation of a functional polymer (bearing thiol or alkene functional groups) with a low-molecular-weight bi-or multifunctional (thiol/ene) system, even if an excess of the low-molecular-weight compound must be used to achieve high conjugation efficiencies 3 . This can be extended even to multifunctional-bifunctional polymer coupling 3 . Usually, side chain alkene functional polymers and small molecular weight cross-linkers bearing two or more thiol groups are employed for such reactions. This ensures homogeneous crosslinking and, as consequence, a certain uniformity of the cross-linked material properties. However, in the case of thiol-ene chemistry for polymer-polymer conjugation systems, there are strong limitations. The conjugation between a polymer bearing multiple thiol functional groups and a polymer bearing multiple alkene functional groups largely fails if the starting materials are employed in (or close to) equimolar ratios. Moreover, the reactivity of the radical thiol-ene reaction can vary considerably depending on the chemical structural environment surrounding the alkene and thiol components. The reason for failure may be a loss of radical species, loss of active SH species due to secondary reactions, or reduced mobility of partially reacted macromolecular chains depending on the functional density and molar masses. Thus, because of the complications mentioned above, the approach where two or more macromolecular compounds are coupled remains insufficiently explored, and only a few papers on this topic can be found in the literature 3,8, 9 .

Polyglobalide (PGL), the hydrophobic polymer considered in our study, may be prepared chemically or enzymatically starting from globalide (GL), an unsaturated cyclic ester with 16 cycle members containing a double bond at the 11 or 12 position 10 . Unsaturated lactones with 14 to 19 members such as GL, ambrettolide, pentadecalactone, and hexadecalactone are extracted from natural sources and used in the fragrance industry for their musky odor 11, 12 . The polymers of these natural lactones are highly crystalline with low melting points, are nontoxic and show low hydrolytic or enzymatic degradability, being characterized by high hydrophobicity 10 . Such properties make them good candidates for biomaterials obtained from natural resources with low degradability. Additionally, the presence of double bonds on the polymer backbone provides the possibility for further expansion of chemical functionalities that may be attached to PGL 13 . Moreover, the double bonds can be used as the initiation sites for other polymers, thus creating polymer brushes on PGL surfaces 14 . The thiol-ene coupling between PGL and a low-molecularweight bis-thiol functional crosslinker, ethylene glycol bis (3-mercaptopropionate), in THF solution has been previously described by Ates and Heise 15 . The reaction led to the formation of compact hydrophobic materials. The approach for thiol-ene coupling was via a radical mechanism using radical photoinitiators, a mixture of (diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide and 2-hydroxy-2-methylpropiophenone. Such materials were also shown to undergo slow enzymatic degradation. Moreover, thiol functionalities were further attached to the PGL network by thermally initiated radical thiol-ene crosslinking 15 . Claudino et al. 16 have studied the potential use of globalide-based copolymers in thermoset synthesis via the photoinduced thiol-ene reaction as well. Also, porous fibrilar materials were prepared by electrospinning of PGL/thiol crossllinkers mixtures followed by in situ UV-initiated thiol-ene crosslinking 17 .

Poly(ethylene glycol) (PEG) is a synthetic polymer with unique physicochemical and biological properties. It is frequently used as a nonionic hydrophilic polymer for functionalization. Biodegradable and biocompatible PEG hydrogels were easily obtained through the thiol-ene "click" reaction under physiological conditions by Zhu et al., who presented a facile method for preparing two PEG copolymers containing multiple thiol or multiple ene functionalities by direct polycondensation of PEG with thiomalic acid (TMA) or maleic anhydride using scandium trifluoromethane sulfonate (Sc(OTf)3) as a highly efficient and chemoselective catalyst 18 . Another example of degradable PEG derivatives with multiple alkene and multiple thiol functionalities is presented by Du and collaborators 19 . Huang et al. 20 also reported the synthesis of a series of biodegradable poly(ether-ester)s with multiple pendant thiol groups by melt polycondensation of diol poly(ethylene glycol), 1,4-butanediol, and TMA using Sc(OTf)3 as the catalyst. The study presented the use of TMA for the preparation of reduction-responsive core-crosslinked micelles via an in situ thiol-ene "click" reaction, which are considered promising candidates as biodegradable and biocompatible nanocarriers for antitumor chemotherapy.

The combination of hydrophilic/hydrophobic polymers in the formation of a crosslinked network represents a challenging task because of the phase separation. Generally, such polymer networks containing two types of polymers present bimodal properties resulting from the differences between the molar mass of the two macromolecular components and from the different properties of each component 21 .

Herein, we report the development of advanced materials through photoinitiated radical thiolene coupling of PGL bearing double bonds in the main chain and poly(ethylene glycol-cothiomalate) copolymers with pendant thiols (PEG-SH). We investigated the particularities of the crosslinking process between two multifunctional polymers in solution, PGL and PEG-SH. The strategy involved the use of polymer-polymer thiol-ene crosslinking in order to prepare potentially nontoxic amphiphilic networks. The obtained cross-linked networks were characterized in order to establish the influence of the synthesis parameters, such as the SH/C=C functionality ratio and total polymer concentration, on the final properties of the crosslinked polymer networks. The structure and properties of the networks were characterized by Raman spectroscopy, Ellman's titration of thiol groups, elemental analysis, rheological analysis, SEM, and swelling analysis. The cytotoxicity of the prepared polymeric networks was evaluated based on ISO:10993-12 using 3T3 fibroblasts. Finally, the loading and in vitro delivery of levofloxacin as a model drug was assessed.

Materials

Poly(ethylene glycol) (PEG) (Mw = 600 g•mol -1 , Sigma Aldrich), scandium trifluoromethanesulfonate [Sc(OTf3)] (Sigma Aldrich), thiomalic acid (TMA, Sigma Aldrich), globalide or 11/12-pentadecen-15-olide (GL), a gift from Symrise, Germany, lipase acrylic resin from Candida Antarctica (25.0000 U/g, Sigma Aldrich), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99 %, Sigma Aldrich), 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, 99 %, Sigma Aldrich), levofloxacin (LEV, ≥98 %, Sigma Aldrich); tetrahydrofuran (THF, Merck); methanol (99.8 %, Sigma Aldrich), diethyl ether (Sigma Aldrich), dichloromethane (DCM, 99 %, Sigma Aldrich) and Milli-Q ultrapure distilled water (Merck) were of analytical grade. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), streptomycin, penicillin, and Lglutamine were obtained from Gibco (ThermoFisher Scientific, Bratislava, Slovakia). 3-(4,5-Dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Calbiochem (Merck Millipore, Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and Dulbecco's phosphate buffered saline (PBS) were purchased from Sigma-Aldrich (Weinheim, Germany). Murine 3T3 fibroblasts were purchased from DSMZ (Braunschweig, Germany). All these chemicals were used as received.

Methods

Preparation of polyglobalide

Globalide (5.0 g, 21 mmol) and enzyme (Lipase acrylic resin from Candida Antarctica) (0.11 g) were added to a 25-mL reaction flask and purged with argon for 10 minutes. Then the flask was immersed in an oil bath heated at 60 °C. After four hours the high viscosity of the mixture prevented proper stirring. THF was added to the reaction mixture to solubilize the product and inhibit the enzyme activity. After removal of the enzyme by centrifugation, the solution was precipitated into ice-cold methanol. The PGL polymer was filtered off and dried at RT in a vacuum oven until constant weight 1-3 . Typical yield was 74 %. Scheme 1 display the synthetic strategy used for the PGL synthesis. Scheme 1. Synthesis of poly(globalide)

Preparation of poly(ethylene glycol-co-thiomalate) copolymer (PEG-SH)

In order to obtain PEG-SH, we considered the protocol reported by Zhu and collaborators 4 . PEG-SH was synthesized via polycondensation process, in bulk, in the presence of [Sc(OTf)3] as a catalyst, under moderate temperature and reduced pressure. Briefly, PEG (20.0 g, 33 mmol), TMA (5.0 g, 33 mmol), and [Sc(OTf)3] (0.16 g, 0.33 mmol) were added to a 100 mL Amber flask immersed in an oil bath preheated to 80 °C under argon atmosphere and vigorously stirred for 30 minutes until the melt system turned transparent. Afterward, the polycondensation reaction was carried out at 100 °C, under reduced pressure and maintained for 24 h to complete the reaction.

The polymer was purified by three times repeated precipitation from CH2Cl2 solution into cold diethyl ether. The final product was dried in a vacuum oven for 48 h to constant weight, yield 85 %. Scheme 2 displays PEG-SH synthesis. Scheme 2. Synthesis of poly(ethylene glycol-co-thiomalate) copolymer (PEG-SH)

Preparation of polymeric networks

PGL based networks (PGN) were prepared by photo-initiated radical thiol-ene reaction of PGL and PEG-SH. PGN materials with various concentrations and SH/C=C functionalities molar ratios were prepared by mixing different amounts of polymers (PEG-SH and PGL) in THF, using glass vials with plastic caps. A calculated amount of DMPA photoinitiator dissolved in THF was added to polymers solution and well homogenized with a Vortex. The concentration of DMPA was kept at the 0.5 wt. % with respect to the total amount polymers, throughout the study. Thiol-ene reactions were carried out using a medium-pressure mercury lamp in a Spectramat apparatus (Ivoclar AG, Liechtenstein, glass filter λ = 350-550 nm). The intensity of the light in the sample position was measured with a Nova Meter Assy, from OPHIR (Israel). We used cylindrical closed vials with a diameter of 20 mm. The thickness of the polymer solution layer was approximately 10 mm. The samples were irradiated from the side and the radiation intensity at the 365 nm wavelength was measured before (65 mW•cm -2 ) and after passing through the vials containing the polymers solutions (57 mW•cm -2 ). Typically, after 25 minutes of light exposure transparent PGN materials, swollen in THF, were obtained. The obtained polymer gels were swollen in THF and flexible enough to be pulled out of the vials using a spatula. They maintained the cylindrical shape after removal from the glass vials. To examine the gelation point, each sample was exposed to UV-vis light and the gelation point was determined visually via the test-tube inversion method 22 , by turning the vials upside down and observing the solution flow inside the glass vial. A chronometer was used to record the gelation time.

The following purification steps were applied: in the first step the PGN samples were immersed into THF (20:1 vol/vol) under stirring for 24 h (THF was changed twice during this period) and then dried overnight in a vacuum oven at 50 0 C; the second step of purification was carried out by washing with water (80:1 vol/vol) during 7 days. During this time the water was changed twice daily, and finally samples were lyophilized. The recovered gel fraction was calculated using the equation (1). Each material was tested in three identical replicas.

𝑔𝑒𝑙 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 (%) = 𝑚 𝑓 𝑚 𝑖 𝑥100 (1)
where: mf -the weight of dry gels after purification step, mi -weight of the initial total amount of polymers.

After the purification steps the samples were considered for characterization: reaction yieldsgravimetric; SH groups titration -Ellman's test 23, 24 ; FT-IR; sulfur content -elemental analysis; morphologyscanning electron microscopy; swelling behavior, cytotoxicity and drug loading and release. The experimental protocol with the observed parameters in the obtaining of PGN materials is presented in Table 1.

Characterization 4.1. Gel permeation chromatography analysis

Polymers molar mass and dispersity were estimated by gel permeation chromatography (GPC), on an Agilent 1200 Infinity Series and Shimadzu instrument equipped with GPC control software and consisted of a Waters 515 pump, two PPS SDV 5 mm columns (d = 8 mm, l = 300 mm; 500 Å + 105 Å) and a Waters 410 differential refractive index detector, with THF as an eluent at flow rate of 1.0 mL/min. Polystyrene standards were used for calibration. Anisole was used as the internal standard to correct for any fluctuation in THF flow rate.

MALDI analysis

Matrix-assisted laser desorption/ionization analysis (MALDI) was performed using an UltrafleXtreme TOF instrument (Bruker) equipped with a 355 nm smartbeam-2 laser, capable of pulsing frequency 1 kHz. The mass spectrometer was operated by FlexControl 3.3 software (Bruker). Polymer samples were dissolved in THF at a concentration of 10 mg/mL. Samples were mixed with a matrix solution (10 mg/mL of 1,8,9-anthracenetriol -dithranol in THF) at a ratio of 1/100 (v/v). 1 μL of this mixture were deposited on polished steel MALDI target (Bruker). Acquired spectra were processed by Flex Analysis 3.3 software (Bruker). The ionization laser power was adjusted just above the threshold in order to produce charged species. The mass spectra were collected in the amount of above 10000 spectra.

Elemental analysis

The amount of sulfur (wt. %) in the synthesized samples was determined using FLASH 2000 Organic elemental analyzer (CHNS-O) from Thermo Fisher Scientific. Three different measurements were performed for each sample.

Determination of thiol groups by Ellman's test

In order to understand the efficiency of the cross-linking process, an analysis of residual thiolgroups content in PGN samples was undertaken via colorimetric sulfhydryl assay using 5,5'dithio-bis-(2-nitrobenzoic acid) (DTNB) Ellman's reagent 5,6 . Ellman's reagent is very useful as a sulfhydryl assay reagent because of its specificity for (-SH) groups at neutral pH, high molar extinction coefficient and short reaction time. The reagent selectively reacts with thiol groups forming a yellow colored product, 5-thio-2-nitrobenzoic acid (TNB 2-), which possesses high absorbance at 412 nm. The intensity of the absorbance at 412 nm increases proportionally with the number of thiol functionalities, thus UV-vis analysis of TNB 2-enables the determination of available thiols according to the absorbance calibration curve. To quantify the available thiol groups after the PGN materials were purified, the following procedure was used: The Ellman's reagent solution was prepared by dissolving 4 mg DTNB in 1 mL of sodium phosphate buffer. 1-3 mg of sample was placed in a vial and was hydrated in 1 mL of 0.5 M sodium phosphate buffer (PBS) at pH=8. After a swelling process of 60 min, 950 µL of PBS and 0.05 mL of Elman's reagent were added and the samples were incubated for 30 min in the dark at RT. After removing the sample by centrifugation (5 min/15.000 rpm), 7.5 µL of the supernatant fluid was transferred to a microtitration plate and the absorbance was measured.

Scanning Electron Microscopy

Surface and section morphology of PGN materials was investigated by Scanning Electron Microscopy (SEM) technique. SEM micrographs were recorded using a HITACHI SU 1510 (Hitachi SU-1510, Hitachi Company, Japan) Scanning Electron Microscope. In order to obtain a representative image of the PGN materials the dry gels were cut transversally using a scalpel; the cut was parallel with the vertical axis of the cylinder. The exposed transversal section was observed. The individual samples were fixed on an aluminum stub covered with double adhesive carbon band. Before observation the samples were placed on the chamber pedestal of a Cressington 108 Sputter Coater device and coated with a 7-nm thick gold layer, under vacuum.

Rheology

Viscoelastic properties of the PGN were evaluated in triplicate by using an Anton Paar MCR 502 (Anton Paar, Austria) a modular compact rheometer fitted with original UV accessory (Anton Paar, Austria) including UV lamp operating at 365 nm and an intensity of 12.5 mW cm -2 . In order to evaluate the kinetics during cross-linking reactions, the viscoelastic moduli, such as storage modulus (G') and loss modulus (G'') were monitored online the cross-linking process. The measurements were performed in the linear viscoelastic region when the strain deformation was 1 % and frequency of 1 Hz and the temperature was set to 23 °C in all cases. The total volume of the reaction was adapted to meet the measurement as follows: for each measurement 250 µL of THF solutions containing polymers mixture composed of PEG-SH and PGL with different polymers concentrations (5 %, 10 %, 20 %, 40 %) and DMPA photoinitiator in specified amounts related to polymer content. The samples were well homogenized and introduced into the measuring cell. During whole measurements, small drops of the THF were placed around the measuring cell covered by protection ring to provide the solvent saturated atmosphere in order to avoid solvent evaporation during the time-dependent reactions.

Spectroscopic analysis

1 H NMR spectra of both polymers were recorded on a 400 MHz VNMRS Varian NMR spectrometer equipped with 5 mm 1 H-19F/15N-31P PFG AutoX DB NB in CDCl3 as a solvent. All chemical structures were analyzed by Fourier Transform Infrared (FTIR) spectroscopy in the solid state on a Nicolet 6700, USA, (Thermo Scientific). Fourier Transform Raman spectroscopy (FT-Raman) spectra were acquired using a Renishaw inVia Raman Microscope. UV absorbance studies were recorded with a UV-Vis NanoDrop ND 1000.

Swelling study

Swelling studies were conducted by gravimetric method. Freeze-dried samples were weighed and then immersed in a vial containing an excess of water or THF, at room temperature. The samples were removed from the water/solvent and weighed. The swelling was monitored at different time intervals until equilibrium swelling was attained (20.5 hours in the case of water swelling and 7.5 hours in the case of THF swelling). The percentage of swollen gels ratio was determined using the equation ( 2 The measurements were made in triplicate and average data were used for plotting swelling curves.

Cytotoxicity

Cell culturing. Murine 3T3 fibroblasts were cultured in a full growth DMEM medium supplemented with 10 % w/w FBS, L-glutamine (2 mM), streptomycin (100 μg/mL), and penicillin (100 IU/mL) in the CO2 incubator with saturated humidity at 37 °C, 5 % CO2.

MTT assay. Cells at concentration 5000 cells per well were seeded into 96 well tissue culture plate and cultured overnight. All hydrogels were sterilized under ultraviolet (UV) light for 30 min in a laminar flow and rinsed with 1 mL of sterile culture media before extraction. Extracts from hydrogels (n=3) were prepared by 24 h extraction of 0.1 g of material to 1 mL of cell growth medium at 37 °C according to ISO 10993-12: 2012. Cytotoxicity of single components was also evaluated. Cytotoxicity of water-insoluble PGL was evaluated from extracts obtained after 24 h at 37 °C according to ISO:10993-12: 2012 and also from powder suspended in culture medium at a concentration range of 0.01 -1 mg/mL. Water-soluble PEG-SH was dissolved in full growth medium in the range of concentrations 0.001-10 mg/mL.

The cytotoxicity of extracts was evaluated after 24 h of incubation with 100 % (100 mg/mL) extracts and their dilutions (50 %, 10 %, and 1 %, corresponding to 50, 10 and 1 mg/mL of extracted material, respectively) in sextuplets (n = 6). After the incubation, the tested extracts and polymers were replaced by full growth medium containing MTT at concentration 0.5 mg/mL and incubated for 2 hours. Afterward, the medium was removed and 100 µL of DMSO was added. The DMSO absorbance was measured at the wavelength of 595 nm by a plate reader (Labsystems Multiskan MS).

Statistical analysis. Cytotoxicity of components and polymeric gels extracts is expressed as the percentage of living cells compared to the untreated control cell which was taken as 100 % survival. All results are presented as mean ± SD from sextuplet (n = 6). The statistical analysis of significance in differences was performed using one-way ANOVA followed by Turkey test.

Levofloxacin loading and release

Drug loading-release process was carried out through a diffusional mechanism. In this regard, levofloxacin (LEV) was used as a model drug. Freshly lyophilized samples (0.030 g) were immersed in 1 mL of 30 mg/mL LEV solution in THF, the drug uptake by the samples was monitored by UV-vis spectrophotometry until equilibrium, which was reached after 72 h of incubation under gently mechanical stirring (100 rpm). After preset times the quantity of LEV retained in the samples was calculated by determining the drug content from the supernatant, based on a calibration curve previously obtained, with the equation from relation (3): 𝑦 = 7.062 • 𝑥; 𝑅 2 = 0.998 (3) LEV loaded gels were dried in a ventilated oven at 50 0 C for one day followed by drying in a vacuum oven at 50 0 C until constant weight was attained. Drug release kinetics in ABS (pH = 5.5) was determined by monitoring the wavelength at 294 nm for LEV. Dried LEV loaded gels were gently rinsed with distilled water (3 mL) to wash the surface bound drug. The investigation of the release kinetics was carried out by immersing the samples loaded with LEV in 15 mL acetate buffer solution (pH = 5.5) at 37 °C, under continuous gentle stirring (100 rpm), after which the drug concentration in the supernatant was spectrophotometrically determined. All measurements were performed in triplicate and averaged. Due to the higher swelling degree of PGN materials in THF comparing with aqueous environment, we decided that different samples to be loaded by drug diffusion from concentrated LEV/THF solutions, and subsequently, the LEV release to be measured ABS (pH=5.5).

Results and discussion

Precursor polymer synthesis

The present work proposes the preparation of polymer networks via photoinitiated thiol-ene coupling between alkene functional polyesters, such as PGL, with a polymer possessing pendant thiol functional groups, such as PEG-SH, in THF solution, as presented in Scheme 1. Similarly, as the other polyesters consist of monomer units with a long aliphatic chain, PGL has high hydrophobicity, making it less interactive with the hydrophilic environment. Thus, we decided to include PGL in a polymer network together with a hydrophilic polymer, such as PEG.

The proposed pathway to obtain PGL is an enzymatic ring-opening polymerization catalyzed by lipase (Scheme S1) in bulk. The average molar mass was calculated using both NMR spectroscopy and GPC. The results (Table 2) showed good agreement among these methods, with an Mn of 6350 g/mol but a high dispersity Ð of 3.4, as revealed by GPC analysis. Such broad dispersity would be expected given the bulk enzymatic ROP used for the preparation. Monomer conversion reached 74 %, which is slightly higher than that described previously for toluene solution polymerization 10 . Scheme 1. Schematic route of PGN synthesis via a radical thiol-ene reaction The 1 H-NMR recorded spectra offer useful information regarding the chemical composition of obtained polymer. As shown in fig. 1 the characteristic proton signals of PGL are presented and marked. Spectra was recorded in CDCl3 using a 400 MHz spectrometer. Poly(globalide): 1 H NMR: δ (ppm) 5.59-5.30 (m, CH=CH), 4.17-3.98 (m, CH2O(C=O)), 3.71-3.60 (CH), 2.43-2.24 (CH2(C=O) O), 2.17-1.93, 1.76-1.55, 1.43-1.19 (m, CH2) MALDI MS (Fig. 2) revealed only a low molar mass fraction due to the broad dispersity of the sample. However, the nature of the PGL chains was revealed by the peak-to-peak difference of the m/z values of 238 Da, which corresponds to the mass of the monomer unit. The MS analysis confirmed that the PGL chains are initiated by water according to the structure presented in Scheme 1. PEG-SH was prepared by a polycondensation reaction similar to that in the method reported by Zhu and coworkers 17 . The molar mass was determined by GPC analysis (Table 2), and the structural characterization was also performed by NMR spectroscopy and MALDI MS. As expected for a polycondensation process that involves polydisperse PEG oligomers, PEG-SH copolymers with broad dispersity were obtained. Therefore, MALDI MS could not be used to measure the molar mass. However, the nature of the macromolecular chains was disclosed by analyzing the MS spectrum (Fig. 3a and3b) and confirmed the presence of both PEG and TMA units, noted as PEGxTMAy. In addition to linear PEG-SH copolymer chains, cyclic species were also observed. The NMR spectrum (Fig. 4) was similar to that obtained by Zhu et al., also confirming the successful synthesis of the PEG-SH copolymers. The MALDI MS characterization (fig. 3) revealed that the PEG-SH sample consists in a mixture of macromolecular chains with different number of PEG macromers and thiomaleate comonomers. Being given the broad dispersity of the polymer, the results were considered only as a qualitative measure of the PEG-SH sample. Thus, there may be observed chains having a certain number of PEG macromers and corresponding number of TMA units, which can be noted as PEGxTMAy.

The analyzed sample is consistent with the typical outcome of such polycondensation reactions.

Figure 3a. MALDI analysis of PEG-SH

The assignment of the peaks from the MS spectrum was performed based on the characteristic m/z increment of 44 (fig. 3b), which may be generally observed for PEG. Thus, the main series of peaks may be assigned to K charged PEG-SH species. The m/z calculation of a PEG oligomer may be performed using the general formula m/z = 18 (H2O) + 44 (C2H4O) + 39 (K). In the case of PEG-SH copolymer, the characteristic m/z = 18 (H2O) + 44 (C2H4O) + 39 (K) + 132 (TMA -C4H4O3S). Unfortunately, the mass increment due to TMA presence is also a multiple of 44, thus making more difficult the direct identification of number of TMA comonomers units by a simple MALDI MS analysis. However, considering the Mn of the starting PEG oligomers (600 g/mol, Ð = 1.37) we may ascertain that the observed peaks are corresponding to PEG-SH structures as depicted in the fig. 3a.

Besides the main series of peaks there may be observed a second series with a consistent difference from the first series of 16 Da. Thus, this second series may be assigned as Na charged adducts, m/z = 18 (H2O) + 44 (C2H4O) + 23 (Na) + 132 (TMA -C4H4O3S).

A third series, with a mass decrement of 18 from the second series, may be assigned to cyclic oligoesters. It seems that the cyclic species have a prevalent formation of adducts with Na, but cyclic adduct with K may be also observed. Both PGL and PEG-SH were further characterized by FT-IR (Fig. 5 and6, respectively) and Raman spectroscopy (Fig. 7). Notably, Raman spectroscopy (Fig. 7) revealed a C=C stretching band around 1673 cm -1 arising from the presence of the alkene functionality in the polymer backbone of PGL. Additionally, in the case of PEG-SH, Raman analysis (Fig. 7) revealed a band at 2570 cm -1 , characteristic of thiol groups (SH). Structural characterization of PGL is also performed by FT-IR spectroscopy which regrettably didn't show relevant chemical modification. Figure 5 illustrates the FT-IR spectra of the starting precursor GL and the polymer synthesized PGL. As we can see the characteristic bands of GL and PGL from the spectra are: a) GL: 712 cm -1 , cis RCH=CHR; 968 cm -1 Trans RCH=CHR; 1170 cm -1 , COC; 1248, 1347 cm -1 , COH; 1443, 1460, 2855 and 2926 cm -1 , CH2; 1733 cm -1 , C=O; b) PGL: 721 cm -1 , cis RCH=CHR; 965 cm -1 Trans RCH=CHR; 1174 cm -1 , COC; 2916, 2849 and 1468 cm -1 , CH; 1731 cm -1 , C=O;

Figure 5. FT-IR analysis of PGL and GL monomer Also, in this case, FT-IR spectroscopy of PEG-SH failed to reveal the important chemical moieties on the polymer backbone, the SH groups, respectively. Figure 6 illustrates the FT-IR spectra of the starting precursor PEG and the polymer synthesized PEG-SH. The presence of characteristic bands of PEG and PEG-SH that appear in this spectrum are: 638 cm -1 , S-S; 1033 cm -1 , C-O; 1143 cm -1 , COC; 1246 cm -1 , COH; 1454cm -1 , CH; 1732 cm -1 , C=O; 2570 cm -1 , S-H Figure 6. FT-IR analysis of PEG-SH Raman spectroscopy (Fig. 7) revealed the functional groups of PGL, a characteristic band around 1673 cm -1 arising from the presence of alkenes functionality in the polymer backbone. FT-Raman spectra of PEG-SH (Fig. 7) revealed the presence of thiol groups. The spectra display the presence of characteristic bands of PEG-SH such as: 638 cm-1, S-S; 1038 cm-1 C-O; 1143 cm-1, COC stretch; 1246 cm-1 COH; 1474cm-1, CH2, CH3CH; 1732 cm-1, O-C=O; 2570 cm-1, S-H.

Synthesis of PGN networks by thiol-ene crosslinking

Previous studies concerning the preparation of cross-linked networks based on PGL used smallmolar-mass thiol bifunctional compounds or bis-thiol-functionalized PEG oligomers 15 . The approach for thiol-ene coupling consisted of a radical mechanism using photoinitiators in THF solution. Herein, polyglobalide network (PGN) samples were synthesized by UV-induced thiolene reactions between two polymers, PGL and PEG-SH, at room temperature (Scheme 1). We aimed to determine the influence of the cross-linking reaction parameters of the photoinitiated thiol-ene coupling on the final structure and properties of the PGN (morphology, swelling degree, and recovered gel fraction of the obtained materials). The photoinitiator DMPA was employed to initiate this reaction due to its high efficiency in the generation of reactive radicals 25 . Importantly, when the DMPA photoinitiator was not introduced in the reaction, gelation was not observed. Additionally, each polymer was separately tested under similar irradiation conditions, with and without DMPA, and no gelation was observed. Effective initiation depends on the amount of absorbed light at the exposure wavelength and on the thickness of the sample. The particular radical initiators chosen for the thiol-ene reaction have a significant effect not only on the rate of the reaction but also on the conversion and byproduct formation 26 . To obtain some information about DMPA photoinitiator efficiency in the studied system, first, the gelation point was determined for various SH/C=C functionality molar ratios and DMPA concentrations and for two significantly different reaction volumes. The results (Table S1) revealed gelation time values in the range of 90-240 s when the DMPA concentration was 1 wt. %, with respect to the total amount of polymers, and a low reaction volume (300 µL) was used. The gelation time was 5-10 times higher when the reaction volume increased to 2.6 mL. It should be mentioned that all THF-swollen PGN samples prepared using the higher reaction volume in the presence of a DMPA concentrations of 0.75 or 1 wt. % were difficult to handle and easily breakable. When a DMPA concentration as low as 0.25 wt. % was used, an extremely long gelation time was determined for both low and high reaction volumes, and in some cases, no gelation was observed. It was found that 0.5 wt. % DMPA led to relatively fast gelation while maintaining the good integrity of the gel. Therefore, for all further studies, the PGN gels were prepared in the presence of 0.5 wt. % DMPA. Thus, a series of PGN samples with different initial polymer concentrations in THF and initial SH/C=C functionality molar ratios were prepared (Table 3). All reactions resulted in tacky and transparent gels in a swollen state. All PGN samples, however, gradually lost their transparency and turned white when contacting the water environment during the second purification step (Fig. 8d). After the purification steps, the recovered PGN gel fraction was found to depend on both the total polymer concentration and initial SH/C=C functionality molar ratio (Table 1). During the purification of PGN in THF, all unreacted polymer chains, PGL and PEG-SH, were expected to be removed. Only a slight decrease in the gel fraction was observed after subsequent purification in water, probably caused by the removal of some residual unreacted PEG-SH and/or THF solvent. A higher percentage of recovered gel fraction (Fig. 9) was obtained for the thiol-ene coupling performed using a total polymer concentration of 20 % (best samples, G9 and G12) compared to concentrations of 10 % and 5 %, regardless of the initial SH/C=C functionality molar ratio. This may be explained by the decreased probability of continuous thiol-ene cycles and the increased probability of side reactions of the thiyl and/or carbon-centered radicals at lower total polymer concentrations.

On the other hand, at the same initial polymer concentration, the yield of PGN gels depended on the SH/C=C molar ratio (Fig. 9). At a 20 wt. % polymer concentration, the recovered PGN gel fraction clearly increased from 39 % to 55 % as the SH/C=C ratio decreased from 3/1 to 1/4. This result can be related to the fact that PGL has a higher molar mass; therefore, the local concentration of alkene functionalities is higher compared to the concentration of thiol groups. Ultimately, this may lead to a higher probability for PGL than PEG-SH to become part of the PGN network. Additionally, the number of functional groups per chain length is higher in the case of PGL because the molar mass of the polymer chain between two consecutive functional groups is lower than its counterpart, PEG-SH. Thus, PGL participates more effectively in polymer network formation.

We further used photo rheology to monitor the in situ evolution of the dynamic rheological properties of the PGN formed through photoinduced thiol-ene coupling using viscoelastic quantities. Fig. 9 shows the in situ rheological profile acquired during the thiol-ene coupling of polymeric solution mixtures with different polymer concentrations for a 1/3 SH/C=C ratio. As can be observed from the inset in Fig. 9, at the beginning of the gelation process, G'' exhibited higher values than G', indicating a liquid-like behavior of the polymer mixture. However, over time, G' started to rapidly increase and later overcame G''. The crossover point of the viscoelastic moduli, represented by dashed vertical lines in the inset of Fig. 9, indicates the starting of the gelation process (increasing amount of cross-links in the system). For all samples, the gelation process was completely finished after 25 min, when both viscoelastic moduli became nearly independent of the time or reached at least 90 % of their maximum value. The gelation processes, as well as the gelation kinetics, were slightly different for various polymer concentrations (5 %, 10 %, 20 %, and 40 %) in the polymer mixtures. It can be seen in the Fig. 9 inset that due to the low polymer concentration, the cross-linking reaction (gelation) started after 4 minutes at a polymer concentration of 5 %. However, as the concentration increased to 10 %, the gelation moment shifted to below 1 minute and then slightly increased as the concentration of polymer in the mixture further increased. This slight increase to 1.2 min or 1.8 min at 20 % and 40 %, respectively, is most likely connected to the decreased possibility of cross-linking reactions due to increased restriction of the polymer chain mobility, even though the polymer concentration increased. The polymer concentration also plays an important role in the overall gel stiffness. The highest toughness, nearly 10 kPa for the storage modulus, was found for the 20 % gel, while nearly the same values were obtained at 40 % as at a 10 % polymer concentration, indicating that for the cross-linking kinetics and overall stiffness, there is an optimum in the range of a 10 %-20 % polymer concentration.

Structural characterization of PGN networks

Another issue to be addressed is thiol and double bond consumption during the crosslinking process. In the case of PGN samples (G1-3) possessing a stoichiometrically equivalent amount of double bonds (PGL) and thiol groups (PEG-SH), ideally, complete consumption of thiol groups would occur. However, due to different factors that may affect the loss of radicals, such as the polymer concentration, steric interactions of the polymer chains, steric crowding around the crosslinking sites and different mobility of the polymer chains, some fraction of the functional groups, both thiols and double bonds, may remain unreacted, and the number of remaining thiol groups could provide information about the deviation from the ideal network structure. Such behavior is typical of polymer-polymer crosslinking 3,24 .

To better understand the network formation in the presence (or absence) of thiol or alkene functional groups, the PGN materials were first investigated by Raman spectroscopy (Fig. 10). FT-Raman analysis of PGN samples were recorded in the solid phase. As we can see from Fig. 10, the characteristic bands of PGN samples are: 2572 cm -1 , S-H; 1734 cm -1 , C=O; 1673 cm -1 , C=C; 1001 cm -1 , C-S-C.

Figure 10. Raman analysis of PGN samples with various SH/C=C molar ratios

The spectra revealed C=C stretching at 1673 cm -1 , representing unreacted double bonds of PGL, and SH stretching at 2570 cm -1 , representing unreacted thiol groups. Previously 15 , Raman spectroscopy was used to quantify the amount of unreacted double bonds from the change in the ratio of the bands corresponding to the ester bonds and double bonds. However, in the current study, the PEG-SH chains contained ester bonds, and such a quantitative determination was not possible. Thus, the Raman spectra were used here only for a qualitative evaluation. The SH stretching band clearly faded as the initial SH/C=C ratio decreased, i.e., as the PGL content in the initial reaction mixture increased. In addition, excess thiol groups in the initial mixture did not lead to complete double bond conversion. Raman analysis of the relative ratios between the bands corresponding to thiol and alkene groups qualitatively confirmed a trend of thiol-ene interactions with the formation of the C-S-C links between PGL and PEG-SH macromolecular chains. The thiol-ene coupling could also be confirmed by the presence of a weak signal band at 1001 cm -1 , which corresponds to C-S-C bonds.

To evaluate the extent of thiol-ene coupling, further characterization of the PGN samples was performed by elemental analysis and titration of the thiol groups using Ellman's method. The results obtained by elemental analysis are listed in Fig. 11 and Table 4. The observed gravimetric ratios between elements (herein, the sulfur content is mainly considered) were not expected to be similar to the initial ones due to the incomplete thiol-ene coupling and high yield of unreacted polymers removed from the PGN by purification. During the removal of unreacted polymers, the existing ratio of the elements in the initial reaction mixtures may be affected by removing nonproportional amounts of unreacted PEG-SH or PGL with respect to the initial ratios. However, as seen from the results of elemental analysis of PGN prepared using a 20 % polymer concentration and various initial SH/C=C ratios, the sulfur content was slightly lower than the initial theoretical content calculated from the initial ratio of the starting polymers to the recovered gel fraction (Fig. 11). This fact suggests that during purification, the relative amount of removed unreacted PEG-SH was only slightly higher than that of PGL. For example, in the case of the G3 sample with an initial SH/C=C ratio of 1/1, there was a decrease of approximately 12 % in the sulfur content (relative to the theoretical sulfur content), which should reflect the proportional relative loss of PEG-SH compared to the original content of the polymer mixture. The deviation of the sulfur content in all PGNs from the theoretical reaction mixtures was generally in the interval of 5 to 10 %. The tendency for the reaction system to yield slightly more unreacted PEG-SH than PGL is in agreement with the higher probability of PGL being involved in the network due to its higher molar mass and higher functional density.

Fig. 11 also shows the relative amount of sulfur present in the PGN samples in the form of unreacted thiol groups, as determined by Ellman's titration. The relative amount of this sulfur decreases with the shift in the initial molar ratio in favor of double bonds (the increasing amount of PGL in the initial polymer mixture). The difference between the total sulfur and the sulfur from unreacted thiol groups is related to the reacted sulfur from transformed thiols, i.e., most likely from the C-S-C bonds between PGL and PEG-SH, as represented in Fig. 12, even though the formation of some S-S connections cannot be completely excluded. Thus, for a 1/1 SH/C=C ratio, 49 % of the sulfur remaining in the polymer network was involved in crosslinking reactions. However, only 42 % of the initial polymer content was recovered after purification, and thus, the proportion of thiol groups involved in crosslinking reactions was only approximately 21 %. The residual 79 % remained as unreacted SH groups either attached to the polymer network or leached during purification.

In the case of PGN obtained using a high initial thiol content, i.e., a SH/C=C ratio of 3/1, it can be observed that the relative amount of thiol groups was lower than theoretically expected. For a SH/C=C ratio of 3/1, at least 66 % of the sulfur should remain in the form of unreacted thiol groups. However, the determined amount of free thiol groups was approximately 48 %. Therefore, we may assume that the thiol groups were also consumed through a process other than thiol-ene coupling.

A main secondary process that can occur is thiol-thiol coupling, resulting in the formation of disulfide bonds (S-S). The formation of disulfide bonds was not quantified and may be considered for all studied PGN materials. However, we may consider that such processes are more prevalent under conditions of excess PEG-SH in the reaction mixture, where the PEG-SH chains have fewer connections to the network and, as consequence, have higher mobility. Thus, the comparison between the sulfur content determined by elemental analysis and thiol titration should be made under the assumption that the free thiol groups may be partially affected by S-S coupling. Higher thiol consumption was observed for samples with higher initial PGL contents (lower SH/C=C ratios). Under conditions of using 3 or 4 times more double bonds than thiol groups, approximately 79 % of the sulfur remaining in the PGN after purification was coupled to a carbon double bond (Fig. 12). Thus, the thiol consumption of the investigated thiol-ene reaction system seems to be limited to a maximum of approximately 46 %, taking into account the PGN fraction after purification. These calculations, however, did not take into consideration the fact that the purification processes may also lead to the removal of partially reacted polymers that are not part of the formed network but are sufficiently mobile to diffuse through the network and to be removed during purification.

The effect of the initial polymer concentration on the amount of unreacted thiol groups in the purified PGN materials was also studied. The analyses of the residual thiol content and the thiol groups consumed during network formation for all PGN samples are presented in Fig. 13 and Table 5.

The evaluation of free thiol groups of PGN samples was done by utilizing Ellman's reagent (5,5'dithiobis-[2-nitrobenzoic acid]) (DTNB). DTNB reacts with thiol groups forming a yellow colored product, 5-thio-2-nitrobenzoic acid (TNB 2-), which possesses high absorbance at 412 nm. The intensity of yellow color increased proportionally with the reduced thiol content. The amount of free thiol groups was calculated by extrapolating the obtained results with an according standard curve. A linear calibration curve for comparison was obtained using solutions of known amount of thiomalic acid, with following equation: 𝑦 = 0.0697 • 𝑥; 𝑅 2 = 0.9956 (4) The results obtained for the PGN samples following the Ellman's test are outlined in Table 5 It can be observed that the amount of free thiol groups consistently decreased with the polymer concentration at all SH/C=C ratios. Thus, the PGN obtained at lower concentrations contained more reacted sulfur. However, the gel recovery also diminished with a decrease in the polymer concentration (Table 1), thus decreasing the total thiol conversion.

On the basis of the obtained results for samples G1-G18, we can conclude that the proportion of unreacted thiol groups is clearly dependent on both the initial total polymer concentration and the feed SH/C=C molar ratio. We may postulate that once the polymer chains start to become a part of the crosslinked network, there might appear a certain phase separation induced by decreased solubility. Such phase separation is not characterized by a change in the transparency of the reacting THF polymer solution during photoinduced thiol-ene coupling probably due to the good swelling properties of both PGL and PEG-SH in THF, as was also observed for the final PGN gels swollen in THF. The transparency of PGL-based crosslinked networks was also noticed by van der Meulen et al. 10 Therefore, if the polymer concentration is lower, at the same volume, the crosslinked polymer phase would be more disperse and, as consequence, more readily available for thiol-ene reactions, thus explaining the lower content of free thiols in the purified PGN prepared with lower feed polymer concentrations.

Morphology

SEM was used to visualize the morphological characteristics of lyophilized polymeric gels. Cross-sections of the prepared PGN materials were analyzed to determine the influence of the SH/C=C molar ratio and polymer feed concentration on the morphology of the materials. First, the cross-sectional morphology of the PGN samples with different SH/C=C feed ratios was considered (Fig. 14). It could be observed for the samples prepared with excess PEG-SH (Fig. 14A-C) that the PGN materials were macroporous with interconnected pores with sizes ranging from 10 to 40 μm. The porous structure was homogenous; however, small insertions of less porous regions could also be observed. The appearance of the porous structures is probably related to the specific thiolene coupling conditions, in particular, the high content of PEG-SH in the polymer feed. The increase in the PGL amount to reach an equimolar SH/C=C ratio led to a decrease in the proportion of the porous region in the PGN materials (Fig. 14D-F). Thus, some large pores with irregular sizes and shapes could be observed at a 1/1 SH/C=C ratio. The PGN obtained with excess PGL did not exhibit a porous morphology. In the last set of images (Fig. 14G-I) obtained at a 1/4 SH/C=C ratio, the pores are no longer present. However, heterogeneity could be observed at the highest magnification (Fig. 14I), which could have been caused by phase separation due to the crosslinking of polymers with different hydrophilic/hydrophobic properties. These microphases were uniformly spread, with an average size of approximately 1 μm. The formation of the porous structures also depended on the initial concentration of the polymers. As can be observed from Fig. 15, the morphology of the PGN obtained at a 3/1 SH/C=C ratio was porous with rather regular pore sizes and shapes at each polymer concentration. The PGN materials obtained at lower concentrations had pores with larger sizes, i.e., approximately 30, 60 and 150 μm for the PGNs obtained at 20, 10 and 5 % feed polymer concentrations, respectively. Thus, the size of the pores can be controlled using different feed polymer concentrations. While it is obvious that the thiol-ene coupling process governs pore formation, a precise description of the mechanism may have to take into consideration the interactions between the polymers and THF environment during the crosslinking reaction. To the best of our knowledge, the process of pore formation during a crosslinking reaction via photoinduced thio-ene coupling of hydrophilic/hydrophobic polymers (such as PEG-SH and PGL) has not yet been elucidated.

As previously described in the literature 26 , polymers containing meso-/macropores can be prepared by reaction-induced phase separation. Thus, the polymers progressively segregate from the solution to form microscale cross-linked phases that can be considered network units, followed by covalent cross-linking in various directions to form a 3D microscale network. Such porous polymer networks are usually prepared by direct polymerization, where the phase separation occurs during the polymerization process. However, in our case, the phase separation probably occurs during crosslinking. The PEG-SH-rich phases in THF seem to have an important porogenic effect since porous structures were obtained with a high PEG-SH content in the initial polymer feed. The observation of the porous structure also supports our hypothesis that a certain crosslinked polymer phase is formed during the thiol-ene coupling process, preventing higher thiol group conversion. Such polymer phase separation would lead to a local entrapment of the reactive species and consumption, while the free polymer chains in the THF solution will not undergo thiol-ene crosslinking.

Swelling behavior

The PGN materials were prepared by crosslinking a hydrophilic polymer with a hydrophobic polymer. Therefore, it could be expected, the prepared materials should possess the capacity to swell in both hydrophilic and hydrophobic environments. Such materials would include the prepared materials in the category of amphiphilic conetworks 20 . The swelling behavior of the PGN samples was studied in water (Fig. 16) and THF (Fig. 18) by recording the liquid uptake at predetermined time intervals until equilibrium swelling was reached.

Figure 16. Water uptake of PGN gels

Swelling behaviors kinetics of PGN samples were studied in water (fig. 17) and THF (fig. 19).

Figure 17. Water uptake kinetics in water (20.5 hours) All PGN gels exhibited quite low water uptake, in the range of 20 % to 70 % (Fig. 16). This behavior could depend on the content of PEG-SH, the hydrophilic polymer present in the reaction mixture. The best swelling ratio in water was achieved for the samples obtained at a high content of PEG-SH and at a higher feed polymer concentration of 20 %. The higher water uptake by PGN obtained at higher polymer feed concentrations is opposite the expected result since at lower concentrations, a looser network should be formed; furthermore, the pores observed in the SEM Water uptake for samples G1,G2,G3

Water uptake for samples G4,G5,G6

Water uptake for samples G7,G8,G9 Water uptake for samples G10,G11,G12

Water uptake for samples G13,G14,G15 Water uptake for samples G16,G17,G18

images were larger at lower polymer concentrations, and, as consequence, better swelling properties could be expected. Therefore, we can propose that water does not properly interact with the PGN materials, even in materials with higher PEG-SH contents. The water uptake increased with the initial content of PEG-SH only for the PGN prepared at a 20 % feed polymer concentration. Nevertheless, given the presence of PGL (hydrophobic polymer) in the PGN materials, the observed interaction with water is a remarkable feature.

In contrast, very good swelling properties were observed in THF, where the swelling degree of the PGN gels was in the range of 400-1000 % (Fig. 18). The swelling degree of gels in THF showed a dependence on both the SH/C=C ratio and total initial polymer concentration. For PGNs obtained at lower polymer concentrations (5 wt. %), we observed a higher swelling degree in THF, probably due to a more relaxed network compared with that obtained at the higher two concentrations (10 and 20 wt. %). The swelling degree also clearly increased with the increase in the feed content of PGL. Thus, unlike water, which probably penetrates the PGN only in the PEG-SH area and loosely packed phases, THF interacts well with all the components of the network, thus revealing its hydrophobic nature.

The swelling properties of PGN in both water and THF suggested that PEG-SH is not fully part of the PGN and loses its unreacted polymer chain ends. This is also justified by the large number of unreacted thiol groups. Moreover, water could be only superficially absorbed by PGN materials and probably does not diffuse fully into the inner polymer structure, which is largely hydrophobic. This behavior was observed even for the PGN prepared with a large excess of PEG-SH in the polymer feed. However, the water-swelling properties of a highly hydrophobic polymer such as PGL can be altered by thiol-ene coupling with a more hydrophilic counterpart. Such features may be highly valuable in altering the hydrophobic character of other polymers by thiol-ene coupling. Swelling behavior in THF for samples G4,G5,G6

Swelling behavior in THF for samples G7,G8,G9

Swelling behavior in THF for samples G10,G11,G12

Swelling behavior in THF for samples G13,G14,G15

Swelling behavior in THF for samples G16,G17,G18

Cytotoxicity study

The possibility of using PGN materials for biomedical applications was also investigated. In this respect, cytotoxicity was assessed based on ISO norm 10993-12 with the pure polymers used for gel preparation as well as the PGN gel extracts. As expected, PEG-SH is a highly biocompatible polymer, and the cell viability of 3T3 fibroblasts treated with concentrations of up to 10 mg/mL was comparable to the viability of untreated control cells (Fig. 21A). Similar PEG derivatives modified with maleic anhydride (MA) or DL-thiomalic acid (TMA) also possessed very low cytotoxicity to human mesothelial cells Met-5A 27 . PGL, as the second polymer used for PGN preparation, was also evaluated. Since the PGL polymer is water insoluble, the powder extract was used for the cytotoxicity evaluation. In the study of van der Meulen et al. 10 , the viability of 3T3 fibroblasts decreased by more than 1/3 in comparison to control cells when incubated with extracts of 0.1 g/mL PGL. In this study, extracts of 0.2 g/mL PGL and corresponding dilutions were used according to ISO10993-12 (Fig. 21B). As shown in this figure, extracts of 0.2 g/mL PGL were highly cytotoxic to 3T3 fibroblasts. Extracts of 0.1 g/mL PGL decreased the cell viability to ca. 50 %, which is slightly lower than that in the study of van der Meulen et al. 10 , corresponding to the inhibitory concentration (IC50). In addition, we also tested the cell viability when PGL powder was directly added to the culture medium (Fig. 21C). At a concentration of 10 mg/mL, the cell viability decreased to approximately 90 %, corresponding to the cytotoxicity of extracts of the same concentration.

The cytotoxicity of three PGN samples differing in their SH/C=C molar ratio was also evaluated (Fig. 20). All tested polymer networks were considered biocompatible up to a 50 mg/mL solid gel concentration in the extracts, as the viability was above 80 %. A decrease in cell viability to ~ 80 % at a statistically significant level (P > 0.001) was valid for extracts of gels with SH/C=C ratios of 1/2 and 1/1. If the cells were treated with extracts obtained from a 100 mg/mL solid gel, the cytotoxicity increased significantly in all cases (P > 0.001). Interestingly, the highest decrease in cell viability was observed for an SH/C=C ratio of 1/1. The most biocompatible gel had an SH/C=C ratio of 1/2, containing the highest content of reacted sulfur. The resulting differences in cytotoxicity between the gels could be assigned to the interplay between several factors: steric effects, the acidity of the thiol group, the stability of the thiolate anion and the thiyl radical, hydrophilicity, protein interactions 28 and precursor polymer purity.

Levofloxacin release study

The possibility of using PGN materials for drug delivery was also investigated. The drug loading and release characteristics of PGN materials were examined using levofloxacin (LEV), a broadspectrum, third-generation fluoroquinolone antibiotic, as a drug model. The influence of the SH/C=C ratio and the feed polymer concentration on the PGN drug loading capacity was investigated. Different samples were loaded with LEV in THF, and subsequently, the released LEV in ABS (pH = 5.5) was measured. The higher swelling ability of PGN materials in THF ensured a higher loading efficiency of LEV, comparing with aqueous solutions, and on the other hand, we assumed that the lower swelling ability in water will provide a reduced rate of LEV release. The loaded LEV amount after 48 hours varied between 0.32 and 0.41 mg LEV/mg PGN (Table 6); thus, no significant difference was observed with respect to the SH/C=C ratio or polymer concentration. The samples with higher PGL contents (SH/C=C ratio of 1/4) demonstrated a higher load capacity probably due to better swelling in THF. To evaluate the LEV release, drug-loaded gels were stripped of their THF and gently rinsed with distilled water before adding them to the release medium. Example kinetic data are presented for samples G3, G12, and G18 in Fig 23 . One can observe a fast phase that is reached within the initial 10 hours, followed by a slower phase (characterized by a constant release) until 48 hours. The PGN samples were analyzed also concerning the LEV release kinetics, wich was accomplished based on the Korsmeyer-Peppas mathematical model 7 following equation:

M t M∞ = k • t n (8)
where: k is a kinetic parameter that characterizes the interactions between drug and polymer, n is the parameter that specifies the release mechanism and was determined from experimental data.

The values of the difusional exponent were close to 0.5 and corresponded to a transport/release mechanism dominated by diffusion. Table 6 present the values obtained for loaded, released amount of LEV and the efficiency of released LEV for the tested samples. The sustained release of LEV can be explained by the fact that the released drug was adsorbed in the internal structure of the gel due to its excellent swelling in THF. By analyzing the influence of the preparation parameters on the release ability of the gels, a behavior similar to the drug loading behavior was observed. The maximum amount of released LEV varied between 0.22 and 0.38 mg/mg gel (Table 6), and no significant difference was noticed among the samples.

Additionally, when calculating the efficiency of LEV release for all samples, values between 62 and 100 % were obtained (Fig. 22), and the highest efficiency was observed for samples prepared with SH/C=C ratio of 3/1, which were shown to be porous and to have the highest water uptake. The analysis of the release kinetics accomplished based on the Korsmeyer-Peppas mathematical model 28 (ESI Eq. 8) demonstrated that the drug transport/release mechanism was dominated by diffusion. The obtained results demonstrated that PGN materials can be a promising and attractive controlled drug delivery systems for a variety of applications.

Conclusions

Successful thiol-ene coupling between two polymers (PGL and PEG-SH) with highly different hydrophilic/hydrophobic properties was achieved via photoinduced thiol-ene coupling. The effect of synthesis parameters such as the feed polymer concentration and feed SH/C=C ratio on the structure and properties of the final PGN gels were studied. The recovered gel fractions were highest at a 20 % initial polymer concentration in the reaction solvent and increased with an increasing initial PGL content (decreasing SH/C=C ratio). The characterization methods employed (Raman, FTIR spectroscopy, elemental analysis and thiol group titration) showed that significant amounts of SH groups remained unreacted. At a 1/4 SH/C=C ratio, approximately 20 % of the thiol groups remained unreacted, and at a 1/1 SH/C=C ratio, only 50 % of the sulfur found in the final product was from reacted thiol groups. This reaction system design led to the formation of macroporous, regulated structures, demonstrated here for the first time for a material prepared by a photoinitiated thiol-ene reaction between two polymers. This phenomenon depends on both the feed polymer concentration and the ratio between the reacting polymers. Thus, excess PEG-SH (3/1 SH/C=C) led to the formation of uniformly distributed, intercommunicating pores, as shown by SEM analysis. By increasing the amount of PGL in the polymer feed, the porous character of the PGN materials was reduced (at 1/1 SH/C=C) and eventually completely disappeared (1/4 SH/C=C). Additionally, the size of the pores depended on the initial polymer concentration. The prepared materials demonstrated good swelling in THF and rather limited water uptake. The cytotoxicity of the crosslinked networks, determined using 3T3 fibroblasts, demonstrated that PGN materials possess high biocompatibility at a solid gel content of up to 50 mg/mL in the extracts and can be considered for biomedical applications. The PGNs were loaded with a model drug in a THF environment, and the water release was studied; fast release was achieved during the initial 10 hours, followed by a slower phase (characterized by constant release) until 48 hours.

Overall, the presented studies demonstrated the possibility of incorporating hydrophilic polymers into hydrophobic polyglobalide networks via thiol-ene polymer-polymer coupling to obtain nontoxic porous biomaterials with possible applications as drug delivery devices. Moreover, the porosity of these materials may be important for further tissue engineering applications.

Introduction

Disorders affecting posterior eye segment like age-related macular degeneration (AMD), diabetic macular edema, viral retinitis, proliferative vitreoretinopathy, posterior uveitis, retinal vascular occlusions, choroid neovascularization (CNV), and diabetic retinopathy are increasing every year at an alarming rate [1], [2], [3]. Generally, drug delivery to the posterior segment is limited due to the difficulty in delivering effective exact doses of drugs to target tissues within the eye. The administration of pharmaceutical agents may be delivered to the eye through four primary pathways of administration: topical, systemic, intravitreal and periocular [1]. Current, proven treatment regimens for ocular neovascularization include ocular photodynamic therapy (PDT) and laser photocoagulation to either directly treat the choroidal neovascular membrane (CNV) as in AMD, or ablate the ischemic retina sparing the macula and non-ischemic areas as in diabetic retinopathy, vein occlusions, retinopathy of prematurity and anterior segment neovascularization. Antiangiogenic therapy relies on a different approach to treat neovascular diseases. The treatment directly targets the angiogenic cascade that it is thought to be initiated by several growth factors such as vascular endothelial growth factor (VEGF), platelet derived growth factor, transforming growth factor, and basic fibroblast growth factor (bFGF). The advantage of antiangiogenic therapy is its potential to preserve the function of retinal tissue while directly targeting the neovascular complexes. The antiangiogenic drugs that are currently known as effective are: monoclonal antibodies that target specific molecular sites; bevacizumab (Avastin)the -,,parent,, molecule of ranibizumab, a full-length monoclonal antibody; corticosteroids (intravitreal triamcinolone acetonide, dexamethasone, fluocinolone acetonide, anecortave acetate) [4], [5]. Bevacizumab was authorized in 2004 for the chemotherapy treatment of metastatic colorectal cancer [6], in 2006 for cell lung cancer [7], in 2009 for glioblastoma multiform [8]. Intravitreal injections of BEV have been used for the treatment of several eye disorders such as AMD; CRVO; PDR; rubeosis iridis; pseudophakia cystoid macular edema (CME); CNV; secondary to pathologic myopia. Studies in vivo revealed the absence of ocular toxicity for BEV administrated by intravenous injection. Due to its short half-life and the necessity of frequent intravitreal injection, a method for sustained delivery is required [9], [10], [11], [12]. Studies concerning BEV usefulness in the field of ophthalmology as an off-label drug for the treatment of wet age-related macular degeneration (AMD) have been reported. Abrishami et al. study reported that liposome loaded with BEV presented a beneficial effect such as sustained release in the vitreous in animal model during 42 days [13]. Nanosystems based on polymeric biomaterials have been extensively investigated because of their numerous advantages, such as protecting sensitive drug molecules from degradation, controlled release properties, and increasing the bioavailability of poorly water-soluble drugs [14]. Hao et al. have prepared NPs based on poly(D,L-lactide-co-glycolide)-loaded BEV, capable to have a prolonged release for 4 weeks [15]. Also, Pan and co-workers have successfully performed two long-lasting formulation based on poly(ethylene-glycol) and poly (lactic-co-glycolic acid). Authors obtained NPs based on poly(lactic-co-glycolic acid)-encapsulated BEV via solid-in-oil-in-water encapsulation method and poly(ethylene-glycol)-BEV conjugate for the treatment of choroid neovascularization. Both formulation presented a long and sustained release of BEV during 8 weeks [16]. Micro/nanospheres were prepared from poly(D,L-lactide-co-glycolide) and poly(ethylene glycol)b-poly(D,L-lactic acid) for the treatment of age-related macular degeneration (AMD) by Fengfu and colobarotors. The study results revealed that BEV loaded micro/nanospheres could be released in a controlled sustained manner over 90 days. The drug release rate could be adjusted by modification of the drug/polymer ratio. Study results indicate that this type of carrier's system loaded with drug could improve the treatment of wet AMD and cancer [17]. Also, the use of cationic polysaccharides such as chitosan and its derivatives as polymeric matrix for drug delivery generate particular interest due to their unique physicochemical and biological properties. Chitosan (CS) is a natural biopolymer obtained by alkaline deacetylation of chitin, an abundant biopolymer isolated from the exoskeleton of crustaceans, such as shrimps. This natural amino polysaccharide stands out due to its properties like biocompatibility, biodegradability, mucoadhesivity [14] and characteristics such as anti-bacterial, anti-tumor, hemostatic, low toxicity and immunogenicity, fungal, analgesic, and film-forming, pH sensitivity. It is suitable to easily prepare targeted drug-delivery nanocarriers [18], [19], [20]. Still, the most important obstacle associated with the use of chitosan for improvement of drug absorption is its limited solubility at pH higher than its apparent pKa of 6.5, meaning is only soluble in some dilute acid solutions such as acetic acid and hydrochloric acid. Thus, a various number of different chemical modification for improving chitosan solubility in water such as sulfonation, quaternarization, carboxymethylation, N-and O-hydroxyalkylation, modification with thiol groups, graft copolymerization etc. have been reported [21]. Chemical modification of chitosan via graft copolymerization is a promising method and provides a wide variety of molecular characteristics. Grafting hydrophilic natural or synthetic polymers onto CS backbone not only enhances the water solubility of CS but also form particles and maintains the stability in dilute conditions [22]. Poly(ethylene glycol) (PEG) is a linear polyether diol, frequently used as a non-ionic hydrophilic polymer for its « camouflage behavior ». PEG is an excellent graft forming polymer because is soluble in water and organic solvents, and possess thermo responsive properties, low toxicity, good biocompatibility and biodegradability [23]. Nevertheless, particular attention has been dedicated to PEGylated chitosan (PEG-g-CS), the grafting process usually being performed on the NH2 group with various approaches available. Generally, one of the PEG-g-CS advantages is the water solubility in a wide range of pH from 1.0 to 11.0, depending on the substitution degree [24]. Nanoparticulate systems-based PEG-g-CS deals with the main problems, such as rapid clearance from the blood stream through phagocytosis after intravenous administration and recognition by the macrophages of the mono-nuclear phagocyte system [23]. PEGylation of natural or synthetic polymers or drugs benefits of nontoxicity, reduced clearance by the reticuloendothelial system, a prolonged circulation time in the blood flow, and an increased stability of the drug against enzymatic degradation and reduce its immunogenicity [24], [23]. PEG grafted CS nanocarriers have been investigated as potential applications in drug delivery [25]. Due to the presence of unmodified amine groups PEGylated chitosan have proprieties such as selfaggregation (by strong inter-or intra-molecular hydrogen bonds) or complexation (by electrostatic interaction with negatively charged compounds), which makes them suitable materials for potential applications in drug delivery [24].

Methods used to obtain nanoparticulate systems-based PEG-g-CS are self-aggregation [26], complexation [25], ionic gelation technique [27], [28], [29], [START_REF] Aktas | Development and brain delivery of chitosan-PEG nanoparticles functionalized with the monoclonal antibody OX26[END_REF], solvent evaporation method [START_REF] Falabella | In vivo validation of biological responses of bfgf released from microspheres formulated by blending poly-lactide-coglycolide and poly(ethylene glycol)-grafted-chitosan in hamster cheek pouch microcirculatory models[END_REF]. For example, Ouchi et al. prepared a copolymer mPEG-g-CS which spontaneously form nanosized (<120 nm) aggregates in aqueous solution by strong intermolecular hydrogen bonds between CS moieties. Authors have investigated the aggregation phenomenon of the copolymer [26]. Bae et al. have synthesized PEG-g-CS/heparin a self-assembly polyelectrolyte complex for inducing apoptotic death of cancer (B16F100) cells in vitro. The micelles (200 nm) obtained were stable under physiological environment due to the PEG shell layer [START_REF] Bae | Intracellular delivery of heparin complexed with chitosan-g-poly(ethylene glycol) for inducing apoptosis[END_REF]. Other study performed by Yang et al. shows the ability of mPEG-g-CS to self-aggregate and forming nanosized carriers (300 nm) with loading efficiency depending of DS (degree of substitution) [25]. Zhang et al. performed a mPEG-g-CS used for prepare NPs (>300 nm) by the ionic gelation method using sodium triphosphate (TPP) as cross-linker. The NPs were loaded with insulin and presented a loading efficiency of 38%. The study results showed that the NPs diameter and insulin release from mPEG-g-CS/TPP/insulin NPs could be controlled just by modifying the composition or temperature [START_REF] Zhang | PEG-grafted chitosan nanoparticles as an injectable carrier for sustained protein release[END_REF]. Herein, we decided to uncover the particularities of novel micro/nanoparticles (MNPs) -based polymer systems for the transport, targeting and controlled release of drugs used to treat diseases of posterior segment of the eye. Thus, in the present work, we study the synthesis of a new polymer chitosan grafted PEG methacrylate and also, the preparation of the micro/nanoparticulate system. The strategy involves the use of CS-g-PEGMA with high solubility in water, obtained via Michael addition, in order to prepare potentially non-toxic MNPs. First, the synthesis and thorough characterization of the polymer and the methods used for CS-g-PEGMA characterization (FT-IR, NMR, TGA, DSC) have been described. Secondly, the obtained MNPs characteristics such as size, polydispersity, the degree of swelling can be controlled by changing the reaction parameters (water/oil ratio, polymer concentration, dispersion speed, amount of cross-linking agent, the viscosity of the two phases, etc.). The MNPs were obtained through a double crosslinking technique (ionic and covalent) applied in reverse emulsion system, previously elaborated by our research group. The NPs have been analyzed structurally by FT-IR spectroscopy, morphologically by SEM, gravimetrically for swelling abilities. Also, hemocompatibility and cytotoxicity were performed to test the applicative potential of the particles. Finally, bevacizumab loading and its in vitro and ex vivo release are studied. Also, the antiangiogenic effect of selected MNPs was studied for diabetes/inflammatory diseases of the eye and central retinal vein occlusion -experimental animal model.

Materials

The following materials were used for the experiments: low molecular weight Chitosan (CS), (degree of deacetylation 82 %, Aldrich); Poly(ethylene glycol) methacrylate (PEGMA, Mn = 500, Aldrich); Acetic acid (99%, Sigma Aldrich); Glutaraldehyde (GA) (Sigma Aldrich, 25% aqueous solution); Sodium tripolyphosphate (TPP, Sigma Aldrich); Sodium sulfate (Na2SO4, Sigma Aldrich); Bevacizumab [(BEV); (25mg/ml, Avastin®, Genentech Inc)] was provided by University of Medicine and Pharmacy "Grigore T Popa" Iasi, Romania; Levofloxacin (LEV; Sigma Aldrich) Tween 80 (Sigma Aldrich); Span 80 (Sigma Aldrich); acetate and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich; lipopolysaccharide, E. coli 055:B5 (LPS, Sigma Aldrich); adult rabbits from Baneasa (Romania); Eritrosein B ( ≥95 %, Sigma Aldrich); ketamine (Sigma Aldrich); xylazine (99 %, Sigma Aldrich); PBS solution Hank (HBSS, Sigma Aldrich); collagenase / dispase ( Sigma Aldrich); bovine serum albumin (Sigma Aldrich); GST tag (26 kDa, Sigma-Aldrich); Milli-Q ultrapure distilled water (Merck) were of analytical grade and used without further purification. The chemicals used in this study were of analytical grade purity and were used without further purification. The human blood samples used were freshly obtained from one healthy nonsmoker volunteer. Fibroblast cells were extracted from rabbit dermis in the Bioengineering Department of "Grigore T. Popa" University of Medicine and Pharmacy, Iasi, Romania.

Equipment

Fourier-transform infrared spectroscopy spectra of CS, PEGMA and CS-g-PEGMA derivative were recorded by a DIGILAB SCIMITAR FTS 2000 spectrometer. The samples were prepared as KBr pellets and scanned over the wave number range of 4000-450cm -1 at a resolution of 4.0 cm -1 . 1 H NMR spectra were obtained by using a Bruker Avance DRX 400. CS, PEGMA (Mn=500) and CS-g-PEGMA derivative were dissolved in D3CCOOD/D2O or D2O according to their solubility. The substitution degrees (DS) were calculated from the peak area at about chemical shift of -CH2b-proton against that of NHAc proton. Thermo gravimetric analysis (TGA) was carried out with a Mettler Toledo model TGA/SDTA 851 (TGA Q 500-1285 system). Samples (2÷5 mg) were loaded in an open aluminum pans, heated under a dynamic N2 atmosphere, with a heating rate of 10 0 C/min, in a temperature range from the room temperature to 800 0 C. CS-g-PEGMA NPs morphology was investigated by SEM technique using a HITACHI SU 1510 (Hitachi SU-1510) scanning electron microscope. Also, MNPs mean diameter and size distribution were analyzed by laser light diffractometry technique (SHIMADZU SALD 7001). Differential scanning calorimetry (DSC) was carried out with a Q20 Series™-DSC. Samples (2÷5 mg) were analyzed under a N2 atmosphere on the temperature range 25 °C -300 °C.

Experimental part 4.1. Synthesis of chitosan grafted poly (ethylene glycol) methacrylate (CS-g-PEGMA)

In order to obtain PEGMA-g-CS via Michael addition reaction, the modified protocol reported by Ma and collaborators was considered [START_REF] Peptu | Double cross-linked chitosangelatin particulate systems for ophthalmic applications[END_REF] , [START_REF] Alupei | New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery[END_REF], [START_REF] Jatariu (cadinoiu | Double crosslinked interpenetrated network in nanoparticle form for drug targeting-Preparation, characterization and biodistribution studies[END_REF]. Briefly, chitosan (1.0 g) and 100 mL acetic acid solution 1% (w/w) were added to a 250-ml reaction flask equipped with a reflux condenser. The reaction flask was immersed into a water bath heated at 40 0 C, purged with nitrogen and allowed to stir for 60 min. Subsequently, PEGMA was added drop wise at a final molar ratio NH2: PEG = 1:2. Afterwards, Michel addition reaction was carried out at 60 0 C, under nitrogen atmosphere and maintained for 24 h to complete the reaction. Finally, the polymer solution was filtered by centrifugation, rotary evaporated, and precipitated into acetone. The polymer was purified by repeated washing with methanol and acetone. The final product was dried in a vacuum oven for 48 h to constant weight. A light brown powder was obtained.

Preparation of MNPs based CS-g-PEGMA

CS-g-PEGMA MNPs were performed by double cross-linking, ionic followed by covalent in reverse emulsion (w/o) according to a method reported by C.A. Peptu and coworkers [START_REF] Peptu | Double cross-linked chitosangelatin particulate systems for ophthalmic applications[END_REF], [START_REF] Alupei | New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery[END_REF], [START_REF] Jatariu | Double crosslinked interpenetrated network in nanoparticle form for drug targeting-Preparation, characterization and biodistribution studies[END_REF]. Therefore, a calculated amount of CS-g-PEGMA (depending of desired concentration) was dissolved in CH3COOH solution (1% w/w). An appropriate quantity of nonionic surfactant Tween 80 (2 % w/w) was added in the polymer solution and well homogenized. Afterwards, the mix was added slowly drop wise in toluene containing the adequate quantity of surfactant (Span 80) (200 mL), under vigorous stirring (different stirring speed). Following the emulsion stabilization time, an amount of ionic cross-linker solution (TPP or Na2SO4, 5%) was added and transferred to a mechanical stirring-equipped reactor, were the process of ionic cross-linking continued at 500 rpm. The covalent cross-linking process, was carried out by adding in the reactor a calculated amount of glutaraldehyde extracted in toluene (c = 1.12 mg/mL). After crosslinking reaction ended, the emulsion was centrifuged and removed the toluene phase. MNPs obtained were repeatedly washed with distilled water, acetone and hexane, before being dried out at room temperature. After the purification steps MNPs samples were considered for chraracterization: diameter and size distribution; morphology; swelling behavior, drug loading and release, zeta potential, hemocompatibility analysis, toxicity and cytotoxicity test, etc.The experimental protocol with the observed parameters for the preparation of MNPs and their average diameter are presented in Table 1

Nanoparticles behavior in aqueous media

In order to predict and understand the behavior of MNPs during loading-release processes, swelling studies were performed by gravimetric method. 0.030 g freeze-dried samples of MNPs freeze-dried were weighted and immersed in vials containing 1 ml acetate buffer solution (ABS) (pH=3,3) or phosphate buffer solution (PBS) (pH=7,4). The vials were maintained at room temperature under mechanical stirring. Swelling process was monitored and after preset times, MNPs suspensions were ultra-centrifuged (15.000 rpm) and weighted after supernatant removal. Swelling process was monitored by recording the liquid uptake at pre-determined time intervals until equilibrium swelling was attained. The percentage of swelling ratio was determined with Eq. ( 1):

Q% = 𝑤 𝑠 -𝑤 0 𝑤 0 × 100 (1)
where: ws-the weight of swollen probe; w0-the weight of dry probe.

Laser light diffractometry

The NPs mean diameter and size distribution were analyzed by laser light diffractometry technique (SHIMADZU SALD 7001). Freeze-dried NPs were immersed in acetone and sonicated for 15 min at room temperature using a sonication bath (Bandelin Sonorex). All measurements were performed in triplicate.

Morphological characterization by SEM (scanning electron microscopy)

MNPs morphology was investigated by SEM technique. SEM images were recorded with a HITACHI SU 1510 (Hitachi SU-1510, Hitachi Company, Japan) scanning electron microscope, NPs were fixed on Aluminium stub and coated with a 7 nm thick gold layer using a Cressington 108 device before observation.

Levofloxacin/Bevacizumab loading kinetic

Drug loading process was carried out through diffusional mechanism. In this regard, BEV was used as model drug. 0.030g CS-g-PEGMA NPs dry powder were suspended in BEV aqueous solution (25 mg/mL) and dispersed by sonication for 30 minutes. The suspensions were maintained at room temperature, under gentle mechanical stirring for 72 hours. After preset times intervals samples were centrifuged (15,000 rpm, 5 min) and freeze-dried. The quantity of BEV retained was calculated by determining the drug content from supernatant based on a calibration curve previously obtained, with the equations from relations (2): 𝑦 = 0.1373 • 𝑥; 𝑅 2 = 0.9846 (2) BEV loaded NPs were dried by lyophilization. All tests were accomplished with a spectrophotometer UV-VIS NanoDrop ND-1000, which allows the analysis of very small sample volumes, in microliter range. Drugs release rate was determined by absorption solution, monitoring the wavelength at 272 nm. Due to the higher swelling degree of NPs in ABS (pH=3.3) comparing with PBS aqueous environment, we decided the different samples to be loaded with BEV in ABS, and subsequently, the BEV release in to be measured PBS (pH=7.4) in order to ensure a lower release rate.

Levofloxacin/Bevacizumab in vitro release kinetic

Drug release kinetic in PBS (pH = 7.4 similar to aqueous humor of the eye) was determined by monitoring the wavelength at 272 nm for BEV. The investigation of the release kinetics, was carry out by immersing the drug loaded NPs in 1.0 mL PBS at 37 0 C, under continuous gentle stirring (100 rpm), after which the drug concentration in the supernatant was spectrophotometrically determined. All measurements were performed in triplicate and averaged.

Nanoparticles hemocompatibility

Hemolysis tests were performed using a method proposed by Vuddanda et al. [START_REF] Vuddanda | Investigations on Agglomeration and Haemocompatibility of Vitamin E TPGS Surface Modified Berberine Chloride Nanoparticles[END_REF] First, 5 mL of blood were centrifuged for 5 min at 2000 rpm. Supernatant plasma surface layer was removed and the red blood cells (RBC) were separated and washed with normal saline solution. The purified RBCs were re-suspended in saline solution to obtain 25 ml of RBC suspension. 2 mL of NPs suspension in saline solution at different concentrations were added to 2 mL of RBC suspension (final concentrations were 100 μg NP/ml, 200 μg NP/ml and 400μg NP/ml). Positive (100% lysis) and negative control samples (0% lysis) were prepared by adding equal volumes of 2% Triton X-100 solution and saline, respectively, over the RBC suspension. The samples were incubated at 37 ° C for 2, 4 and 6 hours. The samples were gently shaken every 30 min for re-suspension. After the incubation time, the samples were centrifuged at 2000 rpm for 5 min, and each 1.5 mL of the supernatant was incubated for 30 min at room temperature to allow hemoglobin oxidation. Oxyhemoglobin absorbance in supernatant was measured with a spectrophotometer (PG Instruments T60 UV-Vis Spectrophotometer) at 540 nm. The haemolysis percentages of RBC were calculated by the following equation (3). All experiments were performed in triplicate.

% 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝐴𝑏𝑠 𝑠𝑎𝑚𝑝𝑙𝑒-𝐴𝑏𝑠 𝑛𝑒𝑔𝑎𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝐴𝑏𝑠 𝑝𝑜𝑧𝑖𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙-𝐴𝑏𝑠 𝑛𝑒𝑔𝑎𝑡𝑖𝑣 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

(3)

Study of antiangiogenic effect of selected NPs on diabetes/inflammatory diseases of the eye and central retinal vein occlusion -experimental animal model

Inflammation of the rabbit eye (5 animals) was induced by injection of lipopolysaccharide intravenously, 10 g/kg/100 l, at 72 hours for 18 days, according to the literature [START_REF] Chen | Experimental Uveitis Induced by Intravitreal or Intravenous Lipoteichoic Acid in Rabbits[END_REF]. Rabbit diabetes (5 animals) was induced by streptozotocin injection, 180 mg / kg i.p. in single injection. Demonstration of onset of diabetes was made by sequential measurements of blood glucose after induction, one day, 3 days, 7 days, 14 days and 21 days [START_REF] Chatzigeorgiou | The Use of Animal Models in the Study of Diabetes Mellitus[END_REF].

Central RVO in the rabbit (5 animals) was induced by intraperitoneally injection, 20 mg / kg, of Eritrosein B solution (2%), according to the literature but slightly adapted [START_REF] Chen | Establishing an experimental rat model of photodynamically-induced retinal vein occlusion using erythrosin B[END_REF].

The central RVO was induced within 5 minutes (pre-experimental interval) by focused use of the laser wavelength of 532 nm, laser (Nikon Eclipse TE-300) present in the confocal laser microscopy setup (Microradiance, BioRad). Thus, the laser beam was intraocularly focused (in rabbits) by detaching the conduction tube from the microscope and setting in front of the open eye. One eye was used for the experiment, and the contralateral eye served as control. With a laser (power of 5 mW) 10-second flashgun train, every 25 seconds was applied to the rabbit's eye. This was the highest success rate (approximately 95%) obtained. For the experiment, adult rabbit was grown under standard laboratory conditions, nourished with standard feed for rabbits, treated under human conditions and anesthetized and sacrificed (without pain).

In the experiments, rabbit retinal endothelial cells were obtained as described. Were used 5 rabbit retinas, immediately after sacrifice, obtained by dissection under the microscope. These were homogenized in HBSS, on ice, without Ca 2 + and Mg 2 + . After centrifugation, the sediment was digested for 1 hour at 37 °C in the presence of a mixture containing 2 mg / ml collagenase / dispase, DNA type 1 from bovine pancreas (20 U / ml) and Tosyl lysine chloromethyl ketone (50 ng / ml) in HBSS and without Ca 2 + and Mg 2 + , but with penicillin, streptomycin and 10 mM HEPES, at pH = 7.4. After digestion, the suspension was filtered through a 70 μm sterile nylon sieve and microvascular structures retained on the filter were washed twice with HBSS and without Ca 2 + and Mg 2 + . In order to reflect the number of endothelial cells, the supernatants obtained in separation processes above were aspirated several times through a 1 ml pipette tip for mechanical dispersion and then incubated at 4° C in PBS containing 1% bovine serum albumin and 20 μg / ml fluorescein bound lectin [START_REF] Rezzola | In vitro and ex vivo retina angiogenesis assays[END_REF].

After 24 hours, the cells were washed by centrifugation and the free fluorescein removed, and the endothelial cells were quantified by confocal laser microscopy, the HQ515 / 530 filter was used as a 488 nm excitation filter. The green fluorescein fluorescence lectin coupled, was quantified to highlight the percentage of the endothelial cells number under conditions of inflammation, diabetes and Central RVO in experimental rabbits, already mentioned. Basically, we quantified the number of pixels associated with endothelial cells. The experiments were performed in the presence of simple micro / nanoparticles or loaded with BEV and monoclonal antibody (mouse) anti-VEGF-A human (equivalent bevacizumab) on cells derived from inflammatory, diabetic models and central RVO.

The immunogenic sequence (VEGF) is a partially recombinant protein with the GST tag. All types of NPs, whether or not loaded with a biologically active substance, were administered in suspension (1%), by intraocular injection, for 72 hours until the animals were sacrificed, after the animal model of inflammation, diabetes or central RVO was achieved.

Results and discussion

Synthesis and characterization of chitosan grafted poly (ethylene glycol) methacrylate

Chitosan characteristics and properties such as non-toxicity towards living organisms, biodegradability, hemocompatibility, biocompatibility and the amino and hydroxyl groups make him suitable for chemical modification. The higher reactivity of the amino group compared to hydroxyl groups, is one of the reasons for which amino groups are the most used in chemical modifications. Published works on PEGylation of chitosan report the PEG was grafted to the amino group of the glucosamine unit. Chitosan was chemically modified through various methods, but reactions such as reductive amination, condensation reaction have disadvantages including organic medium, complex or toxic catalyst, protection and deprotection of groups, which limits chitosan derivatives utilization in biomedical filed. Through Michael addition were synthesized few chitin and chitosan derivatives. Michael addition reaction have advantages such as mild reaction conditions, high functional group tolerance, a large host of polymerizable monomers and functional precursors, high conversions, lack of side reactions, favorable reaction rates and versatility in terms of: monomer selection, solvent environment, reaction temperature. In present work, CS-g-PEGMA derivative was synthesized through Michael addition of poly(ethylene glycol) methacrylate to the amino groups of chitosan. Reaction of PEGMA grafted chitosan (Scheme no. 1) was carried out at 40 0 C.

Scheme 1. Synthesis of chitosan grafted poly (ethylene glycol) methacrylate

A first structural characterization of the grafted chitosan derivative is given by the Fourier Transform-Infrared Spectroscopy analysis. FT-IR spectra's of CS, PEGMA, CS-g-PEGMA and cross-linked NPs are presented in fig. 1. The infrared spectrum of the CS showed a strong peak at 3365 cm -1 that could be attribute to the axial stretching vibration of -OH superimposed to the -NH2 stretching band and inter-and extra-molecular hydrogen bonding of CS molecules which decreased in the acylated CS derivatives. The absorption peaks at 1646, 1423 and 1379 cm -1 are due to the NHAc units, the amide I, -NH2 bending and the amide III [START_REF] Zang | Synthesis and characterization of water-soluble Osuccinyl-chitosan[END_REF]. The vibration at 1076 cm -1 is characteristic to -C-O-C-stretching and its saccharine structure [START_REF] Peniche | Self-curing membranes of chitosan/PAA IPNs obtained by radical polymerization: preparation, characterization and interpolymer complexation[END_REF]. The prominent peak at 1726 cm -1 was assigned to double bond of the PEGMA. Two peaks demonstrate that PEGMA was grafted with CS. The first new peak at 1732 cm -1 appeared and is assigned to C=O of -OCOR group. This peak highlights the presence of PEGMA in the structure of the functionalized chitosan.

The second new peak is at 1646 cm -1 and is typical for the composition of the polysaccharide. A slight displacement of the absorption bands for the grafted product comparative with the baseproducts was observed, the cause being the surrounding groups which influences the wavelength.

The signal for the formed amino group cannot be define because the peak signal it is around 3300 ÷ 3400 cm -1 . Both CS and PEGMA have hydroxyl groups and they give a signal around the same value 3300 ÷ 3400 cm -1 overlapping the secondary amine group (-NH-). The peaks at 2941 and 2879 cm -1 are characteristic for -C-H stretching, but they are not significant because CS and PEGMA give approximately the same signal. The intensity of absorption peaks at 3436 and 1722 cm -1 decreased due to their participation in reaction. It was confirmed that PEGMA group substituted the amino group. Also, results presented in figure 3 indicates that NPs based CS-g-PEGMA obtained are stable. A characteristic band at 3400 cm -1 is attributed to -NH2 and -OH groups stretching vibration and the band for amide I at 1646 cm -1 is seen in the infrared spectrum of modified chitosan.

Preparation and characterization of MNPs

CS-g-PEGMA MNPs were performed by double cross-linking, ionic followed by covalent, in reverse emulsion (w/o). After the purification steps freeze dried NPs samples were considered for chraracterization: diameter and size distribution; morphology; swelling behavior, drug loading and release, zeta potential, hemocompatibility analysis, toxicity and cytotoxicity test, etc. The FT-IR analysis revealed that in case of cross-linked NPs the peak of 1646 cm -1 (NHAc units) disappears and 2 new peaks at 1643 cm -1 and 1548 cm -1 appeared (fig. 3). The disappearance of the band could be attributed to the linkage between the phosphoric, sulfate and ammonium ions.

The FT-IR spectra of crosslinked NPs (MA-8) also showed a absorption band signal corresponding to the bonds formed by the ionic crosslinking process between polyanions tripolyphosphate (P-O-P at 891 cm -1 ) and ammonium cations of CS-g-PEG-MA, respectively the a absorption band signal corresponding to the new P=O bonds formed at 1282 cm -1 appeared. For sample MA-H the FT-IR spectra revealed at 636 cm -1 the absorption band signal corresponding to the bonds formed by the ionic crosslinking process between the sulfate anions from Na2 SO4 and the ammonium cations of CS-g-PEG-MA.

Figure 3. FT-IR spectra's of CS-g-PEGMA and NPs

According to the curves presented in the thermogram presented in figure 4, it can be seen that the melting process shows an endothermic peak of CS which is high at 94.01 0 C, for chitosan derivative at 102.56 0 C and the NPs at 106.2 0 C. By comparing the results obtained, it can be affirm that the NPs thermogram is similar to chitosan and CS-g-PEG-MA thermogram, suggesting a good thermal stability of NPs. Also, the appearance of the exothermic peak at 253.75 0 C indicates the formation of new chemical bonds in the NPs structure, that can be attributed to ionic and covalent crosslinking processes. Furthermore, CS-g-PEGMA NPs morphology was investigated by SEM technique. Scanning electron microscopy pictures for different nanoparticle samples indicates the fact that after optimization steps, the NPs obtained presented a spherical shape, are individualized and the optimized best sample crosslinked with TPP (MA-7,8,9) have size ranged from 200 nm to 900 nm in diameter, also the polydispersity is reduced. In the case of the optimized best sample (MA-G, H, I) crosslinked with Na2SO4 the size is ranging from 1000 to 1500 nm due to a low crosslinking density. SEM photographs for the most representative samples are presented in figure 5,6. For samples MA-8,9,11 and MA-H, I, K the polymer / crosslinker ratio and the stirring rate were kept constant, only the polymer concentration was modified. It was observed that the particle diameter is dependent of the polymer concentration. The highest diameter was recorded in the case of MA-11 samples, respectively MA-K, this behavior was reported also, in other studies [START_REF] Alupei | New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery[END_REF], [START_REF] Peptu | Double cross-linked chitosangelatin particulate systems for ophthalmic applications[END_REF]. Also, we can assert that the viscosity of the polymer solution increases when the concentration is higher, leading to big droplets in the emulsion phase wich need to be crosslinked. As we can see for samples MA-9 and I, the polymer solution is more diluted and irregular particle are formed due to a weaker cross-linking process. For samples MA-11 and K, the concentration of polymer solution was increased to 0.75%, fact which led to an increased particle diameter and polydispersity, and a lower agglomeration tendency.

In the case of MA-10 and J particles, the polymer / ionic crosslinking ratio was modified, which led to a small increasing of particle diameter, due to the increment of the cross-linking density of the polymer matrix. Another important aspect highlighted by SEM photographs is that the particle diameter and polydispersity of the particles is influenced by the nature of the crosslinking agent. Thus, the change of the ionic crosslinker in the case of MA-A ÷ K samples, was used sodium sulphate fact which leads to an increased particle diameter, according to our expectations.

MA-I

concentration and stirring intensity influences the NPs diameter. As expected, both types of MNPs diameter decreases with the increasing of the stirring speed above 9000 rot/min, the smaller mean particle size may be attributed to a greater cross-linking density. Also, the results confirmed that the diameter of the both types of particles is reduced as the initial polymer concentration in the synthesis decreased. Regarding the dimensional polydispersity curves (fig. 7,8,9,10) they have unimodal aspect and NPs size are in concordance with the SEM images previously presented. All measurements were performed in triplicate. In order to evaluate a very important factor the NPs ability to encapsulate/entrap drugs, further characterization of NPs samples was performed by studying swelling behavior in different physiological environments. Swelling behaviors of NPs samples were performed in solutions with different pH values (pH 7.4 and pH 3.3; these pH values were chosen as the suspensions are intended to be used in biological fluids) by recording the liquid uptake at pre-determined time intervals until equilibrium swelling was reached. As expected the results obtained revealed a correlation between the maximal swelling degree and NPs preparation parameters such as stirring speed, polymer concentration (fig 11,12 and tables 6 and 7). All NPs samples were displaying a higher swelling degree in acetate buffer (pH 3.3) between 700 and 1200%, compared with those in phosphate buffer (pH 7.4) between 450 and 800%. We can therefore state that this effect of a higher swelling degree of the NPs in acidic environment involves the protonation of amino/imine groups which leads to strong electrostatic repulsion between the polymer chains, thus to an increased capacity of the network to include a higher amount of water.

As we can observe from fig.

12 by increasing concentration of the polymer solution, while maintaining the constant stirring speed leads to the following changes:

-although a lower polymer concentration (0.35%) was used for MA-9 and MA-I samples, the swelling degree was highest; the reduction of the polymer amount is correlated with the decresing number of amine groups participating in the two types of crosslinking, thus decreasing the crosslinking density, but also should be metioned that the swelling degree values are close to those of MA-8 and MA-H samples (c = 0.5%); As a result, increasing of the swelling degree is a logical consequence.

-increasing the mean particle diameter with the increase of the polymer concentration has led to a decrease in the amount of retained water and hence of the swelling degree; the obtained values can be related with a higher tendency of particle agglomeration (and thus a higher degree of crosslinking). Also, a reduction of the maximum swelling degree with the increase of the amount of TPP or sodium sulfate, in fact of the molar ratio of TPP or sodium sulphate / amino groups was also recorded in the case of MA-10 and MA-J samples, we can state that the result is a natural one due to the increase in crosslinking density. For all samples, the nature of the ionic crosslinker used does not influence the maximum degree of swelling in both aqueous media, the experimental values obtained for both acidic and basic media being quite close, the differences being quite small, for both the TPP and the one in which sodium sulphate was used, contrary to our expectations.

Zeta Potential

MNPs stability in aqueous suspension was evaluated by Zeta potential measurements. Table 4 summarizes the arithmetic average of zeta potentials determined by Smoluchowski equation for optimized CS-PEGMA MNPs synthesized. A large negative or positive zeta potential for suspended particles indicates that the system is stable against flocculation or coagulation. In general, literature studies report that zeta potentials values for MNPs prepared using grafted copolymers have a tendency to be higher compared to those synthesized only from chitosan, this could indicate a low stability of aqueous suspension of synthesized MNPs.

Hemocompatibility analysis

Hemolysis is the destruction of red blood cells to release hemoglobin and other components in the surrounding fluid. Dangerous pathological conditions can be induced by an increase in red blood cell degradation. Therefore, all products intended for intravenous administration biomedical must be assessed to determine their hemolytic potential [START_REF] Dobrovolskaia | Method for Analysis of Nanoparticle Hemolytic Properties In Vitro[END_REF]. The NPs obtained can be used as drug delivery systems and can be administered intravenously and therefore preliminary tests were conducted to determine their interaction with human blood components. Chitosan-based NPs obtained by various methods, ranging in size between 200 and 900 nm and positively charged, were demonstrated to be hemocompatible for concentrations less than 50 mg/ml [START_REF] Lee | Physicochemical properties and blood compatibility of acylated chitosan nanoparticles[END_REF], [START_REF] Nadesh | Hematotoxicological analysis of surface-modified and -unmodified chitosan nanoparticles[END_REF]. The hemolytic potential of CS-g-PEGMA NPs was evaluated for concentrations between 100 and 400 mg/mL, using a spectrophotometric method. The results of hemolysis tests are expressed as average ± SD (n = 3) in fig. 14. If hemolysis percentage is less than 5% when the sample is considered hemocompatible [START_REF] Li | Self-Assembled Polymeric Micellar Nanoparticles as Nanocarriers for Poorly Soluble Anticancer Drug Ethaselen[END_REF]. For all concentrations tested was obtained a percentage of less than 5% hemolysis (Figure 13), which indicates that the NPs are hemocompatible and can be administered intravenously.

Toxicity test

The quantitative analysis of the cellular toxicity of two CS-PEGMA NPs formulations were also evaluated using the arithmetic method Spearman -Kἄrber, equation (5). The average values of LD50 for samples CS-PEGMA 7 is 4922 mg/kg and for CS-PEGMA 8 is 6744 mg/kg. The results show in table 5, demonstrate that both formulation is nontoxic and can be used in biomedical applications.

𝐷𝐿 50 = 𝐷𝐿 100 • [∑ a•b n ] (5)
where: a -the difference between two consecutive doses; b -the average number of dead animals from two successive groups; n -the number dead animals from a group; DL100 -the amount of substance that kills 100% of animals tested.

. MNPs ability of loading Levofloxacin or Bevacizumab

Considering the purpose of using MPs as a possible drug delivery system for the treatment of posterior segment of the eye conditions, drug loading potential has been studied. The drug type selection was dependent on the hydrogel character of the NPs and the encapsulation/release diffusion principle. Furthermore, the diffusional process for loading NPs loaded drug in aqueous solution follows the swelling behavior of the sample. Following the morphological analysis results, sample MA-8 and MA-H were selected as example for the inclusion of LEV/BEV. The results obtained prove that the analyzed sample MA-8 retained the maximum amount of loaded BEV (0.327 mg BEV / mg NPs), respectively LEV (0,81 mg LEV / mg NPs) (figure 14,table 6) after 72 h, with a encapsulation efficiency of 39% (BEV) and 97 % (LEV). In the case of MA-H sample the maximum amount of LEV loaded was 0,62 mg drug/ mg NPs, with a encapsulation efficiency of 74 %.

The results obtained after the spectrophotometric analysis regarding the quantities of drug included in the MNPs and the encapsulation efficiency are presented in table 6. The loading efficiency of the drug was determined by the equation:

Ef. loading = [amount of loaded drug/theoretical drug amount]x100 As can be seen from table 6 the amount of LEV included after 72 h varied between 0.7 and 0.8 mg / mg MNPs for MA-5÷11 samples crosslinked with TPP, respectively 0.55 mg and 0.67 mg / mg MNPs for MA-E÷K samples crosslinked with Na2SO4, the loading efficiency exhibiting high values. Differences in the amounts of drug quantities included in both types of systems are lower when using Na2SO4 as a crosslinker, so we can state the nature of the crosslinker influences the particle loading process, a possible explanation being the agglomeration tendency due to weaker crosslinking. The experimental data obtained are consistent with the values obtained for the maximum inflation rate discussed above, the amounts of drug varying according to it.

MNPs ability to release Levofloxacin or Bevacizumab in vitro

MNPs ability to release Levofloxacin or Bevacizumab in vitro was performed in a basic environment at pH of 7.4, 37° C. 30 mg MNPs loaded with LEV or BEV were suspended in 1 ml of phosphate buffer solution, pH 7.4, the amount of drug released was determined by spectrophotometric analysis at predetermined times. For example, the release kinetics profile and release efficiency of LEV and BEV for MA-8 and H samples are highlighted in figure 15; 16 and table 7.

Regarding the LEV release process (Figure 16 a), a faster phase was observed that is reached in the first 10 hours, followed by a slower phase (characterized by a constant release) up to 120 hours when the maximum amount of released drug was achieved. Additionally, when calculating the efficiency of LEV release for all samples, values between 35 and 84 % were obtained. MA-10 y = 0,4031• x -2,8183 0,2475 0,4031 0,9498 MA-11 y = 0,6427• x -2,6351 0,0269 0,6427 0,910 MA-E y = 0,5708• x -2,8403 0,0444 0,5708 0,9456 MA-F y = 0,5799• x -2,6854 0,0412 0,5799 0,9612 MA-G y = 0,5336• x -2,7954 0,0608 0,5336 0,9638 MA-H y = 0,5023•x -1,7476 0.127 0.5023 0.9968 MA-I y = 0,4593• x -3,1908 0,1320 0,4593 0,9772 MA-J y = 0,4567• x -2,3565 0,1282 0,4567 0,9712 MA-K y = 0,7975• x -3,277 0,0071 0,7975 0,9768

Regarding the process of releasing the biological active principles, literature studies report that it depends on factors such as particles shynthesis, dimension and, last but not least, the physico-chemical properties of the used polymers. After the experimental kinetic data analysis for the interval 0 ÷ 300 minutes, it was possible to calculate the diffusion exponent. The analysis of the release kinetics was accomplished based on the Korsmeyer-Peppas mathematical model ( Eq. 6)

M t M∞ = k • t n (6)
The equation obtained from processing the experimental data of BEV the release kinetics is: -y = 0,4968•x -3,8964, from which were determined the parameters k = 0.053, n = 0.4968 și R 2 = 0.9853. The analysis demonstrated that the BEV transport/release mechanism was dominated by diffusion.

Table 7 shows the values of the n and k exponents obtained for the analyzed samples, and we can conclude that for the MA -5,6,11 and MA-E, F, G, K samples, these indicate an abnormal non-Fickian diffusion the drug transport through the polymer matrix being governed by both diffusion and swelling processes.

It is known that the value of n for a normal Fickian diffusion is 0.5 <n <1.0. In the case of MA-8 and H we can state that we have a Fickian diffusion the LEV transport process through the polymer matrix being governed by diffusion [START_REF] Ritger | A simple equation for description of solute release I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs[END_REF], [START_REF] Ritger | A simple equation for description of solute release II. Fickian and anomalous release from swellable devices[END_REF], [START_REF] Peppas | A simple equation for the description of solute release. III. Coupling of diffusion and relaxation[END_REF].

Study of antiangiogenic effect of selected micro/nanoparticles on diabetes / inflammatory diseases of the eye and central retinal vein occlusion -experimental animal model

The data obtained from the models of inflammatory diseases, diabetes and central RVO on animals (rabbits) is the basis for the extension of research in terms of combined in vivo models by applying the antibodies to the target tissue reactivity as close as possible. As can be seen from Table 6 and fig. 21, 22 we can state that the 1 % suspension of NPs loaded bevacizumab and monoclonal antibody (mouse)anti-VEGF-A human (equivalent bevacizumab) are very efficient as anti-angiogenic treatment in the case of the model of diabetes in rabbits. Due to the fact that there are no significant differences between the obtained results, both NPs suspensions could also be used as anti-angiogenic treatments with moderate efficacy in the case of inflammation with lipopolysaccharide and central RVO models in rabbits.

Conclusions

In this study new polymer CS-g-PEGMA was synthesized by Michael addition reaction, as a new nanocarrier material for drug delivery. The chemical structure of CS-g-PEGMA was confirmed by 1 NMR and FT-IR analysis. Newly synthesized CS-g-PEGMA, presented an improved solubility in aqueous solution. CS-g-PEGMA nanoparticles were prepared through a double crosslinking technique (ionic and covalent) applied in reverse emulsion system. The SEM analysis of the nanoparticles indicated a relative high polydispersity, confirmed by laser diffractometry, but an uniform round shape,. The NPs dimension ranged from 200 to 900 nm and they manifested a pH sensitive behavior in aqueous environments. Moreover, in vitro and ex vivo release study indicated that BEV was released from the nanoparticles in a controlled-release manner for several days and is related to the swelling behavior in aqueous environment. NPs toxicity was similar to the individual polymers, practically nontoxic degree of toxicity. The hemocompatibility tests showed that the NPs are hemocompatibile and can be administered by local injection. Therefore, we believe that CS-g-PEGMA could be a potential carrier for controlled release of BEV as an ophthalmic drug delivery system. The major benefits of bevacizumab loading in micro / nanoparticles would be: a much lower dose to administer (thus reducing the adverse effects of drugs), their prolonged release (less frequent administration), and the effectiveness of local delivery (which may extend up to at 14-30 days), their targeted administration by local injection near the retina.

Chapter 8. Chitosan grafted PEG methyl eter acrylate micro/nanoparticles as carrier for controlled release of active principles

Materials

The following materials were used for the experiments: low molecular weight Chitosan (CS), (degree of deacetylation 82 %, Aldrich); Poly(ethylene glycol) methacrylate (PEGA, Mn = 480, Aldrich); Acetic acid (99%, Sigma Aldrich); Glutaraldehyde (GA) (Sigma Aldrich, 25% aqueous solution); Sodium tripolyphosphate (TPP, Sigma Aldrich); Sodium sulfate (Na2SO4, Sigma Aldrich); Levofloxacin (LEV; Sigma Aldrich); Tween 80 (Sigma Aldrich); Span 80 (Sigma Aldrich); acetate and phosphate buffered saline (PBS) were purchased from Sigma-Aldrich; Milli-Q ultrapure distilled water (Merck) were of analytical grade and used without further purification. The chemicals used in this study were of analytical grade purity and were used without further purification. The human blood samples used were freshly obtained from one healthy nonsmoker volunteer. Fibroblast cells were extracted from rabbit dermis in the Bioengineering Department of "Grigore T. Popa" University of Medicine and Pharmacy, Iasi, Romania.

Equipment

Fourier-transform infrared spectroscopy spectra of CS, PEGA and CS-g-PEGA derivative were recorded by a DIGILAB SCIMITAR FTS 2000 spectrometer. The samples were prepared as KBr pellets and scanned over the wave number range of 4000-450cm -1 at a resolution of 4.0 cm -1 . 1 H NMR spectra were obtained by using a Bruker Avance DRX 400. CS, PEGA (Mn=480) and CS-g-PEGA derivative were dissolved in D2O. CS-g-PEGA NPs morphology was investigated by SEM technique using a HITACHI SU 1510 (Hitachi SU-1510) scanning electron microscope. Also, MNPs mean diameter and size distribution were analyzed by laser light diffractometry technique (SHIMADZU SALD 7001).

Synthesis of chitosan grafted poly (ethylene glycol) methil ether acrylate (CS-g-PEGA)

The chemical modification of poly (ethylene glycol) methyl ether acrylate (CS-g-PEGA) chitosan via Michael addition reaction was performed according to a protocol reported by Han et al. [START_REF] Han | Electrospinning of methoxy poly(ethylene glycol)-grafted chitosan and poly(ethylene oxide) blend aqueous solution[END_REF]. Briefly, chitosan (2.0 g) and 100 mL acetic acid solution 1% (w/w) were added to a 250ml reaction flask equipped with a reflux condenser. The reaction flask was immersed into a water bath heated at 50 0 C, purged with nitrogen and allowed to stir for 30 min. Subsequently, PEGA was added drop wise at a final molar ratio NH2: PEG = 1:0.5; 1:0.75; 1:1. Afterwards, Michel addition reaction was carried out at 50 0 C, under nitrogen atmosphere and maintained for 48 h to complete the reaction. Finally, the polymer solution pH was raised from 3.4 to 8 with NaHCO3 saturated solution. Subsequently, the polymer solution was filtered by centrifugation and precipitated into acetone.The polymer was purified by repeated washing with acetone. The final product was purified during 3 days by dialysis dried. The polymer was isolated by lyophilization.

Preparation of MNPs based CS-g-PEGA

MNPs based CS-g-PEGA were prepared by double cross-linking, ionic followed by covalent in reverse emulsion (w/o) according to a method reported by C.A. Peptu and coworkers [START_REF] Peptu | Double cross-linked chitosangelatin particulate systems for ophthalmic applications[END_REF], [START_REF] Alupei | New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery[END_REF], [START_REF] Jatariu | Double crosslinked interpenetrated network in nanoparticle form for drug targeting-Preparation, characterization and biodistribution studies[END_REF], previously described in chapter 7 subsection 4.2.

5. Results and disscution 5.1. Synthesis of chitosan grafted poly (ethylene glycol) methil ether acrylate (CS-g-PEGA)

Synthesis of the chitosan derivative based on the Michael addition reaction [START_REF] Han | Electrospinning of methoxy poly(ethylene glycol)-grafted chitosan and poly(ethylene oxide) blend aqueous solution[END_REF],. The grafting reaction is shown in Scheme 1. The main justification for the use of PEGA is based on the idea of introducing new properties to the grafted polymer, namely, improving the solubility of chitosan in weakly acidic media by introducing hydrophilic segments (PEGA) to the chitosan linear chain.

Scheme 1. Synthesis of chitosan grafted poly (ethylene glycol) methil ether acrylate

A first confirmation of CS-g-PEGA was obtained by FT-IR spectroscopy. As an example has been chosen, the FT-IR spectra of CS-g-PEGA molar ratio 1:1 (Figure 1 and table 1). Because FT-IR spectrum of CS was described in detail previously, we have focused on the description of FT-IR spectra of PEGA and CS-g-PEGA.

The PEGA FT-IR spectra exhibit the following characteristic peaks: -a prominent peak at 1722 cm -1 which corresponds to the double bond vibrations -at 2862 cm -1 was observed the appearance of an intense signal characteristic to the absorption band of the CH groups, -at 3435 cm -1 notices the axial stretching vibrations of OH groups of PEGA.

-chitosan grafted poly (ethylene glycol) methyl ether acrylate is validated by the appearance of absorption bands at 1720 and 1654 cm -1 . Also, in the CS-g-PEGA spectra, we observed the absorption bands corresponding to the stretching vibrations of the hydroxyl, amino and amide groups of chitosan are displaced slightly, the signal peak at 1720 cm -1 is ascociated double bonds C = C of PEGA and is reduced due to the participation in the reaction, and the absorption band at 1654 cm -1 is specific to the deformation vibrations of the acetylated amine groups corresponding to chitosan.

Similar to CS-g-PEG-MA, the signal for the secondary amine group that has been formed can not be highlighted because it should be displayed in the range 3300 ÷ 3400 cm -1 . CS and PEGA have OH groups in the structure give a signal around the same value of 3300 ÷ 3400 cm -1 which also overlap with secondary amine group (-NH-) values. Signals appeard at 2941 and 2879 cm -1 are characteristic of C-H stretching vibrations, but they are not significant because both CS and PEGA have about the same value. Characterization of the CS-g-PEGA copolymer was also performed by NMR spectroscopy. Various molar ratios of chitosan / poly (ethylene glycol) methyl ether acrylate (1: 0.5; 1: 0.75; 1: 1) were followed on the degree of substitution achieved. As example, in figure 8. 3 (a,b,c) are display the 1 H NMR spectra of CS-g-PEGA (1:1 molar ratio).

Figure 2 a) shows the 1 H NMR spectrum of chitosan, which was recorded in D2O at 80 0 C. The peak corresponding to 1.90 ppm is attributed to the -CH3 protons in the chitin residue specific acetylated group. The peak corresponding to 3.01 ppm is attributed to the H2 protons of the deacetylated groups, and the multiplets from 3.50 to 3.80 ppm are assigned to the H3, H4, H5, and H6 protons of the deacetylated and acetylated groups of chitosan.

The 1 H NMR spectrum of PEG acrylate is illustrated in figure 2 b) and has the following characteristic peaks: 6.44 ppm (CH2=CH-COO), 6.24 ppm (CH2=CH-COO), 6,005 ppm (CH2=CH-COO), 4.35 ppm (-COOCH2-), 3.6-3.83 ppm (-COOCH2CH2-, -OCH2CH2-, -CH2CH2OCH3), 3.38 ppm (-OCH3).

For CS-g-PEGA, compared to CS, the peaks corresponding to the protons -COOCH2CH2and -NH-CH2CH2-COO-appear at 4.33 ppm and 2.60 ppm, respectively. The clear signal at 3.37 ppm corresponds to the protons of the -OCH3 group in the PEG structural unit. Signals of methylene protons of poly (ethylene glycol) overlap with the signal of H3, H4, H5 and H6 protons of CS.

The degree of substitution (table 2) was calculated according to the equation ( 1): an were used the peak intensity of the -COOCH2-protons (4.33 ppm) and the peak intensity of the -CH3 group (2.00 ppm) specific for the chitin residues. The highest grafting degree of 16.5% was reached for the molar ratio 1:1 (CS:PEGA), and also a important thing that was achieved the complete solubilization in the bidistilled water which confirmed our assumption of departure and concluded that the chitosan derivative was optimized from this point of view. However, chitosan derivative has demonstrated also, an increasing solubility in acidic environment with increasing the ratio of polysaccharide to synthetic polymer, thereby understanding first of all reducing the time required for solubilization. For the preparation of MNPs based CS-g-PEGA, the same double-crosslinked reverse phase emulsion described in chapter 7 subsection 4.2. Also, in this case, following the purification steps, the micro/nanoparticle samples were taken into account for the determination of some characteristics such as: diameter and polydispersity; morphology; drug inflating, drug loading and release, Zeta potential, hemocompatibility, toxicity and cytotoxicity test. Experimental protocols with observed parameters for preparation of MNPs are presented in table 3 and4

FT-IR analysis

FT-IR analysis for MNPs has demonstrated the formation of the polymer network through double crosslinking. FT-IR spectra were recorded for all the MNPs obtained, but they showed a similar profile. As representative example, only the FT-IR spectra for samples A8 and B8 are displayed, the difference being the nature of the ionic crosslinker.

Table 8.5 shows the characteristics absorption band of chitosan derivative and samples A8, B8. From the spectra samples outlined in figure 3: -for sample A8, we observed the appearence of a new signal at 896 cm -1 which correspond to the new bond formed due to the ionic crosslinking process, between tripolyphosphate polyanions and CS-g-PEGA ammonium cations.

-in the case of the sample B8, also we observed the appearence of a signal at 617 cm -1 which correspond to the new bond formed due to the ionic crosslinking process, between the sulfate anions from Na2SO4 and the ammonium cations of CS-g-PEGA.

-the absorption bands of imine linkages -C=N-resulting from the covalent crosslinking process are highlighted at 1568 cm -1 for sample A8 and 1575 cm -1 for sample B8. The covalent crosslinking process took place between the amine groups of the chitosan derivative and the carbonyl groups of glutaraldehyde [START_REF] Martins | Polyelectrolyte complexes of chitosan/heparin and N,N,N-trimethyl chitosan/heparin obtained at different pH: I. Preparation, characterization, and controlled release of heparin[END_REF], [START_REF] Facchi | Preparation and cytotoxicity of N-modified chitosan nanoparticles applied in curcumin delivery[END_REF], [START_REF] Jafary | Stability improvement of immobilized alkaline phosphatase using chitosan nanoparticles[END_REF], [56], [START_REF] Contreras | Preparation of Sodium Aluminate From Basic Aluminium Sulfate[END_REF], [START_REF] Han | Electrospinning of methoxy poly(ethylene glycol)-grafted chitosan and poly(ethylene oxide) blend aqueous solution[END_REF]. The absorption band signal corresponding to the bonds formed by the ionic crosslinking process between the sulfate anions from Na2 SO4 and the CS-g-PEGA ammonium cations

Scanning electron microscopy analysis SEM

The morphological characterization of the MNPs made according to the experimental plan detailed in subchapter 5.2, was performed by scanning electron microscopy. Taking into account the main purpose of using particle systems for the treatment of posterior pole diseases, it is important that they have certain characteristics such as nanometric dimensions, stability and individuality.

As mentioned above, the selected method for particle preparation was the double crosslinking (ionic and covalent) technique in water-in-oil emulsion, the advantage being the use of a lower amount of covalent crosslinker, which leads to a decrease of MNPs toxicity. The fundamental principle of the process is the formation of a interconnected / interpenetrated network in the first stage through the majority ionic crosslinking process, followed by the covalent crosslinking step.

Taking into account the optimization steps, namely the increase of the polymer /ionic crosslinking ratio, the MNPs obtained are characterized by a spherical shape, they are well individualized, the diameter and the polydispersity are reduced compared to the first sets of samples (Figure 4 and5). Also, SEM recorded images shows an important morphological differences and in terms of the particle size which is depend on the varied parameters of MNPs shynthesis. Therefore, for samples A-5 ÷ 8, respectively B-5 ÷ 8, the concentration of the polymer solution (0.5%) and the polymer / crosslinker ratio were kept constant, changing the stirring rate from 5,000 to 15,000 rpm / min. Thus, we notice that the stirring speed has a significant influence on the particle size which is inversely proportional to the agitation rate. We can state that the stirring speed is essential to establish the particle diameter, which decreases with increasing stirring intensity which leads to better dispersion and formation of very small droplet in the emulsion phase.

For samples A-8,9,11 and B-8,9,11, the concentration of the polymer solution ranged from 0,35% to 0,75%, the other parameters being kept constant. Also, in this case, an increase of the diameter from 0.5 nm to 1.3 μm was observed for samples A-8,9,11 and from 0.6 nm to 1.5 μm for samples B-8, 9,11. Thus, it can be argued that the diameter particle is clearly influenced by the concentration of the polymer solution. In the case of samples A-11, respectively B-11 (c=0,75 %) the concentration is higher which leads to to an increase in viscosity solution therefore to the formation of larger droplets, fact presented by other studies [START_REF] Alupei | New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery[END_REF], [START_REF] Peptu | Double cross-linked chitosangelatin particulate systems for ophthalmic applications[END_REF]. By increasing the concentration of the polymer solution to 0.75%, was observed a considerable increase in the mean diameter and particle polydispersity, as well as a decrease of the agglomeration tendency.

In the case of sample B9, the concentration of the polymer solution being low, the particleshaped are irregular, and exhibiting a agglomeration tendency due to weaker cross-linking density of the polymer matrix. Changing the polymer / ionic crosslinker ratio (A-10, B-10) caused a slight decrease in particle diameter due to the increase of the cross-linking density of the polymer matrix.

Another important observation highlighted by the SEM images is that the diameter and the polydispersity of the particles is influenced by the nature of the crosslinking agent. Thus, the modification of the type of ionic crosslinker in the case of samples B-1 ÷ 10 using sodium sulphate leads to a slight decrease of the particle diameter and to the formation of capsules.

Laser light diffractometry analysis

The dimensional analysis of MNPs by laser diffraction was performed to highlight the diameter and dimensional polydispersity. The determination was carried out in acetone, thus avoiding the swelling of the particles. Also, the experimental values obtained by this characterization method confirmed that the particle size is dependent on the concentration of the polymer solution and the rate of stirred during the particle synthesis. Thus, a higher speed rotation of ultratraurax implies a higher energy dissipated in the agitated emulsion, i.e. a higher energy transfer, which causes the emulsion to break into smaller droplets, resulting in nanometric particles. The granulometric distribution curves of the MNPs obtained (determined by laser beam diffraction) have a unimodal allure and a nanometric diameter, depending on the synthesis conditions (Figures 6,7 and Tables 6,7). The results are consistent with SEM images. -the increased particle mean diameter and concentration of polymer solution, lead to a decrease of the amount of retained water (A-8 and 11, B-8 and 11) and thus the swelling degree, the obtained results can be attributed to a better packing of chemical bonds between polymer molecules to obtain the network, the water absorption being inversely proportional to the crosslinking degree of the samples. -in the case of A-10 and B-10 samples, it has been found a decrease of the maximum swelling degree when the amount of TPP or sodium sulfate was enhanced (in fact of the molar ratio TPP / amine groups or sodium sulfate / amine groups), being a logic consequence because of a better network cross-linking density. -for MNPs A-5 ÷ 11 the maximum swelling degree in the basic medium are lower than those achieved in the acidic environment, compared to results of the B-5 ÷ 11 samples, where the values for both mediums tested are quite close. Thus, we can assert that the swelling degree is depending on the nature of the crosslinker used and the fact that in PBS most of the chitosan amine groups are deprotonated, therefore, hydrogen bonds are formed in the newly formed polymer network. -We observed also that the obtained experimental data for swelling degree in both acidic and the basic media were lower in the case when TPP was used compared to sodium sulphate. This behavior is a confirmation of a lower cross-linking degree in the case of the use of sodium sulphate, thus a less dense polymer network with a large macromolecule mobility, which leads to an increase in water retention in the polymer matrix compared to TPP. For all prepared MNPs the kinetics of the swelling process was analyzed in both media. As example we chose A9 and B9 samples which are displayed in figure 8. The results revealed two phases for the acidic media: a fast increase to 800% (A9) and 1000% (B9) in the first 90 minutes, followed by a constant increase (90-480 minutes), the maximum value being reached after 24 hours at 1144% for sample A-9 and 1225 % for sample B-9. In the case of basic media, the maximum value for the swelling degree was reached at 669% for sample A-9 and 804% for sample B-9.

MNPs capacity to include LEV

The ability of MNPs to encapsulate active principles was analysed using levofloxacin as a model drug, a broad spectrum antibiotic belonging to the class of fluoroquinolones, generally used to treat antibacterial infections. Levofloxacin is a hydrophobic drug, therefore a suitable candidate for particulate systems designed for drug delivery in the ocular region to reduce the frequency of administration [58], [START_REF] Gupta | Biodegradable levofloxacin nanoparticles for sustained ocular drug delivery[END_REF]. The LEV loading process was performed by diffusion, the protocol being described in detail in Chapter 7, Subchapter 4.6. Thus, the LEV content in the supernatant was determined by UV-Vis spectrophotometry (at 287 nm LEV) by difference based on a calibration curve, and it was determined that the MNPs were able to encapsulate between 0.48 ÷ 0,69 mg drug/ mg particles. Therefore, the drug loading efficiencies values were between 47.0 ÷ 76.0%, showing a high capacity of the micro/nanoparticulate polymer supports to retain a biologically active compound. The amounts of encapsulated drug in MNPs and loading efficiency are outlined in Table 10. Regarding drug encapsulation efficiency was used the equation: The values obtained after the spectrophotometric analysis at different time intervals shows that after 72 h the amount of encapsulated LEV is ranging from 0.48 ÷ 0.75 mg for the case when TPP was used (A5-A11) and between 0.6 ÷ 0.89 mg for MNPs crosslinked with sodium sulphate. Drug encapsulation efficiency presented high values. The experimental data obtained are in agreement with the swelling behavior previously discussed, as we expected, the encapsulate LEV content for MNPs crosslinked with sodium sulfate are higher compared with those with TPP. As example, 4 samples were selected (A8, A9, B8, B9), the loading kinetics of the LEV is shown in figure 12.

drug

The MNPs ability to release LEV

Releasing the drug into the eye region is a difficult task. Only 1-2% of the drug dose is available in the eye for therapeutic action, the rest of the drug being eliminated through the nasolacrimal drainage system and other ocular physiological barriers. According to literature studies, LEV may be administered topically as a drop every 1-2 hours for 3 days and after every 4-5 hours. In order to overcome these problems of conventional administration formulations, new drug delivery systems, such as MNPs systems, have been explored increasing the residence time of the drug at the eye level is the main objective [START_REF] Gupta | Biodegradable levofloxacin nanoparticles for sustained ocular drug delivery[END_REF].

The present study proposes LEV administration for the treatment of diseases specific to the posterior segment of the eye via CS-g-PEGA particle systems. Therefore, was analysed the LEV release capacity of MNPs, by the diffusion method in basic pH = 7.4 at 37 ° C.

The released drug content and the release efficiency for all samples are highlighted in table 11. Regarding the process of LEV releasing from the MNPs the kinetic data are presented as an example for samples A8, A9 and B8, B9 [(Figure 8.15; 8.16a) and b)]. One can observe a fast phase which is reached within the initial 10 hours followed by a slower phase (characterized by a constant release) until 120 hours. The sustained release of LEV can be explained by the fact that the released drug was adsorbed in the intimate structure of MNPs due to excellent swelling in ABS.

By analyzing the influence of preparation parameters on the release ability of the MNPs, similar behavior as for drug loading was observed. The maximum released amount of LEV varied between 0.27 and 0.57 mg/mg MNPs (table 11). Also, when calculating the efficiency of LEV release for all samples, values between 38 and 64 % were obtained (Fig. 13 b and 14 b), the highest efficiency being noticed for the sample A8 and B8 which have been shown the highest water uptake. The analysis of the release kinetic was achieved also based on the Korsmeyer-Peppas mathematical model (Figures 15 and16).

Tabel 11. Released drug from MNPs and the release efficiency According to the literature [START_REF] Ritger | A simple equation for description of solute release I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs[END_REF], [START_REF] Ritger | A simple equation for description of solute release II. Fickian and anomalous release from swellable devices[END_REF], [START_REF] Peppas | A simple equation for the description of solute release. III. Coupling of diffusion and relaxation[END_REF] , the drug release process is dependent on the method of obtaining the particulate systems, their dimensions, and the physical-chemical properties of the polymer support used. After analyzing the experimental kinetic data for the interval 0 ÷ 480 minutes, it was possible to determinate the diffusion exponent based on the equations: -y = 0,4294• x -3,351; k = 0.4214, n = 0.4294 și R 2 = 0.9778.

Conclusions

The experimental results obtained for previously discussed CS-g-PEGA particulate systems lead to the following conclusions:

-A new poly (ethylene glycol) methyl ether acrylate grafted chitosan derivative was prepared by the Michael addition reaction, which was optimized from the point of view of solubility, by achieving a complete solubilization of the derivative in bidistilled water;

-The structural characterization of CS-g-PEGA was performed by FT-IR and by 1 H NMR spectroscopy, both analyzes revealing the formation of the new derivative.

-New MNPs based chitosan derivative were prepared by double-crosslinking in water-inoil emulsion;

-The influence of initial parameters such as the initial polymer concentration and the rate of stirring, the nature of the ionic crosslinker on the final properties of the MNPs supports has been studied; -It has been observed that the concentration of the polymer solution and the stirring rate influence the particle mean diameter, which decreases with the increase of the stirring rate and the decrease of the polymer concentration; -It has been found that by changing the type of ionic crosslinker influences the behavior of the MNPs like swelling behavior, ie the capacity of inclusion / release of the biologically active principles;

-MNPs FT-IR characterization revealed the formation of new bonds resulting from the ionic crosslinking process between polyanions (tripolyphosphate) of Na5P3O10 or sulphate anions of Na2SO4 and CS-g-PEGA ammonium cations, respectively the imine bond formed through the covalent crosslinking process between the amine groups of the polymer and the carbonyl groups of the glutaraldehyde; -Morphological characterization by SEM has confirmed the obtaining of MNPs with spherical shape and submicronic dimensions, suitable for treatment of some diseases of the posterior segment of the eye -MNPs behavior in two different aqueous media: acid (pH = 3,3), basically (pH = 7,4) was studied, demonstrating a higher water retention capacity for the acidic media, respectively basic media;

  weight of swollen samples mdthe weight of dry samples

Figure 1 .Figure 2 .

 12 Figure 1. NMR spectrum of PGL

Figure 4 .

 4 Figure 4. NMR spectrum of PEG-SH

Figure 7 .

 7 Figure 7. Raman analysis of PGL and PEG-SH

Figure 8 .

 8 Figure 8. Photos of PGN materials before (a) and after the photochemical thiol-ene reaction (b) and after their purification in THF (c) and water (d).

Figure 9 .

 9 Figure 9. Dependence of the recovered gel fraction of prepared on the total polymer concentration (20 %, 10 %, and 5 %) and SH/C=C molar ratio

Figure 9 .

 9 Figure 9. Dependence of the storage modulus G' (solid symbols) and loss modulus G'' (open symbols) on time with  5 %,  10 %,  20 %, and  40 % of the polymer in the polymer mixture using an initial SH/C=C ratio of 1/3.

Figure 11 .

 11 Figure 11. Comparative plot of the theoretical sulfur content calculated from the initial polymer composition with respect to the recovered gel fraction, total sulfur content in the PGN samples (20 % concentration in the initial mixture) determined by elemental analysis and sulfur content in the form of unreacted thiol groups in the PGN samples determined by Ellman's method. Both methods were applied after PGN purification.

Figure 12 .

 12 Figure 12. The relative content of reacted sulfur in PGN samples obtained at a 20 % polymer concentration with various SH/C=C ratios.

Figure 13 .

 13 Figure 13. Thiol group content determined by Ellman's titration method in purified PGN samples prepared with various total feed polymer concentrations and SH/C=C molar ratios and the theoretical content of SH calculated from the initial amount of PEG-SH introduced in the synthesis of the gels.

Figure 14 .

 14 Figure 14. SEM images: cross-sections of PGN samples prepared using different SH/C=C ratios (A, B, and C, 3/1; D, E, and F, 1/1; and G, H, and I, 1/4) and a 20 % total feed polymer concentration shown at various magnifications (scale bar length: A, D, and G, 1 mm; B, E, and H, 0.5 mm; and C, F, and I, 0.05 mm).

Figure 15 .

 15 Figure 15. SEM images: cross-sections of PGN samples prepared using a 3/1 SH/C=C ratio and different concentrations in the polymer feed (A and B, 20 %; C and D, 10 %; and E and F, 5 %) at various magnitudes (scale bar length: A, C, and E, 0.5 mm, and B, D, and F, 0.3 mm).

Figure 18 .

 18 Figure 18. Swelling behavior of PGN in THF

Figure 20 .

 20 Figure 20. Cytotoxicity of PGN extracts to mouse 3T3 fibroblasts. Statistically significant differences are shown by asterisks: * P > 0.05, ** P > 0.01 and *** P > 0.001.

Figure 21 .

 21 Figure 21. Cytotoxicity of polymers, used for PGN preparation, to 3T3 fibroblasts: A) PEG-SH, B) extracts obtained from different amounts of PGL powder, C) PGL powder. The dashed line represents the viability of untreated control cells which was considered to be 100%. Significance of differences at different level is shown by asterisks: * P > 0.05, and *** P > 0.001, respectively.

Figure 22 .

 22 Figure 22. Release efficiency of LEV for samples with SH/C=C ratios of 1/4, 1/1 and 3/1 prepared with polymer concentrations of 20 wt. %.

Figure 23 .

 23 Figure 23. In vitro LEV release for sample G3,G12,G18

Figure 1 . 4 )Figure 2 .

 142 Figure 1. FT-IR spectra of PEGMA, CS, CS-g-PEGMA The 1 H-NMR recorded spectra offer useful information regarding the chemical composition of obtained modified polymer. The 1 H NMR spectrum of CS, PEGMA and CS-g-PEGMA in D3CCOOD/D2O or D2O at 80 0 was shown in Fig. 2 a,b,c. A small peaks at 1.795 ppm existed because of the presence of -CH3 of N-alkylated GlcN residue. A singlet at 3.139 ppm assigned to H2 of 2-amino-2-deoxy-β-d-glucopyranosyl (GlcN) and N-alkylated GlcN. The multiplets from 3.5 to 3.8 ppm attributed to H3, H4, H5, H6 of GlcN and N-alkylated GlcN. A small peak at 4.52 ppm attributed to H1 of GlcN and N-alkylated GlcN. While a small signal of formed methylene protons adjacent to the 2-amino group of the GlcN residue was observed at 1.994 ppm (-CH2b-COO-). The typical peak at 4.528 ppm is appeared because of -CH2d of GlcN residue. The DS values based on -CH2b-COOwas determined by the relative intensities of these signals and was calculated from the peak area of CH2b-proton against NHAc proton. The DS calculation was calculated according to the equation (4): 𝐷𝑆 = 3•𝐼 3.1 𝑝𝑝𝑚 •(1-𝐷𝐷) 2•𝐼 2.0 𝑝𝑝𝑚

Figure 5 .

 5 Figure 5. a) Chitosan thermogram b) PEGMA-g-chitosan thermogram

Figure 4 .

 4 Figure 4. Thermogram for chitosan, CS-g-PEG-MA, NPs

Figure 5 .Figure 6 .

 56 Figure 5. SEM pictures of optimizedpoly(ethylene glycol) methacrylate grafted chitosan micro/nanoparticles crosslinked with sodium tripolyphosphate

Figure 7 .Figure 8 .Figure 9 .Figure 10 .

 78910 Figure 7. Polymer concentration influence on particle diameter. Granulometric distribution of MNPs crosslinked with sodium tripolyphosphate for samples MA-8 (0,5 %), 9 (0,35 %), 11 (0,75 %)

Figure 11 . 24 MAFigure 12 .

 112412 Figure 11. Stirring speed influence on swelling process for NPs samples MA-8 and H (15.000 rpm); 7 and G (12.000 rpm); 6 and F (9.000 rpm); 5 and E (5.000 rpm) in acidic (ABS)/alkaline (PBS) environment

Figure 13 .

 13 Figure 13. Hemolysis percentage after 2  , 4  and 6 hours of exposure to NPs

5. 6 .

 6 Cytotoxicity testThe cytotoxicity of MNPs was studied on osteoblastic cell cultures. Few NPs samples were selected and the results are shown in figure14. Colorimetric cytotoxicity assay, MTT was performed on the osteoblastic cell cultures type at intervals of 24, 48, or 72 hours. The assay confirmed that the NPs have a reduced dose of toxicity a fact also demonstrated by DL50 test. After 5 days, cells have proliferated normally in the presence of CS-PEGMA NPs forming a dense cell 0 . 14 a,b,c). The cell viability was 100%, the increased cell viability indicates that cells have proliferated favorable in the presence of NPs compared to control sample (fig.17 d), and that CS-PEGMA NPs are not cytotoxic.

Figure 14 .

 14 Figure 14. Evaluation of osteoblast cell morphology for 5 days in the presence of 5 mg/ml MPs 5.7. MNPs ability of loading Levofloxacin or Bevacizumab

Figura 15 .

 15 Figura 15. Cinetica de încărcare a LEV și BEV în particulele MA-8 și H

Figura 16 .Figure 17 . 8 Figura 18 .

 1617818 Figura 16. In vitro a) release kinetics of LEV; b) LEV release efficiency for MA-8 and H sample

Figure 20 .Figure 21 .Figure 22 .

 202122 Figure 20. The fluorescein fluorescence for control cells without treatment (100%) for: a) No treatment; b) Inflammation with lipopolysaccharide; c) Diabetes; d) Central RVO

Figure 1 .

 1 Figure 1. Comparative FT-IR spectra of CS, PEGA, CS-g-PEGA

DS = 3 •Tabel 2 .

 32 I4,3 ppm•(1-DD)/2•I2,0 ppm (1) Valori ale gradului de substituție CS-g

Figure 2 . 5 . 2 .

 252 Figure 2. Specta of 1 H RMN al a) CS; b) PEGA; c) CS-g-PEGA 5.2. Preparation of MNPs based PEGA by double cross-linking in reverse emulsion (w/o)

  .

Figure 3 .Table 5 .

 35 Figure 3. Comparative FT-IR spectra of CS-g-PEGA and NPs samples A8, B8Table 5. Characteristic signals of CS-g-PEGA and NPs based chitosan derivative

Figure 4 .Figure 5 .

 45 Figure 4. SEM images of optimized MNPs ionic crosslinked with TPP

Figure 6 .

 6 Figure 6. Granulometric distribution curves of A-5÷11 samples Tabel 6. Average diameter of samples A5-11 Sample

Tabelul 8 .

 8 Swelling degree of A

Figure 8 .Figure 9 .Figure 10 .Figure 11 .

 891011 Figure 8. Kinetic of swelling degree in acetate buffer pH = 3.3 and phosphate buffer pH = 7.4for samples A9 and B9

Figure 12 .

 12 Figure 12. Loading kinetic of LEV for A8, A9, B8, B9

Figure 13 .Figure 14 .Figura 15 . 8 Figura 16 .

 131415816 Figure 13. Capacity of samples A-8 și B-8 of a) releasing LEV b) Release efficiency of LEV

Figure 17 .

 17 Figure 17. Influence of nanoparticle concentration on the degree of hemolysis

Table 1 .

 1 Initial parameters for the synthesis of PGN and results of gel fraction analysis of the PGN materials

	Sample	Total polymers	Molar ratio	Gel fraction,	Gel fraction,
	code	concentration,	SH/C=C	after THF	after water
		% (w/v)		washing, %	washing, %

Table 2 .

 2 Molecular characteristics of the synthesized polymers

	Synthesized polymers	Mn, g/mol	Mw, g/mol	Ð	Mn-from NMR data
	PGL	5600	18900	3.4	6350
	PEG-SH	2400	7800	3.3	-

1492.2 1254.0 1730.4 1968.6 2206.8 1015.8 2445.0 2683.2 737.6 2922.4 3160.6 3397.8 3637.0 3875.2 4113.4 4351.6 2262.8 1548.2 1786.4 2024.6 2501.0 4589.8 2743.2 2978.4 3216.6 4828.0 3454.8 5066.2 5304.4 5542.6 5780.8 6020.0 6258.3 0 2 4 6 4 x10 Intens. [a.u.] 1000 2000 3000 4000 5000 6000 m/z

1377.7 1333.6 1289.6 1465.7 1509.7 1201.6 1553.7 1025.5 1597.8 1157.5 1069.5 1641.8 1685.8 1773.8 2154.0 2242.0 2066.0 1933.9 2330.1 1845.9 2418.1 1993.9 1229.5 1009.4 2506.2 2594.2 2814.3 2726.3 2946.4 PegSH Dir Dith 0:K21 MS Raw 0 1 2 3 4 4 x10 Intens. [a.u.] 1000 1500 2000 2500 3000 3500 4000 4500 m/z

  Figure 3b. MALDI analysis of PEG-SH, detailed viewThe 1 H-NMR recorded spectra of PEG-SH (fig.4) point out the characteristic signal of thiol group detected at 2.27 ppm. Overall, the employed characterization methods suggest that the sulfhydryl groups were stable during the polycondensation process. Also, another important signal of PEG end methylnene group (neighboring the carbonyl from TMA) was detected at 4.27 ppm, which indicates the esterification with thiomalic acid. As shown in figureS7the characteristic proton signals of PEG-SH are presented. PEG-SH: 1 H NMR: δ (ppm) 4.

	PEG 2 -TMA 2(1)
	PEG 3 -TMA 3(4)
	PEG 4 -TMA 5
	PEG 5 -TMA 6
	37-4.19 (CH2O(C=O)), 3.85-3.77
	(PEG backbone) (3.12-2.94 and 2.86-2.74 (CH2(C=O) O), 2.38-2.24 (C-SH)

1377.7 1333.6 1421.7 1289.6 1465.7 1245.6 1509.7 1201.6 1553.7 1597.8 1157.5 1113.5 1069.5 1641.8 1493.7 1449.7 1581.7 1537.7 1685.8 1625.7 1405.6 1669.8 1729.8 1475.7 1431.6 1713.8 1361.6 1519.7 1387.6 1343.6 1563.7 1317.6 1607.7 1273.6 1651.8 1695.8 1229.5 1301.6 1739.8 1185.5 PegSH Dir Dith 0:K21 MS Raw 0 1 2 3 4 4 x10 Intens. [a.u.] 1100 1200 1300 1400 1500 1600 1700 m/z 1377.7 1333.6 1289.6 1245.6 1361.6 1387.6 1343.6 1317.6 1299.6 1273.6

	44.02	44.02
		18.02	16.05

PegSH Dir Dith 0:K21 MS Raw 0 1 2 3 4 4 x10 Intens. [a.u.] 1240 1260 1280 1300 1320 1340 1360 1380 m/z

Table 3 .

 3 The influence of the photoinitiator and reaction volume on gelation time

	Molar		Gelation time, seconds
	ratio	Low reaction		High reaction volume
	SH/C=C	volume		(2.6 mL)
		(300 μL)
		2,2-Dimethoxy-2-phenylacetophenone
		1%	1%	0.75% 0.5%	0.25%
		with respect to total amount polymers
	1/1	150	1200	1320	1500	3000÷∞
	1/2	210
	1/3	240

Table 4 .

 4 Elemental analysis of PGN samples prepared using 20 % polymer concentration with various initial SH/C=C ratios

	Sample name	SH/C=C ratio	Weight (mg)	Nitrogen Carbon Hydrogen Sulfur
	Reference		1.330	9.23	40.33	7.39	21.61
	G3	1:1	1.222	0.00	56.70	8.50	2.50
	G6	1:2	1.365	0.00	60.40	9.00	2.40
	G9	1:3	1.333	0.00	62.50	9.30	2.00
	G12	1:4	1.266	0.00	64.90	9.70	1.60

Table 5 .

 5 Content of sulfur in the form of free thiol groups and in the form of sulfur coupled to double bonds in purfied PGN samples with different feed total polymers concentrations and thiol/ene molar ratios.

	Sample code	Total feed polymers concentration, % (w/v)	SH/C=C molar ratio	µM SH/g	SH % free,	C-S-C, %
	G1	5	1:1	173	17	83
	G2	10	1:1	301	29	71
	G3	20	1:1	428	42	58
	G4	5	1:2	98	12	88
	G5	10	1:2	246	30	70
	G6	20	1:2	298	36	64
	G7	5	1:3	88	13	87
	G8	10	1:3	134	19	81
	G9	20	1:3	156	23	77
	G10	5	1:4	49	8	92
	G11	10	1:4	60	10	90
	G12	20	1:4	118	20	80
	G13	5	2:1	274	23	77
	G14	10	2:1	364	31	69
	G15	20	2:1	477	41	59
	G16	5	3:1	435	35	65
	G17	10	3:1	471	38	62
	G18	20	3:1	541	44	56

Table 6 .

 6 Loaded and released amount of LEV for the analyzed samples Sample code

		Concentration,	SH/C=C Loaded	Released	Efficiency
		%		LEV,	LEV	of LEV
				mg/mg	mg/mg	released,
				gel	gel	%
	G1	5	1/1	0.380	0.30	79
	G2	10	1/1	0.330	0.245	74
	G3	20	1/1	0.327	0.223	68
	G10	5	1/4	0.380	0.370	97
	G11	10	1/4	0.383	0.290	76
	G12	20	1/4	0.410	0.270	66
	G16	5	3/1	0.386	0.380	98
	G17	10	3/1	0.325	0.320	98
	G18	20	3/1	0.353	0.350	99

Table 2 .

 2 Protocol for the synthesis of chitosan grafted poly (ethylene glycol) methacrylate MNPs crosslinked with sodium sulfate

	, 2.
	Table1. Protocol for the synthesis of chitosan grafted poly (ethylene glycol) methacrylate
	MNPs crosslinked with sodium tripolyphosphate
	Sample	Polymer	Ratio	NH3 + /	NH3 + /	Surfactants	Speed, rpm	Ionic/Co	Averange
	code	solution concentrat ion, %	W/O	Na5P3O10, molar ratio	C5H8O2, ratio molar	with polymer the respect to		valent duration, linking Cross-	Diameter, µm
						solution%		h
						w/w
	MA-5	0.5		1:2			5.000			3.00
	MA-6	0.5		1:2			9.000			2.90
	MA-7	0.5		1:2			12.000			1.30
	MA-8	0.5		1:2			15.000			0.50
	MA-9	0.35		1:2			15.000			0.50
	MA-10	0.5		1:3			15.000			0.80
	MA-11	0.75	1:4	1:2	1:2	2	15.000	1		1.10
	Sample	Polymer	Ratio	NH3 + /	NH3 + /	Surfactant	Speed,	Ionic	Covale	Averan
	code	solution concentratio n, %	W/O	Na5P3O10 , molar ratio	C5H8O2, molar ratio	s with respect to polymer the	rpm	Cross-linkin durati g	nt Cross-duratio linking	ge Diamete r, µm
						solution%		on, h	n, h
						w/w
	MA-E	0.5		1:2			5.000			4.40
	MA-F	0.5		1:2			9.000			2.60
	MA-G	0.5		1:2			12.000			1.60
	MA-H	0.5		1:2			15.000			1.50
	MA-I	0.35		1:2			15.000			1.10
	MA-J	0.5		1:3			15.000			1.30
	MA-K	0.75	1:4	1:2	1:2	2	15.000	1	2	1.10

Table 3 .

 3 Results for thermal analysis in isothermal conditions

	Sample	Degradation stages Temperature	Mass loss (%)
			(ᴼC)
	CHITOSAN	I	30÷100	7.22
		II	255÷465	45.98
		III	465 ÷800	28.25
		Calcination	˃ 800	18.54

Table 4 .

 4 Average diameter of samples MA-5 ÷ 11, obtained after analysis by laser

	diffractometry
	Sample	Average diameter, nm
	MA-5	83
	MA-6	585
	MA-7	763
	MA-8	854
	MA-9	924
	MA-10	857
	MA-11	585

Table 5 .

 5 Average diameter of samples MA-E ÷ K obtained from laser diffractometry analysis

	Sample	Average diameter, nm
	MA-E	4654
	MA-F	457
	MA-G	105
	MA-H	179
	MA-I	143
	MA-J	190
	MA-K	585

Table 4 .

 4 Zeta potential for optimized CS-PEGMA samples

	Samples	Zeta potential [mV]
	MA-5	2.275
	MA-6	0.543
	MA-8	0.583

Table 5 .

 5 Toxicity

	test averange values

Table 6 .

 6 Amounts of drug included and loading efficiencies

	Samples mg LEV/ mg NPs	loading
			efficiency, %
	MA-5	0,787	94,52

Table 7 .

 7 The evolution of the BEV release (fig 17a) is almost linear, excepting the reduced burst effect at 30 min. One can also observe that 51 % of BEV has been released in 168 hours, fact which is suggesting a slow release rate. Also, experimental data of release kinetics were analyzed based on the mathematical model Korsmeyer-Peppas(Figures 18,19,20). The drug quantities released and the release efficiency

	Sample	mg Drug /	Release
		mg MNPs	efficiency, %
	MA-5 (LEV)	0,66	84,53
	MA-6 (LEV)	0,57	78,69
	MA-7 (LEV)	0,52	66,5
	MA-8 (LEV)	0,63	78,12
	MA-8 (BEV)	0,323	99
	MA-9 (LEV)	0,51	63,5
	MA-10 (LEV)	0,3	42,07
	MA-11 (LEV)	0,27	53,56
	MA-E (LEV)	0,24	42,4
	MA-F (LEV)	0,24	36,7
	MA-G (LEV)	0,23	35,18
	MA-H (LEV)	0,37	59,31
	MA-I (LEV)	0,26	44,07
	MA-J (LEV)	0,25	40,76
	MA-K (LEV)	0,28	46,73

Table 7 .

 7 Values of n and k obtained based on the experimental kinetic data analysis for the range 0 ÷ 300 minutes

	0.00000
	0.00	0.50	1.00	1.50	2.00	2.50	3.00
	-0.20000
	-0.40000
	-0.60000
	-0.80000
	1.00000

Table 8 .

 8 Results obtained for the fluorescence of lectin-bound fluorescein for evidence of retinal endothelial cells in control rabbits with Inflammation with lipopolysaccharide, Diabetes and central RVO in the presence of micro/nanoparticle treatments, whether or not loaded with the active substance, at 72 hours (%). P <0.05

			Inflammation with
	Applied treatment	Control	lipopolysaccharide	Diabetes	Central RVO
	No treatment	100	113,45±4,63	132,74±7,19 120,74±5,12
	1% Suspension of BEV	101,12±3,12	105,77±7,47	117,59±4,91 111,79±3,14
	loaded NPs

Table 1 .

 1 The characteristic absorption bands of CS, PEGA, CS-g-PEGA

	Compound	Wavelength (cm -1 )	Absorption band
		1082	stretching vibrations of -C-O-C
	Chitosan	1379,1423,1653	specific signals for deformation vibrations of acetylated amine (NHAc) (amide I, -NH2 and amide III)
		2875	Vibration of CH groups
		3365	Axial stretching vibrations of OH and NH2 groups
		1107	stretching vibrations -C-O-C
	PEGA	1722	The vibration signal corresponding to the C = O bonds
		2862	Vibration of CH groups
		3435	Axial stretching vibrations of OH groups
		1076	stretching vibrations -C-O-C
		1377,	specific signals for deformation vibrations of acetylated
	Cs-g-PEGA	1562,1654	amine groups
		1720	The vibration signal corresponding to the C = O bonds
		2855	Vibration of CH groups
		3466	Axial stretching vibrations of secondary amine groups

Table 3 .

 3 Variation of parameters for the synthesis of MNPs crosslinked with sodium tripolyphosphate.Tabel 4. Variation of parameters for the synthesis of MNPs crosslinked with sodium sulphate

						Surfactants
		Polymer		NH3 + /	NH3 + /	with respect to the		Ionic Cross-	Covalent
		solution		Na5P3O10	C5H8O2	polymer		linking	Cross-	Averange
		concentrati	Ratio	, molar	, molar	solution%	Speed,	duration,	linking	Diameter,
	Sample	on, %	W/O	ratio	ratio	w/w	rpm	h	duration, h	µm
	A1	0.5					5.000	10		-
	A2	0.5					5.000	30		-
	A3	0.5					5.000	120		-
	A4	0.5		1:1			5.000	150		-
	A5	0.5					5.000			3,0
	A6	0.5					9.000			1,7
	A7	0.5					12.000			0,97
	A8	0.5	1:4	1:2	1:1	2	15.000	60	60	0,96

Table 9 .

 9 Swelling degree of B-5÷11

	Sample	Swelling degree in	Swelling degree in
		acidic medium (ABS), %	basic medium (PBS), %
	B-5	864	714
	B-6	894	732
	B-7	855	737

  encapsulation efficiency = [encapsulate LEV content / theoretical LEV content] x100 (2) Tabel 10. Encapsulated drug in MNPs and loading efficiency

	Sample mg LEV/ mg MNPs Encapsulation
			efficiency, %
	A-5	0,63	62,34
	A-6	0,69	66,36
	A-7	0,62	58,76
	A-8	0,75	76,41
	A-9	0,67	66,36
	A-10	0,48	47,32
	A-11	0,66	66,53
	B-5	0,63	75,00
	B-6	0,66	69,37
	B-7	0,65	66,31
	B-8	0,89	75,76
	B-9	0,88	69,05
	B-10	0,60	71,88
	B-11	0,65	64,38

Micro/Nanoparticles behavior in aqueous media

The study of swelling behavior led to interesting results, the values obtained for the maximum degree of swelling being in good correlation with the parameters of the MNPs synthesis. The maximum swelling degree the particles in aqueous media is clearly dependent on the nature of the internal architecture of the newly formed polymer network. Hydrophilicity of MNPs provide important information for predicting their behavior for loading / releasing drugs by diffusion process.

The analysis results of the swelling degree in acid (pH = 3.4) and basic (pH = 7.4) environment after 24 hours (figures 8, 9, 10, 11 and tables 8, 9) lead us to the following conclusion: -After a first analysis of the experimental data obtained, the swelling degree in acetate buffer (ABS, pH = 3.3) is higher than those recorded in the phosphate buffer solution (PBS, pH = 7.4), and was observed the influence of parameters like concentration of the polymer solution, the stirring rate and the polymer / ionic crosslinking ratio; -although at a low polymer solution concentration (0.35%, samples A9, B9) the swelling degree exhibited the highest values as a result of: a) the retention of water in the formed polymer agglomerates (which is mainly evident in sample B9) and b) the lower crosslinking degree of the network;

-y = 0,5182• x -3,2; k = 0.189, n = 0.5182 și R 2 = 0.9845. One can observ that in both cases (A8, B8), the value of the exponent n which characterized the release mechanism is quite close to 0.5 (0.5 <n <1.0), indicating a transport / release mechanism dominated by diffusion.

Table 12. highlights the values of the n and k exponents obtained for the rest of the analyzed samples, and we can state that samples A 11, B5, B6 and B11 exhibit an abnormal, non-Fickian diffusion, the drug transport process through the polymer matrix being governed by both diffusion and swelling.

Hemocompatibility

The method of hemocompatibility analysis was described in detail in chapter 7 subchapter 4.8. The hemolysis test is a mandatory requirement for blood-borne materials, since their interaction with blood components may lead to erythrocyte lysis. For this reason, the effects of the prepared nanoparticles on the blood were evaluated using a hemolysis assay. The results obtained from this study revealed that the degree of hemolysis increases with the increase of the nanoparticle concentration (Figure 17). It has been found that the nanoparticles obtained have good compatibility with the normal blood (<10% compared to the positive control) [431]. Also, an increase in hemolysis percentage for B8 samples was observed in this case being used as ionic crosslinker Na2SO4, a possible cause being the higher amount required for network crosslinking (molar ratio NH 3 + / Na2SO4 -1/4) compared with sample A8 (Molar ratio NH 3 + / TPP-1/2). All samples were analyzed in duplicate.

-Nanoparticles have been tested also by hemocompatibility analysis, the results showing a hemolysis percentage of less than 5%, indicating that they are hemocompatible and can be administered intraocularly.

-Considering the main objective of MNPs, namely drug delivery system for the treatment of posterior segment eye diseases, the ability to include / release LEV in MNPs by diffusion was tested; after loading from acetate buffer, a rapid release was observed within the first 10 hours, followed by a slower phase (characterized by a constant release) up to 120 hours.

Taking into account the results of the research and the conclusions mentioned above, we consider that the objectives of the proposed doctoral thesis have been fulfilled and that the realized polymer systems have structural, morphological characteristics, are indicated for the transport of biologically active principles and have potential for use both in applications of tissue engineering and in the therapy of posterior segment of the eye.