Acta Biomaterialia xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications Xuan Pang a,b, Jun Wu c, Chih-Chang Chu b,c,⇑, Xuesi Chen a,⇑ a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853-4401, USA c Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853-4401, USA b

a r t i c l e

i n f o

Article history: Received 16 November 2013 Received in revised form 22 March 2014 Accepted 2 April 2014 Available online xxxx Keywords: Poly(ester amide)s Amino acids Hydrogel Biodegradable

a b s t r a c t A series of biodegradable and biocompatible cationic hybrid hydrogels was developed from water-soluble arginine-based unsaturated polymer (Arg-AG) and poly(ethylene glycol) diacrylate (PEG-DA) by a photocrosslinking method. The physicochemical, mechanical and biological properties of these hydrogels were intensively examined. The hydrogels were characterized in terms of equilibrium swelling ratio (Qeq), compression modulus and interior morphology. The effects of the chemical structure of the two Arg-AG precursors and the feed ratio of these precursors on the properties of resulting hybrid hydrogels were investigated. The crosslinking density and mechanical strength of the hybrid hydrogels increased with an increase in allylglycine (AG) content in the Arg-AG precursor, as the gelation efficiency (Gf) increased from 80% to 90%, but the swelling and pore size of the hybrid hydrogels decreased as the equilibrium swelling weight (Qeq) decreased from 1890% to 1330% and the pore size from 28 to 22 lm. The short-term in vitro biodegradation properties of hydrogels were investigated as a function of Arg-AG chemical structures and enzymes. Hybrid hydrogels showed faster biodegradation in an enzyme solution than in a phosphate-buffered saline solution. Bovine serum albumin and insulin release profiles indicated that this cationic hydrogel system could significantly improve the sustained release of the negatively charged proteins. The cellular response of the hybrid hydrogels was preliminarily evaluated by cell attachment, encapsulation and proliferation tests using live–dead and MTT assay. The results showed that the hybrid hydrogels supported cell attachment well and were nontoxic to the cells. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Hydrogels are materials that can swell and hold a large amount of water in their hydrated state. Due to their high water contents, similar to that of body tissues, and high biocompatibility, hydrogels have attracted significant attention for use as surgical implants, diagnostics, biosensors, bioreactors, bioseparators and matrices for drug delivery and tissue engineering scaffolds [1–4]. Chemically, hydrogels can be broadly divided into two categories: non-biodegradable and biodegradable. Biodegradable hydrogels are more useful in biomedicine because they are fabricated from biodegradable polymer-based precursors and are degraded in the human body and hence do not cause a permanent foreign-body

⇑ Corresponding authors. Address: Department of Fiber Science and Apparel Design, Cornell University, Ithaca, NY 14853-4401, USA. Tel.: +1 607 255 1938; fax: +1 607 255 1093 (C.-C. Chu). Address: Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China. Tel./fax: +86 431 85262112 (X. Chen). E-mail addresses: [email protected] (C.-C. Chu), [email protected] (X. Chen).

inflammatory response [5–13]. Both synthetic biodegradable polymers, e.g. polylactide, poly(lactide-co-glycolide), poly(e-caprolactone), and natural biodegradable polymers, e.g. dextran [14], chitosan [15,16] and hyaluronic acid [17], can be used to prepare biodegradable hydrogels for drug delivery and tissue engineering scaffold applications. By controlling the feed ratios of the hydrophilic and hydrophobic precursors, biodegradable hydrogels with a wide range of swelling ratios, mechanical strengths, internal morphologies and degradabilities can be obtained [18–21]. Amino acid-based poly(ester amide) (AA-PEAs), which are prepared from amino acids, fatty alcohols and aliphatic acids, have both ester and amide linkages on their backbones. They have been introduced as a new family of biodegradable materials [22–38] as they combine the good mechanical, thermal and processing properties of polyamides with the degradability of polyesters into a single entity. Our laboratory has very recently developed a new advanced generation of water-soluble and cationic functional AA-PEAs called Arg-AG (where Arg is the positively charged amino acid arginine and AG is DL-2-allylglycine) that have a pendant vinyl functional group for subsequent photo or non-photo reactions [39].

http://dx.doi.org/10.1016/j.actbio.2014.04.002 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

2

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

This new family of water-soluble and cationic functional Arg-AG PEAs was made from two amino acid building blocks: L-Arg (a positively charged and water-soluble a-amino acid) and a derivative of L-Gly (DL-2-allylglycine). The incorporation of AG in the synthesis of this Arg-AG PEA conferred photoreactivity on the resulting AAPEAs via the pendant unsaturated carbon–carbon double bonds in the AG unit. The pendant double bond content of Arg-AG PEAs is tunable by adjusting the feed ratio of Arg-based to AG-based monomers. This strategy is different to that used for previously reported unsaturated AA-PEAs which were synthesized from unsaturated fatty alcohols/acids, and hence the resulting unsaturated AA-PEAs (UPEAs) have an unsaturated unit in the AA-PEA backbone [22,38,40,41]. As for UPEA polymer, the carbon–carbon double bonds are embedded in the polymer backbone, and the unsaturated content can only be controlled by tuning the molecular weight of the UPEA [22,41]. However, the new approach proposed here allows us to control the unsaturated contents of AAPEAs by adjusting the feed ratio of two amino acid derivatives (Arg- and Gly-based) during the synthesis of the Arg-AG precursor. The aim of this work is to demonstrate the feasibility of forming a series of new biodegradable cationic hybrid hydrogels through photocrosslinking the pendant carbon–carbon double bonds in the Arg-AGs with other non-AA-PEA co-precursors such as poly(ethylene glycol) diacrylate (PEG-DA). Compared with our previously reported phenylalanine-based hybrid hydrogel systems [42,43], the Arg-based hydrogels have the ability to carry positive charge due to the strong basic guanidino group, which provides the potential to condense negatively charged DNA. The electrostatic interaction between the positive charge on the arginine of the hydrogels and the negative charge on DNA should result in better interactions with cells and achieve sustained release of ionic drugs [25]. The wide variety of hydrogel components and parameters should allow engineering of hydrogels with a wide range of cytokine release kinetics. In our very recent arginine-based hydrogel research [38], double bonds of Arg-PEA were introduced into the PEA backbone via the Arg-UPEA precursor, instead of the pendant double bond groups investigated in this current work. The embedded backbone double bond contents could only be controlled by the molecular weight of the Arg-UPEAs, and the equilibrium swelling ratio (Qeq) of the resulting hydrogels decreased with an increase in the Arg-UPEA precursor. The incorporation of ArgUPEA precursor into the F127 hybrid hydrogels lowered their overall compressive moduli dramatically. In this work, the Arg-AG/ PEG-DA hybrid hydrogels were characterized by their gel fraction (Gf), equilibrium swelling ratio (Qeq), compressive modulus and internal morphology. The effects of the precursor feed ratio on the property of the cationic hybrid hydrogels were studied and compared in detail with our very recent research [38]. Negatively charged proteins were loaded into the hydrogels to evaluate the sustained release performance of this hydrogel system. A preliminary assessment of the cell interaction with these cationic hybrid hydrogels was conducted using a bovine aortical endothelial cell proliferation assay. 2. Experimental 2.1. Materials DL-2-Allylglycine (AG), L-Arginine (Arg), p-toluenesulfonic acid monohydrate (TosOHH2O), diethylene glycol, tetraethylene glycol, hydroquinone, sebacoyl chloride, succinyl chloride, 1,4-butanediol, 1,6-hexanediol (Alfa Aesar, Ward Hill, MA) and p-nitrophenol (J.T. Baker, Phillipsburg, NJ) were used without further purification. Bovine serum albumin (BSA) and insulin were purchased from Sigma–Aldrich (St. Louis, MO) and used directly. PEG-DA (PEG Mn = 4000) was synthesized as described previously [42].

2.2. Synthesis of Arg-AGs polymer The functional Arg-AGs with pendant double bonds were synthesized through the solution polycondensation of di-p-nitrophenyl diester (monomer II) with a mixture of di-p-toluenesulfonic acid salts of bis-L-Arginine (Arg-y) (monomer I) and bis-DL-2-allylglycine diesters (AG-nEG) (monomer III) in a predetermined feed ratio [39]. The combinations used in this work were listed in Table 1 and illustrated in Fig. 1. 2.3. Preparation of Arg-AG-based hybrid hydrogels Arg-AG/PEG-DA Arg-AG/PEG-DA biodegradable hydrogels were prepared by the photopolymerization of both Arg-AG and PEG-DA precursors. The PEG-DA precursor (PEG Mn = 4000) was synthesized as described previously [42]. Irgacure 2959Ò was added as a photoinitiator. The Arg-AG itself, however, could not form hydrogels via photocrosslinking. An example of the synthetic method for such a hydrogel from 8-Arg-4-AG-4EG and PEG-DA (designated as 8-Arg-4-AG-4EG-G) is given below. A weight ratio of 1:3 of 8-Arg-4-AG-4EG to PEG-DA (0.08 g 8-Arg-4-AG-4EG, 0.24 g PEG-DA) was added to a vial and dissolved in 2 ml of dimethylacetamide to form a clear, homogeneous solution with a light yellow color. The photoinitiator Irgacure 2959Ò (0.016 g, 5 wt.% of the total amount of the precursors) was added to the solution of the precursors and dissolved completely at room temperature. The mixed solution was poured into a Teflon mold and irradiated by a long-wavelength UV lamp (365 nm, 100 W) for 15 min at room temperature, resulting in gel formation. The resultant hydrogel (diameter 25 mm, thickness 4 mm) was carefully removed from the mold and washed with distilled water to remove any residual chemicals. The distilled water was replaced periodically. After this purification process, the hydrogels were soaked in distilled water until swelling equilibrium and then removed and dried in vacuo at room temperature for 48 h before subsequent characterization. The gel fraction, Gf, was used to describe the extent of the hydrogel formation through the following equation:

Gf ¼

Wd  100%; Wp

ð1Þ

where Wd is the weight of the dry hydrogel and Wp is the total feed weight of the two macromer precursors and the photoinitiator. 2.4. Internal morphology of hydrogels Scanning electron microscopy (SEM) was used to analyze the internal morphology of the Arg-AG/PEG-DA hydrogels as a function of the precursor feed ratio. A cryofixation technique was used to observe the swollen hydrogel structure with minimal artifacts. Typically, an individual hydrogel was immersed in distilled water at room temperature for 3 days to reach its equilibrium swelling. The hydrogel was then gently removed and immediately transferred into liquid nitrogen to freeze and retain its swollen structure. The sample was subsequently freeze-dried for 72 h in a

Table 1 List of monomers and polymers synthesized by different monomer combinations. Monomer I

Monomer II

Monomer III

Polymer

Arg-4 Arg-6 Arg-4 Arg-6

N-2 N-8 N-2 N-8

AG-2EG

2-Arg-4-AG-2EG 8-Arg-4-AG-2EG 2-Arg-4-AG-4EG 8-Arg-4-AG-4EG

AG-4EG

2-Arg-6-AG-2EG 8-Arg-6-AG-2EG 2-Arg-6-AG-4EG 8-Arg-6-AG-4EG

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

3

Fig. 1. Synthetic pathway for the preparation of poly(ester amide)s.

Virtis (Gardiner, NY) freeze-drier in vacuo at 40 °C and finally fixed on aluminum stubs and coated with gold for 60 s for SEM observation with a Hitachi S4500 microscope (Mountain View, CA). Image analysis of SEM micrographs was performed with the public domain NIH Image program (http://rsb.info.nih.gov/nihimage/). 2.5. Swelling kinetics The swelling kinetics of the Arg-AG/PEG-DA hydrogels was measured over a period of 7 days at room temperature. Dry ArgAG/PEG-DA hydrogels were weighed and immersed in 15 ml of distilled water or buffers at room temperature for predetermined periods; they were then removed, the surface water blotted by filter paper and weighed until there was no further weight change. The swelling ratio Qt at time t was calculated as follows:

Qt ¼

Ws  Wd  100%; Wd

ð2Þ

where Ws is the weight of the swollen hydrogel at time t and Wd is the weight of the dry hydrogel at t = 0. 2.6. Mechanical testing Mechanical testing of the Arg-AG/PEG-DA hydrogels was performed by dynamic mechanical analysis on a DMA Q800 (TA Instruments Inc., New Castle, DE) in ‘‘controlled force’’ mode. The disc-shaped swollen hydrogel samples were submerged in distilled water and mounted between a movable compression probe (diameter 15 mm) and a fluid cup. A compression force from 0.01 to 0.05 or 0.30 N (depending on the gel strength) at a rate of 0.02 or 0.05 N min1 was applied at room temperature. The compression elastic modulus (E) of the swollen hydrogel was calculated by plotting the compressional stress vs. strain [41].

2.7. Short-term in vitro biodegradation of Arg-AG/PEG-DA hybrid hydrogels The biodegradation of Arg-AG/PEG-DA hybrid hydrogels was carried out in a small vial containing a small piece of dry hydrogel sample (80 mg) and 10 ml of phosphate-buffered saline (PBS) (pH 7.4, 0.1 M) with trypsin at different concentrations (0.1 or 0.2 mg ml1). Pure PBS was used as control. The vial was then incubated at 37 °C with constant reciprocal shaking (100 rpm). The incubation media were refreshed daily in order to maintain enzymatic activity. At predetermined immersion durations, hydrogel samples were removed from the incubation medium, washed gently with distilled water, and then lyophilized in vacuo using a FreeZone Benchtop and Console Freeze Dry System (Model 7750000, LABCONCO Co., Kansas City, MO) at 40 °C for 72 h to a constant weight. The degree of biodegradation was estimated from the weight loss of the hydrogel based on the following equation:

W l ð%Þ ¼

Wo  Wt  100; Wo

ð3Þ

where Wo is the original weight of the dry hydrogel sample before immersion, and Wt is the hydrogel sample weight after incubation for t days (with or without the enzyme). The weight loss averaged for three specimens was recorded. 2.8. In vitro release of BSA and insulin from Arg-AG/PEG-DA hydrogel Negatively charged BSA and insulin were chosen as model protein drugs for controlled release from these newly synthesized hydrogels. BSA solution (5.0 wt.%) was prepared by dissolving 2 g BSA in 40 ml of PBS solution (pH 7.4, 0.1 M). Insulin solution (2.0 wt.%) was prepared by dissolving insulin in a low pH solution first, then the solution was neutralized to 7.4 and diluted with PBS

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

4

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

solution to obtain a final insulin concentration of 2.0 wt.%. Before drug loading, both pure PEG-DA and Arg-AG/PEG-DA hybrid hydrogels were dried in vacuo for 2 days. The drug was then loaded into these hydrogels by immersing them into the protein solution at 25 °C for 2 days to reach equilibrium. The BSA release study was performed by placing the BSA-loaded hydrogel samples into a glass bottle filled with a 50 ml PBS solution (pH 7.4, 0.1 M) at 37 °C. At predetermined periods, 5 ml aliquots of the buffer solution were removed from the glass tube and 5 ml of fresh buffer solution was added to maintain the same total solution volume. The concentration of the BSA in the aliquot removed was measured by comparing the UV spectra (Lambda Bio40 UV–Vis spectrometer, Perkin-Elmer) at 280 nm vs. a BSA calibration curve. The results are presented in terms of the cumulative release as a function of time, and the cumulative BSA release (%) was calculated as: cumulative BSA release (%) = (Mt/M0)  100, where Mt is the amount of BSA released from the hydrogel at time t, and M0 is the initial BSA loaded into the hydrogel. In this study, the amount of BSA initially loaded into the hydrogel was calculated as: M0 = (We  Wd)  5.0 wt.%, where We is the weight of swollen hydrogel in the BSA solution, Wd is the initial weight of the dried hydrogel before immersion, and 5.0 wt.% is the concentration of BSA aqueous solution. The insulin release study was performed in the same way as the BSA release study, except that the concentration of insulin was measured by bicinchoninic acid assay. 2.9. Evaluation of cellular interaction of Arg-AG/PEG-DA hydrogels Bovine aortic endothelial cells (BAECs) were purchased from VEC Technologies. BAECs were maintained at 37 °C in 5% CO2 in Medium199 (Invitrogen, Carlsbad, CA) supplemented with 10% Fetal Clone III (HyClone, Logan, UT) and 1% each of penicillin– streptomycin, minimal essential medium amino acids (Invitrogen, Carlsbad, CA) and minimal essential medium vitamins (Mediatech, Manassas, VA). BAECs were used between passages 8 and 12. The medium was changed every 2 days. Cells were grown to a minimum of 70% confluence before splitting or harvesting. Cell culture plates were treated with 2 wt.% gelatin aqueous solution before use. Detroit 539 human fibroblast cells were purchased from ATCC. The fibroblast cells were maintained at 37 °C in 5% CO2 in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (Germini, Woodland, CA) and 1% each of

penicillin–streptomycin, MEM amino acids (Invitrogen, Carlsbad, CA) and 0.1% lactalbumin hydrolysate. Fibroblast cells were used from passages 10 to 20 and media was changed every 2 days. Cells were grown to 70% confluence before splitting or harvesting. The evaluation of the cellular interaction of Arg-AG/PEG-DA hydrogels was performed by live–dead assay (Invitrogen) using a representative 8-Arg-4-AG-4EG-G hybrid hydrogel. The purified hydrogels were cut into discs (1 cm diameter) and put into 24-well cell culture plates after 30 min UV sterilization. Cultured BAECs or fibroblast cells were then seeded at an appropriate cell density concentration (10,000 or 20,000 cells per well) and incubated overnight. After 24 h treatment and incubation, the live–dead assay was performed according to the manufacturer’s protocol (LIVE/ DEADÒ Cell Viability Assays, Invitrogen). Black and white microscope images were also taken to record the cell morphology. For quantitative evaluation, the attached BAECs were first detached from the hydrogel surface by washing or quick trypsin treatment (0.1 mg ml1), then the detached cells were transferred into a new 96-cell culture plate. After incubation (12 h), MTT assay was performed and the optical density was used as an index of the cell density. 2.10. Statistics Where appropriate, the data are presented as mean ± standard error of the mean calculated over at least three data points. Significant differences compared to control groups were evaluated by unpaired Student’s t-test or Dunnett’s test at P < 0.05, and between more than two groups by Tukey’s test with or without one-way analysis of variance (ANOVA). JMP software (v. 8.0, SAS Company) was used for data analysis. 3. Results and discussion 3.1. Arg-AG/PEG-DA hydrogel formulation The double bonds in the Arg-AG side chain make it feasible to crosslink this precursor with other macromer precursors upon photoirradiation. In order to demonstrate their crosslinkable functionality and furthermore investigate the potential applications of Arg-AG in biomedical and pharmaceutical fields, a series

Fig. 2. One of the possible photocrosslinked chemical structures and an optical image of Arg-AG/PEG-DA hydrogel.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

5

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx Table 2 Properties of Arg-AG/PEG-DA hydrogels.a

a

Sample label

Mole feed ratio of Arg to AG

Gf (%)

Qeq (100%)

Compressive modulus (kPa)

Pure PEG-DA 8-Arg-4-AG-4EG-25-G 8-Arg-4-AG-4EG-33-G 8-Arg-4-AG-4EG-50-G 2-Arg-4-AG-4EG-25-G 2-Arg-4-AG-4EG-50-G

N/A 3:1 2:1 1:1 3:1 1:1

92 80 85 88 85 90

13.5 ± 0.5 18.9 ± 0.5 16.0 ± 0.4 13.6 ± 0.2 14.7 ± 0.4 13.3 ± 0.3

72 ± 3 51 ± 5 58 ± 6 66 ± 6 60 ± 8 67 ± 9

The weight ratio of PEA to PEG-DA was kept at 1:3.

of hybrid hydrogels (Arg-AG/PEG-DA) was prepared with PEG-DA co-precursor by UV irradiation. 8-Arg-4-AG-4EG was used as a representative Arg-AG precursor. One of the possible chemical structures of these hybrid hydrogels and optical image is shown in Fig. 2. Our laboratory has successfully established a viable strategy for the synthesis of saturated and unsaturated phenylalanine-based PEAs [22,23,40,44,45]. These UPEAs have carbon–carbon double bonds embedded in the UPEA polymer backbone, and have been used as the precursors to fabricate AA-PEA-based hydrogels via photocrosslinking with PEG-DA co-precursor [41,42] for the delivery of paclitaxel [46]. These UPEA polymers showed controllable biodegradability by changing their chemical structure, and could be desirable candidates for future biomedical and pharmaceutical applications. However, the embedded double bond contents could only be controlled by the molecular weight of the UPEAs, and these phenylalanine-based hybrid hydrogels did not contain any charge. Very recently, we reported a new generation of water-soluble and cationic functional AA-PEAs, called Arg-AG, that have a pendant unsaturated vinyl group for subsequent photo- or non-photo reactions [39]. This new generation of water-soluble and cationic functional Arg-AG PEAs possessing photoreactivity was made from two amino acid building blocks: L-Arg and AG, a derivative of L-Gly. The double bond contents were adjustable by controlling the Arg to AG feed ratio in the synthesis of the Arg-AG precursor. In comparison with our previously reported phenylalanine-based hydrogel systems [42,43], the Arg-based hydrogels have the advantage that they can carry positive charge due to the strong basic guanidino group, which provides the potential to condense negatively charged DNA, improve interactions with cells or perform the sustained release of ionic drugs [25]. To evaluate the effect of the Arg-AG precursor types on the properties of the formed hybrid hydrogels, a family of Arg-AGs with different Arg-4 to AG-4EG mole ratios (3:1, 2:1, 1:1) were used as precursors to fabricate hybrid hydrogels with PEG-DA coprecursor at the constant weight feed ratio of Arg-AG to PEG-DA (1:3). As shown in Table 2, the gelation efficiency, Gf, of the hybrid hydrogels increased with an increase in the AG contents in the ArgAG precursor. For example, the Gf values of the 8-Arg-4-AG-4EG-G hydrogels increased from 80% for 8-Arg-4-AG-4EG-25-G, 85% for 8Arg-4-AG-4EG-33-G to 88% for 8-Arg-4-AG-4EG-50-G. The increasing Gf values indicate a higher conversion yield of the precursor, and therefore a higher level of crosslinked hydrogel structure formation. An increase in the AG content in the Arg-AG precursor would result in a corresponding increase in the pendant double bonds located in the AG unit, i.e. more crosslinkable sites and hence a higher Gf. This relationship between AG and Gf was different from our laboratory’s very recent Arg-based hydrogel research [38]. When hybridized with co-precursor PEG-DA or Pluronic-DA, the location of the double bonds in the unsaturated Arg-based PEA precursor could have a profound effect on the hydrogel properties. When the double bonds were introduced into the AA-PEA backbone as Arg-UPEA precursor [38], the Gf values of the hydrogels obtained

decreased with an increase in Arg-UPEA content. It is believed that, in our current Arg-AG-based PEA systems, the pendant double bonds in the Arg-AG precursor are more accessible than the backbone double bonds in Arg-UPEA. Therefore, it is suggested that the reactivity of the pendant double bonds in Arg-AG is higher than those Arg-UPEAs with double bonds in the backbone. Steric hindrance to the backbone double bonds due to the bulky guanidino group may be the major cause behind this difference in reactivity. 3.2. Swelling of the hydrogels Fig. 3 shows the swelling kinetics of all the hydrogels at room temperature. All the hydrogel samples swelled fast, and within 7 h they all reached 90% of their final equilibrium swelling weight (Qeq); the swelling rates became significantly lower thereafter. All the hydrogel samples reached their swelling equilibrium in 3– 4 days. It was found that the hybrid hydrogels had swollen far more than a pure PEG-DA hydrogel (1350 ± 50%) due to the higher hydrophilicity of Arg in spite of their higher crosslinking density. The effect of Arg-AG architecture on swelling was studied in detail. 3.2.1. Effect of the AG segment content on swelling The Qeq values of the hybrid hydrogels (Table 2) decreased with an increase in the AG-nEG to Arg-y feed ratio. For example, the Qeq values of 8-Arg-4-AG-4EG-G hydrogels decreased from 1,890 ± 50% for 8-Arg-4-AG-4EG-25-G at an AG-4EG:Arg-4 ratio of 1:3, 1,600 ± 40% for 8-Arg-4-AG-4EG-33-G at an AG-4EG:Arg-4 ratio of 1:2 to 1,360 ± 20% for 8-Arg-4-AG-4EG-50-G at an AG-4EG:Arg-4 ratio of 1:1 (Fig. 3a). As the AG-4EG to Arg-4 feed ratio increased, the number of AG units increased which resulted in a higher pendant double bond content, i.e. providing more crosslinkable sites to produce a tighter network structure and hence a lower Qeq. Similar trends in Qeq were also observed in our prior phenylalanine-based hydrogel Phe-AG-G research [37,43]. The Qeq value of the Phe-AG-G decreased with an increase in the Phe-AG precursor from 860 ± 20% (at a Phe-4:AG-4 ratio of 3:1) to 430 ± 30% (at a Phe-4:AG-4 ratio of 1:1). Compared with the corresponding PEA-AG-G hydrogel, the Arg-AG/PEG-DA hydrogels had higher Qeq values than the Phe-AG-G hydrogels due to the higher hydrophilicity of arginine. 3.2.2. Effect of methylene chain length in the Arg-AG precursor on swelling The effect of the methylene chain length in the Arg-AG backbone on the swelling ratios of the Arg-AG/PEG-DA hydrogels is shown in Fig. 3b. At a constant Arg-AG to PEG-DA feed ratio (1:3), shortening the diacid segment –CH2– (i.e. x value) from C8 (8-Arg-4-AG-4EG-G) to C2 (2-Arg-4-AG-4EG-G) decreased the Qeq value from 1,890 ± 50% to 1,470 ± 50% (Fig. 3b). A similar trend was also found for he diester segment –CH2– (i.e. y value) as shortening the diester segment from C6 (8-Arg-6-AG-4EG-G) to C4 (8-Arg-4-AG-4EG-G) also resulted in a lower Qeq value. Qeq ranged from 1,980 ± 50% (8-Arg-6-AG-4EG) to 1,890 ± 50%

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

6

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

Fig. 3. Swelling behaviors of Arg-AG/PEG-DA: (a) with different allylglycine segment contents; (b) with different backbone lengths; (c) with different pH values.

(8-Arg-4-AG-4EG-G). This x and y methylene chain length effect on Qeq was also found in other amino acid–based PEA hydrogels such as phenylalanine-based PEA hybrid hydrogels [43]. Pang et al. reported that a shorter CH2 segment from C8 to C2 in the y methylene chain length decreased the Qeq value slightly from 860% to 750% [43]. They attributed this to the formation of a tighter network structure of the hybrid hydrogels due to a denser double bond content resulting from a shorter methylene chain spacer between two adjacent double bonds. 3.2.3. Effect of pH on Arg-AG hydrogel swelling The effect of the pH values on the swelling ratios of the Arg-AG/ PEG-DA hydrogels is shown in Fig. 3c. The Qeq values of the hybrid hydrogels slightly decreased with an increase in pH. For example, the Qeq values of 8-Arg-4-AG-4EG-33-G hydrogels decreased from 1,730 ± 50% at pH 5, 1,600 ± 40% at pH 7, to 1,240 ± 20% at pH 10 (Fig. 3c). This change in Qeq could be due to the osmotic effect, which was the chief factor affecting hydrogel swelling. The osmotic effect is related to the salt concentration since the guanidinium group of arginine could be in salt form. Another minor effect could be due to the change in electronic repulsion among the guanidinium groups of arginine. It is important to know that when hybridized with a co-precursor, the location of the carbon–carbon double bonds in AA-PEA could have a profoundly different effect on the hydrogel property. In very recent arginine-based hydrogel research, double bonds were introduced into the Arg-PEA backbone via the Arg-UPEA precursor, and the Qeq value of the resulting hydrogels increased with an increase in the amount of Arg-UPEA precursor [38]. In this study, the double bonds were introduced as pendant groups to the Arg-PEA backbone, and their corresponding Qeq values decreased with an increase in the Arg-AG content instead. The only difference between Arg-UPEA and Arg-AG precursors is the double bond location: Arg-UPEA has double bonds located in backbone, and Arg-AG in the side chain. The pendant double bonds in Arg-AG are more accessible than the backbone double bonds in Arg-UPEA due to the fact that the bulky guanidino groups of Arg

in Arg-UPEA can sterically hinder the backbone double bond from participating in the photocrosslinking reaction. Based on the Gf and Qeq data, we conclude that the reactivity of the pendant double bonds in Arg-AG is higher than those with double bonds in the AA-PEA backbone. 3.3. Mechanical property of the hydrogels The compressive moduli of the tested hydrogels are summarized in Table 2. A pure PEG-DA hydrogel is listed as a control. The data in Table 2 show that Arg-AG-based hybrid hydrogels have lower compressive moduli than a pure PEG-DA hydrogel control (72 ± 3 kPa) due to the higher hydrophilicity of Arg unit in spite of their higher crosslinking density. An increase in the AG contents in the Arg-AG precursor resulted in hybrid hydrogels having higher compressive moduli. For example, the compressive moduli of the series of Arg-AG/PEG-DA hybrid hydrogels increased from 51 ± 5 kPa for 8-Arg-4-AG-4EG-25-G (Arg-4:AG-4EG = 3:1), 58 ± 6 kPa for 8-Arg-4-AG-4EG-33-G (Arg-4:AG-4EG = 2:1), to 66 ± 6 kPa for 8-Arg-4-AG-4EG-50-G (Arg-4:AG-4EG = 1:1), i.e. the compressive modulus exhibited a 29% increase as the Arg:AG feed ratios of these hybrid hydrogels changed from 3:1 to 1:1. These mechanical data are also consistent with the equilibrium swelling data Qeq indicating that a tighter network structure due to a higher AG content led to hybrid hydrogels having lower equilibrium swelling and a higher compression modulus. There is no obvious x effect observed in this paper between x = 2 and x = 8, which is not consistent with rubber elasticity theory. One possible reason could be the different structure and properties of the prepared polymer precursors, which may cause the difference in crosslinking efficiency. Further investigation will be carried out on this phenomenon. In our prior Arg-UPEA-based hydrogel research [38], the incorporation of Arg-UPEA precursor into F127 hydrogels lowered their overall compressive moduli dramatically. In the current research, however, the compressive moduli of Arg-AG/PEG hybrid hydrogels decreased slightly with the incorporation of Arg-AG into PEG hydrogels. This suggested that the reactivity of the pendant

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

7

Fig. 4. SEM images of Arg-AG/PEG-DA hydrogels: (a) pure PEG-DA hydrogel; (b) 8-Arg-4-AG-4EG-25-G; (c) 8-Arg-4-AG-4EG-50-G.

double bonds in Arg-AG was higher than that of the Arg-UPEAs with double bonds in the backbone. 3.4. Interior morphology of hydrogels SEM was used to investigate the internal morphology of the Arg-AG/PEG-DA hydrogels as a function of AG content in the Arg-AG precursor. SEM images of a pure PEG-DA hydrogel and Arg-AG/PEG-DA hybrid hydrogels are shown in Fig. 4. Both the pure PEG-DA and Arg-AG/PEG-DA hybrid hydrogels having higher amounts of AG-nEG monomer III (e.g. 8-Arg-4-AG-4EG-50-G) exhibited well-defined, regular, more structured 3-D porous network structures (Fig. 4a,c), while those Arg-AG/PEG-DA hybrid hydrogels having higher Arg-y content (monomer I) in the feed ratio showed a less regular and structured 3-D porous network (Fig. 4b). Compared with the PEG-DA hydrogel (average diameter 20.4 ± 4.0 lm), Arg-AG/PEG-DA hybrid hydrogels had a relatively

bigger pore size mainly due to the higher hydrophilicity of the Arg component. For example, 8-Arg-4-AG-4EG-25-G had a pore size of 28 lm. The pore size of the hybrid hydrogels reduced slightly with an increase in the AG content (monomer III) in the hydrogel systems (22 lm for 8-Arg-4-AG-4EG-50-G vs. 28 lm for 8-Arg-4-AG-4EG-25-G). This was because as the AG-nEG monomer III content in the feed ratio increased, more active double bonds were introduced, resulting in a higher crosslinking density and hence a tighter network structure. The change in methylene chain length had little effect on the interior morphology of the hybrid hydrogels, even though a different methylene chain length could lead to a different double bond density. Compared with the PEA-AG-G hydrogels [43], Arg-AG/PEG-DA hydrogels had larger pores. This is consistent with the equilibrium swelling data Qeq, indicating that the hydrophilicity of Arg-AG is higher than that of Phe-AG due to the more hydrophilic amino acid arginine.

Fig. 5. Biodegradation behaviors of 8-Arg-4-AG-4EG-G: (a) in different trypsin concentrations and pure PBS; (b) with different allylglycine segment contents in 0.1 mg ml trypsin solution.

Fig. 6. Controlled BSA (left) and insulin (right) release from the pure PEG-DA hydrogel, 8-Arg-4-AG-4EG-50-G and hybrid 8-Arg-4-AG-4EG-33-G hydrogel in PBS solution (pH 7.4, I = 0.1 M) at 37 °C.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

8

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

3.5. Short-term in vitro biodegradation of Arg-AG/PEG-DA hybrid hydrogels The short-term in vitro biodegradation property of the Arg-AG/ PEG-DA hybrid hydrogels was investigated in both PBS buffer and trypsin solutions of pH 7.4 in terms of the weight loss over 2 weeks. As shown in Fig. 5a, the weight loss of 8-Arg-4-AG-4EG-G in the trypsin solution was faster than in pure PBS buffer. The weight loss increased with an increase in the trypsin concentration. A 25 ± 2% weight loss in pure PBS buffer was observed for 8-Arg-4-AG-4EG-G during the 2 week incubation. The weight loss increased to 74 ± 3% and 82 ± 2% in 0.1 and 0.2 mg ml1 trypsin solutions, respectively. The weight loss rate decreased with an increase in the AG content in the Arg-AG precursor (Fig. 5b). The 8-Arg-4-AG-4EG-50-G sample had a 60 ± 3% weight loss, compared with the 74 ± 3% and the 81 ± 4% weight loss of the 8-Arg-4-AG-4EG-33-G and 8-Arg-4-AG4EG-25-G samples, respectively, in 0.1 mg ml1 trypsin solution. A decrease in AG content was followed by a decrease in the density of pendant double bonds located in the AG unit. Hence, a reduction in the number of crosslinkable sites would result in a looser network structure, making it easier for trypsin to degrade the network [47]. 3.6. In vitro release of BSA and insulin Fig. 6 shows the release profiles of BSA and insulin from the pure PEG-DA hydrogel and 8-Arg-4-AG-4EG-G hybrid hydrogel in

a PBS solution (pH 7.4, 0.1 M) at room temperature. It was found that there was a burst of BSA or insulin release at the initial stage for both of the hydrogels, but the level of burst release strongly depended on the type of hydrogel. 8-Arg-4-AG-4EG-G hybrid hydrogel showed significantly lower burst BSA or insulin release than the pure PEG-DA hydrogel control. This could be due to the positively charged guanidine groups of Arg-AG PEA in the hydrogels. The pKa of arginine is 12.5, while the isoelectric points of BSA and insulin are 4.7 and 5.4, respectively. Therefore, the cationic guanidine groups in the Arg-AG moiety of the hybrid hydrogels could attract the anionic BSA or insulin at a physiological pH, 7.4, resulting in a smaller burst release than the one from a pure PEG-DA hydrogel. After the initial stage, most of the loaded BSA or insulin was quickly released from the pure PEG-DA hydrogel within 12 h, while the cationic hydrogel showed a sustained BSA and insulin release for 2–3 days due to the strong cationic moiety. Moreover, it was found that the release rate of the hybrid hydrogels increased with an increase in the AG contents as 8Arg-4-AG-4EG-50-G had a higher release rate than 8-Arg-4-AG4EG-33-G. As the AG-4EG to Arg-4 feed ratio increased, the number of positively charged Arg units decreased and the interaction with negatively charged BSA or insulin became weaker. Therefore, the release rate increased. Due to the intrinsic differences of BSA and insulin, their release profiles also showed some difference. In general, for the hydrogel systems used in this paper, insulin always showed a relatively faster rate than BSA. One possible reason could

Fig. 7. Cellular interaction of Arg-AG/PEG-DA hydrogels: (a,b) live–dead assay for BAECs cultured on hydrogel surface (10,000 per well) after 24 h incubation; (c,d) live–dead assay for BAECs encapsulated inside hydrogel (10,000 per well) after 24 h incubation; (e,f) black and white microscope images of fibroblasts and BAECs cultured on hydrogel surface (20,000 per well) after 48 h incubation.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

9

Fig. 8. Proliferation assay of BAECs on hydrogel surface: cell proliferation activity on the surface of (a) PEG-DA hydrogel and (b) 8-Arg-4-AG-4EG-50-G hydrogel. Optical density is used as an index of the living cell density.

be the significant difference in molecular weight between insulin and BSA: a small molecular weight (size) will cause weak electrostatic interaction and therefore a faster release rate. 3.7. Cellular interaction evaluation of hydrogels To preliminarily evaluate the cellular interaction of the hydrogels, the cell attachment, encapsulation and proliferation behavior on the hydrogel surface or inside hydrogel were tested and recorded via the live–dead assay and by microscopy. As shown in Fig. 7a and b, most of the BAECs attached on the surface of the 8Arg-4-AG-4EG-G hybrid hydrogel after 24 h incubation were still alive (stained in green) with good morphology, and almost no dead cells (stained in red) were observed on the hydrogel surface, implying that this type of hybrid hydrogel was not toxic to the BAECs. For the encapsulated BAECs inside hydrogels, Fig. 7c and d indicate that most of the encapsulated BAECs inside the 8-Arg-4-AG-4EG-G hybrid hydrogel after 24 h incubation were still alive (stained in green dots), and very few dead cells (stained in red dots) were observed, also implying that this type of hybrid hydrogel was not toxic to the encapsulated BAECs. Fig. 7e and f are black and white microscope images of fibroblasts and BAECs (cultured cell density is twice that in Fig. 7a–d) attached and proliferated on the hydrogel surface after 48 h. Almost all the cells showed good morphology, indicating that this hydrogel system can well support the cell attachment for multiple types of cells. The qualitative data in Fig. 8 also confirmed that the 8-Arg-4-AG-4EG-50-G hydrogel can significantly increase BAEC attachment and proliferation on the surface compared to a pure PEG-DA hydrogel. Thus, chemical incorporation of 8-Arg-4-AG-4EG-G into PEG-DA can significantly promote cell proliferation of PEG-DA-based hydrogels. 4. Conclusions A series of novel biodegradable cationic hybrid hydrogels were fabricated from a new functional Arg-based poly(ester amide) precursor and a commercial poly(ethylene glycol) diacrylate co-precursor by UV photocrosslinking. These hybrid hydrogels were characterized in terms of their internal morphology, equilibrium swelling ratio (Qeq) and compression modulus. All these hybrid hydrogels showed 3-D porous network structures and their pore size and equilibrium swelling ratio reduced with an increase in AG content (i.e. double bond content) in the Arg-AG precursor; however, the compression modulus increased with an increase in AG content. The effects of the Arg-AG chemical structures on hydrogel biodegradation properties (in terms of weight loss) were investigated in trypsin media at different concentrations. The short-term in vitro biodegradation data suggested that the hybrid

hydrogels biodegraded faster in an enzyme solution than in a PBS solution. Preliminary cellular interaction tests of these hybrid hydrogels using fibroblasts and BAECs were conducted, and live– dead cell assay and microscopy images showed that these hybrid hydrogels could support cell attachment/encapsulation and were nontoxic to the BAECs and fibroblasts. BSA and insulin were chosen to demonstrate the controlled drug release capability, and the cationic hybrid hydrogel showed reduced burst release and sustained release for 72 h in a PBS solution, i.e. significantly better sustainability than a pure PEG-DA hydrogel. Acknowledgments The National Natural Science Foundation of China (21004061, 21074018, 51173183, 51021003, 51103058 and 51203155), the Cornell University Morgan Tissue Engineering Seed Grant Program and the Jilin Province Youth Fund (201101059) are greatly appreciated for their financial support. We also thank Prof. C.A. Reinhart-King at Cornell University for the use of her laboratory for some cell culture work. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 2, 6 and 7 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio. 2014.04.002. References [1] Inoue T, Chen G, Nakamae K, Hoffman AS. A hydrophobically-modified bioadhesive polyelectrolyte hydrogel for drug delivery. J Controlled Release 1997;49:167–76. [2] Patterson J, Hubbell JA. SPARC-derived protease substrates to enhance the plasmin sensitivity of molecularly engineered PEG hydrogels. Biomaterials 2011;32:1301–10. [3] Pal K, Banthia AK, Majumdar DK. Polymeric hydrogels: characterization and biomedical applications. Des Monomers Polym 2009;12:197–220. [4] Wu DQ, Wu J, Chu CC. A novel family of biodegradable hybrid hydrogels from arginine-based poly(ester amide) and hyaluronic acid precursors. Soft Matter 2013;9:3965–75. [5] Uhrich KE, Cannizzaro SM, Langer RS, Shakesheff KM. Polymeric systems for controlled drug release. Chem Rev (Washington, DC) 1999;99:3181–98. [6] Albertsson A-C, Varma IK. Recent developments in ring opening polymerization of lactones for biomedical applications. Biomacromolecules 2003;4:1466–86. [7] Chiellini E, Solaro R. Biodegradable polymeric materials. Adv Mater (Weinheim, Germany) 1996;8:305–13. [8] Drumright RE, Gruber PR, Henton DE. Polylactic acid technology. Adv Mater (Weinheim, Germany) 2000;12:1841–6. [9] Eguiburu JL, Fernandez-Berridi MJ, Cossio FP, San Roman J. Ring-opening polymerization of L-lactide initiated by (2-methacryloxy)ethyloxy-aluminum trialkoxides. 1. Kinetics. Macromolecules 1999;32:8252–8.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002

10

X. Pang et al. / Acta Biomaterialia xxx (2014) xxx–xxx

[10] Mooney DJ, Organ G, Vacanti JP, Langer R. Design and fabrication of biodegradable polymer devices to engineer tubular tissues. Cell Transplant 1994;3:203–10. [11] O’Keefe BJ, Breyfogle LE, Hillmyer MA, Tolman WB. Mechanistic comparison of cyclic ester polymerizations by novel Iron(III)-alkoxide complexes: single vs multiple site catalysis. J Am Chem Soc 2002;124:4384–93. [12] Takeuchi D, Nakamura T, Aida T. Bulky titanium bis(phenolate) complexes as novel initiators for living anionic polymerization of e-caprolactone. Macromolecules 2000;33:725–9. [13] Zhong Z, Dijkstra PJ, Birg C, Westerhausen M, Feijen J. A novel and versatile calcium-based initiator system for the ring-opening polymerization of cyclic esters. Macromolecules 2001;34:3863–8. [14] Pitarresi G, Palumbo FS, Giammona G, Casadei MA, Moracci FM. Biodegradable hydrogels obtained by photocrosslinking of dextran and polyaspartamide derivatives. Biomaterials 2003;24:4301–13. [15] Tachaboonyakiat W, Serizawa T, Akashi M. Hydroxyapatite formation on/in biodegradable chitosan hydrogels by an alternate soaking process. Polym J 2001;33:177–81. [16] Shantha KL, Harding DRK. Preparation and in-vitro evaluation of poly[N-vinyl2-pyrrolidone-polyethylene glycol diacrylate]-chitosan interpolymeric pHresponsive hydrogels for oral drug delivery. Int J Pharm 2000;207:65–70. [17] Ito T, Fraser IP, Yeo Y, Highley CB, Bellas E, Kohane DS. Anti-inflammatory function of an in situ cross-linkable conjugate hydrogel of hyaluronic acid and dexamethasone. Biomaterials 2007;28:1778–86. [18] Zhang XZ, Wu DQ, Sun GM, Chu CC. Novel biodegradable and thermosensitive Dex-AI/PNIPAAm hydrogel. Macromol Biosci 2003;3:87–91. [19] Wu J, Chu C-C. Water insoluble cationic poly(ester amide)s: synthesis, characterization and applications. J Mater Chem B 2013;1:353–60. [20] Yan M, Du J, Gu Z, Liang M, Hu Y, Zhang W, et al. A novel intracellular protein delivery platform based on single-protein nanocapsules. Nat Nanotechnol 2010;5:48–53. [21] Nochi T, Yuki Y, Takahashi H, Sawada S-i, Mejima M, Kohda T, et al. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines. Nat Mater 2010;9:572–8. [22] Guo K, Chu CC, Chkhaidze E, Katsarava R. Synthesis and characterization of novel biodegradable unsaturated poly(ester amide)s. J Polym Sci, Part A: Polym Chem 2005;43:1463–77. [23] Katsarava R, Beridze V, Arabuli N, Kharadze D, Chu CC, Won CY. Amino acidbased bioanalogous polymers. Synthesis, and study of regular poly(ester amide)s based on bis(alpha-amino acid) alpha, w-alkylene diesters, and aliphatic dicarboxylic acids. J Polym Sci, Part A: Polym Chem 1999;37:391–407. [24] Deng M, Wu J, Reinhart-King CA, Chu C-C. Synthesis and characterization of biodegradable poly(ester amide)s with pendant amine functional groups and in vitro cellular response. Biomacromolecules 2009;10:3037–47. [25] Wu J, Mutschler MA, Chu CC. Synthesis and characterization of ionic charged water soluble arginine-based poly(ester amide). J Mater Sci – Mater Med 2011;22:469–79. [26] Fan YJ, Kobayashi M, Kise H. Synthesis and biodegradation of poly(ester amide)s containing amino acid residues: the effect of the stereoisomeric composition of L- and D-phenylalanines on the enzymatic degradation of the polymers. J Polym Sci, Part A: Polym Chem 2002;40:385–92. [27] Paredes N, Rodriguez-Galan A, Puiggali J. Synthesis and characterization of a family of biodegradable poly(ester amide)s derived from glycine. J Polym Sci, Part A: Polym Chem 1998;36:1271–82. [28] Paredes N, Casas MT, Puiggali J. Packing of sequential poly(ester amide)s derived from diols, dicarboxylic acids, and amino acids. Macromolecules 2000;33:9090–7.

[29] Asin L, Armelin E, Montane J, Rodriguez-Galan A, Puiggali J. Sequential poly(ester amide)s based on glycine, diols, and dicarboxylic acids: thermal polyesterification versus interfacial polyamidation. Characterization of polymers containing stiff units. J Polym Sci, Part A: Polym Chem 2001;39:4283–93. [30] Han SI, Kim BS, Kang SW, Shirai H, Im SS. Cellular interactions and degradation of aliphatic poly(ester amide)s derived from glycine and/or 4-amino butyric acid. Biomaterials 2003;24:3453–62. [31] Fan YJ, Kobayashi M, Kise H. Synthesis and specific biodegradation of novel polyesteramides containing amino acid residues. J Polym Sci, Part A: Polym Chem 2001;39:1318–28. [32] De Wit MA, Wang Z, Atkins KM, Mequanint K, Gillies ER. Syntheses, characterization, and functionalization of poly(ester amide)s with pendant amine functional groups. J Polym Sci, Part A: Polym Chem 2008;46:6376–92. [33] Botines E, Casas MT, Puiggali J. Alternating poly(ester amide)s of glycolic acid and omega-amino acids: crystalline morphology and main crystallographic data. J Polym Sci, Part B: Polym Phys 2007;45:815–25. [34] Armelin E, Paracuellos N, Rodriguez-Galan A, Puiggali J. Study on the degradability of poly(ester amide)s derived from the alpha-amino acids glycine, and L-alanine containing a variable amide/ester ratio. Polymer 2001;42:7923–32. [35] Rodriguez-Galan A, Pelfort M, Aceituno JE, Puiggali J. Comparative studies on the degradability of poly(ester amide)s derived from L- and L,D-alanine. J Appl Polym Sci 1999;74:2312–20. [36] Vera M, Puiggali J, Coudane J. Microspheres from new biodegradable poly(ester amide)s with different ratios of L- and D-alanine for controlled drug delivery. J Microencapsul 2006;23:686–97. [37] Pang X, Chu CC. Synthesis, characterization and biodegradation of functionalized amino acid-based poly(ester amide)s. Biomaterials 2010;31:3745–54. [38] Wu J, Wu DQ, Mutschler MA, Chu CC. Cationic hybrid hydrogels from aminoacid-based poly(ester amide): fabrication, characterization, and biological properties. Adv Funct Mater 2012;22:3815–23. [39] Pang X, Wu J, Reinhart-King C, Chu CC. Synthesis and characterization of functionalized water soluble cationic poly(ester amide)s. J Polym Sci, Part A: Polym Chem 2010;48:3758–66. [40] Guo K, Chu CC. Synthesis, characterization, and biodegradation of copolymers of unsaturated and saturated poly(ester amide)s. J Polym Sci, Part A: Polym Chem 2007;45:1595–606. [41] Guo K, Chu CC. Biodegradation of unsaturated poly(ester-amide)s and their hydrogels. Biomaterials 2007;28:3284–94. [42] Guo K, Chu CC. Synthesis and characterization of novel biodegradable unsaturated poly(ester amide)/poly(ethylene glycol) diacrylate hydrogels. J Polym Sci, Part A: Polym Chem 2005;43:3932–44. [43] Pang X, Chu CC. Synthesis, characterization and biodegradation of poly(ester amide)s based hydrogels. Polymer 2010;51:4200–10. [44] Guo K, Chu CC. Synthesis, characterization, and biodegradation of novel poly(ether ester amide)s based on L-phenylalanine and oligoethylene glycol. Biomacromolecules 2007;8:2851–61. [45] Guo K, Chu CC. Copolymers of unsaturated and saturated poly(ether ester amide)s: synthesis, characterization, and biodegradation. J Appl Polym Sci 2008;110:1858–69. [46] Guo K, Chu CC. Controlled release of paclitaxel from biodegradable unsaturated poly(ester amide)s/poly(ethylene glycol) diacrylate hydrogels. J Biomater Sci, Polym Ed 2007;18:489–504. [47] Ohkawa K, Kitsuki T, Amaike M, Saitoh H, Yamamoto H. Biodegradation of ornithine-containing polylysine hydrogels. Biomaterials 1998;19: 1855–60.

Please cite this article in press as: Pang X et al. Development of an arginine-based cationic hydrogel platform: Synthesis, characterization and biomedical applications. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.04.002