Dextran Injectable Hydrogel as an Optically Trackable Drug

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A sericin/ dextran injectable hydrogel as an optically trackable drug delivery system for malignant melanoma treatment Jia Liu, Chao Qi, Kaixiong Tao, Jinxiang Zhang, Jian Zhang, Luming Xu, Xulin Jiang, Yunti Zhang, Lei Huang, Qilin Li, Hongjian Xie, Jinbo Gao, Xiaoming Shuai, Guobin Wang, Zheng Wang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00959 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 24, 2016

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ACS Applied Materials & Interfaces

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A sericin/ dextran injectable hydrogel as an optically trackable drug delivery system for malignant melanoma treatment Jia Liu1,#, Chao Qi1,#, Kaixiong Tao2,#, Jinxiang Zhang3, #, Jian Zhang1, Luming Xu1, Xulin Jiang4, Yunti *

Zhang4, Lei Huang1, Qilin Li1, Hongjian Xie1, Jinbo Gao2, Xiaoming Shuai2, Guobin Wang2, , Zheng *

Wang1,2, , Lin Wang1,5,6,

*

1

Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 2

Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022. 3

Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022. 4

Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, China 430072. 5

Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 6

Medical Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022.

#

, These authors contributed equally to this work.

*

Correspondence to:

Lin Wang, Phone: 86-27-85726612. E-mail: [email protected] Guobin Wang, Phone: 86-27-85726612. E-mail: [email protected] Or to: Zheng Wang, Phone: 86-27-85726612. E-mail: [email protected]

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ACS Paragon Plus Environment


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Page 2 / 38 ABSTRACT Severe side effects of cancer chemotherapy prompt developing better drug delivery systems. Injectable hydrogels are an effective site-target system. For most of injectable hydrogels, once delivered in vivo, some properties including drug release and degradation, which are critical to chemotherapeutic effects and safety, are challenging to monitor. Developing a drug delivery system for effective cancer therapy with in vivo real-time non-invasive trackability is highly desired. Although fluorescence dyes were used for imaging hydrogels, the cytotoxicity limits their applications. By using sericin, a natural photoluminescent protein from silk, we successfully synthesized a hydrazone crosslinked sericin/dextran injectable hydrogel. This hydrogel is biodegradable and biocompatible. It achieves efficient drug loading and controlled release of both macromolecular and small molecular drugs. Notably, sericin’s photoluminescence from this hydrogel is directly and stably correlated with its degradation, enabling long-term in vivo imaging and real-time monitoring of the remaining drug. The hydrogel loaded with Doxorubicin significantly suppresses tumor growth. Together, the work demonstrates the efficacy of this drug delivery system, and the in vivo effectiveness of this sericin-based optical monitoring strategy, providing a potential approach for improving hydrogel design towards optimal efficiency and safety of chemotherapies, which may be widely applicable to other drug delivery systems. KEYWORDS: Sericin, Dextran, Injectable hydrogel, Photoluminescence, Cancer chemotherapy, In vivo trackability

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Page 3 / 38 INTRODUCTION Cancer is a leading cause of death accounting for 8 million deaths worldwide in 2012.1 Among various cancer treatment options (surgery, chemotherapy, radiotherapy, and immunotherapy), 2 , 3 chemotherapy remains the most common. Chemotherapy is often challenged by severe cytotoxic side effects, fluctuating blood concentrations, and limited drug assess to cancer regions.4 These limitations motivate the development of controlled, target-specific drug delivery systems. A rational design of these systems is crosslinked injectable hydrogels. As an effective, localized delivery vehicle, injectable hydrogels serve as an in situ depot allowing minimally invasive delivery, avoiding surgical implantation with reduced infection risk, reaching higher drug levels at cancer sites, controlling drug release rate, sustaining duration of therapeutic concentrations, circumventing poor solubility of drugs, thereby improving therapeutic efficacy while minimizing side effects. 5 Some properties/advantages of these injectable hydrogels, including drug release dynamics and hydrogel degradation, which are critical to chemotherapeutic effects and safety, can be affected by the interactions of hydrogels with surrounding microenvironment. It is important to be able to monitor hydrogels non-invasively over time for optimizing therapeutic efficacy and safety. However, most of hydrogels lacks in vivo real-time trackability, leaving the dynamic status of injectable hydrogels unknown and challenging to monitor after implantation. A common way to impart trackability to hydrogels is to incorporate fluorescence dyes into hydrogels. The substantial cytotoxicity and potential impairment to biomaterials’ properties6 caused by organic fluorescent dyes and inorganic materials is a major concern when used in vivo. This limitation might be overcome if backbone materials are biocompatible and fluorescent, which would spare the needs of including exogenous toxic fluorescence dyes. 3



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Page 4 / 38 Sericin, a natural protein from silk, envelops and glues fibroin (another component of silk) to form cocoons (Figure 1A).7 Unlike silk fibroin that has been extensively studied for biomedical applications,8 sericin has just begun to be explored in a growing number of tissue engineering applications.9,10 Given sericin’s biodegradability, biocompatibility, low-immunogenicity and newly-recognized diverse bioactivities, such as anti-apoptosis, oxidation resistance, and natural adhesive to cells,11-13 sericin has been used to form films, scaffolds and hybrid gels14-17 in conjunction with other natural or synthetic polymers.18-21 Importantly, we recently identified the intrinsic photoluminescent property of sericin.22 We hypothesize that taking sericin’s photoluminescence as the basis of the tracking property design would avoid the introduction of extrinsic organic dyes/ probes. The sericin reportedly emitting photoluminescence is the one extracted from cocoons of a fibroin-deficient mutant silkworm. Such sericin contains a well-preserved protein profile (we term this type of sericin “sericinfull”). However, the stability of sericinfull is low as it in aqueous solution lasts only for several days (at 4oC) and readily degraded when sterilized by commonly-used methods, such as autoclaving. Moreover, sericinfull ’s particular source requiring genetic mutants limits its availability. These drawbacks may confine the scope of its applications. An alternative ample source for sericin is the silk industry that each year produces approximately 50,000 tons of sericin that is however discarded as a waste after degumming, causing environment pollution.23 Despite being extracted from wild-type silkworm cocoons, this sericin from silk industry is degraded due to the harsh degumming conditions involving high heat and alkaline treatments (we thus term this degraded sericin “sericindegraded”). Sericindegraded is known to contain a large quantity of degraded soluble polypeptides with low molecular weight (Figure S1), which poses a challenge for crosslinking to form a hydrogel.7 4


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Page 5 / 38 To overcome these limitations, we propose a design of a crosslinked sericin composite hydrogel using the two biocompatible backbone components, dextran (polysaccharide) and sericindegraded (protein) via the hydrazone-crosslinking chemistry. This crosslinking strategy employing reactions between hydrazide and aldehyde would offer fast crosslinking. Sericin with abundant polar side chains are designed to bear hydrazide groups that are complimentarily reactive to aldehyde-functionalized dextran. To date, no studies report exploiting chemical functionalization to sericin for tissue engineering applications. Here we report that sericindegraded inherits the photoluminescent property. Using functionalized sericindegraded (hereinafter we refer sericindegraded as “sericin”) and dextran, we have successfully synthesized a hydrazone crosslinked sericin/dextran hydrogel with injectablility and controllable biodegradablility. The chemical and physical properties of this sericin/dextran injectable hydrogel are comprehensively characterized towards tissue engineering applications. This composite hydrogel exhibits in vivo long-term stable photoluminescence, tightly correlated with in vivo weight loss of the hydrogel. This photoluminesce can be conveniently used to non-invasively monitor both in vivo degradation and the amount of drug remaining within the hydrogel. The in vivo application of this hydrogel as a sustained drug carrier for cancer therapy is assessed in a mouse malignant melanoma model. Our data show that this hydrogel can serve as an effective drug delivery platform for improving cancer chemotherapy. The in vivo effectiveness of this optical tracking strategy suggests that it may be broadly applicable to hydrogel design for improving chemotherapeutic efficacy and safety.

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Figure 1. Schematics for preparation and utilization of a sericin/dextran composite hydrogel. (A) The extraction process of degraded sericin from Bombyx mori, Baiyu cocoons (wild-type). (B) Chemical modifications of sericin and dextran (oxidation). (C) The utilization of a sericin/dextran crosslinked hydrogel as an injectable and photoluminescence-trackable drug delivery system.

EXPERIMENTAL SECTION Materials Adipodihydrazide was purchased from Aladdin Industrial Corporation (Shanghai, China). N, N'-carbonyldiimidazole were purchased from Sigma-Aldrich (USA). Dextran, Sodium periodate, sodium carbonate, calcium sulfate, glutaraldehyde solution (25%) and dimethyl sulfoxide (DMSO) were purchased from Sinopharm chemical regent Co., Ltd (Shanghai, China). Doxorubicin hydrochloride was purchased from Meilun Biology Technology Co., Ltd (Dalian, China). Horseradish peroxidase（HRP）was purchased from Aladdin Industrial Corporation (Shanghai, China). Cellulose dialysis membranes 6


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Page 7 / 38 (MWCO 3.5 kDa, 8-14 kDa) were purchased from Spectrum Laboratory, Inc (Rancho Dominguez, CA). The silk cocoons (Bombyx mori，Baiyu) were provided by the Sericultural Research Institute, China Academy of Agricultural Sciences (Zhenjiang, Jiangsu, China). High molecular weight (HMW) alginate was obtained from FMC Biopolymer (Philadelphia, PA, USA). Low molecular weight (LMW) alginate was degraded from HMW alginate by gamma irradation at 50 kGry for 4 hours. Chitosan was purchased from Sigma-Aldrich with a degree of deacetylation higher than 75%.

Isolation of silk sericin Sericin was extracted by heat and alkaline degumming method from cocoons (Bombyx mori，Baiyu) as previously described.18 In brief, 10 g cocoons were cut into pieces and washed with deionized water. The cut pieces of cocoons were immersed in 0.02 M Na2CO3 solution (200 mL) and boiled for 1 hour. The silk fibroin was removed by filtration and the supernatant was dialyzed (MWCO: 8-14 kDa) against deionized water to remove Na2CO3 for 4 days at room temperature. The silk sericin was obtained by lyophilization.

Synthesis of sericin-ADH Sericin (5.6 g) was dissolved in 100 mL DMSO at 35oC for 2 hours. Subsequently, N, N'-carbonyldiimidazole (4.1g, 25 mmol) in DMSO (50 mL) was added. After stirring overnight at room temperature, this solution was added dropwise into the adipodihydrazide solution (43.55 g, 0.25 mol, 200 ml DMSO) and stirred for 24 hours at 45oC. After reaction, the solution was dialyzed against water (MWCO 3.5 kDa) for 3 days. The resulting sericin derivative (Sericin-ADH) was obtained by lyophilization. The structure of product was analyzed by FTIR, and the amine content was determined by the ninhydrin colorimetry assay as previously described.22,24 Briefly, sericin solution (100 µL, 2 wt%) mixed with solution A (50 µL; 0.0536 mol/L Na2HPO4, 0.0134 mol/L KH2PO4) and solution B (50 µL; 20 g/L ninhydrin, 0.8 g/L SnCl2), and then incubated at 100oC for 15 minutes. After cooling down to room temperature, the absorbance of the mixed solution at 570 nm was measured using a microplate reader 7



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Page 8 / 38 (Infinite F50, Tecan, Switzerland). The adipodihydrazide solutions with different concentrations were used to plot the reference standard curve.

Synthesis of dextran dialdehyde (DEX-Al) Dextran (10.0 g, 61.75 mmol glucose unit) was dissolved in 25 mL deionized water. NaIO4 (1.3 g, 2.6 g or 5.2 g) was then added and stirred for 24 hours at 4oC in dark. After reaction, the solutions were dialyzed against water (MWCO: 8-14 kDa) for 2 days at 4oC. The oxidized polymers (DEX-Al) were obtained by lyophilization. The structure was analyzed by FTIR and 1H-NMR. The aldehyde content of oxidized dextran was determined by the oxidation reduction titration method as previously described.25 The

molecular

weight

of

dextran

and

derivatives

was

determined

by

size-exclusion

chromatography-multiangle light scattering (SEC-MALLS) as described below.

Fabrication of sericin/dextran composite hydrogels Sericin-ADH was dissolved in PBS with the concentration of 20% (w/v), and DEX-Al was dissolved in PBS at concentrations of 10%, 20% and 35% (w/v), respectively. The crosslinked sericin/dextran composite hydrogels (SDH) were prepared by mixing equal volume of Sericin-ADH and DEX-Al solution. The gelation time of sericin/dextran composite hydrogels were determined by a flow test using a glass tube inverting method at different temperature (4oC, 25oC and 37oC).26 The amine contents of hydrogels were determined by the ninhydrin colorimetry assay as described above.

1

H NMR and FTIR analysis of polymers 1

H NMR spectra were measured with a Mercury VX-300 MHz spectrometer (300 MHz, Varian,

USA) using D2O as solvent. FTIR spectra were recorded on Spectrum One spectrometer (Perkin-Elmer, USA). The molecular weight and polydispersity of dextran and its derivatives were determined by a SEC-MALLS system with a Waters 2690D separation module, a Waters 2414 refractive index detector (RI) and a Wyatt DAWN EOS MALLS detector. Two chromatographic columns (ShodexOHpak 8


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Page 9 / 38 SB-802.5 and SB-803, Showa Denko, Japan) with a pre-column (Shodex SB-G) were used in series. A sodium nitrate (NaNO3) solution (0.1 M) was used as the eluent at a flow rate of 0.6 mL/min. The eluent was filtered by a 0.22 µm filter before use. The molecular weight of sericin-ADH was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as previously described.27

Evaluation of rheological behavior Rheological measurements of the sericin/dextran composite hydrogels were carried out using Rheostress6000 (Thermo Scientific, Germany) with parallel plates at 15oC in an oscillatory mode. The temperature of 15°C instead of 37°C was chosen for this experiment was because the gelation of the sericin/dextran composite hydrogels was very fast 37°C (for example, 5 seconds for SDH-3), which prevented the smooth loading of the samples onto the plate of the rheometer before gelation and the thorough examination of modulus. In brief, sericin-ADH and DEX-Al solution were mixed quickly at an ice bath and applied immediately on the lower plate of the rheometer. The upper plate was lowered to a gap size of 1 mm. The storage modulus (G’) and loss modulus (G”) were recorded over time at 15oC. The gelation time was defined as the time when G’/ G” equaled to 1.

Scanning electron microscopy and porosity measurement A scanning electronic microscope (SEM) was used to examine the morphology of lyophilized hydrogels. Cross-sections of sericin/dextran composite hydrogels were mounted onto aluminum studs and sputter-coated with gold. They were examined using an SEM (JSM-5610LV) at an accelerating voltage of 25 kV. The average pore sizes of different specimens were determined by quantifying 25 random pores per sample (5 samples for each analysis) with Image Pro Plus version 6.0.0.260. The porosity of the hydrogels were measured by the method of liquid displacement. 28 Sericin/dextran composite hydrogels were frozen at -196oC and then dried by lyophilization. The samples were then immersed in a known volume (V1) of water in a graduated cylinder. The volume of water after impregnation into the sample was recorded as V2. The volume of the water remaining in the cylinder after 9



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Page 10 / 38 removing the sample was recorded as V3. The porosity of the sample (ε) was obtained by:

ε(%) =

V1−V 3 ×100 % V 2 −V 3

Evaluation of swelling ratio The swelling behavior of the hydrogels was evaluated using conventional gravimetric mehods. Lyophilized hydrogels were immersed in phosphate buffered saline at different pH conditions (pH 6.0, pH 7.4 and pH 11.0) at 37oC. At various time intervals, the samples were carefully taken out and weighted unitl the swelling equilibrium was reached. The swelling ratios of the lyophilized hydrogels at equilibrium were calculated using the following equation:

swelling (%) =

Ws − Wd ×100 % Wd

where Wd was the dry weight of the construct and Ws was the swollen weight of the construct.

Degradation in vitro The degradation assay of the hydrogels was investigated with respect to weight loss in phosphate buffer solution for 65 days. Briefly , 200 µL of the hydrogel made from sericin-ADH (20%) and DEX-Al with different concentrations (10%, 20% and 35%) was immersed in a sealed tube containing 1 mL phosphate buffered saline (pH 6.0, pH 7.4, or pH 11.0) at 37oC. At specified time points, the weight loss was measured and calculated the percentage of degradation compared with the initial dry weight.

Cell culture and animal care Mouse myoblast cells (C2C12) were obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). Human liver cells (HL7702) were provided by the China Center for Type Culture Collection (Wuhan, China). These cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37oC 10


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Page 11 / 38 under a humidified atmosphere of 95% air and 5% CO2. All animal treatments and procedures were approved by the animal care and use committee of Huazhong University of Science and Technology, Wuhan, China. All animal experiments were conducted according to the guidelines for the care and use of laboratory animals approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Cytotoxicity assay The cytotoxicity of a hydrogel was evaluated using CCK8 assay on C2C12 and HL7702 cells.29 The cells were seeded in 96-well plate with a density of 6000 cells/well in 200 µL medium. After incubation for 24 hours in an incubator (37°C, 5% CO2), the culture medium was replaced by 200 µL of medium containing hydrogel with the given weight (final hydrogel concentrations ranging from 4 to 64 g/L), and the cells were incubated for another 48 hours. Then the medium with hydrogel was substituted by 100 µL fresh DMEM, and 10 µL of CCK-8 solution (DOJINDO, Japan) was added to each well for 1-hour incubation at 37oC. The absorbance was measured at 452 nm with a reference of 620 nm using a microplate reader (Infinite F50, Tecan, Switzerland). The cell viable rate was calculated by the following method: Cell viability (%) = (ODsample/ODcontrol) ×100%, where ODsample was the absorbance of the solution with the cells treated with the hydrogel and ODcontrol was the absorbance for the untreated cells.

Degradation behavior in vivo The sericin/dextran composite hydrogel (SDH-2, 200 µL) was subcutaneously injected into the dorsal skin of C57BL/6 mice to evaluate in vivo degradation. At given time intervals (Day 4, Day 8, Day 12, Day 16, Day 22, Day 40 and Day 70), the mice were imaged using a Xenogen IVIS Lumina II (Caliper Life Sciences, USA) to visualize the hydrogel and then sacrificed to isolate the remaining hydrogels. After lyophilization, the weight loss of the hydrogels was measured to evaluate in vivo degradation. 11



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In vivo biocompatibility The sericin/dextran composite hydrogels (SDH-1, SDH-2 and SDH-3) were administered by dorsal subcutaneous injection in male C57BL/6 mice to evaluate biocompatibility in vivo. Each injection was 0.2 mL in volume and performed through a hypodermic syringe. Alginate/CaSO4 hydrogel and glutaraldehyde-crosslinked chitosan hydrogel were used as controls. HMW alginate and LMW alginate (weight ratio of 1:3) were dissolved in ddH2O at the concentration of 2% (w/v) for 12 hours. Subsequently, 200 µL alginate solution was mixed with 8 µL CaSO4 (0.21 g/mL) in a syringe and rapidly injected. The chitosan solution (0.5%, W/V) was prepared according to the literature.30 Then 200 µL chitosan solution was mixed with 4 µL glutaraldehyde solution (25%, W/V) in a syringe and directly injected. After two weeks, the animals were sacrificed. The gels and surrounding tissues were isolated and fixed in 4% paraformaldehyde. The paraffin-embedded gel implants were sectioned through the center, and were subject to hematoxylin and eosin (H&E) staining and CD68 immunostaining for identifying infiltrated inflammatory macrophages.

The release of HRP and doxorubicin in vitro Release kinetics of sericin/dextran composite hydrogels was evaluated using horseradish peroxidase (HRP) and doxorubicin (DOX) as macromolecular and small model therapeutic agents, respectively. HRP was dissolved in 150 µL sericin-ADH solution (20%) at the concentration of 0.026 mg/mL. Subsequently, equal volume of DEX-Al-3 solution at different concentrations (10%, 20% or 35%) was added and mixed at 4oC. The mixture solutions were incubated at room temperature for 2 hour to form HRP-loaded hydrogels. The HRP-loaded hydrogels (0.3 mL) were immersed in PBS (1 mL, pH 7.4). The media were replaced with fresh PBS at predetermined time points and HRP contents of the release media were determined by ELISA as previously described.31 The release of DOX was performed with the same method described above. DOX concentration was determined by Nanodrop (Thermo Scientific, Germany) at the wavelength of 490 nm. 12


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Photolumiscent properties and tracking in vivo The photoluminescence of sericin solution, sericin-ADH solution and sericin/dextran composite hydrogels were analyzed using an RF-5301 PC fluoro spectrophotometer (Shimadzu, USA) with the excitation walvelength ranging from 320 to 600 nm. The photoluminescence of lyophilized hydrogels (SDH-1, SDH-2 and SDH-3) was investigated using a confocal laser scanning microscope (Nikon A1Si, Japan). For in vivo imaing analysis, SDH-1, SDH-2 and SDH-3 were subcutaneously implanted into the dorsal skin in C57BL/6 mice. Two hours after implantation, the mice were imaged using a Xenogen IVIS Lumina II (Caliper Life Sciences, USA). The in vivo release of doxorubicin (DOX) from DOX-loaded sericin/dextran composite hydrogels was also monitored. DOX-loaded hydrogels (SDH-1, SDH-2 and SDH-3, 200 µL, 0.4 mg/mL) were subcutaneously injected into the dorsal skin of C57BL/6 mice. At different time points, the mice were imaged using a Xenogen IVIS Lumina II (Caliper Life Sciences, USA) to visualize the hydrogel and DOX in vivo. The fluoroscence intensity of hydrogel and DOX within the hydrogel site was quantified using the software Living Image ® 4.3.1.

In vivo antitumor test The tumor model was established by subcutaneously inoculating approximately 2×105 B16-F10 cells in 0.2 mL into the back of C57BL/6 mice (6~8 weeks old). When the tumor size reached 30~40 mm2 (width × length), the C57BL/6 mice were randomly divided into four groups receiving the injection of PBS, the SDH-2 hydrogel, free DOX (4 mg/kg), and the DOX-loaded SDH-2 hydrogel (4 mg/kg of DOX in 200 µL hydrogel) into the vicinity of established tumors, respectively. The tumor size, animal body weight and survival period were monitored to evaluate antitumor efficacy. Fourteen days after the treatments, mice were sacrificed for the isolation of the tumors that were subject to hematoxylin and eosin (H&E) staining and the Terminal Transferase dUTP Nick-End Labeling (TUNEL) analysis using an assay kit (In Situ Cell Death Detection Kit, Roche, Switzerland). 13



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Statistical analysis All results were expressed as mean ± standard deviation with at least three independent tests. The statistical analysis was performed using Student t-tests. P < 0.05 was considered to be significant; P < 0.01 was highly significant; n.s. was not significant.

Table 1. Aldehyde content and molecular weight of dextran and derivatives.

Sample

NaIO4/glucose units (mole ratio)

% Oxidation

Mw b

PDI

DEX-Al-1

0.1:1

8.5±1.6

2.80×104

1.73

DEX-Al-2

0.2:1

18.9±2.2

2.50×104

1.60

DEX-Al-3

0.4:1

35.5±0.5

2.67×104

1.72

Dextrana

/

/

3.53×104

1.78

a

Commercial dextran with molecular weight (Mw) of 40 kDa. Molecular weight of dextran and derivatives determined by SEC-MALLAS. Oxidation was determined by iodinetry with oxidation degree recorded as percentages of dialdehyde groups per 100 glucose units. b

RESULTS AND DISCUSSION Chemical functionalization of sericin and dextran Silk from wild-type silkworms contains two components: fibroin at the center of silk fibers and sericin in the periphery (Figure 1A). After sericindegraded was extracted from wild-type cocoons using the conventional high heat/ alkaline treatments (Figure 1A), hydrazide groups were introduced to this sericin by adipodihydrazide (ADH) conjugation (Figure 1B). After the reaction (see “Experimental Section” for details), the free amino content was increased by 2.5 folds (from 1.14 to 2.84 mmol/g), indicating a successful conjugation of adipodihydrazide to sericin. This functionalized sericin was termed “sericin-ADH” in this study and designed to react with aldehyde-functionalized dextran to form a hydrogel carrying a drug for cancer treatment (Figure 1C). The aldehyde groups were introduced to dextran through oxidizing vicinal hydroxyl groups with 14


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Page 15 / 38 NaIO4. Depending on the feed ratios of NaIO4 to glucose, the dextran derivatives with different amount of aldehyde groups were termed DEX-Al-1, DEX-Al-2 and DEX-Al-3, accordingly (Table 1). DEX-Al-3 was chosen for the following four analyses to verify the occurrence of a hydroxyl-aldehyde conversion. (1) The 1H NMR spectra with the presence of new peaks between δ 5.0 to 5.7 ppm indicated the existence of the protons from aldehyde groups (Figure S2A).32,33 (2) The FTIR analysis revealed a new peak at 1745 cm-1 that was assigned to C=O stretching vibration, suggesting the presence of aldehyde groups (Figure S2B). (3) The iodinetry analysis showed that the aldehyde content of oxidized dextran (DEX-Al) increased as periodate increased (Table 1). (4) The molecular weight of DEX-Al decreased, suggesting the cleavage of partial dextran backbone by oxidation.

Figure 2. The FTIR spectra of sericin-ADH, sericin, and a sericin/dextran composite hydrogel (SDH-2).

Fabrication of a sericin/dextran crosslinked hydrogel and its FTIR analysis DEX-Al-3 with the highest aldehyde content was used to prepare sericin/dextran composite hydrogels. The hydrogel was fabricated by simply mixing sericin-ADH and DEX-Al-3 (see “Experimental Section” for details) (Table 2). The resulting hydrogels were termed as SDH-1, SDH-2 15



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Page 16 / 38 and SDH-3 according to their different DEX-Al-3 content (Table 2). After gelation, the free amine content of sericin-ADH was decreased from 2.84 to 0.77 mmol/g (SDH-1), 0.51 mmol/g (SDH-2) and 0.26 mmol/g (SDH-3), respectively, indicating that the hydrazide bonds of sericin-ADH are reacted with aldehyde groups of DEX-Al-3 forming crosslinked networks and the crosslinking degree increases as the content of DEX-Al-3 increases.34 The hydrogels were then analyzed by FTIR (Figure 2). The protein and polypeptide lead to nine characteristic absorption bands in FTIR analysis. Among them, amide I (C=O stretching, 1600-1690 cm-1), II (N-H bending and C-N stretching, 1480-1575 cm-1) and III (C-N stretching and N-H in-plane bending, 1299-1301 cm-1) are widely used for analyzing protein secondary structures.35 The sericin, used in this study, had the peak of amide I at 1662 cm-1, indicating the presence of β-sheet structure.36 After conjugation with adipodihydrazide, the peaks of amide I, II and III of sericin-ADH had no significant changes, indicating the chemical modifications do not lead to obvious secondary structure transformations. Upon crosslinking with DEX-Al-3, the peak of amide III of the composite hydrogel shifted from 1264 cm-1 to 1285 cm-1, suggesting the crosslinking process may cause a slight transformation from random coil structure to β-turn.37 Table 2. The formulation of sericin/dexran composite hydrogels fabricated with different concentrations of DEX-Al-3.

Composite hydrogels

Sericin-ADH (wt%)

DEX-Al-3 (wt%)

SDH-1

10%

5%

SDH-2

10%

10%

SDH-3

10%

17.5%

Gelation time and mechanical properties The gelation time of these composite hydrogels was determined in PBS (pH 7.4) at the different 16


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Page 17 / 38 temperatures (4oC, 25oC and 37oC) (Figure 3A). The gelation time exhibited two features: temperature-dependent and dextran-content-dependent. Specifically, gelation time reduced as the temperature increased, which was independent of the types of the composite hydrogels. At any given temperature, the gelation time of these three hydrogels was shortened as their DEX-Al-3 content increased (Figure 3A). These results indicate that the gelation time of this sericin/dextran composite hydrogel

can

be

conveniently

controlled

by

adjusting

temperature

or

the

content

of

aldehyde-functionalized dextran to meet various requirements of biomedical applications. However, although rapid gelation is thought to reduce in vivo non-specific diffusion of encapsulated cells or therapeutic agents, thereby minimizing the possibility of initial burst release of therapeutic agents,38 the second-scaled gelation time at 37oC appeared to be slightly fast for this purpose. The further chemical modifications might be needed towards improving gelation time under physiological conditions. The viscoelastic properties of these three sericin/dextran composite hydrogels were investigated in oscillatory rheological experiments. The in situ sol-gel transition was analyzed by monitoring storage modulus (G’) and loss modulus (G”) over time at 15oC (Figure 3B). Consistent with the above gelation analysis, the comparisons of the intersection points of G’ and G” (G’=G”), the gelling points, indicate that increasing DEX-Al-3 content within the hydrogels significantly accelerates the gelation process. Furthermore, the equilibrium storage and loss moduli of these three sericin/dextran hydrogels were also positively correlated with their DEX-Al-3 content. The higher content of DEX-Al-3 resulted in higher crosslinking density, which would in turn increase the gelation rate and mechanical strength of the hydrogels.39

17



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Figure 3. Gelation time, microstructure and swelling ratios of the sercin/dextran composite hydrogels. (A) Gelation time of the composite hydrogels (SDH-1, SDH-2 and SDH-3) formed at 4oC, 25oC and 37oC (mean ± SD, n=3; *P

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