Dextran Injectable Hydrogel as an Optically ... - ACS Publications

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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*,†,⊥,#

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Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022 ‡ Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022 § Department of Emergency Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 430022 ∥ Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, China 430072 ⊥ Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022 # Medical Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022 S Supporting Information *

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 noninvasive trackability is highly desired. Although fluorescence dyes are used for imaging hydrogels, the cytotoxicity limits their applications. By using sericin, a natural photoluminescent protein from silk, we successfully synthesized a hydrazone cross-linked 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 toward 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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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 access to cancer regions.4 These limitations motivate the development of controlled, target-specific drug delivery systems. A rational design of these systems is cross-linked injectable hydrogels. As an effective, localized delivery vehicle, injectable hydrogels serve as an in situ depot allowing minimally invasive © 2016 American Chemical Society

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 Received: January 24, 2016 Accepted: February 22, 2016 Published: February 22, 2016 6411

DOI: 10.1021/acsami.6b00959 ACS Appl. Mater. Interfaces 2016, 8, 6411−6422

Research Article

ACS Applied Materials & Interfaces

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 4 °C) and readily degrades 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 50000 tons of sericin that is however discarded as a waste after degumming, causing environmental pollution.23 Despite being extracted from wild-type silkworm cocoons, this sericin from the 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 cross-linking to form a hydrogel.7 To overcome these limitations, we propose a design of a cross-linked sericin composite hydrogel using the two biocompatible backbone components, dextran (polysaccharide) and sericindegraded (protein) via the hydrazone-cross-linking chemistry. This cross-linking strategy employing reactions between hydrazide and aldehyde would offer fast cross-linking. Sericin with abundant polar side chains are designed to bear hydrazide groups that are complimentarily reactive to aldehydefunctionalized 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 cross-linked sericin/ dextran hydrogel with injectablility and controllable biodegradablility. The chemical and physical properties of this sericin/dextran injectable hydrogel are comprehensively characterized toward 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 noninvasively 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.

by the interactions of hydrogels with the surrounding microenvironment. It is important to be able to monitor hydrogels noninvasively over time for optimizing therapeutic efficacy and safety. However, most of the hydrogels lack 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 are 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. Sericin, a natural protein from silk, envelops and glues fibroin (another component of silk) to form cocoons (Figure 1A).7

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 cross-linked hydrogel as an injectable and photoluminescence-trackable drug delivery system.

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 antiapoptosis, 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 fibroindeficient mutant silkworm. Such sericin contains a well-

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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 (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 6412


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°C was chosen for this experiment because the gelation of the sericin/ dextran composite hydrogels was very fast at 37 °C (for example, 5 s 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 15 °C. 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 sputtercoated 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 −196 °C 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 removing the sample was recorded as V3. The porosity of the sample (ε) was obtained by

(Zhenjiang, Jiangsu, China). High molecular weight (HMW) alginate was obtained from FMC Biopolymer (Philadelphia, PA). Low molecular weight (LMW) alginate was degraded from HMW alginate by gamma irradation at 50 kGry for 4 h. 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 h. 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 35 °C for 2 h. Subsequently, N,N′-carbonyldiimidazole (4.1 g, 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 h at 45 °C. 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 100 °C for 15 min. After cooling down to room temperature, the absorbance of the mixed solution at 570 nm was measured using a microplate reader (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 or 5.2 g) was then added and stirred for 24 h at 4 °C in the dark. After reaction, the solutions were dialyzed against water (MWCO: 8−14 kDa) for 2 days at 4 °C. 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 cross-linked sericin/dextran composite hydrogels (SDH) were prepared by mixing equal volume of sericinADH 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 (4, 25, and 37 °C).26 The amine contents of hydrogels were determined by the ninhydrin colorimetry assay as described above. 1 H NMR and FTIR Analysis of Polymers. 1H 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 (PerkinElmer). 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 SB802.5 and SB-803, Showa Denko, Japan) with a precolumn (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 15 °C in an oscillatory mode. The temperature of 15 °C instead of 37

ε(%) =

V1 − V 3 × 100% V2 − V3

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, 7.4, and 11.0) at 37 °C. At various time intervals, the samples were carefully taken out and weighed until 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, 7.4, or 11.0) at 37 °C. At specified time points, the weight loss was measured and calculated and 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 37 °C 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 h in an incubator (37 °C, 5% CO2), 6413


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ACS Applied Materials & Interfaces 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 h. 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 h incubation at 37 °C. 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 (days 4, 8, 12, 16, 22, 40, and 70), the mice were imaged using a Xenogen IVIS Lumina II (Caliper Life Sciences) 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. 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-cross-linked 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 h. 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 2 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. 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 4 °C. The mixture solutions were incubated at room temperature for 2 h 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. 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) with the excitation wavelength 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 imaging 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). 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) 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). 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, and n.s. was not significant.

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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 fold (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 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 Table 1. Aldehyde Content and Molecular Weight of Dextran and Derivatives sample

NaIO4/glucose units (mole ratio)

% oxidation

DEX-Al-1 DEX-Al-2 DEX-Al-3 dextrana

0.1:1 0.2:1 0.4:1 −

8.5 ± 1.6 18.9 ± 2.2 35.5 ± 0.5 −

Mwb

PDI

× × × ×

1.73 1.60 1.72 1.78

2.80 2.50 2.67 3.53

104 104 104 104

a

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

b

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 6414


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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 nonspecific 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 37 °C appeared to be slightly fast for this purpose. The further chemical modifications might be needed toward 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 15 °C (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 DEXAl-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 cross-linking density, which would in turn increase the gelation rate and mechanical strength of the hydrogels.39 Porous Microstructure and Swelling Properties. The morphology of the composite hydrogels was examined using scanning electron microscope (SEM). The sericin/dextran hydrogels possessed porous network structures (Figure 3C). The pore size and porosity across these three SDH hydrogels decreased (Table 3) as DEX-Al-3 content increased. This was likely because the higher amount of aldehyde-functionalized dextran allowed the formation of more hydrazone linkages with sericin-ADH, thus resulting in a denser network with smaller pore sizes. The swelling ratios of the sericin/dextran hydrogels were measured in PBS in different pH conditions (6.0, 7.4, and 11.0). At the same pH, the equilibrium weight swelling ratios of these composite hydrogels decreased as DEX-Al-3 content (5% for SDH-1, 10% for SDH-2, and 17.5% for SDH-3) increased (Figure 3D), possibly due to their reduced pore size and porosity (Table 3). Interestingly, regardless of the types, the swelling ratio of each composite hydrogel in the acidic condition (pH 6.0) was significantly lower than those in the neutral and alkaline conditions (pH 7.4 and pH 11.0). The relative low swelling ratio of the hydrogel in the acidic condition might be because the pH value for the test was close to the isoelectric point of sericin, pH 3.8.40 The pH-dependent swelling property may allow these hydrogels to serve as a potential pH-responsive drug carrier or biomedical device, such as a microfluidic pH sensor.41−43 In Vitro and in Vivo Degradation and Biocompatibility. Controlled degradation of a hydrogel is important for drug release, as degradation causes the structure disassembly allowing the release of encapsulated drugs and avoiding surgical retrieval after use. Degradation of the sericin/dextran composite hydrogels was first evaluated in vitro under different pH conditions over time. The sericin/dextran composite

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. Fabrication of a Sericin/Dextran Cross-Linked 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 Table 2. 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 SDH-2 SDH-3

10% 10% 10%

5% 10% 17.5%

SDH-1, SDH-2, and SDH-3 according to their different DEXAl-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 sericinADH are reacted with aldehyde groups of DEX-Al-3 forming cross-linked networks and the cross-linking 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

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

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 sericinADH had no significant changes, indicating the chemical modifications do not lead to obvious secondary structure transformations. Upon cross-linking with DEX-Al-3, the peak of amide III of the composite hydrogel shifted from 1264 to 1285 cm−1, suggesting the cross-linking process may cause a slight transformation from random coil structure to β-turn.37 Gelation Time and Mechanical Properties. The gelation time of these composite hydrogels was determined in PBS (pH 7.4) at the different temperatures (4, 25, and 37 °C) (Figure 3A). The gelation time exhibited two features: temperature6415


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Figure 3. Gelation time, microstructure, and swelling ratios of the sericin/dextran composite hydrogels. (A) Gelation time of the composite hydrogels (SDH-1, SDH-2, and SDH-3) formed at 4, 25, and 37 °C (mean ± SD, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; and student’s t-tests). (B) Time evolution of storage modulus (G′) and loss modulus (G″) of SDH-1, SDH-2, and SDH-3 at 15 °C. (C) Scanning electron micrographs of SDH-1 (left), SDH-2 (middle), and SDH-3 (right). Scale bars, 10 μm. (D) The swelling ratios of the sericin/dextran hydrogels (SDH-1, SDH-2, and SDH-3) in PBS at different pH conditions (6.0, 7.4, and 11.0) at 37 °C (mean ± SD, n = 3).

degradation. The nearly complete degradation was observed 70 days after the injection. To investigate in vitro cytotoxicity of the sericin/dextran composite hydrogels, the CCK-8 assay, a method assessing cell proliferation by measuring the activity of mitochondrial dehydrogenase in living cells,29 was used to examine the cell viability of mouse myoblasts cells (C2C12) and human liver cells (HL7702) incubated with the hydrogels. After coincubating C2C12 and HL7702 with the hydrogels (from 4 to 64 g/L, hydrogel/culture medium, w/v, see Experimental Section for details), the cell viability of both cell lines was higher than 90% (Figure 4B), suggesting a good in vitro cytocompatibility. To assess in vivo biocompatibility of the sericin/dextran composite hydrogels, they were subcutaneously injected into the dorsal skin of C57BL/6 mice. Alginate/CaSO4 hydrogel, a widely used biocompatible hydrogel, was employed as the negative control as it is known to induce low inflammation;45 a glutaraldehyde-cross-linked chitosan hydrogel was chosen as the positive control because it can elicit inflammation.46,47 Two weeks after implantation, a large number of inflammatory cells infiltrated into the chitosan hydrogel implanted tissue, while the region implanted with the sericin/dextrin composite hydrogels (SDH-1, SDH-2, and SDH-3) showed limited cell infiltration, similar to that in the tissue with the alginate hydrogel implanted (Figure 4C). Consistently, the number of infiltrated macro-

Table 3. Pore Size and Porosity of the Sericin/Dextran Composite Hydrogels composite hydrogels average pore size (μm) porosity (%)

SDH-1

SDH-2

SDH-3

128.83 ± 34.88

47.13 ± 27.77

10.41 ± 4.83

81.06 ± 0.46

76.58 ± 4.00

73.82 ± 3.28

hydrogels were degraded faster in the alkaline condition (pH 11.0) than in the acidic (pH 6.0) and neutral (pH 7.4) conditions (Figure 4A). The acidic nature of sericin44 would make sericin more hydrophilic in an alkaline condition than an acidic condition, which might facilitate a more rapid hydrolysis of the amide groups in sericin via base catalysis. Although the hydrazone bond is a reversible linkage, its cleavage would be limited by sericin’s hydrophobicity to a greater extent in the acidic condition (pH 6.0) than the neutral and alkaline conditions. Along this line, the degradation of sericin protein rather than the hydrolysis of hydrazone bonds should presumably be the primary driving force underlying hydrogel degradation. In vivo degradation was analyzed by the subcutaneous injection of SDH-2 to the dorsal skin of C57BL/6 mice (also see Photoluminescent Property for in Vivo Degradation Monitoring for details). The significant weight loss was observed after 12 days followed by a slowed 6416


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Figure 4. In vitro degradation and in vitro and in vivo biocompatibility of the sericin/dextran composite hydrogels. (A) In vitro degradation profiles of SDH-1 (left), SDH-2 (middle), and SDH-3 (right) in PBS at different pH (6.0, 7.4 and 11.0) at 37 °C. (B) The relative cell viability of human liver cells HL7702 (left) and mouse myoblasts C2C12 (right) after a 48 h coincubation with the sericin/dextran composite hydrogels (SDH-1, SDH2, and SDH-3). (C) The hematoxylin-eosin (H&E) staining of the subcutaneous tissue where chitosan hydrogels, alginate hydrogels, and the sericin/ dextran composite hydrogels (SDH-1, SDH-2, and SDH-3) were implanted for 2 weeks. (D) The immunohistological staining of the CD68 marker for macrophages in (C). The black dotted boxes in the upper panel were enlarged in the lower panel. Scale bars, 50 μm. (E) Quantification of macrophages (CD68+) in (D). ***P < 0.001, N.S., not significant; n = 3 animals per group per condition, 6 random fields per animal; and student’s t-tests.

were rather low provides a strong support for the notion that pure sericin induces low immune responses. In Vitro Release of Horseradish Peroxidase (HRP) and Doxorubicin (DOX). To test the drug release property of the sericin/dextran hydrogels, a protein enzyme (horseradish peroxidase, HRP) and an antitumor drug (doxorubicin, DOX) were chosen as macromolecular and small molecular model drugs, respectively. The hydrogels sustained HRP release for over 50 days (Figure 5A). During a 50-day period, the cumulative HRP release from SDH-1, SDH-2, and SDH-3 was 95%, 71%, and 51%, respectively, suggesting a negative correlation between DEX-Al-3 content and the cumulative release. The release of DOX, a small molecular drug, was much faster than that of HRP (Figure 5B). At day 32, over 90% of

phages (CD68 positive cells) in the area where the sericin/ dextran composite hydrogels were implanted (SDH-1, 340 cells/mm2; SDH-2, 375 cells/mm2; and SDH-3, 341 cells/ mm2) was close to that found for the alginate negative control (337 cells/mm2), and much lower than that for the chitosan hydrogel (1012 cells/mm2) (Figure 4, panels D and E). Together, these results indicate that sericin/dextran composite hydrogels (SDH-1, SDH-2, and SDH-3) have a good biocompatibility in vivo. Although sericin’s immunogenicity suggested by several early studies has concerned sericin studies toward biomedical applications for a decade,48 some recent studies demonstrate that pure sericin has low immunogenicity unless physically associated with fibroin.49,50 Our data showing that the inflammatory responses to the sericin/dextran hydrogel 6417


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release. In addition, the higher content of DEX-Al-3 might have some reactive aldehyde groups forming imine with amine of HRP proteins, which might also contribute to the decreased HRP release. This tunable release property would enable the hydrogels to serve as a drug delivery system meeting various treatment requirements. Sericin-ADH Hydrogels Inherits Sericin’s Photoluminescent Property. Although sericinfull with a well-preserved protein profile was found to be photoluminescent,22 it was not clear whether sericindegraded that we used here was also photoluminescent. We next determined whether this degraded sericin still had the photoluminescent property. Surprisingly, this sericin did exhibit fluorescence (Figure 6A). We then determined whether this property was inherited by the functionalized sericin protein, the sericin/dextran hydrogels, and their corresponding lyophilized scaffolds. The sericin-ADH emitted fluorescence with the emission spectra falling into two wavelength ranges, from 340 to 520 nm (high-intensity peaks), and from 560 to 800 nm (low-intensity peaks) (Figure 6B). The emission spectra of the sericin/dextran hydrogels mainly

Figure 5. Cumulative release of (A) HRP or (B) DOX from SDH-1, SDH-2, and SDH-3 in PBS at 37 °C (mean ± SD, n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; and student’s t-tests).

encapsulated DOX in SDH-1 and SDH-2 was released. Similarly, a negative correlation between DEX-Al-3 content and the cumulative release was also observed for DOX. This may be explained by the fact that the hydrogel with higher DEX-Al-3 content had higher cross-linking density with smaller pore size and lower porosity,51 which would restrict drug

Figure 6. Photoluminescent property of the sericin/dextran hydrogels in vitro. (A−E) The emission spectra of sericin protein extracted using the (A) high heat/alkaline conditions, (B) sericin-ADH, (C) SDH-1, (D) SDH-2, and (E) SDH-3. (F and G) Each emission spectral curve corresponds to a specific excitation wavelength. SDH-1, SDH-2, and SDH-3 hydrogels were imaged under the light with the excitation wavelength of (F) 405 nm and (G) 488 nm. Scale bars, 100 μm. 6418


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Figure 7. Photoluminescent property for monitoring hydrogel degradation and drug release in vivo. (A) In vivo fluorescence imaging of C57BL/6 mice subcutaneously injected with SDH-1, SDH-2, and SDH-3 (white arrows) 2 h after injection. (B) Quantification of in vivo weight loss and fluorescence intensity reduction of the composite hydrogel (SDH-2) over 70 days. (C) Correlation of fluorescence intensity and weight of the SDH2 hydrogel during in vivo degradation. (D) SDH-2 (upper panel) loaded with DOX (middle panel) was injected subcutaneously and degraded over 17 days monitored by a small animal imaging device using the green fluorescence of SDH-2 (excitation wavelength 420 nm; emission wavelength 530 nm). DOX was observed by its red fluorescence (excitation wavelength 430 nm; emission wavelength 600 nm). The merged images of SDH-2 and DOX were shown in the lower panel. The images outlined by white dotted lines at the right upper corner are the enlargement of the merged images. (E and F) Quantification of fluorescence intensity reduction of (E) SDH hydrogels and DOX in vivo at (F) the SDH hydrogel sites over 17 days (n = 3 per group per time point; *P < 0.05, **P < 0.01; and student’s t-tests). (G−I) Correlation of the fluorescence intensity of DOX with the fluorescence intensity of the (G) SDH-1, (H) SDH-2, and (I) SDH-3 hydrogels during in vivo degradation.

and at 488 nm (for conventional green fluorescence dyes; Figure 6G). The porous structures were clearly visualized (Figure 6, panels F and G). Given that noninvasive ways of monitoring the inner structures of scaffolds are limited,52 the photoluminescent property of the sericin offers a potential alternative for examining the inner structures. Photoluminescent Property for in Vivo Degradation Monitoring. To determine whether this fluorescence is in vivo

ranged from 360 to 560 nm, while the emission from 560 to 800 nm nearly disappeared (Figure 6, panels C and E), suggesting that cross-linking with DEX-Al-3 alters the photoluminescent spectra of sericin protein. These remaining spectra covered the excitation wavelengths of commercially available blue and green fluorescence dyes or proteins. Consistently, the lyophilized hydrogels were observed under the excitation light at 405 nm (for conventional blue fluorescence dyes; Figure 6F) 6419


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injection (Figure 7, panels D and E). These changes on the hydrogels and DOX were also observed for SDH-1 and SDH-3 (Figure S3). Intriguingly, regardless of SDH hydrogel composition, the remaining DOX intensity was highly correlated with the intensity from the remaining hydrogel (R2 > 0.9) (Figure 7, panels G−I), revealing a dominant role of hydrogel degradation in determining in vivo DOX release rate. Of note, several other mechanisms, including diffusion and swelling, are thought to also control drug release from a hydrogel.53 Which mechanism is a driving force promoting drug release along with their relative contribution to drug release might depend on hydrogel composition, physical/ chemical properties, and types of encapsulated drugs. Our drug monitoring strategy offers a simple and precise means dissecting the interactions among these drug-release-influencing factors, thereby assisting the optimization of hydrogel design for drug release to meet various treatment requirements. Antitumor Efficiency of the DOX-Loaded Sericin/ Dextran Composite Hydrogels. The antitumor efficacy of the DOX-loaded SDH-2 was investigated using a B16-F10 melanoma model in male C57BL/6 mice. Two ×105 B16-F10 cells in 0.2 mL were subcutaneously injected into the dorsal skin of mice. When the tumor size reached 30−40 mm2 (14 days after injection), the mice were treated with a single injection of PBS, SDH-2, free DOX (4 mg/kg), and DOXloaded SDH-2 (4 mg/kg of DOX), respectively, into the vicinity of the tumors. The DOX-loaded SDH-2 hydrogel significantly inhibited tumor growth by approximately 50% and improved the survival rate by roughly 33% in comparison to the free DOX group (Figure 8, panels A, C, and D). No significant differences in body weight were found across the four groups (Figure 8B). To further examine the antitumor effects, the histological and apoptosis analyses were performed on isolated tumors. The tumors treated with the DOX-loaded SDH-2 exhibited severe tumor cell death (Figure 8E). Consistently, the DOX-loaded SDH-2 led to more apoptotic cells within tumor tissues than the other groups (Figure 8F). These therapeutic effects were likely because the sericin/dextran hydrogel sustains DOX release and maintains the relatively high concentration of DOX in tumor local regions. Importantly, these results demonstrate that SDH-2 can be a drug carrier for in vivo effective cancer treatment.

long-lasting and allowing effective transmission after sericin’s chemical modifications and cross-linking, we examined in vivo detection efficacy of the photoluminescent property for the sericin/dextran hydrogels with the excitation wavelength at 420 nm. These hydrogels were readily detected and visualized after subcutaneous injection into the dorsal skin of C57BL/6 mice with the emission wavelength at 530 nm (Figure 7A), suggesting that the photoluminescence of these hydrogels is sufficiently strong for effective transdermal transmission. To test whether the photoluminescence could be an indicator of the hydrogel degradation, we assessed the relationship between the weight and the fluorescence intensity of the hydrogels. Surprisingly, the weight loss and the fluorescence reduction were tightly correlated (R2 = 0.87) over the time period of 70 days during in vivo degradation (Figure 7, panels B and C), suggesting that the photoluminescence of the hydrogel can not only reliably reflect hydrogel’s quantity, but also is in vivo longterm stable without being quenched in complicated in vivo microenvironment. Thus, the photoluminescence of these hydrogels offers a noninvasive, reliable way of monitoring hydrogel amount in a real-time manner. We next took advantage of this fluorescence to examine in vivo degradation of three SDH (SDH-1, 2, and 3) hydrogels. The quantification on the hydrogel fluorescence (Figure 7, panels D and E) reduction showed that their degradation rates reduced as their DEX-Al-3 content increased, consistent with their in vitro degradation. However, the in vivo degradation rate of each of these SDH hydrogels was much faster than the in vitro degradation rate of the corresponding hydrogel at pH 7.4 (Figure 4A). Specifically, while SDH-1 took 45 days to degrade by 50% in vitro (pH 7.4), it took approximately 2 days to lose 50% in vivo. Similarly, in contrast to 65 days for SDH-2 and SDH-3 to reach 50% degradation in vitro, approximately 6 and 10 days were needed to degrade 50% in vivo, respectively. These 6−20-fold faster in vivo degradation rates might be attributed to complex in vivo microenvironment with more degradation-promoting factors, such as enzymes and cells, than in vitro conditions. Thus, the photoluminescence of the hydrogels made it possible to noninvasively, quantitatively compare the differences between in vitro and in vivo degradation, which would otherwise involve invasive methods and manipulation inconvenience. Our monitoring approach may be applicable to quantitative in vivo/in vitro comparative analyses on other properties of hydrogels toward hydrogel design improvement. Photoluminescent Property for in Vivo Drug Monitoring. We next tested whether this photoluminescence property could be used to monitor hydrogels’ other dynamic changes, such as drug release, which is important to chemotherapeutic efficacy and safety. To this end, we used doxorubicin (DOX) as the model drug. The sericin/dextran composite hydrogel (SDH-2) loaded with DOX was subcutaneously injected into the dorsal skin of C57BL/6 mice. The injection site was visually located by detecting the emission light of the hydrogels at the wavelength of 530 nm (green fluorescence) (Figure 7D). The remaining DOX within the hydrogel was readily observed and quantified by its red fluorescence (600 nm) (Figure 7, panels D and F). By quantifying the remaining amount of DOX within the hydrogels simply via fluorescence imaging, we circumvented the difficulty of precisely measuring the total amount of released drug in the body outside of the implantation site. The fluorescence intensity of DOX at the implantation site of the hydrogel gradually decreased during the period of 17 days after

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CONCLUSION

We have designed and developed a new cross-linked sericin/ dextran hydrogel via a Schiff base reaction as a versatile platform for cancer chemotherapy. This hydrogel is injectable, permitting a facile administration via minimally invasive approaches. This hydrogel not only exhibits weight-correlated, transdermal, biostable fluorescence that enables long-time in vivo tracking, but also sustains controlled drug release that is required for effective site-specific cancer therapy. With these integrated functions, this multifunctional drug delivery system offers a convenient noninvasive approach of monitoring hydrogel degradation and drug release, which would help improve the hydrogel design toward optimized chemotherapeutic safety and efficacy. Our tracking/monitoring strategy may be applied to other drug delivery systems. Further, tunable properties, such as gelation kinetics, degradation, and drug release rates, provide more opportunities for on-demand cancer therapy, paving the way to personalized medicine. 6420


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ACS Applied Materials & Interfaces Author Contributions @

J.L., C.Q., K.T., and Jin.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work is supported by the National Natural Science Foundation of China Programs 81272559, 81441077, 81572866 and 81402875, the International Science and Technology Corporation Program of Chinese Ministry of Science and Technology S2014ZR0340, the Science and Technology Program of Chinese Ministry of Education 113044A, the Frontier Exploration Program of Huazhong University of Science and Technology 2015TS153, and the Natural Science Foundation Program of Hubei Province 2015CFA049.

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Figure 8. In vivo antitumor activities of the DOX-loaded SDH-2 hydrogel. (A−C) Quantification of (A) tumor size, (B) body weight, and (C) the survival rate in B16−F10-bearing mice receiving PBS, SDH-2, free DOX (DOX), and the DOX-loaded SDH-2 hydrogel (SDH-2 + DOX) [n = 7−12 per group per time point; *P < 0.05 (DOX-loaded SDH-2 relative to free DOX); and student’s t-tests]. (D) Representative image of the isolated tumors on Day 14 from the mice receiving the indicated treatments. (E) H&E histological staining of the tumors isolated on Day 14 after the indicated treatments. (F) TUNEL staining of the isolated tumors in (E).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00959. Detailed information on the SDS−PAGE pattern comparison of sericin, characterization of chemically modified dextran and its derivative, and the remaining DOX in SDH-1 and SDH-3 during in vivo hydrogel degradation (PDF)

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REFERENCES

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