Hydrazone-Linkage-Based Self-Healing and Injectable XanthanâPoly

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Biological and Medical Applications of Materials and Interfaces

Hydrazone Linkage-based Self-healing and Injectable XanthanPEG Hydrogels for Controlled Drug Release and 3D Cell Culture peeyush sharma, Sagarika Taneja, and Yashveer Singh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07310 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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ACS Applied Materials & Interfaces

Hydrazone Linkage-based Self-healing and Injectable Xanthan-PEG Hydrogels for Controlled Drug Release and 3D Cell Culture Peeyush Kumar Sharma, Sagarika Taneja, and Yashveer Singh* Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar-140001, Punjab, India KEYWORDS: Hydrogel, pH-responsive, injectable, self-healing, drug delivery, 3D cell culture

ABSTRACT: Polymeric hydrogels have been extensively explored for controlled drug delivery applications but there is an increasing demand for smart drug delivery combined with tunable physicochemical attributes and tissue engineering potential. In this work, novel xanthan-PEG hydrogels were developed by crosslinking polysaccharide, oxidized xanthan, and 8-arm PEG hydrazine through dynamic, pH-responsive, and biodegradable hydrazone linkages. Aqueous solutions (pH 6.5) of oxidized xanthan and PEG hydrazine were mixed together at 37 oC to obtain hydrogels within minutes and formation of hydrazone linkages was ascertained using FTIR spectroscopy. Fabrication of xanthan-PEG hydrogels using hydrazone linkages has not been reported previously. The 3% hydrogels exhibited the storage modulus of 194 Pa, which increased to 770 Pa for 5% hydrogels. When subjected to alternating cycles of varying strains of 1 and 800% (5 cycles), hydrogels demonstrated instant recovery each time the extreme strain

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was relieved, thus suggesting excellent self-healing capabilities. Doxorubicin (DOX), chemotherapeutic agent, was loaded onto hydrogels and release studies were carried out at pH 5.5 (tumoral) and 7.4 (physiological). The cumulative release from 3, 4, and 5% hydrogels at pH 5.5 was 81.06, 61.98, and 41.67%, while the release at pH 7.4 was 47.43, 37.01, and 35.34% at 30 days. MTT assay showed that oxidized xanthan and PEG hydrazine are not toxic to mammalian cells (NIH-3T3), as the cell viabilities were found to be 84.66 and 102% for concentrations up to 1 mg/mL. The live/dead assay with encapsulated NIH-3T3 cells showed no significant dead cell population, suggesting excellent compatibility of hydrogels in 2D and 3D culture. DOX-loaded hydrogels exhibited cytotoxicity against A549 cells when exposed to media released from hydrogels. Overall, hydrogels developed in this work may have potential applications in drug delivery and 3D cell culture for cell delivery.


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1. INTRODUCTION

Hydrogels are crosslinked polymer networks formed by covalent crosslinking or non-covalent interaction of polymer chains, which swell in water1–4. Polymeric hydrogels have emerged as most sought-after biomaterials owing to their close resemblance to soft human tissues and extracellular matrix along with their tuneable physicochemical characteristics. Hydrogels, on account of their versatility, have been developed for diverse applications, including sensing, imaging, controlled drug delivery, and 3D cell culture5–7. It is possible to incorporate stimuliresponsive triggers in hydrogels and these biomaterials are inherently biocompatibile due to the use of biopolymers. Thus, it is possible for hydrogels to deliver drugs in controlled manner, which is responsive towards physiochemical changes in body, but remaining compatible with host tissues at the same time8–12. Although extremely promising, clinical translation of hydrogels as drug delivery depots will require material optimization in terms of ease of administration and patient compliance7. Minimally invasive administration of drug-eluting hydrogels requires them to possess viscoelastic self-healing capability to ensure reversible sol-to-gel transition during parenteral administration4,13,14. This mechanical adaptability can be induced into matrix by employing dynamic covalent linkages to fabricate hydrogels. A variety of covalent linkages including amide, disulfide, thioether, Schiff base (imine15, oxime and hydrazone) along with click approaches like azide-alkyne cycloaddition, Diels-Alder reaction, thiol-ene, and Michael addition have been used to develop hydrogels5,16. In particular, hydrazone linkages formed by condensation of carbonyl and hydrazine groups are an example of dynamic covalent linkages, which can provide self-healing attributes to hydrogel owing to their kinetically reversible nature17–20. Hydrazone bond formation is highly efficient (click chemistry)


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and orthogonal (occurs with minimal interference) in nature, and hydrogels formed through hydrazone linkages have been reported to be mechanically robust and homogenous21. These linkages have been explored in tissue engineering applications with promising outcomes18,22,23. The hydrazone linkages have thermodynamic bias towards slightly acidic or basic environment and it can be exploited for designing pH-responsive hydrogels24–26 for localized controlled delivery of chemotherapeutic agent in solid tumors27. In this context, in situ forming, injectable hydrogels could be more efficient in providing precise local delivery of chemotherapeutic agent28–32. Most hydrogels entrap chemotherapeutic agent by passive entrapment and mainly offer short-term release. What has completely eluded researchers to date is the development of strategies employing in situ covalent interactions between chemotherapeutic agent and components of matrix for the controlled release of chemotherapeutic agent over a period of months, resulting from pH-responsive, slow bond cleavage30,33. Hydrogels can also be employed for constructing scaffolds mimicking the 3D environment around cells (extracellular matrix) and facilitate their proliferation in vitro. The major requirements are immobilized aqueous environment, analogous viscoelasticity, and dynamic nutrient transport34–36. Hydrogels fabricated using dynamic hydrazone linkages can efficiently cater to these requirements37. The reversible nature of hydrazone linkages can provide adaptable viscoelastic properties, efficient nutrient transport, and tunability in terms of swelling and degradation behavior38,39. Besides linkages, choice of polymers is equally important. Xanthan gum is a high molecular weight heteropolysaccharide derived from Xanthomonas campestris40 and a FDA-approved pharmaceutical aid (21CFR172.695) and release retarding polymer in drug delivery systems41– 43

. The presence of vicinal diols at cellulosic backbone and trisaccharide side chains of xanthan44


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provides an easy opportunity for generation of aldehyde functionality in the structure and subsequent crosslinking with polymers containing hydrazine groups. Poly(ethylene glycol) or PEG is a FDA-approved biocompatible hydrophilic polymer, which has been explored for a variety of biomedical applications due to its tuneable characteristics and proven safety profile. With the advent of efficient synthetic strategies, highly monodisperse PEG with a variety of morphologies and functionalities can be easily obtained for further modifications45,46. Thus, hydrogel based on cheap and biocompatible polysaccharides and click crosslinking strategy can provide a scalable biomaterial for cell therapy, with potential in tissue engineering47–49. Keeping the challenges discussed above in mind, we designed and developed xanthan-PEG hydrogels using dynamic and biodegradable hydrazone linkages and their viscoelastic, selfhealing, thixotrophic, swelling, and degradation characteristics were investigated. DOX-loaded hydrogels were fabricated and controlled delivery was measured in buffers mimicking tumoral and physiological pH. The cytotoxicity of polymers and hydrogel extract against mammalian cells (NIH-3T3) was assessed using MTT and live/dead assays, and cell encapsulation studies. Finally, the toxicity of DOX released from hydrogels and its intracellular distribution in A549 NSCLC cells was also investigated. 2. EXPERIMENTAL 2.1. Materials and Methods. All reagents used were of analytical reagent grade and used without further purification. The 8-arm PEG amine (Mw = 20,000 Da) was procured from Jenkem,

USA

and

HATU

(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]

pyridinium 3-oxid hexafluorophosphate), tri-boc-hydrazinoacetic acid, TFA (trifluoro acetic acid), and sodium metaperiodate were procured from Sigma Aldrich. DIEA (N,Ndiisopropylethylamine) was purchased from TCI Chemicals, India and xanthan gum, MTT assay


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reagents, and DAPI were purchased from Himedia. Doxorubicin hydrochloride (DOX) was purchased from Dalian Meilun Biotech, China and the live/dead assay kit was obtained from Life Technologies Corp. The NIH-3T3 mouse embryonic fibroblast cell lines were a generous gift from Dr. Javed N Agrewala, Chief Scientist, CSIR- Institute of Microbial Technology, Chandigarh and A549 NSCLC cell lines were a gift from Dr. Vishwajeet Mehandia, Assistant Professor, Department of Mechanical Engineering, IIT Ropar, Rupnagar. 1H NMR spectra were obtained on a JEOL JNM-ECS 400 MHz system and FT-IR spectra were obtained on a Bruker Tensor 27 in ATR mode. The UV-Vis absorbance was recorded using a Tecan Infinite Pro multiple plate reader and fluorescence microscopy images were obtained on a Leica DMi8 fluorescence microscope (20x). 2.2. Preparation of Oxidized Xanthan. A xanthan gum solution (0.5%, w/v) was prepared in DI water using constant stirring and heating at 80 oC for 30 min. Next, an aqueous solution of sodium metaperiodate (200 mg, 10 mL) was added drop wise and mixture was stirred at room temperature for 4 h in dark. Finally, reaction was quenched using ethylene glycol solution (1%). The mixture was dialyzed against DI water for 3 days with sink media being replaced every 12 h and the dialyzed solution was freeze-dried to obtain pure xanthan containing aldehyde groups50. 2.3. Preparation of 8-arm PEG Hydrazine. The 8-arm PEG amine (1 G) was dissolved in anhydrous DMF (5 mL), and HATU (200 mg) and triboc hydrazine acetic acid (200 mg) were added to it. Next, DIEA (100 µL) was added and reaction mixture was stirred at room temperature for 4 h under nitrogen atmosphere. After completion, the reaction mixture was precipitated from excess of cold diethyl ether, centrifuged, and vacuum dried to obtained tribocprotected 8-arm PEG hydrazine. The triboc group was deprotected using a 30% TFA solution in DCM for 2 h and precipitated again from cold diethyl ether to obtain 8-arm PEG hydrazine23.


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The product was purified by gel-permeation chromatography on a Sephadex LH-20 column using methanol as eluent. 1H NMR (DMSO-D6): δ = 3.59 (s, PEG), (Figure S2, see the supporting information). The conversion of amine groups into hydrazine groups was quantified using fluorescamine assay51. A calibration curve was obtained using varied concentrations of 8arm PEG amine and the fluorescence intensity was measured for triboc-protected 8-arm PEG hydrazine and 8-arm PEG hydrazine (Figure S3). 2.4. Fabrication of Hydrogels. Hydrogels were fabricated by mixing aqueous solutions of oxidized xanthan and 8-arm PEG hydrazine at 37 oC. The oxidized xanthan (50 mg, 1% w/v) was dissolved in phosphate buffer (5 mL, pH 6.5) by heating at 70 oC for 1 h. A stock solution of 8arm PEG hydrazine (500 mg, 10% w/v) was prepared in phosphate buffer (5 mL, pH 6.5) and diluted to obtain 3, 4, and 5% (w/v) solutions. To fabricate hydrogels, equal volumes (150 µL) of aqueous solution of oxidized xanthan gum and appropriate PEG hydrazine were mixed using vortex and allowed to stand at 37 oC. The hydrogels were considered formed when the solution ceased to flow and time taken to form hydrogels were recorded. Hydrogels were categorized as 3, 4, or 5% based on the amount of PEG hydrazine used in hydrogel formation. The formation of covalent crosslinks between two polymers was ascertained using FT-IR spectroscopy (Figure S1). The cross-linking efficiency was determined using TNBS assay as reported by Lee et al.52 (Figure S4). 2.5. Rheological Studies. The rheological parameters were measured on an MCR-102 modular rheometer (Anton Paar, Austria) equipped with a parallel plate at 37 o C and the gap was kept at 0.5 mm across all experiments. Liquid paraffin was applied around the plates to prevent water evaporation during measurements. The hydrogels were formed on the peltier plate itself and allowed to incubate for 1 h. Initially, an amplitude sweep study was performed to determine the


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linear viscoelastic range (LVE) for hydrogels. Next, hydrogels were subjected to variable amplitude of strain and variations in values of storage and loss modulus were measured to understand the mechanical resilience of hydrogels. Hydrogels were then subjected to time sweep studies (amplitude value kept at 1% as per the LVE) to investigate concentration-dependent gelation kinetics of hydrogels. The frequency sweep measurements, where angular frequency was varied from 0.01-100 Hz at constant amplitude of 1%, was also carried out. Finally, the selfhealing characteristics were measured by subjecting hydrogels to 5 cycles of contrasting strain of 800 and 1%, each for 60 s. 2.6. Self-healing Studies. A 3% hydrogel was fabricated in a mould to obtain it in circular disk shape. The hydrogel was cut into two semi-circular pieces using a surgical blade. One of the semi-circular disk was loaded with methylene blue dye and the other disk was left untreated. Both disks were kept together in the same orientation as they were cut and incubated at 37 oC and observed visually at different time points to look for self-healing and diffusion of dye across the seam to the unloaded half of the hydrogel. After 24 h, the hydrogel was observed under microscope. The self-healing studies were carried out at different pH by fabricating hydrogels at pH 5.5 and 7.4. At pH 5.5, one disk was loaded with DOX, whereas the other disk was left untreated. At pH 7.4, one disk was loaded with methylene blue dye and other disk was kept unloaded (Figure S5). 2.7. Drug Loading and Controlled Release. The oxidized xanthan solution (1% w/v) was mixed with 3, 4, and 5% PEG hydrazine solution containing DOX (0.43 mg) to obtain DOXloaded hydrogels. The hydrogels were suspended in phosphate (pH 7.4) and acetate (pH 5.5) buffers (1 mL) to mimic physiological and tumor microenvironments and transferred to a shaking incubator (120 rpm, 37 oC). The release media (1 mL) was collected at specified time


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points and replaced with fresh media. The amount of DOX released was determined by measuring absorbance at 485 nm using a microplate reader. Release experiments were done in triplicates. 2.8. Cytotoxicity Assay of Polymers. The cytotoxicities of both polymers were assessed by MTT assay. The NIH-3T3 mouse fibroblasts were cultured up to confluency (4th passage) and seeded in a 96 well plate at an initial density of 2 x 104 cells/well in a DMEM medium, supplemented with 10% FBS and 1% penicillin/streptomycin, followed by incubation for 24 h. Oxidized xanthan and PEG hydrazine were dissolved in DMEM and passed through a 0.22 µM syringe filter and further diluted to different concentrations using complete DMEM media, as described earlier. The media in wells was replaced with fresh media containing different concentrations of polymer and unspiked media was taken as a control. The cells were further incubated for 24 h and culture medium was removed and replaced with MTT solution and incubated again for 4 h. The MTT solutions were discarded and DMSO was added to each well to dissolve formazan crystals. Absorbance of the dissolved formazan crystals was measured at 570 nm using a microplate reader. The relative cell viability was estimated by comparing the absorbance in control wells with culture medium. The data presented are mean±standard deviation (SD) of six experiments. 2.8. Live/dead Assay of Hydrogels. The cytotoxicity of hydrogel was assessed using live/dead assay. Both polymers (oxidized xanthan and PEG hydrazine) were dissolved in complete DMEM and passed through a 0.22 µM syringe filter. Hydrogels (0.2 mL, 5%) were formed by mixing equal volumes of polymer solutions (0.1 mL each) in a sterile environment and further sterilized by keeping under UV radiation for 2 h. The hydrogel was placed on top of the membrane of a transwell insert and incubated with DMEM (2 mL) in a 24 well plate.


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Meanwhile, the cells were seeded onto wells of a 24 well plate at a density of 2 x 104 cells/well and incubated for 24 h. The transwell inserts containing hydrogels were introduced into wells containing cells and incubated for 24 h. After incubation, the transwell inserts were removed along with media. After washing twice with PBS, sufficient amount of live/dead assay dyes were introduced into wells and cells were observed under fluorescence microscope and compared with control (complete DMEM media). 2.9. 3D Cell Culture. The NIH-3T3 cells were used for 3D encapsulation studies. The polymer solutions were prepared as described above. The cells were suspended into the PEG hydrazine solution (5% w/v, 0.2 mL) at a density of 5 x 104 cells per 0.1 mL solution and oxidized xanthan solution (1%, 0.2 mL) was added carefully to avoid bubbling. Following the formation of hydrogel within a minute, DMEM (2 mL) was added to the well and plates were incubated at 37 oC for 24 h, under humid conditions (5% CO2). After 24 h, the culture media was removed and hydrogel was washed with PBS twice and stained with live/dead assay dyes. After 30 min of incubation, the dye solution was removed and hydrogel was washed with PBS twice. Finally, PBS (2 mL) was added to the well and visualized under fluorescence microscope. Simple 2D as well as Z stack images were taken. 2.10. Cytotoxicity Assay of DOX-loaded Hydrogels. The cytotoxicity of DOX-loaded hydrogel was assessed using MTT assay, as described above. In this study, A549 (NSCLC) cell lines were used and DOX (0.43 mg) was added to hydrogels. The transwell was kept onto a new well containing fresh media every 5 days to obtain hydrogel release extracts. The extracts were diluted with DMEM to obtain equivalent DOX concentrations of 2 µg/mL. After 24 h of culture, the cells were treated with extracts obtained at different time points, free DOX solution (2 µg/mL, positive control), and DMEM complete media (negative control). The cells were


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incubated for another 24 h and subjected to MTT assay protocol. The data are presented as mean ± standard deviation (SD) of six experiments. 2.11. Live/dead Assay of DOX-loaded Hydrogels. The experiments were done as described earlier. The A549 (NSCLC) cells were used and exposed to the release media obtained from DOX-loaded hydrogels (3% w/v) after 5 days and free DOX (5 µg/mL). The cells were incubated for 0 and 24 h periods. At each time point, the media was removed and hydrogels were washed twice with PBS and treated with dyes for 30 min. It was washed again twice and imaged using a fluorescence microscope. 2.12. Intracellular Distribution of Released DOX. For intracellular distribution studies, the cells were seeded in a 24-well plate at a density of 2 x 104 cells per well and incubated for 48 h. The cells were treated with free DOX (2 µg/mL, 1 mL) and equivalent extract solutions (5 µg/mL) and incubated for 4 h. These were washed with DPBS twice and fixed with 3.7% glutaraldehyde and counterstained with DAPI for 30 min. The cells were washed twice again with DPBS and observed under fluorescence microscope. 3. RESULTS AND DISCUSSION

In the present work, we have designed and developed robust polysaccharide hydrogels by intermolecular covalent crosslinking between oxidized xanthan polymer containing aldehyde groups and 8-arm PEG hydrazine through hydrazone linkages (Scheme 1). The presence of hydrazone linkages makes hydrogel pH-responsive and biodegradable and, to the best of our knowledge, it is the first report of employing hydrazone chemistry to develop hydrogels from xanthan. Xanthan was chosen as the polysaccharide component because of its proven safety record in food industry as a thickening agent40. Also, the polysaccharide backbone provides opportunity to induce multiple functional groups within a single monomer unit. A high molecular weight is expected to improve the biodegradation profile53,54. In the present work, xanthan


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polymer containing vicinal diols was partially oxidized using sodium metaperiodate to obtain oxidized xanthan polymer containing aldehyde groups. The oxidized xanthan was purified by dialysis and freeze-dried to obtain a fluffy powder and the degree of oxidation was estimated to be 27.71% by hydroxylamine hydrochloride assay method55. The FTIR spectrum of oxidized xanthan showed a new peak at 1720 cm-1, which was not present in the xanthan polymer (Figure S1), and a mild peak an 898 cm-1 that was attributed to hemiacetal groups.

Scheme 1. Fabrication of oxidized xanthan-PEG hydrogels from oxidized xanthan and 8-arm PEG hydrazine using hydrazone linkages and sustained and pH-responsive delivery of doxorubicin. An 8-arm PEG polymer containing multiple hydrazine groups was used to crosslink oxidized xanthan polymer. PEG, being the hydrophilic synthetic polymer, is available in variety of forms


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and functionalities. An 8-arm, homofunctional amine terminated PEG was selected to get a higher density of grafting sites with minimal steric hindrance56. The 8-arm PEG amine (20 kDa) was reacted with triboc-hydrazinoacetic acid in presence of HATU/DIEA, followed by deprotection of Boc- groups with TFA. The product was characterized using 1H NMR (Figure S2). The amine to hydrazine conversion was monitored using fluorescamine assay and found to be 93.6% (Figure S3). A series of hydrogels were fabricated by mixing aqueous solutions of oxidized xanthan (1% w/v) and PEG hydrazine (3-5% w/v) at 37 oC (Scheme 1). The oxidized xanthan gum polymer was dissolved in phosphate buffer (pH 6.5) by heating at 70 oC for 1 h, whereas 3, 4, and 5% PEG hydrazine solutions were prepared in phosphate buffer (pH 6.5). Both solutions were incubated at 37 oC. The oxidized xanthan solution was mixed with appropriate PEG hydrazine solution to obtain hydrogels at 37 oC. In this work, hydrogels have been identified as 3, 4 or 5% (w/v) based on the amount of PEG hydrazine used to fabricate hydrogels. The molar ratios of xanthan polymer and 8-arm PEG hydrazine were 1:0.95, 1:1.26, and 1:1.58 for 3, 4, and 5% hydrogels. The hydrogels were considered formed when the solution ceased to flow and time taken to form hydrogels were recorded. The gelation time was found to decrease with increase in the concentration of PEG hydrazine, which is on expected lines

57

. The hydrogels fabricated

using 3% PEG hydrazine took few minutes to form gel; whereas those formed using 5% PEG hydrazine formed gel within few seconds. The FTIR spectrum of hydrogels gave a new peak at 1671 cm-1 due to the formation of C=N linkages, thus confirming the formation of hydrazone linkages (Figure S1). The cross-linking efficiencies were estimated using TNBS assay and found to be 80.56% for 3% hydrogels and it increased to 83.44% for 4% hydrogels (Figure S4). It


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dropped to 68.59 % for 5% hydrogels due to the presence of molar excess of 8-arm PEG hydrazine.

Figure 1. Rheological properties of hydrogels: (A) time sweep studies indicating the evolution of storage modulus (G’) over time, (B) amplitude sweep studies depicting G’/G” at different hydrogel concentrations and corresponding crossover values, (C) frequency sweep studies indicating G’/G” at different hydrogel concentrations, (D) storage modulus (G’) at alternating strains of 1 and 800% for 60 seconds each (5 cycles). The rheological studies were performed to understand the viscoelastic characteristics of hydrogels (Figure 1). The linear viscoelastic range (LVE) of hydrogel with least PEG hydrazine concentration was measured by an amplitude sweep test and found between 0.1 to 10% strain values. Therefore, 1% strain value was taken for all gelation kinetics experiments. The gelation kinetics studies were conducted to understand the change in storage modulus immediately after


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mixing, as a function of time (curing of hydrogels with time). The hydrogels were prepared on the stage of rheometer and immediately subjected to time sweep measurements, which were continued at 37 oC for 4000 s. A time dependent increment in the values of G’ was observed in all cases (Figure 1A). The initial value for 3% hydrogel was 74.5 Pa, which increased to 192.07 after 4000 s, whereas for 5% hydrogel, the initial value was 165.95 Pa and it increased to 730.72 Pa, in same time. The dynamic nature of the storage modulus values provides an idea about the time-dependent nature of cross linking reactions. Amplitude sweep studies were conducted for hydrogels by subjecting these to variable strains, ranging from 0.1 to 800% (Figure 1B). A higher value of strain was taken because hydrogels did not show any sign of crossover at conventionally used strain values of 100%18. It could be due to the excellent viscoelastic characteristic of hydrogels, which resisted structure failure at moderate strains. Hydrogels consistently exhibited higher storage modulus (G’) values than loss modulus (G”) at varying strains and crossover appeared only at 256% for 3% hydrogel and 471% for 5% hydrogel, which was far higher as compared to 80% reported by Wei et al.18 and 72% by Qu et al.31 but comparable to mussel-inspired hydrogels reported by Li et al.58, with a crossover of around 400%. Thus, a higher PEG hydrazine concentration resulted in higher elasticity, which is likely to equip these hydrogels with capability to withstand the formulation and administration-related strains as an injectable formulation. In dynamic frequency sweep studies, variable angular frequency shear was applied to hydrogels at constant amplitude of 1% (Figure 1C). The hydrogels were found to be mechanically robust across the frequency range tested and these withstood their elastic solid-like character. The G’ values followed a pattern similar to that of amplitude sweep.


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Figure 2. Self-healing and injectability characteristic of hydrogels: (A) freshly fabricated hydrogel, (B) cut into two halves (one loaded with methylene blue and other unloaded), (C-E) healing and dye diffusion at 2, 6, and 24 h, (F) self-healed hydrogel with equilibrium dye diffusion, (G) microscopic evidence of healing, (H-I) injectability and extrudability, and (J) mechanism of self-healing. Finally, 5% hydrogels were subjected to alternating cycles of extreme (800%) and mild (1%) strain to understand the extent and swiftness of recovery achieved by hydrogels after it has undergone structural failure (Figure 1D). It was observed that hydrogels recovered almost instantaneously every time it was relieved of the extreme strain and the recovery process was


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consistent for 5 alternating cycles of extreme and mild strains. The quick recovery is resulting from dynamic hydrazone linkages employed to form hydrogels. It is likely to improve the selfhealing and thixotropic (time-dependent shear thinning) characteristics of hydrogels, which could be of great advantage while exploiting these biomaterials for injectable intratumoral delivery of chemotherapeutic agent, as it will provide precise localization and retention of drug in the target tissue59. The self-healing characteristic of hydrogels was investigated by visual method to understand the propensity of hydrogels to self-heal into unity when forcibly cut into two pieces in a seamless manner (Figure 2). A 3% hydrogel was prepared in a silicon mould and taken out after curing (Figure 2A) and cut into two semi-circular pieces using a surgical blade. One half of the hydrogel was loaded with methylene blue dye and another half kept untreated (Figure 2B). The two halves were kept in contact along the cut and incubated at 37 oC in a closed environment. The hydrogels were visually observed after 2, 6, and 24 h (Figure 2C-E) for diffusion and healing. As evident from images, the dye diffused across to the unloaded half in a timedependent manner and healing along the cut was observed at each time point. At the end of 24 h period, the hydrogel pieces healed into a self-sustaining unit, which endured its weight against gravity when hanged (Figure 2F). The hydrogels were observed under microscope and no visible seam was observed indicating efficient healing of two halves (Figure 2G and J). Self-healing studies were also carried out by fabricating hydrogels at pH 5.5 and 7.4 and it was observed that gels fabricated at slightly acidic pH exhibited more efficient distribution of drug/dye than those fabricated at neutral pH (Figure S5). SEM images of freeze dried hydrogel samples just after the cut was made (before self-healing) and after 6 h of self-healing were also taken and images clearly reveal efficient self-healing (Figure S6).


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The 3% hydrogels containing methylene blue were prepared in a syringe and forced to extrude through the needle to observe the thixotropic characteristic of these biomaterials, which is desirable for an injectable formulation. As can be seen from the accompanying image, hydrogels immediately formed but were easily extrudable through the needle and instantaneously restored their viscoelastic gel character (Figure 2H-I).

Figure 3. DOX release from hydrogels in vitro: (A) pH 5.5 and (B) pH 7.4. Hydrogels also demonstrated pH-responsive swelling and slow degradation profiles (Figure S7). At pH 7.4, maximum swelling of 23.18% was observed for 4% hydrogels, while the maximum degradation of 9.17% was observed for 3% hydrogels after 30 days. Similarly at pH 5.5, the maximum swelling of 68.12% and degradation of 36.77% were observed for 4 and 3% hydrogels. Owing to the self-healing and injectability characteristics of hydrogels developed in this work, we decided to explore it first for controlled delivery of a chemotherapeutic agent such as doxorubicin (DOX), which is used to treat breast and ovarian cancers60. DOX (0.43 mg) was loaded onto hydrogels by mixing it with PEG hydrazine solutions. The DOX-loaded hydrogels were suspended in acetate and phosphate buffers of pH 5.5 and 7.4 mimicking the tumoral and physiological environments and incubated in a shaking incubator (120 rpm, 37 oC). Aliquots


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were withdrawn at specified time points and amount of chemotherapeutic agent released was measured by recording UV-Vis absorbance at 485 nm (Figure 3). As evident from release profiles, hydrogels exhibited a pH-responsive release pattern. At pH 5.5, 81.06, 61.98, and 41.67% DOX was released from 3, 4, and 5% hydrogels after 30 days (Figure 3A). It clearly indicates that higher crosslinking density impedes the drug release, owing to slower degradation of matrix as well as DOX conjugates7. At pH 7.4, 47.43, 37.01, and 35.34% chemotherapeutic agent was released from 3, 4, and 5% hydrogels during the same period (Figure 3B). The slower release can be attributed to higher hydrolytic stability of hydrazone linkages at neutral pH and release phenomenon predominantly reliant only on diffusion of passively entrapped DOX out of the matrix. This slower release at physiological pH is desirable to prevent accidental leakage at non-target sites. The pH-sensitive release of DOX could be due to the fact that its aqueous solubility is higher at lower pH. Consequently, we loaded the hydrogels with 5-fluorouracil (5-FU), which is pH-independent. Similar pHresponsive release was observed with 5-FU, thus confirming that pH-sensitive release of DOX is not primarily due to the increased aqueous solubility at lower pH (Figure S8). Also, the release was much faster in case of 5-FU due to the passive entrapment of drug employing noncovalent interactions, resulting in release by diffusion and swelling. The release data was fitted with multiple kinetic models to understand the mechanism of release and it was found that the release data fit better with Korsemeyer-Peppas exponential model with better regression, which is considered most suitable for drug release from polymeric matrices61 . The diffusion coefficients for all release data was found to be around 0.38 at pH 7.4, which suggested that the principal mechanism of DOX release was pseudo-fickian or diffusionbased release. The diffusion coefficients for release at pH 5.5 were around 0.3, which suggested


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there are other mechanisms which are contributing to slower release of DOX. The controlled release of chemotherapeutic agent from hydrogels for 30 days is resulting from the unique chemical architecture of these hydrogels, which allows DOX to interact with matrix by multiple mechanisms. DOX, being a small molecule, can be passively entrapped within the matrix62. The hydrogels possess free aldehyde and hydrazine groups, which can interact with amine (imine linkages)63 and carbonyl groups present on DOX (hydrazone linkages)30,64. It must be mentioned that all interactions are responsive towards acidic microenvironment of tumor tissues and, thus can provide a preferable release at tumour sites. The main prerequisite for hydrogels as biomaterial for drug delivery and tissue culture applications is the biocompatibility towards host tissue. Therefore, the cytotoxicity of polymers employed in hydrogel formation, oxidized xanthan and PEG hydrazine, and hydrogel extract was investigated towards mammalian cells (NIH-3T3 fibroblasts cells). The MTT assay of polymeric precursors showed that 8-arm PEG hydrazine exhibit minimal toxicity, with cell viability being 102% even at 1 mg/mL concentration (Figure 4A). The viability dropped to 84.66% at 1 mg/mL concentration for oxidized xanthan. A slight decrease in cell viability with oxidized xanthan can be attributed to possible interactions of carbonyl groups in the polymer with proteins65. Next, live/dead assay was carried out to assess the effect of hydrogel extracts on survival of fibroblast cells. Fluorescence microscopy images revealed no significant dead cell population upon incubation with hydrogel extract for 24 h and compared to cells incubated with PBS (Figure 4BC). The hydrogels were further investigated as a potential scaffold for cell therapy through in situ encapsulation of fibroblast cells in a 3D matrix. The cells were added into the 8-arm PEG hydrazine solution before hydrogel fabrication (addition of oxidized xanthan) and incubated for


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24 h. Fluorescence microscopy images of hydrogel encapsulating cells after staining with live/dead reagent (2D and 3D Z-stacked) are shown in Figure 4D-E. The images showed that cells maintain their round unadhered morphology and viable state even after 24 h of encapsulation. Thus, hydrogel matrix is compatible with cells and it allows for nutrient transport. Dynamically crosslinked matrices are considered as better mimics to the ECM and can be explored as 3D scaffolds for cell therapy and tissue regeneration applications66.

Figure 4. Cytotoxicity, live/dead, and cell encapsulation studies of polymers and hydrogel: (A) viability of NIH-3T3 cells incubated with oxidized xanthan and PEG hydrazine, (B-C) live/dead assay of NIH-3T3 cells incubated with PBS and hydrogel extract for 24 h, (D-E) fluorescence


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images of NIH-3T3 cells encapsulated within the hydrogel in 2D and corresponding 3D zstacked projection. The in vitro cytotoxicity studies were conducted to assess the efficacy of released DOX from DOX-loaded hydrogels (Figure 5). The viability of A549 cells in presence of release media (at different time points) was evaluated using MTT assay. For this study, 3 and 5% hydrogels containing 0.43 mg of DOX were fabricated and transferred to the upper chamber of transwell inserts. DOX was allowed to release for different time intervals in DMEM. Cells were then incubated with free DOX (2 µg/mL) and the release media with equivalent DOX concentration. The release media retained its activity even after 25-day, which indicates a sustained release of DOX from hydrogels (Figure 5A). Release media from 5% hydrogel reduced the cell viability to 57.44% after 5-day and it sustained it to 84% after 20-25 days. A considerably higher inhibition was observed for 3% hydrogels (45.47% cell viability) for initial 5-day and it sustained it to 71.5% after 20-25 days period. The 3% hydrogels gave better result due to faster drug release. Cell viability for free Dox was found to be only 11.23% at 2 µg/mL concentration. These results suggest that the DOX-loaded hydrogels were active against cancer cells for a longer duration of time, which is desirable for efficient management of solid tumours and minimizing the probability of recurrence67. The results were also consistent with the in vitro release data as the cytotoxicity effect of hydrogels must have translated from the sustained release of DOX in the release media. Wu et al.68 reported the retention of cytotoxicity up to 10 days while Yamada et al.28 reported 60% cytotoxicity even after 56 days of release. Although, the methods employed for in vitro efficacy assessment of DOX-loaded hydrogels vary a lot, hydrogels still demonstrated a capacity to kill A549 cells for at least 25 days which can be further fine-tuned as per the requirement.


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Figure 5. Cytotoxicity and live/dead assay of DOX-loaded hydrogels: (A) cell viability of A549 cells when incubated with release media obtained from DOX-loaded hydrogels against time, and (B) fluorescence microscopy images of live/dead staining of A549 cells incubated with PBS, free DOX, and release media at 5-day, after none and 24 h incubation. The cytotoxicity activity of release media from DOX-loaded hydrogels was confirmed using the live/dead assay (Figure 5B). The images revealed visibly significant dead cell population upon incubation with 5-day release media obtained from 3% DOX-loaded hydrogels. As


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expected, cells incubated with free DOX for same duration exerted higher toxicity while the control group showed no apparent existence of dead cell population.

Figure 6. Intracellular distribution studies. DIC and fluorescence microscopy images of A549 cells incubated with PBS (negative control), free DOX (positive control), and release media obtained from DOX-loaded hydrogels showing intracellular localization of DOX. The cells were counterstained with DAPI. The 5-day release media from 3% hydrogels was employed for assessing the intracellular distribution of DOX in A549 cells (Figure 6). Since DOX is a fluorescent molecule, its intracellular localization can be visualized using a fluorescence microscope. The cells were incubated with release media for 2 h and in parallel with free DOX and PBS. The cells were observed under the fluorescence microscope after fixing and counterstaining with DAPI. The


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cells incubated with release media showed cellular internalization of DOX, similar to free DOX but at lower intensity, thus indicating lower concentration of DOX in the release media. 4. CONCLUSIONS

In summary, we have for the first time developed self-healing, injectable polysaccharide hydrogels by intermolecular crosslinking of oxidized xanthan containing aldehyde groups and 8arm PEG hydrazine, through dynamic, pH-responsive, and biodegradable hydrazone linkages. Hydrogels exhibited strong viscoeleastic, self-healing, and thixotrophic characteristics, making it aptly suited for application as injectable drug depot. Hydrogels upon loading with DOX, chemotherapeutic agent, provided a pH-responsive drug release for up to 30 days, which was significantly faster at tumoral pH, in comparison to physiological pH. MTT assay showed that oxidized xanthan and PEG hydrazine are not cytotoxic to mammalian (NIH-3T3) cells and live/dead assay with encapsulated cells depicted the excellent cytocompatibility of hydrogels in 2D and 3D culture. The release media obtained from DOX-loaded hydrogels were effective in causing inhibition of A549 cancer cell lines and DOX released from hydrogels was internalized into cells, similar to free DOX. The hydrogel platform developed in this work may be further exploited for intratumoral controlled delivery of chemotherapeutic agent, cell therapy, and tissue engineering. ASSOCIATED CONTENT Supporting Information Experimental procedure for swelling and degradation, and 5-FU release studies. FTIR spectra of oxidized xanthan, PEG hydrazine, and hydrogel; 1H NMR spectrum of PEG hydrazine; calibration curves for fluorescamine and TNBS assays, self-healing at different pH, swelling and


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degradation profiles of hydrogel; and in vitro 5-FU release from hydrogel. These materials are available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *

Email: [email protected]. Tel: 91-1881-242246.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Authors gratefully acknowledge financial assistance (to YS) from the CSIR, New Delhi [grant # 02(0245)/15/EMR-II] and PKS received the institute fellowship from IIT Ropar. ST is an MSc project student supported by the Department of Chemistry, IIT Ropar. Thanks to Neelam Chauhan for carrying out SEM studies and TNBS assay.


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REFERENCES (1) (2) (3) (4) (5)

(6) (7) (8)

(9) (10) (11)

(12) (13)

(14)

(15)

(16) (17)

(18)

Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Deliv. Rev. 2012, 64, 18–23. Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Hydrogels in Biology and Medicine: From Molecular Principles to Bionanotechnology. Adv. Mater. 18, 1345–1360. Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869–1879. Yu, L.; Ding, J. Injectable Hydrogels as Unique Biomedical Materials. Chem. Soc. Rev. 2008, 37, 1473–1481. Jiang, Y.; Chen, J.; Deng, C.; Suuronen, E. J.; Zhong, Z. Click Hydrogels, Microgels and Nanogels: Emerging Platforms for Drug Delivery and Tissue Engineering. Biomaterials 2014, 35, 4969–4985. Rosales, A. M.; Anseth, K. S. The Design of Reversible Hydrogels to Capture Extracellular Matrix Dynamics. Nat. Rev. Mater. 2016, 1, 15012. Li, J.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. Mater. 2016, 1, 16071. Jalili, N. A.; Jaiswal, M. K.; Peak, C. W.; Cross, L. M.; Gaharwar, A. K. Injectable Nanoengineered Stimuli-Responsive Hydrogels for on-Demand and Localized Therapeutic Delivery. Nanoscale 2017, 9, 15379–15389. Willner, I. Stimuli-Controlled Hydrogels and Their Applications. Acc. Chem. Res. 2017, 50, 657–658. Hoare, T. R.; Kohane, D. S. Hydrogels in Drug Delivery: Progress and Challenges. Polymer 2008, 49, 1993–2007. Appel, E. A.; Tibbitt, M. W.; Webber, M. J.; Mattix, B. A.; Veiseh, O.; Langer, R. SelfAssembled Hydrogels Utilizing Polymer–Nanoparticle Interactions. Nat. Commun. 2015, 6, 6295. Qiu, Y.; Park, K. Environment-Sensitive Hydrogels for Drug Delivery. Triggering Drug Deliv. Syst. 2001, 53, 321–339. Zhao, X.; Wu, H.; Guo, B.; Dong, R.; Qiu, Y.; Ma, P. X. Antibacterial Anti-Oxidant Electroactive Injectable Hydrogel as Self-Healing Wound Dressing with Hemostasis and Adhesiveness for Cutaneous Wound Healing. Biomaterials 2017, 122, 34–47. Deng, Z.; Guo, Y.; Zhao, X.; Ma, P. X.; Guo, B. Multifunctional Stimuli-Responsive Hydrogels with Self-Healing, High Conductivity, and Rapid Recovery through Host– Guest Interactions. Chem. Mater. 2018, 30, 1729–1742. Dong, R.; Zhao, X.; Guo, B.; Ma, P. X. Self-Healing Conductive Injectable Hydrogels with Antibacterial Activity as Cell Delivery Carrier for Cardiac Cell Therapy. ACS Appl. Mater. Interfaces 2016, 8, 17138–17150. Kharkar, P. M.; Kiick, K. L.; Kloxin, A. M. Designing Degradable Hydrogels for Orthogonal Control of Cell Microenvironments. Chem Soc Rev 2013, 42, 7335–7372. Apostolides, D. E.; Sakai, T.; Patrickios, C. S. Dynamic Covalent Star Poly(Ethylene Glycol) Model Hydrogels: A New Platform for Mechanically Robust, Multifunctional Materials. Macromolecules 2017, 50, 2155–2164. Wei, Z.; Yang, J. H.; Liu, Z. Q.; Xu, F.; Zhou, J. X.; Zrínyi, M.; Osada, Y.; Chen, Y. M. Novel Biocompatible Polysaccharide-Based Self-Healing Hydrogel. Adv. Funct. Mater. 2015, 25, 1352–1359.


27


Page 28 of 31

(19) Yang, X.; Liu, G.; Peng, L.; Guo, J.; Tao, L.; Yuan, J.; Chang, C.; Wei, Y.; Zhang, L. Highly Efficient Self-Healable and Dual Responsive Cellulose-Based Hydrogels for Controlled Release and 3D Cell Culture. Adv. Funct. Mater. 2017, 27, 1703174. (20) Deng, G.; Li, F.; Yu, H.; Liu, F.; Liu, C.; Sun, W.; Jiang, H.; Chen, Y. Dynamic Hydrogels with an Environmental Adaptive Self-Healing Ability and Dual Responsive Sol–Gel Transitions. ACS Macro Lett. 2012, 1, 275–279. (21) El-Mahdi, O.; Melnyk, O. α-Oxo Aldehyde or Glyoxylyl Group Chemistry in Peptide Bioconjugation. Bioconjug. Chem. 2013, 24, 735–765. (22) Boehnke, N.; Cam, C.; Bat, E.; Segura, T.; Maynard, H. D. Imine Hydrogels with Tunable Degradability. Biomacromolecules 2015, 16, 2101–2108. (23) McKinnon, D. D.; Domaille, D. W.; Cha, J. N.; Anseth, K. S. Biophysically Defined and Cytocompatible Covalently Adaptable Networks as Viscoelastic 3D Cell Culture Systems. Adv. Mater. 2014, 26, 865–872. (24) McKinnon, D. D.; Domaille, D. W.; Cha, J. N.; Anseth, K. S. Bis-Aliphatic HydrazoneLinked Hydrogels Form Most Rapidly at Physiological PH: Identifying the Origin of Hydrogel Properties with Small Molecule Kinetic Studies. Chem. Mater. 2014, 26,23822387. (25) Cinay, G. E.; Erkoc, P.; Alipour, M.; Hashimoto, Y.; Sasaki, Y.; Akiyoshi, K.; Kizilel, S. Nanogel-Integrated PH-Responsive Composite Hydrogels for Controlled Drug Delivery. ACS Biomater. Sci. Eng. 2017, 3, 370–380. (26) Cevik, O.; Gidon, D.; Kizilel, S. Visible-Light-Induced Synthesis of PH-Responsive Composite Hydrogels for Controlled Delivery of the Anticonvulsant Drug Pregabalin. Acta Biomater. 2015, 11, 151–161. (27) Norouzi, M.; Nazari, B.; Miller, D. W. Injectable Hydrogel-Based Drug Delivery Systems for Local Cancer Therapy. Drug Discov. Today 2016, 21, 1835–1849. (28) Oliva, N.; Conde, J.; Wang, K.; Artzi, N. Designing Hydrogels for On-Demand Therapy. Acc. Chem. Res. 2017, 50, 669–679. (29) Subha, M. C. S.; Mallikarjuna, B.; Pallavi, K.; Rao, Kc.; Rao, Km. Biodegradable Interpenetrating Polymer Network Hydrogel Membranes for Controlled Release of Anticancer Drug. Asian J. Pharm. 2015, 9, 129-136. (30) Li, L.; Gu, J.; Zhang, J.; Xie, Z.; Lu, Y.; Shen, L.; Dong, Q.; Wang, Y. Injectable and Biodegradable PH-Responsive Hydrogels for Localized and Sustained Treatment of Human Fibrosarcoma. ACS Appl. Mater. Interfaces 2015, 7, 8033–8040. (31) Qu, J.; Zhao, X.; Ma, P. X.; Guo, B. PH-Responsive Self-Healing Injectable Hydrogel Based on N -Carboxyethyl Chitosan for Hepatocellular Carcinoma Therapy. Acta Biomater. 2017, 58, 168–180. (32) Shi, J.; Guobao, W.; Chen, H.; Zhong, W.; Qiu, X.; Xing, M. M. Q. Schiff Based Injectable Hydrogel for in Situ PH-Triggered Delivery of Doxorubicin for Breast Tumor Treatment. Polym Chem 2014, 5, 6180–6189. (33) Yamada, Y.; Schneider, J. P. Fragmentation of Injectable Bioadhesive Hydrogels Affords Chemotherapeutic Macromolecules. Biomacromolecules 2016, 17, 2634–2641. (34) Tibbitt, M. W.; Anseth, K. S. Hydrogels as Extracellular Matrix Mimics for 3D Cell Culture. Biotechnol. Bioeng. 2009, 103, 655–663. (35) Cai, L.; Heilshorn, S. C. Designing ECM-Mimetic Materials Using Protein Engineering. Acta Biomater. 2014, 10, 1751–1760.


28

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science 2009, 324, 59–63. (37) Li, Y.; Rodrigues, J.; Tomas, H. Injectable and Biodegradable Hydrogels: Gelation, Biodegradation and Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2193–2221. (38) McKinnon, D. D.; Domaille, D. W.; Brown, T. E.; Kyburz, K. A.; Kiyotake, E.; Cha, J. N.; Anseth, K. S. Measuring Cellular Forces Using Bis-Aliphatic Hydrazone Crosslinked Stress-Relaxing Hydrogels. Soft Matter 2014, 10, 9230–9236. (39) Nguyen; Ivan Huc, R. Optimizing the Reversibility of Hydrazone Formation for Dynamic Combinatorial Chemistry. Chem Commun 2003, 8, 942–943. (40) Shalviri, A.; Liu, Q.; Abdekhodaie, M. J.; Wu, X. Y. Novel Modified Starch–Xanthan Gum Hydrogels for Controlled Drug Delivery: Synthesis and Characterization. Carbohydr. Polym. 2010, 79, 898–907. (41) Verhoeven, E.; Vervaet, C.; Remon, J. P. Xanthan Gum to Tailor Drug Release of Sustained-Release Ethylcellulose Mini-Matrices Prepared via Hot-Melt Extrusion: In Vitro and in Vivo Evaluation. Eur. J. Pharm. Biopharm. 2006, 63, 320–330. (42) Tao, Y.; Zhang, R.; Xu, W.; Bai, Z.; Zhou, Y.; Zhao, S.; Xu, Y.; Yu, D. Rheological Behavior and Microstructure of Release-Controlled Hydrogels Based on Xanthan Gum Crosslinked with Sodium Trimetaphosphate. Food Hydrocoll. 2016, 52, 923–933. (43) Pooja, D.; Panyaram, S.; Kulhari, H.; Rachamalla, S. S.; Sistla, R. Xanthan Gum Stabilized Gold Nanoparticles: Characterization, Biocompatibility, Stability and Cytotoxicity. Carbohydr. Polym. 2014, 110, 1–9. (44) Becker, A.; Katzen, F.; Puhler, A.; Ielpi, L. Xanthan Gum Biosynthesis and Application: A Biochemical/Genetic Perspective. Appl. Microbiol. Biotechnol. 1998, 50, 145–152. (45) Peppas, N. A.; Keys, K. B.; Torres-Lugo, M.; Lowman, A. M. Poly(Ethylene Glycol)Containing Hydrogels in Drug Delivery. J. Controlled Release 1999, 62, 81–87. (46) D’souza, A. A.; Shegokar, R. Polyethylene Glycol (PEG): A Versatile Polymer for Pharmaceutical Applications. Expert Opin. Drug Deliv. 2016, 13, 1257–1275. (47) Kirker, K. R.; Luo, Y.; Nielson, J. H.; Shelby, J.; Prestwich, G. D. Glycosaminoglycan Hydrogel Films as Bio-Interactive Dressings for Wound Healing. Biomaterials 2002, 23, 3661–3671. (48) Dahlmann, J.; Krause, A.; Möller, L.; Kensah, G.; Möwes, M.; Diekmann, A.; Martin, U.; Kirschning, A.; Gruh, I.; Dräger, G. Fully Defined in Situ Cross-Linkable Alginate and Hyaluronic Acid Hydrogels for Myocardial Tissue Engineering. Biomaterials 2013, 34, 940–951. (49) Oommen Oommen P.; Wang Shujiang; Kisiel Marta; Sloff Marije; Hilborn Jöns; Varghese Oommen P. Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering. Adv. Funct. Mater. 2012, 23, 1273–1280. (50) Ma, Y.-H.; Yang, J.; Li, B.; Jiang, Y.-W.; Lu, X.; Chen, Z. Biodegradable and Injectable Polymer–Liposome Hydrogel: A Promising Cell Carrier. Polym. Chem. 2016, 7, 2037– 2044. (51) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W.; Weigele, M. Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines in the Picomole Range. Science 1972, 178, 871–872. (52) Lee, K. Y.; Bouhadir, K. H.; Mooney, D. J. Degradation Behavior of Covalently CrossLinked Poly(Aldehyde Guluronate) Hydrogels. Macromolecules 2000, 33, 97–101.


29


Page 30 of 31

(53) Bueno, V. B.; Bentini, R.; Catalani, L. H.; Petri, D. F. S. Synthesis and Swelling Behavior of Xanthan-Based Hydrogels. Carbohydr. Polym. 2013, 92, 1091–1099. (54) Bueno, V. B.; Petri, D. F. S. Xanthan Hydrogel Films: Molecular Conformation, Charge Density and Protein Carriers. Carbohydr. Polym. 2014, 101, 897–904. (55) Zhao, H.; Heindel, N. D. Determination of Degree of Substitution of Formyl Groups in Polyaldehyde Dextran by the Hydroxylamine Hydrochloride Method. Pharm. Res. 1991, 8, 400–402. (56) Zhao, H.; Rubio, B.; Sapra, P.; Wu, D.; Reddy, P.; Sai, P.; Martinez, A.; Gao, Y.; Lozanguiez, Y.; Longley, C.; et al. Novel Prodrugs of SN38 Using Multiarm Poly(Ethylene Glycol) Linkers. Bioconjug. Chem. 2008, 19, 849–859. (57) Anumolu, S. S.; Singh, Y.; Gao, D.; Stein, S.; Sinko, P. J. Design and Evaluation of Novel Fast Forming Pilocarpine-Loaded Ocular Hydrogels for Sustained Pharmacological Response. J. Controlled Release 2009, 137, 152–159. (58) Li, L.; Yan, B.; Yang, J.; Chen, L.; Zeng, H. Novel Mussel‐Inspired Injectable Self‐Healing Hydrogel with Anti‐Biofouling Property. Adv. Mater. 27, 1294–1299. (59) Qiu, M.; Wang, D.; Liang, W.; Liu, L.; Zhang, Y.; Chen, X.; Sang, D. K.; Xing, C.; Li, Z.; Dong, B.; et al. Novel Concept of the Smart NIR-Light–Controlled Drug Release of Black Phosphorus Nanostructure for Cancer Therapy. Proc. Natl. Acad. Sci. 2018, 115, 501–506. (60) Tacar, O.; Sriamornsak, P.; Dass, C. R. Doxorubicin: An Update on Anticancer Molecular Action, Toxicity and Novel Drug Delivery Systems. J. Pharm. Pharmacol. 65, 157–170. (61) Korsmeyer, R. W.; Gurny, R.; Doelker, E.; Buri, P.; Peppas, N. A. Mechanisms of Solute Release from Porous Hydrophilic Polymers. Int. J. Pharm. 1983, 15, 25–35. (62) Ta, H. T.; Dass, C. R.; Larson, I.; Choong, P. F. M.; Dunstan, D. E. A Chitosan– Dipotassium Orthophosphate Hydrogel for the Delivery of Doxorubicin in the Treatment of Osteosarcoma. Biomaterials 2009, 30, 3605–3613. (63) Saito, H.; Hoffman, A. S.; Ogawa, H. I. Delivery of Doxorubicin from Biodegradable PEG Hydrogels Having Schiff Base Linkages†. J. Bioact. Compat. Polym. 2007, 22, 589– 601. (64) Ossipov, D.; Kootala, S.; Yi, Z.; Yang, X.; Hilborn, J. Orthogonal Chemoselective Assembly of Hyaluronic Acid Networks and Nanogels for Drug Delivery. Macromolecules 2013, 46, 4105–4113. (65) LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27, 1081–1091. (66) Sivashanmugam, A.; Kumar, R. A.; Priya, M. V.; Nair, S. V.; Jayakumar, R. An Overview of Injectable Polymeric Hydrogels for Tissue Engineering. Eur. Polym. J. 2015, 72, 543– 565. (67) Exner, A. A.; Saidel, G. M. Drug-Eluting Polymer Implants in Cancer Therapy. Expert Opin. Drug Deliv. 2008, 5, 775–788. (68) Wu, H.; Liu, S.; Xiao, L.; Dong, X.; Lu, Q.; Kaplan, D. L. Injectable and PH-Responsive Silk Nanofiber Hydrogels for Sustained Anticancer Drug Delivery. ACS Appl. Mater. Interfaces 2016, 8, 17118–17126.


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