Article pubs.acs.org/Biomac
Sulfated Glycosaminoglycan-Based Block Copolymer: Preparation of Biocompatible Chondroitin Sulfate‑b‑poly(lactic acid) Micelles André R. Fajardo,†,‡,§ Alexandre Guerry,† Elizandra A. Britta,∥ Celso V. Nakamura,∥ Edvani C. Muniz,‡ Redouane Borsali,† and Sami Halila*,† †
Centre de Recherches sur les Macromolécules Végétales (CERMAV, UPR-CNRS 5301), Université Grenoble Alpes, BP 53, 38041 Grenoble Cedex 9, France ‡ Grupo de Materiais Poliméricos e Compósitos (GMPC) and ∥Department of Clinical Analysis, Maringá State University, 87020-900, Maringá, PR Brazil § Centro de Ciências Químicas, Farmacêuticas e de Alimentos (CCQFA), Federal University of Pelotas, P.O. Box 354, 96010-900, Pelotas, RS Brazil S Supporting Information *
ABSTRACT: Despite a growing interest in amphiphilic polysaccharide-based diblock copolymers as functional polymeric drug delivery nanosystems, biologically relevant sulfated glycosaminoglycan systems were not yet investigated. Here, we report the synthesis and the self-assembly properties in water of chondroitin sulfate-bpoly(lactic acid) (CS-b-PLAn). The CS-b-PLAn were synthesized using click-grafting onto method implying reducing-end alkynation of low-molecular weight depolymerized CS (Mw = 5000 g·mol−1) and azide-terminated functionalization of PLAn (Mw = 6500 g·mol−1 (n = 46) and Mw = 1700 g·mol−1 (n = 20)). The diblock copolymer selfassembled in water giving rise to spherical micelles that were characterized in solution using dynamic/static light scattering and at dry state by TEM technique. In vitro assays on healthy cells showed that at high concentrations, up to 10 μg·mL−1, CS-b-PLAn were noncytotoxic. Those preliminary studies are promising in the perspective to use them as biocompatible nanovehicles for anticancer drug delivery.
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dextran,6−10 maltodextrins,2,11−13 cyclodextrin,13−15 xyloglucan,16 and pullulan.17,18 Interestingly, polyanionic hyaluronic acid or hyaluronan-based BCPs nanoparticles19,20 have been recently developed because of their binding to overexpressed tumor cell surface receptors such as CD44, a family member of hyaladherins.21,22 Sulfated glycosaminoglycans (GAG), such as heparin, dermatan, and chondroitin sulfate, are other fascinating anionic polysaccharides that have attracted considerable attention in biomedicine due to their numerous and enhanced biological activities and physicochemical properties.23 Chondroitin sulfate (CS)24 is found as proteoglycans (CS side chains attached to a core protein) of the extracellular matrix and it is composed of alternating units of (β-1,3)-linked glucuronic acid (GlcUA) and (β-1,4) N-acetyl galactosamine (GalNAc), with sulfate at either the 4- or 6-position of GalNAc or at 2-position of GlcUA. Its structural similarity with hyaluronic acid makes CS as a
INTRODUCTION Recently, amphiphilic oligo-/polysaccharide-based diblock copolymers (BCPs)1,2 have gained increasing attention due to their ability to self-assemble, in solution3 or in thin films,4 in various naturally occurring nanostructured materials. Moreover, a significant interest of such diblock copolymers, over largely studied graft copolymers,5 is the respect of structural integrity of constitutive blocks and subsequently of their inherent bioand physicochemical properties. In a selective solvent for carbohydrates, such as aqueous solutions, the self-assembled nanoparticles or the so-called micelles usually form spherical core−shell structures in which the hydrophobic core serves as matrice for the encapsulation of a wide range of hydrophobic active agents and the hydrophilic shell, herein the carbohydrate molecules, plays a critical role in the steric stabilization of the micelles and in the interaction with the surrounding environment. While a myriad of functional hydrophobic blocks have been studied so far in micelle-forming amphiphilic oligosaccharidesbased BCPs, only few carbohydrate blocks have been investigated, promoting exclusively their highly hydrophilic feature. Most of those blocs are neutral carbohydrates such as © XXXX American Chemical Society
Received: April 11, 2014 Revised: May 19, 2014
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Propargyloxyaniline (30 mg, 204 μmol) was solubilized in a small portion of methanol and then added dropwise to CS solution. NaBH3CN (10 mg, 159 μmol) was added to the reaction mixture and the system was heated up to 50 °C and kept under magnetic stirring for 5 days. After the reaction mixture was cooled to room temperature, it was diluted with 50 mL of distilled water. A liquid−liquid extraction with dichloromethane (2 × 25 mL) was performed. The aqueous phase was filtered in a Millipore Minaton ultrafiltration unit (Millipore, U.S.A.) using a disc-membrane with MWCO of 5000 and then concentrated (final volume 40 mL). The resulting solution was lyophilized to recover the LMW-CS-alkyne (2) as a white powder. Yield: 290 mg (97%). FTIR (cm−1): 3422 (O−H and N−H), 2912 (C−H), 2121 (CC), 1632 (CO, amide), 1565 (N−H, bending), 1514 (CC, aromatic), 1420 and 1382 (C−H, bending), 1345 (C− N), 1238 (SO2), 1054 (C−O), and 885 (C−H, out-of-plane bend). 1H NMR (400 MHz, D2O, 353 K) δ (ppm): 6.99 and 6.88 (bb′ and aa′ pattern, d, C6H4), 4.79 (m, H-4 GalNAc, C4-sulfated), 4.74 (m, H-1 GalNAc), 4.61 (m, H-6 GalNAc, C6-sulfated), 4.49 (m, H-1 GlcUA), 4.16−3.34 (m, H-2,3,4,5,6 GlcUA and H-2,3,4,5,6 GalNAc, NH, and CH2), 2.98 (s, CCH), and 2.03 (s, CH3). 13C NMR (400 MHz, D2O, 353 K) δ (ppm): 175.4 (O-CO GlcUA), 174.4 (NH-CO GalNAc), 117.8 (>CCHCH), 116.9 (CHCHCCCHCH), 128.48 (CHCHCCCH3), 18.91 (CH3CHSO2), and 17.09 (CH3CHO). Step 2: Azidation Reaction. Compound 4a or 4b and NaN3 (10 equiv) were stirred in DMF anhydrous (1 mL) at room temperature and under Argon atmosphere for 24 h. After, the reaction mixture was diluted in DCM (15 mL) and washed with brine (10 mL) and distilled water (3 × 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated to a few milliliters (ca. 3 mL) under reduced pressure. The PLAn-N3 (5a, 5b) was precipitated in cold methanol and recovered by centrifugation (4 °C, 8000 rpm, 15 min). The final materials were dried under reduced pressure. Yield: 5a (61%) and 5b (80%). FTIR (cm−1): 2991, 2950, and 2851 (C−H), 2118 (N3), 1754 (CO, ester), 1085 (C−O). 1H NMR (400 MHz, CDCl3, 298 K) δ (ppm): 5.15 (m, CH), 4.26 (t, CH2), 4.01 (m, CH), 3.57 (m, CH2), 3.55 (s, CH3), and 1.55 (d, CH3). 13C NMR (75 MHz, CDCl3, 298 K) δ (ppm): 170.04 and 169.36 (O−CO), 70.56 (OCH2CH2), 69.10 (OOCCHO), 64.77 (CH2CH2O), 59.34 (CH3O), 62.13 (CHN3), and 16.45 (CH3CHO). Synthesis of CS-b-PLAn Block Copolymers. The CS-b-PLA1700 (6b) and CS-b-PLA6500 (6a) block copolymers were synthesized by “click” chemistry approach. Compounds 5b (110 mg, 64.7 μmol) and 2 (540 mg, 97.1 μmol) or 5a (130 mg, 20 μmol) and 2 (170 mg, 30 μmol) were placed in two different round flasks and then solubilized in DMF/water (10 mL, 3:1 v/v). Sodium ascorbate (2 equiv) and CuSO4 (2 equiv) were added, and the reaction mixture was stirred and heated at 60 °C for 48 h. The solutions were diluted with distilled water (50
potential candidate for site-specific drug delivery vectors to cancer cells via CD44 receptor-mediated endocytosis.25,26 Herein, we expanded our study on the preparation of amphiphilic oligosaccharide-based BCPs and their self-assembly in aqueous solution by reporting CS-block-poly(lactic acid) (PLA), obtained using a click-grafting onto approach, and their nanoprecipitation. It should be mentioned that up to now, CSbased amphiphilic copolymers have been successfully synthesized exclusively by grafting hydrophobic polymers onto CS backbone.27,28 In the perspective to propose such BCPs as potential active-targeting drug delivery systems it was of great interest to use biodegradable or at least biocompatible (bio)polymers, that is why PLA with different molecular weight were preferred.29,30 Finally, the cytotoxicity of the CS-b-PLAn was investigated against healthy cells.
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MATERIALS AND METHODS
Materials. Chondroitin sulfate (raw CS) was kindly supplied by Solabia (Brazil). Hydroxyl end-functionalized poly(lactic acid)s (D,Lform) with different molar masses (Mw 1700 and 6500 g·mol−1) were purchased from PolymerSource (Canada). Hyaluronidase from sheep testes (type II, ≥ 300 units/mg), 4-nitrophenol, p-toluenesulfonyl chloride (TsCl), 4-dimethylaminopyridine (DMAP), tetrabutylammonium bromide, tin(II) chloride dehydrate, triethylamine (Et3N), and sodium azide were purchased from Sigma-Aldrich (U.S.A.). All the reagents have analytical grade and were utilized without previous purification step. Characterization Techniques. Infrared (IR) spectra were recorded using a PerkinElmer Spectrum RXI FTIR Spectrometer. 1 H NMR, 13C NMR, and DOSY NMR spectra were recorded using 400 MHz Bruker Avance DRX400. Fluorescence spectra were recorded on a LS-50B PerkinElmer spectrofluorimeter, equipped with a thermostated cell holder. For functionalized PLAn and block copolymers, gel permeation chromatography (GPC) measurements were performed at 60 °C using a Agilent 390-MDS system (290-LC pump injector, ProStar 510 column oven, 390-MDS refractive index detector) equipped with Knauer Smartline UV detector 2500 and two Agilent PolyPore PL1113−6500 columns (linear, 7.5 × 300 mm; particle size, 5 μm; exclusion limit, 200−2000000) in DMF containing lithium chloride (0.01 M) at the flow rate of 1.0 mL min−1, and GPC for CS was performed using water as solvent and at 25 °C with a Waters 150C system equipped with a multiangle laser light scattering detector (DAWN DSP-F, Wyatt) and two Shodex columns in series (OHPack 802 and 803). Light scattering experiments were carried out using an ALV setup. Microscopic images were recorded in a field emission scanning electron microscope (SEM-FEG, Zeins Ultra 55 FEG) and in a transmission electron microscope (TEM, JEOL 2010 FEF). The viscometric measurements were done at 25 °C using a Schott viscometer (diameter 0.63 mm), model 531-10, equipped with a Schott AVS360 pump for automatic dilution. The solution concentrations were adjusted based on sample viscosity. The viscometric molar mass (Mv) was calculated using the Mark− Houwink−Sakurada equation31 and the constants used were K = 3.26 × 10−5 (dL·g−1) and a = 0.96. Synthesis of Alkyne End-Functionalized CS. Preparation of Low Molecular Weight CS (LMW-CS) (1). The enzymatic method to prepare LMW-CS was adapted from the literature.32 Briefly, CS (1 g, 50 μmol) was digested with hyaluronidase (24 mg, 300 units·mg−1) from sheep testes in 10 mL of phosphate buffer (50 mM, pH 6) containing 150 mM NaCl at 37 °C for 48 h. The enzyme was inactivated was by boiling the solution for 20 min. The solution was fractionated by ultrafiltration in a Millipore Minaton ultrafiltration unit (Millipore, U.S.A.) between membranes of molecular weight cutoff (MWCO) 10000 and 5000 Da then lyophilized to give LMW-CS (1; yield, 0.595 g (60%)), as a white solid. Synthesis of LMW-CS-alkyne (2). Compound 1 (300 mg, 53 μmol) was solubilized in 20 mL of NH4OAc buffer (5 mM, pH 5) in a oneneck flask under magnetic stirring at room temperature. 4B
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Scheme 1. Synthesis by Click-Grafting onto Approach of CS-b-PLAn Block Copolymers
mL) and centrifuged (4 °C, 8000 rpm, 15 min) to remove the unreacted PLAn-N3. While the residual LMW-CS-alkyne in the aqueous phase was removed using two different techniques according to the block copolymer; for CS-b-PLA1700 (6a) the aqueous phase was dialyzed against distilled water for 48 h using a dialysis membrane (Spectra/Por) with MWCO of 3500. For CS-b-PLA6500 (6a), the aqueous phase was ultrafiltered in a Millipore Minaton ultrafiltration unit (Millipore, U.S.A.) for 24 h using a disc-membrane of MWCO 5000. The solutions were lyophilized for 24 h to recover the CS-bPLAn copolymers. Yield: 6b (168 mg, 56%) and 6a (200 mg, 67%). Typical characteristic data for both CS-b-PLAn; FTIR (cm−1): 3422 (O−H and N−H), 2991 and 2950 (C−H), 1754 (CO, ester), 1632 (CO, amide), 1565 (N−H, bending), 1514 (CC, aromatic), 1420 and 1382 (C−H, bending), 1381 (C−N), 1238 (SO2), 1085 and 1054 (C−O), and 885 (C−H, out-of-plane bend). 1H NMR (400 MHz, DMSO-d6/D2O (6:1), 353 K) δ (ppm): 8.20 and 8.10 (2× s, Htriazole), 6.77 and 6.59 (bb′ and aa′ pattern, H-aromatic), 5.62 (s, 2H, CH2), 5.13 (m, CH from PLAn block), 4.89−3.14 (m, H-1, 2, 3, 4, 5, 6 GlcUA and GalNAc, 2× CH2, CH, CH3 from PLAn block), 1.82 (m, 3H, CH3 from PLAn block), and 1.44 (s, CH3 GalNAc). Determination of Critical Micelle Concentration (CMC) Value by Fluorescence. The CMC value for CS-b-PLA6500 (6b) and CS-b-PLA1700 (6a) block copolymers were determined using pyrene as hydrophobic fluorescence probe. Stock pyrene solution was prepared by addition of pyrene (3 mg) in acetone (30 mL). A
predefined volume of stock pyrene solution was transferred into several vials then the acetone solvent was removed by evaporation. After different volumes of diblock copolymers stock solution (6a and 6b in DMF/water mixture (80:20, v/v)) were added and diluted with PBS buffer (10 mM, pH 7.4) to give various final concentrations (ranging from 5 to 0.001 mg·mL−1) and a final pyrene concentration of 3 × 10−4 mg·mL−1. The mixture were stirred and left 24 h to equilibrate before performing the measurement. The excitation wavelength was fixed at λ = 339 nm, and the monomer emission was read at λ = 372 (I1) and λ = 383 (I3). Preparation and Characterization of Self-Assembled CS-bPLAn Micelles. Mean size and the light scattering intensity of the nanoparticles suspension were determined, both by static and dynamic light scattering (SLS and DLS) methods. Measurements were performed using an ALV laser goniometer, which consists of 25 mW HeNe linear polarized laser operating at a wavelength of 632.8 nm, an ALV-5004 multiple τ digital correlator with 125 ns initial sampling time, and a temperature controller. All samples were systematically studied at 60, 90, and 120° and some of them were studied at different scattering angles varying from 50 to 140°. Nanoprecipitation was preferred for 6a and 6b self-assembly by adding slowly water to the well-dissolved copolymer solution. Typically, 6a or 6b (30 mg) was solubilized in a DMF/water mixture (500 μL, 60:40 v/v-%) and remained under magnetic stirring for 24 h at room temperature. Then water (4.5 mL) was slowly added dropwise (1.02 C
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mL·h−1) via an automatic syringe pump, to give a final concentration 3.0 mg·mL−1. The solution was kept under magnetic stirring for 2 h. Before the SLS and DLS measurements, the aqueous solutions of 6a and 6b were dialyzed against distilled water using dialysis membrane (Spectra/Por, MWCO 3500) for 48 h to remove the DMF and then filtered through 0.20 μm Millipore Millex PES hydrophilic filters. The data were collected using the ALV Correlator Control software. The relaxation-time distributions A(t) were obtained using CONTIN analysis of the acquired autocorrelation functions C(q,t).34 The relaxation times (τ) correspond to the local maxima of the distributions A(t), and the relaxation frequencies (Γ) are equal to 1/ τ.34 The apparent diffusion coefficient (Dapp) of the nanoparticles at a given copolymer concentration is calculated from the following relation: Γ q2
azide−alkyne ligation reaction, termed as click chemistry,34,35 was performed between size-controlled alkyne reducing-end modified CS (2) and two azide-terminated poly(lactic acid) (5a and 5b) differentiated by their molecular weight. Preparation of LMW-CS (1). The enzymatic depolymerization of high molecular weight raw CS (1) has been preferred for two main reasons. From a synthetic point of view, the selective reducing end functionalization is greatly facilitated with the reduction of saccharidic chain length. And from a bioactivity point of view, low molecular weight CS (LMW-CS) demonstrates generally a better bioavailability and pharmacokinetic.36,37 LMW-CS was performed by a controlled enzymatic depolymerization of raw CS using a nonspecific testicular hyaluronidase, which is able to cleave β-1,4-glycosidic bond between GlcUA and GalNAc of CS.38,39 The molecular weight of LMW-CS (2), obtained by ultrafiltration fractionation between 10000 and 5000 MWCO membranes, was determined by aqueous gel-permeation chromatography (GPC) and viscometry. The measurements of intrinsic viscosity [η] informs about the viscometric molecular mass (Mv) thanks to the Mark−Houwink−Sakurada equation.31 According to Table 1,
= Dapp (1)
q→0
where q is the modulus of the scattering vector that is defined as
q=
⎛θ⎞ 4πn sin⎜ ⎟ ⎝2⎠ λ
(2)
where λ is the wavelength of the incident laser beam, n is the refractive index of the pure solvent (1.33 for water), and θ is the scattering angle. The hydrodynamic radius (RH; or diameter, 2RH) was calculated from the Stokes−Einstein relation: k Tq2 RH = B 6πη Γ
q→0
kBT = 6πηDapp
Table 1. Molar Mass Distribution for the Raw CS and LMWCS Evaluated by GPC Analysis and Viscometry Data sample raw CS LMWCS
(3)
where kB is the Boltzmann constant, T is the temperature of the sample, and η is the viscosity of the pure solvent (herein, water).33 TEM Images. TEM images were recorded in a JEOL microscope (JEM 2010 FEF) operating at 120 kV. The TEM samples were prepared as follows: 4 μL of aqueous solution of 6a or 6b (0.1 mg· mL−1) was dropped on Formvar-coated grids, which were rendered hydrophilic by glow discharge treatment. Before the complete drying of the TEM samples, 4 μL of uranyl acetate solution (2 w/v-%), a negative stain, was dropped over the grids. The liquid excess was removed by filter paper and then the grids were dried at room temperature. From TEM images, the average size of the self-assembled nanoparticles was determined by the Size Meter software with differentiation threshold set according to the image scale. The average size was calculated from the sizes measured for 150 nanoparticles chosen randomly (n = 150). In Vitro Cytotoxic Assays. The cytotoxic effects of the block copolymer 6a or 6b on Vero and L-929 cells were evaluated by MTT assay. The Vero cells were isolated from kidney epithelial cells extracted from African green monkey, while L-929 cells were isolated from mouse fibroblasts. Both cell lines were cultured in DMEM, Dulbecco’s Modified Eagle Medium (Gibco, U.S.A.), supplemented with 10% FBS, Fetal Bovine Serum (Gibco, SA), and 50 μg·mL−1 getamicin (oven, 37 °C, 5% CO2). Cell viability was detected using the 5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay. For this assay, a total of 2.5 × 105 Vero or L-929 cells/wells were seeded in 96-well plates with DMEM supplemented with 10% FBS and 50 mg·mL−1 gentamicin and then incubated in oven (37 °C, 5% CO2). After 24 h, the block copolymers 6a or 6b were added to cells with final concentrations ranging from 1 to 1000 μg·mL−1 for an additional incubation of 72 h. After, the cells were treated with MTT (concd 1.0 mg·mL−1) and incubated in an oven for 4 h (37 °C, 5% CO2). The purple formazan in supernatant was solubilized in DMSO and quantified by measuring 570 nm absorbance using an ELISA plate reader (BioTek, model Power Wave XS, U.S.A.).
a
Mwa (kDa)
Mna (kDa)
Mz a (kDa)
Mvb (kDa)
Mw/Mn
[η]c (dL· g−1)
19.28 5.66
14.11 3.89
27.65 8.23
18.7 5.1
1.366 1.457
0.44 0.12
GPC data. bViscometry data. cCalculated from viscometry data.
the raw CS (1) was promptly depolymerized by hyaluronidase to yield after purification 60% of narrow distributed LMW-CS (2) of around 5000 Da, which corresponds to a chain length of 12 repeating disaccharide units. It should be noted that viscometry and GPC, which are both based on the hydrodynamic volume of the biopolymer, gave roughly similar results. Synthesis and Structural Characterization of AlkynylEnd LMW-CS (2). To introduce the alkyne function to the reducing-end of LMW-CS (1), we have applied a recent methodology developed in our laboratory,40 consisting in the use of alkyne-modified aniline, herein, the 4-propargyloxyaniline, in reductive amination conditions (see Scheme 1). By this way, the reductive amination results in ring-opening of the reducing-end sugar and we assume that the alkyne-modified LMW-CS (2) is enough elongated to be not impacted on its biological activities. After 5 days of reaction, 97% of the compound 2 was recovered after ultrafiltration with a 5000 Da MWCO and lyophilization. The slight modification at the reducing end of LMW-CS (1) was confirmed by analysis of the FTIR spectrum (Figure S1) showing a broad band at 2121 cm−1 that indicates the presence of alkyne group, and bands at 1514, 1345, and 885 cm−1 assigned to the characteristic features of the aromatic group. Moreover, the 1H NMR spectrum (Figure 1) clearly revealed the aromatic peaks around 6.8 ppm (Figure 2b; H-a, a′, b, and b′) and the terminal alkyne group (Figure 2b; H-c) with the presence of the characteristic acetylenic proton at 2.98 ppm. The degree of functionalization was calculated from the integral ratio of the aromatic protons (δ 6.99 and 6.88 ppm) to the H-2GlcUA proton (δ 3.34 ppm), and the result was close to 94%.
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RESULTS AND DISCUSSION Scheme 1 describes the general strategy adopted for the synthesis of amphiphilic CS-b-PLAn. The copper(I)-catalyzed D
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Figure 1. 1H NMR spectra of (a) LMW-CS (1) and (b) LMW-CS-alkyne (2; 400 MHz, D2O, 353 K).
Figure 2. 1H NMR spectra of (a) PLAn (3a, 3b); (b) PLAn-tosyl (4a, 4b), and (c) PLAn-N3 (5a, 5b; 400 MHz, CDCl3, 298 K).
(3a) and 1700 g·mol−1 (3b), which correspond to an average DPn of 86 for 3a and 20 for 3b, were used to convert them into clickable azide-terminated PLA (PLAn-N3, 5a and 5b). As depicted in Figure 1, a two-step procedure was developed where PLAn−OH (3a and 3b) were tosylated under basic conditions to give intermediates 4a (64% yield) and 4b (78% yield) and subsequently substituted with sodium azide in DMF to give, after purification, PLAn-N3, 5a (61%) and 5b (80%). The conversion was quantitative for each step, as indicated by 1 H NMR spectra, where the peak f of the methine proton next
This high degree of conversion further indicated that our strategy using aniline derivative is a powerful method to achieve reductive amination on a large set of relatively low-molecular weight polysaccharides. It is worthy to report that although the reducing-end functionalization of CS has already been done,41,42 their derivatives were not well purified, quantified, and structurally characterized. Synthesis and Structural Characterization of PLAn-N3. Well-defined and commercially available hydroxyl-terminated PLA (PLA−OH, 3) with two different molecular weight, 6500 E
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Figure 3. 1H NMR spectra of (a) CS-b-PLA1700 (6b) and (b) CS-b-PLA6500 (6a) (400 MHz, DMSO-d6/D2O 8:2 v/v, 353 K).
to the terminal chemical function in PLAn−OH at δ 4.30 ppm (Figure 2a) shifted completely at δ 5.15 ppm (Figure 2b) after tosylation and δ 4.01 ppm (Figure 2c) after azidation. The success of the azidation reaction was also confirmed by FTIR evidenced with strong absorbance peaks representing the valence vibration of azide groups at 2118 cm−1 (Figure S4). SEC chromatograms of 4 and 5 were unimodal with low PDIs ( 90%) on Vero cells at a concentration below 10 μg·mL−1 after 72 h of incubation. While for L-929 cells, 6a and 6b concentrations below 50 μg·mL−1 did not show cytotoxic effect. The cytotoxic concentration needed to reduce the cell viability by 50% (CC50 parameter) was calculated for CS-bPLAn against both cells (Vero and L-929). CC50 values were calculated by regression analysis of the dose−response curves (data not shown here) after 72 h of incubation. The CC50 values of 6a and 6b against Vero cells were 140 and 740 μg· mL−1. For the L-929 cells, the CC50 values of 6a and 6b were 518 and 1000 μg·mL−1. From these data is possible to infer that CS-b-PLAn showed higher cytotoxic effect on Vero cells than on L-929 cells. Additionally, for both cell lines tested the copolymer CS-b-PLA1700 (6b) showed the lowest cytotoxicity, probably due to the lowest content of synthetic PLA block. For L-929 cells, even in the highest 6b concentration (1000 μg· mL−1) the cell viability remains superior to 50%.
ACKNOWLEDGMENTS The authors thank CNPq, CAPES (Brazil), and CNRS (France) for the financial support and A.R.F. thanks CAPES for the Ph.D. sandwich fellowship (PDSE, Process No. 857511-8). The authors thank CAPES/CNPq/FAPs for financial support through the Proc. 400702/2012-6 (PVE). The authors are also grateful to COMCAP/UEM for the TEM images and I. Jeacomine and C. Travelet for their technical assistance in NMR spectroscopy and light scattering, respectively. CNRS, Institut Carnot PolyNat, and Labex Arcane ANR-11-LABX0003-01 funding are acknowledged.
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CONCLUSIONS Through this work, a step forward was achieved by completing the existing portfolio of “hybrid” oligosaccharide-based block copolymers by incorporating an important class of biologically relevant polysaccharide as natural block, for example, sulfated glycosaminoglycan. Thus, chondroitin sulfate-b-poly(lactic acid), with varying PLA lengths, were synthesized via clickgrafting onto approach requiring end-modification of both blocks. Reducing-end CS-alkynyl was efficiently prepared by I
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Biomacromolecules
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dx.doi.org/10.1021/bm5005355 | Biomacromolecules XXXX, XXX, XXX−XXX
