Synthesis and Characterization of PEGylated and Fluorinated

Article pubs.acs.org/Biomac

Synthesis and Characterization of PEGylated and Fluorinated Chitosans: Application to the Synthesis of Targeted Nanoparticles for Drug Delivery Yamina Belabassi,† Juliette Moreau,*,† Virginia Gheran,‡ Celine Henoumont,§ Anthony Robert,† Maité Callewaert,† Guillaume Rigaux,† Cyril Cadiou,† Luce Vander Elst,§,∥ Sophie Laurent,§,∥ Robert N. Muller,§,∥ Anca Dinischiotu,‡ Sorina N. Voicu,‡,⊥ and Françoise Chuburu*,† †

Institut de Chimie Moléculaire de Reims, CNRS UMR 7312, University of Reims Champagne-Ardenne URCA, 51685 Cedex 2 Reims, France ‡ Faculty of Biology, Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest 030018, Romania § NMR and Molecular Imaging Laboratory, University of Mons UMons, B-7000 Mons, Belgique ∥ Center for Microscopy and Molecular Imaging, Rue Adrienne Bolland 8,B-6041 Charleroi, Belgium ⊥ Faculty of Pharmacy, Department of Pharmacy, Titu Maiorescu University, Bucharest 040441, Romania S Supporting Information *

ABSTRACT: To synthesize chitosan nanoparticles (CS NPs), ionic gelation is a very attractive method. It relies on the spontaneous supramolecular assembly of cationic CS with anionic compounds, which leads to nanohydrogels. To extend ionic gelation to functionalized CS, the assessment of CS degree of substitution (DSCS) is a key step. In this paper, we have developed a hyphenated strategy for functionalized CS characterization, based upon 1H, DOSY and, when relevant, 1D diffusion-filtered 19F NMR spectroscopies. For that, we have synthesized two series of water-soluble CS via amidation of CS amino groups with mPEG2000-COOH or fluorinated synthons (TFB-COOH). The aforementioned NMR techniques helped to discriminate between ungrafted and grafted synthons and finally to determine DSCS. According to DSCS values, the selection of CS−mPEG2000 or CS−TFB copolymers can be made to obtain, in the presence of hyaluronic acid (HA) and tripolyphosphate (TPP), CS−mPEG2000−TPP/HA or CS−TFB−TPP/HA nanohydrogels suitable for drug delivery.

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INTRODUCTION Development of nanocarriers is of uppermost importance for the administration of therapeutic or imaging agents and for theranostic applications.1 They can reduce the unwanted toxic side effects of active substances, prolong their circulation time, and reduce their uptake by the reticuloendothelial system (RES) leading to an increase of drug therapeutic index.2 Materials used for preparing nanoparticles (NPs) for drug delivery must be at least biocompatible and biodegradable. For these reasons, polysaccharides3 are of huge interest, and among them chitosan (CS) is probably one of the most used for biomedical applications.4−12 CS is a linear, random copolymer constituted of β-(1−4)-linked D-glucosamine and N-acetyl-Dglucosamine units and obtained from a partial deacetylation of chitin.13,14 CS is known for its biocompatibility, biodegradability, and low toxicity.9,15 Due to the presence of primary amino groups along the CS backbone, CS exhibits a cationic character (CS is a weak base (pKa = 6.4)) that confers properties such as pH-dependent behavior, muco-adhesiveness, and ability to open epithelial tight junctions.8,9,11 For these © 2017 American Chemical Society

reasons, CS and its derivatives are frequently used to design drug delivery systems.6−12,16 As the only natural positive polysaccharide, CS is also especially advantageous to form stable complexes with anionic compounds in the presence of cross-linkers such as polyphosphates.16 The corresponding socalled ionic gelation process offers the advantage of producing spontaneously ionically cross-linked NPs under mild conditions, that is, without involving high temperature or harmful organic solvents since as far as CS is concerned water is the solvent.17,18 The corresponding hydrogel NPs (also called nanogels) exhibit a high water absorption ability, meaning that CS NPs can be used for hydrophilic molecule entrapment.19 However, in basic or neutral conditions, CS suffers from poor water solubility so additional modification with small molecules or polymers is needed. For that, CS functional groups available for chemical modification include generally hydroxyl groups Received: May 11, 2017 Revised: July 18, 2017 Published: August 4, 2017 2756

DOI: 10.1021/acs.biomac.7b00668 Biomacromolecules 2017, 18, 2756−2766

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Biomacromolecules Scheme 1. Synthesis of (i) CS−mPEG2000 and (ii) CS−TFB

fluorination was attempted on the basis of the strategy developed for PEG moieties. Corresponding fluorinated CS NPs were finally synthesized by ionic gelation and fully characterized from morphological and toxicological points of view.

(primary or secondary) as well as the primary amino group of the deacetylated CS units. The higher reactivity of the electronic lone pair of the CS primary amino group at C-2 (Scheme 1) in comparison with that of hydroxyl groups at C-6 and C-3 results in the specific involvement of the amino groups in most chemical modifications.14,20 Amidation is one of the most attractive techniques to functionalize the CS amino group. Among all the groups used to functionalize CS, poly(ethylene glycol) (PEG) is one of the most popular, not only because it helps to improve CS solubility over a wide pH range (from 1 to 11, depending on the degree of substitution, DS) but also because of its nonionic hydrophilic character, it improves CS pharmacokinetic and pharmacodynamic properties (such as prolonged body-residence time).21−23 Nevertheless, a special attention has to be paid to CS degree of substitution (DSCS). Indeed with a high DSCS, only a few CS positive charges are remaining, which could be a drawback for the development of subsequent electrostatic interactions between functionalized CS and anionic species (for the formation of polyelectrolyte complexes or for ionic gelation for instance). Therefore, CS functionalization has to be carefully characterized.24 A wide variability is reported for the DSCS of PEGylated-CS copolymers (0.1% < DSCS < 93%), according the protocols and methods used for DSCS determination20 and even when similar initial (mPEG-COOH/NH2 CS) molar ratios are used, DSCS fluctuating between 0.56%25 and 5.4%26 can be reported. These discrepancies show that a combination of techniques is required to characterize CS functionalization, especially when no specific marker of the amide bond can be unambiguously identified. In this context, we wish to reinvestigate CS PEGylation by amidation with mPEG2000-COOH using DOSY experiments to determine DSCS. Actually, DOSY experiments helped to discriminate between the free PEG chains and the grafted ones because diffusion coefficients of PEG chains drastically decrease27 when they are grafted to a CS backbone. Therefore, a series of CS−mPEG2000 conjugates were synthesized in which the level of PEG substitution was systematically varied (by increasing the initial mPEG-COOH/NH2 CS) molar ratio) and quantified by DOSY. To evaluate the effects of CS degree of substitution by PEG moieties on nanoparticle production, CS− mPEG2000 conjugates were involved in nanoparticle synthesis by an ionic gelation process. The corresponding NPs were fully characterized by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), and their toxicity was evaluated in RAW 264.7 cell lines. Our objective was also to design chitosan drug delivery systems for the lymphatic system. As the introduction of fluorinated side chains is known to favor the accumulation in vivo of imaging probes in lymph nodes,28 CS

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EXPERIMENTAL SECTION

Materials. Chitosan (CS, low viscosity, from shrimp shells) was purchased from Sigma-Aldrich. A deacetylation degree (DD) of 86% was determined by 1H NMR spectroscopy according to published procedures.14,29,30 For calculations, CS repetitive unit (rep unit) molecular mass, in which CS DD was taken into account, was considered to be MWin‑avg(CSrep‑unit) = 200 g·mol−1. Hyaluronic acid sodium salt, HA (1000 kDa extracted from Streptococcus equi sp), methoxy-poly(ethylene)glycol (mPEG2000-OH) (Mw = 2000 g·mol−1), succinic anhydride, pyridine, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. 4,4,4-Trifluorobutyric acid (TFB-COOH) and sodium tripolyphosphate (TPP) were purchased from FluoroChem and Acros Organics, respectively. DCl (35 wt % in D2O) and D2O were purchased from Sigma-Aldrich and Euriso-top, respectively. Fetal bovine serum (FBS), heat inactivated, was purchased from Gibco by Life Technologies (New Zealand), Dulbecco’s modified Eagle’s medium (DMEM) was from Gibco (Invitrogen, Grand Island, N.Y., USA), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and antibiotics (penicillin, streptomycin, and amphotericin B) were provided by SigmaAldrich (St. Louis, MO, USA). All products were used as received, without further purification. Sterile water for injections (Laboratoire Aguettant, Lyon, France) was systematically used for polymer, nanoparticle preparations, and analyses. Polymers and copolymers (mPEG2000-COOH, CS−mPEG2000, and CS−TFB) were characterized by means of FTIR (Nicolet IS 5 spectrometer equipped with an ATR ID 5module), 1H and 19F NMR spectroscopies (Bruker 250 DPX (1H − 250 MHz), and Bruker Avance III (1H, 500 MHz; 19F, 470.6 MHz) spectrometers) at 318 K with D2O/DCl (700/1, v/v) as the solvent. Thermogravimetric analysis (TGA) was performed using a thermogravimetric analyzer (TG209 F3, Netzsch) at a heating rate of 10 °C/min from 30 to 600 °C. Ultrafiltration experiments were realized with an Allegra X-30 Centrifuge (Beckman-Coulter) (4500 rpm, between 45 and 60 min, at room temperature). The diffusion coefficients of different materials (mPEG2000-COOH, CS−mPEG2000, CS−TFB, and CS) were determined by DOSY experiments (diffusion ordered spectroscopy) on an Avance II 500 spectrometer (Bruker). Preparation, IR and 1H NMR Characterizations of PEGylated and Fluorinated CS. Synthesis of mPEG2000-COOH. mPEG2000COOH was obtained from methoxy-poly(ethylene)glycol (mPEG2000−OH) according to a protocol adapted from Jeong et al.31 Briefly, mPEG2000-OH (4.6 g, 2.30 mmol) was dissolved in anhydrous CH2Cl2 (30 mL) at room temperature under an argon atmosphere. Succinic anhydride (1.2 g, 12 mmol) was added and 2757


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Biomacromolecules stirred until complete dissolution, and then pyridine (200 μL, 2.5 mmol) was added dropwise to the solution. The mixture was stirred for 72 h at room temperature under argon. After concentration, diethyl ether was added, and the mixture cooled at 4 °C for 1 h to allow mPEG2000-COOH precipitation. The solid was recovered by filtration as a white powder (90% yield). For further calculations, averaged MW (mPEG2000-COOH) was considered to be equal to 2000 g·mol−1.1H NMR (250 MHz, 298 K, D2O) δ (ppm): 2.67 (s, 4H), 3.36 (s, 3H, -OCH3), 3.5−3.8 (m, 166H), 4.27 (m, 2H). MALDI MS spectrum of mPEG2000-COOH: distribution around a central peak at m/z = 2005.1 corresponding to [CH3(OCH2CH2)42OCOCH2CH2CO2H − Na]+. CS Grafting with mPEG2000-COOH (CS−mPEG2000) and Determination of CS Degree of Substitution by DOSY Experiments (DSmPEG2000/CS). CS (200 mg, 1.0 mmol of NH2 function) was dissolved in 10 mL of an aqueous solution of acetic acid 1% (v/v). After complete CS dissolution, the pH of the solution was increased to 5 by addition of NaOH (1 M). mPEG2000-COOH was then added to the CS solution at different stoichiometric ratios (mPEG2000-COOH/ NH2 CS molar ratio expressed as mol % (COOH/NH2) of 10%, 20%, 50%, and 100% corresponding to 200, 400, 1000, and 2000 mg of mPEG2000-COOH respectively). In a first series of experiments, mPEG2000-COOH conjugation was performed in the presence of NHS (22 mg, 0.2 mmol for a mol % (COOH/NH2) of 10%, that is, 2 equiv compared to mPEG2000-COOH), while in a second series of experiments, no NHS was used. Each mixture was stirred for 15 min at room temperature before gradual addition of EDC·HCl (for instance, for an initial mol % (COOH/NH2) of 10%, 38 mg of EDC· HCl, 0.2 mmol, was added). Each reaction was then heated at 60 °C for 15 h before being dialyzed against water for injection (Spectra/ Por6 dialysis membrane with a MW cutoff of 15 kDa, 25 °C, 3 dialysis cycles of 2, 4, and 24 h, respectively). The resulting solution was ultrafiltered twice with Vivaspin tubes (Sartorius, cutoff 10 kDa, 4500 rpm, 1 h) before freeze-drying to finally recover CS−mPEG2000 as a white foam. FT-IR (ATR, cm−1): 3362 (νOH and νNH), 2879 (νCH), 1651 (amide I), 1557 (amide II), 1075 (pyranose ring). 1H NMR (500 MHz, 318 K, D2O/DCl, 700 μL: 1 μL), δ (ppm): 2.07 (s, CH3COO−), 2.11 (s, CH3 of CS acetyl units), 2.70 (s, mPEG2000), 3.19 (s, CS), 3.40 (s, -OCH3 of mPEG2000), 3.50−4.00 (m, mPEG2000 + CS), 4.31 (m, mPEG2000), 4.90 (s, 1H of CS anomeric carbon). Determination of DSmPEG2000/CS. Bipolar gradient pulses with two spoil gradients were used to measure the diffusion coefficients (BPPLED pulse sequence). The value of the gradient pulse length δ was 4 ms, while the value of the diffusion time Δ was set to 500 ms. The pulse gradients were increased in 16 steps from 2% to 95% of the maximum gradient strength (53.5 G/cm) in a linear ramp, and the temperature was set at 30 °C. Under these conditions, preliminary DOSY experiments were performed to determine CS and mPEG2000COOH diffusion coefficients (DCS and DmPEG2000‑COOH respectively). (It has to be noted that a δ of 2 ms was used for the measurement of the PEG-COOH diffusion coefficient.) CS and mPEG2000-COOH diffusion curves were then extracted from DOSY spectra of CS (for the peak at δ = 2.1 ppm) and mPEG2000-COOH (for the peak at δ = 3.7 ppm). In each case, the monoexponential diffusion curves were fitted with eq 1 to obtain DCS and DmPEG2000‑COOH values of 5 × 10−12 m2.s−1 and 1.4 × 10−10 m2.s−1, respectively.

I = Io exp[− γ 2g 2Dδ 2(Δ − (δ /3) − (τ /2))]

where IG and IUG are the intensities at 0% gradient of grafted and not grafted PEG, respectively, γ is the gyromagnetic ratio, g is the gradient strength, DG and DUG are the diffusion coefficients of grafted and ungrafted PEG, respectively, δ the gradient pulse length, Δ is the diffusion time, and τ is the interpulse spacing in the BPP-LED pulse sequence. Assuming that CS−mPEG2000 molecular weight must be close to that of CS (due first to the large difference between PEG2000 and CS molecular weights and second assuming that in the experimental conditions used in this study a DSCS in the range 0.56−5.4% was expected25,26), one can consider that PEGG (and then CS−mPEG2000) has the same diffusion coefficient as CS. During the fitting, DG and DUG were then fixed to values measured independently on chitosan and PEG, respectively: DCS = 5 × 10−12 m2.s−1; DmPEG2000‑COOH = 1.4 × 10−10 m2.s−1. The values of IG and IUG extracted from the fitting allowed us to calculate the percentage of grafted PEG over the total amount of PEG (PEGG/PEGT):

PEGG IG = × 100 PEGT IG + IUG

The percentage of mPEG2000 grafted to CS chains (DSPEG/CS) was then calculated from 1H NMR and DOSY experiments:

DSPEG/CS = % ×

(I(3H, mPEG2000)3.4ppm )/3 PEGG = CS I(1H, CS)3.2ppm

IG × 100 IG + IUG

(4)

where I represents the integration of the peaks indicated in brackets, whereas IG and IUG stand for the intensities extracted from the DOSY experiments for grafted and ungrafted PEG, respectively. CS Grafting with TFB-COOH (CS−TFB) and Determination of CS Degree of Substitution DSTFB/CS. CS (200 mg, 1.0 mmol of NH2 function) was dissolved in 10 mL of an aqueous solution of acetic acid 1% (v/v) at room temperature. After complete CS dissolution, the pH of the solution was increased to 5 by addition of NaOH (1 M). In parallel, TFB-COOH (TFB-COOH/NH2 CS expressed as mol % (COOH/NH2) of 10%, 20%, 40%, and 100% corresponding to 14, 28, 57, and 142 mg of TFB-COOH, respectively) was dissolved in DMF (1 mL) in the presence of EDC (19 mg, 0.1 mmol for mol % (COOH/NH2) of 10%, for instance) and stirred for 1 h at room temperature. Then, TFB-COOH/EDC solution was slowly added to the CS solution, and each reaction was heated at 60 °C for 15 h. Workup similar to the one used for CS PEGylation was applied, and CS−TFB was finally recovered as a white foam after freeze-drying. FTIR (ATR, cm−1): 3354 (νOH and νNH), 2926 (νCH), 1633 (amide I), 1538 (amide II), 1072 (pyranose ring). 1H NMR (500 MHz, 318 K, D2O/DCl, 700:1), δ (ppm): 2.06 (s, CH3COO−), 2.08 (s, CH3 of CS acetyl units), 2.45−2.70 (m, TFB units), 3.18 (s, CS), 3.60−4.00 (m, CS), 4.87 (s, 1H of CS anomeric carbon).19F NMR (470.38 MHz, 318 K, D2O/DCl, 700:1), δ (ppm): −66.1, −66.3 ppm. These two can be attributed to grafted and ungrafted TFB respectively. The unambiguous assignment of these two peaks was performed thanks to the use of 1D 19F diffusion-filtered experiments recorded on a Avance II 500 spectrometer with the following parameters: δ = 4 ms, Δ = 500 ms and g = 2 or 95% of the maximum available gradient (53.5 G/cm). Determination of DSTFB/CS. The integration of both peaks at −66.1 and −66.3 ppm allowed us to determine the respective percentages of grafted and ungrafted TFB, and the percentage of TFB grafted to CS chains could thus be calculated from 1H and 19F NMR spectroscopies as follows:

(1)

Similar DOSY experiments were then performed with CS−mPEG2000 copolymer to characterize the diffusion coefficients of ungrafted and grafted mPEG2000 chains. The diffusion curves were extracted from CS−mPEG2000 DOSY spectra for the more intense peak of PEG at 3.7 ppm and were characterized by two contributions: one coming from the ungrafted PEG (PEGUG), which diffuses fast, and the other coming from the grafted PEG (PEGG). Diffusion curves can thus be fitted with a biexponential equation taking into account the two contributions (eq 2).32,33 I = IG exp[− γ 2g 2DGδ 2(Δ − (δ /3) − (τ /2))] + IUG exp[ − γ 2g 2DUGδ 2(Δ − (δ /3) − (τ /2))]

(3)

(2) 2758


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Figure 1. 1H NMR spectrum of CS−mPEG2000 in D2O/DCl (700/1, v/v), 500 MHz, 318 K, mol % (COOH/NH2)initial = 10%.

DSTFB/CS = % ×

(3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide) according to the manufacturer’s instructions.34 RAW 264.7 cells were seeded in 24-well plates at a density of 5 × 104 cells.mL−1 and allowed to adhere for 24 h. Then, they were treated with CS−mPEG2000− TPP/HA or CS−TFB−TPP/HA (and for comparison CS−TPP/HA NPs) at NP concentrations between 5 and 200 μg·mL−1 and incubated for 24 and 48 h. After these exposure times, the culture medium was removed and 500 μL MTT (1 mg·mL−1) was added in each well. After 2 h, the MTT solution was removed, and formazan crystals were solubilized in 100% isopropanol. The optical density was measured at 595 nm using a TECAN GENios Multireader. The cell viability was expressed in percentage considering 100% viability for control cells related to the treated samples. RAW 264.7 cells treated with trehalose (10% (m/v)) were used as the control.

(I(4H, TFB)2.5ppm )/4 TFBG = CS I(1H, CS)3.2ppm I(CF3 , TFBG)−66.1ppm

I(CF3 , TFBG)−66.1ppm + I(CF3 , TFBUG)−66.3ppm

× 100 (5)

where I represents the integration of the peaks indicated in brackets and G and UG standing for grafted and ungrafted, respectively. Preparation by Ionic Gelation and Physicochemical Characterizations of CS−TPP/HA, CS−mPEG2000−TPP/HA, and CS− TFB−TPP/HA Nanoparticles. Stock solutions of CS were prepared by dissolution of the unfunctionalized CS, CS−mPEG2000, or CS−TFB powders (2.5 mg·mL−1) in a 16 mM HCl solution. Insoluble residues were removed by centrifugation at 3800 rpm for 4 min at room temperature. CS−TPP/HA nanoparticles were obtained by an ionotropic gelation process. The polyanionic phase containing HA (0.8 mg· mL−1) and TPP (1.2 mg·mL−1) dissolved in water (4.5 mL) was added dropwise to the CS solution (9 mL) under sonication (750 W, amplitude 32%) to obtain stable nanosuspensions. At the end of the addition, magnetic stirring was maintained for 10 min. Similar protocol was used for CS−mPEG2000−TPP/HA and CS−TFB−TPP/HA nanoparticles. pH correction was achieved by dialysis against water (3 × 12 h) for CS−TPP/HA NPs or by NaOH addition (0.1 M) for CS−mPEG2000−TPP/HA and CS−TFB−TPP/HA NPs to reach physiological pH. Nanosuspensions were then freeze-dried, using trehalose (10% (m/v)) as a cryoprotectant. The nanoparticle averaged hydrodynamic diameters (Z-avg) were determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). Each nanosuspension was analyzed in triplicate at 20 °C at a scattering angle of 173°, after a 1/20 dilution in water. Water was used as a reference dispersing medium. ζ-Potential data were collected through electrophoretic light scattering (ELS) at 20 °C, 150 V, in triplicate for each sample, after a 1/20 dilution in water. The instrument was calibrated with a Malvern −68 mV standard before each analysis cycle. Cell Culture and Cell Exposure to CS and Functionalized-CS Nanoparticles. Adherent RAW 264.7 (American Type Culture Collection TIB-71) cells were grown in Dulbecco’s modified Eagle medium (DMEM), pH 7.4, supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotics (penicillin, streptomycin, and amphotericin B) and maintained at 37 °C in a humidified atmosphere (95%) with 5% CO2. Cell viability was measured by the MTT assay

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RESULTS AND DISCUSSION Preparation and Characterizations of CS Grafted with mPEG2000-COOH (CS−mPEG2000). mPEG2000-COOH grafting onto the amino group of the CS glucosamine residue was performed by a two-step procedure involving the conversion of monofunctionalized mPEG2000-OH into the carboxylic acidterminated mPEG2000-COOH.31 Provided that the PEG is monofunctional, no cross-linking will occur upon reaction with CS. Then CS was reacted with mPEG2000-COOH (either in this form35 or in its NHS ester form) to give the desired CS− mPEG2000 conjugate, this reaction being mediated by EDC (Scheme 1). The initial molar ratio (COOH/NH2) was set at 10%, which corresponded to an intermediate value in the range of percentages encountered in similar works.25,26 All experiments were conducted at 60 °C for 15 h without observing any CS degradation.36 CS−mPEG2000 was then recovered after extensive dialysis against water for injection and subsequent freeze-drying. To characterize the CS−mPEG2000 copolymer, FT-IR and TGA analyses were performed. Whether or not PEG acted as its NHS ester, FT-IR spectra of CS−mPEG2000 were similar and corresponded to the superimposition of CS and mPEG2000COOH FT-IR spectra (data not shown). CS−mPEG2000 thermogram exhibited three decomposition steps (Figure S1, Supporting Information) and were similar to thermograms obtained for similar CS−mPEG2000 copolymers.37,38 The first 2759


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Figure 2. Diffusion curves and diffusion coefficients extracted from DOSY spectra (ref δ (1HPEG) = 3.7 ppm) for CS and mPEG2000-COOH (a) in the absence of EDC and (b) in the presence of EDC and (c) PEG2000-COOH alone and (d) CS alone as controls. See eq 2 for IUG and IG definitions (UG corresponds to ungrafted PEG chains, while G corresponds to grafted PEG chains)

ring at δ = 3.2 ppm, H3 to H6 of pyranose ring at δ = 3.5−4.0 ppm, anomeric H1 of pyranose ring at δ = 4.9 ppm and acetyl Ha proton at δ = 2.11 ppm).39 The degree of substitution of mPEG2000 to the amino groups of chitosan (DSPEG/CS = PEGG/CS, where PEGG stands for grafted PEG chains) was determined by quantitative 1H NMR analysis. Integral ratio of He to H2 signal, for which integration of H2 was set to 1 and integration of He was divided by 3,31,39 led to a DSCS of 10% whether mPEG2000 acted as its Oacylisourea intermediate or as its NHS ester. This result indicated that the entire PEG amount introduced in the medium was still present, in spite of extensive dialysis. Moreover, in the absence of a spectroscopic marker specific to the amide bond, it was difficult to conclude that PEG chain grafting on CS backbone was quantitative. Indeed, mPEG2000-

degradation stage occurred in the range 60−120 °C and was the consequence of the evaporation of water adsorbed by the polymer. The second one in the range 200−290 °C resulted from the CS backbone decomposition, and the third one in the range 340−430 °C corresponded to the degradation of PEG units. These results suggested the presence of mPEG2000 associated with CS chains, without providing evidence for a covalent linkage between them. To characterize this linkage, CS−mPEG2000 was subjected to 1H NMR analysis at 318 K (D2O/DCl as a solvent, Figure 1 and Figure S2, Supporting Information). The 1H NMR spectrum indicated the presence of the chemical shifts corresponding to PEG2000 chain protons (Hc at δ = 4.3 ppm, Hd at δ = 3.6−3.8 ppm, He at δ = 3.4 ppm, Hb at δ = 2.7 ppm) and CS backbone or acetyl protons (H2 of pyranose 2760


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Biomacromolecules COOH chains could also be associated with CS by chain−chain or by electrostatic interactions that are known to be efficient with these systems.14,24 In order to properly evaluate the ratio PEG G /CS, experimental conditions corresponding either to CS and mPEG2000-COOH in the absence or in the presence of EDC were tested. For that, the initial molar ratio (COOH/NH2) was set at 10%, and as previously indicated, quantitative 1H NMR analysis indicated that PEGT/CS ratio was equal to 10% for both conditions. The resulting mixtures were then subjected to DOSY experiments (Figure 2).27 When mPEG2000-COOH and CS were reacted in the absence of EDC, the resulting diffusion curve was fitted by a monoexponential function (Figure 2a), which led to a diffusion coefficient D of 1.45 × 10−10 m2 s−1. This D value is similar to the one of mPEG2000-COOH alone (DmPEG2000‑COOH = 1.40 × 10−10 m2 s−1, Figure 2c), which indicated that as expected under these conditions no grafting was detected by DOSY. When mPEG2000-COOH and CS were reacted in the presence of EDC, the obtained diffusion curve was clearly nonlinear (Figure 2b). A biexponential fitting of this curve with eq 2, for which two diffusion coefficients of 1.40 × 10−10 m2 s−1 and 5 × 10−12 m2 s−1 were used, was performed. The first coefficient corresponded to ungrafted PEG chains that diffused rapidly, and the second one, corresponding to PEG chains that diffused much more slowly, was attributed to grafted PEG chains. For the latter, the diffusion coefficient was the same as that of CS (see Experimental Section). This was expected because PEG and CS chains having very different molecular weights, PEG grafting should not restrict CS chain mobility. In a second time, this fitting allowed the extraction of the percentage of grafted PEG over the total amount of PEG (PEGG/PEGT, eq 3, Experimental Section). For an initial molar ratio (COOH/NH2) of 10%, the PEGG/PEGT ratio was estimated by DOSY at 3%, leading to a final DSCS of 0.3% (Table S1, according to eq 4, Experimental Section). This value was similar whether mPEG2000 reacted as its NHS ester or in its O-acylisourea form, which indicated that NHS activation was not necessary to improve mPEG2000 grafting. Since only 3% of the initial amount of mPEG2000 chains were grafted to CS, most mPEG2000 chains were associated with CS, probably via ionic mPEG2000-COO−/CS-NH3+ or chain−chain mPEG2000/CS interactions (Figure S3 and Table S2, Supporting Information). This could be a drawback for the forthcoming formulations as CS−mPEG2000 NPs. Indeed, the percentage of PEG chains ungrafted to NPs should be as minimal as possible to avoid their leakage in vivo. For that, purification of dialyzed CS−mPEG2000 copolymers was completed by membrane ultrafiltration (Figure S4, Supporting Information), and the stability of the CS−mPEG2000 construct was checked over a period of 22 days by DOSY (Figure S5, Supporting Information). CS−mPEG2000 workup having been developed, the initial molar percentage, mol % (COOH/NH2), was varied in the range 10% to 100%, in order to improve the final PEGG/CS ratio. After dialysis and two ultrafiltration cycles, grafting efficiency was determined (Table 1). As the initial ratio increased, PEGG/CS ratio increased linearly leading to a maximum mPEG2000 grafting percentage of 20%. The corresponding CS−mPEG2000 copolymers with respective DSCS of 0.3%, 0.9%, 8% ,and 20% were subsequently involved in the preparation of CS−mPEG2000 nanoparticles by ionic gelation.

Table 1. PEG2000,total/CS (PEGT/CS), PEG2000,grafted/ PEG2000,total (PEGG/PEGT, eq 3), and PEG2000,grafted/CS (PEGG/CS, eq 4) Ratiosa % mol (COOH/ NH2)initial 10 20 50 100

(1) PEGT/ CS [%] 2 12 47 83

± ± ± ±

1 1 3 2

(2) PEGG/ PEGT [%] 14 7 16 25

± ± ± ±

4 1 2 2

(3) DSPEG/CS = PEGG/CS [%] 0.3 0.9 8 20

± ± ± ±

0.1 0.1 1 2

a ±sd, n = 3 experiments per ratio; after extensive dialysis and 2 UF cycles determined by (1) 1H NMR and (2) DOSY experiments, respectively. (3) (PEGT/CS) × (PEGG/PEGT).

Preparation and Characterizations of CS Grafting with TFB-COOH (CS−TFB). CS functionalization by fluorinated derivatives was attempted in order to confer to CS a lymphotropic character.28 TFB-COOH was chosen as the fluorinated synthon. Indeed, because of the chemical shift equivalence of the fluorine atoms of the −CF3 group, the TFB moiety exhibited one single resonance peak in 19F NMR, which could be useful to report on the TFB-COOH covalent grafting ratio. CS functionalization with TFB-COOH was initially conducted under conditions similar to the ones optimized for the mPEG2000 synthon (Scheme 1), with an in situ activation of TFB-COOH by EDC. Unfortunately in these conditions, no TFB-COOH grafting was observed. To circumvent this limitation, external EDC activation of TFB-COOH was performed. TFB-COOH was then solubilized in a minimum of DMF and activated by EDC before being added to an acetic solution of CS. As in previous experiments, the initial (COOH/ NH2) molar ratio was set to 10%. To characterize the CS−TFB copolymer, FT-IR and TGA analyses were performed. As for CS−mPEG2000 analogue, FT-IR spectrum of CS−TFB corresponded to the superimposition of CS and TFB-COOH signals (data not shown). CS−TFB thermogram (Figure S6, Supporting Information) presented two decomposition steps, the first one corresponding to water evaporation (40−140 °C) and the second one being assigned to chitosan decomposition (230−350 °C).37,38 As previously mentioned for the CS− mPEG2000 homologue, these results suggested the presence of TFB associated with CS, without demonstrating the existence of a covalent linkage between them. To go further into the copolymer characterization, CS−TFB was characterized by 1H and 19F NMR spectroscopies. 1H NMR spectrum exhibited peak resonances of CS and TFB-COOH (Figure 3 and Figure S7, Supporting Information). The 1H NMR spectrum confirmed the presence of the chemical shifts corresponding to proton pyranose ring (anomeric H1 at δ = 4.9 ppm, H3−H6 at δ = 3.6−4.0 ppm, and H2 at δ = 3.2 ppm).31,39 The broad signals observed between 2.4 and 2.7 ppm were attributed to protons of the TFB group, on the basis of 1H NMR spectrum of TFBA starting material (spectrum b, Figure S7 Supporting Information). The 19F NMR spectrum of CS−TFB exhibited two peak resonances, a broad one at −66.1 ppm and a more intense one at −66.3 ppm, which was indicative of two fluorinated species in solution (Figure S8, day 0, Supporting Information). The broad shape of the signal at −66.1 ppm suggested that the corresponding species was in low molecular motion by comparison to the other one. In order to assign those signals, two 1D diffusion-filtered 19F NMR spectra were recorded, the first one with an applied gradient of 2% (Figure 4a), the second 2761


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Figure 3. 1H NMR spectrum of CS−TFB in D2O/DCl (700/1, v/v), 500 MHz, 318 K, mol % (COOH/NH2)initial = 10%.

Table 2. TFBtotal/CS (TFBT/CS), TFBgrafted/TFBtotal (TFBG/ TFBT), and TFBgrafted/CS (TFBG/CS) Ratiosa % mol (COOH/ NH2)initial 10 20 40 100

(1) TFBT/ CS [%] 9 9 14 34

± ± ± ±

3 1 1 1

(2) TFBG/ TFBT [%] 16 17 24 36

± ± ± ±

1 4 6 5

(3) DSTFB/CS = TFBG/CS [%] 1.5 1.6 3 12

± ± ± ±

0.5 0.5 1 2

±sd, n = 3 experiments per ratio); determined after workup by (1) H NMR and (2) 19F NMR. (3) (TFBT/CS) × (TFBG/TFBT).

a

Figure 4. 1D diffusion-filtered 19F NMR spectra with a gradient g of (a) 2% and (b) 95%.

1

percentage of around 12%. Previous investigations on fluorinated chitosan systems have demonstrated that fluorine substitution below 40% is optimal for biological applications.40 The CS degree of substitution found here fell in this range, rendering the corresponding CS−TFB polymers suitable for nanoparticle formulations. Formation of CS−TPP/HA Nanoparticles by Ionic Gelation from the Functionalized-CS. CS, CS−mPEG2000, and CS−TFB, in association with sodium hyaluronate (HA) were used to produce nanoparticles by physical gelation in a one-step procedure. This method relied upon the establishment of multivalent electrostatic interactions between CS derivatives (polycationic) and HA (polyanionic). The resulting supramolecular network could be reinforced by cross-linking mediated by small anionic cross-linkers such as sodium tripolyphosphate (TPP).41−45 PEGylated or fluorinated CS with DSCS ranging from 0.3% to 20% were then evaluated for their ability to produce functionalized CS−TPP/HA NPs by ionic gelation. The average hydrodynamic diameters of functionalized CS−TPP/HA NPs were determined by DLS as well as the polydispersity index of the NP population (Table 3). Nanoparticle zeta potential (ζ), which was indicative of their outermost surface charge, was determined by ELS. For PEGylated chitosans (CS−mPEG2000), stable and homogeneous nanosuspensions were obtained for PEGG/CS grafting ratios less or equal to 0.9%, afterward flocculation was systematically observed. CS−mPEG2000−TPP/HA NP sizes were comparable to those of unfunctionalized CS NPs (220−

one with an applied gradient of 95% (Figure 4b). The 2% gradient condition is not able to discriminate low- and rapiddiffusion species; therefore the corresponding spectrum was similar to the 19F CS−TFB NMR spectrum. For the 95% gradient spectrum (Figure 4b), a sole resonance at −66.1 ppm remained. In this spectrum, the applied filtering conditions removed species diffusing rapidly. Consequently, the peak observed at −66.1 pm was unambiguously assigned to the signal of TFB grafted to the CS backbone. At last, the two resonances observed in the CS−TFB 19F NMR spectrum did not evolve over a period of time of 19 days, indicating a good stability for the CS−TFB construct (Figure S8, Supporting Information). The comparison of 19F signal intensity associated with TFB grafted to CS (−66.1 pm) and 19F signal intensity associated with ungrafted TFB (−66.3 pm) allowed us then to calculate the DSCS. For an initial molar ratio (COOH/NH2) of 10%, the TFBT/CS ratio was estimated by 1H NMR at 9%, while the quantitative 19F NMR analysis indicated that TFBG/TFBT ratio was equal to 16% leading to a final DSTFB/CS = TFBG/CS of 1.5% (eq 5, experimental section). In order to explore the stoichiometry of CS functionalization with TFB-COOH, initial molar ratios (COOH/NH2) comprised between 10% and 100% were used and the evolution of the final TFB percentage grafting was followed by 19F NMR spectroscopy (Table 2). Once again, as the initial ratio increased, TFBG/CS final ratio increased linearly leading to a maximum TFB grafting 2762


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For fluorinated chitosan (CS−TFB) whatever the TFBG/CS grafting ratio, nanoparticles were obtained with sizes ranging between 230 and 300 nm, that is, superior to the CS− mPEG2000 NPs. This might be attributed to an increase of the hydrophobicity inherent to the fluorinated synthons, which led to less stable nanosuspensions.25 Furthermore, the highest TFBG/CS ratios led to a smaller number of CS protonated amino functions available for ionotropic gelation and then to less packed nanostructures. Regarding CS−TFB NP ζ potential, these values did not evolve significantly according to CS grafting ratio increase. One should however notice that since the TFB synthon size was smaller than the mPEG2000 one, a similar grafting density should rationally induce a lower shielding of CS charges. This could also indicate that TFB conformation at the NP surface is different than the conformation previously proposed for PEG chains.46 Finally, regarding the nanoparticle stability, after 2 days of storage at 37 °C a slight swelling was observed for PEGylated and fluorinated nanoparticles (as expected from the hydrogel structure of the nano-objects), after which the values remained constant over time (Figure S9, Supporting Information). In the meantime, almost no evolution of nanoparticle ζ potentials was observed. These data suggested that there was no degradation of the nanosuspensions in these conditions. Cytotoxicity Studies of Functionalized CS−TPP/HA Nanoparticles. Evaluation of cell viability was a first stage in understanding of how nanoparticles interact in vitro with cells. Because macrophages have recently emerged as a model for in vitro toxicological study of various nanoparticles,47−53 RAW 264.7 murine macrophage cell line was selected as a relevant model for biocompatibility evaluation of CS−mPEG2000 and

Table 3. Intensity Weighted (Z-Average) Diameters, Polydispersity Indexes (PdI), and ζ Potential of Functionalized CS−TPP/HA Nanoparticles According to CS Degree of Substitution (DSCS) synthon

DSCS [%]

mPEG2000

0.3 0.9 8 20 1.5 1.6 3 12 0

TFB

no synthon a

Z-avg (nm) 236 222 a a 292 230 270 304 225

±2 ±1

± ± ± ± ±

3 2 3 3 2

ζ (mV)

PdI 0.20 0.20 a a 0.30 0.30 0.20 0.20 >0.3

± 0.01 ± 0.01

± ± ± ±

0.02 0.02 0.01 0.01

+22 +24 a a +30 +30 +30 +29 +31

±3 ±4

± ± ± ± ±

4 4 4 4 3

Flocculation.

230 nm).41,42 Polydispersity indexes (PdI) of the corresponding nanosuspensions were however lowered by comparison to unfunctionalized CS NP PdIs. This point is indicative of the presence of PEG at the NP surface, which induced a steric barrier and helped to prevent NP aggregation. This result was corroborated by CS−mPEG2000−TPP/HA NP ζ potential of around +23 mV, that is, 10 mV lower than the one of unfunctionalized CS NPs (+31 mV). This decrease indicated that grafted mPEG2000 chains are mainly orientated at the outside of the NPs. Due to the low PEG grafting density on the CS backbone, one can propose that PEG chains at the NP surface could acquire a mushroom conformation.46 Therefore, even if PEG density is low, such a conformation could account for an efficient screening of CS positive charges at the NP surface.

Figure 5. RAW 264.7 cells viability after exposure to 5, 25, 50, 100, and 200 μg/mL of (a) CS−mPEG2000−TPP/HA and (b) CS−TFB−TPP/HA NPs and for comparison (c) CS−TPP/HA for 24 and 48 h according to MTT assay. Cells exposed to solution of trehalose 10% (m/v) were used as control. Results are calculated as means ± sd (n = 3) and expressed as % from controls. 2763


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Biomacromolecules CS−TFB−TPP/HA NPs. The cytotoxicity induced in RAW 264.7 murine macrophages due to their treatment with different concentrations of CS−mPEG2000 and CS−TFB−TPP/HA NPs was analyzed after 24 and 48 h of exposure by the widely used MTT assay (Figure 5).54 Whatever the nanogels, the applied dose, and the exposure time, MTT assay revealed that PEGylated or fluorinated nanogels did not modify the functions of RAW 264.7 macrophages, suggesting a good nanogel biocompatibility with this type of cells.

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AUTHOR INFORMATION

Corresponding Authors

CONCLUSION To sum up and conclude, our objective was to obtain PEGylated and fluorinated chitosan for the design of nanoparticles targeting the lymphatic system. The main issue was to characterize as precisely as possible the copolymers and especially the formation of the covalent amide linkage between the chitosan backbone and the PEG/fluorinated chains. TGA and IR analyses gave an overall description of the copolymers and were not able to distinguish between a blend of chitosan with additional chains and chitosan grafted with chains. For fluorinated chitosans, 19F NMR and 1D diffusion filtered 19 F NMR experiments helped to circumvent the problem since two signals were observed, when the fluorinated synthon was grafted or not. For a series of CS−TFB conjugates, the chitosan degree of substitution DSCS was determined between 1.5% and 12%. For PEGylated chitosans, no specific marker of the amide bond was identified by 1H NMR spectroscopy, and therefore no discrimination between a blend of chitosan plus PEG chains and chitosan grafted with PEG chains was possible either. To solve this problem, which was mandatory for the application, DOSY has proved to be the method of choice. Exploiting DOSY spectra of copolymers has allowed first to distinguish ungrafted chains, which diffused fast, from the grafted ones, which diffused much slower, and second to calculate the percentage of grafted PEG chains. The DSCS was then determined between 0.3% and 20% for a series of CS− mPEG2000 conjugates. Ionic gelation from CS−mPEG2000 conjugates proved to be efficient to produce CS−mPEG2000−TPP/HA nanohydrogels having morphological characteristics compatible with subcutaneous injection, as soon as DSCS was low (lower than 1%). For CS−TFB conjugates, CS−TFB−TPP/HA nanogels were obtained for all the DSCS tested. Finally, when RAW 264.7 murine macrophages were exposed to the nanogels, no modification of cell functions was observed, which suggested that these nanogels exhibited a good biocompatibility toward this cell line. As RAW 264.7 murine macrophages are considered to be a relevant model to test nanoparticle safety toward the lymphatic system, these results are encouraging with regard to the use of CS−mPEG2000−TPP/HA and CS−TFB− TPP/HA nanocarriers for encapsulation of probes and MRI lymphography experiments. Further work is currently in progress to achieve this goal.

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TGA analyses, 1H and 19F NMR characterizations of CS−mPEG2000 and CS−TFB polymers, DOSY experiments on CS and mPEG2000COOH mixtures, PEGT/CS ratio evolution according to purification steps, stability of CS−mPEG2000 and CS−TFB evaluation by DOSY and 19 F NMR experiments, CS−mPEG2000−TPP/HA and CS−TFB−TPP/HA nanoparticle stabilities monitored for 16 days at 37 °C (PDF)

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Cyril Cadiou: 0000-0002-2737-9976 Françoise Chuburu: 0000-0002-4937-173X Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the ANR, the Region Wallone, the Romanian National Authority for Scientific Research CCCDIUEFISCDI and the EuroNanoMed framework for financial support to the project Gadolymph. The Region Champagne Ardenne, the DRRT Champagne Ardenne (through MESR), the EU-program FEDER (Project NanoBio2, Nano’Mat Platform), the ARC program, the FNRS, the Holocancer program, COST actions, the UIAP VII program, the Center for Microscopy and Molecular Imaging (CMMI, supported by the European Regional Development Fund and the Region Wallone), and the PlAneT platform (supported by the European Regional Development Fund and the Region Champagne Ardenne) are thanked for their support. The work was funded by ANR (Gadolymph Project No. ANR-13ENM2-0001-01, through the EuroNanoMed 2013 framework), by the Région Wallone (Gadolymph Project No. 1317980 through the EuroNanoMed 2013 framework), and by the Romanian National Authority for Scientific Research CCCDIUEFISCDI (Project Number 4-006/2014, EuroNanoMed II),

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00668. 2764


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