Funda Demirhan

Bahar Çağatay

Deniz Demir

Miguel Baya

Jean-Claude Daran

Rinaldo Poli
email: poli@lcc-toulouse.fr

Prof Rinaldo Poli

Reduction of Cp*2Mo2O5 in an aqueous medium. Structure and properties of a triangular mixed oxo-hydroxo-bridged product, [Cp*3Mo3(-O)2(-OH)4](X)2, with X = CF3CO2 or CF3SO3

Keywords: Molybdenum, aqueous organometallic chemistry, metal-metal bonds, oxo ligands, hydroxo ligands, hydrogen bonding

The reduction of Cp*2Mo2O5 with Zn in a MeOH-H2O solution that is acidified with either CF3COOH or CF3SO3H leads to the formation of [Cp*3Mo3(-O)2(-OH)4] 2+ ion as a trifluoroacetate or trifluoromethylsulfonate salt. The structure of the compound is confirmed by X-ray analyses. The anions establish hydrogen-bonding interactions with all four bridging OH groups. DFT calculations afford bonding parameters in close agreement with the observed structure and indicate that the cluster is best described as a valence-delocalized Mo3 13+ species. The 5 metal electrons are distributed among an a-type (z 2 ) orbital, which insure most of the metal-metal attraction, and two essentially metal-metal nonbonding e-type (xy) orbitals with a slight Mo-(-O) *-type contribution. Because of the C2 symmetry, the latter orbitals are not degenerate. The calculations show that the unpaired electron is located in a MO with equal contribution from two Mo atoms, in agreement with the experimental observation of coupling of the unpaired electron to two Mo atoms in the isotropic EPR spectrum.

Introduction

We have recently initiated a research program aimed at developing the chemistry of high oxidation state organometallic compounds in water, in the spirit of "green" chemistry [1][2][3] and with the long term goal of exploring the catalytic and electrocatalytic potential of organometallic compounds in water. [4,5] Unlike the low-valent organometallic systems, which are generally supported by hydrosoluble modified phosphine or cyclopentadienyl ligands, [6][7][8] the high-oxidation state complexes are supported by oxo ligands, which make them hydrosoluble by virtue of hydrogen bonding and protonation equilibria, yielding charged hydroxo and aqua complexes.

Although oxo-supported high oxidation organometallics are now well established and have interesting catalytic properties, [9,10] their systematic investigation in water has received little attention. We further argue that the greater metal electronegativity in the higher oxidation states confers a higher degree of covalency to the metal carbon bonds with odd-electron carbon ligands (i.e. alkyls, allyls, cyclopentadienyls, etc.), which consequently may become quite resistant toward hydrolytic decomposition. For a redox-active metal, reduction of a high oxidation state oxo complex should favour the generation of aqua ligands, opening the way to the generation of open coordination sites for substrate activation, catalytic and electrocatalytic applications. [11] We have so far focused our attention on the speciation [12] and electrochemical behaviour [13,14] of compound Cp*2Mo2O5. The Cp*-Mo bond is quite hydrolytically stable, being split only quite slowly at very low pH. [15] By chemical reduction with zinc in an acidic MeOH/H2O mixed solvent medium, we have obtained novel oxo-supported complexes, the nature of which seems to delicately depend on the nature of the acid used. Namely, when using acetic acid, we isolated the dinuclear Mo IV complex Cp*2Mo2(-O)2(-O2CCH3)2, I, [16] whereas the use of CF3COOH under the same conditions (mixed MeOH/H2O solvent, Zn as reducing agent) led to the crystallization of the Mo V trinuclear complex [Cp*3Mo3(-O)(-O)3(-O2CCF3)3] + , II, as a salt of the Zn2(O2CCF3)6 2-dianion. [17] The formation of these compounds demonstrates the removal of water ligands (from the expected protonation of oxo ligands, following the reduction process), generating vacant positions that are filled by the anions of the acids used. In previous contributions, [11,[START_REF] Collange | Perspectives in Organometallic Chemistry[END_REF] we have mentioned that another product may also be crystallized from the reaction mixture obtained by reduction in the presence of CF3COOH, depending on the solvent and conditions used for the crystallization.

This is another triangular cluster to which the formula [Cp*3Mo3(-OH)x(-O)6-x] 2+ was assigned on the basis of an X-ray structural analysis. However, this product could not be properly characterized because of severe disorder problems in the cation as well as in the CF3COO -anion. This situation could not even allow the unambiguous identification of the x value (number of hydrogen atoms on the bridging oxygen atoms), and thus of the number of electrons in the cluster. We have now repeated the same reaction with a related strong acid, CF3SO3H, and crystallized the corresponding product. We report here the structure of these compounds and a description of their properties.

Results

(a) Structure of the trifluoroacetate salt

The general procedure for the reduction of Cp*2Mo2O5, i.e. zing reduction in a watermethanol solution acidified by CF3COOH, has been described previously. [START_REF] Saurenz | [END_REF] Numerous subsequent syntheses and crystallization of the resulting product consistently gave the same kind of crystals, always affected by the initially observed severe disorder problem (see Introduction). [20] Unfortunately, the same type of crystals, showing the same kind of disorder, were consistently obtained. Interestingly, the same product (as revealed by the EPR study, vide infra) was also obtained in the presence of other weaker acids, in an attempt to obtain other kinds of carboxylato derivatives analogous to the previously reported Cp*2Mo2(-O)2(-O2CCH3)2, when working at the same low pH. [16] This observation attests to the thermodynamic stability of this product.

Although badly disordered, the structure of the trifluoroacetate salt serves to identify the structural motif and to set the stage for the discussion of this compound. A view of the central Mo3O6 core, including the interaction with the two CF3CO2 anions, is shown in Figure The reduction process described in the previous section was repeated in the presence of triflic acid, with two objectives in mind. First, the different anion may allow a less problematic crystal structure determination. Second, and most important, the triflate anion has three equivalent oxygen atoms available for hydrogen bonding. Therefore, should the two additional bridging functions that are not involved in hydrogen bonding with the trifluoroacetate anions (i.e. O3 and O5 in Figure 1a) also bear hydrogen atoms, the triflate ions would be expected to establish an interaction with them. Conversely, the lack of such interactions may be taken as strong indication for the absence of H atoms in these positions.

Complex

<Figure 2>

The relative arrangement of the ions in the structure is shown in 2+ fragment). [22] They are identical, on the other hand, to the average distance reported for compound [Cp*3Mo3(-OH)n(-O)6-n]Cl2 (namely 2.78(3) Å). The latter structure, however, was poorly defined because of solvent disorder. [21] (c) Mass spectrometric characterization

Both salts (trifluoroacetate and triflate) have been investigated by electrospray mass spectrometry (positive mode) in mixed H2O-MeOH solvents. In both cases, the spectrum shows the presence of trinuclear species and the absence of mono-and dinuclear species. For the trifluoroacetate salt, the major envelope is satisfactorily simulated on the basis of the formula [Cp*3Mo3(-O)5(-OMe)] + , see Figure 3. This shows that, under the conditions of the electrospray MS experiment, the dication readily loses one proton and exchanges the residual bridging OH group with the methanol solvent. The shape of the simulated envelope matches very closely the experimental one, but the major peak m/z value leaves a doubt as to the exact number of protons. This question is resolved by the additional experiments that will be presented in later sections. The minor envelope (Figure 3c

<Figure 4> (e) Magnetic susceptibility

The compound exhibits a simple Curie behaviour, consistent with the present of a single unpaired electron. Measurements carried out in the 6-60 K range show a linear  -1 vs.

T plot, with a negligible value for the Curie temperature (-0.38 K), see Figure 1. The corresponding effective magnetic moment, which is approximately temperature independent, averages 1.66B after correction for the ligands diamagnetism. This is rather close to the expected spin-only value for one unpaired electron (1.73 B). This result is consistent with the EPR spectrum and confirms that the latter is representative of the bulk sample.

<Figure 5>

(f) DFT Calculation

In order to further confirm the assignment of the chemical composition and electronic structure, we have carried out a theoretical study. Before describing the computational results, it is useful to qualitatively analyze the electronic structure on the basis of first principles and relevant previous knowledge. [21,23,24] The cluster may be viewed as resulting from the assembling of three "four-legged piano stools" of the Cp*MoO4 type, whose electronic structure is well known, [25,[START_REF] Albright | Orbital Interactions in Chemistry[END_REF] as shown in Scheme 1. Taking a D3h-symmetrical structure with six identical bridging ligands as a first approximation, it is possible to envisage that the interaction of the z 2 -type orbitals will lead to one bonding (of type a) and two degenerate antibonding (of type e) combinations, and that the interaction of the xy-type orbitals will equally lead to one a-type bonding and two degenerate antibonding combinations of e-type. The overlap between the xy orbitals being much weaker, the energy of the MOs resulting from these orbitals will be less perturbed from that of the starting fragment orbitals.

However, as well documented for other bridging systems, * mixing with the bridging ligands lone pairs should destabilize the xy orbital combinations. [START_REF] Abugideiri | [END_REF] It is easily predictable that this destabilization should be strongest for the symmetric (a-type) orbital, as shown in Scheme 1. The asymmetric nature of the bridging ligands (four OH and two O), however, is expected to alter this scheme, particularly by removing the degeneracy of the e-type combination.

<Scheme 1>

The DFT optimized geometry agrees quite well with the experimental geometry, see Table 1. The minor variations could partly be attributed to the different environment (hydrogen-bonded solid state structure with the CF3SO3 anion for the experimental geometry, free "gas phase" dication for the DFT optimized one), and partly to the well known

<Table 1>

The calculated electronic structure, see Figure 6, confirms the qualitative expectations.

The bonding a(z 2 ) orbital is located just above the group of the six MO's describing the three double-sided Mo-Cp*  interactions. Above it, we find the pair of e(xy) orbitals, which contains three electrons. The calculation converges with two electrons in the e1-type orbital and one in the e2-type orbital, whose degeneracy (in the ideal C3 symmetry) is substantially split by the different nature of the bridging groups (O vs. OH). It is rewarding to see that the unpaired electron, according to the calculations, chooses to reside in the orbital from the e set that has the nodal plane parallel to the C2 symmetry axis (e2), rather than in the orbital with the nodal plane perpendicular to it (e1). The half-occupied orbital has contributions only from the two Mo' atoms and a symmetry imposed zero contribution from the Mo atom, in perfect agreement with the result of the EPR analysis above. Furthermore, the DFT calculation suggests that the cluster is completely valence-delocalized. Indeed, the computed natural charge [28] is very similar on atoms Mo (+1.075) and Mo' (+1.100 and 1.101), and so is the computed Mulliken spin density (-0.820, 0.849 and 0.847, respectively). We note that the mean value of S 2 from the spin-unrestricted calculation is high (see Computational Details), suggesting mixing of an excited [a(z 2 )] 2 [e1(xy)] 1 [e2(xy)] 1 [a(xy)] 1 configuration into the ground state. However, according to the result of the EPR experiment, this contribution must be negligible for the real system, because the isotropic resonance is relatively sharp at room temperature and unaffected by cooling.

< Figure 6>

A somewhat related triangular Mo3 cluster, [Mo3CuS4(H2O)10] 4+ , which was described as having a Cu I ion and a Mo3 11+ core, [29] has an electronic structure similar to that described here, although there are two additional electrons, thereby also involving occupation of the atype orbital. In that case, the experimental observation of electronic coupling to a single Mo nucleus in the EPR spectrum was interpreted as resulting from a valence-trapped

Mo IV Mo IV Mo III structure, the unpaired electron being mostly localized on the Mo III center, although the calculations (at the EHMO level) indicated a delocalized shape for the MOs, similar to that reported here.

Discussion

It is useful to compare the salts reported here with a closely related one reported earlier by Bottomley et al., namely [Cp*3Mo3(-O)6-n(-OH)n] 2+ (Cl -)2. [21] This compound was obtained by a synthetic strategy closely related to ours (zinc reduction of Cp*MoO2Cl in CHCl3 in the presence of concentrated HCl). The X-ray structure presented for this compound was affected by severe disorder, preventing the unambiguous determination of the number of bridging OH groups, like for the trifluoroacetate complex reported here. We have already pointed out above that the reported Mo-Mo distances for the dichloride salt are very similar to those reported here for the [Cp*3Mo3(-O)2(-OH)4] 2+ (CF3SO3 -)2 salt. For compound [Cp*3Mo3(-O)6-n(-OH)n] 2+ (Cl -)2, the authors proposed 5 as the most likely n value, but also suggested the presence of a redox disproportionation equilibrium in solution, which involves other species characterized by n = 4 and n = 6. This suggestion was based on magnetic, EPR, and NMR observations. In particular, the EPR spectrum was assigned to the n = 6 species, whereas two NMR resonances, a sharp one and a broad one, where assigned to the diamagnetic n = 5 species and to the paramagnetic n = 4 species, respectively. The room temperature isotropic EPR spectrum reported for solutions of the dichloride salt seems very similar to that of the trifluoromethylsulfonate and trifluoroacetate salts reported here, but it was interpreted in that case as arising from coupling to three equivalent Mo nuclei. An argument given in favor of the above scheme was the fact that the n = 4 species could not yield an observable isotropic EPR spectrum, given the expected (e) 3 electronic configuration. [21] Our DFT calculation, however, shows that the asymmetry associated to the distribution of the two -O and four -OH groups significantly splits this orbital degeneracy.

The trifluoromethylsulfonate and trifluoroacetate salts reported here (at least batches of clean single crystals) did not show any sharp or even broadened NMR signal that could be assigned to the Cp* ligand in a diamagnetic or paramagnetic compound. All evidence, therefore, points to the fact that the trifluoromethylsulfonate salt reported here maintains its identity as a single compound in solution.

Another useful comparison involves compounds Cp*4Mo5O11 [21] and Cp*6Mo8O16, [22] both being described as containing the central [Cp*3Mo3(-O)6] -unit (vide supra). In this unit, there are only 4 cluster electrons (Mo3 IV,V,V ) and the structures are stabilized by capping one triangular O3 face with either Mo V or Mo VI ions. Therefore, it seems possible that the salts of [Cp*3Mo3(-O)2(-OH)4] 2+ exhibit interesting electrochemical properties, which may dependent on pH and/or on the presence of other metal ions.

Conclusion

We have shown in this paper that the zinc reduction of Cp*2Mo2O5 in an acidic aqueous environment leads to the triangular [Cp*3Mo3(-O)2(-OH)4] 2+ cluster, containing a paramagnetic (5-electron) trimetallic core. The spectroscopic evidence points to a solution structure identical to the solid state structure, with the unpaired electron located in a delocalized molecular orbital that has atomic orbital participation from only two Mo atoms.

A study of the redox properties and acid/base properties of this system, followed by an investigation of its chemical reactivity, are now in progress and will be reported in due course.

Experimental Section

General Methods

All preparations and manipulations were carried out with Schlenk techniques under an oxygen-free argon atmosphere. All glassware was oven-dried at 120 C. Solvents were dried by standard procedures and distilled under dinitrogen prior to use. EPR spectra were measured on a Elexsys E500 BRUKER spectrometer (X-band) equipped with both a frequencemeter and gaussmeter. Mass spectra were recorded with a NERMAG R10-10 instrument. The starting compound, Cp*2Mo2O5, was prepared as described in the literature. [START_REF] Saurenz | [END_REF] 3.

X-ray analyses

Single crystal of compounds [Cp*3Mo3(-O)2(-OH)4](X)2 (X = CF3CO2, CF3SO3)
were mounted under inert perfluoropolyether at the tip of glass fibre and cooled in the cryostream of the Oxford-Diffraction XCALIBUR CCD diffractometer. Data were collected using the monochromatic MoK radiation (= 0.71073). The structure was solved by direct methods (SIR97 [30] ) and refined by least-squares procedures on F 2 using SHELXL-97. [START_REF] Sheldrick | SHELXL97. Program for Crystal Structure refinement[END_REF] Although the model could be easily defined by direct methods, its refinement appeared to be very poor with large elongated ellipsoids for the Cp* and rather high R and wR2 values. The elongated ellipsoids suggest a free rotation of the Cp ring around the Mo-Cp* centroid axis.

However, all attempts to refine a disordered Cp* model failed. The occurrence of a twinned structure by pseudo merohedry, considering a monoclinic cell, was also attempted but again without improvement of the refinement. Trying to refine using a non centrosymmetric space group did not improve the result either. Careful examination of the reciprocal space revealed that the crystal used is certainly not single but an attempt to integrate using different domains failed. There is, however, no doubt about the reality of the model as confirmed by different analytical methods. All H atoms attached to carbon were introduced in the calculation in idealised positions and treated as riding models. Owing to the poor quality of the refinement, the H atom attached to the hydroxyl groups could not be located. The drawings were obtained with the help of ORTEP32. [START_REF] Farrugia | [END_REF] The structures was solved by direct methods (SIR97) [30] and refined by least-squares procedures on F 2 using SHELXL-97. [START_REF] Sheldrick | SHELXL97. Program for Crystal Structure refinement[END_REF] All H atoms attached to carbon were introduced in calculation in idealised positions and treated as riding models. The drawing of the molecules was realised with the help of ORTEP32. [START_REF] Farrugia | [END_REF] The statistically distributed disulfide and dichloromethane molecules were freely refined. The crystal data and refinement parameters are shown in Table 2 and selected geometric parameters are collected in Table 1. <Table 2>

Computational details

The DFT calculation was carried out on a model system where the Cp* ligands were replaced on the simpler Cp rings.

The starting geometry was based on the crystallographically determined structure of the triflate salt and no symmetry restrictions were imposed. The geometry was fully optimized and the resulting minimum of the potential energy surface (PES) was verified by the positive value of all second derivatives of the energy. The calculation, which used the spin-unrestricted formulation, was performed using the B3LYP three-parameter hybrid density functional method of Becke, [33] as implemented in the Gaussian03 suite of programs. [START_REF] Frisch | Gaussian 03, Revision B.04[END_REF] The basis sets used for the geometry optimizations are the standard 6-31G** for C, H and P atoms, and the standard LANL2DZ basis set, which included the Hay and Wadt effective core potentials (ECP), [START_REF] Hay | [END_REF] for the metal atoms. The value of <S 2 > resulting from the calculation is 1.39 (0.81 after annihilation). This shows that there is significant spin contamination, indicating mixing with low-lying excited states. The correspondence between the resulting Kohn-Sham spin-orbitals was verified by displaying contour plots with the aid of the program MOLDEN. [START_REF] Schaftenaar | CAOS/CAMM Center Nijmegen[END_REF] No. MRTN-CT-2003-503864) and the COST D29 program (working group 0009-03). FD thanks the University of Celal Bayar (FEF 2002/101) for a research grant.

Captions for Figures

  1a. The three Mo atoms identify a nearly ideal equilateral triangle (vide infra), each edge of which is symmetrically bridged by two oxygen atoms, one above and one below the Mo3 plane. Thus, the three O atoms above the metal plane, and the three below, define two additional approximately equilateral triangles that are parallel to and staggered with respect to the Mo3 triangle. The two anions are located above and below the Mo3 plane and establish hydrogen bonding interactions with the bridging O atoms, which desymmetrize the triangle. Both bridging O atoms of one particular Mo3 edge (namely Mo1-Mo2 in Figure 1a) are engaged in hydrogen bonding, each with an O atom of a different trifluoroacetate anion. The second O atom of each CF3COO -anion interacts with a bridging O atom of a different edge (O4, bridging Mo1-Mo3, and O6, bridging Mo2-Mo3

Figure 1 (

 1 b). Like in the related trifluoroacetate salt, only two of the three bridging groups on each triangular face establish hydrogen bonds with the oxygen atoms of the CF3SO3 anions. The third O atom of each trifluoromethylsulfonate anion does not point toward the third bridging O atom. Specifically, the CF3SO3 anion located on the same side occupied by the bridging O1, O2 and O3 atoms places the third SO3 oxygen atom away from (exo) and the CF3 group on the same side as (endo) the triangle of the bridging O atoms. One CF3 fluorine atom is in proximity of atom O1, but the F•••O distance (3.22 Å) is too long to envisage a hydrogen bond (sum of van der Wall radii of F and O = 2.75 Å). The second CF3SO3 anion, which is located on the same side as O4, O5 and O5 from the Mo3 triangle, places the third O atom endo and the CF3 group exo, but the anion is tilted in such a way as to place the third O atom at 4.02 Å from O5, clearly indicating a repulsive interaction (sum of van der Wall radii of the two O atoms = 2.80 Å). This evidence allows us to propose the molecular formula of the dicationic trimetallic cluster as having four bridging hydroxo and two bridging oxo groups. Although the structure of the trifluoroacetate salt (vide supra) is much less precisely determined than that of the trifluoromethylsulfonate salt, a comparison of the hydrogen bonded O•••O contacts shows that these are tighter with the former anion and looser with the latter one, in agreement with their relative basicity scale. Although the structure has no crystallographically imposed symmetry, the trimetallic dication possesses an approximate C2 local symmetry, the pseudo-symmetry axis passing through the Mo2 atom and the middle of the Mo1-Mo3 bond. We shall refer to all averaged bonding parameters by using the symbol Mo for the unique atom Mo2 and the symbol Mo' for the two pseudo-symmetry related Mo1 and Mo3 atoms. The unique Mo'-Mo' bond is marginally but significantly longer than the two Mo-Mo' bonds, possibly as a consequence of the different nature of the bridging groups. Indeed, the bridging Mo-(-O) and Mo'-(-O) bonds are significantly shorter than the corresponding Mo-(-OH) and Mo'-(-OH) bonds. The alternative interpretation as assigning a greater oxidation state (V) to atom Mo and a lower one (IV) to atoms Mo' seems excluded by the DFT calculations (vide infra). There is no significant difference between the Mo-(-O) and Mo'-(-O) bonds on one side [average 1.95(1) Å], and between the Mo-(-OH) and Mo'-(-OH) bonds on the other side [average 2.05(1) Å], in agreement with an insignificant difference in metal ionic radius between Mo and Mo'. This analysis further serves to confirm the interpretation of atoms O1 and O5 as (-O) rather than (-OH) groups. The Mo-Mo distances are slightly longer than those found in related triangular Cp*3Mo3(-O)6-type clusters, e.g. 2.730(13) Å in Cp*4Mo5O11 (where the central [Cp*3Mo3(-O)6] -unit is capped by a [Cp*Mo VI O2(-O)MoVI O2] + fragment),[21] and 2.744(9) Å in Cp*6Mo8O16 (where two equivalent [Cp*3Mo3(-O)6] -units are bridged by a [Mo V 2O2(-O)2]

Figure 4

 4 Figure4shows, simulations were carried out using hyperfine coupling constant, shape and

  overestimation of bond distances by DFT. In particular, it is comforting to see the same trend of Mo-O bond distances [Mo-(-O) < Mo-(-OH)] in the optimized and experimental parameters, once again confirming the structural assignment.

  Figure 1. An ORTEP view of the central Mo3O6 core of the [Cp*3Mo3(-O)2(-OH)4] 2+ in the acetate salt (left, the disordered F atoms are not shown) and trifluoromethylsulfonate salt (right), showing the hydrogen bonding interactions with the two anions.

Figure 2 .

 2 Figure 2. An ORTEP view of the [Cp*3Mo3(-O)2(-OH)4] 2+ ion from the structure of the trifluoromethylsulfonate salt. For clarity, the hydrogen atoms of the Cp* ligands are not shown and the ellipsoids of the Cp* C atoms are replaced by spheres of arbitrary radius. The ellipsoids of the Mo and O atoms are shown at the 30% probability level.

Figure 3 .

 3 Figure 3. (a) Electrospray mass spectrum (positive mode) of compound [Cp*3Mo3(-O)4(-OH)2](O2CF3)2 in MeOH. Excerpts of this spectrum are shown in (b) and (c), and a simulation of the envelope corresponding to the [Cp*3Mo3(-O)5(-OMe)] + ion is given in (d).

Figure 4 .

 4 Figure 4. EPR spectrum of [Cp*3Mo3(-O)2(-OH)4](CF3SO3)2 in CH2Cl2 solution. (a) Experimental spectrum. (b) Simulated spectra with coupling to one, two, and three equivalent Mo nuclei (simulation parameters: Gaussian line shape, linewidth = 18 G). All spectra are scaled to the same peak-to-peak height for the central resonance.

Figure 5 .

 5 Figure 5. Magnetic properties (molar magnetic susceptibility, M, and effective magnetic moment, eff) of compound [Cp*3Mo3(-O)2(-OH)4](CF3SO3)2.

Figure 6 .

 6 Figure 6. Electronic structure of the [Cp*3Mo3(-O)2(-OH)4] 2+ cluster as resulting from a gas-phase DFT calculation. The shape of the spin-orbitals connected by a dashed line is essentially identical (only one for each pair is shown).

Figure 1

 1 Figure 1

  Figure 2

(b) Structure of the trifluoromethylsulfonate salt

	). The four O•••O distances, in the
	range 2.59-2.64 Å, unambiguously show the presence of 4 OH groups. However, the quality
	of the structure is not sufficient to establish whether the two remaining bridging O atoms
	carry additional H atoms or not. Although of low quality, the structure of the trifluoroacetate
	salt indicates that the unique Mo1-Mo2 distance is slightly longer (2.797 Å) than the other
	two (Mo1-Mo3, 2.786 Å; Mo2-Mo3, 2.785 Å). It is important to stress that the spectroscopic
	and magnetic properties are insufficient to make an unambiguous choice, because both a
	(OH)4(O)2 and a (OH)6 structure correspond to an odd number of electrons (paramagnetic).
	Thus, they would not be easily distinguishable by EPR spectroscopy. They are likewise
	difficult to distinguish by infrared spectroscopy.
	< Figure 1>

  The mixture was stirred under argon at room temperature for two-days during which time it changed from a clear yellow solution to a green suspension. The mixture was filtered and the solid was dried in vacuo. The solid was extracted with THF and, after filtration, Average eff in the 6-60 K range: 1.66 B (diamagnetic correction: -7.91•10 -4 cgs).

	2. Synthesis and crystallization of compound [Cp*3Mo3(-O)2(-OH)4](CF3SO3)2
	To a solution of [Cp*2Mo2O5] (0.05 g; 0.092 mmol) in MeOH-H2O (1:1, 6 mL), after
	acidification with concentrated CF3SO3H (10 drops), was added metallic zinc (0.48 g; 7.38
	mmol). addition of n-hexane yielded a green precipitate (0.021 g; 31.3%). A single crystal for the X-
	ray analysis was obtained by diffusion of a n-hexane layer into a green CH2Cl2 solution at
	room temperature. Elemental analysis. Calcd. for C32H49F6Mo3O12S2.2CH2Cl2 (MW =
	1261.53): C, 32.37; H, 4.23. Obtained: C, 32.92; H, 3.65. EPR (CH2Cl2): g = 1.962, aMo =
	26.2 G.

Acknowledgements

This research was supported by the CNRS in France and by TUBITAK in Turkey.

Supplemental travel support by a bilateral CNRS-TUBITAK (TBAG-U/62-102T211)

program is also gratefully acknowledged. In addition, RP acknowledges support from the European Commission through the Research Training Network "AQUACHEM" (contract


Isabelle Bertholon

Gilles Ponchel

Denis Labarre

Patrick Couvreur

Christine Vauthier
email: christine.vauthier@u-psud.fr

Bioadhesive Properties of Poly(alkylcyanoacrylate) Nanoparticles Coated with Polysaccharide

Keywords: bioadhesion, gastrointestinal tract, mucus, chain conformation, nanoparticles, polysaccharides

come   L'archive ouverte pluridisciplinaire

Introduction:

The oral route is often preferable for drug administration because of improved compliance of the patients to their treatment, better comfort and lowered medical costs. Unfortunately, for various reasons, many compounds administered by this route are poorly bioavailable, due to low permeability of the intestinal membrane or chemical instability in the gastrointestinal tract. To solve these problems, encapsulation of fragile or poorly bioavailable molecules can improve these characteristics and thus the development of suitable encapsulation techniques is of considerable interest. Drug encapsulation in nanoparticulate systems may result in the following advantages: (i) protection of the molecules against the harsh conditins prevaling in the gastrointestinal medium, (ii) direct translocation of the particles through the intestinal barrier and (iii) immobilization of the particles at the intestinal surface due to a bioadhesion mechanism, which is of interest for prolonging the time period available for drug absorption 1,2 . This last property can be obtained with systems which allow an increased time of contact between delivery systems and epithelial cells, and the control of the delivery profile of the drug from the particles. Bioadhesive systems are often formulated with hydrophilic polymers which strongly interact with the mucus standing over the intestinal epithelium [3][4][5] . The mucus is mainly constituted by various glycoproteins ("mucins") composed of a protein backbone and carbohydrate side chains 6,7 , rendering these molecules extremely hydrophilic in nature and capable of forming coherent and elastic hydrogels. Its physiologically function is to protect the cells of the intestinal epithelium from the luminal content and catch nutriments to be absorbed. These glycoproteins are negatively charged at physiological pH. The thickness of the mucus layer depends on the region of the intestine and decreases from 50 to 500 µm in the stomach to 16-50 µm in the colon 6 . The mucus layer is one of the barriers to drug absorption. Thus it may be interested to retain the drug as close as possible to the epithelial cells by artificial means 8 . To this aim, nanoparticles are able to diffuse into the mucus layer.

They can be better retained at the surface of enterocytes brush border thanks to their small size compared to microparticles 6 . However, their retention may be further improved by coating the nanoparticles with hydrophilic polymers like chitosan or poly(ethylen glycol) to develop bioadhesive formulation.

Chitosan has been widely used in bioadhesive formulations intended to the oral route. It is known as a low toxic excipient with strong bioadhesiveness, due to the presence of positive charges which can interact with the negatively charged glycoproteins of mucus. Chitosan was suspected to promote the oral absorption of hydrophilic drugs by opening the tight junctions of the epithelial membrane 1,[9][10][11] .

Mechanisms and parameters affecting the bioadhesive behaviour of nanoparticles in the gastrointestinal tract were investigated in several works [12][13][14][15] . It was proposed that bioadhesion of particles in the nano-size range resulted from two phenomenon: A diffusion of the nanoparticles occured at first from the suspension to the intestinal surface. Then the nanoparticles can interact with the surface of the intestine resulting in the immobilization of the particles on the mucosa 5 . Obviously, the attachment of the nanoparticles on the mucosa depends on the physico-chemical properties of the particles. For example, it has been suggested that the particles could diffuse into the mucus layer, depending on their size according to a size exclusion process 16 . Once in close contact with the mucus glycoproteins, various types of interactions may occur between the surface of the particles and the glycoproteins, including electrostatic or hydrophobic bonding. Although it is obvious that the bioadhesiveness of the nanoparticles on mucosa depends greatly on their surface characteristics, there is no work so far which has considered the effect of the conformation of hydrophilic polymer chains at the nanoparticles surface. Thus the purpose of our work was to investigate the effect of the characteristics of the hydrophilic coating of poly(alkylcyanoacrylate) nanoparticles in their bioadhesiveness using a series of nanoparticles coated with different polysaccharides.

The nanoparticles prepared by two methods of polymerization also differed by the conformation of the polysaccharide chains grafted at the surface of the nanoparticles allowing for the first time to investigate the effect of this parameter on the bioadhesion properties of the nanoparticles 17 .

Materials and methods:

Materials:

Isobutylcyanoacrylate (IBCA), was used as monomer and was kindly provided as a gift by Loctite (Dublin, Ireland). Dextran 66 900 g/mol, were purchased from Sigma (Saint-Quentin Fallavier, France). Dextran 40 000 g/mol and chitosan medium molecular weight were purchased from Fluka (Saint-Quentin Fallavier, France). 9-anthracenyl methacrylate was purchased from Biovalley (Conches, France). All chemicals were reagent grade and used as purchased.

Preparation of chitosan of various molecular weights:

Depolymerization of chitosan:

Chitosan was selectively depolymerized by reaction with sodium nitrite at various concentrations 18 . Typically, 100 mL of a solution of chitosan (2%) in acetic acid (6%) was depolymerized during 1 hour with 10 mL of sodium nitrite, which concentration was either 0.04 or 0.08 mol/L, at room temperature and under magnetic stirring.

Chitosan was precipitated by raising the pH to 9 with sodium hydroxide. The precipitate was recovered by filtration and washed extensively with acetone before being dried. Chitosan dissolved in acetic acid (0.1 M) was further purified by dialysis against water during 24 hours.

The resulted solutions were freeze dried before storage. The degree of deacetylation of the parent chitosan was 79±2%. According to the literature 19,20 , it is not modified by depolymerization reaction performed with nitrites.

Measurement of molecular weight:

The molecular weight of chitosan was determined from capillary viscosity measurements. Briefly, the reduced viscosity of solutions of chitosan of various concentrations (0.1 to 2.5 g/L) in acetic acid 0.1M, NaCl 0.2M was measured in a Ubbelohde tube (53710/1 Schott Geräte) at 25°C (Bath CT1450 Schott Geräte and cooling system CK100 Schott Geräte) using a viscometer AVS400 (Schott Geräte). The intrinsic viscosity [] was then deduced from the reduced viscosity measured for each solution of chitosan by extrapolation at zero concentration. The molecular weight (M) was determined by using the Mark Houwink Sakurada equation: [η] = K*M a , with K = 1.81*10 -3 and a = 0.93 21 .

Preparation of nanoparticles:

Redox radical emulsion polymerization (RREP):

The radical polymerization was carried out according to 22,23 . Briefly, dextran or chitosan (0.1375g) was dissolved in 4 mL of nitric acid (0.2M) at 40°C under gentle stirring and argon bubbling. After 10 minutes, 1 mL of cerium (IV) ammonium nitrate (8*10 -2 M in nitric acid 0.2 M) and 0.250 mL of IBCA were successively added. Argon bubbling was maintained for another 10 min and the reaction was continued for 50 min. Preparations were cooled to room temperature. When preparations were done with dextran, 0.63 mL of trisodium citrate dihydrate dissolved in water (1.02M) was added to the polymerization medium. The pH of all suspensions was then adjusted to 7.0 with NaOH 1N. In preparations performed with chitosan, the addition of trisodium citrate induced bulk precipitation of the nanoparticles and the pH was directly adjusted to 7.0 with NaOH 1N.

Anionic emulsion polymerization (AEP):

The polymerization media was prepared by dissolving at 40°C Chitosan (0.1375g) in 5 mL of nitric acid (0.2M), whose pH was lower than 1. IBCA (0.250 mL) was added under strong magnetic stirring and left to polymerize at 40°C during 1h. Chitosane and IBCA concentrations were the same than in RREP but these polymerizations were performed in the absence of ceric ions to avoid the radical polymerization.

Labeling of nanoparticles with Rhodamine : 9-anthracenyl methacrylate (Rhod) was

dissolved in acetonitrile at a concentration of 4 mg/mL. Polymerization in presence of Rhodamine labeled monomer was achieved in the dark. In all cases, 1mL of Rhod solution was added two minutes after adding IBCA and reaction was let to continue following method without Rhod.

Purification of nanoparticles:

All the polymer suspensions were purified by dialysis (Spectra/Por  membrane 100 000 g/mol molecular weight cut off (MWCO), Biovalley, Marne la Vallée, France) two times 1h30 and one time overnight against 500 mL of distilled water. The purified suspensions were stored at 4°C until use.

Nanoparticles characterization: 2.5.1 Particle size:

The diameter of the nanoparticles was measured at 20°C by quasi-elastic light scattering using a Nanosizer N4 PLUS (Beckman-Coulter, Villepinte, France) operating at the angle of 90°C. The samples were diluted in Milli Q water by 1/300 (v/v) for nanoparticles made by RREP and by 1/150 (v/v) for nanoparticles formed by AEP pH1. The results were expressed as the average of the mean hydrodynamic diameter of the dispersed particles obtained from three determinations. The standard deviation of the size distribution and the polydispersity index were also given. The polydispersity index given by the apparatus is equivalent to the variance of the log-normal distribution. A polydispersity index lower than 0.1 indicates a monodisperse dispersion.

Zeta potential:

The electrostatic surface charge of the polymer particles was deduced from the electrophoretic mobility using a Zetasizer nanoseries Nano 2S (Malvern Instruments Ltd., Orsay, France). Dilution of the suspensions (1/200 (v/v)) was performed in NaCl 1mM.

Concentration of nanoparticles in suspension:

Concentration of nanoparticles was evaluated by gravimetric measurement after freeze-drying of a known amount of suspension.

For freeze-drying, the suspensions were frozen at -18°C and freeze-dried during 48h (Christ Alpha 1-4 freeze dryer, bioblock Scientific, Illkrich, France) without using cryo-protecting agent. If the weighted amounts were insufficient, suspensions were concentrated by evaporation of water with a breath of air prior to sampling and lyophilization.

Ex-vivo study of bioadhesion:

Adsorption experiments were performed using a method developed by Durrer et al. 15 . The fresh small intestine of sacrificed male Wistar rats (Charles River, L'Arbresle, France) was excised, rinsed with physiological saline solution (NaCL 0,9%), and cut into segments of 5cm length. Each segment was opened lengthwise along the mesentery with scissors and spread on an aluminum plate. Another plate of aluminum with a slit (surface 2 cm²) in the center was fixed on the mucosa sample.

For bioadhesive experiments, 500 µL of each solution of rhodamine labeled nanoparticles was deposited in the split and covered with an aluminum sheet to protect from light. After one hour, the suspension was removed and mucosa was washed three times with 500 µL of phosphate buffer (pH 6) during one minute to remove nonadsorbed nanoparticles. After washing, mucosa was scraped from intestinal wall for dosage. Concentrations of nanoparticles in tested suspensions used were 5, 10 and 20 mg/mL and all experiments were repeated 3 or 4 times

Dosage of nanoparticles by fluorimetry:

Mucosa with nanoparticles were dissolved with 3 mL of a solution of sodium dodecyl sulfate (SDS) 2% and NaOH 1% by ultrasound during 2 hours and on planetary agitation overnight.

A calibration curve was obtained from sample containing 500 µL of nanoparticules suspensions of various concentrations ranging from 0.5 to 20 mg/ml, 100 mg of mucus with 3 mL of a solution of SDS 2% and NaOH 1%. Treatment of calibration samples was the same as for the treatment of experimental samples.

Assays were made with a fluorimeter (spectrometer L550B, Perkin Elmer, Norwalk, USA), the emission wavelength was fixed at 575 nm and the excitation wavelength at 555 nm corresponding to the fluorescence characteristics of Rhodamine. Concentration of nanoparticles in mucus was deduced from the calibration curve.

Statistical analysis:

The results obtained were statistically analyzed by using Mann-Whitney's t-test with a 95% confidence level (p<0.05) or a 90% confidence level (p<0,1).

Results and discussion:

In this study, bioadhesive properties of PIBCA nanoparticles coated with different polysaccharides (dextran, chitosan) were investigated to better understand the influence of the surface properties of nanoparticles on their bioadhesion to intestinal epithelium. In this purpose, ex-vivo bioadhesion experiments were achieved on rat intestinal membrane according to a previously described technique 15 . The core of the nanoparticles has been labeled with rhodamine and the amounts of adhering nanoparticles were determined by fluorimetry.

Characteristics of the nanoparticles

Nanoparticles were obtained by two methods of polymerization (anionic and radical) leading to the formation of amphiphilic copolymers made of hydrophobic PIBCA blocks and hydrophilic polysaccharidic blocks which structure differ depending on the method of polymerization 24 . With the radical redox emulsion polymerization (RREP) nanoparticles coated with either dextran or chitosan where produced whereas only nanoparticles coated with chitosan were prepared by the anionic emulsion polymerization (AEP). The nanoparticles which formed showed a size range between 210 nm to 575 nm. In agreement with previous results 23 , diameters of nanoparticles mainly depended on molecular weight of the polysaccharide. This may be explained by an increase of the polysaccharide layer thickness at the surface of the nanoparticles as the molecular weight of the polysaccharide increases.

Nanoparticles of almost the same size were obtained with and without Rhodamine by the RREP while the diameter slightly increased for the nanoparticles prepared by AEP. For a same molecular weight of the polysaccharide, the size of the nanoparticles depended on the charge of the polysaccharide and on the method of polymerization. The higher diameter was obtained with nanoparticles prepared by RREP with chitosan. The zeta potential of the nanoparticles was clearly affected by the type of the polysaccharide whereas no effect of the method of polymerization could be highlighted in agreement with previous work 23 . The introduction of Rhodamine in the nanoparticles did not affect the zeta potential of the nanoparticles (table 1). According to recent studies, Electronic Paramagnetic Resonance (EPR) spectroscopy showed that conformation of polysaccharide chains grafted at nanoparticles surface was different in function of method of polymerization (Figure 1). Dextran at the surface of nanoparticles obtained by RREP can fold at a molecular weight of 18,000 g/mol and chitosan at a molecular weight of 100,000 g/mol whereas in the case of the nanoparticles prepared by AEP the conformation of the polysaccharide chains at the surface of nanoparticles didn't allow the complete folding of polysaccharide chains whatever the molecular weight of the polysaccharide was 17,25 .

A B C D Figure 1: Hypothesized configuration of polysaccharide chains at the surface of nanoparticles prepared either by AEP with polysaccharide of low molecular weight (A) or high molecular weight (B) or RREP with polysaccharide of low molecular weight (C) or high molecular weight (D).

Polysaccharide chains: gray line, PIBCA chains: black line.

Effect of the size of the nanoparticles on bioadhesion

Mucoadhesion of nanoparticles have been described previously 13,15 . When a particulate suspension is placed in contact with an intestinal mucosa, it has been shown that mucoadhesion was a two steps mechanism, involving primarily the diffusion of the particles from the suspension to the mucosal surface and secondly, the attachment of the particles to the mucosa. As shown previously with model polystyrene particles, the kinetic of adhesion depended mainly on the size of the particles and thus on their diffusion coefficient in the suspending medium. Size decrease resulted in faster adhesion. Whatever the kinetics, adhesion was maximal after 10-20 min for particles in the range of 200-600 nm and did not vary anymore for larger contact times. The second step, corresponding to particles attachment to the mucosa is thought to depend on many parameters and its intensity has been related to various physico-chemical factors. The present study aims to investigate more particularly these adhesion factors explaining why all adhesion experiments were performed at an incubation duration of 1 hour. This incubation time was chosen to avoid any influence of the size of the nanoparticles which is predominant during the step of diffusion of the particles in the suspension medium but before their adhesion on the membrane. Under these conditions, it was assumed that the intensity of adhesion which was measured solely depended on the processes of attachment at the mucosal surface.

During the bioadhesion process and when the particles enter in contact with the mucosal surface, the particles firstly encounter a mucus layer, which can be compared to a thick hydrogel beside the size of the particles. It has been shown previously that the diffusibility of the nanoparticles in this hydrogel depended strongly on the size of the particles 6 . As expected, the attachment of the nanoparticles depended on the diameter of our nanoparticles.

The number of particles found in mucus layer was plotted against the diameter of the nanoparticles for all the particles included in this study (Figure 2). Whatever the initial concentration of the suspensions placed in contact with the intestinal mucosa was, the number of particles attached to the mucosa increased considerably when the particle size decreased.

However, at low concentration (Figure 2A), dextran-coated nanoparticles exhibited less adhesion than the corresponding chitosan-coated nanoparticles. When the particle concentrations of the suspensions increased (Figure 2 B,C), the number of nanoparticles adsorbed tended to follow the same evolution for all the type of nanoparticles, particularly at 20 mg/mL. This evolution was in agreement with the work of Norris et al., which showed that the permeability of particles through mucus layers followed similar exponential curves 6 . Furthermore, it was found that the 575 nm nanoparticles diffused very poorly, in agreement with the calculations of permeability coefficients in the mucus for nanoparticles in this size range 16 . However, these curves reflected not only the diffusion of the particles in the mucus layer, but also their immobilization at different sites of attachment. This was clearly shown by the fact that dextran-coated nanoparticles prepared by RREP behaved differently from the other particles (compare figure 2 A and 2 B).

The hypothesis of a diffusion of the particles in the mucus hydrogel was suggested by the following calculations. When considering the number of nanoparticles adherent to the mucus, the surface occupied by the particles (considering a square packing of the particles) has been calculated and further divided by the experimental surface offered by the intestinal fragment.

It resulted from this calculation for the experiments made at the highest concentration of nanoparticles (20 mg/L) that the amount of adhering nanoparticles would require the formation of many layers of particles in the mucus. The calculated number of layers ranged from 4 to 81 (figure 2C), suggested that nanoparticle diffusion inside mucus layer necessarily occurred to represent the experimentally determined amounts of adsorbed nanoparticles.

Indeed, if nanoparticles didn't diffuse into the gel, nanoparticles at the surface would have been removed during the washing process following adhesion experiments and multi-layer adsorption could be highlighted. This profile can be linearized (Figure 3) with a logarithm function (R² = 0,9635), leading to the following equation which represents the adsorption of the nanoparticles as a function of their diameter: N =5,9 * 10 12 * e (-0,0145D ) (

with N : number of nanoparticles adsorbed in mucus layer and D : diameter of nanoparticles.

Figure 3: Linearization of evolution of number of nanoparticles absorbed on mucus layer in function of diameter by representation of ln(number nanoparticles) in function of diameter for nanoparticles obtained by RREP in presence of dextran (circle) or chitosan (diamond) or obtained by AEP in presence of chitosan (triangle),

Additionally, these results suggested that the possibility for the smallest particles to strongly diffuse in the mucus layer allows the particles to reach much more attachment sites compared to the one available at the mucus layer surface, resulting in very efficient adhesion. However, from a practical point of view, one has to keep in mind that the number of particles adhering to the mucosa is not the most relevant parameter in drug delivery. Indeed for pharmaceutical applications, the most important is the amount of adhering particles expressed in mass which defines the amount of encapsulated and immobilized drug at the nanoparticle surface.

Influence of the surface properties on bioadhesion intensity

Because it was assumed that the duration of the contact between the particle suspensions and the mucosa was high enough (1 hour), it was expected that adhesion had reached equilibrium at the end of adhesion experiments. For further analysis, the amounts of particles which adhere on the mucosa were plotted as isotherms. As can be seen on figures 4, isotherms highlighted differences between nanoparticles depending on their surface characteristics.

Figure 4 A depicts the adhesive behavior of nanoparticles coated with dextran and prepared by RREP. Amounts of adhering particles continued to increase and no plateau could be reached at the highest concentration of nanoparticles in the bulk suspensions (20 mg/L). At this concentration, adhesion reached about 9 g/m², which corresponded to the highest adhesion values obtained in this study. The quasi-linearity of the isotherms suggested that the adhesion sites were still not saturated with these nanoparticles. The effect of the molecular weight of dextran was moderate. Compared to dextran-coated nanoparticles, the adhesion of the chitosan coated nanoparticles increased rapidly at low concentration of the suspensions and a pseudo-plateau was reached for a concentration of 10 mg/mL. This suggested that particles adhesion sites were rapidly saturated. In contrast to dextran-coated nanoparticles, the molecular weight of chitosan influenced the adhesion of the nanoparticles on the mucosa. At the maximal concentration tested (20 mg/L), the amounts of adherent nanoparticles were about 8 and 4 g/m² for chitosan 30 000 g/mol and 70 000 g/mol , respectively. Figure 4 C shows the adhesion isotherms of chitosan-coated nanoparticles prepared by anionic polymerization. Although the general form of the isotherms was not very precisely defined, due to the limited number of experimental points, it can be seen that the isotherms were quite similar to those observed for the nanoparticles prepared by radical polymerization.

Pseudo-plateau could be obtained and the molecular weight of chitosan seemed to modulate the adhesion of the particles to the mucus. The amounts of particles adsorbed were typically lower than 5 g/m² whatever the concentrations of the bulk suspensions placed in contact with the intestinal mucosa were. Even, at the concentration of 5 mg/L, the amount of nanoparticles which adsorbed on mucosa was almost nil. Differences between these nanoparticles can be attributed to both the nature and the conformation of polysaccharide chains at the surface of nanoparticles. The nature of the adhesive bonds which formed could depend on the type of polymer. Glycoproteins of mucus are negatively charged 26 and allowed electrostatic interactions with positively charged compounds like chitosan at the surface of the nanoparticles. The number of negative charges in the mucus is finite so does the number of interacting sites with positively charged substances. This can explain the pseudo-plateau shown by the adhesion isotherms observed with the nanoparticles coated with chitosan, which could correspond to a saturation of the negatively charge interacting sites of the mucus. In addition, the intensity of the interaction as evaluated at the plateau level was unexpectedly low in the case of the high molecular weight chitosan compared to the low molecular weight chitosan (Figure 4B). It can be assumed that a higher local viscosity in the high molecular weight chitosan corona can result in a lower accessibility of the interaction sites with the mucus and therefore to a lower adhesion of the nanoparticles. In the case of dextran which is a neutral polysaccharide, no electrostatic interactions can be expected. Although the intimate adhesion mechanism with glycoproteins is unknown, this is in favor to an enhanced diffusion of the dextran-coated nanoparticles in the mucus gel. Indeed, adhesion was in this case concentration dependant (Figure 4A). The marked differences in adhesion profiles for chitosan-coated and dextran-coated nanoparticles clearly supported that adhesion was due to the superimposition of different mechanisms.

The conformation of the chains at the surface of the particles depended on the preparation method. For the nanoparticles prepared by RREP the polysaccharide chains which are grafted at the surface of the nanoparticles in a end-on configuration formed a brush at the surface of the nanoparticles while in the AEP, polysaccharide chains grafted in the side-on configuration formed loops and trains at the nanoparticle surface (figure 1). It is likely that loops of chitosan at the surface of nanoparticles prepared by AEP were quite compact and could restrain interpenetration and interaction of polysaccharide chains with the network formed by the glycoprotein chains of the mucus. This effect could lower the adhesive interactions.

Obviously, when the molecular weight of chitosan increased, the length of loops also increased, which could facilitate to some extent the interactions between mucus and nanoparticles prepared by AEP. This can explain that a higher adhesion with the mucosa was observed when chitosan molecular weight increased. For nanoparticles made by RREP, chitosan chains were extended at the surface of nanoparticles and were likely to interact freely with the glycoproteins of the mucus layer. It can be suggested that chains interpenetration occurred more easily in this case. In summary, not only the size, but also the nature and conformation of the polysaccharides at the surface of nanoparticles influenced the adhesion of the nanoparticles on the mucus layer.

Adhesion of the nanoparticles to the intestinal mucus can be viewed as a complex process, including the diffusion of the particles in the hydrogel, depending on their size, followed by their interactions with the mucus glycoproteins. It is likely that the molecular interactions played a key role in this latter process. For getting a better understanding of these effects, differences in polymer chain flexibility and local modification of viscoelasticity should probably be considered. Indeed, as shown previously at a macroscopic scale, these polymers can interact with mucus. For example, the viscoelasticity of mucus can be decreased in presence of dextran as shown elsewhere using the airway mucus 27 . Dextran was also found to diffuse through mucus layer in rat distal colon mucus 28 . However, a more detailed investigation of the dynamic of polymer chains at the nanoparticle surface would be required for being able to fully describe these effects. Finally, from a practical point of view, it is interesting to point out that the bioadhesive properties of the nanoparticles could be adjusted to some extent by varying the nature of the coating polymer and probably its conformation.

Conclusion:

Influence of the coating of nanoparticles by polysaccharides with varying molecular weight and molecular conformation on their bioadhesive properties has been investigated. In agreement with the literature, accumulation of the smallest nanoparticles in the mucus layer could be attributed to a diffusion mechanism. Further, interaction of nanoparticles with mucus was promoted by electrostatic interactions in the case of chitosan coated nanoparticles and non electrostatic interactions in the case of dextran coated nanoparticles. Interestingly, differences in bioadhesion were observed, which depended on the nature of the polysaccharide and its conformation at the surface of the nanoparticles, suggesting that bioadhesive properties of nanoparticulate systems can be modulated by a fine tuning of the surface properties of the particles.

Aknowledgements

Authors wish to thank K. Broadley from Loctite (Dublin, Ireland) for providing the isobutylcyanoacrylate used in the preparation of the nanoparticles and Irene Bravo-Usuna for helpful discussion. I. Bertholon-Rajot was a fellow from the French Ministery of Research. This work was part of the Integrated Action Program (Amadeus Program 06645SH).

Figure 2 :

 2 Figure 2: Number of particles fixed on 2 cm² of mucosa after washing. Nanoparticles made by RREP (circle, diamond) and AEP (triangle) in presence of dextran (circle) and chitosan (diamond, triangle) A: 5mg/ml, B: 10 mg/ml, C: 20 mg/ml, in brackets calculated number of nanoparticles layer at 20 mg/ml. All experiments were repeated 3 or 4 times and for clarity standard deviation were not superimposed.

Figure 4 B

 4 Figure 4 B shows the adhesion isotherms of chitosan-coated nanoparticles prepared by RREP.

Figure 4 :

 4 Figure 4: Adsorption isotherms of nanoparticles obtained by RREP: A, B or AEP: C, in presence of dextran: A or chitosan: B,C. Open symbol : low molecular weight (40,000 g/mol for dextran, 30,000 g/mol for chitosan), black symbol : medium molecular weight (67,000 g/mol for dextran, 70,000 g/mol for chitosan); Influence of molecular weight statistics * : p<0,1; **: p< 0,05

Table 1 :

 1 Properties of nanoparticles with and without labeling with rhodamine in pure water for diameter measurements and in NaCl 1mM solution for zeta potential measurements

		Without Rhodamine	With Rhodamine
		Diameter	Zeta potential	Diameter	Zeta potential (mV)
		(nm)	(mV)	(nm)
	Dex 40k -RREP	246  25	-9.2  0.6	225  22	-8.0  0.1
	Dex 67k -RREP	272  27	-5.0  0.4	275 27	-4.6  0.3
	Chito 30k -RREP 286  29	ND	271  27	+ 30.3  0.1
	Chito 70k -RREP 365  36	+ 42.0  0.7	344  34	+ 36.5  0.2
	Chito 200k -RREP 575  57	+ 43.0  0.3	550  55	+ 48  1.0
	Chito 30k -AEP	210  21	+ 40.3  0.5	260  26	+ 42.9  0.3
	Chito 70k -AEP	251  25	+ 42.4  0.4	340  34	+ 44.4  0.3
	ND : not determined


Khairallah Alhareth

Christine Vauthier
email: christine.vauthier@u-psud.fr

Fanchon Bourasset

Claire Gueutin

Gilles Ponchel

Fathi Moussa
email: fathi.moussa@u-psud.fr

Conformation of surface-decorating dextran chains affects the pharmacokinetics and biodistribution of doxorubicin-loaded nanoparticles

Keywords: Stealth nanoparticles, nanoparticle surface, dextran, polysaccharide-coating, anionic emulsion polymerization

come

Introduction:

Many types of nanoparticles were developed as drug carriers to modify tissue distribution of drugs. Among those, poly(alkylcyanoacrylate) nanoparticles (PACA-NPs) were the most studied nanoparticles [START_REF] Vauthier | Design aspects on poly(alkylcyanoacrylate) nanoparticles for targeted drug delivery[END_REF].

The first PACA-NPs prepared by anionic emulsion polymerization (AEP) show a very short lifetime in blood and accumulate in the liver and the spleen after intravenous administration [START_REF] Grislain | Pharmacokinetics and distribution of a biodegradable drug-carrier[END_REF]]. They are suitable carriers to target drugs to organs of the mononuclear phagocyte system (MPS) [START_REF] Chiannilkulchai | Hepatic tissue distribution of doxorubicin-loaded nanoparticles after i.v. administration in reticulosarcoma M 5076 metastasis-bearing mice[END_REF]. Their accumulation in the liver was explained by their recognition and uptake by macrophages of the MPS in relation with their strong capacity to activate the complement system [START_REF] Chiannilkulchai | Hepatic tissue distribution of doxorubicin-loaded nanoparticles after i.v. administration in reticulosarcoma M 5076 metastasis-bearing mice[END_REF][START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF].

Capacity to activate the complement system and the more general phenomenon of opsonisation consisting in protein adsorption on nanoparticles in contact with blood depends strongly on nanoparticle surface properties. It is well known that these phenomena are affected by the nanoparticle size, the charge and the hydrophilicity of the nanoparticle surface [START_REF] Moghimi | Nanomedicine: current status and future prospects[END_REF][START_REF] Vonarbourg | Evaluation of pegylated lipid nanocapsules versus complement system activation and macrophage uptake[END_REF][START_REF] Alexis | Factors affecting the clearance and biodistribution of polymeric nanoparticles[END_REF]. More recently, it was reported that it also depends on the conformation of polysaccharide chains stranded on nanoparticle surface to achieve stability of nanoparticle dispersion [START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF]. The most unexpected result was an opposite capacity of activating the complement system reported with dextran-coated PACA-NPs of the same composition but obtained from two methods of polymerization. It was explained by a difference of the chain conformation arrangement in the coat of dextran due to the mechanism of polymerization used to produce the nanoparticles. Indeed, it was demonstrated that the polysaccharide chains form "loops" on the surface of PACA-NPs produced by AEP (AEP-PACA-NPs) while they form a dense "brush" on the surface of PACA-NPs produced by redox radical emulsion polymerization (RREP) (RREP-PACA-NPs) [START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Bertholon | Complement activation by core-shell poly(isobutylcyanoacrylate)-polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide[END_REF] (Figure 1). This last configuration of the dextran chains hampered adsorption of large proteins on the surface of nanoparticles including the protein C3 of the complement system [START_REF] Vauthier | Protein adsorption and complement activation for di-block copolymer nanoparticles[END_REF]. It was found suitable to obtain nanoparticles with a low capacity to activate the complement system in contrast with the conformation found on the surface of the AEP PACA-NPs [START_REF] Vauthier | Protein adsorption and complement activation for di-block copolymer nanoparticles[END_REF][START_REF] Vauthier | Configuration of Bovine Serum Albumin adsorbed on polymer particles with grafted dextran corona[END_REF]. All previous data suggested that RREP-PACA-NPs were a new drug carrier with distinct pharmacokinetic and biodistribution profile compared to those described for the well-known AEP-PACA NPs.

The aim of the present work was to investigate the pharmacokinetic and tissue distribution of a drug formulated with the RREP-PACA-NPs and to compare the results to those obtained for the free drug and for the drug associated with AEP-PACA-NPs. For this work, doxorubicin was chosen as the model drug because the pharmacokinetic and tissue distribution were already well known for the free drug and for the drug formulated in AEP-PACA-NPs. The association and stability of doxorubicin formulated as AEP PACA-NPs and RREP PACA-NPs were described and compared in a recent work [START_REF] Alhareth | Doxorubicin loading and in vitro release from poly(alkylcyanoacrylate) nanoparticles produced by Redox Radical Emulsion Polymerization[END_REF]. Additionally, based on the knowledge available on these two nanoparticle formulations, we believed that they were relevant models to study the influence of the conformational characteristics of dextran in their corona on the pharmacokinetic and biodistribution of an associated drug.

Materials and Methods

Preparation of nanoparticles

Dox-loaded RREP and AEP-PACA NP were prepared according to previously described methods [START_REF] Alhareth | Doxorubicin loading and in vitro release from poly(alkylcyanoacrylate) nanoparticles produced by Redox Radical Emulsion Polymerization[END_REF]. The mean hydrodynamic diameter of the Dox-loaded RREP-PACA-NPs was 301 ± 82 nm (PDI = 0.3 ± 0.05) and their zeta potential was 1.1 ± 0.1 mV as determined using a Malvern Zetasizer nano ZS90 (Malvern Instruments, Orsay, France). The mean hydrodynamic diameter and zeta potential of the Dox-loaded AEP-PACA-NPs were 196 ± 48 nm (PDI 0.05 ± 0.02) and. 0.7 ± 0.1 mV, respectively.

Pharmacokinetics and biodistribution study of the free Dox and the Dox associated with nanoparticles

Animals: Experiments were performed on male Wistar rats (Janvier, Le Genest Saint Isle, France) weighing 240-300 g. Animals were stabilized under normal conditions for 1 week prior to experimentation and had free access to food and water. Experiments were carried out in compliance with the guidelines of the European Community (Recommendation 2007/ 526/ EC). The protocol ethics were institutionally approved.

Pharmacokinetics: Under general anesthesia by pentobarbital (50 mg/kg bw), a catheter was introduced into the rat jugular vein, positioned subcutaneously with the tip in the inter scapular region. The rat was allowed to recover all night, and the blood catheter was flushed with heparinized saline solution (NaCl 0.9%, heparin 50 UI /ml). The three formulations, free Dox (f-Dox), Dox loaded-AEP PACA-NPs (Dox-AEP) and Dox loaded-RREP PACA NPs (Dox-RREP), were administered at the dose of 6 mg/kg to three groups of 5 rats each. All formulations were administered in the non catheterized jugular vein by slow perfusion (0.25 mL/min). During the perfusion, the animal was maintained under isoflurane anesthesia (1 % of isoflurane in air). Blood samples were collected at time 10, 15, 30, 45 min and 1, 2, 4, 6, 10 and 24 h, after administration. After each collection, the volume of the collected sample was replaced by the same volume of 0.9% NaCl solution. Immediately after collection, blood was centrifuged (2000 g for 10 min) to recover plasma samples which were then stored at -20 °C until analysis.

Biodistribution: To study tissue biodistribution, 45 rats were randomly divided in three groups of 15 rats. Each group received an injection of one of the three formulations in the jugular vein. At time 1, 4, 10, 24 and 48 h after dosing, three rats of each group were sacrificed by cardiac puncture under general pentobarbital anesthesia. Heart, lungs, liver, kidneys, spleen and brain were collected and stored at -20 °C until analysis. Concentration in Dox in each organ was analyzed in duplicate.

Dox analysis in plasma and tissues

Dox analyses in plasma and tissue were performed using a HPLC method after liquid-liquid extraction as reported elsewhere (12)

Pharmacokinetic analysis and statistics

Statistical studies were made using t tests of unequal variance performed by Origin 8 software, (Origin Lab corp., USA). For pharmacokinetic analysis, the plasma concentrations of each rat were analyzed with the WinNonlin ® professional version 5.2 software (Pharsight, Sunnyvale, CA, USA), using a bicompartmental model. The estimation method was the weighted last squares (WLS) weighting by the inverse of the variance 1/σ 2 . The distribution (α) and elimination (β) rate-constants and the maximal concentrations A and B were determined by nonlinear regression of the observed data. The area under the plasma concentration-versus-time curve (AUC) and the area under the first moment curve (AUMC) were determined by the equations ( 1) and (2), respectively.

AUC0- = A/α + B/β (1) AUMC0- =A/α 2 + B/β 2 (2)
Total clearance (CL) was calculated from the ratio dose/AUC0- . The steady state volume (Vdss) was calculated from (dose * AUMC0- / (AUC0- ) 2 ). The distribution and elimination half-lives (t1/2α and t1/2β) were calculated from Ln2/α and Ln2/β, respectively. The concentrations of Dox-RREP were significantly higher than those of f-Dox and Dox-AEP (p < 0.05) at the distribution phase. While there was no significant difference between Dox-AEP and f-Dox in the elimination phase until 10 hours, there was a significant difference between Dox-RREP and Dox-AEP. Twenty four hours after administration, the difference between Dox-RREP and the other formulations was significant. Table 1 summarizes the main pharmacokinetic parameters calculated using a bi-compartmental pharmacokinetic analysis.

RESULTS AND DISCUSSION

Pharmacokinetics:

The main difference between the three formulations was in the distribution phase. The Dox-RREP showed the longer distribution phase compared with the other formulations. The distribution half-life (t1/2  ) of Dox-RREP was about twice that of f-Dox (Table 1; p = 0.001) and 3.5 fold higher than that of Dox-AEP (p = 0.0001). As compared to f-Dox, a faster distribution rate was obtained with the AEP-NPs (p = 0.042) while a slower one was obtained with the RREP-NPs (p = 0.001). Results obtained with Dox-AEP can be explained by the rapid uptake of the AEP-NPs by the macrophages of the mononuclear phagocyte system (MPS) in agreement with previous reports [START_REF] Grislain | Pharmacokinetics and distribution of a biodegradable drug-carrier[END_REF][START_REF] Chiannilkulchai | Hepatic tissue distribution of doxorubicin-loaded nanoparticles after i.v. administration in reticulosarcoma M 5076 metastasis-bearing mice[END_REF][START_REF] Reddy | Pharmacokinetics and biodistribution studies of doxorubicin loaded poly(butylcyanoacrylate) nanoparticles synthesized by two different techniques[END_REF]. Results obtained with the Dox-RREP were in agreement with the low capacity of the RREP-NPs to trigger the activation of the complement system reported earlier [START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Vauthier | Protein adsorption and complement activation for di-block copolymer nanoparticles[END_REF].

The increase of blood circulation time of Dox by RREP nanoparticles was also evidenced by the increase of AUC0- value and the decrease of the total clearance (Cl) in comparison with f-Dox and Dox-AEP (Table 1; p = 0.003, 0.009, 0.023 and 0.029, respectively). The steady state volume of distribution (Vss) of Dox-RREP was significantly smaller than that of f-Dox (p = 0.028) that showed a less extensive distribution of Dox in tissue when it was administered with RREP-NPs. In contrast, there was no difference between the Vss of Dox-RREP and Dox-AEP as well as between Dox-AEP and f-Dox.

The pharmacokinetics parameters of Dox-loaded RREP nanoparticles showed that these nanoparticles behaved like stealth nanoparticles. Compared to f-Dox or Dox-AEP our results

show that Dox-RREP has a higher distribution half-life and a higher AUC with a slower clearance. Although the concentrations of Dox during the elimination phase were significantly higher in the Dox-RREP treated rats, there was no significant difference between the elimination half-life (t1/2  ) between the three formulations. This result suggested that the elimination of Dox concerned only the free fraction of the drug. The biodistribution of Dox found with the formulation Dox-AEP agreed with results reported in previous works [START_REF] Grislain | Pharmacokinetics and distribution of a biodegradable drug-carrier[END_REF][START_REF] Chiannilkulchai | Hepatic tissue distribution of doxorubicin-loaded nanoparticles after i.v. administration in reticulosarcoma M 5076 metastasis-bearing mice[END_REF][START_REF] Reddy | Pharmacokinetics and biodistribution studies of doxorubicin loaded poly(butylcyanoacrylate) nanoparticles synthesized by two different techniques[END_REF]. It confirmed the rapid uptake of the Dox-AEP by macrophages of the MPS and possibility to target the associated drug in the liver. Results from both the pharmacokinetics and the biodistribution study of the Dox-RREP formulation supported the hypothesis that the RREP-PACA NPs were stealth and agreed with their low capacity to activate the complement system reported earlier [START_REF] Labarre | Interactions of blood proteins with poly(isobutylcyanoacrylate) nanoparticles decorated with a polysaccharidic brush[END_REF][START_REF] Vauthier | Protein adsorption and complement activation for di-block copolymer nanoparticles[END_REF]. However, based on the criteria of the size of the nanoparticles, results were in contrast with those expected from the literature [START_REF] Alexis | Factors affecting the clearance and biodistribution of polymeric nanoparticles[END_REF].

Tissue distribution

Indeed, the larger nanoparticles (PPER-PACA NPs) appeared stealth while it was expected to be highly susceptible to recognition mechanisms of the defense systems of the organisms because of their large size. Since all experimental results were in agreement with each others and with previous findings, it can be concluded that surface properties of nanoparticles were predominant parameters to determine the in vivo fate of the present nanoparticles. Thus, the difference in the pharmacokinetics and biodistribution of Dox highlighted between the two nanoparticle formulations considered in the present work can be attributed to their difference in the conformation of the dextran chains decorating the surface of the nanoparticles. It is noteworthy, that uptake of foreign bodies by the spleen occurred irrespectively of their surface properties. A similar fate was reported by other authors considering PACA-NPs decorated with poly(ethylene-glycol) chains [START_REF] Peracchia | Stealth PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting[END_REF][START_REF] Calvo | Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery[END_REF]. It can be suggested that a different mechanism of recognition was involved in the spleen compared to that used by liver macrophages.

In conclusion, results from this study showed that conformation of polysaccharide chains decorating the nanoparticle surface are influencing the pharmacokinetic and biodistribution of nanoparticles. The stealth property described for the first time for the RREP-NPs was different from the know stealth property described for pegylated nanoparticles which generally show a much longer time of persistence in the blood circulation during the distribution phase of the pharmacokinetic [START_REF] Vonarbourg | Evaluation of pegylated lipid nanocapsules versus complement system activation and macrophage uptake[END_REF][START_REF] Peracchia | Stealth PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting[END_REF][START_REF] Calvo | Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery[END_REF]. The rather short half life in the blood circulation was explained by the rapid accumulation of the nanoparticles in organs which occurred in parallel with the reduction of the plasma concentration of the drug. Thus, the RREP-NPs are stealth with a short circulation time. By analogy of the accumulation of the drug carried by the RREP nanoparticles in highly vascularised tissued, it might be expected that these nanoparticles may enhance drug delivery in tumors thanks to the enhanced permeation and retention effect. In contrast, the delivery of drugs in the brain will probably be

Figure 1 :

 1 Figure 1: Scheme of the conformation taken by dextran chains at the surface of the PIBCA nanoparticles prepared by the AEP (AEP) and the RREP method (B) as deduced from previous works (Vauthier et al. 2009, 2011). The capacity of these nanoparticles to trigger the activation of the complement system was well correlated with the characteristics of the nanoparticle corona regarding their property to let large proteins such as the protein C3 of the complement system to diffuse across and to reach the nanoparticle core surface (Labarre et al. 2005).

Figure

  Figure 2A reported the mean Dox plasma concentration as a function of time following intravenous administration of the three formulations. The Dox plasma concentration-time profiles showed biphasic curves with a first rapid distribution phase followed by a slow elimination phase. Each formulation of Dox showed a single pharmacokinetic profile

Figure 2 :

 2 Figure 2: Pharmacokinetics of doxorubicin after intravenous administration of different formulations to rats. (A) Data are given as the mean concentration of doxorubicine in plasma of rats (n = 5). (Full square): free Dox (f-Dox), (empty diamond): Dox loaded RREP PACA-NPs (Dox-RREP), (empty triangle): Dox loaded AEP PACA-NPs (Dox-AEP). Lines only serve as guides for the eyes. (B) Example of the analysis of the pharmacokinetic data using a bicompartmental model performed on one animal of each group having received a different formulation of Dox by the intravenous route. Experimental data were given as the symbols on the graph: (full square): free Dox (f-Dox), (empty diamond): Dox loaded RREP PACA-NPs (Dox-RRP) and (empty triangle): Dox loaded AEP PACA-NPs (Dox-AEP). Curves were drawn from the predicted values calculated on the basis of the assumption of a bi-compartmental pharmacokinetic model with the help of the WinNinLin software: (-) f-Dox, ( ---) Dox-RREP, ( _._._ ) Dox-AEP.

Figure 3 reported

 3 Figure 3 reported Dox concentrations found in different organs of the rats after intravenous administration of the three types of Dox formulations. As shown on this figure, significant amount of Dox was found in the different organs including the liver, spleen, heart, lungs and kidneys. The concentrations varied with time and were greatly influenced by the type of injected formulation. No Dox was found in the brain of rats treated by the different formulations.This confirmed that Dox followed the biodistribution of the drug carrier with which it was associated. Dox concentrations found in liver of rats treated with Dox-RREP were significantly lower than that found in animals of the group treated with Dox-AEP. It is noteworthy that this was observed over the whole period of time of the pharmacokinetic experiment which lasted for 48 hours. These results agreed with those of the pharmacokinetic study and indicated a lower uptake of Dox by the MPS when it was associated with the RREP-PACA-NPs as compared to Dox-AEP and f-Dox. Expressed as the percentage of the dose administered, the amount of Dox found in the liver after 1 hour of the administration of Dox-RREP (5 ± 1%) was two times lower than that found following the administration of Dox-AEP (10 ± 2%) and the f-Dox (9 ± 1%). Concentrations of Dox found in the spleens were similar for the three formulations at all studied time points (figure3.b). The group of rats treated with Dox-RREP showed significantly higher concentrations of Dox in the heart and lungs during the first 4 h of the experiment than those observed in groups treated with f-Dox and Dox-AEP (figure3.c, 3.d). For instance, the fractions of the dose which reached these organs with the Dox-RREP formulation were 1.8 ± 0.4% in the heart and 1.1 ± 0.2 in the

Figure 3 :

 3 Figure 3: Body distribution of doxorubicin after intravenous administration of different formulations, free Dox (f-Dox) (white columns), Dox loaded RREP PACA-NPs (Dox-RREP) (black columns) and Dox loaded AEP PACA-NPs (Dox-AEP) (hashed columns), to rats. Concentrations of doxorubicin in organs (y axis) were determined in different organs: liver (A), spleen (B), heart (C), lungs (D) and kidneys (E) at different times (x axis). Statistics: : p < 0.05 Dox-RREP vs f-Dox, #: p < 0.05 Dox-RREP vs Dox-AEP, ¥: p < 0.05 f-Dox vs Dox-AEP, n = 3.

Table 1 :

 1

	Dox-RREP vs Dox-AEP, ¥ p<0.05 Dox-AEP vs f-Dox)
	Parameter	f-Dox	Dox-AEP	Dox-RREP
	t1/2 (h)	0.11 ± 0.03	0.06 ± 0.03 ¥	0.21 ± 0.03 #
	t1/2 (h)	9.69 ± 1.09	7.26 ± 2.09	8.56 ± 0.71
	AUC0- (h.µg/mL)	1.45 ± 0.21	1.59 ± 0.40	2.50 ± 0.43 #
	Cl (L/h)	1.14 ± 0.32	1.03 ± 0.27	0.65 ± 0.09 #
	Vss (L)	12.0 ± 4.84	6.96 ± 1.89	4.98 ± 1.76 

Pharmacokinetic parameters of Dox after i.v. administration to rats in different formulations: free Dox (f-Dox), Dox loaded RREP PACA-NPs (Dox-RREP) and Dox loaded AEP PACA -NPs (Dox-AEP). (n = 5),  p<0.05 Dox-RREP vs f-Dox, # p<0.05

hampered as suggested by the total absence of distribution of Dox in this organ and because of the rapid clearance from the blood compartment.

Acknowledgments

The authors wish to thank Henkel Biomedical for having provided the monomer used to synthesize the nanoparticles used in this study. K.A. thanks the Ministry of Health of Syria for providing with a Ph.D. scholarship. We thank Pharsight Corporation (Sunnyvale, CA, USA) for allowing our University to benefit from the Academic Licensing Program.

Declaration of Interest

The authors report no declarations of interest.