Article pubs.acs.org/Biomac
In Situ Cross-Linkable Hydrogel of Hyaluronan Produced via CopperFree Click Chemistry Akira Takahashi,† Yukimitsu Suzuki,† Takashi Suhara,† Kiyohiko Omichi,‡ Atsushi Shimizu,‡ Kiyoshi Hasegawa,‡ Norihiro Kokudo,‡ Seiichi Ohta,§ and Taichi Ito*,†,§ †
Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Center for Disease Biology and Integrative Medicine and ‡Department of Surgery, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
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ABSTRACT: Injectable hydrogels are useful in biomedical applications. We have synthesized hyaluronic acids chemically modified with azide groups (HA−A) and cyclooctyne groups (HA−C), respectively. Aqueous HA−A and HA−C solutions were mixed using a double-barreled syringe to form a hydrogel via strain-promoted [3 + 2] cycloaddition without any catalyst at physiological conditions. The hydrogel slowly degraded in PBS over 2 weeks, which was accelerated to 9 days by hyaluronidase, while it rapidly degraded in a cell culture media with fetal bovine serum within 4 days. Both HA−A and HA−C showed good biocompatibility with fibroblast cells in vitro. They were administered using the double-barreled syringe into mice subcutaneously and intraperitoneally. Residue of the hydrogel was cleared 21 days after subcutaneous administration, while it was cleared 7 days after intraperitoneal administration. This injectable HA hydrogel is expected to be useful for tissue engineering and drug delivery systems utilizing its orthogonality.
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INTRODUCTION Injectable hydrogels are useful in biomedical engineering applications such as drug delivery,1 prevention of postoperative adhesions,2,3 and tissue engineering.4 For example, fibrin is a natural injectable hydrogel used therapeutically for hemostasis.5 Sodium alginate extracted from seaweed is well-known to form a hydrogel by the addition of calcium ions.6 Hyaluronan (HA)7 is a major component of the extracellular matrix found in various tissues such as cartilage and the eye. Thanks to its origin and unique characteristics, in situ crosslinked hydrogels composed of HA have been studied intensively.8 Various chemical reactions, such as the formation of Schiff bases,9 disulfide bonds,10 and Michael additions,11,12 have been evaluated, and these HA hydrogels showed good biocompatibility. However, the nonspecific reactions between biomolecules of tissues and reactive functional groups are unavoidable and affect the performance of in situ cross-linked hydrogels. For instance, in the case of Schiff bases, the chemical reactions between membrane proteins or gangliosides of cells and the aldehyde groups of dextran derivatives were hazardous in the dextran-based antiadhesive materials,13 where the nonspecific reaction might have prevented healing of traumas. Recently, in situ cross-linking by Schiff bases had been controlled more precisely.14 That is to say, the stability of hydrozone-linkages was dramatically improved.14 However, nonspecific reactions between aldehydes and biomolecules are not avoidable yet. Therefore, other orthogonal reactions,15,16 in which the components react together in high yield and in the presence of many other functional groups, were considered for in situ cross-linking of HA to avoid nonspecific reactions; the © XXXX American Chemical Society
thiol−disulfide exchange reaction using pyridine-2-thione was utilized.17 A Diels−Alder reaction between furan-modified HA and dimaleimide poly(ethylene glycol) was reported to achieve in situ cross-linking.18 A thiol−ene reaction was also used, combined with photopolymerization.19,20 In a specific enzyme reaction, tyramine-modified HA was cross-linked by peroxidasecatalyzed oxidative coupling in situ.21 Potential medical applications of these compounds are versatile, including peritoneal adhesion prevention barriers,2,3,22 drug delivery for peritoneal dissemination23 and sciatic nerve blockade,24 and injectable scaffold materials for tissue engineering.11,25−28 In addition to these injectable HA hydrogels, a click reaction between azido- and alkynyl-amide derivatives of HA was reported recently.29−31 Although yeast cells were encapsulated in these normal clickable HA gels,30 animal cells were not evaluated. In spite of the orthogonality of the click reaction, the need for copper ions as a catalyst is still a concern.32 However, strain-promoted [3 + 2] cycloaddition using cyclooctyne derivatives32,33 enabled the click reaction without catalysis under normal physiological conditions. For in vivo imaging, the reaction between N-azidoacetylmannosamine and the Alexa Fluor 488 derivative of difluorinated cyclooctyne was studied; there was reaction specificity even in vivo between azide groups and cyclooctyne groups.34,35 The reaction was applied to a peptide and polyethylene glycol hydrogel for in vitro cell encapsulation.36,37 Thus, the copper-free click reaction has Received: July 2, 2013 Revised: August 21, 2013
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Figure 1. Schematic diagram of in situ cross-linking hydrogels of hyaluronan by copper-free click chemistry (cHA hydrogel). DMSO and stirred for 1 h at room temperature.38 Then, CDI and ABA were added to HA−TBA dropwise at room temperature, and the mixture was stirred for 20 h at 60 °C. The solution was dialyzed against distilled water and aqueous sodium chloride solution and then lyophilized. Synthesis of Hyaluronan-Modified with Cyclooctyne Groups (HA−C). Cyclooct-1-yn-3-glycolic acid (CGA) was synthesized as reported.32 HA−TBA (0.63 g) was dissolved in 20 mL of DMSO. CGA (0.36 g) and CDI (0.32 g) were dissolved in 3 mL of DMSO. The remainder of the procedure was the same as that for HA−A. Preparation of Disk Hydrogels. HA−A (3 wt %) and HA−C (3 wt %) in PBS were injected into a rubber mold sandwiched between two slide glasses using a double-barreled syringe (Baxter, Deerfield, IL). The prepared hydrogel disks were 1.2 cm in diameter and 3.5 mm thick. Below, these cross-linked hydrogels are termed cHA hydrogels. Characterization of Polymers and Hydrogels. The syntheses of HA−A and HA−C were confirmed by 1H NMR (α500, JEOL, Japan) and FT-IR (FT/IR 4000, JASCO, Japan). The polymers were dissolved in D2O for 1H NMR analysis. FT-IR spectra of HA−A and HA−C were determined using a potassium bromide (KBr) tablet of each polymer. Reaction kinetics between ABA and CGA were measured by FT-IR. ABA and CGA (100 μL, 10 mM in chloroform) were mixed in an optical monocrystal NaCl cell (JASCO, Japan), and the absorbance at 2100 cm−1 was tracked immediately after the mixing for 20 min at room temperature. Reaction conversion was calculated by normalizing the absorbance by the initial absorbance at each time point. Gelation time was measured by the following protocol for different polymer concentrations. Aqueous HA−A solution (100 μL) was added to aqueous HA−C solution (100 μL) and mixed with a magnetic stirrer bar in a Petri dish at 155 rpm using a hot plate/stirrer (Asone, RS-1DR, Japan). The gelation time was the time until the mixture became a globule; it was measured four times per sample.2 The degradation of the gel disks over time was measured gravimetrically in PBS, PBS with 10 units/mL of hyaluronidase (HAse), or Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 °C. The weight of hydrogel after
potential for in situ cross-linking of injectable HA hydrogels. However, to the best of our knowledge, no injectable hydrogels cross-linked by strain-promoted [3 + 2] cycloaddition have been administered in vivo. Here, we have for the first time prepared an in situ crosslinked HA hydrogel (cHA hydrogel) using strain-promoted [3 + 2] cycloaddition, as shown in Figure 1. HA was chemically modified with azide and cyclooctyne groups to prepare precursor polymers of HA−azide (HA−A) and HA−cyclooctyne (HA−C), respectively. The HA−A and HA−C solutions were mixed and reacted with each other by injection using a double-barreled syringe. They formed a rigid hydrogel by a reaction between the azide groups of HA−A and the cyclooctyne groups of HA−C. Finally, the hydrogels’ biocompatibility was evaluated in vitro and in vivo.
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EXPERIMENTAL SECTION
Materials. HA was kindly gifted from Kikkoman Biochemifa Co. The molecular weight of HA was 80 or 800 kDa. The 80 kDa was the main form used in all the experiments, except the measurement of gelation time using both molecular weights of HA. All the other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Wako Pure Chemical Industries (Tokyo, Japan) and used without further purification. Synthesis of Hyaluronan-Modified with Azide Groups (HA− A). The synthesis of HA−A is shown in Figure 2. HA (0.994 g, MW = 80 kDa) was dissolved in 300 mL of distilled water, subsequently changing Na+ to H+ by cation-exchange resin. Then, tetrabutylammonium (TBA) hydroxide was added and stirred at a room temperature for 2 h. The resin was removed by filtration, and aqueous tetrabutylammonium HA (HA−TBA) was lyophilized.38 In addition, 4-azidebutanoic acid (ABA) was synthesized as reported.39,40 HA−TBA (0.63 g) was dissolved in 20 mL of DMSO. ABA (0.26 g) and carbonyldiimidazol (CDI, 0.32 g) were dissolved in 3 mL of B
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Figure 2. Synthetic route of precursor polymers: (A) HA−azide (HA−A) and (B) HA−cyclooctyne (HA−C). gelation, Ws, was measured at each time point, and the incubation solution was replaced. The measurement was continued until the complete degradation of the hydrogels over 14 days. The ratio of the volume of hydrogel at each time point to the initial volume, W, of Ws to the initial weight of hydrogel after gelation, Wi, was calculated as W = Ws/Wi. Cytotoxicity Assay. In vitro cell viability in the presence of HA−A or HA−C was determined using the MTT assay (Promega) using a mouse fibroblast cell line (NIH-3T3, Riken Cell Bank). Fibroblast cells were grown and maintained in DMEM with 10% FBS at 37 °C in 5% CO2. About 105 cells were put into each well of a 96-well plate and incubated at 37 °C in 5% CO2 overnight. The medium was replaced with media containing different concentrations of HA−A, HA−C, or unmodified HA. On the second day after adding the materials, the MTT assay was performed. Tetrazolium salt solution (15 μL) was added to each well and incubated at 37 °C for 4 h. The purple formazan produced by active mitochondria was solubilized using 1 mL of detergent solution and then measured at 570 nm by a plate reader (2030 ARVO V3, Perkin Elmer). The absorbance values were normalized to wells in which no test materials were added to the media. In vitro cell viability in the cHA hydrogel was evaluated by calcein acetoxymethyl ester (AM) staining after encapsulating using NIH-3T3. HA−A (3 wt %) and HA−C (3 wt %) in PBS were injected with fibroblast cells into a glass-bottomed dish (D110300, Matsunami Glass Ind., Ltd.) using a double-barreled syringe. The encapsulated cell density was 2.0 × 106 cells/hydrogel·mL, and the volume of the hydrogel was 200 μL. The cell-encapsulated hydrogels were incubated in 3 mL of DMEM with 10% FBS at 37 °C in 5% CO2 for 2 days. Then, the hydrogels were washed carefully with PBS to remove serum proteins, and stained using 2 μM calcein AM with 1 μM ethidium homodimer-1 (Promega). The hydrogel was observed by confocal
laser microscopy (LSM510, Carl Zeiss) at 495 nm/520 nm (ex/em) or 550 nm/650 nm (ex/em) 30 min after the staining. Subcutaneous and Intraperitoneal Administration of cHA Hydrogels by a Double-Barreled Syringe. The experiments were performed at the Section for Animal Research, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, the University of Tokyo. The Animal Care Committee of the University of Tokyo approved all procedures in this study before it began. ICR mice (3 weeks old, male) weighing 20 g were purchased from CLEA Japan, Inc. (Japan), and housed in groups in a 6 AM−6 PM, light−dark cycle. The polymers were sterilized by UV irradiation for 12 h and then dissolved in PBS at 3 wt % concentration. Anesthesia was induced with 50 mg/kg ketamine and 10 mg/kg xylazine. HA−A and HA−C dissolved in PBS (0.5 mL each) were administered intraperitoneally using a syringe and a 24-gauge needle (Terumo, Japan). A further 0.5 mL of HA−A and HA−C dissolved in PBS were injected subcutaneously to the posterodorsal wall using a double-barreled syringe. Six mice were administered intraperitoneally and six subcutaneously. The mice were sacrificed 1, 2, and 3 weeks after the injections, and the presence of residues and adhesions in the peritoneum were evaluated. Residual hydrogels with surrounding tissues and abdominal contents were sampled as needed, fixed in 10% formalin, and processed for histology (hematoxylin−eosin [HE] stained slides) using standard techniques.
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RESULTS Azide Cycloaddition Rate Constant between ABA and CGA. Conversion of the copper-free click reaction between ABA and CGA is shown in Figure 3. The apparent kinetic constant was determined as 6 × 10−4 M−1 s−1, assuming a C
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shift region between 1.4 and 2.4 ppm, by comparing with unmodified HA. The methylene protons of CGA from glycolic acid residues were also confirmed at 4.28 ppm (singlet). The degree of modification was calculated from the ratio of the area of the peak for N-acetyl-D-glucosamine residue of HA (singlet peak at 2.00 ppm) to that for the ring skeleton protons of CGA; the degrees of modification were 25.3% in HA of 80 kDa and 14.6% in HA of 800 kDa. Because of the higher reactivity of the primary hydroxyl group at the sixth position of N-acetyl-D-glucosamine residue than other secondary hydroxyl groups of HA,42 we speculate that the primary hydroxyl groups were preferentially conjugated and the chemical structures shown in Figures 1 and 2 were the major structures of HA-A and HA-C, though further characterizations are needed. Figure 5 shows the FT-IR spectra of HA−A and HA−C. In the spectrum of HA−A, absorbance at 2107 cm−1 from the Figure 3. Copper-free click reaction rate between 4-azidebutanoic acid and cyclooct-1-yn-3-glycolic acid measured by FT-IR spectra (n = 1).
second-order reaction. Azide cycloaddition rate constants between various substituted cyclooctynes and benzyl azide were reported in previous studies. Although difluorocyclooctyne showed the highest reaction rate because of its electronrich substituents,41 methoxy-substituted cyclooctenes showed a high reaction rate, 24 × 10−4 M−1 s−1.41 The order of the reaction rate constant coincided with the reported value, so the in situ cross-linking reaction is expected to be rapid enough for gelation. Synthesis and Characterization of HA−A and HA−C. Because ABA and CGA did not dissolve in water, the sodium ion of HA was exchanged by TBA, and HA became solubilized in DMSO. Conjugation between HA and ABA or CGA was successfully performed in DMSO, as shown in Figure 4 of 1H NMR spectra of HA, HA−A, and HA−C. Figure 5. FT-IR spectra of HA, HA−A, and HA−C. Arrows indicate new peaks resulting from modifications.
azide group and absorbance at 1732 cm−1 from the ester bonds were confirmed. In the spectrum of HA−C, absorbance at 1751 cm−1 from the ester bonds was confirmed. Therefore, the syntheses of HA−A and HA−C were confirmed by both 1H NMR and FT-IR. Dependency of Gelation Time on Polymer Concentration. The gelation time was measured using a stirring plate according to the reported procedure. As shown in Figure 6, only the 800 kDa form of cHA formed a hydrogel 100 min after starting stirring with 1 w/v% of cHA, while the 80 kDa form of cHA did not form a hydrogel. With 5 w/v%, cHA gelled in less than 5 min for both molecular weights. Gelation time depended strongly on the polymer concentration. For previous in situ HA hydrogels using Schiff base formation,2,3 gelation time measured in the same manner was below 30 s when polymer concentrations were 2 w/v%. For previous carboxymethyl dextran and carboxymethyl cellulose hydrogel using Schiff base formation, gelation time was below 2 min when the polymer concentration was 3 w/v%.13 Thus, gelation times of cHA were slower than those of previous similar materials using Schiff base formation. Thanks to the high-enough reaction rate between ABA and CGA as shown in Figure 3, we speculate that the reason for the
Figure 4. 1H NMR spectra in D2O of HA, HA−A, and HA−C. Arrows indicate new peaks resulting from modifications.
In the spectrum of HA−A, the methylene protons of ABA were confirmed at 3.37, 3.39, 3.40 ppm (triplet, α position of ABA), 1.90, 1.92, 1.93 ppm (triplet, β position of ABA), and 2.53 ppm (singlet, γ position of ABA). The degree of modification was calculated from the ratio of the area of the peak for N-acetyl-D-glucosamine residue of HA (singlet peak at 2.00 ppm) to that for the methylene protons of ABA at 2.53 ppm; the degrees of modification were 10.8% in HA of 80 kDa and 12.6% in HA of 800 kDa. In the spectrum of HA−C, 10 protons of the cyclooctyne ring skeleton from CGA were confirmed in the low chemical D
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Figure 7. Degradation kinetics of cHA hydrogels at 37 °C in PBS that contained no enzymes or 10 unit/mL of HAse, or in DMEM with 10% FBS. Weight ratios expressed as average ± standard deviation (n = 4).
Figure 6. Gelation times of the cHA hydrogels. The results were expressed as averages ± standard deviation (n = 4).
difference of gelation time between cHA and other in situ hydrogels is the low degree of modification of HA−A and HA− C. The degree of modification of hydrazide-modified HA was about 50%, while that of HA−A or HA−C was below 20%. In the present synthetic scheme, 3 mol equiv of ABA or CGA were conjugated to HA, while 10 mol equiv of adipic dihydrazide were added for the synthesis of hydrazide-modified HA in the previous studies. Thus, the increase in the degree of modification of HA−A and HA−C is expected to be achieved by increasing the molar ratio of ABA of CGA in the conjugation reaction in further studies. Compared with another copper-free clickable hydrogel,36 the gelation by mixing four-arm poly(ethylene glycol) tetra-azide and difluorinated cyclooctyne moiety-functionalized polypeptide occurred at 5 min and was completed in about 1 h, which was measured by viscoelasticity even without stirring. However, the total polymer concentration was 13.5 wt %.36 The tendency of our research coincided well with that of previous studies. In terms of viscosity and the gelation time of HA−A and HA−C, 80 kDa cHA hydrogel was evaluated in the following results. Degradation Kinetics of the cHA Hydrogels. The cHA hydrogels, with polymer concentrations of 1.5, 2.0, or 3.0 w/v %, were degraded slowly over about 2 weeks in PBS without any enzyme, as shown in Figure 7. The hydrogels with high polymer concentrations degraded slower than those with low polymer concentrations. Although the triazole cross-links are not degraded in PBS, the cHA hydrogels showed the hydrolytic degradation in PBS. In addition, the visual change of the hydrogels was observed during the incubation of the cHA hydrogels in PBS, as shown in Figure 8. The slightly clouded hydrogel became transparent 1 week after the starting point of the incubation. The aqueous HA−C solution was also cloudy; thus, cyclooctyne modification might lower the solubility of HA and the hydration of cHA hydrogels, and we hypothesized that the hydrolysis of ester bonds between the triazole cross-links and HA promoted the degradation of the hydrogel and increased the transparency of the hydrogels during their incubation in PBS. HAse, found in many tissues and body fluids, accelerated the degradation of the cHA hydrogels. Compared with the effects of PBS, degradation by HAse occurred without burst swelling
Figure 8. Physical appearances of cHA hydrogel. (A) Initial hydrogels before incubation and (B) 1 week after starting incubation. The diameter of the hydrogel in (A) was 1.2 cm.
before complete degradation, as shown in Figure 7. The gradual decrease of the mass of the hydrogels without swelling is probably because the HA backbone might be degraded from the outside of the hydrogels by HAse without the hydrolysis of the inside cross-links of the hydrogels, due to the slow hydrolysis of the ester bonds in PBS. On the other hand, the hydrogels degraded in DMEM with FBS much faster than in PBS containing HAse. In addition, burst swelling before complete degradation was observed. The difference between the degradation rate in DMEM with FBS and PBS containing HAse probably occurred because serum esterases43 degraded the ester bonds in the hydrogel. Therefore, cHA hydrogel is expected to have degradation potential in vivo, though the triazole cross-links are not degraded in PBS. NIH-3T3 Cell Viability after Incubation with HA−A, HA−C, and the cHA Hydrogel. Unmodified HA showed mild dose-dependent toxicity in the present study. Both HA−A and HA−C showed the same cell viability at 0.5 w/v% of polymer concentration, as shown in Figure 9. At 1.0 w/v%, HA−A and HA−C were slightly toxic compared with unmodified HA. In addition, the cHA hydrogels were also used for encapsulation of NIH-3T3 cells. All the cells showed good cell viability by calcein AM staining 2 days after encapsulation, as shown in Figure 10, while almost no cells were stained by ethidium homodimer-1 (data not shown). E
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Table 1. Adhesions Following Intraperitoneal Injection of Hydrogels in Mice postoperative day (days)
administration route
material residue
peritoneal adhesion
7 14 21 7 14 21
intraperitoneally intraperitoneally intraperitoneally subcutaneously subcutaneously subcutaneously
0/2 0/2 0/2 2/2 1/2 0/2
0/2 0/2 0/2
gathered in the hydrogels, as shown in Figure 11C, and the density of lymphocytes near the boundary between surrounding tissues and the hydrogel is slightly higher than that inside the hydrogel, as shown in Figure 11D. Although inflammation from the cHA hydrogel appeared slightly more severe than that of the in situ cross-linked HA hydrogels by Schiff base formation,44 it was better than that caused by the in situ cross-linked dextran-based hydrogels.13
Figure 9. Effect of the unmodified HA and synthesized polymers HA− A and HA−C on cell viability of mouse fibroblast cell line, NIH-3T3, measured by MTT assay. Values are averages ± standard deviations (n = 8).
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DISCUSSION The orthogonality of the copper-free click reaction shows its potential for biomedical applications. Some hydrogels using the copper-free click reaction have already been reported.36,37,45 However, the HA hydrogels have only been reported via a click reaction using a copper catalyst.29−31 Injectable HA hydrogels via strain-promoted [3 + 2] cycloaddition were successfully prepared in this research. Generally speaking, conjugation with cyclooctyne derivatives raises the low solubility of the conjugate because of the hydrophobicity of cyclooctyne rings. However, HA is well-known to be extremely water retentive and biocompatible; therefore, the combination of HA and the copper-free click reaction was ideal to achieve high water content. Both the backbone of HA and the ester linker of the cHA hydrogels enabled their biodegradation, which differs from the behavior of the clickable PEG-based hydrogels.36,37,45 The biocompatibility of cyclooctyne derivatives has not yet been studied in detail. The high biocompatibility of HA−C shown in this study was positive data in this field. In a previous paper, 0.2 mM of benzoate cyclooctynes (BCs) was applied to Jurkat cells as a maximum dose,32 and the cells showed good cell viability. Based on an MTT assay protocol, 0.2 mM of BCs is converted into 1.8 × 1010 molecules/cell. We applied 1 wt/v % of HA−C to NIH-3T3, which is converted into 2.3 × 1013 molecules/cell of the cyclooctynes based on the degree of modification of HA−C. Although our study exposed ca. 1000 times as much cyclooctynes to the cells, cell viability was almost the same as that with unmodified HA. We speculate that the cyclooctyne compound is not toxic, even when considering the effect of conjugation. Because of the high reactivity of cyclooctynes and azides, in situ gelation was performed in vivo, and the biocompatibility evaluated. For medical applications, cell viability assay in vitro is not enough to show the safety of materials. As a result, all the mice were healthy, and the weight of the mice increased as they grew for 3 weeks after the material administration. Formulations using copper-free click reactions have strong potential for clinical uses in the future. Inflammation of surrounding tissues was observed in the subcutaneous injection. Degradation kinetics and biocompatibility in vivo closely relate to each other. Based on the degradation over time of the cHA hydrogel, cyclooctyne
Figure 10. Confocal microscopic observation of NIH-3T3 cells encapsulated in cHA hydrogels. The hydrogel and cells were stained by calcein AM 2 days after encapsulation and culture in DMEM with 10% FBS: (A) Calcein AM staining and observed by 490/515 (ex/ em); (B) Optical image; (C) Merge image.
Subcutaneous and Intraperitoneal Administration of the cHA Hydrogels. No mice generated peritoneal adhesion after intraperitoneal administration of the cHA hydrogel, as shown in Table 1, despite the formation of trauma induced by the injection. The hydrogels were all cleared perfectly from the peritoneum 1 week after the injection. These results mean that cHA hydrogel is safe in the peritoneum. In addition, no pathological appearance was observed in liver, spleen, or kidney by HE staining (data not shown). To evaluate the effect of the cHA hydrogel on surrounding tissues, it was injected subcutaneously, as shown in Figure 11A. The hydrogels were degraded 3 weeks after injection, while the hydrogel residues were visually confirmed 1 week after injection, as shown in Figure 11B. HE staining of the hydrogel residues and surrounding tissues shows that many lymphocytes F
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Figure 11. Subcutaneously administered cHA hydrogel 7 days after injection. Residues of the injected cHA hydrogels were found in two mice after 1 week (n = 2), while no residue was found after 2 and 3 weeks (n = 2, respectively). (A) Injection site shortly after injection. (B) Residues of the injected cHA hydrogels 1 week after injection. (C) Hematoxylin−eosin (HE) stained cHA of (B), 10×. (D) Magnification of the boundary between cHA hydrogel and adipose tissue of (C), 20×.
synthetic hydrogel niche52 will become more important in tissue engineering. The cHA hydrogels can easily be modified with various cell adhesion ligands via the copper-free click reaction. Therefore, cHA hydrogels can be expected to construct a hydrogel niche in the future as an injectable scaffold.
compounds may be released to the surrounding tissues both as the carboxyl cyclooctynes by the cleavage of ester bonds by serum esterases and in the conjugated form with HA by the cleavage of glycoside bonds by HAse. The predicted value of log P of ABA, CGA, and the trijazole compound between ABA and CGA were calculated as 0.31, 2.03, and 1.25 by ChemAxon (ChemAxon Ltd., U.K.). Thus, the carboxyl cyclooctynes can penetrate cell membranes. Also, degradation fragments of cHA hydrogel can be taken up endocytically by cells through the interaction between HA and CD44.46 Thus, optimization of conjugation47 may lead to better biocompatibility and less inflammation in the future. One of the possible approaches in further research is to utilize both hydrophilic linker composed of polyethylene glycol and amide bonds instead of the current hydrophobic ester-linker, which may prevent the release of the free carboxyl cyclooctynes from hydrogels and the uptake by cells. Because the HA backbone of the cHA hyderogels were degraded by HAse in this study, stable linkers composed of amide bonds may be utilized in future. In addition, triazole compounds formed by cross-linking are expected to be biocompatible, although the metabolism of triazole rings has not yet been studied sufficiently. Some triazole derivatives were recently proposed as pharmacological agents for inhibitors of human cytochrome P45048,49 or antitubercular agents50 because of the ease of synthesis by click chemistry. Therefore, further research on the metabolism of these materials will be necessary. Because hydrogels were applied to scaffolds, cell adhesion ligands such as the RGD motif were attached to hydrogels mainly via carbodiimide chemistry.4,6 The interactions between various cell adhesion peptides and integrin receptors have been clarified to control stemness, the fate of developing stem cells, or various functions of somatic cells. Microarray techniques51 will accelerate the progress of research in this field. Thus, the
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CONCLUSIONS We have prepared in situ cross-linkable HA (cHA) hydrogels via a copper-free click reaction. Azido-modified HA (HA−A) and cyclooctyne-modified HA (HA−C) formed a hydrogel by mixing for 5 min. The hydrogel was administered intraperitoneally and subcutaneously and showed good biocompatibility. Biodegradation was observed in vitro and in vivo probably because of the degradation of ester linker and the HA backbone. Therefore, this cHA hydrogel is expected to create an injectable hydrogel niche for tissue engineering and drug delivery matrix for conjugate drugs.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +81-3-5841-1696. Fax: +81-3-5841-1697. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We deeply thank Kikkoman Biochemifa Co. for supplying hyaluronan and Baxter for supplying a double-barreled syringe. We also appreciate the aid of a Health Labor Sciences Research Grant from the Ministry of Health, Labor and Welfare of Japan. We also thank the Japan Society for the Promotion of Science (JSPS) for a Research Fellowship for S.O. We also thank the G
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Biomacromolecules
Article
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Center for NanoBio Integration, the University of Tokyo, for confocal laser microscopy.
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dx.doi.org/10.1021/bm4009606 | Biomacromolecules XXXX, XXX, XXX−XXX
