Biocompatible and Biodegradable Ultrafine Fibrillar Scaffold Materials

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Biomacromolecules 2008, 9, 1902–1908

Biocompatible and Biodegradable Ultrafine Fibrillar Scaffold Materials for Tissue Engineering by Facile Grafting of L-Lactide onto Chitosan Maciej Skotak,† Alexei P. Leonov,†,‡ Gustavo Larsen,*,†,§ Sandra Noriega,† and Anuradha Subramanian† Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Nebraska 68588-0643, and LNK Chemsolutions, LLC, 4701 Innovation Drive, Lincoln, Nebraska 68521 Received February 13, 2008; Revised Manuscript Received April 12, 2008

A chitosan derivative was prepared with good yields using a “one pot” approach by grafting L-lactide oligomers via ring opening polymerization. Side chains are primarily attached to hydroxyl groups located on carbons 3 and 6 of the glucosamine ring, while the amine group remains nonfunctionalized. By increasing the L-lactide to chitosan ratio, side chain length is controlled. This allows the manipulation of the biodegradation rate and hydrophilicity of the tissue engineering scaffold material. This general synthetic route renders functionalized chitosan soluble in a broad range of organic solvents, facilitating formation of ultrafine fibers via electrospinning. Cytotoxicity tests using fibroblasts (L929 cell line) performed on electrospun L-lactide modified chitosan fibers showed that the specimen with the highest molar ratio of L-lactide (1:24) investigated in this study is the most promising material for tissue engineering purposes, while less stable formulations might still find application in drug delivery vehicles.

Introduction Chitosan is a deacetylated derivative of chitin (N-acetyl-2amino-2-deoxy-D-glucopyranose units, linked by β-D(1-4) bonds), the second most abundant polysaccharide after cellulose. For the glucosamine chains to be considered “chitosan”, a degree of N-deacetylation of at least 50% is normally suggested. Due to its ubiquitous presence and unique properties (biodegradability, antibacterial, antifungal, mucoadhesive, and wound healing activity) it motivates research in many areas1–3 and is found as an ingredient in several commercial products.4–7 Intra- and intermolecular hydrogen bonds created among hydroxyl groups of the glucosamine (or acetylglucosamine) rings render chitosan a partially crystalline substance, insoluble in water at neutral pH.8,9 Solubility of chitosan in diluted acids is due to protonation of the amine group, which gives it a polyelectrolyte character in solution, while on a molecular level it still remains a fairly rigid polymeric chain.6 This is likely one of the primary reasons why very few reports on the processing of pure chitosan solutions via electrospinning (without the use of plasticizers)10–13 exist, while electrospinning of chitosan-based formulations containing good fiber-forming polymers like polyethylene oxide (PEO),14–17 polyvinylalcohol (PVA),18–21 or polyvinylpyrrolidone (PVP) (with quaternized chitosan)22 have been widely documented. The fact that additivefree chitosan aqueous acidic solutions normally have electric conductivities and surface tensions that are too high to process via electrospinning or electrospray presents another challenge. Putting aside for a moment the intrinsic value of making modified chitosans for a variety of applications, from a mere electrospinning processing standpoint, it is advantageous to make * To whom correspondence should be addressed. E-mail: glarsen@ unlserve.unl.edu. † University of Nebraska-Lincoln. ‡ Current Address: Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084. § LNK Chemsolutions, LLC.

such derivatives if they can be dissolved in less electrically conducting, lower surface tension solvents. Sachiwa and co-workers23 used organic acids with relatively bulky substituents (e.g., p-toluenesulfonic, salicylic, camphorsulfonic, and methanesulfonic acids) to make the corresponding chitosan salts. After lyophilization these salts were soluble in dimethylsulfoxide (DMSO), but we were unable to produce electrospun fibers using these solutions directly: The reason is they still had unsuitably high conductivities and surface tension for electrospinning. At room temperature, the surface tension of DMSO is 43.50 dyn/cm, while for ethanol, a frequently used solvent for electrospinning of polymer solutions, it is only 22.40 dyn/cm. However, these salts and their DMSO solubility opened up an opportunity to develop a relatively simple L-lactide chitosan modification route and, consequently, materials that are easily processed into ultrafine fibrous mats via electrospinning. As a starting point for our research, the rationale was that the backbone needed to remain relatively intact to facilitate this type of processing in light of the polymer entanglement theory adapted to electrospinning.24 To our knowledge, the only available account on electrospinning of a similar grafted polymer type chitosan material was that of a hexanoyl chitosan form and its blends with polylactic acid (PLA).25,26 Polylactic acid is a biodegradable, resorbable aliphatic ester that in recent years received significant attention in the biomedical research field.27,28 It is also generally viewed as a “polymer of the future” because cost of production keeps on decreasing and it could be disposed easily without any significant environmental penalty.27 There are two general methods for polylactic acid (PLA) manufacturing: condensation of lactic acid and ring opening polymerization of lactides. The first one results in low molecular weight polymers with properties that are not suitable for a number of practical uses.27 On the other hand, ring opening polymerization (ROP) yields high molecular weight materials with narrow molecular weight distributions.29 ROP of lactides can be carried out with the aid of four different

10.1021/bm800158c CCC: $40.75  2008 American Chemical Society Published on Web 06/10/2008

Fibrillar Scaffold Materials for Tissue Engineering

Figure 1. 1H NMR spectrum of HMWCHIT-PLA 1:6 in DMSO-d6. Assignment of peaks and structural features of grafted chitosan are presented. Poly-L-lactide oligomers attached to their respective hydroxyl groups on acetylglucosamine are not drawn for clarity.

classes of initiators and reaction mechanisms: (a) cationic, (b) anionic, (c) coordination insertion, and (d) catalyzed by enzymes.26 Of crucial importance, however, is that for biomedical applications only nontoxic and biocompatible catalysts need to be selected because some catalysts may terminate a polymer chain and remain attached to it or simply be hard to remove due to entrapment inside the bulk of the polymer material, thereby contributing to its cytotoxicity after hydrolysis in vivo.30,31 In our effort to design a synthetic route for making chitosan-PLA polymeric chains for tissue engineering scaffolds via electrospinning, the issue of minimizing or eliminating altogether the use of a strongly cytotoxic catalyst for the PLA grafting process was clearly a crucial one. Due to the presence of hydroxyl and amine groups in chitosan, we reasoned these could in principle act as protolytic agents (initiators) in the ROP reaction of lactides just like cellulose does.32 In the literature, there are a few successful examples of this approach for grafting chitosan with lactides.9,33 However, these reactions were normally carried out at elevated temperatures (80 °C33 and 90-130 °C9), and this likely contributes to racemization of polylactide chains, which takes place above 50 °C.28 Reactions carried out under heterogeneous conditions gave rather moderate reaction yields, despite the fact that usually very effective lactide polymerization metallorganic catalysts were used. Condensations of L-lactic acid34 or D,L-lactic acid35 were also evaluated for the grafting of the chitosan chains. In this study, we demonstrate a simple and effective “onepot” grafting type modification of chitosan by ring opening polymerization of L-lactide, with methanesulfonic acid playing a dual solvent and catalyst role. Due to protective protonation of the amine group in acidic medium, grafting of L-lactide oligomers takes place predominantly on hydroxyl groups. Thus, three types of functional groups become evident upon characterization of the grafted polymer: primary amine and two types of hydroxyls (terminal L-lactide and terminal chitosan, located on the 1 and 4 carbon atoms, see Figure 1). The modified chitosans were found to be soluble in a variety of common organic solvents, and processing them into micron-range fibers via electrospinning was a relatively simple task. Cytotoxicity tests using fibroblasts (L929 cell line) performed on L-lactide modified chitosan showed that the sample with the highest molar ratio of L-lactide (1:24) was the most promising material for tissue engineering purposes.

Experimental Section Materials. Chitosan powder HMWCHIT (high molecular weight, Aldrich, Brookfield viscosity as 1% w/w solution in 1% acetic acid:

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800 cP) or LMWCHIT (low molecular weight, Vanson-Halo, Brookfield viscosity: 80 cP) was vacuum-dried at 50 °C for 3 h using a vacuum line equipped with liquid nitrogen traps and then stored in a desiccator. Both chitosan samples revealed a degree of deacetylation (%DD) of 80% via analysis of their 1H NMR spectra in 1% CD3COOD/D2O (5 mg in 0.5 mL) collected at 70 °C.6,36 After characterizing the CHITPLA samples with single detector GPC system, it was realized that the HMWCHIT-based samples were the most attractive ones (better processability, narrow polydispersities, etc.). Thus, we submitted one sample of HMWCHIT to Viscotek, Inc. for further characterization with their Triple Detection System technology. The sample was thus analyzed using two ViscoGel C-MBHMW-3078 columns with 0.02 M ammonium acetate buffer (pH ) 4) as solvent, at a 1.0 mL/min flow rate, and injection volume 0.1 mL. Column temperature was 30 °C and chitosan concentration is ∼1.5 mg/mL. Results are listed in Table 1. Poly-L-lactide (MW 175,000 Da, spherical granules OD 2-4 mm) was purchased from Cargill Inc. (Minneapolis, MN). Prior to use, L-lactide (98%, Alfa) was recrystallized from toluene, vacuum-filtered in an argon atmosphere, vacuum-dried, and stored in a desiccator. Methanesulfonic acid (>99.5%, Aldrich), chloroform (99.9%, Sigma-Aldrich), acetonitrile (99.99%, EM Science), and THF (min. 99.0%, Mallinckrodt) were used as received. Chitosan Grafting with L-Lactide. In a typical procedure, a flamedried 50 mL round-bottom flask equipped with a Teflon-coated stirbar was used to dissolve 600 mg of chitosan in 10 mL of methanesulfonic acid. For stoichiometry purposes, this mass of chitosan translates into 3.5 mmol of glucosamine units, each with a “weighted” MW of 169.61 g/mol, taking into account %DD. The mixture was stirred for 10 min at 40 °C to allow the solid material to dissolve, followed by the addition of 3.05 g of the L-lactide monomer (21.2 mmol, 6 equiv). This reaction mixture was stirred for 4 h at 40 °C under an argon atmosphere and then transferred by plastic pipette to a 250 mL beaker containing 100 mL of 0.2 M KH2PO4, 16 mL of 10 M NaOH, and crushed ice. Such an excess of cooled solution was used to neutralize the methanesulfonic acid, effectively avoiding excessive heat release (which was found later to lead to degradation of the polymer chains). The buffering properties of the monobasic phosphate ion avoid rapid and uncontrolled pH changes during the quenching of the reaction. Isolation of a product suitable for electrospinning and with good yields (70-80%) was not possible if this last step was skipped. Precipitated L-lactide chitosan grafts were vacuum-filtered and washed with four 100 mL aliquots of deionized water and then vacuum-dried overnight at room temperature using a vacuum line with two liquid nitrogen traps. FTIR, UV-Vis, and NMR Spectroscopy. FTIR spectra were recorded in the transmittance mode using a Nicolet 20XB spectrometer, in the range of 4000-400 cm-1, usually with a spectral resolution of 2 cm-1. A sealed liquid cell with KBr windows with circular aperture and a 0.015 mm optical path length was used. After normalization and conversion to absorbance units, the solvent (chloroform or acetonitrile) spectrum was subtracted from all spectra. UV-vis spectra were collected using a Beckman DU-640 spectrophotometer. In a typical experiment, 0.5 mL of solution was transferred into the cuvette with the aid of a Pasteur pipet. The cell was made of quartz (“Q” type) and has a 2 mm optical path. Spectra were recorded in the 200-800 nm range with resolution of 1 nm, with respective solvents (methanesulfonic acid or THF) used as the blank. 1 H and 13C NMR spectra were collected on a Bruker Avance 500 NMR spectrometer at 500.13 and 125.75 MHz, respectively. A 5 mm triple-resonance Cryoprobe (1H, 13C, 15N) with XYZ gradients was used. All experiments with L-lactide grafted chitosan samples were performed at 25 °C, with DMSO-d6 (D 99.9% atom, Cambridge Isotope Laboratories) as a solvent and tetramethylsilane (TMS, 99.9+%, NMR grade, Aldrich) as a reference. Pulse repetition delay of 10 s was used to facilitate full relaxation of the molecules. Peak assignments were based on HMQC (1H-13C) spectra of selected samples in DMSO-d6 and were supported by simulated 1H NMR spectra generated by the ACD/HNMR Predictor (ACD Laboratories).

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Table 1. Characterization of Poly-L-lactide Grafted Chitosans by GPC and 1H NMR 1

GPC analysis sample ID

a

yield %

HMWCHIT poly-L-lactide

H NMR analysis

b

Mn

Mw

Mz

Mp

PI

313522 61816

517758 161726

1296000 251828

228879

1.62 2.62

c

d

MU

%DD

DSe

SCLf

HMWCHIT-PLA 1:4 1:6 1:12 1:18 1:24

69.0 72.0 83.0 75.0 74.6

2731 4524 8232 8152 8946

6006 13246 27213 21613 26347

10663 30162 63565 45142 57849

4687 8210 13004 11663 13075

2.20 2.93 3.31 2.65 2.95

4.6 9.1 12.1 7.6 8.1

72.0 68.9 66.9 65.0 62.8

1.8 2.0 1.8 2.0 2.4

4.6 5.7 10.2 12.8 14.7

8212 35778 13643 11386 11656

3.83 4.16 4.35 3.15 3.62

13.8 46.8 17.2 8.3 7.0

73.4 70.8 66.0 68.4 64.4

1.4 1.7 1.4 1.8 2.0

4.4 5.9 9.9 12.8 16.0

LMWCHIT-PLA 1:4 1:6 1:12 1:18 1:24

70.5 78.0 82.0 76.5 81.0

4633 37627 8695 7446 6350

17748 48382 37802 23449 22955

48027 63751 92610 52783 53902

a Reaction yield based on the mass of isolated, dried product and masses of substrates. b Polydispersity index (Mw/Mn). c Number of monomer units per molecule of grafted chitosan; this calculation was based on Mw and a rounded-off value of SCL obtained from 1H NMR analysis. d Degree of deacetylation of poly-L-lactide grafted chitosans; average value estimated from integrated peak areas of acetal proton signals (1/(1 + 1′) × 100%) and integrated peak areas of protons located on the carbon atom to which the amine group is attached, and one-third of the integral of methyl group of acetylamine (2/(2 + (2′:CH3/3)) × 100%). Refer to Figure 1 for more details. e Degree of substitution of a single glucosamine unit of chitosan by L-lactide oligomers; average value estimated from integrated peak areas of the proton on R carbon signals of terminal L-lactide group versus integral of acetal protons of glucosamine (8′′/(1 + 1′) × 100%), and integral of protons located on carbon atom to which amine group is attached, and one-third of the integral of methyl group of acetylamine (8′′/(2+(2′:CH3/3)) × 100%). Refer to Figure 1 for more details. f Side chain length, with average values estimated using two sets of peaks (8”/(8” + 8,8′) × 100%, or 8”:CH3/(8”:CH3 + 8,8′:CH3) × 100%).

Gel Permeation Chromatography. GPC data were collected with HPLC grade THF as an eluent, using a Viscotek GPCmax integrated pump, autosampler and degasser module system, equipped with Viscotek Model 3580 Differential Refractive Index detector. The column used was a Jordi Gel DVB Organic Solvent (catalog # 15023; pore size, 10000 Å). The retention times were calibrated against a set of Viscotek PolyCAL Universal Calibration Standards, which consisted of a mixture of seven polystyrene standards with molecular weights of 410, 170, 116, 29.6, 13.4, 6.04, and 1.05 kDa. Typically, 20 mg of each sample was dissolved in 2.0 mL THF. Column temperature was set to 35 °C, eluent flow rate was 1 mL/min, and injection volume was 100 µL. Electrohydrodynamic (EHD) Processing, SEM Imaging. The L-lactide grafted chitosan solution was loaded into a Hamilton series 1000 gastight syringe (model 1001, 1 mL). A 19 gauge blunt needle was attached by connecting it to the syringe through an appropriate fitting to serve as electrospinning nozzle. The syringe was placed on the syringe holder of a digital syringe pump (Cole-Parmer 74900-00, Vernon Hills, IL). Voltages in the range of 5-15 kV, (Gamma High Voltage Research ES30P-5W/PRG, Ormond Beach, FL) were applied to the nozzle. In the course of a typical experiment, various flow rates in the range of 0.2-2 mL h-1 were tested; collection time was limited to 10 min. The distance between the tip of the nozzle and the collector plate was fixed to 10 cm. Fibers were collected over the surface of an aluminum foil wrapped around the circular copper disk (OD ) 15 cm) serving as collector/counterelectrode. After EHD processing, the fibers were left for 24 h on the aluminum foil. Subsequently, SEM imaging experiments were performed with the aid of a Hitachi S-3000N microscope, using an accelerating voltage of 15 kV. Samples were coated before SEM characterization with metallic gold for 2 min, while keeping the electrical current inside the chamber of the Technics Hummer II sputter coater constant (10 mA). Statistical analysis was performed on sets of at least 50 counts within each specimen. Extracting Leachables for Cell Culture Tests. A total of 300 mg of each sample were contacted with 5 mL of DMEM cell culture medium in a 15 mL centrifuge tube and placed in a shaker incubator (37 °C, 60 rpm) for 3 days. After 3 days, medium was filtered with 0.45 µm sterile filter into a sterile container. Medium was stored at 4 °C in a refrigerator before use.

Cell Culture and Proliferation. Fibroblast cells (L929, ATCC) were seeded in a 96-well polystyrene tissue culture plate (TCP, Falcon) at 3 × 104/well. Cells were left in the cell culture incubator overnight to settle and attach to the bottom of the plate. Cell culture medium (DMEM, Invitrogen CA) was replaced by the extracted leached medium from the corresponding material, 100 µL/well. Cells were allowed to grow for 3 days before performing cell viability and proliferation tests. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) Viability Test. Wells were rinsed with sterile phosphate buffered saline (PBS) to remove phenol red from the culture media. A total of 50 µL of MTT solution (1 mg/mL in phenol red-free medium) was added to each well. Plates were incubated for 4 h in a CO2 cell culture incubator, MTT solution was removed, and 100 µL of isopropanol was added to each well. Plates were incubated for 10 min in the incubator, followed by incubation for 15 min at room temperature. Absorbance at 595 nm was taken by a microplate reader (Elx800, Bio-Tek Instrument, Clarkston, MI). Results were expressed in percentage of viability compared to control (% of control). MTT Solution Preparation. A total of 50 mg of MTT (Sigma Aldrich, St. Louis, MO) was dissolved in 10 mL of sterile PBS solution, then taken up to 50 mL with DMEM/F-12 phenol red-free medium (1:5 dilution). The final concentration was 1 mg/mL. Cell Proliferation in Leachables (Cell Count). Culture media was removed and wells were rinsed with PBS. Cells were released from the bottom of the plates by adding 100 µL of trypsin solution (0.05% trypsin in HBSS-Ca, -Mg) to each well. After 10 min, 100 mL of DMEM medium was added. Cells count was performed by using a hemocytometer and back calculating the total cells in each well. Cell number was compared with control wells.

Results and Discussion Chitosan Grafting with L-Lactide. Grafting of L-lactide onto chitosan, as indicated earlier, targeted the hydroxyl groups located on carbon atoms 3 and 6 (Figure 1) to initiate the polymerization process. This occurred in spite of the fact that primary amines are known to be effective initiators for the 37,38 L-lactide ROP reaction. A similar approach was recently used to produce water-soluble8 and photosensitive chitosan.39



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Figure 3. 13C NMR spectrum of HMWCHIT-PLA 1:12 in DMSO-d6. Assignment of peaks is based on the drawing shown in Figure 1. Figure 2. ROP grafting of L-lactide onto chitosan. (A) Ex situ reaction screening of HMWCHIT-PLA 1:6 sample (600 mg of chitosan was dissolved in 10.0 mL of methanesulfonic acid). (B) UV-vis spectra of various LMWCHIT-PLA samples in THF (100 mg dissolved in 2.0 mL).

At the relatively mild temperature of 40 °C, chain growth was still initiated easily, and just after a few minutes, a noticeable color change in the reaction mixture was observed (Figure 2). As the reaction progressed, a viscosity increase was also noticed, and it affected more significantly those mixtures with higher ratios of chitosan to L-lactide. Side chain growth was assumed to be complete after 4 h, and it was controlled by varying the molar excess of L-lactide relative to the chitosan ring equivalents. Desired products were synthesized with good reaction yields (Table 1) and observed within the whole set of formulations developed in this study. Water molecules generated during the esterification reaction are most probably consumed during degradation of chitosan ether type linking groups, because no noticeable amounts of pure poly-L-lactide chains were detected. The water content increase during the course of reaction is below the level needed to compete as an initiator with hydroxyl groups of chitosan, but it might nevertheless influence the stability of the chitosan backbone toward the end of the reaction. Structural Characterization of Poly-L-lactide Grafted Chitosan by NMR and FTIR Spectroscopy. The 1H NMR spectrum of HMWCHIT-PLA 1:6 in DMSO-d6 is shown in Figure 1. Signals from protons 4, 5, 6, and 6′ of the glucosamine ring did not experience chemical shift changes with respect to unmodified chitosan (see Figure S1 in Supporting Information for more details). Protons 1 and 2 experience upfield shifts, while the proton 3 signal is downfield shifted by about 0.7 ppm relative to those of unmodified chitosan. The presence of just four glucosamine ring protons in grafted chitosans instead of the five in pure chitosan was evident by integration of these NMR signals (3.5-4.00 ppm region). The detailed position of the NMR signals of the glucosamine ring protons is not crucial to extract structural information about the grafts, because there are at least two internal reference signals that can be used for this purpose: (A) The sum of integrals of acetal carbon protons (1 and 1′), or (B) amine group protons (2 and 2′, i.e., 2′ as one-third of integral of 2′:CH3 signal). Poly-L-lactide side chains give distinct 4.20 and 5.20 ppm R-carbon proton signals from the terminal and internal groups, respectively. Integration of these two peaks correlate very well with the integrals of methyl group bands at 1.47 and 1.29 ppm (Figure 1, denoted as 8, 8′: CH3 and 8′′:CH3). The 13C NMR spectrum (Figure 3) is relatively simple to interpret, with a signal that is characteristic of chitosan acetal carbon 1 located at 100.0 ppm. The presence of only two peaks

Figure 4. FTIR spectra of HMWCHIT-PLA (A) and LMWCHIT-PLA (B) grafts in acetonitrile (solvent spectrum was subtracted).

in the “methyl” region of the spectrum indicates that all methyl groups of poly-L-lactide side chains have similar chemical environments. Thus, their proposed ordering is depicted in Figure 1. Carbonyl carbons (7, 7′, and 7′′) are particularly sensitive toward rearrangements of their chemical environment,28 such as stereochemical modifications. In our case, it is apparent that no racemization occurred during the reaction. The FTIR spectra of grafted chitosans in two solvents with different polarities are presented in Figures 4 and 6. Characteristic infrared absorption bands for chitosan are present in the 1500-1700 cm-1 range: (A) the amide I (CdO stretching) band at 1682 cm-1, shifted toward higher wave numbers with respect to solid state IR spectra of chitosan (see Supporting Information, Figure S2), which is characteristic of amides in solution,40 (B) N-H bending from the primary amine group at 1610 cm-1, and (C) the amide II band at 1535 cm-1 (N-H bending from the amide group). Oligo-L-lactide side groups are characterized

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Figure 5. FTIR spectra of HMWCHIT-PLA (A) and LMWCHIT-PLA (B) grafts in chloroform (solvent spectrum was subtracted).

Figure 6. FTIR spectra of HMWCHIT-PLA (A) and LMWCHIT-PLA (B) grafts in chloroform (solvent spectrum was subtracted). CdO stretching region, complementary to Figure 5, is presented. Signal intensity increases with increasing length of poly-L-lactide side chain.

by one very strong (CdO) band from ester groups at 1758 cm-1 and three (C-H) bands at 2996, 2943, and 2879 cm-1. The band at 3030 cm-1, present on spectra collected using chloroform, is a residual band from the solvent, which was impossible to remove (Figure 5). It is not present when acetonitrile was used as solvent instead (Figure 4). The intensity of the band at 1757 cm-1 increases with increasing PLA/CHIT molar ratio (Figure 6), which was an anticipated result in light of NMR observations. Different solvent polarities cause different folding of chitosan-L-lactide grafts, that is, hydroxyl terminal groups tend to “hide” in chloroform, creating inter- or intramolecular bonds (Figure 5), as they appear as two well-resolved bands at

Skotak et al.

3500 and 3635 cm-1. The presence of two bands indicates that two types of hydroxyl groups are present. Table 1 compiles values of calculated structural parameters, extracted from GPC and NMR measurements. Chitosan from two different sources, with different molecular weights but similar %DD, was used to evaluate its extent of degradation during the course of reaction. It is apparent that after L-lactide grafting, all samples have similar remaining chitosan chain length based on MU numbers. A very high MU value can be explained by nonhomogeneous distribution of acetyl-glucosamine units within the whole batch, which translates into different hydrolysis rates of glycosidic bonds. Relative hydrolysis rates of glycosidic bonds connecting at least one acetylglucosamine are approximately 103 times more susceptible to hydrolysis than their glucosamine linking counterparts (see Scheme 3 in ref 41, for more details). Upon grafting, the average degree of deacetylation does not decrease by more than 10%, which indicates protonation of the amine group is sufficient to shield its nucleophilicity. On average, there are two side chains of oligo-L-lactide per glucosamine ring, and their length depends on the initial reagents ratio. If this ratio increases, additional oligo-L-lactide graft might be attached to hydroxyl groups located on carbons 1 or 4 (they are generated in situ during degradation of chitosan, when water content increases as a byproduct of the main esterification reaction). The polydispersity of newly generated grafts, especially those based on HMWCHIT samples, are comparable to those of commercially available high molecular weight poly-L-lactide (Table 1). Neither NMR nor FTIR spectra gave any indication of the presence of residual methanesulfonic acid. Thus, only hydrolytic degradation of oligo-L-lactide side chains is expected to contribute the most to cytotoxicity tests. Electrospinning and Cytotoxicity Tests. Ethyl acetate (EtOAc) and 2-butanone (MEK, methyl ethyl ketone), two solvents with very similar properties (Table 2), were selected to perform spinnability tests; main selection criterion was surface tension value needs to be comparable with that of ethanol. Low health hazard rating according to NFPA 704 classification eliminated possible solvent toxicity concerns from our studies. Typically, it was necessary to prepare relatively concentrated solutions (at least 40% w/w) of grafted chitosans to obtain fibers with well-defined morphologies. This may suggest during synthesis degradation of chitosan in the presence of a harsh solvent environment could be important. Nonetheless, regardless of what solvent was used, it was not possible to produce fibers with either CHIT-PLA 1:4 samples. With the LMWCHIT-PLA 1:6 sample dissolved in 2-butanone, some difficulties in processing were experienced (Figure 7). The influence of applied voltage on fiber diameter was evaluated for sample HMWCHIT-PLA 1:6 (Figure 8). Interestingly and as opposed to what is typically found in the literature, fiber diameter increased (p < 0.05) with voltage.42–45 This may be an artifact, as the actual morphology of collected strands observed by SEM is ribbon-like (Figure 7). Collapse of soft, solvent-filled fibers into ribbons may have just happened post modum. Similar morphologies and characteristic dimensions were reported with electrospun hexanoyl chitosan samples.25 Cytotoxicity tests performed on “as prepared” chitosan grafts were compared to the pure high molecular weight chitosan and poly-L-lactide. Results are summarized in Figure 9. After 3 days of incubation in cell culture media, the most lethal to cells is the material with the lowest ratio of chitosan to L-lactide. With an increase of this characteristic parameter, the number of cells surviving the test gradually increases, with HMWCHIT-PLA



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Table 2. Properties of Solvents Used for Electrospinning of L-Lactide Grafted Chitosans

ethyl acetate 2-butanone a

Health.

b

NFPA 704 standards

BP, °C

surface tension, dyn/cm

dielectric constant

dipole moment

H

Fb

Rc

78 80

23.97 24.53

6.02 18.56

1.78 2.78

1 1

3 3

0 0

a

Flammability. c Reactivity.

Figure 8. Fiber diameter of HMWCHIT-PLA 1:6 as a function of processing voltage. Electrospinning was performed using 44.0% (w/ w) solution in ethyl acetate (EtOAc). Flow rate, 0.2 mL/h; nozzlecollector distance, 10 cm.

Figure 7. SEM images of electrospun fibers using solutions of 44% (w/w) HMWCHIT-PLA in ethyl acetate and 50% (w/w) LMWCHITPLA in 2-butanone (left and right panels, respectively). Samples with particular original chitosan to L-lactide ratios were used: (A) 1:6; (B) 1:12; (C) 1:18; (D) 1:24. Scale bar: 20 µm. Samples with CHIT-PLA 1:4 were not processable at such high concentrations due to gelation.

1:24 sample almost achieving the performance of poly-L-lactide and also becoming comparable to that of pure medium. The leachates-based instability for tissue engineering purposes of the remaining samples would demand more frequent medium replacements if they were still under consideration to ultimately fine-tune the hydrophobicity and biodegradability of the tissue engineering scaffold. This practice would reduce the amount of free lactic acid, which appears to be the major culprit of lethality: We observed a rapid and marked decrease of pH in the case of 1:4 sample, from 7.45 to 4.4 in 3 days (pKa of lactic acid is 3.85, pH of pure DMEM is 7.80) and decomposition of just 6.5 wt % of initial sample mass is responsible for this effect. This corresponds to an L-lactic acid concentration of 35 mM. Indeed, even for linear poly(D,L-lactides; PLDA, Mw range 95300-132800 Da), a pH value of about 2 after six weeks was reported in studies of osteoblast viability and proliferation. The adverse effect of in situ generated D,L-lactic acid was greatly suppressed by modification with maleic anhydride (MA) and

Figure 9. Viability (A) and proliferation (B) test results carried on a series of selected HMWCHIT-PLA samples, poly-L-lactide (PLA), and untreated high molecular weight chitosan (CHIT). Dubelco’s modified Eagle medium was used as a control, while fresh medium allowed the checking of the effect of incubation (9 indicates mean value).

subsequent immobilization of butylene diamine (via N-acylation of anhydride),46 due to the proton acceptor properties of the later. However, liberation of L-lactic acid alone cannot be considered as the only source of cell proliferation inhibition: These might also include unfavorable effects associated with the presence of glucose47 or increased levels of phosphate ions, which are nowadays considered as a major cause for muscle fatigue, instead of lactic acidosis.48 During intense muscle activity, intracellular pH can fall by ∼0.5, and such a decline was considered acidification in ref 48. In our case, glucose levels were the same in each case, thus, its negative influence on fibroblast viability can be ruled out. If increased phosphate concentrations have any adverse effect on viability of fibroblasts,

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this is strongly correlated with a decrease in pH, which in turn is directly correlated with transient L-lactic acid release.

Conclusions Using a one-pot approach we synthesized a range of L-lactide grafted chitosan polymers with different side chain lengths controlled by the molar ratio of the substrates. Success achieved by using low temperatures and short reaction times point to the versatility of our approach, with the main challenge being establishing an appropriate isolation procedure for the product. Graft chitosan-L-lactides are soluble in a broad range of organic solvents (see Table 2 in Supporting Information), which facilitated ample choice of solvent for electrospinning based on their biocompatibility and nontoxicity. Terminal functional groups and branched architecture also enable a versatile route for further functionalization of this material to tune its physical and chemical properties for a specific application. In tissue engineering, in particular, the fine-tuning of the hydrophobicity and bioabsorbability of the material is of crucial importance. The relatively short molecular fragments present in L-lactide grafted chitosan are expected to contribute greatly to enhanced and controlled biodegradability.49,50 Indeed, L-lactide grafted chitosan samples display a wide range of cytotoxicities, as demonstrated during cell culture tests. This is expected to be a strong function of the oligo-L-lactide chain length (SCL), which is a key parameter controlling biodegradation, as it was demonstrated for star-shaped, polylactide grafted poly(amidoamine) dendrimers, PAMAM-g-PLA.51 Thus, besides tissue engineering, these materials might be quite interesting for other applications, such as controlled release and drug delivery, where hydrolysis rate control is of key importance. To further explore the issues of biodegradability and hydrophobicity control, research is currently underway in our group to also graft the chitosan chains with -caprolactone and produce similar electrospinning amenable biopolymers under reaction conditions similar to those used for L-lactide grafting. Acknowledgment. The authors thank financial support from the Nebraska Research Initiative, and the National Institutes of Health. Valuable comments made by Mrs. Sara Basiaga during NMR data acquisition are gratefully acknowledged. The GPC analysis was carried out thanks to Dr. Redepenning at UNL Chemistry Department. We are also grateful to Dr. Noureddini for facilitating use of his group’s lyophilizer. Supporting Information Available. 1H NMR and FTIR spectra of chitosans, FTIR spectrum of poly(lactic acid), solubility table of HMWCHIIT-PLA grafts in various organic solvents, HMQC spectra of HMWCHIT-PLA 1:6 in DMSOd6, after 3 and 4 h of reaction. This information is available free of charge via the Internet at http://pubs.acs.org.

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