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Zinc-stabilized chitosan-chondroitin sulfate nanocomplexes for HIV-1 infection inhibition application Danjun Wu, Agathe Ensinas, Bernard Verrier, Charlotte Primard, Armelle Cuvillier, Gaël Champier, Stephane Paul, and Thierry Delair Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00568 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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Molecular Pharmaceutics

Zinc-stabilized chitosan-chondroitin sulfate nanocomplexes for HIV-1 infection inhibition application Danjun Wu,† Agathe Ensinas,‡ Bernard Verrier,‡ Charlotte Primard ,§ Armelle Cuvillier,ǁ Gaël Champier,ǁ Stephane Paul,⊥ Thierry Delair*† †

Ingénierie des Matériaux Polymères, UMR CNRS 5223, Université Claude Bernard Lyon 1, 15 Bd. André Latarjet, 69622 Villeurbanne Cedex, France ‡ Institut de Biologie et Chimie des Protéines UMR 5305, CNRS/Université de Lyon, France § ADJUVATIS - 7, passage du Vercors - 69367 Lyon Cedex 07 - France ǁ B-Cell Design, 98 Rue Charles Legendre, 87000 Limoges, France ⊥

Groupe Immunité des Muqueuses et Agents Pathogènes, INSERM Centre d’Investigation Clinique en Vaccinologie 1408, Université de Lyon, 15 rue Ambroise Paré, 42023 Saint-Etienne Cedex 2, France Supporting Information

ABSTRACT: Polyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g. degree of depolymerization, charge mixing ratio, the concentration and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II)ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir(TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particles interfaces could efficiently be functionalized with anti-OVA or anti-α4β7 antibodies with conservation of the antibody bio-recognition properties over one week of storage in PBS at 4 °C. In-vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were non-cytotoxic to human peripheral blood mononuclear cells (PBMCs) and in-vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decrease the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 µmol·L-1 to 1.95 µmol·L-1. Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection. KEYWORDS:Chitosan; Chondroitin sulfate; Polyelectrolyte Complexes; Stabilization; HIV infection inhibition

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ABBREVIATIONS: HYA, hyaluronan; ChonS, chondroitin sulfate;CS, chitosan; DA, degree of acetylation; PECs, polyelectrolyte complexes; TF, tenofovir ; PBMCs, human peripheral blood mononuclear cells; HAART, highly active antiretroviral therapy; the IC50, 50% inhibitory concentration; FITC, fluorescein isothiocyanate; OVA, ovalbumin; SEC, size exclusion chromatography; PBS, phosphate buffer saline. 1. INTRODUCTION The advent of highly active antiretroviral therapy (HAART), in which three or more antiretroviral drugs are combined in the treatment regimen, allowed a drastic reduction of the plasma viremia in HIV-infected individuals which resulted in a decrease in the death rate attributable to AIDS.1 Nonetheless, HIV-1 exists in intra-cellular reservoir of latently infected resting memory CD4+T cells, which shows minimal decay in a majority of patients on HAART regimens.2 Thus, the persistence of stable viral reservoirs remains a major issue in the eradication of HIV.3 These viral reservoirs are distributed predominantly in the lymphocytes of the peripheral lymphoid organs, especially the spleen, lymph nodes, and gut-associated lymphoid tissue (GALT).2 Among them, the GALT seems to be an important site of active viral replication, probably reflecting high levels of T cell activation.4 Lately, Arthos et al. indicated a gut mucosal homing receptor α4β7 can mediate the migration of lymphocytes to GALT, and they also demonstrated that HIV-1 envelope protein gp120 could bind to an activated form of integrin α4β7, through interaction mediated by a tripeptide in the V2 loop of gp120 5. This specific affinity of gp120 for α4β7 provides a target for clearing virus present in these latent viral sanctuaries.6 Though antiretroviral agents have contributed to improve disease management, HAART remains inaccessible to HIV viruses, located in the viral reservoirs, resulting in the failure of clearing HIV from these reservoirs and also the development of multidrug resistance.7 Nanotechnology-based systems for antiretroviral drug delivery offer a real potential of therapeutic improvement, as the nanosystems protect or promote the absorption of anti-HIV drugs, improve their bioavailabilities, prolong drug residence in desired cellular or anatomic sites, thus reducing needed doses and prolonging time between administrations.8 Colloidal polyelectrolyte complexes (PECs) offer a great interest as they are formed in water instead of any organic solvents, and also without using chemical cross-linker nor surfactant.9; 10 The fabrication process is simple to implement such as the one-shot addition11, and is also energy efficient (room temperature, low shear rate). However, PECs suffer from a lack of stability in physiological conditions due to the presence of electrolytes and of pH values responsible for a decrease in the charge density of polyions. The effect of electrolytes was well described by Dautzenberg and Kriz.12 According to this paper, complexes from weak polyelectrolytes redissolved at a critical salt concentration and those from strong polyelectrolytes irreversibly precipitated. Improvement of the stability of nanoPECs in physiological conditions is of great important for applications as efficient drug delivery systems. For chitosan-dextran sulphate based colloidal complexes, we preserved the colloidal stability in physiological conditions by using 2


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polyelectrolytes of highly different relative degree of depolymerizations and a high molar fraction of N-acetyl groups in the chitosan chains to ensure a steric stabilization.13; 14 Chitosan-hyaluronan (HYA) colloidal PECs, stable in physiological conditions, were obtained by using a nontoxic zinc(II) cations to stabilize the nano-complexes.15 To exemplify the versatility of the colloidal PECs approach, we investigated this technique with chondroitin sulphate A (ChonS), a glycosaminoglycan from bovine trachea. Apart from its biological relevance similarly to HYA (both of them are important components of many cartilaginous tissues), ChonS also displays a similar structure and chain flexibility as HYA (persistence length of the order of 4.5-5.5 nm)16;17, but different charged groups and densities (One carboxylic group/disaccharide unit for HYA with a pKa~2.9, but one carboxylic group and 1~2 sulfate groups per disaccharide unit for ChonS with lower pKa(sulfate)=2-2.5 due to the presence of the sulfate group)18 to investigate the influence of the lateral charged groups on PECs formation and stability in physiological medium. ChonS is a copolymer of N-acetyl-β-D-galactosamine and β-D-glucuronic acid that can be sulphated at C4 or C6 (scheme S1 of supporting information). In this work, nanoPECs composed of chitosan and ChonS were formed and the colloidal stability in physiological media was evaluated with or without using zinc (II) stabilizer. The stable nanoPECs were used to load tenofovir(TF), a novel nucleotide analogue reverse transcriptase inhibitor, with favorable anti-HIV activity 19 and low cytotoxicity in various human cell types in comparison with other nucleoside reverse transcriptase inhibitors.20 To target the viral reservoirs of intestinal sub-mucosal lymphocytes, the drug-loaded nanoPECs were surface functionalized with anti-α4β7 immunoglobulin type A, and the antiviral potency toward human peripheral blood mononuclear cells (PBMCs) in vitro were evaluated. 2. MATERIALS AND METHODS 2.1. Materials. Chitosan (CS) obtained from squid pens, was provided by Mahtani Chitosan PVT, Ltd., India, batch 114 (degree of acetylation (DA)~4%, average molar mass (Mw)~5.8×105 g·mol-1). Chondroitin sulfate A sodium salt (ChonS, Assay:≥60%, balance is chondroitin sulfate C) obtained from Sigma-Aldrich (extracted from bovine trachea) and characterized by size exclusion chromatography coupled on line with light scattering. The antiretroviral drug model Tenofovir(TF) (purity >99%) was purchased from R&D Systems company (Lille, France). The monoclonal chimeric immunoglobulins A (IgA) anti-Ovalbumin (anti-OVA) clone A1, anti-α4β7 integrin clone 7G3 and 1E1 were provided by B-Cell Design (Limoges, France) after development and production using HAMIGA® mice model. The concentration was determined by BCA Assay according to the procedure provided by Pierce (Thermo Fischer Scientific, Courtaboeuf, France). Phytohemagglutinin (PHA-M) was provided by Sigma Aldrich Laboratories. HIV-1 p24 antigen capture assay was provided by Advanced Bioscience Laboratories, Inc. USA. Phosphate buffer saline (PBS) and ultrapure water were from Invitrogen® (Paisley, UK).

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2.2. Preparation and characterization of chitosan. Prior to use, chitosan was purified, N-acetylated in homogeneous media at different DAs with acetic anhydride, and then depolymerized to produce low-molar-mass chitosans, with a control of the reaction kinetics (see supporting information) 21,22. For the characterization, the degree of acetylation (DA) was determined on purified chitosans by 1H-NMR spectroscopy (Varian, 500MHz), according to the method developed by Hirai, Odani and Nakajima.23 The water content was determined by thermogravimetric analysis (DuPont Instrument 2950). The weight-average molar mass (Mw) and the polydispersity index (Ip) were measured by an aqueous size exclusion chromatography (SEC) system (supporting information). Refractive index increments (dn/dc) were determined from a master curve previously established for each degree of acetylation under identical conditions.24 2.3. Formation and characterization of polyelectrolyte complex Chitosan was dispersed in ultrapure water at 0.1 or 0.2% (w/v) concentration, taking into account the initial water content. Dissolution was achieved under moderate stirring by adding a stoichiometric amount of acetic acid, with respect to the free amines for each chosen degree of acetylation. Then, the pH of the solutions was adjusted to the desired value (4.0, 4.5, 5.0, and 5.5) with 0.1M sodium hydroxide or hydrochloric acid. Chondroitin sulfate (ChonS) solutions, at 0.1 or 0.2% (w/v) concentration, were prepared directly in ultrapure water under magnetic stirring. Zinc chloride (ZnCl2) was added to the previous chitosan and ChonS solutions at a final ZnCl2 concentration of 1.0 mM. These solutions were stirred for 30 min before use. Colloidal polyelectrolyte complexes (PECs) were formed in non-stoichiometric conditions at predetermined charge mixing ratios (R = n+/n-) by the one-shot addition of the polymer solution in default to the polymer in excess under magnetic stirring (1,200 rpm) at room temperature. Fluorescent nanoparticles (F-NPs) were prepared as above, with FITC labelled chitosan (0.1%, w/v) instead. For the preparation of tenofovir (TF) loaded CS-ChonS nanoparticles, TF was firstly dissolved in 1M NaOH, at a concentration of 1% (w/v). The TF solution was dropped into ZnCl2-chitosan solution under magnetic stirring, and the nanoparticles spontaneously formed upon one-shot addition of the ZnCl2-ChonS solution. The developed formulation was characterized for particle size, polydispersity index (PDI), zeta potential and morphology (supporting information). 2.4. Evaluation of the drug encapsulation efficiency. For the determination of drug encapsulation efficiency (EE), TF loaded nanoparticles were separated from the suspension by centrifugation at 20,000g for 60 min. The free amount of TF in the supernatants was determined by measuring the absorbance of the supernatant at 259 nm after subtraction of the blank value, using a calibration curve established in the same conditions. Each sample was measured in triplicate. EE was calculated by the following equations:

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EE (%) =

− [TF ] [TF] input residual ×100 [TF ] input

Where [TF]input is the total concentration of TF which was put into the nanoparticles, [TF]residual is the concentration titrated from the supernatant, taking into account the background signal from a blank experiment representing particles dispersion without drug. 2.5. Evaluation of colloidal stability. The colloidal stabilities of 0.05 % (w/v) PECs dispersions were monitored over time at +4°C, 20°C, or 37°C. The average particles diameters and the zeta potentials were measured with the Malvern Nanosizer SZ at 25°C. 2.6. Protein sorption onto colloidal PECs. Various protein solutions (anti-OVA (Clone A1), anti-α4β7 integrin (Clone 7G3 and 1E1)), in twice isotonic PBS buffer, were mixed with equal volumes of water dispersions of ZnCl2 stabilized CS-ChonS particles. The mixtures were stirred moderately end-over-head for predefined time. Proteins loaded CS-ChonS particles were centrifuged at 28,000g for 10 min. The quantification of unbound protein in the supernatants was achieved by BCA protein assay titration, according to the manufacturer’s instructions, using a calibration curve obtained via serial dilutions in the same experimental buffer. The sorption yields (SY) of proteins were calculated as follows. % = Protein − Protein /Protein × 100 Where [Protein]input is the total concentration of protein which was put into the nanoparticles, [Protein]residual is the concentration titrated from the supernatant, taking into account the background signal from a blank experiment representing particles dispersion without proteins. 2.7. In-vitro HIV virus infection inhibition test. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from three healthy donors and stimulated with phytohemagglutinin (PHA-M, 2 µg/mL) at 37°C in humid atmosphere with 5%CO2 for 72 hours. In 96 well conical bottom plates, 100 µL of stimulated PBMC were distributed at 3×106 cells/mL, 50 µL of different formulations of chitosan-ChonS nanoparticules were added, followed by 50 µL of 92US660 primary HIV-1 strain (10 TCID50) which was diluted to 1/120 in the final concentration. After 24 hours of infection in 5%CO2 at 37°C, the cells were collected after low-speed centrifugation at 1,200 rpm for 5 min and the supernatant were pipetted off, to remove the excess of virus, and cells were resuspended in 200 µL of the culture medium and the washing process was repeated once. After that, cells were incubated for 7 additional days and culture supernatants were harvested after 8 days. The amount of the p24 antigen in the culture supernatants 5



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was determined by a commercial sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody against HIV-1 p24 antigen. The detection of the bound antigen was achieved with a peroxidase-conjugated human anti-p24 polyclonal antibodies conjugate (HIV-1 p24 Antigen Capture Assay, Advanced Bioscience Laboratories, Inc. USA). Cells infection inhibition efficiency was calculated as followed: Infection inhibition efficiency% = 1 − '

A)* + . × 100 A,- +,, +

[A]NPs treated cells is the absorbance of p24 antigen titrated from infected cells treated with the different formulations of nanoparticles at 450nm; [A]positive control cells is the absorbance of p24 antigen titrated from infected, untreated control cells incubated after 8 days. The absorbance of non-infected, untreated cells was used as negative control. 3. RESULTS AND DISCUSSION 3.1. Physico-chemical properties of chitosan and ChonS. After analysis by SEC, the dn/dc of ChonS was 0.1275 ± 0.0013 ml·g-1, and the average Mw of ChonS used was 3.66 ×104 g·mol-1 and polydispersity index Ip of 1.31. To study the influence of charge density and chain length on the formation of polyelectrolyte complexes, chitosans of various degrees of acetylation and molar masses were prepared according to Wu & Delair (Wu & Delair, 2015), which are described in Table 1. Table 1 Physicochemical characteristics of chitosans determined by 1H-NMR (DA) and SEC (weight-average molar mass Mw, number-average molar mass Mn and polydispersity index Ip) Mn ×104 Mw ×104 DA (%) Ip (g·mol-1) (g·mol-1) 4 58.6 ± 0.9 35.9 ± 1.0 1.63 ± 0.05 36.5 ± 0.4 23.6 ± 0.4 1.54 ± 0.03 18.6 ± 0.3 12.3 ± 0.3 1.51 ± 0.04 8.8 ± 0.1 6.0 ± 0.2 1.49 ± 0.05 2.1 ± 0.1 1.6 ± 0.1 1.34 ± 0.10 16 61.5 ± 1.0 38.2 ± 1.1 1.61 ± 0.05 36.9 ± 0.5 24.1 ± 0.5 1.53 ± 0.04 21.5 ± 0.3 14.1 ± 0.4 1.52 ± 0.05 11.4 ± 0.2 7.2 ± 0.2 1.59 ± 0.05 4.3 ± 0.1 3.1 ± 0.1 1.40 ± 0.05 28 61.6 ± 0.8 38.4 ± 1.3 1.60 ± 0.06 31.8 ± 0.5 21.8 ± 0.4 1.45 ± 0.04 17.9 ± 0.2 13.0 ± 0.1 1.38 ± 0.02 9.2 ± 0.1 6.3 ± 0.1 1.47 ± 0.04 48 62.2 ± 1.6 38.9 ± 0.9 1.60 ± 0.05 6


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26.5 ± 0.4 14.8 ± 0.2 7.6 ± 0.1 1.5 ± 0.2

15.6 ± 0.5 10.8 ± 0.1 6.4 ± 0.2 1.3 ± 0.2

1.70 ± 0.06 1.37 ± 0.02 1.18 ± 0.04 1.17 ± 0.02

3.2 Formation of polyelectrolyte complexes. According to preceding investigation25; 26, factors impacting the particle formation process were the pH of polymer solutions, the positive to negative charge ratio, the polyelectrolyte concentration. Despite these previous works, to the best of our knowledge no systematic investigation on the impact of chitosan molar mass and DA on the formation of nano-PECs was clearly reported. Moreover, the stability issue of the colloidal PECs in physiological conditions has never been addressed before this work though it is a major parameter for further applications. 3.2.1. Degree of polymerization of polyelectrolytes Chitosans of low, medium, high molar mass with DAs ranging from 4% to 48% were complexed with ChonS. A same concentration of 1×10-3 g·mL-1 was used for both chitosan and ChonS in this experiment. The definition of the particle formation domain as a function of Mw and DA of chitosan was achieved by DLS and naked-eye observation. From the particle formation diagram, Figure 1a, colloidal systems were obtained for chitosan Mw up to ca. 3.5×105 g·mol-1 for DAs 4% and 16%. This upper limit decreased when DA increased up to 48%. This behaviour can be linked to the polyelectrolyte character of chitosan, as defined by Domard and collaborators24; 27. For a DA range of 0-25% chitosan behaves as a polyelectrolyte where electrostatic interactions have a great impact on the conformation of the macromolecules. For the 25-50% DA range, hydrophilic and hydrophobic interactions are balanced. 24; 27 24; 27 So, the role of the DA of chitosan on the decrease in the upper limit in Mw of the colloidal domain in Figure 1a can be understood as follows. For highly charged samples (low DA) the neutralisation of the polycation by ChonS resulted in a condensation of the macromolecules into nanoparticles. For higher DAs, the charge density of the chitosan chains was reduced, hence, the charge neutralization with ChonS occurred by interacting with several polycations into aggregates. The particle formation diagram of Figure 1a is quite different from that obtained with chitosan and hyaluronan (HYA).15 With HYA, the upper chitosan molar mass range of the colloidal domain of was limited to 1~2.5 ×105 g·mol-1 and increased with DA. These differences cannot be attributed to variations of the degrees of polymerization(DP) of the polyanions which were closely related in molar masses (HYA Mw=5.9×104 g·mol-1 and 3.9×104 g·mol-1; ChonS Mw=3.66×104 g·mol-1). Thus structural differences between the polyanions should be taken into consideration. Though both polymers have a disaccharide as repeat unit, HYA only bears one carboxylate moiety whereas ChonS has one carboxylate group and two sulphate moieties per repeat unit. So HYA has a much lower charge density than ChonS whose neutralization with chitosan probably took place via a zip process preventing 7



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interactions with other molecules. For HYA due to its low charge density, the charge neutralization with chitosan required several chains to react with each other, leading to aggregates even at low DPs. When DA increases, the charge density of chitosan was reduced and so was the probability of cross-linking. Hence, the increase in Mw observed with DA in Figure 1b. The impact of chitosan Mw on particle size was scrutinized in more details. As shown in Figure S1 of supporting information, an increase in chitosan Mw resulted in an increase in the particle size, which was consistent with other previous results.15; 28

Figure 1 (a) CS-ChonS and (b) CS-HYA based nanoparticles formation available diagram in terms of the molar mass of chitosan as a function of DA at polymer concentration of 0.1% and pH 4: ( ) aggregates; ( ) colloidal particles. 3.2.2 Effect of the charge ratio (R) and DA of chitosan The charge molar ratio represents the excess of one polymer versus the other, the higher the n+/n- ratio, the greater the excess in chitosan. As seen in Figure 2, particle sizes increased with increasing R for all the DA of chitosan. The increase in size with R can be attributed to the presence of increasing excess of non-complexed polymer segments at the interface and/or also to a less dense complex core containing more uncomplexed ammonium groups. The impact of DA of chitosan on particle size is quite clear: the higher the DA, the higher the particle mean diameter, which is related to the increase of hydrodynamic radius of chitosan, and the influence of polymer-water interaction with increasing DA. These factors will be discussed in the next section. Moreover, with increasing DA to maintain the charge ratio constant the amount of chitosan increased. Hence, the observed effect may also be attributed to an increased amount of material per particle.

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Figure 2 Influence of the charge ratio R on (a) average sizes and (b) polydispersity index (PDI) of PECs particles. Conditions: ChonS (0.1% (w/v), Mw 3.66×104 g·mol-1), CS: 0.1% (w/v), pH 5: ( ) CS (DA 4%, Mw 8.86×104 g·mol-1); ( ) CS (DA 16%, Mw 1.14×105 g·mol-1); ( ) CS (DA 28%, Mw 9.20×104 g·mol-1); ( ) CS (DA 48%, Mw 1.48×105 g·mol-1) 3.2.3 Influence of the pH of polymer solutions on particles sizes The impact on particle sizes of the pH of the complexation medium was investigated for DAs 4, 16, 28 and 48% and for increasing R ratios. pH values were 4.0, 4.5, 5.0 and 5.5 in order to ensure protonation of chitosan. As seen in Figure 3, an increase in the chitosan solution pH led to a decrease in the particle hydrodynamic radii at most ratios. On increasing the pH, the concomitant reduction of the charge density of chitosan will induce a decrease in the macromolecule stiffness, as a result of the decreased repulsive electrostatic interactions. Therefore, the conformational adaptation required for the charge matching should be easier, thus leading to more compact complexes. However, it is worth noting that at pH 5.5, the colloidal PECs were prone to flocculation, related to the low charge density of the chitosan amino groups. At low charge density, the decrease in intensity of intramolecular repulsive electrostatic forces induced the collapse of the polysaccharide chains and, concomitantly the reduction in colloidal stability.

Figure 3 Influence of the pH of the CS solutions on (a) average sizes and (b) polydispersity index (PDI) of PECs particles. Conditions: ChonS (0.1% (w/v), Mw3.66×104 g·mol-1), CS: 0.1% (w/v): ( ) CS (DA4%, Mw8.86104 g·mol-1); ( ) CS (DA 16%, Mw1.14105 g·mol-1); ( ) CS 9



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(DA 28%, Mw9.20104 g·mol-1); ( )CS (DA 48%, Mw1.48105 g·mol-1) At this stage, it is worth commenting the apparently contradictory results, concerning the opposite effects of the increase of DA or pH of the complexation media on particle size. If we consider that both the increase in DA and in pH can be regarded as a decrease in the charge density of chitosan, in the case of the DA increase, particle size increased and, for the pH increase, particle size decreased. This difference can be attributed to the steric hindrance induced by the N-acetyl groups preventing an optimal conformational adaptation to yield compact complexes. Indeed, Sorlier et al. showed that, at constant pH, the hydrodynamic radius Rh, measured by light scattering, increased with DA 29. Moreover, one has to take into account that the solubility of chitosan in water decreases with pH as shown by Schatz et al. based on the decrease of the second virial coefficient with increasing pH till the macromolecules precipitated out 30. Interestingly, the critical pH at which chitosan chains precipitated out increased from 6.1 to 7.45 when DA increased from 1% through 50%. This illustrates that an increase in DA cannot be considered only as a decrease in charge density because the presence of the acetamide groups in the polymer chain influence the polymer-water interactions. 3.2.4 Influence of the polyelectrolyte concentration on particles sizes The impact of the polymer concentration on the particle formation was assessed using polyelectrolyte solutions of concentrations ranging from 0.1% to 1%. Two chitosans were used (DA 4%, Mw 8.86104 g·mol-1) and (DA 48%, Mw7.6104 g·mol-1), and complexed with ChonS at identical concentrations. Polyelectrolyte concentrations higher than 0.5%(w/v) lead to aggregates (data not shown). For concentrations of 0.5%(w/v) and lower, an increase in the polyelectrolyte concentration led to PECs particles with high average diameters (Figure 4). The upper polyelectrolyte concentration allowing the formation of colloidal PECs was 0.3% for a chitosan at DA48%, and 0.5% for a chitosan at DA4%.

Figure 4 Influence of the concentration of the both polyelectrolytes (%, w/v) on (a) average sizes and (b) polydispersity index (PDI) of PECs particles at R(n+/n-)=2. Conditions: ChonS (0.1% (w/v), Mw3.66×104 g·mol-1): ( ) CS (DA 4%, Mw 8.86104 g·mol-1, pH4); ( ) CS (DA 48%, Mw1.48105 g·mol-1, pH4).

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3.2.5 Optimal conditions of the particles formation From the previous results on the production of colloidal PECs, the impact of each studied parameter on particle size and polydispersity are summarized in Table 2. The formation of small and isodisperse particles required a low molar mass of chitosan, relatively low n+/n- ratio (1.5~2), relatively higher pH of chitosan solution (pH~5), and low concentration of polyelectrolytes (0.1%, w/v). Under these conditions, colloidal PECs with 0.1% (w/v) of chitosan (DA=48%, Mw =1.50104 g·mol-1, pH 5) and of ChonS (Mw 3.66×104 g·mol-1) Rn+/n-=2 displayed an average diameter 140 ± 0.3 nm and a polydispersity index about 0.09 ± 0.02. Table 2 Effect of an increase in various parameters on the properties of PEC particles with an excess of chitosan: (+) increase, (-) decrease, (×) no significant effect Mw of chitosan Size Polydispersity index

DA of chitosan

Ratio (n+/n-)

pH of chitosan

Conc. of polyelectrolytes

+

+

+

-

+

+

×

+

-

+

3.2.6 Morphology The morphology of the chitosan-ChonS complexes formed in the optimal conditions was examined by TEM (Figure 5). The particles showed a dense, spherical structure with particles size around 100-140 nm, which was consistent with the result by DLS.

Figure 5 Transmission electron micrographs of chitosan/ChonS nanoPECs: chitosan (DA=48%, Mw =1.5104 g·mol-1), ChonS (Mw 3.60×104 g·mol-1), Rn+/n-=2. (a) morphology of nanoparticles in the dry state, (b) is a higher magnification of (a). 3.3 Formation and colloidal stability of CS-ChonS nanoparticles. PECs from the optimal conditions were stable in water for at least one month at room temperature. However, in physiological conditions (phosphate buffer saline, PBS), the nanocomplexes were disrupted due to the deleterious effects of ionic strength and pH of 7.4, with partly solubilized and with partly aggregated colloidal dispersion. Irreversibly aggregation was also observed for PECs obtained with chitosan at DA 4%.These results were not anticipated on the basis of our results with 11



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another sulphated polysaccharide, namely dextran sulphate.13 To stabilize the chitosan-ChonS colloidal PECs, the cross-linking of the colloidal particles with zinc (II) was attempted, similarly to the chitosan-HYA nanoPECs.15 Zinc(II), as an electron deficient ion, can coordinate with electron rich amines and hydroxyl groups of one or more polysaccharide chains. Moreover, zinc cation can also co-react with the negatively charged ChonS by electrostatic attraction. These two processes jointly ensure the cross-linking of the polysaccharide responsible for the stabilization of the colloids (see Figure S2 of supporting information). In this study, zinc chloride (ZnCl2) or zinc sulfate (ZnSO4) was selected as stabilizer to be added during the formation of the complexes. It is noted that the optimized nanoPECs (using low Mw of chitosan, Mw =1.5104 g·mol-1) with ZnCl2 or ZnSO4 stabilization precipitated in PBS, whereas nanoPECs formed with chitosans of higher Mw featured a better stability in PBS. The observed flocculation in PBS of colloidal PECs obtained from close Mw of chitosan and ChonS, or from close DP (chitosan: Mw =1.5104 g·mol-1, DP=83, ChonS Mw=3.60×104 g·mol-1, DP=60), was attributed to the fact that low Mw of polyanion complexed with polycation in a flat conformation31 leading to a mostly neutralized interface unable to ensure the stabilization of the colloids. Whereas the polyelectrolytes largely differing in Mw or DP (by using chitosan Mw=1.48105g·mol-1), lead to more stable PECs in PBS. This is consistent with the guest-host model in which the polymer in excess (host) displays a higher DP than the polymer in default (guest)11. To compare the stability effect of zinc ions on the chitosan-HYA system, the same Mw of chitosan as in chitosan-HYA system of 1.48105g·mol-1 with DA 48% was selected, and 1.0 mM of Zn(II) was added in the complex system according to the turbidity test result of nanocomplexes in water and PBS media. The stability evaluation of the nanoparticles dispersion, stabilized by ZnCl2 or ZnSO4, was carried out at three temperatures. One volume of dispersion was mixed with the same volume of PBS (2×) in order to monitor, by DLS, the particle size evolution in physiological salt and pH conditions (1× PBS). In figure 6, the evolution with time of the particle mean diameter, observed by quasi elastic light scattering, was due the formation of aggregates in the dispersion, indicating the onset of the aggregation process. Figure 6 reports the changes in nanoPECs particles sizes and zeta potentials, for 0.05% solid content, over 35 days of storage at the temperatures of 4, 20 and 37 °C. The average PEC diameter remained constant on storing in water, irrespective of the storing temperature and the nature of the stabilizing salt. In PBS, colloidal PECs particles stabilized with ZnCl2 or ZnSO4 remained stable for approximately 3 weeks at 4 °C, while without zinc, particles destabilized within three days. At 20 and 37 °C, zinc-stabilized colloids remained stable in PBS for two and one week, respectively. These results confirm the efficiency of the stabilizing strategy, allowing colloidal PECs to be used in physiological environment without any chemical cross-linker nor stabilizers. Particles stabilized with ZnSO4 showed slightly lower in particle diameter than those obtained with ZnCl2 (compare a with b in Figure 6) and, though the differences are small, we regularly observed this trend over a large number of batches. This size 12


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contraction can arise from the fact that sulfate ions can also complex with part of amino groups of chitosan 32, hence condensing the chitosan outer shell. This complexation also neutralized the charges of chitosan, which also explains the lower zeta potentials of PECs stabilized by ZnSO4 than that by ZnCl2 counterpart. The mode of stabilization by zinc ions can be attributed to the formation of co-ordinate bonds between electron rich atoms, like nitrogen or oxygen, and the free bonding levels of zinc, ensuring thus a cross-linking of the polysaccharide chains which provided the stabilization. This binding may take place at the nitrogen atoms, and that would account for the fact that the zeta potentials measured for the stabilized particles were systematically lower than without stabilization. Worth noting that the charge density of ChonS is higher than that of HYA, hence the stabilizing salt dosage in this system is lower than the preceding chitosan-HYA system.15 Besides, the zinc(II) stabilization in this system was less efficient than chitosan-HYA system in the stability in PBS whatever the three storing temperatures.

Figure 6 Stability studies of PECs dispersions (0.05%, w/v) in H2O and PBS at temperatures of 4, 20 and 37 °C: Average particle size of PECs (a) stabilized by ZnCl2, (b) stabilized by ZnSO4, (c) Zeta potential of PECs in H2O (I. stabilized by ZnCl2; II. stabilized by ZnSO4 (mean ±S. D., n = 3). To achieve an oral delivery route objective, the colloidal stability was also investigated by storing the particle dispersions at pH 1.2 of hydrochloric acid aqueous solution at 37 °C, to mimic a gastric environment. As reported in Figure S3 of Supporting Information, the average particle size and the zeta potential showed limited variation within 6 days, which is much longer than stomach transit. As a 13



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matter of fact, the residual charge on chondroitin sulphate is very low and may not be sufficient to maintain the integrity of the colloids, suggesting that other interactions are involved like hydrogen bonding for instance. A similar phenomenon had already been observed by Costalat et al.33 when they obtained chitosan/dextran sulphate nanocomplexes stable at pH 7.4. At this pH the ionization of chitosan was very low too. This good stability of nanoPECs in various media prompted the further investigation of these colloids for biomedical applications. 3.4 Evaluation of the drug encapsulation efficiency. TF was selected as a model drug in this feasibility study because it is part of the whole HAART protocol. TF loaded CS-ChonS nanoparticles, with varying drug concentration from 5 to 40 µg·mL-1 were obtained for PECs solid content of 0.1% (w/v). In preliminary experiments, involving a model molecule adenosine 5’-monophosphate monohydrate, no deleterious impact on the encapsulation efficiency was observed by the presence of the zinc ion (data not shown). Table 3 Effects of different TF concentration on the physicochemical properties of the zinc stabilized CS-ChonS nanoparticles with polyelectrolyte concentration of 0.1% (w/v) (mean ± SD, n = 3) Theoretical final conc. of TF(µg·mL-1)

Charge ratio(NH3+/PO3-)

EE/% -

TF loaded, (µg·mL-1) (particles)

Particles size (nm)

PDI

-

307 ± 4.1

0.2 ± 0.02

Zeta potential (mV)

0

-

5

57

93 ± 5.3

4.6

305 ±7.6

0.2 ± 0.04

36.6 ± 0.7 36.2 ± 0.8

10

28

52 ± 4.1

5.2

301 ± 3.7

0.2 ± 0.01

36.0 ± 0.9

20

14

28 ± 1.8

5.6

315 ± 7.1

0.2 ± 0.03

35.4 ± 0.2

40

7

19 ± 3.2

7.6

299 ± 7.2

0.2 ± 0.02

31.1 ± 0.7

As shown in Table 3, the encapsulation efficiency (EE) decreased when the initial TF concentration in the mixture increased, but the loading capacity increased. The association of TF to chitosan nanoparticles was mediated by an electrostatic interaction between the phosphate moiety of TF and the amino groups of chitosan. The particle sizes and zeta potentials of PECs nanoparticles slightly decreased as the concentration of TF increased from 5 to 40 µg·mL-1. The size compaction could be attributed to a decrease in repulsive electrostatic charges within the nano-complexes due to the consumption of the excess of un-complexed ammonium groups by increasing amount of incorporated TF molecules. The same argument can be used to explain the reduction in zeta potential. Nevertheless, one should also note that increasing the volume of TF solution in the mixture of oppositely charged polymers led to a slight increase in the pH, which may also partly impact the particle sizes and zeta potentials. 14


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3.5 Antibody sorption onto colloidal PECs. Multi-functionalized nanoPECs, especially those surface-decorated by targeting species allow the drug release to take place at specific locations. Immunoglobulins type A, secreted at the mucosa level, are well tailored for the delivery of drugs to these tissues. In this study, two humanized chimeric IgAs, anti-ovalbumin (anti-OVA), and anti-α4β7 IgA (with two different types: Clone 1E1 and Clone7G3) were selected. To maintain their colloidal character in PBS, the nanoPECs were stabilized by ZnCl2 during the formation process. The antibody sorption onto PECs was investigated in PBS (pH 7.4) to mimic a physiological environment, and the sorption kinetics are shown in Figure 7. The adsorption kinetics differed according to the investigated protein. Examining the early stage of the processes, inserts of Figure 7, the adsorption behaviour of anti-OVA IgA and anti-α4β7 IgA clone C1E1 (inserts Figure 7a and 7c) are similar, characterized by a fast initial step (after one hour of contact) and a second step with a slower adsorption rate up to completion. The difference between Figure 7a and 7c is that the fast adsorption step of the former was of higher amplitude than the later. Clone C7G3 of anti-α4β7 IgA displayed a different sorption profile (insert Figure 7b) characterized by a very low amount of IgA associated at the onset of the process. In more details, considering the time point at 1 hour, the adsorption efficiencies of anti-OVA IgAs was of 85, 50 and 25% for the three initial anti-OVA protein concentrations (insert Figure 7a), whereas 50, 45 and 40% for clone 1E1 IgA (insert Figure 7c) and less than 10% of anti-α4β7 clone 7G3 IgA (insert Figure 7b). The sorption plateau reached 90%-100% of the initial protein input and the time to completion increased with the protein input and depended on the nature of the IgA. Interestingly, the saturation of the available binding sites of the colloids was not observed for the investigated protein concentrations, affording maximum amounts of assembled anti-OVA and anti-α4β7 IgA achieved around 80mg protein/g nanoparticles. So, to summarize this important step of the elaboration of the targeted nanodelivery systems, the binding capacity of the carriers was very high probably as a result of the diffusion of the protein within the soft shell of the colloids, as already observed by Polexe et al. with chitosan/hyaluronan nanocomplexes34. The nature of the binding protein mainly impacted the assembly kinetics at the early stages of the process, but not on the longer term yielding 90-100% assembly.

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Figure 7 Sorption kinetics of IgAs on CS-ChonS particles with solid content 0.05% (w/v) in PBS medium: (a) mixed anti-OVA/CS-ChonS NPs; (b) C7G3 anti-α4β7 IgA/ CS-ChonS NPs; (c) C1E1 anti-α4β7 IgA/ CS-ChonS NPs. ( ) Ig A 10µg·mL-1, ( ) Ig A 20µg·mL-1, ( ) Ig A 40µg·mL-1. (mean ±S. D., n = 3). 3.6 In vitro assessment of IgA recognition properties. The recognition capability of the immunoglobulins immobilized on chitosan-ChonS nanoparticles was evaluated by a specific solid phase enzyme-linked immunosorbent assay (ELISA). As shown in Figure 8, antibody bio-recognition properties were calculated by the ratio of the absorbance measured with anti-OVA or anti-α4β7 IgA immobilized onto particles vs the same concentration of free antibodies, taking into account the background signal from a blank experiment representing particles dispersion without IgA. After one day, the antibodies had retained around 95% of their activities. After storing for one week in PBS at 4°C at particles solid content of 0.05% and IgA concentration of 40 µg·mL-1, 84% and 83% of anti-OVA and anti-α4β7 IgA bioactivities respectively were maintained.

Figure 8 Relative bioactivity of anti-OVA (orange bars) and anti-α4β7 IgAs (green bars) adsorbed onto chitosan-ChonS nanoparticles in PBS. Each bar corresponds to the mean plus the standard error of the mean. 3.7 In vitro cell viability/cytotoxicity studies after exposure of PBMC cells to nanoparticles. 16


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WST-8 assay was performed to quantify the cell viability after exposure of human peripheral blood mononuclear cells (PBMCs) to chitosan-ChonS nanoparticles. The cells were treated with varied concentration of blank and IgA adsorbed chitosan-ChonS nanoparticles with particles concentration varied from 25 to 500 µg·mL-1. All the components (including chitosan, ChonS, Zn2+ with original concentration of 1.0mM) were chosen as control. As illustrated in Figure 9, all the components showed very limited toxicity to PBMCs all through the investigated concentrations up to 500 µg·mL-1. The addition of 1.0 mM of zinc ions caused slight promotion of the cell viability. Interestingly, equivalent high survival rate was observed with chitosan-ChonS blank nanoPECs as that of all of the components up to 500 µg·mL-1, and the sorption of anti-α4β7 IgA on the chitosan-ChonS nanoparticles had no major alteration of the cell viability. 100 Cell Viability (% of Control)

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CS ChonS

80

Zn2+ CS-ChonS NPs

60

IgA/CS-ChonS NPs 40 20 0 25

50

100

125

250

500

Concentration of Solution or Nanoparticles (μg/mL)

Figure 9 Cell viability of chitosan, ChonS, zinc (II), chitosan-ChonS blank nanoparticles, and anti-α4β7 IgA adsorbed chitosan-ChonS nanoparticles after incubation for 24 h (Cell numbers were counted using a Cell Counting Kit-8). 3.8 Interaction of chitosan-HYA nanoparticles with PBMCs. The interaction of chitosan-ChonS nanoPECs with PBMCs was studied by confocal microscopy. FITC-labeled chitosan-ChonS nanoparticles appeared as nanometer-sized, evenly distributed green fluorescent dots inside the cells (Figure 10A). The bright green dots observed in Figure 10A and C should be ascribed to nanoparticle aggregates (arrow shown). In Figure 10B the actin filaments were stained in red and Figure 10C shows Figure 10A and B merged, proving that the nanoparticles were internalized within the cells. By taking with the confocal microscope, six serial images at 0.8-µm intervals along the z-axis, from the top to the bottom of the cell monolayer, we confirmed the intracellular presence of the chitosan-ChonS nanoparticles (Figure 11). The extent of cell-internalized chitosan-ChonS nanoPECs increased as the cytoplasmic area increased progressively along the z-axis.

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Figure 10 Confocal laser scanning imaging of PBMCs incubated with FITC-labelling chitosan-ChonS nanoPECs for 4 h. (A): green fluorescence arising from FITC-labelled chitosan-ChonS nanoparticles internalized in PBMCs; (B): rhodamine phalloidin stained actin filaments of PBMCs; (C): merged image of A and B (Scale bar: 20 µm).

Figure 11 Confocal serial images along the z-axis of PBMCs. Cells were exposed for 4h to 0.5 mg·mL-1 FITC-labelled chitosan-ChonS nanoPECs. This is a representative gallery of six serial micrographs showing the green fluorescence at 0.8-µm intervals along the z-axis, from the top to the bottom of the cell monolayer. Green fluorescent images were merged with images of red rhodamine phalloidin stained actin filaments. Scale bar: 20 µm. 3.9 In-vitro HIV virus infection inhibition test. The objective of this section is to demonstrate the capability of chitosan-ChonS nanoparticles to deliver a model drug under its active form. In accordance with our 18


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global objective, we selected the inhibition of the HIV-1 infection of human peripheral blood mononuclear cells (PBMCs) by chitosan-ChonS nanoparticles as a model of application. PBMCs were infected with 92US660 primary HIV-1 strain (10 TCID50) and then treated with the TF/chitosan-ChonS nanoPECs formulations with varying amounts of loaded TF and with or without anti-α4β7 IgA at their interface. The inhibition of viral replication was determined by measuring the HIV-1 capsid p24 production, by specific ELISA in the cell culture supernatant after 8 days of incubation, as shown in Figure 12. As compared with the chitosan control, both the chitosan+zinc(II) mixture and the chitosan-ChonS blank nanoparticles at a concentration of 250µg·mL-1 exhibited up to 100% infection inhibition, which could be attributed to the presence of zinc ions as reported by Haraguchi et al.35. Free TF aqueous solution control showed limited infection inhibition potency, with an IC50 (50% inhibitory concentration) of 4.35 µmol·L-1 (1.25 µg·mL-1, Figure 12). TF loaded chitosan-ChonS nanoPECs exhibited dose-dependent inhibition activities, at nanoparticles concentration of 100 µg·mL-1, with decreased IC50 value to 1.95µmol·L-1. The inhibition of infection increased with the amount of loaded TF (see the loading TF in Table 3 and the effective TF concentration at different particles concentrations can be seen in Table S1 in Supporting Information). This improved anti-viral potency can be regarded as the consequence of the release of TF from nanoparticles. Compared with the untargeted particles at the concentration of 100 µg·mL-1, the presence of the anti-α4β7 IgA at the interface of the TF loaded particles (125 µg·mL-1, N° 8, 9, 10 and 11) has no major deleterious effect on the availability of the drug and even slightly improved the inhibition efficacy. The dose dependence is not monotonous in the presence of the targeting IgAs, but more experimental work would be required to get a better understanding of the phenomenon.

Figure 12 HIV-1 Infection inhibition of PBMCs by TF-loaded chitosan-ChonS nanoparticles. Cells were cultured in the presence of the HIV-1 virus and exposed to various concentrations of nanoPECs (orange bar: 250 µg·mL-1, green bar: 100 µg·mL-1, blue bar: 125 µg·mL-1, and purple bar: 50 µg·mL-1. TF control represents the inhibition efficiency of free TF aqueous solution with respective concentration of 0.312, 0.625 and 1.25 µg·mL-1). On day 8 of cell culture, virus production was monitored by measuring the HIV-1 p24 content in the cell culture supernatant by 19



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ELISA. The results are presented as percent inhibition efficiency of HIV antigen compared with that of infected, untreated controls. 4. CONCLUSIONS Our work aimed at using a safe and simple to implement assembly process to manufacture efficient drug delivery systems, within a long-term objective of developing a strategy to efficiently address the elimination of the HIV-1 virus from its natural reservoirs. We showed that chitosan-ChonS nanoPECs were easily elaborated by a one-shot addition of default amounts of ChonS to chitosan of varied molar masses and degrees of acetylation. The mean diameters of the colloidal PECs proved dependent on the molar mass (Mw) of chitosan and charge ratio R (n+/n-) between chitosan and ChonS. Relative low Mw of chitosan (Mw 1.5×104 g·mol-1) and low charge ratio (R 1.5~2) favoured the formation of small and relatively isodispersed particles. In physiological salt and pH conditions the colloidal stability was greatly improved via interactions with zinc (II) ions during the PECs formulation process. Tenofovir (TF), an HIV-1 reverse transcriptase inhibitor, was successfully encapsulated, with a limited impact on the alteration of their colloidal stability. For targeting purposes, the colloids could be efficiently surface decorated by the anti-α4β7 IgA, whose recognition ability was preserved up to 83% over one week of storage in PBS at 4 °C. Once we obtained nanocarriers loaded with an anti-HIV drug, coated with a targeting specie corresponding to our global strategy and with an established colloidal stability in physiological conditions, we demonstrated that the drug was efficiently released in vitro. Three very positive results were obtained i) each of the tested formulations proved non cytotoxic; ii) the particles had an intrinsic infection inhibition capability due to the presence of low amounts of zinc in the formulations; iii) on dilution of the formulation, the inhibition capability of the nano-carriers was dose dependent with the amount of tenofovir encapsulated. In more details, a high survival rate of human peripheral blood mononuclear cells (PBMCs) was obtained with chitosan-ChonS nanoPECs decorated or not with the anti-α4β7 IgA. The particle internalization within PBMCs was evidenced by confocal microscopy, using fluorescent nanoPECs. In-vitro, the infection of PBMC by the HIV-1 virus was inhibited by the adjunction of chitosan-ChonS colloids. This effect could be attributed to the presence of Zn(II) ions in the formulation, but upon dilution, the inhibition was greatly reduced and a TF dose dependent effect was observed with the TF loaded colloidal PECs. In comparison to free TF in solution, a clear beneficial effect was observed from the use of a nanoparticulate formulation. These important preliminary result point out the high potential of these new carriers and open up new perspectives of applications in the fight against viral infections and in particular against HIV.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge via the Internet. 20


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AUTHOR INFORMATION Corresponding Author * E-mail address: [email protected]. Tel.: +33 (0)4 72 44 85 87; Fax: +33 (0)4 72 43 85 87 Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors would like to acknowledge Agnès Crépet and also the Centre for the Characterization of Polymers by Liquid Chromatography of the Institut de Chimie de Lyon for their expertise and assistance in molar mass determination by SEC measurements. We are also in debt to Pierre Alcouffe and the Centre Technologique des Microstructures-Université Claude Bernard Lyon 1 for their kind assistance in transmission electron microscopy and confocal microscopy characterizations. This work was financed by the PECSDDeli ANR project. D.Wu is grateful to China Scholarship Council (CSC) for providing a doctoral scholarship.

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Graphical abstract of chitosan-chondroitin sulfate nanocomplexes for HIV-1 infection inhibition application 84x63mm (300 x 300 DPI)


Zinc-Stabilized Chitosan-Chondroitin Sulfate ... - ACS Publications

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