Franceline Reynaud

Nicolas Tsapis
email: nicolas.tsapis@u-psud.fr

Michel Deyme

Tibiriça G Vasconcelos

Claire Gueutin

Sílvia S Guterres

Adriana R Pohlmann
email: pohlmann@iq.ufrgs.br

Elias Fattal

SPRAY-DRIED CHITOSAN-METAL MICROPARTICLES FOR CIPROFLOXACIN ADSORPTION: KINETIC AND EQUILIBRIUM STUDIES

Keywords: spray-drying, chitosan, metal, ciprofloxacin, adsorption

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Introduction

Recently, the scientific community started to consider residual pharmaceuticals as potential pollutants and research about the behavior of these compounds and their metabolites has constantly grown [1][2][START_REF] Kümmerer | Pharmaceuticals in the Environment: Sources, Fate Effects and Risks[END_REF][4] . It has been shown that concentrations of drugs/metabolites in hospital wastewater, sewage treatment plants (STPs), surface water or even in drinking water range from ng.L -1 to g.L -1 [5][6][7] . In drinking water, although concentrations are lower than those administered therapeutically, chronic effects on humans when exposed are still unknown and drugs often present low or no biodegradability [8][9][10] .

Antibiotics [11][12] as well as resistant bacteria 13 have been found in environmental compartments. Indeed, the intestinal tract is the site where most antibiotic resistance develops [14][15] . In major cases, residual colonic antibiotics provided by of orally administered or parenterally administered followed by intestinal secretion can give rise to the emergence of resistance to antibiotics by a process in which commensal resistant bacteria are first selected in the natural human or animal ecosystems. These bacteria can then transfer resistance mechanisms horizontally to pathogenic species either in the same host in whom the resistant commensals have emerged, or in another host after interindividual transmission of the resistant commensals [16][17][18] . Another possibility is the rejection of the antibiotic itself in the ecosystem which can as well induce the selection of resistant bacteria but also induce other adverse effects behaving such as signaling molecules that may regulate the homeostasis of microbial communities 17,[19][20][21] . Moreover, common receptors have been identified in plants for a number of antibiotics affecting number of biological process such as chloroplast replication, transcription and translation, folate biosynthesis, fatty acid synthesis and sterol biosynthesis 22 . Among all antibiotics, ciprofloxacin, a synthetic fluoroquinolone, is a broad-spectrum antibacterial agent for oral administration, used to treat various bacterial infections 23 .

However, ciprofloxacin presents a low biodegradability even in the presence of sewage treatment plants in effluents [24][25] , and can still exert its deleterious effect on the environment 26 27 . Moreover, as it is the antibiotic reaches surface water 8,11 . A study on the ecotoxicological effects of ciprofloxacin antibiotic in the aquatic environment demonstrated significant impact on the rate of algal biomass production and algal community structure 28 .

Investigations of technologies to treat effluents containing ciprofloxacin are therefore crucial. Advanced oxidation processes (AOPs) like ozone-and photo-based ones have demonstrated good potential 29 in transforming ciprofloxacin. Although these AOPs are promising alternatives, they generate by-products with low biodegradability 30 and more research on the environmental characteristics of these compounds is needed. As an alternative to AOPs, we intend to develop a system that combines the ability of chitosan to bind metal ions with the natural complexation of antibiotics such as ciprofloxacin with the same metal ions. Chitosan-metal microparticles were therefore designed for ciprofloxacin removal.

The polyaminosaccharide chitosan is the deacetylated derivative of chitin, a naturally occurring polymer found in the shells of crustacean, the cuticles of insects and cell walls of some fungi 31 32 . Due to its biocompatibility, biodegradability, bioactivity and low toxicity, this biopolymer has been used for flocculation and coagulation in food processing, in heavy-metal ion recovery from wastewaters and in fabrication of structural matrices for food, cosmetic, biotechnological and biomedical applications 33 . Considering the extra electron pair of nitrogen, chitosan can form polymer-metal complexes with many metal cations [34][35] and is thus used as a metal-ion sorbent. Studies have shown that the metal ion is complexed with chitosan through the oxygen and the nitrogen of the aminosaccharide group [34][35][36][37] . Possible applications of the metal binding property are wastewater treatment for heavy metal and radio isotope removal with valuable metal recovery, and drinking water purification for reduction of unwanted metals 38 Ciprofloxacin is able of forming strong complexes with multivalent metal ions. The stability of the complex formed is greatly dependent on the nature of the metal ion involved, and the environmental conditions. In the case of ciprofloxacin, the complex formation constant with iron ions is very high and the complex ciprofloxacin:metal stability declines in order: Fe(III) ≈ Al(III) > Cu(II) > Fe(II) ≈ Zn (II) > Mg(II) > Ca(II) [39][40][41] In this paper, the efficiency of chitosan-metal microparticles to adsorb ciprofloxacin was evaluated as a function of the type of metal ions, the contact time and the concentration of ciprofloxacin. More important, we tried to give insight into the mechanism of adsorption by confronting experimental data for both isotherm and kinetics with theoretical models.

2.Materials and methods

Materials

Chitosan (CH), zinc acetate dehydrate, and ciprofloxacin were obtained from Fluka (Switzerland). Glutaraldehyde and sodium acetate were provided by VWR (France). Iron (II) chloride was from Acros Organic (Belgium). Hydroxyethylpiperazine-ethanesulfonic acid (HEPES), 1-(2-pyridylazo)-2-naphthol (PAN), ammonia buffer (pH 10) for complexometry, hydroxylamine hydrochloride, 1,10-phenantroline and iron(III) nitrate were obtained from Sigma-Aldrich (France). HPLC grade solvents were supplied by Carlo Erba (Italy).

Preparation of Chitosan-metal microparticles

Chitosan-metal (CH-metal) complexes were prepared according to a modification of a procedure previously described 34 . Briefly, solutions were obtained by dissolving chitosan (1% w/v) in a metal ion aqueous solution (FeCl 2 0.1M; Fe(NO 3 ) 3 0.1M and Zn(CH 3 COO) 2 0.1M). They were maintained upon magnetic stirring for 12 h at room temperature (20ºC)

to yield CH-metal complexation. The metal complexation capacity of chitosan is strongly affected by the pH of the solution. For each of these metals, there is an optimal complexation pH which is rather acidic in order to reduce the formation -NH +3 groups unfavorable for complexation. Therefore, in our conditions, the pH was fixed at 5 as a compromise that permits to compare the different chitosan-metal complexes prepared under the same conditions.

CH-metal microparticles were then prepared by spray-drying. The chitosan-metal complex solutions prepared above (200 mL) were spray-dried using a Buchi ® Mini Spray Dryer, type B191 (Switzerland) with a standard 0.7 mm nozzle at a feed rate of 5 ml.min -1 . The atomizing air flow rate was 600 L.h -1 and the aspiration of 100%. The inlet temperature was controlled at 150°C and the outlet temperature varied between 80-100°C.

Microparticles cross-linking

After spray-drying, CH-metal microparticles were suspended into a 0.025M glutaraldehyde (GLA) solution to obtain a 1:1 molar ratio with chitosan (mol GLA: mol NH 2 ). Non-cross-linked microparticles (1 g) were suspended under magnetic stirring for 4 h in a glutaraldehyde solution (25% in acetone, 100 mL). Microparticles were then filtered and washed with ethanol-water (2:1, v/v) solutions to remove the excess of metal ions and glutaraldehyde, and finally vacuum-dried for at least 24 h.

Microparticles size distribution

A Mastersizer 2000 granulometer equipped with a Scirocco dry disperser (Malvern Instruments, France) was used to determine CH-metal microparticle size distribution.

Average particle size was expressed as the volume mean diameter (D 4,3 ). Polydispersity was given by the span index, which was calculated according to Eq. (1):

0.9 0.1 0.5 ( ) / Span D D D  (1)
where D 0.9 , D 0.5 and D 0.1 are the particle diameters determined respectively at the 90 th , 50 th and 10 th percentile of undersized particles.

Scanning Electron Microscopy (SEM)

Microparticle morphology was evaluated by scanning electron microscopy using a LEO 9530, Gemini (France) at an accelerating voltage of 3kV. Prior to observation, samples were mounted on metal stubs, using carbon-conductive double-sided adhesive tape, and coated with a 4 nm platinum/palladium layer under vacuum (Cressington 208 HR, Eloise, France).

Degree of deacetylation

The degree of deacetylation of the chitosan and CH-metal microparticles was determined by infrared spectrometry. A thin film of microparticles was used to record an infrared spectrum using a Spectrum One FT-IR and a Diamond ATR accessory (Perkin Elmer).

The absorbance at 1.655 cm -1 for amide-I and at 3.450 cm -1 for OH groups in chitosan were determined. The degree of deacetylation (DDA) was then calculated using Eq. ( 2) 42

1.655 3.450 (%) [1 ( / ) /1.33] 100 DDA A A x  (2)
where factor 1.33 represents the A 1.655 /A 3.450 ratio for fully N-acetylated chitosan.

Metal determination 2.7.1 Iron determination

The quantity of iron associated to cross-linked CH-iron microparticles was determined by a spectrophotometric method using 1,10-phenantroline for colorimetry as described [START_REF]Standard Methods for the Examination of Water and Wastewater[END_REF] .

Cross-linked CH-Fe(II) and CH-Fe(III) microparticles were first submitted to acid hydrolysis (100 mg/ 5 mL HCl:HNO 3 1:1 v:v). Iron Standards solutions containing different concentrations of iron (0.1-2.5 µg.mL -1 ) were prepared. Briefly, 1 µl of samples containing iron ions, 2 mL of hydroxylamine hydrochloride solution (1 g.mL -1 ) and 5 mL acetate buffer solution were placed in volumetric flasks of 10 mL. 2 mL of 1,10-phenantroline (1g.L -1 ) stock solution were added and the volume was adjusted to 10 mL with water. After 20 min, an orange-red complex was formed. Absorbances were measured at 510 nm against free iron solution using a UV-2101PC spectrophotometer (Shimadzu, Japan).

Zinc determination

The quantity of Zn(II) associated to chitosan was determined according to a modification of procedure described in the literature 18,[START_REF] Khoder | [END_REF] . Briefly, cross-linked CH-Zn(II) microparticles were submitted to acid hydrolysis (100 mg/ 5 mL HCL:HNO 3 1:1 v:v). Standard zinc solutions containing known concentrations of zinc ions (25 -300 µg.mL -1 ) were prepared.

1µl of samples containing zinc ions and 1 mL of ammonia buffer solution (pH 10) were placed into a volumetric flask. 3mL of ethanolic PAN stock solution (5.0x10 -4 M) were added and the volume was adjusted to 10mL with ethanol. The sample was then shaken and left to stand in a thermostated water bath for 10 min. at 40°C to allow complete formation of the PAN-Zn complex. The amount of zinc was determined by measuring the absorbance at 550nm against free zinc solution (UV-2101PC Shimadzu, Japan). The uptake of ciprofloxacin by unit mass of adsorbent (q) at any time was determined from Eq. ( 3):

Ciprofloxacin adsorption by chitosan-metal microparticles

(

) 3
where C o and C are the initial and remaining unadsorbed ciprofloxacin concentration (µg.mL -1 ) in solution at any time and  is the microparticle concentration (mg.mL -1 ).

Chromatographic system and conditions

The HPLC system consisted of a Waters 510 pump (with an on-line degasser), a Waters 717plus autosampler, a Waters 470 fluorescence detector and column heater. The wavelength of excitation was set at 280 nm and the wavelength of emission at 460 nm, Chromatographic separations were performed in 10 min (flow rate 1mL.min -1 ), at 40°C using a C18 Symmetry ® column (5 µm, 150 x 4.6 mm; Waters, France). The mobile phase consisted of a mixture of acetonitrile and phosphate buffer solution 10 mM (15:85 v/v), acidified at pH 3 with orthophosphoric acid. Data acquisition and analysis was performed using the Azur ® software.

2.9 Modeling of sorption equilibrium and kinetics of ciprofloxacin

Modeling of sorption equilibrium

Equilibrium data, commonly known as adsorption isotherms, are basic requirements for the design of adsorption systems used for removal of organic compounds from solutions. The Langmuir and Freundlich equations are the most frequently used for data interpretation. The mathematical description of Langmuir model follows Eq. ( 4):

  max 1 L eq L eq K C M q KC   (4)
where C eq is the unadsorbed ciprofloxacin concentrations, and q the uptake of ciprofloxacin by unit mass of adsorbent given by Eq. 3. M max represents a practical limiting adsorption capacity when the surface is fully covered with antibiotic and K L is a constant related to the affinity of the binding sites. K L and M max can be determined from the linear plot of 1/q versus 1/ C eq . The Langmuir model assumption is that one adsorption site can bind one molecule.

The mathematical description of Freundlich model follows Eq. ( 5):

F eq q K C   (5)
where K F and  are the Freundlich constants related to the adsorption capacity and adsorption intensity of the sorbent, respectively. This model can be linearized in logarithmic form and Freundlich constants can be determined.

Modeling of adsorption kinetics of ciprofloxacin

The adsorption kinetics of ciprofloxacin may be described by pseudo-first-order 45 and pseudo-second-order 46 kinetic models. The pseudo-first-order model is based on sorbent capacity and considers that the rate of occupation of adsorption sites is proportional to the number of unoccupied sites. The pseudo-first-order equation follows in

Eq. ( 6):

1, ( ) log 2.303 ad eq eq K Log q q q t   

where q eq and q refer to the amount of ciprofloxacin adsorbed (both in µg.mg -1 ) at the equilibrium time and time t (min), respectively. K 1,ad (min -1 ) is the equilibrium rate constant of pseudo-first-order sorption. A straight line of Log(q eqq) versus t suggests the applicability of this kinetic model.

The pseudo-second-order equation is also based on the adsorption capacity of the solid phase and expressed as follows in Eq. ( 7):

2 2,
11 ad eq eq t t q K q q  (7

)
where K 2,ad (mg.µg -1 .min -1 ) is the rate constant for pseudo-second-order adsorption kinetics. If second-order kinetics is applicable, the plot of t/q versus t should give a linear relationship, from which q eq and K 2,ad can be determined from the slope and the intercept of the plot respectively.

3 Results and discussion

Characteristics of chitosan and chitosan-metal microparticles

When suspended in aqueous solution, non-cross-linked chitosan microparticles do not maintain the form of spheres, especially in an acidic environment: they swell and dissolve 47 . The swelling and dissolution behavior of the microparticles depend on the degree of deacetylation of chitosan 42 . In order to prepare insoluble/stable chitosan microparticles suited for drug adsorption, cross-linking agents such as glutaraldehyde, are generally used 32 . The morphology of the chitosan and CH-metal microparticles (crosslinked and non-cross-linked) was examined by scanning electron microscopy (SEM) (Figure 1). Microparticles are polydisperse in size and present spherical collapsed shapes, independently of cross-linking. The collapse shape is characteristic of buckled shells due the quick drying process 48 , 49 , 50 . Cross-linking does not influence surface roughness.

Surface roughness of CH microparticles is more important than for CH-metal microparticles, probably due to differences in mechanical properties of CH and CH-metal solutions as they dry. Characteristics of CH and CH-metal microparticles, either crosslinked or not are listed in Table 1. Particle size distribution of non-cross-linked microparticles is rather polydisperse with span values between 2 and 68. The polydispersity may arise from two phenomena: first, the viscosity of the solutions may lead to an inhomogeneous droplet distribution in the spray-dryer, second, surface properties may induce particle sticking. Non-cross-linked CH-Zn(II) microparticles present the lower span. Microparticle size distribution is strongly influenced by the cross-linking process.

Indeed, after cross-linking, microparticle size distribution becomes narrower except for CH-Fe(II) which remains polydisperse and for CH-Zn(II) which was already narrow. Crosslinking may modify the chemistry of particles surface and therefore their tendency to aggregation.

To assess the efficacy of metal complexation and of cross-linking, the degree of deacetylation (DDA) of CH and CH-metal microparticles was determined by an infrared technique. The FT-IR spectra of CH and CH-metal microparticles (crosslinked and non-cross-linked) are given in Figure 2. The bands at 900 and 1050 cm -1 , characteristic of saccharide structure, are present in all spectra. The bands corresponding to C-H stretching are observed at 2864 cm -1 and 2932 cm -1 and free hydroxyl at 3290 cm -1 .The ratio of absorbance of amide I at 1655 cm -1 on that of hydroxyl group at 3450 cm -1 in the CH-metal microparticles is related to the degree of polymer deacetylation by Eq. 2. The DDA of the microparticles decreases from 75% to 19-25% after complexation with metal ions for non-cross-linked microparticles, indicating that complexation of metal ions with chitosan polymer takes place by reaction with amine groups of chitosan (Table 1). For all cross-linked samples the presence of a weak band at 1560 cm -1 corresponding to imine bond (C═N) confirms the reaction between the aldehyde (CHO) and the NH 2 sites of chitosan. This reaction may decrease the cytotoxicity as shown previously 51 . Furthermore, there is no evidence of a carbonyl band near 1720cm -1 indicating that all unreacted glutarldehyde is removed after the washing procedure. In addition, the DDA decreases after the cross-linking process.

Metal determination

Before cross-linking, CH-Fe(II) and CH-Fe(III) microparticles contain 66 mg.g -1 and 191 mg.g -1 of iron respectively and Ch-Zn microparticles contain 114mg.g -1 of zinc. It correspond to 17.6, 53 and 28.8% of the initial metal. After cross-linking, the metal concentration decreases: cross-linked chitosan CH-Fe(II) microparticles contain 45 mg.g -1 iron whereas cross-linked chitosan CH-Fe(III) microparticle contain 130 mg.g -1 iron, corresponding respectively to 12% and 36% of initial Fe(II) and Fe(III). Cross-linked CH-Zn(II) showed 87 mg.g -1 , corresponding to 22% of Zn(II) added in the reaction. The decrease of the metal associated to microparticles after cross-linking indicates that the amine groups implicated in the cross-linking are also implicated in the association with metal ions. This result confirms the decrease of the DDA observed after cross-linking.

Ciprofloxacin adsorption by chitosan-metal microparticles

Uptake capacity of the chitosan-metal microparticles

The effect of contact time on ciprofloxacin adsorption was analyzed by plotting uptake capacity (q) versus time, for 25, 50, 100, 200, 300 and 400 µg.mL -1 initial ciprofloxacin concentrations, at constant temperature (37 o C). Initially, a large number of adsorption sites are free and ciprofloxacin adsorption occurs quickly. The adsorption equilibrium of ciprofloxacin onto CH and CH-Fe(II) microparticles is reached after 30 min only (Fig. 3 (1) and ( 2)). Almost no significant improvement is observed for longer contact times. For these microparticles, the initial ciprofloxacin concentration does not influence the equilibrium time.

By contrast, the adsorption equilibrium of ciprofloxacin onto CH-Fe(III) and CH-Zn(II) microparticles seems to be influenced by the initial ciprofloxacin concentration. For low ciprofloxacin concentrations (25; 50 and 100 µg.mL -1 ) the equilibrium state was reached around 120 min and 180 min for CH-Fe(III) and CH-Zn(II), respectively [Fig. 3 (3) and ( 4 Indeed the initial ciprofloxacin concentration can influence the adsorption process.

To verify this influence, the effect of initial ciprofloxacin concentration was studied between 25 to 400 µg.mL -1 , for all microparticles and the uptake capacity (q 300 ) and adsorption yields at 300min are presented in Table 2. The equilibrium ciprofloxacin uptake was significantly enhanced as the initial concentration of ciprofloxacin increased from 25 to 400 µg.mL -1 . For CH microparticles, the uptake capacity increases from 2.8 to 32.9 µg.mg -1 whereas for CH-Fe(II) it increases from 8.7 to 105.7 µg.mg -1 . The presence of the metal strongly enhances microparticle adsorption capacity. This increase is even more obvious for CH-Fe(III) and CH-Zn(II) for which the uptake capacity increases from 20.5 to 305.3 µg.mg -1 and from 15.5 to 302 µg.mg -1 , respectively. The equilibrium uptakes and adsorption yields of CH-Fe(III) and CH-Zn(II) were much higher than those of CH-Fe(II) and CH microparticles (Figure 4). This variation in the sorption capacity between the sorbents is related to the amount of metal ions associated to microparticles: the higher the metal associated to chitosan, the higher the ciprofloxacin adsorption. However, although there is less Zn(II) than Fe(III) associated to CH, the affinity of ciprofloxacin with Zn(II) is higher, therefore leading to a better adsorption capacity for CH-Zn(II) microparticles.

Adsorption isotherms

In order to optimize the design of an adsorption system it is important to establish the most appropriate model for the equilibrium isotherms. These isotherms were determined by incubating microparticles (1 mg.mL -1 ) in a HEPES buffer solution containing ciprofloxacin at various concentration (25 -400 µg.mL -1 ) and plotting log(q eq ) (adsorption capacity at time 300 min = at equilibrium) versus log(C eq ) (equilibrium concentration of ciprofloxacin) (Figure 5). Neither the Langmuir model nor the Freundlich one fit experimental data. For CH and CH-Fe(II) microparticles, experimental data can be fitted by a straight line whose slope is equal to one. Saturation seems to start for CH-Fe(II) around 100 g.mg -1 . For CH-Fe(III) and CH-Zn(II) microparticles, the plot is bilogarithmic with a n=2 initial slope, followed by a plateau indicating saturation around 200 g.mg -1 . These data suggest that the adsorption site of CH or CH-Fe(II) can bind a single ciprofloxacin molecule whereas the adsorption site of CH-Fe(II) and CH-Zn(II) microparticles can bind two ciprofloxacins.

Adsorption kinetics

Adsorption kinetics has been proposed to elucidate the origin of adsorption. The mechanism of adsorption depends on the physical and chemical characteristics of the sorbent as well as on the mass transport process. To investigate whether the mechanism of ciprofloxacin adsorption on CH-metal microparticles occurs by mass transfer (physisorption) or chemical reaction (chemisorption), two kinetic models were examined:

the pseudo-first-order model 45 and the pseudo-second-order model 46 . The first-order rate constant (K 1,ad ) and q 300,1,calc values are presented with the correlation coefficients in Table 4. The K 1,ad values do not seem to be influenced by ciprofloxacin initial concentration or the particles used. In addition, theoretical q 300 values obtained from the first-order kinetic model do not give reasonable values. Using Eq (7), t/q was plotted against t, and secondorder adsorption rate constant (K 2,ad ) and equilibrium uptake values (q 300,2,calc ) were determined from the slope and the intercept of plots (data not shown). Values of K 2,ad and q 300,2,calc and of correlation coefficients are presented in Table 4. The correlation coefficient of pseudo-second-order equation is always higher than that of pseudo-first-order. In addition the theoretical q 300 value calculated from the pseudo-second-order model is closer to the experimental q 300 value than the one obtained from the pseudo-first-order model.

The pseudo-second-order adsorption mechanism is predominant for the adsorption of ciprofloxacin on CH-metal microparticles indicating that the adsorption process is mainly due to chemisorption. The chemisorption of ciprofloxacin on CH-metal microparticles may arise from the presence of the carboxylic group and the keto oxygen group as observed by others 39 . For Fe(III) and Zn(II) that are hexavalent one CH-Metal complex may bind two ciprofloxacins and for Fe(II) that is tetravalent only one ciprofloxacin may be bound to CH-Fe(II) in agreement with the slope of the adsorption isotherms. Additional structural studies using techniques such as X-ray absorption spectroscopy should be performed to determine the exact organization of the CH-metal-quinolone complexes 52 .

Conclusion

Chitosan-metal microparticles were prepared by a conventional spray-drying method followed by crosslinking using glutaraldehyde. Their capacity to adsorb ciprofloxacin, an antibiotic often responsible of the emergence of antibiotic resistance, was investigated as microparticles present a high adsorption capacity of ciprofloxacin, revealing that these particles could be employed as promising adsorbent for ciprofloxacin removal either from hospital waste water or from the gastrointestinal tract to prevent the emergence of antibiotic resistance. Table 3: Comparison of the first-and second-order rate constants and calculated and experimental q 300 values obtained at different initial ciprofloxacin concentration for CH and CH-metal microparticles.

a
Microparticles C o (µg.mL -1 ) q 3oo (µg.mg -1 ) Pseudo-first-order kinetic Pseudo-second-order kinetic K 1,ad (x10 -3 min -1 ) q 300,1, calc (µg.mg -1 ) R 1 2 K 2,ad (x10 -3 mg . g -1. min -1 ) q 300,2, calc (µg.mg -1 ) R

  Adsorption experiments were carried out by a batch technique. CH or CH-metal microparticles (CH, CH-Fe(II), CH-Fe(III) or CH-Zn(II)) were incubated at 1mg/mL in HEPES buffer (pH = 6.5) containing ciprofloxacin at various concentrations(25, 50, 100, 200, 300 and 400 µg.mL -1 ). The flasks were sealed to prevent change in volume of the solution during the experiments and they were submitted to a gentle tangential stirring, at 37±2 o C for 300 min. Samples were taken at pre-determined time intervals(30, 60, 90, 120, 180, 240 and 300 min.), centrifuged at 4000 rpm for 3 min and the supernatant was analyzed for the remaining ciprofloxacin concentration by high performance liquid chromatography (HPLC) following method described in section 2.8.1. All experiments were carried out in duplicates.

  function of contact time and initial drug concentration. Results show that the adsorption isotherms are fitted either by monologarithmic model for CH and CH-Fe(II) microparticles with one ciprofloxacin per adsorption site, or by a bilogarithmic model with a initial slope of two for CH-Fe(III) and CH-Zn(II) with two ciprofloxacins per adsorption site. In addition, the adsorption kinetics are well described by a pseudo-second-order kinetic model, indicating that the adsorption mechanism is controlled by chemisorption. CH-Fe(III) and CH-Zn(II)

Figure 1 :

 1 Figure 1: SEM figures of chitosan and CH-metal microparticles obtained by spray drying from aqueous solutions followed by cross-linking: (A) CH; (B) CH-Fe(II); (C) CH-Fe(III); (D) CH-Zn(II).

Figure 2 :

 2 Figure 2: FT-IR spectrum of CH (1), CH-Fe(II) (2), CH-Fe(III) (3) and CH-Zn(II) (4) microparticles; (a) non-cross-linked and (b) cross-linked, for determining the degree of deacetylation using transmittance ration of amide-I (1.655 cm -1 ) and hydroxyl group (3.450 cm -1 ).

Figure 3 :

 3 Figure 3: Effect of contact time and initial ciprofloxacin concentrations (25 -400 µg.mL -1 ) on ciprofloxacin adsorption on CH (1), CH-Fe(II) (2), CH-Fe(III) (3) and CH-Zn(II) (4) microparticles, in HEPES buffer (pH = 6.8, at 37 o C).

Figure 5 :

 5 Figure 5: Adsorption isotherms of ciprofloxacin on CH, CH-Fe(II), CH-Fe(III) and CH-Zn(II) microparticles, in HEPES buffer (pH = 6.8, at 37 o C). Dotted line represent the position of the adsorption saturation.

  )]. For higher ciprofloxacin concentrations (200; 300 and 400 µg.mL -1 ) the equilibrium state was achieved at 300 min. No significant adsorption improvement can be observed for longer contact times (8 and 24h, data not shown). For example for an initial ciprofloxacin concentration of 400 µg.mL -1 , one milligram of CH-Fe(III) microparticles can adsorb up to 305, 320 and 304 µg after 5, 8 and 24h, respectively. One milligram of CH-

	Zn(II) microparticles can adsorb up to 330, 325 and 328 µg ciprofloxacin after 5, 8 and
	24h, respectively.

Table 1

 1 Physiochemical characteristics of non-cross-linked and cross-linked chitosan and CH-

	metal microparticles
				Non-cross-linked					Cross-linked
	Microparticle	D0.1	D0.5	D0.9	D4,3	Span	DDA (%)	D0.1	D0.5	D0.9	D4,3	Span	DDA (%)
	CH	0.73 ±0.01	2.66 ±0.02	183.71 ±12.93	36.1 ±1.61	68.7 ±5.38	75	0.74 ±0.01	2.88 ±0.08	8.68 ±1,29	27.47 ±4.83	2.7 ±0.37	26
	CH-Fe(II)	2.69 ±0.02	9.85 ±0.34	185.46 ±8.65	54.84 ±2.05	18.5 ±0.34	23	1.59 ±0.01	4.34 ±0.01	151.64 ±4.60	36.71 ±0.39	34.5 ±1.03	22
	CH-Fe(III)	1.95 ±0.13	6.02 ±0.36	167.72 ±20.98	42.96 ±4.21	27.4 ±2.10	19	1.47 ±0.01	4.63 ±0.01	30.56 ±2.46	29.51 ±0.54	6.2 ±0.53	14
	CH-Zn(II)	0.71 ±0.01	3.85 ±0.04	9.68 ±0.33	20.88 ±1.59	2.3 ±0.07	25	1.12 ±0.02	4.58 ±0.10	12.50 ±2.07	20.12 ±2.07	2.4 ±0.05	13

Table 2 :

 2 Comparison of the equilibrium uptake and adsorption yields of ciprofloxacin at different initial concentration to the CH, CH-Fe(II), CH-Fe(III) and CH-Zn(II) microparticles.

	Ciproflo-xacin (µg.mL -1 )	q300 (µg.mg	-1 )	CH	Adsorption (%)	CH-Fe(II) Adsorption (µg.mg q300 -1 ) (%)	CH-Fe(III) Adsorption (µg.mg q300 -1 ) (%)	CH-Zn(II) Adsorption (µg.mg q300 -1 ) (%)
	25	2.8±0.13		11.2	8.7±1.75	34.8	20.5±1.22	82.0	15.5±0.36	62.0
	50	4.1±1.00		8.2	17.2±0.47	34.4	43.4±4.68	86.8	39.4±5.10	78.8
	100	8.2±2.17		8.2	33.8±3.67	33.80	90.6±6.19	96.6	86,7±4,58	86.7
	200	16.1±3.33	8.1	69.9±2.78	35.0	186.5±1.23	93.2	180.1±1.21	90.1
	300	23.1±4.41	7.7	99.2±0.65	33.1	262.3±9.73	87.4	241.2±2.25	80.4
	400	32.9±1.51	8.2	105.7±4.61	26.4	305.3±24.8	76.3	302.1±1.51	75.5

AcknowledgementsF. Reynaud acknowledges financial support from CAPES/COFECUB. Authors would like to thank M.F. Trichet (ICMPE, Thiais) for access to the SEM facility and CNRS, Univ Paris-Sud and CNPq-MCT/Brazil for funding.


Letícia Cruz

Elias Fattal

Leandro Tasso

Gabrielle C Freitas

Adriano B Carregaro

Sílvia S Guterres

Adriana R Pohlmann

Nicolas Tsapis
email: nicolas.tsapis@u-psud.fr

Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery

Keywords: Sodium Alendronate, Lung Toxicity, Spray Drying

Spray-dried powders for lung delivery of sodium alendronate (SA) were prepared from hydroalcoholic solutions. Formulations display geometric particle size below to 12 µm and spherical shape associated to a hollow structure. The addition of leucine and ammonium bicarbonate leads to porous particles with rough surfaces. The tapped density ranges from 0.016 to 0.062 g/cm 3 , decreasing with the increase of the leucine concentration. For all formulations, the calculated aerodynamic diameters are lower than 5 µm. The in vitro aerodynamic evaluation shows that all powders present a high emitted fraction of 100%, a fine particle fraction ranging from 34.4% to 62.0% and an alveolar fraction ranging from to 23.7% to 42.6%. An optimized sample was evaluated regarding sodium alendronate acute pulmonary toxicity and lung bioavailability. The bronchoalveolar lavage study shows that the intratracheal administration of sodium alendronate dry powder and sodium alendronate aqueous solution do not induce significant increases of lung toxicity indicators as compared with the positive control. Moreover, the intratracheal administration of sodium alendronate dry powder results in a 6.23 ± 0.83% bioavailability, a 3.5-fold increase as compared to oral bioavailability. Finally, these results suggest that sodium alendronate pulmonary delivery could be a new and promising administration route.

1-Introduction

Sodium alendronate belongs to a class of drugs called bisphosphonates known for their effectiveness in the treatment of bone diseases [START_REF] Lin | Bisphosphonates: A review of their pharmacokinetic properties[END_REF][START_REF] Perez-Lopez | Postmenopausal osteoporosis and alendronate[END_REF]. The pharmacological action of sodium alendronate is based on its affinity for the hydroxyapatite crystals of bone and selective inhibition of osteoclastic bone resorption during the remodeling cycle [START_REF] Lin | Bisphosphonates: A review of their pharmacokinetic properties[END_REF][START_REF] Russell | Bisphosphonates: pharmacology, mechanisms of action and clinical uses[END_REF]. As a potent antiresorptive drug, sodium alendronate was approved by the FDA in 1995 for the oral treatment of Paget's disease and postmenopausal osteoporosis [START_REF] Shinkai | New drugs -Reports of new drugs recently approved by the FDA -Alendronate[END_REF]. The clinical dose of sodium alendronate is about 0.2 mg/Kg orally (10 mg tablet daily). However, oral administration of sodium alendronate has been reported to be associated with gastrointestinal intolerance [START_REF] Graham | Alendronate gastric ulcers[END_REF]. Gastric and esophageal lesions such as erosions and ulcers have been observed as a result of a topic irritant effect of the drug [START_REF] Marshall | A randomized controlled trial to assess alendronate-associated injury of the upper gastrointestinal tract[END_REF][START_REF] Lanza | Bisphosphonate mucosal injury--the end of the story?[END_REF]. Patients presenting gastrointestinal problems or esophageal abnormalities such as achalasia have to avoid treatment with sodium alendronate [START_REF] Lanza | Bisphosphonate mucosal injury--the end of the story?[END_REF]. In addition, oral bioavailability of sodium alendronate is very low, around 1.7% [START_REF] Lin | Bisphosphonates: A review of their pharmacokinetic properties[END_REF][START_REF] Lin | Effects of dose, sex, and age on the disposition of alendronate, a potent antiosteolytic bisphosphonate, in rats[END_REF]. Intake together with meals and beverages other than water further reduces its bioavailability. High hydrophilicity and complexation with divalent cations, such as Ca ++ , are the reasons for poor sodium alendronate absorption [START_REF] Lin | Bisphosphonates: A review of their pharmacokinetic properties[END_REF]. Studies on alternative routes of administration have been conducted to overcome the adverse gastrointestinal effects and the poor bioavailability of sodium alendronate. However, intravenous administration of sodium alendronate presents the risk of nephrotoxicity [START_REF] Karie | Drud-induced nephrotoxicity: Bibliography from January 2003 to December 2005[END_REF]. Indeed, after intravenous injection in rats an accumulation of sodium alendronate in non-calcified tissues was observed [START_REF] Lin | Effects of dose, sex, and age on the disposition of alendronate, a potent antiosteolytic bisphosphonate, in rats[END_REF]. Subcutaneous and intramuscular administrations are associated with local tissue damage and irritation at the site of injection [START_REF] Lin | Bisphosphonates: A review of their pharmacokinetic properties[END_REF][START_REF] Monkkonen | Comparison of the Distribution of 3 Bisphosphonates in Mice[END_REF]. As an alternative to the peroral and parenteral administrations, Sutton et al. studied the nasal absorption of sodium alendronate in dogs and rats [START_REF] Sutton | Intranasal Delivery of the Bisphosphonate Alendronate in the Rat and Dog[END_REF]. Although the intranasal administration showed a greater absorption than the peroral route, no formulations concerning the nasal delivery of sodium alendronate have been reported in the literature. In recent years, the pulmonary route has gained attention as a potential route of administration for the treatment of local and systemic diseases [START_REF] Edwards | Large porous particles for pulmonary drug delivery[END_REF][START_REF] Tsapis | Direct lung delivery of para-aminosalicylic acid by aerosol particles[END_REF][START_REF] Labiris | Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications[END_REF][START_REF] Wichert | The lung, an organ for absorption?[END_REF][START_REF] Patton | Inhaling medicines: delivering drugs to the body through the lungs[END_REF]. Lung is an attractive target for drug delivery due to its large absorptive surface area, thin pulmonary epithelium and rich blood supply [START_REF] Wichert | The lung, an organ for absorption?[END_REF]. A large number of drugs which are poorly absorbed from enteral route are well absorbed from the lung [START_REF] He | Improvement of pulmonary absorption of insulin and other water-soluble compounds by polyamines in rats[END_REF]. In addition, inhaled drugs bypass the GI tract avoiding hepatic first-pass metabolism and dietary complications [START_REF] Labiris | Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications[END_REF]. Considering the advantages of pulmonary delivery, the development of a formulation of sodium alendronate intended for inhalation would be another interesting strategy to circumvent the disadvantages presented by other routes. Reducing the incidence of GI irritation and improving the absorption may increase the patient convenience and compliance to sodium alendronate therapy. In this study, we report the development of spray-dried powders for the lung delivery of sodium alendronate. The influence of leucine concentration, a dispersibility enhancer, on the physical properties of the powders, including particle size, morphology, density and in vitro deposition, was investigated in order to determine an optimized formulation to deliver sodium alendronate into the lungs via inhalation. Lung toxicity and bioavailability of the optimized formulation was also assessed.

2-Materials and methods

2-1 Materials

Sodium alendronate (SA) was provided by Chemos (Germany), ammonium bicarbonate (AB), DL-leucine (Leu), o-phthalaldehyde (OPA) and 2-mercaptoethanol were purchased from Sigma-Aldrich (France). Ethanol 96.2% was obtained from Carlo Erba Réactifs (France). 9-Fluorenylmethyl chloroformate (FMOC) was purchased from Sigma-Aldrich (Brazil). Acetonitrile and methanol were HPLC grade and obtained from Tedia (Brazil). All other chemicals and solvents were of analytical grade.

2-2 Preparation of the spray-dried powders

Sodium alendronate and leucine at different concentrations (Table 1) were dissolved into 200 mL of water. Ammonium bicarbonate (2.5 g) was dissolved into 100 mL of water and then added to the first solution. The resultant aqueous solution was mixed with 200 mL ethanol prior to atomization to form a 40% ethanolic solution with final solid content of 2 g/L. The ammonium bicarbonate, a volatile pore forming agent, was omitted in the final concentration of solids since this compound sublimes during the drying process. The prepared solutions were spray-dried using a BÜCHI Mini Spray Dryer B-191 (Büchi, France) equipped with a 0.7 mm two-fluid nozzle, using the following operational conditions: inlet temperature of 160 °C, aspiration at 100%, feed rate of 15 mL/min and atomizing air flow rate of 500 L/h. Powders samples were stored in a dessicator at room temperature until analysis. The outlet temperature was around 70 °C.

2-3 Granulometry and tap density

The volume median geometric diameter (d) was measured by light diffraction using Mastersizer 2000 equipped with a Sirocco dry disperser (Malvern Instruments, France) at dispersing pressures of 1, 2 and 3 bar. Each sample was measured in triplicate. Tap densities of the samples were determined using a Pharma test PT-TD1 (Copley Scientific, Switzerland). The powder density () was measured after 2500 taps because preliminary results have shown that there was no change in the volume after this number of taps. Measurements were performed in duplicate. The theoretical aerodynamic diameter of the particles (d aer ) was calculated from the particle size and tap density using the following equation [START_REF] Edwards | Large porous particles for pulmonary drug delivery[END_REF][START_REF] Tsapis | Trojan particles: large porous carriers of nanoparticles for drug delivery[END_REF]

2-4 Scanning Electron Microscopy

The morphology of the particles was evaluated by scanning electron microscopy (SEM) using a LEO 1530 (LEO Electron Microscopy Inc, Thornwood, NY) operating between 1 kV and 3 kV with a filament current of about 0.5 mA. Samples were deposited on carbon conductive double-sided tape (Euromedex, France) and dusted to remove the excess. They were sputter-coated with a layer of palladium-platinum alloy of about 4nm (Cressington sputter-coater 208HR) before analysis.

2-5 Aerodynamic properties

The aerodynamic properties of the powders were assessed using a multi-stage liquid impinger (MSLI, Apparatus C, European Pharmacopoeia 2000, Copley Scientific, Switzerland) through a dry-powder, breath-activated inhaler device (Aerolizer ® , Novartis). Twenty milliliters of water were added into each of the four stages of the impinger. A size 4 capsule containing the sample was placed into the inhaler device. The capsule was punctured and the powder was delivered at a flow rate of 60 L/min for 4 s to simulate an inspiration. Depending on the sample, this process was repeated at maximum 10 times to ensure adequate quantities of powder on each stage for dosing. Particles were separated between the stages based on their aerodynamic diameters. The cut-off diameters were comprised between 3.1-6.8 µm, 1.7-3.1 µm and < 1.7 µm for stages 3, 4 and filter, respectively. The amount of powder deposited on the impinger stages, as well as the amount retained by the filter was assayed spectrophotometrically by derivatization of the samples using the ophthalaldehyde reagent which forms a UV-absorbing derivative with sodium alendronate at 333 nm [START_REF] Al Deeb | Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine[END_REF]. Stock solutions of each sample were prepared by dissolving 50 mg of the powder in 50 mL of 0.2 M NaOH. Calibration curves were prepared in the range of 10-60 g.mL -1 by adding aliquots of the stock solutions in the 50 mL volumetric flasks. Four milliliters of the derivatizing agent were added to each flask and the volume was completed to 50 mL with 0.2 M NaOH. The absorbance values were measured after 30 min at 333 nm. The solvent of each stage was collected, the filter was rinsed with water and the respective volumes were transferred to 50 mL volumetric flasks. Then, 4 mL of derivatizing reagent were added and the volume was completed with 0.2 M NaOH. After 30 minutes, the absorbance was measured at 333 nm. The fine particle fraction (FPF) corresponds to the percentage of powder with an aerodynamic diameter lower than 5 m, which expresses the fraction of powder reaching the deep lung. The alveolar fraction (AF) was calculated in percent by dividing the dose collected on the fourth stage and the filter by the initial mass in the capsule. The emitted fraction (EF) defined as the percent of total loaded powder mass exiting the capsule was determined gravimetrically and calculated using the following equation:

.100

full empty powder mm EF m      
where m full and m empty are the masses of the capsule before and after simulating the inhalation and m powder the mass of the powder.

2-6 In vivo lung toxicity evaluation

Male Wistar rats weighing 280-350 g were used (Biotério Central, UFRGS, Brazil). The protocol has been approved by the Ethical Comittee of the Universidade Federal do Rio Grande do Sul (protocol number 2006637). After arrival, animals were kept in separate cages at maximum 5 animals per cage, and maintained on a diet of tap water and standard laboratory rat food until the beginning of the experiments. On the day of the experiments, animals were divided into four groups of six, and were intraperitoneally anesthetized with urethane (1.25 g/Kg) before the experiments. The trachea was exposed and an incision was made between the fifth and sixth tracheal rings caudal to thyroid cartilage. The rats were maintained at an angle of 70° and animal body temperature was maintained at 37°C with a 40 W incandescent heat lamp. The following treatments were administered intratracheally: (i) insufflation of 10 mL air (negative control group); (ii) instillation of 0.1% sodium dodecyl sulfate aqueous solution (100 µL, positive control group); instillation of 5.0 g/mL sodium alendronate aqueous solution (2.0 mg/Kg); and insufflations of sodium alendronate dry powder (Sample 4, 2.0 mg/Kg). In the case of insufflation, the dry powder was put in the body of a needle and dispersed in the incision of rat trachea by releasing air compressed in a syringe. The amount of dry powder administered was calculated by subtracting the needle weight after administration from before administration. In the case of instillation, the solution was injected into the lungs through the needle of a calibrated syringe. Four hours after treatment administration, animals were sacrificed and bronchoalveolar lavage was performed by cannulating the trachea and infusing lungs 3 times with 3 mL physiological saline. The bronchoalveolar lavage fluid (BALF) was centrifuged (1000 rpm, 10 min, 4°C) and the supernatant was analyzed for lactate dehydrogenase activity (LDH) and total protein content using commercial kits (Roche Diagnóstica ® ). Preliminary studies have shown that the 4 hours time point was relevant to assess membrane toxicity (data not shown). Statistical analysis was performed using one way analysis of variance (ANOVA). When the differences in the means were significant, Tukey test was used to compare the treatments. The significance level was set at P < 0.01.

2-7 Lung bioavailability evaluation

Four male mixed-breed dogs weighing 6-12 Kg were used (Biotério Central, UFSM, Brazil). The protocol has been approved by the Ethical Comittee of the Universidade Federal de Santa Maria (protocol number 23081.003008/2010-51). After arrival, animals were kept in individual cages and maintained on a diet of tap water and standard laboratory dog food until the beginning of the experiments. All the animals were fasted for 18 h before the experiments but they were allowed free access to water. On the day of the experiment, dogs were intravenously administered with a saline solution of sodium alendronate (2.0 mg/Kg) via the jugular vein. For the intratracheal administration, the same animals were previously anaesthetized with an intravenous injection of propofol (4 mg/Kg) and a tube was inserted through the throat in order to make an insufflation of the sodium alendronate dry powder (Sample 4, 2.0 mg/Kg) directly into the trachea, using the same device described above. After the administrations, dogs were returned to cages for 36-hr urine collection via catheter inserted in the urethra. Total urinary excretion of sodium alendronate following i.v. and intratracheal administrations was assayed by an HPLC method with fluorescence detection [START_REF] Kang | HPLC method validation and pharmacokinetic study of alendronate sodium in human urine with fluorescence detection[END_REF]. Briefly, sodium alendronate was extracted by using diethylamine solid phase extraction (Bond Elut®-DEA, Varian, Brazil). The sample was precipitated with sodium hydroxide and derivatized with FMOC in sodium carbonate buffer at pH 11.9. Separation was performed on a Gemini C18 column (150 x 4.6 mm, 5 m particles, Phenomenex, Brazil), using a gradient method starting with mobile phase acetonitrile/methanol-citrate/pyrophosphate buffer (28:72, v/v). The fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). Sodium alendronate bioavailability was calculated using the following equation:

.

.100 Div Ait F Dit Aiv  where D iv and D it are the doses and A iv and A it are the total milligrams of sodium alendronate excreted in the 36-hr urine following intravenous and intratracheal administration, respectively. Comparisons were performed using one way analysis of variance (ANOVA) and Student's t test and the differences were considered significant at P < 0.05.

3-Results and discussion

Aqueous ethanol spray-drying feedstocks containing sodium alendronate in the presence of leucine as a dispersibility enhancer and ammonium bicarbonate as a pore-forming agent were prepared with ethanol concentration of 40% v/v. It has been accepted that the inclusion of a nonaqueous solvent in the feedstock is advantageous because generally both the outlet temperature and the yield increase whereas the moisture content and particle size decrease [START_REF] Seville | Spray-dried powders for pulmonary drug delivery[END_REF]. Since sodium alendronate presents high water solubility, solutions with ethanol concentration higher than 40% are not stable due to the precipitation of the drug. Previous studies have demonstrated that leucine is a useful additive for dry powders designed for pulmonary delivery due to the improvement of the aerosolization properties [START_REF] Lucas | Enhancement of small particle size dry powder aerosol formulations using an ultra low density additive[END_REF][START_REF] Najafabadi | The effect of vehicle on physical properties and aerosolisation behaviour of disodium cromoglycate microparticles spray dried alone or with L-leucine[END_REF][START_REF] Chew | Effect of amino acids on the dispersion of disodium cromoglycate powders[END_REF][START_REF] Rabbani | The influence of formulation components on the aerosolisation properties of spray-dried powders[END_REF][START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF][START_REF] Seville | Amino acidmodified spray-dried powders with enhanced aerosolisation properties for pulmonary drug delivery[END_REF]. The effect of leucine on the aerodynamic properties is often explained by its distinct hydrophilic and lipophilic regions, which leads to an accumulation of leucine molecules at the air-water interface [START_REF] Seville | Amino acidmodified spray-dried powders with enhanced aerosolisation properties for pulmonary drug delivery[END_REF][START_REF] Vehring | Pharmaceutical particle engineering via spray drying[END_REF][START_REF] Lechuga-Ballesteros | Trileucine improves aerosol performance and stability of spray-dried powders for inhalation[END_REF]. In this study, a total of six samples were prepared with leucine concentration ranging between 5% and 20%, except for sample 1 which was prepared with sodium alendronate only and sample 2 which was prepared without leucine. The volume median geometric diameter of all samples varies from 4.8 to 11.7 µm (Table 1). Regarding the literature, a large number of factors and process conditions can influence particle size such as nozzle diameter, inlet temperature, air flow rate, feed rate, feedstock concentration, non-aqueous solvent and excipient materials [START_REF] Seville | Amino acidmodified spray-dried powders with enhanced aerosolisation properties for pulmonary drug delivery[END_REF]. In this work, we observed that an increase of leucine concentration in the formulations led to an increase of particle diameter, despite the fact that all formulations were prepared with the same solid content (2 g/L) and using the same operating conditions. Increasing the concentration of leucine above 10% results in doubling particle size. This effect is totally different from the one observed with similar formulations, in which a reduction of particle size with increasing leucine concentration was observed [START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF]. The slight hydrophobicity of leucine, which has a tendency to stand at the air/liquid interface reducing the size of the droplets produced at the spray-drying nozzle, was used to explain particle size reduction. However, the study of Gervelas et al. used high ethanol concentration (70% v/v), whereas here ethanol volume fraction was only 40% v/v. Spray-drying solutions with different ethanol water ratios present distinct surface tension, vapor pressure and viscosity, which will influence the size of the droplet produced during atomization, and thus the size of resultant dried particle. Taking into account that leucine presents a low water solubility, (22 mg/mL), and even a lower one in ethanol [START_REF] Nozaki | The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale[END_REF][START_REF] Glinski | Surface properties of aqueous solutions of Lleucine[END_REF], leucine surface activity probably differs for each solvent mixture as previously suggested by others [START_REF] Rabbani | The influence of formulation components on the aerosolisation properties of spray-dried powders[END_REF]. Moreover, sodium alendronate can interact with leucine affecting its surfactantlike properties as well as changing its initial saturation in the droplet.

Sample SA (g/L) Leu (g/L) AB (g/L) The tap density data are showed in Table 1. Sodium alendronate spray-dried alone (sample 1) presents a tap density of 0.101 g/cm 3 . When ammonium bicarbonate is added in the formulation, the tap density value decreases down to 0.062 g/cm 3 , proving the pore forming properties of this excipient (sample 2). This is further confirmed by SEM images of samples 1 and 2: particles of sample 2 are obviously more porous than particles of sample 1 (Figure 1). When ammonium bicarbonate is added in the formulation, the tap density values range between 0.016 g/cm 3 and 0.062 g/cm 3 diminishing with the raise of leucine concentration. A low density is much more desirable for powders designed for pulmonary delivery because it improves dispersibility and delivery efficiency [START_REF] Vehring | Particle formation in spray drying[END_REF]. It has been demonstrated recently that the addition of ammonium bicarbonate to formulations intended for inhalation reduces their density by formation of porous particles [START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF]. A sodium alendronate formulation prepared with 20% leucine, but without ammonium bicarbonate was spray-dried under the same operating conditions and showed a tap density of 0.584 g/cm 3 (data not shown) demonstrating the influence of ammonium bicarbonate on powder density. The laser diffraction and tap density data were used to calculate the theoretical aerodynamic diameter (d aer ). For all formulations the d aer are lower than 5 µm (Table 1), suggesting that the powders are suitable for deposition in the deep lungs.

Figure 1 shows the scanning electron micrographs of the dry powders. As a general rule, all samples are spherical in shape and possess a hollow structure. Particles of sodium alendronate (sample 1) present a smooth surface without pores. In contrast, with the addition of leucine and ammonium bicarbonate, particles surface becomes rougher and porous confirming the tendency of leucine to enhance surface roughness and the ability of ammonium bicarbonate to form large pores or voids. It was previously demonstrated that an increase of surface roughness reduces the cohesion between particles and therefore improves powder aerosolization properties [START_REF] Najafabadi | The effect of vehicle on physical properties and aerosolisation behaviour of disodium cromoglycate microparticles spray dried alone or with L-leucine[END_REF][START_REF] Rabbani | The influence of formulation components on the aerosolisation properties of spray-dried powders[END_REF][START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF]. However, the formulations present a tendency to form aggregates which could increase the values of particle sizes. To confirm this hypothesis, the ratio between the volume median geometric diameter of particles measured at different dispersing pressures (1, 2 and 3 bars) and the volume median geometric diameter of particles measured at a dispersing pressure of 1 bar d(pressure)/d(1bar) for each powder was calculated (Figure 2). All samples show a decrease of the d(pressure)/d(1bar) values as the dispersing pressure increases. For samples 2, 3 and 4, results suggest the presence of a few aggregates. On the other hand, for sample 1, results show that aggregates are numerous and need higher pressure to be individualized since the volume median geometric diameter decreases by 50% at 3 bars. Similarly, samples 5 and 6 present particles that are either aggregated or very fragile and break as dispersion pressure increases. In order to verify the in vitro aerosolization properties, powders were tested on a MSLI. Table 2 displays the values obtained for EF, FPF and AF for each sample. All powders present a high emitted fraction of 100%. In spite of the excellent emitted fraction, there is a considerable variation in FPF and AF values. The FPFs of the powders range from 34.4% to 62.0% and the AF values range from to 23.7% to 42.6%. Since analyses were carried out at least three times with different batches, and under the same operating conditions, differences observed in FPF and AF can be related to the different proportions of leucine. Sample 4, prepared with 10% leucine, displays the best aerosolization properties with a FPF around 80% and an AF around 42%. Contrarily to previous results that demonstrated an improvement of the aerosolization properties with an increase of leucine content [START_REF] Lucas | Enhancement of small particle size dry powder aerosol formulations using an ultra low density additive[END_REF][START_REF] Rabbani | The influence of formulation components on the aerosolisation properties of spray-dried powders[END_REF][START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF] Given that sodium alendronate treatment has been associated with topical irritant effects following oral, subcutaneous and intramuscular administration [START_REF] Lin | Effects of dose, sex, and age on the disposition of alendronate, a potent antiosteolytic bisphosphonate, in rats[END_REF][START_REF] Monkkonen | Comparison of the Distribution of 3 Bisphosphonates in Mice[END_REF][START_REF] Aki | Gastrointestinal side effect profile due to the use of alendronate in the treatment of osteoporosis[END_REF], it seemed relevant to evaluate whether the pulmonary delivery of the drug could lead to lung damage. Bronchoalveolar lavage studies have proven to be useful as a rapid method to investigate damage of the airways epithelial cells by the analysis of biochemical content of BALF [START_REF] Henderson | Use of bronchoalveolar lavage to detect respiratory tract toxicity of inhaled material[END_REF]. High protein content in the lavage fluid is associated with alterations in the permeability of alveolar-capillary barrier [START_REF] Henderson | Use of bronchoalveolar lavage to detect respiratory tract toxicity of inhaled material[END_REF]. LDH, an indicator of cell damage or inflammation, is a cytoplasmic enzyme whose extracellular presence indicates cell death [START_REF] Drent | Usefulness of lactate dehydrogenase and its isoenzymes as indicators of lung damage or inflammation[END_REF]. In this work, the anionic surfactant sodium dodecyl sulphate (SDS) was used as a positive control because of its harsh effect on biological membranes. The acute toxicity of sodium alendronate was compared with SDS by determining the changes in the markers of lung injury in BALF. The LDH activities with sodium alendronate dry powder, sodium alendronate solution and negative control group are significantly lower than those with the positive control group (P < 0.01) (Figure 3). SDS administration produces a five-fold increase in LDH levels 4h after the treatment as compared to the negative control. It is important to note that groups treated with sodium alendronate present enzymatic activities 2.2- 6-fold lower than the positive control group, respectively. Comparing both treated groups, no significant difference was observed (P= 0,143). Regarding total protein, the positive control group shows protein levels three-fold greater than the negative control group suggesting lung injury. On the other hand, sodium alendronate treated groups show significant decrease in this marker as compared to SDS (P < 0.01) (Figure 4). Regardless of the formulation (dry powder or solution), no significant increase of indicators for acute lung injury seem to be induced by sodium alendronate intratracheal administration. Then, we evaluated whether the dry powder was able to promote the lung absorption of sodium alendronate. Sodium alendronate was therefore administered either intravenously as a solution, or insufflated in dog lungs as a powder (sample 4). The total dose of sodium alendronate excreted in the 36-hr urine following intravenous and intratracheal was used to calculate the lung biovailability. The administration of the powder results in a 6.23 ± 0.83% bioavailability. The incomplete absorption could be explained by the high pharyngeal deposition and the hydrophilicity of the drug, combined with the negative charge of sodium alendronate. In contrast, when sodium alendronate was administered to dogs by oral route the bioavailability was only of 1.76 ± 0.07% [START_REF] Lin | Physiological Disposition of Alendronate, a Potent Anti-Osteolytic Bisphosphonate, in Laboratory-Animals[END_REF]. Thus, the absorption of sodium alendronate by lungs was 3.5-fold greater than by oral route. This substantial improvement of sodium alendronate bioavailability arises from the good alveolar deposition of the powder. In alveoli, the epithelium is very thin around a few hundreds of nanometers and even a charged molecule such as sodium alendronate is able to diffuse into the blood stream [START_REF] Rytting | Biodegradable polymeric nanocarriers for pulmonary drug delivery[END_REF]. These results confirm the pulmonary route is a promising for sodium alendronate delivery.

Conclusion

A dry powder prepared by spray-drying offers great promise as a formulation for the lung delivery of sodium alendronate. The optimized formulation developed in this study exhibits good in vitro aerosolization properties. The bronchoalveolar lavage study shows that the intratracheal administration of sodium alendronate dry powder and sodium alendronate aqueous solution does not induce significant increase of lung toxicity markers as compared with the positive control. Moreover, the pulmonary administration of sodium alendronate dry powder leads to a lung bioavailability 3.5fold greater than oral administration. These results suggest that the pulmonary delivery of sodium alendronate could be a new and safe administration route.

  :

Figure 1 .

 1 Figure 1. SEM images of typical particles from the different formulations of sodium alendronate spraydried powders. Scale bars represent 2 µm on all images.

Figure 2 .

 2 Figure 2. Evolution of the ratio of the volume median geometric diameter over the volume median geometric diameter measured at 1 bar d(pressure)/d(1bar) as a function of the dispersing pressure.

Figure 3 .

 3 Figure 3. Lactate dehydrogenase activity (LDH) in the bronchoalveolar lavage fluid 4 hours after intratracheal administration of air, sodium dodecyl sulfate (SDS), sodium alendronate dry powder (SA powder) and sodium alendronate solution (SA solution). The asterisk (*) denotes significant differences as compared with SDS.

Figure 4 .

 4 Figure 4. Total protein content in the bronchoalveolar lavage fluid 4 hours after intratracheal administration of air, sodium dodecyl sulfate (SDS), sodium alendronate dry powder (SA powder) and sodium alendronate solution (SA solution). The asterisk (*) denotes significant differences as compared with SDS.

Table 1 .

 1 Composition and characteristics of sodium alendronate spray-dried powders. SA=sodium alendronate, Leu=leucine, AB=ammonium bicarbonate, d=volume median geometric diameter (dispersing pressure of 1 bar), =tap density, d aer =aerodynamic diameter. Data are represented as mean±S.D.

	d (µm)	g/cm 3 )	d aer (µm)

Table 2 .

 2 samples 5 and 6 showed lower values of FPF and AF than sample 4. Doubling leucine concentration (sample 6) causes approximately a 50% reduction in the FPF and AF values. This result may be due to the presence of lot of aggregates which deposit via inertial impaction higher in the MSLI, leading to a reduced FPF and AF. Since sample 4 possesses the best aerodynamic properties, this formulation was chosen to evaluate sodium alendronate powder toxicity following pulmonary administration. Aerodynamic evaluation of sodium alendronate spray-dried powders. EF=emitted fraction, FPF=fine particle fraction, AF=alveolar fraction. Data are represented as mean±S.D.

	Sample	EF (%)	FPF (%)	AF (%)
	1	101.5 ± 2.3	46.6 ± 3.7	30.5 ± 2.8
	2	100.3 ± 1.5	53.1 ± 10.2	34.7 ± 8.3
	3	100.7 ± 1.1	42.2 ± 7.2	26.2 ± 9.8
	4	100.0 ± 0.4	62.0 ± 5.5	42.6 ± 8.6
	5	100.1 ± 0.3	48.9 ± 1.7	39.7 ± 3.0
	6	100.1 ± 0.1	34.4 ± 1.4	23.7 ± 1.8

Acknowledgements:

L. Cruz acknowledges financial support from CAPES/COFECUB. Authors would like to thank A. Allavena-Valette (ICMPE, Thiais) for access to the SEM facility and CNRS, Univ Paris-Sud and CNPq-MCT/Brazil for funding.


Regina Reul

Nicolas Tsapis
email: nicolas.tsapis@u-psud.fr

Hervé Hillaireau

Lucie Sancey

Simona Mura

Marion Recher

Julien Nicolas

Jean-Luc Coll

Elias Fattal

Near Infrared labeling of PLGA for in vivo imaging of nanoparticles

Keywords: PLGA, NIR imaging, covalently labeled conjugate, nanoparticles, biodistribution

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Introduction

Nanosystems such as nanoparticles (NPs) have been applied to the formulation of nanomedicine designed for controlled and targeted delivery of drugs as well as for diagnostic applications [1][2][3] .

Translation to the clinics will potentially improve drug therapeutic efficiency since nanomedicines reduce both drug side effects and toxicity 4 . However, the very same properties that make NPs exciting devices, might induce harmful effects as they interact with specific cells. NPs are solid colloidal systems smaller than 1 m. Due to their small size, they exhibit a large specific surface area which makes their reactivity to biological systems very high and might consequently induce undesired effects and severe toxicities 5 . Most toxicity studies have been focusing on inorganic NPs: carbon nanotubes, fullerenes, urban particulate matter, silica and metal NPs [6][7][8] . The evaluation of nanomedicine toxicity has started to raise attention of the community but most studies still consider that due to their biodegradability, polymer NPs do not lead to side-effects or toxicity. However, previous results contrast with this general view: specific risks might arise from the nanoparticle form itself. For example, after intravenous administration, biodegradable poly(alkyl cyanoacrylate) NPs can trigger an inflammation and oxidative stress during both acute and chronic treatments 9 .

However, this effect was reversible after interruption of the treatment while it was not the case for non-biodegradable polystyrene NPs. In order to study the potential toxicity of biodegradables NPs, it is crucial to follow their biodistribution and to check for undesired organ accumulation [10][11][12] . Near infrared (NIR) optical imaging is as a powerful tool for in vivo experiments, being a minimally invasive, non-ionizing method allowing sensitive deep tissue imaging 13 . In monitoring applications in whole-body living tissue, the advantageous long emission of NIR is preferred, as most tissues generate little NIR fluorescence 14,15 . Labeling NPs with NIR moieties could be used to monitor the fate of NPs after administration. For biodegradable polymer NPs, although encapsulation of quantum dot has shown promising results [16][17][18][19] , covalent labeling has recently started to attract attention 20 since artifacts have been observed with dye encapsulation 21,22 .

Poly(lactide-co-glycolide) (PLGA), an FDA approved polymer, is the component of many biodegradable nanosystems 2,[23][24][25][26][27][28][29][30][31] , however, covalent labeling with a NIR moiety has only been recently tested 20 . Although the strategy we propose is based on the similar coupling chemistry, we report herein a comprehensive study in which surface properties, colloidal stability, label integrity, potential cytotoxicity, fluorescent properties as well as in vivo and ex vivo biodistributions following intravenous administrations were thoroughly detailed.(Scheme 1).

Materials and methods

Materials

Poly(lactide-co-glycolide) PLGA Resomer RG502 H (50:50 lactic to glycolic acid ratio, M n 7900 g.mol -1 , M w 14600 g.mol -1 ) and PLGA Resomer RG756 S (75:25 lactic to glycolic acid ratio) were provided by Boehringer-Ingelheim (Germany). Polyvinyl alcohol (PVA) (MW 30000-70000 g.mol - 1 , 89% hydrolyzed) was purchased from Sigma-Aldrich (France). The NIR label DY-700 amine was purchased from Dyomics (Dyomics, Germany). Solvents were obtained from Carlo Erba (Italy). Water was purified using a Synergy set-up from Millipore (France). Cell culture reagents such as DMEM (Dulbecco's modified Eagle's medium), RPMI 1640 (Roswell Park Memorial Institute medium), FBS (Fetal Bovine Serum), Trypsine-Versene (EDTA) and PBS (Ca² + and Mg² + free phosphate buffer) were purchased from Lonza (Belgium). All other chemicals of analytical grade were purchased from Sigma (Sigma Aldrich, France).

Synthesis of the DY-PLGA conjugate

Briefly, PLGA 502H was dissolved into dichloromethane (DCM) at a concentration of 0.01 mmol.mL -1 . 5.6 molar equivalents of N,N'-dicyclohexylcarbodiimide (DCC) with respect to PLGA and 5.6 molar equivalents of N-hydroxysuccinimide (NHS) predissolved into DCM (0.5 mmol.mL - 1 ) were then added. The reaction was maintained upon magnetic stirring at room temperature (20ºC) until precipitation was observed and then for an additional hour. The solution was then filtered through a pipette tightly packed with cotton to remove the precipitated dicyclohexylurea (DCU).

The production of DCU is indicative of successful activation of PLGA. The appropriate amount of PLGA-NHS was taken for the subsequent conjugation reaction. DY-700 amine was dissolved into a small amount of dimethylformamide (DMF) (0.027 mmol.mL -1 ) in an amber vial, then one equivalent of PLGA-NHS in DCM was added, as well as 4 equivalents of triethylamine (TEA) diluted in DMF (0.13 mmol.mL -1 ). The conjugation reaction was maintained upon magnetic stirring at room temperature (20ºC) for 60 h. Finally, the mixture was precipitated into ice-cold methanol at a 1:10 volume ratio and centrifuged for 1 h at 4°C at 20000 rpm (maximum 37000 × g) using a

Beckman Coulter Optima LE-80K ultracentrifuge (Beckman Coulter Inc., France). The precipitate was redissolved into DCM and the procedure was repeated three times more. To ensure its efficacy, purification was monitored by measuring the supernatants by fluorimetry to assess the presence of free dye. The conjugate was finally washed with methanol. DY-PLGA was protected from light continuously using amber glassware or aluminum foil. Labeling efficacy was measured by fluorimetry using an excitation wavelength of 700 nm and an emission wavelength of 725 nm in a mixture of DCM:ethanol 2:1 (v:v).

Nanoparticle preparation

NPs were prepared using a solvent emulsion evaporation method using PVA as stabilizer 32,33 .

Briefly 25 mg of PLGA Resomer 756 were dissolved into 1.25 mL of a DCM:acetone (1:1 v:v) mixture. This organic solution was pre-emulsified by vortexing for 1 min with 5 mL of a pre-cooled 0.25 % (w/v) PVA aqueous solution (4°C). The pre-emulsion was kept on ice and sonicated for 1 minute using a VibraCell Sonicator (Fischer Scientific, Illkirch, France) at 40% of power. The organic phase was then evaporated at room temperature upon magnetic stirring (600 rpm) under a fume hood. To ascertain that the presence of the dye would not modify the nanoparticle surface chemistry, the NIR conjugate has been introduced in very small quantities (1% w/w) as compared with the work of Tosi et al. 20 : 1% of DY-PLGA (w/w) was introduced into the formulation replacing 1% of PLGA Resomer 756. NPs were then purified by ultracentrifugation (maximum 37000 × g, 1 h, 4 °C, Beckman Coulter Optima LE-80 K, Beckman Coulter, Inc., France) to remove the excess of PVA. After centrifugation, NPs were redispersed at concentrations of 5 mg.mL -1 and 25 mg.mL -1 in water or in cell culture medium.

Size exclusion chromatography

Size exclusion chromatography (SEC) was performed at 30°C with two columns from Polymer Laboratories (PL-gel MIXED-D; 300 × 7.5 mm; bead diameter: 5 μm; linear part: 400 -4 × 10 g.mol -1 ) calibrated against PS standards in chloroform (CHCl 3 ) at a flow rate of 1 mL.min -1 . A SEC system equipped with a Waters 501 HPLC pump (Millipore, France), a waters 717 plus autosampler (Milipore France), a differential refractive index detector (Spectrasystem RI-150 from Thermo Electron Corp., France) and a UV detector (Waters 486 tunable absorbance detector, Milipore, France) was used.

Fluorescence measurements

Fluorescence was measured using a Perkin Elmer Luminescence Spectrometer LS50B (Perkin Elmer, France) and a 1 cm optical path cuvette. Fluorescence measurements were performed in DCM:ethanol 2:1 (v:v), in water or in rat serum fixing the excitation at 700 nm and collecting the emitted fluorescence at 725 nm.

Size and zeta potential

Average diameter and polydispersity index (PDI) were obtained using a Malvern Zetasizer Nano ZS (Malvern Instrument, UK) based on quasi-elastic light scattering. Size measurements were performed at 20°C, at an angle of 173° to avoid multiple scattering. Zeta potential was measured using the same instrument. Measurements were performed in triplicates from three independent batches. Transmission electron microscopy (TEM) was performed using a Philips EM208 operating at 60 kV. The suspension of NPs (1 mg.mL-1) was deposited on copper grids covered with a formwar film (400 mesh) for 2 min. Negative staining (30 seconds) with phosphotungstic acid (1%) was performed. The excess solution was blotted off using filter paper and grids were air dried before observation. Image acquisition was performed using a high-resolution camera, Advantage HR3/12GO4 (AMT-Hamamatsu).

Stability studies

NP stability in terms of average diameter, particle size distribution, zeta potential and fluorescence was tested at 4°C in water (storage temperature of the NP suspension) and at 37°C in PBS for several time points. Samples from three independent batches were measured in triplicates. Label stability was investigated by incubating DY-NPs and NPs at 2.5 mg.mL -1 in PBS at 37 °C. At predetermined time intervals (0, 1, 2, 3 and 7 days), samples were ultracentrifuged (maximum 37000 g, 1 h, 4 °C, Beckman Coulter Optima LE-80 K, Beckman Coulter, France) and supernatants were freeze dried (alpha-12-LD, Christ, France). The lyophilisate was then dissolved in DCM and centrifuged for 5 min at 800 × g (MR1812, Jouan, France) to remove PBS salts. The supernatant was then measured in DCM ethanol 2:1 (v:v) fluorimetrically to determine the dye content.

Measurements were performed in triplicate for two different batches.

Cell culture and cell viability (MTT assay)

To analyse the impact of labeling on cell viability, three different cell lines were used. Embryonic murine fibroblast (NIH/3T3) and human endothelial umbilical vein cells (HUVEC) were cultured in DMEM supplemented with 50 U.mL -1 penicillin, 50 U.mL -1 streptomycin, and 10% FBS. The J774.A1 murine macrophage-monocyte cell line was cultured in RPMI 1640 medium supplemented with 50 U.mL -1 penicillin, 50 U.mL -1 streptomycin, and 10% heat inactivated FBS. Cells were splitted twice a week, either by trypsinisation for HUVEC and NIH/3T3 cells were trypsinized or by scraping for J774.A1 cells. All cell lines were maintained at 37ºC and 5% CO 2 in a humidified atmosphere. In order to evaluate the in vitro cytotoxicity, NPs were resuspended in cell culture medium and filtered (PVDF, 0.45 µm) before being added onto the cells. Cell viability was evaluated using the MTT assay as described previously 34 . Briefly, cells were seeded (8  10 3 cells/well) in 96-well plates (TPP, Switzerland) and pre-incubated for 24 h. Then, the culture medium was replaced with serial dilutions of NP suspensions or equivalent amounts of pure DY-700 (n = 14) in culture medium. After 24 h, the incubation medium was replaced by fresh medium containing 0.5 mg.mL -1 (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, France). After 4 h of incubation, the culture medium was gently aspirated and replaced by 200 µL dimethylsulfoxide (ACS grade, BioBasic Inc, France) to dissolve the formazan crystals. The absorbance of the solubilized dye, which correlates to the number of living cells, was measured with a microplate reader (LAB Systems Original Multiscan MS, Finland) at 570 nm. Cell viability [%] relative to non-treated (control) cells: [A] test /[A] control ×100. Polyethyleneimine (PEI) (25 kDa), a cationic polymer, was used as positive control. The IC50 was calculated as the sample concentration which inhibits growth of 50% of cells relative to the control cells according to Unger et al. 34 . Data are presented as a mean of four measurements ± standard deviation. IC50 was calculated using the sigmoidal fitting function from Origin® v 8.0 (OriginLab, Northampton, USA) when possible.

Confocal microscopy

The three types of cells were seeded onto glass cover slips (25 mm) inside 6 well plates at a density of 250 000 cells per well with 2 mL of medium and grown for 24 h. Medium was replaced by 2 mL of fresh medium containing NPs (1 mg.mL -1 ) and incubated at 37°C for further 24 h. Then, the medium was gently removed and the cover slips were covered with 1 mL of fresh medium and analysed using a confocal laser scanning microscope LSM 510 META (Zeiss, Germany) equipped with a 1mW Helium Neon Laser and a Plan-Apochromat 63X objective lens (Numerical Aperture 1.40, oil immersion) coupled with an LSM 5 Image Browser (Zeiss, Germany). The cover slips were maintained at 37ºC during image acquisition. Red fluorescence was observed with a 650 nm long pass emission filter under a 633 nm laser illumination.

Fluorescence Reflectance Imaging (2D-FRI)

Imaging of mice was performed as described previously 14,35 . All animal experiments were performed in agreement with the EEC guidelines and the Principles of laboratory animal care (NIH publication 14, No. 86-23, revised 1985). The investigator (L.S.) possesses the authorization number 38-09-22. Female NMRI nude mice (6 weeks old, n = 14, Janvier, Le Genest-Saint-Isle, France) were used and randomly separated into six different groups. Anesthetized mice (isoflurane/oxygen 3.5/4% for induction and 1.5/2% thereafter, CSP, Cournon, France) were injected intravenously with 200 L of 25 mg.mL -1 NPs or equivalent dye concentration alone (n = 3 mice/group), 5 mg.mL -1 NPs or equivalent dye concentration alone (n = 3 mice/group) or 1 mg.mL - 1 NPs or equivalent dye concentration alone (n = 1 mouse/group). NP suspensions were filtered on a 0.45 µm PVDF filter prior to injection. Mice were illuminated by 675 nm light-emitting diodes equipped with interference filters and emission was collected by 720/20 nm pass-band filter (IVIS Kinetic, Caliper, Villepinte, France). Fluorescence images were acquired during 1 sec at 90 min, 3 h, 5 h (n = 3 mice/group) and 24 h (n = 1 mouse/group) after injection. At the end of the experiment, 5 h or 24 h after injection, mice were euthanized to visualize the ex vivo fluorescence in the different organs (heart, lung, brain, skin, muscle, kidney, adrenal, bladder, intestine, spleen, pancreas, fat, stomach, uterus/ovary and liver).

Results and Discussion

DY-700 amine, a hemicyanine dye, was chosen for the conjugation to PLGA due to its characteristics in the NIR range and its primary amine group which allows a convenient coupling reaction (Figure 1, inset). The absorption spectrum of DY-700 amine in DCM:ethanol 2:1 (v:v) presents a maximum at 700 nm, a shoulder around 650 nm and an additional maximum in the visible range at 485 nm When excited at 700 nm the emission maximum is situated at 725 nm (Figure 1). These absorption/emission characteristics are similar in water and in serum. Besides from being used for NIR animal experiments, it also allows visualization in vitro for example by confocal laser microscopy due to these fluorescence characteristics.

For the sake of simplicity, we propose a 2 step-one pot functionalization strategy whereby the PLGA is end-functionalized with a NHS moiety using DCC/NHS and further reacted in situ with the amino NIR dye (Figure 2a). The carboxylic acid group of PLGA RG502 H was activated via DCC/NHS and the resulting PLGA was reacted in situ with the amino group of the DY-700 in a DMF:DCM mixture. The synthesis via NHS activated ester avoids intramolecular and intermolecular esterification between hydroxyl and carboxylic acid groups of PLGA 36 . To ensure purification was complete, several precipitation/centrifugation cycles were performed and supernatants were monitored via fluorimetry. The precipitate was dark green proving the effective linkage. Indeed, the fluorescent spectrum of the DY-PLGA in DCM:ethanol 2:1 (v:v) is identical to the spectrum of the DY-700 amine (Supplementary material, Figure S1). It appears that 4 precipitation/centrifugation cycles were sufficient to remove unreacted dye, allowing a 92% yield to be obtained. Fluorimetry shows that the labeling efficacy was 38%, which is about 4-fold higher than what was obtained by Tosi et al. 20 Given the molecular weights of DY-700 (747 g.mol -1 ) and of PLGA RG502 H (M n 7900 g.mol -1 , M w 14600 g.mol -1 ), DY-PLGA therefore contains 3.5% DY-700 (w/w). The conjugate was also analyzed via SEC. DY-PLGA presents an intense signal at 485 nm which overlaps with its RI signal whereas PLGA does not show any signal at 485 nm, which proves the successful covalent coupling of the dye to PLGA (Figure 2b). PLGA NPs were then formulated replacing 1% of PLGA RG756 S by DY-PLGA conjugate. In the following, the resulting fluorescent NPs will be noted DY-NPs whereas the non-fluorescent counterparts will be noted NPs. A DY-NP suspension of 1 mg.mL -1 therefore corresponds to an equivalent dye concentration of 0.35 g.mL -1 . DY-NP fluorescence in water gives the same spectrum as the DY-PLGA in DCM:ethanol 2:1 (v:v) (Supplementary material, Figure S1). DY-NPs were characterized and compared to NPs (Table 1). All NPs present average diameters between 205 and 220nm with a monomodal size distribution and a low polydispersity (PDI below 0.2) and slightly negative zeta potentials, independently of the presence of DY-PLGA conjugate. TEM images confirm that particles are spherical, rather monodisperse and around 200nm (Figure 3b, inset). The absence of modifications has already been observed for rhodamine B-labeled PLGA NPs 37 . In addition, incorporation of 1% of DY-PLGA conjugate hardly affects size, PDI and zeta potential either before or after centrifugation, when redispersed at 5 mg.mL -1 and 25 mg.mL -1 .

Moreover, XPS spectra of NPs and DY-NPs are identical (data not shown): the absence of modifications after incorporation of the NIR conjugate further confirms that down to 10 nm thickness, the surface chemistry of DY-NPs does not differ from NPs. Therefore, the in vivo fate of DY-NPs after intravenous administration can be assumed to be identical to the fate of NPs.

According to our calculations, 1% DY-PLGA for labeling was chosen as a balance between fluorescence emission and dye self-quenching. Since DY-NPs are prepared using 24.75 mg PLGA and 0.25 mg DY-PLGA, one formulation therefore contains 7.05 x 10 15 labels for an available surface of a 0.505 m 2 (PLGA density = 1.35 g.cm -3 , NP radius = 110 nm). Assuming that all dyes are positioned at the surface of DY-NPs, the available surface for each label is 71.6 nm 2 , which corresponds to an average distance of 17 nm between each fluorescent molecule. This is larger than the Förster radius that is above 6 nm for cyanine dyes 38 . The fluorescence intensities of the dye and of the DY-NPs for equivalent dye concentrations were compared as a function of NP concentration (Figure 3), both in water or in serum. Since DY-700 is not very soluble in water or aqueous buffer and tends to precipitate, fluorescence of DY-NPs in water is much higher than the one provided by an equivalent concentration of DY-700. Indeed, the linkage of DY-700 on NP surface prevents selfaggregation of the DY-700 molecules. This kind of behavior has been described for other NIR dye conjugated systems such as fluorescent silica NPs or fluorescent probes with proteins 39,40 . In serum, the fluorescence of the DY-700 is much higher than in water. The presence of proteins containing hydrophobic domains enhances the solubility of the dye and might prevent its aggregation. This effect has been described for cyanine dyes in bovine serum albumin solution 39 .

There is no difference of fluorescence intensity between DY-NPs in water or in serum. Importantly, one should notice that the fluorescence intensity of the DY-NPs or the dye itself in serum were comparable. Therefore for in vivo experiments, we chose to use the equivalent dye concentration when comparing with DY-NPs.

Stability of NPs and DY-NPs was tested at 4°C (Figure 4). Both types of NPs exhibit a strong storage stability over a month. Size and zeta potentials remained stable and PDIs were below 0.1.

Good storage stability facilitates studies as NPs do not to have be produced directly prior to their use. In addition, fluorescence was also monitored over a month at 4°C at different concentrations, measuring the very same samples repeatedly (Figure 5). Different NP concentrations were used to take into account the slight quenching observed at high concentration. No significant fluorescence decrease occurred over a month.

Stability was then investigated in PBS buffer at 37°C over a week. The concentrations of 2.5 and 0.5 mg.mL -1 , that correspond to 1/10 dilution that should occur after NP intravenous injection in mice, show very good stability with no modification of the colloidal characteristics. The lowest concentration 0.1 mg.mL -1 seems less stable with a slight increase of the PDI which however remained below 0.2 until the third day. At this high dilution, the increase of polydispersity may arise from a removal of the PVA layer stabilizing nanoparticles. Stability was also assessed in cell culture media and in serum over 24 h during which particle size remain stable. The good NP stability in all tested media provides a proof of the absence of aggregation, and anticipates what would happen when NPs in vitro on cells or in vivo in mice. One the main advantage of covalently labeling of the polymer is that it avoids dye leakage that could produce artifacts 21,22 . The stability of the labeling was verified by incubating DY-NPs in PBS at 37ºC over a week. Figure 6 shows that only 2.5% of the label is released over week, proving the good stability of the conjugate.

Before in vivo evaluation, NP cytotoxicity was assessed in vitro for three different cell lines (Figure 7). Free dye was also tested at equivalent concentrations found in DY-NPs. For HUVEC cells, a human umbilical vein endothelial cell line, and NIH/3T3, a mouse embryonic fibroblast cell line, cell viability remains above or around 50% for both NPs and DY-NPs and the IC50 cannot be determined. The cell viability was very slightly affected by the dye itself even at dye concentrations up to 7 g.mL -1 . In J774.A1, a murine macrophage-like cell line, cell viability decreases as a function of NP concentration. The IC 50 is ~35-40 g.mL -1 for both NPs and DY-NPs.

Interestingly, the cell viability decreases quickly after the IC50 concentration, but remains around 30 % between 0.25 and 1 mg.mL -1 . Incubation of J774.A1 with the free dye leads to a decrease of cell viability around 80% for dye concentration up to 7 g.mL -1 . These different toxicity profiles may arise from differences in the type of cells, as a macrophage-like cell line would probably take up particles much more actively than other cell types. In addition, apart from macrophages, many cell types do not internalize neutral particles in large quantities as they show only limited interaction with the cell membrane 41 . To verify this hypothesis, DY-NP uptake was visualized with confocal laser microscopy. HUVEC and NIH/3T3 cells indeed do not seem to significantly internalize DY-NPs. On the other hand, J774.A1 cells actively internalize DY-NPs due to their well-known tendency for endocytosis. These observations confirm cytotoxicity results. NPs remain in the cytoplasm and do not seem to enter the nucleus. DY-NPs and equivalent concentrations of free dye were then injected intravenously to mice (Figure 9 and 10). In vivo, fluorescence can be observed in the guts, arising from autofluorescent food. A difference in fluorescence distribution can be observed quickly after injection. The dye, which is a low molecular weight compound, shows a much faster clearance, whereas DY-NPs are found more importantly in the liver. Indeed, as DY-NPs were not PEGylated NPs (i.e., stealth), they were quickly opsonized and recognized by macrophages and ended-up in organs of the MPS such as liver, bone marrow and spleen 42 . This result is confirmed by the evaluation of the fluorescence intensity of the different organs ex vivo 5 h after injection. A higher fluorescent signal is recovered from the spleen and the liver for DY-NPs than for the dye itself, whereas the levels of fluorescence are comparable for other organs (Figure 10, top). Evaluation of the ex vivo fluorescence of the liver confirms this tendency 24 h hours after injection and for the different concentrations injected (Figure 10, bottom). In addition, after 24 h, the liver fluorescence decreased as compared with the 5 h time point, indicating that clearance has started (Figure 10, bottom). Importantly, these NPs exhibit intense fluorescence signal in vivo after intravenous administration using as low as 1 % DY-PLGA conjugate in the NP formulation, which allow them to be finely traced.

Conclusion

We have successfully synthesized a covalent conjugate of PLGA with a NIR dye (DY-700, Dyomics) with 38 % coupling efficiency, using DCC/NHS chemistry. The conjugate was incorporated at 1% (w/w) into PVA-stabilized PLGA NPs without modifying major NP features such as size, zeta potential, stability and cytotoxicity on HUVEC, NIH/3T3 or J774.A1 cell cultures. The absence of modifications after incorporation of the NIR conjugate further confirms that the surface chemistry of DY-NPs does not differ from NPs. Therefore, the in vivo fate of DY-NPs after intravenous administration can be assumed to be identical to the biodistribution of NPs. In vivo, after intravenous injection to nude mice, DY-NPs fate was followed down to concentrations of 1 mg.mL -1 . NP accumulation in the liver was easily monitored by NIR imaging providing a proof of concept for in vivo tracking the fate of PLGA-based nanosystems using the DY-PLGA conjugate at rather low concentration. Data are presented as mean ± SEM.

Tables and Figures

Scheme 1 :

 1 Scheme 1: Synthesis of NIR-emitting PLGA nanoparticles and their used for in vitro, in vivo and ex vivo imaging purposes.

Figure 1 :

 1 Figure 1: Fluorescence and UV spectrum of DY-700 amine in DCM:ethanol 2:1 (v:v). The inset presents the chemical structure of the precursor of DY-700 amine NIR dye.

Figure 2 :

 2 Figure 2: Synthesis scheme of the DY-PLGA conjugate (a) and SEC chromatogram of PLGA and DY-PLGA (b).

Figure 3 :

 3 Figure 3: Fluorescence characterization of the nanoparticles. Fluorescence intensity ( ex =700 nm,  em =725 nm) of DY-NPs in comparison with NPs (a). DY-NP fluorescence corrected by the effect of NPs in comparison with pure dye expressed in dye concentration, in water and in serum (b). The inset in (b) presents a typical TEM image of the DY-NPs. Data are presented as mean ± SEM.

Figure 4 :

 4 Figure 4: Stability of NPs and DY-NPs (5 mg.mL -1 ) at 4°C over 30 days: Top: Average diameter, Middle: PDI, Bottom: Zeta potential. Data are presented as mean ± SEM.

Figure 5 :

 5 Figure 5: Fluorescence stability of DY-NPs at 4°C at different concentrations (fluorescence intensity corrected from NP fluorescence). Data are presented as mean ± SEM.

Figure 6 :

 6 Figure 6: NPs and DY-NPs stability at 37°C in PBS over a week. Top: Average diameter, Middle: PDI, Bottom: Label degradation. Data are presented as mean ± SEM.

Figure 7 :

 7 Figure 7: Cell viability as a function of concentration DY-NPs, NPs, equivalent dye concentrations (1mg/mL DY-NIH/3T3 and Bottom: J774.A1. PEI results are fitted by a sigmoidal curve. Data are presented as mean ± SD.

Figure 8 :

 8 Figure 8: CLSM (left), Nomarsky (center) and merge (right) images of 1 mg.mL -1 of DY-NPs incubated with A) HUVEC, B) NIH/3T3 and C) J774.A1.

Figure 9 :

 9 Figure 9: In vivo NIR images of DY-NPs (left) and equivalent concentration of free dye (right) in nude mice as a function of time.

Figure 10 :

 10 Figure 10: Ex vivo organ distributions of DY-NPs and equivalent amounts of pure DY in nude mice measured by NIR 5 h post-injection. Top: all organs, Bottom: liver 5 and 24 hours post-injection.

Table 1 :

 1 Average diameters, Polydispersity Index (PDI) and Zeta Potential of NPs and DY-NPs before and after centrifugation at 5 and 25 mg.mL -1 . Data are presented as mean ± SEM.

	Nanoparticles		Average Diameter (nm)	PDI	Zeta potential (mV)
	Before centrifugation	NP DY-NP	206 ± 11 213 ± 6	0.035 ± 0.02 0.062 ± 0.026	-4.9 ± 0.5 -4.3 ± 0.5
		NP 5 mg.mL -1	207 ± 5	0.048 ± 0.024	-4.5 ± 0.6
	After centrifugation	DY-NP 5 mg.mL -1 NP 25 mg.mL -1	214 ± 2 218 ± 2	0.039 ± 0.002 0.190 ±0.069	-5.1 ± 0.4 -4.5 ± 0.2
		DY-NP 25 mg.mL -1	219 ± 4	0.140 ±0.064	-4.8 ± 0.3

Acknowledgements

Authors would like to acknowledge B. Le Droumaguet and N. Mackiewicz for fruitful discussions, V. Nicolas (IPSIT, Univ Paris-Sud, Châtenay Malabry) for confocal experiments, S. Zanna (Chimie Paris Tech) for XPS analysis, D. Jaillard (CCME, Orsay, Univ Paris-Sud) for TEM, M. Henry for animal experiments and R. Skanji for help collecting rat serum. This study was supported by AFSSET ("Emerging risks" program) and by ANR (under reference 2009-CESA-011). R. Reul and S. Mura acknowledge the support of ANR for their fellowship.