Genipin-cross-linked poly(l -lysine)-based hydrogels: Synthesis, characterization, and drug encapsulation

Colloids and Surfaces B: Biointerfaces 111 (2013) 423–431

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Genipin-cross-linked poly(l-lysine)-based hydrogels: Synthesis, characterization, and drug encapsulation Steven S.S. Wang a , Ping-Lun Hsieh b , Pei-Shan Chen b , Yu-Tien Chen b , Jeng-Shiung Jan b,∗ a b

Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 70101, Taiwan

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 11 June 2013 Accepted 12 June 2013 Available online 21 June 2013 Keywords: Hydrogel Polypeptide Drug delivery pH-sensitive Genipin

a b s t r a c t Genipin-cross-linked hydrogels composed of biodegradable and pH-sensitive cationic poly(l-lysine) (PLL), poly(l-lysine)-block-poly(l-alanine) (PLL-b-PLAla), and poly(l-lysine)-block-polyglycine (PLL-bPGly) polypeptides were synthesized, characterized, and used as carriers for drug delivery. These polypeptide hydrogels can respond to pH-stimulus and their gelling and mechanical properties, degradation rate, and drug release behavior can be tuned by varying polypeptide composition and cross-linking degree. Comparing with natural polymers, the synthetic polypeptides with well-defined chain length and composition can warrant the preparation of the hydrogels with tunable properties to meet the criteria for specific biomedical applications. These hydrogels composed of natural building blocks exhibited good cell compatibility and enzyme degradability and can support cell attachment/proliferation. The evaluation of these hydrogels for in vitro drug release revealed that the controlled release profile was a biphasic pattern with a mild burst release and a moderate release rate thereafter, suggesting the drug molecules were encapsulated inside the gel matrix. With the versatility of polymer chemistry and conjugation of functional moieties, it is expected these hydrogels can be useful for biomedical applications such as polymer therapeutics and tissue engineering. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Hydrogels have been widely investigated for many biomedical applications including drug delivery, cell culture, tissue engineering, and intelligent devices [1–10]. They can be derived from a variety of materials including natural and synthetic polymers. Natural biopolymers including artificial proteins, silk fibroin, chitosan, and hyaluronic acid (HA) have been utilized to generate hydrogels [1,6,11–15]. Despite their innate biocompatibility and biodegradability, these materials suffered from their batch-to-batch variation, possible immunogenicity, and limited structural and functional modification [12,16]. In contrast, synthetic polymer-based hydrogels have advantages over natural counterparts because of their versatility in tailoring macromolecular chemistry to tune the material property and functionality. This provides an exciting opportunity to develop functional hydrogels with suitable material property to meet the criteria for specific biomedical applications. Though hydrogels can be derived from a variety of synthetic polymers, many of them are composed of non-biocompatible or non-biodegradable building blocks, leading to their

∗ Corresponding author. Tel.: +886 6 2757575x62660; fax: +886 6 234 4496. E-mail addresses: [email protected], [email protected] (J.-S. Jan). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.06.028

limitation in biomedical applications. Among these synthetic systems, poly(ethylene glycol) (PEG)-based hydrogels that developed via various chemistries have been extensively studied for cell culture and tissue engineering applications [17–25]. Despite their excellent features including non-toxicity, stealth property, hydrophilicity, and anti-fouling properties, PEG hydrogels exhibit poor cell viability and limited sites for functionalization [26–28]. It would be desirable to prepare hydrogels from polymers that combine the advantages of both natural and synthetic systems. One important strategy to achieve this goal is to utilize natural building blocks such as saccharides and amino acids, which could generate polymers with inherent biocompatibility and biodegradability, and rich functionalities for facile modification. Particularly, peptides can alter molecular organization and ionizing state in response to the external environment [29], which can afford the peptide-based biomaterials to undergo macroscopically observable changes in material properties such as gelation [30–34]. Also, the gelation process can be dictated by the chain conformation and amphiphilicity of the block copolypeptides, resulting in the formation of hydrogels at a low concentration [35–37]. These physically cross-linked hydrogels employ reversible physical interactions including ionic or hydrophobic interactions between polymer chains to form gel matrix. Hydrogels can also be prepared via chemical cross-linking to form covalently bonded matrix networks, which would exhibit better stability

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in vivo and control of properties than physically cross-linked hydrogels. Recent developments on biodegradable and pH-sensitive hydrogels for drug delivery application have paid more attention on utilizing polypeptides and natural biopolymers [38–41]. Comparing with natural biopolymers, polypeptides can adopt ordered conformations such as ␣-helices and ␤-sheets, and their molecular weight and composition can be precisely controlled via the live polymerization of N-carboxyanhydrides (NCAs) [42–44]. Though poly(l-lysine) (PLL) is a commonly known pHsensitive polypeptide, PLL-based hydrogels have received little attention, which is mainly attributed to the concern on the reported cytotoxicity of free PLL to cells [45,46]. Recent study has shown that poly(l-lysine)-block-poly(l-leucine) (PLL-b-PLLeu) hydrogels exhibited good cytocompatibility in vitro, showing that the cationic polypeptide segments in the intact and rigid hydrogel state were inert [47]. Moreover, the in vivo biocompatibility tests indicated that the PLL-b-PLLeu hydrogels exhibited good biocompatibility comparable to physiological saline, good integration with peripheral tissues, and in-growth of cells into the hydrogels [48]. These studies suggested that PLL-based hydrogels are promising biomaterials for biomedical applications. Herein, we investigated the genipin-cross-linked polypeptide hydrogels using natural amino acids including l-lysine, l-alanine, and glycine as the building blocks. Genipin, which is a natural and non-toxic cross-linking agent, was utilized to cross-link the lysine side chain to form hydrogels. While there were a few studies on the genipin-cross-linked polypeptide hydrogels based on elastin [49,50], the chemically cross-linked polypeptide hydrogels with tunable properties have not been reported. The live polymerization of NCAs can warrant the synthesis of polypeptides with well-defined chain length and composition [42–44], which is one approach to tune hydrogel properties. The pH-sensitive swelling behavior, mechanical properties, enzymatic degradation, and drug release behavior of the as-prepared hydrogels were studied by varying the polypeptide chain length and composition as well as the cross-linking degree. To evaluate the feasibility of using these materials for biomedical applications, a preliminary investigation on the cytotoxicity of the hydrogels was carried out, as well as the cell attachment and proliferation of the hydrogels. It was expected that the polypeptide segments can be covalently tethered together via cross-linking and the undesired release of the polypeptide segments, which would cause cytotoxicity, can be prevented. Additional biofunctionality can be incorporated onto the polypeptide chains simply by conjugation of functional moieties. One can envision that these genipin-cross-linked hydrogels would not only be biocompatible and biodegradable but also possess highly structural and functional properties.

2.2. Polypeptide synthesis Homopolypeptides and block copolypeptides were synthesized using the nickel initiator 2,2 -bipyridyl-Ni(1,5-cyclooctadiene) (BpyNiCOD) by following the previously reported procedures [43,51]. The polymerization was carried out under inert conditions at room temperature. The Z group used to protect the amino group on the side chain of PLL was removed by using hydrogen bromide. These as-prepared polypeptides were then dissolved in deionized (DI) water and dialyzed against DI water using a cellulose membrane dialysis tube (MWCO 6000–8000; Sigma, St. Louis, MO, USA). The water was exchanged two to three times per day over the next 3 days. Finally, these polypeptides were lyophilized using a freeze dryer to obtain white spongy materials. The notations for homopolypeptides and block copolypeptides used throughout were Lysm and Lysm Xan (Xa = Ala or Gly), where m and n are the number of amino acids in the respective block).

2.3. Material characterization Gel permeation chromatography (GPC) measurements were performed before deprotection of the polypeptides using a Viscotek system equipped with three detectors, which are RI (VE3580, Viscotek), right angle light scattering, and viscometer (Dual 270, Viscotek). Two ViscoGEL I-Series columns (catalog number: I-MBHMW-3078 and I-MBLMW-3078, Viscotek) for efficient separation, eluted with 0.1 M LiBr in DMF at 55 ◦ C. The eluent flow rate was 1 mL/min. 1 H NMR spectra were recorded at 300 MHz on a Mercury 300 Varian spectrometer using TFA-d1 or D2 O as solvent. Field-Emission Scanning Electron Microscopy (FE-SEM) was performed using a JEOL JSM-6700F microscope operating at 1–10 kV. Samples were collected via lyophilization, and mounted on carbon tape for imaging. Fourier transform infrared (FTIR) spectra were recorded on a Thermo Nicolet Nexus 670 FTIR spectrometer. Circular dichroism (CD) spectra were measured over the wavelength range of 190–260 nm using a 0.1 cm quartz cell on a JASCO J-815 spectrometer (JASCO Inc). The polypeptide hydrogels were ground into small particles and suspended in DI water for CD measurements. The compressive strength of polypeptide hydrogels was determined using MTS machine (AGS-X 500N, SHIMADZU, Japan). Hydrogels with uniform rectangular shapes (n = 4) were prepared in a Teflon mold and placed on the metal plate. The samples were then pressed at a speed of 0.5 mm/min to obtain the load–displacement curves.

2.4. Preparation of hydrogels 2. Experimental 2.1. Materials THF (ACS Reagent, Merck) was dried using Na metal and hexane (ACS Reagent, EM Science) was dried using calcium hydride. N␧ -benzyloxyl carbonyl-l-lysine (H-Lys(Z)-OH, ∼99%) and bromelain were supplied by Sigma–Aldrich. l-Alanine (>99%) and glycine (>99%) were used as received from Fluka and Merck, respectively. Trifluoroacetic acid (99%) and doxorubicin hydrochloride (Dox) were supplied by Alfa Aesar and SeedChem Pty Ltd, respectively. Triphosgene (98%) and hydrogen bromide (33 wt% in acetic acid) were used as received from Fluka. Ethyl ether (ACS Reagent) and genipin (98%) were supplied by ECHO and Challenge Bioproducts Co. (CBC), respectively.

The polypeptide hydrogels were prepared by the cross-linking reaction between genipin and the amino group in polypeptides. The freeze-dried polypeptides were dissolved in DI water and the resultant solutions were stirred for at least 12 h to ensure that the polypeptides were dissolved in DI water completely. Genipin was mixed with the polypeptide solution and then the polypeptides were cross-linked to form dark blue hydrogels. The gelation time of the cross-linked polypeptide hydrogels was investigated using the vial tilting method [52,53]. The polypeptide solutions were prepared by dissolving polypeptides in DI water for one day in the vials with an inner diameter of 13 mm. After one day, a designated amount of genipin (genipin to lysine molar ratio, R = 0.5, 0.25, or 0.125) was added to these solutions. The gelation time was determined as the solutions did not flow for 1 min after inverting the vials, indicating the lost of fluidity.

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2.5. Cross-linking degree determination The cross-linking degree, which was defined as the percentage of free amino groups in the cross-linked polypeptide hydrogels, was determined by ninhydrin assay [54]. In the ninhydrin assay, the lyophilized sample was weighed and subsequently heated with a ninhydrin solution for 20 min. The solution was cooled down to room temperature and diluted with 50% isopropanol. Then the optical absorbance of the solution was recorded with a spectrophotometer (SCINCO S-3100) using glycine at various known concentrations as standard. It is known that the amount of free amino groups in the test sample, after heating with ninhydrin, is proportional to the optical absorbance of the solution. 2.6. Swelling of hydrogels Swelling ratios of the cross-linked polypeptide hydrogels were determined using a gravimetric method. The hydrogel sample was first lyophilized and its dry mass (Wdry ) was measured. The freezedried hydrogel was then immersed in 1 mL of phosphate buffered saline (PBS) solutions with different pH values (4.0, 6.0, 7.4, 8.0, and 10.0) for 2 days. All samples were found to reach equilibrium swelling after 2 days, evidenced by the constant weights of the swelled samples. All experiments were carried out in triplicate. The degree of swelling at equilibrium was determined by the ratio of the sample weight before and after swelling as shown in Eq. (1): Swelling ratio =

(1)

Wdry

where Wwet represents the weight of the swollen state at equilibrium. 2.7. In vitro enzymatic degradation of hydrogels Biodegradation of hydrogels was performed in a glass vial containing the dried hydrogel sample and 2 mL of PBS buffer (pH 7.4, 0.15 N ionic strength) with bromelain at a concentration of 0.2 mg/mL. The vial was incubated at 37 ± 1 ◦ C with constant shaking (100 rpm). The samples were then taken out at different time interval and rinsed thoroughly with DI water before lyophilization. The solution was replaced everyday in order to maintain enzymatic activity. The percentage of weight loss was determined from Eq. (2): Weight loss (%) =

W − W  i f Wi

the absorbance intensity at 570 nm (n = 6). For the control experiment, the cells were grown in the culture medium under the same conditions. For cell adhesion test, fibroblast cells were seeded at 1 × 104 cells/mL onto the wells coated with the polypeptide hydrogels. Cells cultured on the bare wells under the same condition acted as controls. After 72 h of culture, the adherent cells were observed using an inverted phase-contrast microscope. 2.9. Preparation of Dox-loaded hydrogel and evaluation of Dox release in vitro Dox was employed as a hydrophilic model drug. The polypeptide solutions were prepared by dissolving 50 mg of polypeptide in 0.95 mL of DI water and Dox (1 mg) was added to the solutions to form Dox-loaded polypeptide solutions. A fixed amount of genipin (R = 0.5, 0.25, 0.125, or 0.083) was added to the Dox-loaded polypeptide solutions. Subsequently, the resultant solutions were poured into Teflon molds and sealed off to prevent the evaporation of water. After 2 days, the polypeptides were cross-linked to form Dox-loaded polypeptide hydrogels. To calculate the encapsulation efficiency, the Dox-loaded hydrogels were rinsed with DI water for 1 min and the amount of Dox rinsed out into the aqueous solution (M2 ) was determined by UV–vis spectrophotometer (SCINCO S-3100) at a wavelength of 485 nm. The Dox encapsulation efficiency (EE%) was determined by Eq. (3): EE% =

Wwet − Wdry

× 100

(2)

where Wi is the initial weight of the dried gel sample and Wf is the weight of the dried gel sample after degradation at certain time interval. All experiments were carried out in triplicate. 2.8. In vitro cytotoxicity tests The fibroblast cells (3T3) were cultured onto a 48-well plate (1 × 104 cells/mL) using DMEM (Dulbecco’s modified eagle medium, Gibco) with 10% FBS (fetal bovine serum, Gibco) under a humidified atmosphere of 5% CO2 at 37 ◦ C. After culturing for 24 h, the medium was replaced with extract fluids obtained by placing the polypeptide hydrogels (0.1 g/mL of culture medium) in the cell culture medium. Then the viability of adherent cells was determined using the MTT reduction assay after 24 h [55,56]. By adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to the cells, the tetrazolium salt was converted into an insoluble purple formazan salt. After 4 h incubation, the formazan salt was dissolved by a detergent solution. The resulting solution was measured at 570 nm using an ELISA plate reader (Sunrise, Tecan). The number of viable cells can be quantified by measuring

425

M − M  1 2 M1

× 100

(3)

where M1 is the initial loading amount of Dox. The Dox release experiments were performed by placing the Dox-loaded hydrogel (∼200 mg) in a glass vial containing 20 mL of phosphate buffer saline (PBS, pH 7.4, 0.15 N ionic strength). The vial was incubated at 37 ± 1 ◦ C with constant shaking (100 rpm). The release medium (2 mL) was taken at specific time intervals and characterized by using UV–vis spectrophotometer at a wavelength of 485 nm and then the solution was poured back to the vial. All release experiments were carried out in triplicate. 3. Results and discussion 3.1. Synthesis and characterization of polypeptides The homopolypeptides and block copolypeptides were prepared by ring-opening polymerization. Table S1 summarized the list of homopolypeptides and block copolypeptides studied in this paper. The molecular weight (MW) and polydispersity (PDI) of the as-prepared polypeptides were determined by GPC (Table S1 and Fig. S1). 1 H NMR measurements of these block copolypeptides in d1 -TFA were performed to determine their block ratios (Table S1). The MW and block ratio of these polypeptides can be tuned by controlling the monomer and initiator concentrations as well as the monomer/initiator ratio [43]. The typical yield of Z group-protected homopolypeptides and block copolypeptides ranged between 75 and 90%. Poly(l-lysine) (PLL) and poly(l-lysine)block-poly(l-alanine) (PLL-b-PLAla) were used for the preparation of hydrogels. For comparison, the gelation of the block copolypeptide with a different hydrophobic block, polyglycine (PGly), was also studied. 3.2. Gelation of polypeptides It has been previously demonstrated that hydrogels can be prepared via either genipin or glutaraldehyde coupling reaction with the amino group on polysaccharides [54,57–60]. We recently reported that the vesicles assembled by alkyl chain grafted PLL can encapsulate biomolecules and subsequently the hydrophilic

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Fig. 1. (a) Preparation of polypeptide-genipin derivative and (b) photographs of Lys120 polypeptide solution (5 wt%) mixing with genipin before (left) and after (right) gelation. The genipin to lysine molar ratio (R) is 0.5.

PLL can be cross-linked by genipin to form stable hydrogel particles [61,62]. This study turned to a different aspect to prepare hydrogels via genipin coupling reaction with the amino group on the PLL side chain as shown in Fig. 1a. The cross-linking reaction was carried out under mild conditions including aqueous solution, room temperature, and neutral pH. All polypeptides cannot form hydrogels at the concentration of 5 wt% if no genipin was added. However, their solutions of 5 wt% formed hydrogels after addition of genipin at various R values (0.125–0.5). The solution of Lys250 (5 wt%) with added genipin (R = 0.5) was monitored by UV–vis analysis. The results revealed that the decrease in the characterization absorption of genipin at 240 nm was accompanied by an increase in absorption at 290 nm (Fig. S2), indicating the crosslinking reaction between genipin and the amino group [59,61]. It is worth noting that the polypeptide solutions with lower concentration (<3 wt%) cannot form hydrogels after the addition of genipin. The solutions became dark blue within minutes of mixing genipin and polypeptide together in solution and then the polypeptides were cross-linked to form interconnected gel matrices (Fig. 1b). Poly(l-lysine)-block-poly(l-phenylalanine) (PLL-b-PLPhe) and PLLb-PLLeu block copolypeptides were also synthesized and used for preparing genipin-cross-linked hydrogels. However, the solutions of these block copolypeptides were either too viscous or form physical hydrogels. Subsequently, it is hard to mix them with genipin homogeneously and consequently the mixed solutions cannot form hydrogels with reproducible properties. To investigate the effect of polypeptide MW and block ratio on the gelation of the as-prepared solutions, the gelation time was firstly measured using the vial tilting method [52,53]. The gelation time for homopolypeptides and block copolypeptides with different MWs and block ratios at various polypeptide concentrations and R values was investigated. By varying polypeptide MW and block ratio, the gelation time changed at the wide ranges from tens to hundreds of minutes (Table 1). For homopolypeptides and PLL-b-PLAla block copolypeptides with 8:1 block ratio, the gelation time decreased with the increase of the polypeptide MW. The gelation times of Lys120 and Lys120 Ala15 were rather slow and measured to be ∼415 and ∼290 min, respectively. However, the

gelation times of polypeptides with higher MW (that is, Lys250 , Lys385 , and Lys225 Ala28 ) were much faster (<100 min) than those of low MW counterparts. The results can be attributed to the fact that polypeptides with higher MW have the greater chance to undergo cross-linking reaction per chain, which might facilitate the gelation to proceed at shorter time. In addition, incorporating a low fraction of hydrophobic block can facilitate the formation of hydrogels, evidenced by the gelation times of Lys120 Ala15 and Lys225 Ala28 shorter than those of Lys120 and Lys250 , respectively. In contrast, the gelation times of Lys330 Ala55 or Lys225 Gly38 were found to be longer than those of homopolypeptides with comparable MW (that is, Lys385 and Lys250 ). These block copolypeptides tend to form aggregates due to hydrophobic interaction between hydrophobic blocks [35–37,51]. Upon cross-linking, it is harder for the partially cross-linked polypeptide chains to orient themselves in the confined environment for cross-linking reaction to proceed, leading to the hindrance of cross-linking reaction. It was found that the gelation of polypeptides was also influenced by the polypeptide concentration and R value (Fig. S3a). For example, the gelation times of Lys120 and Lys120 Ala15 hydrogels decreased from hundreds to tens of minutes as the polypeptide concentration increased from 5 to 9 wt%. The gelation times of Lys250 and Lys225 Ala28 hydrogels increased with the decrease of genipin to lysine molar ratio (Fig. S3b). 3.3. Structure of hydrogels and polypeptide chain conformation For hydrogels to be useful as cell culture substrates and tissue engineering scaffolds, hydrogel matrices are required to be highly porous to ensure the efficient mass transfer through the matrices. Hence matrix morphology is a very important physical parameter of hydrogels. In this study, the samples were prepared by freezedrying the cross-linked hydrogels (5 wt%) with R = 0.5. Previously, it has been shown that the native morphology of hydrogels can be preserved by the freeze-drying procedure [63,64]. SEM analysis revealed that the freeze-dried polypeptide hydrogels exhibit interconnected membranes (Figs. 2 and S4). The sizes of the pores ranged mostly from 10 to 50 ␮m for all of these samples. Once

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Table 1 Gelation time, percentage of free amino group, and compressive strength of polypeptide hydrogels (n = 4) prepared at the genipin to lysine molar ratio (R) of 0.5. Polypeptide

Weight percentage (%)

Gelation time (min)

Lys120 Lys250 Lys385 Lys120 Ala15 Lys225 Ala28 Lys150 Ala25 Lys330 Ala55 Lys225 Gly38 Lys250 a Lys330 Ala55 a

5 5 5 5 5 5 5 5 2.8 2.1

415 84 73 284 62 39 124 91

a b

± ± ± ± ± ± ± ±

7 5 3 8 7 1 6 2

Free amino group (%)b

Compressive strength (kN/m2 )

7.5 ± 9.5 ± 8.3 ± 10.2 ± 10.1 ± 10.9 ± 7.8 ± 7.5 ± – –

227.4 228.4 214.0 188.3 163.9 182.3 256.4 238.1 90.2 66.3

1.03 0.83 0.25 0.83 0.53 1.88 1.02 0.60

± ± ± ± ± ± ± ± ± ±

2.8 2.4 3.8 3.0 1.4 1.1 3.5 4.3 9.4 5.4

The hydrogel samples (5 wt%) were swelled to reach equilibrium in DI water for 3 days. The percentages of free amino group in hydrogels were determined by ninhydrin assay (n = 3).

the hydrogels (5 wt%) were swelled in DI water to reach equilibrium, the average pore sizes became larger than 50 ␮m. These amphiphilic diblock copolypeptides can aggregate to form assemblies at low concentrations in aqueous solution [35–37,51]. Upon increasing the concentration (>3 wt%), these block copolypeptides form loosely interconnected assemblies. With the addition of genipin, the polypeptide assemblies can be cross-linked to produce hydrogels with defined microstructures and membranous networks. In order to understand the influence of cross-linking reaction on the secondary structures adopted by the polypeptides, FTIR

Fig. 2. FE-SEM images of (a) Lys120 and (b) Lys225 Ala28 freeze-dried hydrogels. The genipin to lysine molar ratio (R) is 0.5 and the polypeptide concentration is 5 wt%.

analysis was performed to investigate the conformation of the polypeptide chains before and after cross-linking. FTIR spectra of the polypeptide and cross-linked polypeptide hydrogels showed that the main amide I characteristic peaks are at 1649–1652 cm−1 and the amide II characteristic peaks are at 1540–1544 cm−1 (Figs. 3 and S5), indicating that the polypeptides adopted mainly random coil conformation. In addition, the shoulder at 1630–1634 cm−1 suggested that the ␤-sheet conformation with much lower

Fig. 3. FTIR spectra of (a) polypeptides and (b) freeze-dried polypeptide hydrogels. The samples are (1) Lys120 , (2) Lys385 , (3) Lys225 Ala28 , and (4) Lys225 Gly38 . The genipin to lysine molar ratio (R) is 0.5 and the polypeptide concentration is 5 wt%.

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percentage was also present in the polypeptides. The IR absorbance between 1670 and 1674 cm−1 was probably from the lysine side chains. It suggested that the polypeptides did not undergo significant conformational changes after cross-linking. Furthermore, CD analysis was performed to investigate the conformation of the polypeptide chains before and after cross-linking. CD spectra for both polypeptides and cross-linked polypeptide hydrogels did not change significantly and exhibited well-known doubly inflected curves, suggesting that the polypeptides adopted mainly random coil conformation (Fig. S6). For cross-linked polypeptide hydrogels, the positive band at higher wavelength (>230 nm) is the characteristic band of genipin. The results from CD analysis are consistent with those from FTIR analysis. It is worth to note that PLAla and PGly block adopted ␣-helical (or ␤-sheet) and random coil conformations, respectively. Due to the low fraction of the hydrophobic blocks in the polypeptide chains, the FTIR and CD spectra exhibited mainly the signals from the PLL block. 3.4. Swelling behavior and mechanical properties The swelling behavior of the freeze-dried polypeptide hydrogels at different pH was assessed. The results revealed that the hydrogels exhibited pH-sensitive swelling behavior and the degree of swelling was dependent on the polypeptide MW and block ratio (Fig. 4). The swelling ratios of these polypeptide hydrogels mostly ranged from 5 to 22, and were correlated with the protonation of the amino group. It is well known that the pKa value of PLL is generally between 9.0 and 9.5. The degree of protonation decreased with the increase of pH. The deprotonated side chain (NH2 ) is less prone to be hydrated and hence the polypeptide hydrogels swelled with a lower degree at basic condition than at acidic condition. Notably, Lys330 Ala55 hydrogel exhibited greater pH sensitivity as comparing with other hydrogels. For all polypeptides except Lys330 Ala55 , these as-prepared hydrogels possessed comparable swelling ratio at acidic conditions and rather the swelling ratio at basic condition decreased with the increase of MW. At neutral condition, all homopolypeptide hydrogels possessed comparable swelling ratio and, however, the swelling ratio of the block copolypeptide hydrogels decreased with the increase of MW for samples with given block ratio. Lys330 Ala55 hydrogel, which possessed the longest polypeptide chain and hydrophobic block, exhibited the highest swelling ratio at various pHs among all of these samples. It can be attributed to the relative high compaction of the polypeptide aggregates due to the hydrophobic interactions, resulting in the high accommodation of water for the freeze-dried Lys330 Ala55 hydrogel upon swelling. The analogous trend was also observed for the hydrogels composed of chitosan bearing alkyl chains. The hydrogels composed of chitosan bearing longer alkyl chains (carboxymethyl-palmityl chitosan) exhibited greater pH sensitivity as compared with those composed of carboxymethyl-hexanoyl chitosan [65]. The obtained mechanical property of the hydrogels varied with the polypeptide MW, block ratio, and R was investigated. For 5 wt% of hydrogels with R = 0.5, the compressive strengths for these samples ranged between 150 and 280 kN/m2 (Table 1). The hydrogels (5 wt%) prepared with R = 0.5 was found to exhibit comparable cross-linking degree, evidenced by the comparable mole percentage of free amino group (7–11 mole%) based on ninhydrin assay (Table 1). It can be found that the compressive strengths of these hydrogels were correlated with the MW and chain composition. Lys120 , Lys250 , and Lys385 hydrogels have comparable compressive strength. The hydrogels prepared with PLL tethered a short PLAla chain (DP < 30) exhibited poor mechanical properties. The compressive strengths of Lys330 Ala55 and Lys225 Gly38 hydrogels were higher than those of others. The enhancement of mechanical property for Lys330 Ala55 and Lys225 Gly38 hydrogels resulted from the

Fig. 4. Equilibrium swelling ratios of hydrogels at different pH values. The genipin to lysine molar ratio (R) is 0.5 and the polypeptide concentration is 5 wt% (n = 3).

formation of relatively compact aggregates due to the hydrophobic interactions. For comparison, the compressive strengths of the swelled Lys250 and Lys330 Ala55 hydrogels were also characterized. These hydrogels were swelled in aqueous solution for 3 days and the weight percentages of Lys250 and Lys330 Ala55 in the hydrogels were 2.8 and 2.1 wt%, respectively. The compressive strengths for the swelled Lys250 and Lys330 Ala55 hydrogels were measured to be 90 and 66 kN/m2 , respectively (Table 1). The compressive strengths of these hydrogels were also found to vary with the R value (Fig. S7). For Lys250 and Lys330 Ala55 hydrogels, their compressive strengths decreased drastically upon decreasing the genipin to lysine ratio from 0.5 to 0.25. 3.5. In vitro enzymatic degradation and cytotoxicity The hydrogel samples can be stored in PBS without significant weight loss for more than one month, suggesting the hydrolytic degradation of cross-linked polypeptide hydrogels was very slow under this condition. The enzymatic degradation of the materials was carried out by using a model enzyme, bromelain, which is an analog of cathepsin. It has preferences for scission at the amide bonds between lysine (as well as arginine or glutamine) and amino acids with hydrophobic side chains. As shown in Table 2, the polypeptide hydrogels cross-linked at R = 0.125 degraded

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Table 2 Degradation of polypeptide hydrogels in the percentage of weight loss (n = 3). The polypeptide concentration is 5 wt%. Day

Genipin/Lys molar ratio (R) Lys250

Lys330 Ala55

0.5 1 3 5 7 10

9.7 10.5 10.3 13.5 19.5

0.125 ± ± ± ± ±

1.8 2.7 2.8 2.1 7.2

6.1 ± 2.0 10.3 ± 4.4 85.4 ± 7.0 100

0.5 10.2 11.5 10.2 13.5 19.6

0.125 ± ± ± ± ±

0.6 1.9 2.2 2.1 3.5

completely after 7 days, which were much faster than those crosslinked at R = 0.5. The hydrogels with higher R were digested slower due to the small swelling ratio and less enzymatic accessibility. At R = 0.5, it was found that Lys250 and Lys330 Ala55 hydrogels were degraded slower than Lys120 , Lys150 Ala25 , and Lys225 Gly38 hydrogels, suggesting that the degradation of hydrogels was also dependent on MW and composition. The in vitro cytotoxicity of selected polypeptide hydrogels was investigated. It has been reported that highly positively charged polypeptides were found to be cytotoxic to various types of mammalian cells [42,45]. It was attributed to the electrostatic interaction between the cationic polypeptides and the anionic phospholipids in the cell membrane, leading to the cytotoxicity of polycations such as the lysine containing copolymers. In contrast, Pochan and co-workers reported that the hydrogels formed by PLL-bPLLeu block copolypeptides were non-cytotoxic, showing that the cationic polypeptide segments in the intact and rigid hydrogel state were inert [47]. In the present study, it was expected that the charge density on the polypeptide chains can be lowered and the undesired release of the polypeptide segments can be prevented after cross-linking, leading to the enhancement of the biocompatibility of the hydrogels. As shown in Fig. S8, the percentage of cell survival detected for the cross-linked polypeptide hydrogels was higher than 90% of those for the control after 24 h. The population of surviving cell detected between the Lys120 hydrogel (or Lys150 Ala25 hydrogel) and control showed no statically difference (p > 0.01). The enhanced biocompatibility of the crosslinked hydrogels resulted from lowering the charge density on the polypeptide chains and preventing the undesired release of the polypeptide segments, consistent with previous study [47]. In addition to biocompatibility, the ability for these hydrogels to promote cell attachment and proliferation is also a vital requirement for a feasible cell growth substrate. Fibroblast cells were seeded on the polypeptide hydrogels. After seeding, fibroblast cells were found to grow on the polypeptide hydrogel-modified wells and were elongated spindle-shape (Fig. 5), indicating the promotion of cell attachment and proliferation by the polypeptide hydrogels. Moreover, the hydrogels were fully covered with the cells after culturing for 5 days. The results supported that the hydrogels were not cytotoxic to cells and can promote cell attachment and proliferation. Previous studies have shown that cell adhesion on substrates can be affected by the factors including hydrophobicity/hydrophilicity, electric charge, surface morphology, and the nature of surface functional groups [66,67]. The promotion of cell attachment and proliferation by the polypeptide hydrogels may be because of the electrostatic attraction between the positively charged PLL and negatively cell membrane. It is also possible that the surface was not highly swollen, which can facilitate the formation of cell anchorage [65]. As can be seen, the difference in cell morphology can be observed on Lys250 and Lys330 Ala55 hydrogels, revealing that the composition in hydrogels can affect cell adhesion and growth. The increase in hydrophobicity for Lys330 Ala55 hydrogel due to the presence of PLAla block may favor the formation of initial cell contact and hence the cells spread extensively on the

11.5 ± 1.5 12.5 ± 3.8 87.6 ± 0.6 100

Lys120 Ala15

Lys150 Ala25

Lys225 Gly38

0.5

0.5

0.5

5.3 10.8 13.8 17.8 29.7

± ± ± ± ±

1.5 2.7 5.8 5.8 8.8

4.7 6.8 10.5 19.6 24.6

± ± ± ± ±

1.5 4.6 1.7 2.6 3.2

13.0 ± 0.6 – 13.4 ± 6.2 – 25.5 ± 5.5

surface [66]. Moreover, the difference in surface morphology, for example the difference in the average pore size (Fig. S3a and d), for Lys250 and Lys330 Ala55 hydrogels also played a role on the cell adhesion. 3.6. Drug encapsulation and in vitro drug release In order to evaluate these hydrogels as a carrier system, the Lys120 , Lys150 Ala25 , Lys250 , and Lys330 Ala55 polypeptide hydrogels were selected for encapsulating a model water-soluble drug Dox. The effects of polypeptide composition and R on the drug encapsulation efficiency and release profile were investigated (Table S2). It was found that the encapsulation efficiency was higher than 75%. For the Lys120 and Lys150 Ala25 hydrogels, the encapsulation

Fig. 5. Optical microscopy images of fibroblast cells grown on (a) Lys250 and (b) Lys150 Ala25 hydrogel-modified wells after seeding for three days.

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compared with that for the initial release period. As can be seen, the samples prepared with different MWs and R exhibited different release profiles. For the samples prepared at R = 0.125, it was notable that the drug loaded in the Lys120 hydrogel released much faster than the other three samples (Fig. 6a). The initial release profile of the Lys250 hydrogel was comparable with those of the block copolypeptide hydrogels, while after 24 h the drug released slightly faster for the Lys250 hydrogel. It was found that the drug release rate increased with the decrease of the R, which was correlated with the change of cross-linking degree in the hydrogels (Figs. 6b and S9). The results indicated that the drug-release rate of the Dox-loaded hydrogels can be adjusted by the both the polypeptide composition and cross-linking degree. 4. Conclusion We report the synthesis and characterization of genipin-crosslinked, pH-sensitive hydrogels composed of PLL, PLL-b-PLAla or PLL-b-PGly polypeptides. The polypeptide chemistry can influence the pH-sensitive swelling behavior, gelation time, mechanical properties, enzymatic degradation, and drug release behavior of the as-prepared hydrogels. The pH sensitivity was determined by the interplay between the hydrophobic/hydrophilic block ratio and chain length. Greater pH sensitivity was observed in samples with longest polypeptide chains and highest hydrophobic/hydrophilic block ratio (Lys330 Ala55 ). The synergy between cross-linking, hydrophobic interactions, and chain entanglement led to the enhancement of the mechanical properties for Lys330 Ala55 . The polypeptide hydrogels demonstrated good cell compatibility and can support cell attachment/proliferation, potentially making them useful biomaterials for cell culture substrates and tissue engineering scaffolds. The commonly used anticancer drug, Dox, was selected as a model water-soluble drug and loaded into the hydrogels. The data indicated that mild burst release of the drug was observed and the drug release properties can be tuned by the polypeptide composition and cross-linking degree. Acknowledgements Fig. 6. In vitro drug-release profiles of Dox-loaded (a) Lys120 , Lys250 , Lys150 Ala25 , and Lys330 Ala55 hydrogels (5 wt%) cross-linked at R = 0.125, and (b) Lys150 Ala25 hydrogel (5 wt%) cross-linked at different genipin to lysine molar ratios at 37 ◦ C (n = 3).

efficiencies were constant regardless varying R and calculated to be about 87 and 91%, respectively. As the samples were prepared with R = 0.125, the encapsulation efficiencies for the Lys250 and Lys330 Ala55 hydrogels can reach almost 100%. The results revealed that the polypeptide MW and R would influence the encapsulation efficiency. Incorporation of the hydrophobic block instead did not affect the drug encapsulation efficiency to any greater extent as the samples were prepared at fixed R. The drug release experiments of Dox-loaded hydrogels at 37 ◦ C under neutral condition (pH 7.4) were investigated (Fig. 6). The drug-release profile showed a biphasic pattern with an initial burst release (0–6 h) and a moderate release rate thereafter. The undesired burst effect in the initial release period was not significant for all of the samples, evidenced by the low exponent values (<0.5) based on the power law model [68]. It is consistent with the fact that the hydrogels were unlikely hydrolyzed in PBS. Moreover, the swelling of the hydrogels did not facilitate the drug release in the initial stage. The mild burst effect in the initial release period suggested that the drug molecules were encapsulated in the cross-linked polypeptide networks instead of the surface of the gel matrix, and the hydrophobic and hydrogen bonding interactions between the drug and polypeptides as well as genipin led to the low drug mobility. After 24 h, the release rate decreased as

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