Article pubs.acs.org/journal/abseba
Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Collagen Hydrogel Functionalized with Collagen-Targeting IFNA2b Shows Apoptotic Activity in Nude Mice with Xenografted Tumors Jun-Gen Hu,† Jin-Kui Pi,† Yan-Lin Jiang,† Xiao-Fan Liu,‡ Jesse Li-Ling,†,§ and Hui-Qi Xie*,† †
ACS Biomater. Sci. Eng. Downloaded from pubs.acs.org by YORK UNIV on 12/07/18. For personal use only.
Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, No. 1, Keyuan Fourth Road, Chengdu, Sichuan 610041, P. R. China ‡ West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, No. 17, Third Section, People’s South Road, Chengdu, Sichuan 610041, P. R. China § Institute of Genetic Medicine, School of Life Science, Sichuan University, No. 17, Third Section, People’s South Road, Chengdu, Sichuan 610041, P. R. China S Supporting Information *
ABSTRACT: Interferon alpha 2b (IFNA2b) has been used in immunotherapy for cancers with certain success. To reduce fast diffusion of IFNA2b and consequent dose-dependent side effects, we constructed a collagen hydrogel loaded with IFNA2b fused to collagen-binding domain by using methods of tissue engineering. The fusion protein showed apoptotic activity similar to that of native IFNA2b against MCF-7 cells in vitro, but with relatively higher affinity for collagen type I. Accordingly, the former diffused out of the collagen matrix slower than the latter. Importantly, collagen hydrogels loaded with the fusion protein possessed apoptotic activity in vitro and released the engineered cytokine in a controlled manner. In addition, such hydrogels reduced tumor size and extended the survival of the mouse model with xenografted tumors, which suggested a moderate antitumor activity in vivo. KEYWORDS: IFNA2b, collagen-binding domain, apoptotic, injectable, hydrogel
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INTRODUCTION Conventional treatments for solid tumors include surgical excision, chemotherapy, and radiotherapy.1 Although such treatment can be effective, the side effects may also be significant. In recent years, new approaches such as targeted drugs and immunotherapy have emerged as alternatives, alone or in combination with other treatments. 1,2 A widely investigated immunotherapeutics has been interferon alpha 2b (IFNA2b), a type I interferon cytokine,3 which has been approved by the FDA and is commonly used against hepatitis and cancer.3−7 IFNA2b can directly inhibit the growth of cancer cells by cell cycle arrest, apoptosis, or differentiation. It may also target cancer cells indirectly by activating immune cells, inducing secretion of other cytokines, and inhibiting angiogenesis.3 However, IFNA2b can also elicit dose-dependent side effects, including flu-like symptoms, hematological toxicity, elevated transaminases, nausea, fatigue, and psychiatric sequelae.3,8 Hence, lowering the systemic dose, but maintaining therapeutic local concentrations, may help reduce side effects and achieve better outcomes. For this purpose, some strategies have been explored, including delivery via microparticles.9−12 Past lessons and experiences from tissue engineering research suggested that biomaterials are preferable to achieve controlled release of bioactive agents. One such biomaterial, collagen, as the main component of extracellular matrix,13 has become widely used because of its excellent biocompatibility. However, © XXXX American Chemical Society
simple infusion of IFNA2b into collagen-based matrices is unlikely to fully resolve the aforementioned issues because IFNA2b may simply diffuse out and thereby increase systemic concentrations. On the other hand, fusion of IFNA2b to the collagen-binding domain, a short peptide (TKKTLRT) derived from collagenase,14 may anchor the resulting protein, IFNA2bCBD, to collagen. In this manner, locally high but systemically low concentrations may be achieved without loss of apoptotic activity. In addition, IFNA2b-CBD may still bind with host collagen upon degradation of the implanted collagen matrix, thereby preventing uncontrolled diffusion. In this study, we have generated a collagen hydrogel biomaterial infused with IFNA2bCBD, and tested their biological activities in vitro and in nude mice with xenografted tumors.
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EXPERIMENTAL SECTION
Preparation and Characterization of Recombinant Proteins. Plasmid Construction. XhoI and XbaI sites were engineered by overlapping PCR into the open reading frames of human IFNA2b and IFNA2b-CBD. IFNA2b and IFNA2b-CBD were then digested by XhoI and XbaI (New England Biolabs, Ipswich, MA, U.S.A.) and inserted into pET28a(+) (Novagen, Merck KGaA, Darmstadt, Germany) using a DNA Ligation Kit (Takara, Shiga, Japan). The resulting plasmids were Received: April 24, 2018 Accepted: November 23, 2018 Published: November 23, 2018 A
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering transformed into E. coli DH5α (TransGen Biotech, Beijing, China). Transformants were selected on appropriate LB-agar media, screened by PCR and DNA gel electrophoresis, amplified in LB media, and confirmed by sequencing (Genewiz, Suzhou, Jiangsu, China). Expression and Purification of IFNA2b and IFNA2b-CBD. pET28a(+) plasmids encoding IFNA2b and IFNA2b-CBD were transformed into E. coli Transsetta DE3 (TransGen Biotech, Beijing, China). Transformants were selected on appropriate LB-agar media, and they were screened by PCR and DNA gel electrophoresis. To induce expression, IPTG was added to cultures to a final concentration of 1 mM. Cultures were then harvested, resuspended in PBS, and sonicated in ice−water bath at 300W with 10 s pulse and 10 s pause cycles for 1 h. Inclusion bodies were collected by centrifugation at 2000g for 20 min at 4 °C, dissolved in Inclusion Body Denature Reagent (Cwbio, Beijing, China), and purified on BeaverBeads IDA-Nickel (BeaverNano Technologies, Suzhou, Jiangsu, China). Purified proteins were bufferexchanged to PBS by ultrafiltration (Merck Millipore, Merck KGaA, Darmstadt, Germany) and filtered using a 0.22 μm filter (Merck Millipore, Merck KGaA, Darmstadt, Germany) for in vivo experiments. Coomassie Blue Staining and Western Blot. Protein samples were boiled for 5 min in loading buffer, and resolved by SDS-PAGE. Gels were stained at 37 °C for 1 h with Coomassie blue, and destained overnight. For Western blotting, resolved samples were transferred to PVDF membranes, which were then blocked at 37 °C for 1 h in TBST supplemented with 5% milk, and probed at 4 °C overnight with 1:1,000 primary antibodies against IFNA (ab45272, Abcam, Cambridge, MA, U.S.A.) and His tags (66005-1-Ig, Proteintech, Rosemont, IL, U.S.A.). Membranes were then washed four times with TBST and labeled at 37 °C for 1 h with goat antimouse IgG conjugated to horseradish peroxidase (Cwbio, Beijing, China). After the membranes were washed in TBST another four times, they were visualized with Immun-STAR HRP Substrate Kit (Bio-Rad, Hercules, CA, U.S.A.) in ChemiDoc Imaging System (Bio-Rad, Hercules, CA, U.S.A.). Quantification of IFNA2b and IFNA2b-CBD by ELISA. IFNA2b and IFNA2b-CBD were quantified with an IFNA ELISA kit (Neobioscience Technology, Shenzhen, Guangzhou, China), following the manufacturer’s protocol. Briefly, samples were added to plates supplied with the kit at 100 μL/well, and they were incubated for 90 min at 37 °C. After the samples were washed five times, biotinylated antibodies were added at 100 μL/well, and they were incubated for 60 min at 37 °C. After they were washed another five times, avidin-peroxidase was added at 100 μL/well and incubated for 30 min at 37 °C. Plates were then washed five times and reacted for 15 min at 37 °C with 100 μL/well TMB. Finally, termination reagent was added at 100 μL/well, and absorbance at 450 nm was measured on a spectrophotometer (Biotek Instruments, Winooski, VT, U.S.A.). Cell Culture. MCF-7 cells were obtained from Cellbank, Shanghai, China, and cultured in a humidified incubator at 37 °C and 5% CO2 in complete medium consisting of RPMI-1640 (Hyclone, GE Healthcare Bio-Sciences, Pittsburgh, PA, U.S.A.) and 10% v/v fetal bovine serum (Biological Industries, Beit Haemek, Israel). Cells 70−80% confluent were subcultured at split ratio 1:3. Basal medium consisting of RPMI1640 only was used as needed. To assess the apoptotic activity of IFNA2b and IFNA2b-CBD, MCF7 cells were seeded in 96-well plates at 2.5 × 103 cells/well in 100 μL complete medium. After attachment, the culture medium was replaced with complete medium supplemented with 1 μM IFNA2b, IFNA2bCBD, or an equal volume of PBS as control. The culture medium was changed daily, and cultures were assayed by CCK-8 daily to generate growth curves. To further compare the apoptotic activities of IFNA2b and IFNA2bCBD without the influence of serum, MCF-7 cells were exposed for 24 h to different concentrations of IFNA2b or IFNA2b-CBD in basal medium, and assayed by CCK-8 to quantify surviving cells, using no cell or cells treated with the same volume of PBS as positive and negative control, respectively. CCK-8 Assay. Following the manufacturer’s protocol, culture supernatants were replaced with 110 μL/well of CCK-8 reagent (Dojindo Molecular Technologies, Kumamoto, Japan) diluted 1:10 v:v in culture medium. After incubation for 2 h at 37 °C, 100 μL samples
from each well were transferred to a new 96-well plate, and they were measured at 450 nm on a spectrophotometer (Biotek Instruments, Winooski, VT, U.S.A.), using as a blank diluted CCK-8 reagent incubated for 2 h at 37 °C without cells. Scatchard Analysis. Scatchard data were collected from a modified ELISA as described previously.15 In brief, rat-tail collagen (Sybio, Hangzhou, Zhejiang, China) was diluted to 20 μg/mL in carbonate buffer pH 9.6, of which 100 μL/well was used to coat a high-binding plate (Corning, NY, U.S.A.) at 4 °C overnight. Wells were then blocked at 37 °C for 1 h with 1% BSA, washed with TBST three times, and incubated at 37 °C for 2 h with 100 μL/well IFNA2b and IFNA2bCBD. After they were washed with TBST five times, wells were probed at 37 °C for 1 h with 100 μL/well 1:5,000 biotinylated anti-IFNA (ab45272, Abcam, Cambridge, MA, U.S.A.). After they were washed with TBST another five times, wells were labeled at 37 °C for 1 h with 100 μL/well 1:10 000 streptavidin conjugated to horseradish peroxidase (Beyotime, Shanghai, China). Finally, wells were washed with TBST five times, and stained at 37 °C for 15 min with 100 μL/well TMB. Reactions were terminated with 100 μL/well 0.5 M H2SO4, and absorbance at 450 nm was measured on a spectrophotometer (Biotek Instruments, Winooski, VT, U.S.A.). Scatchard analysis was performed in GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, U.S.A.). Construction and Characterization of Collagen Hydrogels Infused with IFNA2b and IFNA2b-CBD. Preparation of Collagen Hydrogels Infused with IFNA2b and IFNA2b-CBD. Hydrogels of type I collagen was prepared from rat-tail collagen (Sybio, Hangzhou, Zhejiang, China) according to the manufacturer’s protocol. Briefly, 200 μL of collagen, 100 μL of 10× PBS, 12 μL of 0.1 M NaOH, and 690 μL water, all ice-cold, were mixed to prepare 1 mL of 1 mg/mL collagen on ice. Hydrogels infused with IFNA2b or IFNA2b-CBD were prepared by replacing the water in mother liquor with solutions of corresponding proteins. Finally, soluble collagens were gelled by incubation at 37 °C. Rheology. The rheology tests were performed by HAAKE RheoStress 6000 rheometer (Thermo Scientific, Thermo Fisher Scientific, Inc., Waltham, MA, U.S.A.). For each test, 400 μL samples were placed onto the sample plate. with a P20 Ti L and a gap of 1 mm measuring geometry. For a temperature sweep test, samples were warmed from 15 to 37 °C at the rate of 1 °C/min, and the storage modulus G′ and loss modulus G′′ were measured with a frequency of 1 Hz in controlled stress mode (1 Pa). G′ and G′′ curves were plotted, and the gelation temperature was defined as the crossover point of the G′ and G′′ curves. For time sweeping tests, the G′ and the G′’ of the sample were monitored as functions of time at a frequency of 1 Hz in controlled stress mode (1 Pa) at 37 °C, and the gelation time was defined as the crossover point of the G′ and G′′ curves. Gelation Kinetics. Gelation was assessed by turbidity as previously described.16 In brief, soluble collagen mixed with or without IFNA2b and IFNA2b-CBD was added to 96 well plates (n = 8 wells each) on ice. The plates were set in spectrophotometer prewarmed to 37 °C (Biotek Instruments, Winooski, VT, U.S.A.), and immediately absorbance at 405 nm was measured every 1 min for 1 h. The gelation percentage was calculated as (A − A0)/(Amax − A0), where A, A0, and Amax are the absorbance, initial absorbance, and maximum absorbance at 405 nm. Maximum velocity, time to maximum velocity, and lag time were calculated in Gen5 software (Biotek Instruments, Winooski, VT, U.S.A.). Scanning Electronic Microscopy. Samples were lyophilized by a freeze-dryer (Scientz Biotechnology Co., Ltd., Zhejiang, China). SEM images were acquired by JSM-7500F scanning electronic microscope (JEOL, Tokyo, Japan). Swelling Behavior. Hydrogels were prepared, and after gelation occurred, the swelling behavior was evaluated by immersing the gels in PBS for 24 h at 37 °C. Then, excess PBS was removed with filter paper, and the weight of each sample was measured. The swelling ratio of the hydrogel was calculated as the following formula, swelling ratio = [(Ws − Wi)/Wi] × 100%, where Ws was the wet weight of the swollen hydrogel and Wi was the initial dry weight of the hydrogel after lyophilization by a freeze-dryer (Scientz Biotechnology Co., Ltd., Zhejiang, China). B
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 1. Design and preparation of IFNA2b and IFNA2b-CBD. (A) Schematic diagram of the structures of IFNA2b and IFNA2b-CBD. (B) Coomassie blue staining after SDS-PAGE. Target proteins are framed in dotted lines. (C) Western blot using antibodies against His tag and IFNA2b. (D) Representative proliferation curves of MCF-7 cells supplemented with PBS, IFNA2b, and IFNA2b-CBD. Cells exposed to IFNA2b and IFNA2bCBD showed similarly reduced proliferation in comparison to cells supplemented with PBS. (E) Representative and comparable dose-dependent normalized survival of MCF-7 cells exposed to IFNA2b and IFNA2b-CBD. (F) Binding curves of IFNA2b and IFNA2b-CBD to collagen, fitted by group. IFNA2b-CBD has higher affinity to collagen than IFNA2b. (G) Scatchard analysis of binding between collagen type I and IFNA2b or IFNA2bCBD. The slopes of the lines correspond to −1/Kd. Immunofluorescence. Collagen matrices were cryosectioned at 8 μm, fixed in formalin, washed with PBS, and blocked with 5% BSA. Sections were then probed at 4 °C overnight with 1:100 dilutions of rabbit antibodies to collagen type I (abs120555, Absin Bioscience, Shanghai, China) and mouse antibodies to IFNA2 (MAB9345, RD system, Minneapolis, MN, U.S.A.). Sections probed without primary antibodies were used as control. After washing with PBS five times, sections were labeled at 37 °C for 1 h with 1:200 dilutions of AffiniPure goat antirabbit IgG conjugated to Alexa Fluor 488 (111-545-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, U.S.A.) and AffiniPure goat antimouse IgG conjugated to Alexa Fluor 647 (115605-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, U.S.A.). Finally, sections were washed with PBS five times and imaged on an Axio Imager Z2 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Protein Release Assay. To assess IFNA2b and IFNA2b-CBD release in vitro, collagen with IFNA2b and IFNA2b-CBD were gelled at 100 μL/well in a 96-well plate, mixed with 100 μL/well PBS, and incubated at 37 °C. The total load of IFNA2b or IFNA2b-CBD was 0.69 nmol in 100 μL gel. Samples were collected at 50 μL/well/day, replaced with fresh PBS, and assayed for released protein by ELISA. Biological Evaluations of Constructed Hydrogels. Live/Dead Staining. To assess the direct apoptotic activity of collagen hydrogel with IFNA2b or IFNA2b-CBD, collagen was gelled in a 48-well plate at 200 μL/well (with 1.38 nmol IFNA2b or IFNA2b-CBD), seeded with 2 × 104 MCF-7 cells/well in complete medium, and stained for live and dead cells after 24 h. Cells were washed with PBS and stained at 37 °C for 15 min with 200 μL/cm2 of 2 μM calcein-AM and 2 μM propidium iodide in PBS, washed with PBS to remove excess stain, and imaged on an IX71 microscope (Olympus, Tokyo, Japan). Transwell Cocultures. Transwell cocultures were set up using polycarbonate transwell inserts with diameter 6.5 mm and pore size 0.4 μm (Corning, NY, U.S.A.). Collagen without or with IFNA2b and IFNA2b-CBD were gelled in the upper chamber at 100 μL/well (0.69 nmol IFNA2b or IFNA2b-CBD), and another 100 μL complete culture medium was supplied. MCF-7 cells were seeded in the lower compartment at 40 000 cells/well in 500 μL complete culture medium and grown in a humidified incubator at 37 °C and 5% CO2. Animal Experiments. Animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan University
(No. 2016050A), and they were compliant with the NIH Guideline for the Care and Use of Laboratory Animals. Balb/c nude mice were purchased from Chengdu Dossy Experimental Animals Co., Ltd., and raised with free access to food and water in a specific pathogen-free facility at West China Hospital Science Park, Sichuan University. To establish mouse tumor models, MCF-7 cells were harvested and resuspended in PBS at 2 × 107 cells/mL, of which 200 μL was subcutaneously injected at the axilla of the right forelimb. Tumors were palpable after a week. Collagen solutions (200 μL) with or without 1.38 nmol IFNA2b and IFNA2b-CBD were injected into the tumor mass in six mice each, and hydrogel formation was confirmed after injection. Survival was recorded daily. The major axis A and minor axis B of the tumor were also measured daily with a Vernier caliper by two independent observers, and tumor volume V was calculated as V = A × B2/2.17 Tumor volume at each time point was normalized to the initial tumor volume. Statistics. Data were analyzed in SPSS 22.0 (International Business Machines Corp., Armonk, NY, U.S.A.). Differences among groups were evaluated by one-way ANOVA, using Tukey’s test post hoc. EC50 was determined in GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, U.S.A.). During analysis of in vivo data, the highest and lowest number of days of survival were excluded from each group, along with the corresponding tumor volume. Kaplan−Meier analysis was performed to analyze survival, and log-rank test was used to compare survival curves. Differences were considered statistically significant at P < 0.05.
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RESULTS Preparation and Characterization of IFNA2b-CBD Protein. Preparation of IFNA2b-CBD Protein. The structures of IFNA2b and IFNA2b-CBD are illustrated in Figure 1A. The corresponding open reading frames were inserted into pET28a(+) (Figure S1A), verified by DNA sequencing, expressed as inclusion bodies in E. coli (Figure S1B and S1C), purified (Figure 1B), and verified by Western blot (Figure 1C) using antibodies to IFNA2b and His tags. Due to fusion with CBD, IFNA2b-CBD has higher molecular weight than IFNA2b (Figure 1B). Purified proteins were quantified by ELISA, and C
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 2. Rheology profiles of CH, CH-IFNA2b, and CH-IFNA2b-CBD. (A) Representative zoomed in rheology temperature sweep test results of CH, CH-IFNA2b, and CH-IFNA2b-CBD. CH-IFNA2b and CH-IFNA2b-CBD had increased phase transition temperature than CH. (B) Statistics results of the phase transition temperature of CH, CH-IFNA2b, and CH-IFNA2b-CBD. *, P < 0.05. (C) Representative zoomed in rheology time sweep test results of CH, CH-IFNA2b, and CH-IFNA2b-CBD. CH-IFNA2b and CH-IFNA2b-CBD had longer gelation time than CH. (D) Statistics results of the gelation time of CH, CH-IFNA2b, and CH-IFNA2b-CBD. *, P < 0.05.
Figure 3. Gelation turbidity dynamics of CH, CH-IFNA2b, and CH-IFNA2b-CBD. (A) Normalized gelation percentage over time. (B) Maximum velocity (Max V), which was highest for CH-IFNA2b, but lowest for CH-IFNA2b-CBD. (C,D) Time to Max V (C) and lag time (D), which were highest for CH-IFNA2b-CBD, but lowest for CH. *, P < 0.05.
manner,18 IFNA2b and IFNA2b-CBD were tested for apoptotic activity against MCF-7 cells. As shown in Figure 1D, cells supplemented with IFNA2b or IFNA2b-CBD proliferated at comparable rates (P > 0.05, n = 6) over the course of the
diluted to the same concentration for in vitro and in vivo experiments. IFNA2b-CBD and IFNA2b Have Similar Apoptotic Activities in Vitro. As IFNA2b induces apoptosis in a p53-dependent D
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Morphology and swelling behavior. (A) Representative SEM images of CH, CH-IFNA2b, CH-IFNA2b-CBD. All the materials had internal porous network structure. (B) CH-IFNA2b and CH-IFNA2b-CBD had decreased swelling ratio than CH. *, P < 0.05.
0.09 min, respectively) compared with CH (0.81 ± 0.10 min) (Figure 2C and 2D). The gelation process was further assessed by monitoring the turbidity. The gelation was delayed both in CH-IFNA2b and CH-IFNA2b-CBD (Figure 3A), which was consistent with the results of rheology tests. The maximum gelation velocity was 34.95 ± 0.52 mOD/min for CH, but 38.40 ± 1.80 mOD/min and 26.85 ± 0.89 mOD/min for CH-IFNA2b and CH-IFNA2bCBD (P < 0.05, n = 8, Figure 3B). The times to maximum velocity were 675.00 ± 27.77 s for CH, 862.50 ± 59.46 s for CHIFNA2b, and 967.5 ± 95.88 s for CH-IFNA2b-CBD (P < 0.05, n = 8, Figure 3C). The corresponding lag times were 492.38 ± 33.20 s, 642.38 ± 49.82 s, and 752.88 ± 42.55 s, respectively (P < 0.05, n = 8, Figure 3D). The extended lag time and time to maximum velocity in the presence of IFNA2b and IFNA2bCBD confirm the delayed gelation shown in Figure 3A. It is possible that IFNA2b sterically hinders the interactions among collagen molecules, and that the stronger affinity of IFNA2bCBD for collagen amplifies this effect, essentially turning IFNA2b-CBD into a noncompetitive inhibitor of collagencollagen interactions. However, it is unclear how IFNA2b increases the maximum velocity. Morphology and Swelling Behavior of CH, CH-IFNA2b, and CH-IFNA2b-CBD. The morphologies of CH, CH-IFNA2b, and CH-IFNA2b-CBD were characterized with SEM. As shown in Figure 4A, like other collagen-based hydrogel materials,19 all of the three groups had internal porous network structures. The fibrils in CH can be as thick as 3.20 μm, but in CH-IFNA2b or CH-IFNA2b-CBD the fibrils can only be as thick as 1.31 or 1.40 μm. The swelling behavior of the three hydrogels were also characterized. As shown in Figure 4B, CH had a swelling ratio of 6349 ± 332%, CH-IFNA2b had a swelling ratio of 3391 ± 303%, and CH-IFNA2b-CBD had a swelling ratio of 3948 ± 644%. This may be due to the change of net weight, as well as the change of porosity in hydrogels. Dispersion of IFNA2b and IFNA2b-CBD in Collagen Hydrogels. Frozen sections of collagen hydrogels with or
experiment, but more slowly than cells supplemented with PBS (P < 0.05, n = 6). This result indicates that IFNA2b-CBD and IFNA2b have similar apoptotic activities against MCF-7 cells. The apoptotic activities of IFNA2b and IFNA2b-CBD were further compared in absence of serum. As shown in Figure 1E, dose-dependent normalized survival was similar between cells exposed to IFNA2b and IFNA2b-CBD, with EC50 0.80 ± 0.03 μM for IFNA2b and 0.82 ± 0.08 μM for IFNA2b-CBD under the experimental conditions tested (P > 0.05, n = 6). This result confirms that IFNA2b and IFNA2b-CBD have similar apoptotic activities against MCF-7 cells. IFNA2b-CBD Has Higher Affinity for Collagen than IFNA2b in Vitro. A modified ELISA was used to measure the ability of IFNA2b and IFNA2b-CBD to bind to collagen in vitro. As shown in Figure 1F, absorbance at 450 nm was significantly higher for IFNA2b-CBD than for IFNA2b (P < 0.05, n = 6) from 10 nM to 0.3 mM, indicating that more IFNA2b-CBD was captured by collagen. Kd values were calculated from Scatchard analysis to be 1.738 μM and 1.053 μM for IFNA2b and IFNA2bCBD, respectively, in the experimental conditions tested (Figure 1G). The lower Kd value for IFNA2b-CBD signifies higher affinity for collagen. Construction and Characterization of Collagen Hydrogels Infused with IFNA2b and IFNA2b-CBD. Gelation of Collagen without or with IFNA2b and IFNA2b-CBD. Collagen hydrogel (CH) functionalized with IFNA2b (CH-IFNA2b) or IFNA2b-CBD (CH-IFNA2b-CBD) was prepared by neutralizing the pH and incubation at above phase transition temperature point. The rheology profiles of the hydrogels were first characterized. The storage modulus (G′) and loss modulus (G′′) crossed at a point indicating gel formation. As shown in Figure 2A, collagen solutions with or without IFNA2b or IFNA2b-CBD gelated when the temperature reaches a critical point of phase transition. CH-IFNA2b and CH-IFNA2b-CBD had increased phase transition temperature (30.60 ± 0.03 °C and 30.55 ± 0.04 °C, respectively) compared with CH (22.90 ± 1.11 °C) (Figure 2B). Besides, CH-IFNA2b and CH-IFNA2bCBD had prolonged gelation time (2.46 ± 0.13 min and 2.23 ± E
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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Figure 5. Immunofluorescence images of CH, CH-IFNA2b, and CH-IFNA2b-CBD. IFNA2b was detected both bound to (overlapping fluorescence) and apart from collagen (white arrow), while most IFNA2b-CBD was colocalized with collagen. Scale bar, 100 μm.
without IFNA2b and IFNA2b-CBD were analyzed by immunofluorescence to assess how the cytokines were incorporated. As shown in Figure 5, IFNA2b was detected both bound to and apart from collagen, while most IFNA2bCBD has colocalized with collagen. This also suggested that IFNA2b diffused more easily than IFNA2b-CBD in collagen hydrogels. Controlled Release of IFNA2b-CBD from Collagen Hydrogels in Vitro. Collagen hydrogel with or without IFNA2b or IFNA2b-CBD were prepared to assess protein release in vitro. Samples were collected and assayed for released protein by ELISA. We found that IFNA2b was released noticeably more quickly than IFNA2b-CBD in the first week (P < 0.05), especially in the first 3 days (Figure 6A). The data of cumulative released percentage over 40 days also demonstrated that IFNA2b was released more quickly than IFNA2b-CBD from collagen hydrogel (Figure 6B). This highlighted the greater affinity of IFNA2b-CBD for collagen and indicated bettercontrolled release from gelled collagen. Biological Evaluations of Constructed Hydrogels. Collagen Hydrogel with IFNA2b-CBD Shows Apoptotic Activity in Vitro. To assess the apoptotic activity of collagen hydrogel with IFNA2b or IFNA2b-CBD, MCF-7 cells were seeded onto CH, CH-IFNA2b, and CH-IFNA2b-CBD, and stained for live and dead cells. As shown in Figure 7A, few dead cells were observed in hydrogels of pure collagen, which highlighted their biocompatibility. However, dead cells were
Figure 6. Release of IFNA2b and IFNA2b-CBD from collagen hydrogels. (A) Daily release percentage of IFNA2b and IFNA2bCBD in the first week. (B) Cumulative release percentage of IFNA2b and IFNA2b-CBD over 40 days.
F
DOI: 10.1021/acsbiomaterials.8b00490 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 7. Live/dead staining of MCF-7 cells seeded on or cocultured with collagen hydrogels. (A) Representative live/dead staining of MCF-7 cells seeded on collagen hydrogels. (B) Quantification of dead cells seeded on collagen hydrogels. (C) Representative live/dead staining of MCF-7 cells cocultured with collagen hydrogels. (D) Quantification of dead cells cocultured with collagen hydrogels. Scale bar, 200 μm. *, P < 0.05.
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DISCUSSION IFNA2b has been used for treating solid tumors in the clinic with certain success,3,7 but this treatment has been associated with dose-dependent side effects.3,8 Hence, controlled released, especially controlled release from biomaterials, may help to mitigate undesirable side effects while maintaining local high concentrations that elicit the desired biological effects. Controlled release of protein from biomaterials, such as IFNA2b may be achieved by modifying the structure of delivery vehicles.12 Accordingly, it could be also achieved by modifying the protein itself. Many growth factors and cytokines, including BDNF,20−22 bFGF,23,24 BMP2,25,26 EGF,27 NGF,28,29 NT-3,30 PDGF,31,32 SDF-1α,15 and VEGF33−36 have been fused with CBD to increase binding affinity to collagen,37 a potential biomatrix for controlled release. Of note, such constructs generally enhance tissue repair in animals due to increased local concentration of growth factors or cytokines.15,20−26,28−30,32−36 In this light, we fused CBD with IFNA2b to investigate, for the first time, the value of an apoptotic molecule delivered via collagen. In vitro experiments demonstrated that fusion of CBD to IFNA2b enhances affinity to collagen without loss of apoptotic activity. Indeed, IFNA2b and IFNA2b-CBD have similar apoptotic activities against MCF-7 cells, as shown in Figure 1D,E, although the latter exhibits higher affinity to collagen (Figure 1F,G). When preparing collagen hydrogel materials, both IFNA2b and IFNA2b-CBD increased the critical phase transition temperature point and delayed gelation process (Figure 2). Moreover, IFNA2b-CBD appears to interfere with collagen gelation more effectively, lowering the maximum velocity and extending the lag time and time to maximum velocity in comparison with IFNA2b (Figure 3). Indeed, less thick collagen fibrils could be observed in CH-IFNA2b and CHIFNA2b-CBD (Figure 4A), indicating the interaction or involvement of IFNA2b and IFNA2b-CBD in collagen fibril
observed in collagen matrices with IFNA2b and IFNA2b-CBD, indicating that both proteins have retained apoptotic activity following incorporation into collagen hydrogels. Indeed, only 0.61 ± 0.16% of cells were apoptotic in wells with CH, while 16.11 ± 0.86% and 6.05 ± 2.34% were apoptotic in wells with CH-IFNA2b and CH-IFNA2b-CBD, respectively (Figure 7B). Apoptotic activity was also assessed in transwell cocultures, in which collagen without or with IFNA2b and IFNA2b-CBD was gelled in the upper chamber, while the lower chamber was seeded with MCF-7 cells. After 24 h, cocultures were stained for live and dead cells. As shown in Figure 7C, few dead cells were observed in cocultures with CH, but several were found in cocultures with CH-IFNA2b and CH-IFNA2b-CBD. Indeed, 0.75 ± 0.43%, 15.89 ± 2.68%, and 9.69 ± 3.28% of cells in these cocultures were apoptotic (Figure 7D). Collagen Hydrogel Infused with IFNA2b-CBD Reduces Tumor Volumes and Extends Survival in Nude Mice with Xenografted Tumors. MCF-7 tumors xenografted into nude mice were injected with soluble collagen supplemented without or with IFNA2b and IFNA2b-CBD. Tumor size and survival were recorded daily thereafter. Tumor volume and its ratio to the initial volume were also calculated. As shown in Figure 8A, an obvious shrinkage of tumor volume was observed in collagen hydrogel with IFNA2b-CBD group. Quantitatively as plotted in Figure 8B, the tumor volume decreased on day 1 in mice injected with collagen and IFNA2b, in comparison to other animals. On the other hand, the tumor volume generally decreased from day 3 in mice injected with CH-IFNA2b-CBD, and, indeed, was significantly lower in these mice than in others from day 3 to day 7 (P < 0.05). In addition, median survival was 22 ± 3.50 days, 32 ± 1.33 days, and 44 ± 0.43 days, respectively (Figure 8C), in mice injected with CH, CH-IFNA2b, and CH-IFNA2b-CBD. A log-rank test also indicated that survival curves were significantly different (P < 0.05). G
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Figure 8. Tumor volume and survival in tumor-bearing mice. (A) Representative images of nude mice with tumor xenografts on treatment day 1 and day 5. Dot circles pointed out the tumors. (B) Tumor volume, in ratio of the initial volume, during the first week. Dotted curves show tumor volume in each mouse, while solid curves represent mean and SD values by treatment group. *, P < 0.05. (C) Survival curves.
istration.38 In addition, collagen gels may fill irregularly shaped cavities left by surgical excision of solid tumors and thereby improve appearance,19 so that the collagen hydrogel with IFNA2b-CBD can be an adjuvant therapy after surgical excision to affect residual tumor cells for solid tumor treatment. However, this will naturally depend on gel strength as well as resistance to degradation (Figure S2). Whether collagen hydrogels should be chemically modified or cross-linked to enhance strength and delay degradation remains to be investigated.40 In mice, injection of collagen supplemented with IFNA2bCBD reduced the volume of xenografted tumors in the first week after treatment (Figure 8A,B). Median survival also increased to 44 ± 0.43 days, in line with a significantly different survival curve, as assessed by log-rank test (Figure 8C). Injection with collagen and IFNA2b also decreased the tumor volume on day 1 after treatment and extended survival, but not to the same extent. It is possible that collagen hydrogels infused with IFNA2b has the same apoptotic activity as gels loaded with IFNA2b-CBD, except that IFNA2b diffuses out of the tumor mass more rapidly. In this study, we initially explored whether
assembly. IFNA2b diffused more easily than IFNA2b-CBD in collagen hydrogels (Figure 5), while release of the latter from the matrix was better controlled as a result of higher affinity (Figure 6). Gels of pure collagen exhibited good biocompatibility as expected, while collagen matrices infused with IFNA2b-CBD exhibited apoptotic effects, suggesting that incorporation into collagen preserves bioactivity (Figure 7). According to the results of Figure 6, released IFNA2b-CBD from collagen hydrogel should be too small to cause such high apoptotic cell ratio in Figure 7; thus, this may have occurred because cells promoted the degradation of collagen hydrogel and the release of proteins. Collectively, these in vitro results show that the fusion protein functions as designed. Collagen solutions with or without IFNA2b or IFNA2b-CBD are fluid at low temperatures, but they form porous gels that trap an abundance of water (Figure 4) when the temperature reaches a critical point of phase transition by rheological tests (Figure 2). Indeed, IFNA2b and IFNA2b-CBD increase this phase transition temperature (Figure 2), possibly because the proteins lower the hydrophobicity of collagen.38,39 In any case, the injectability of collagen gel minimizes trauma during adminH
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Whether IFNA2b-CBD can also activate immune cells, induce secretion of other cytokines, or inhibit angiogenesis as well as IFNA2b was not evaluated. Hence, collagen gels infused with IFNA2b-CBD would have to be characterized specifically for indirect apoptotic activity, if such is preferred or is more suitable as treatment.
IFNA2b-CBD with collagen hydrogel was effective, and the side effects had not been evaluated. In any case, detailed investigation of how cytokine-infused collagen gels should be used to maximize therapeutic effects and minimize side effects would likely require larger animal experiments and more accurate methods of assessment, such as in vivo fluorescence, microCT, or ultrasound imaging.41−43 Similarly, more experiments are needed to evaluate whether IFNA2b-CBD, by itself or incorporated in collagen matrices, is suitable to treat other diseases such as hepatitis,4−6,11 against which IFNA2b is already used. Currently, biomaterials have shown potentials in antitumor therapies,44 mostly as drug carriers in the forms such as hydrogels45,46 and nanoparticles.47−49 Biomaterial designs usually take into account the characteristics of the microenvironment at the tumor site, for example, the redox,50 pH,50,51 charge,52 and proteomics.53,54 In addition to chemotherapeutic drugs like DOX and PTX,44 the protein drug can also be delivered via biomaterials. Kim et al. reported loading of TRAIL protein in PEG-HSA hydrogel,46 and the controlled release was achieved by different chemical groups for gelation without modification on the apoptotic protein. Besides, some studies have shown that biomaterials in antineoplastic therapies can not only inhibit tumor cells but also promote tissue regeneration.55,56 In this study, we fused the IFNA2b protein to a collagen binding domain, and used collagen hydrogel as the delivery system. We believe that this study shows a new strategy with respect to antitumor drug delivery via biomaterials; that is, controlled release is achieved by modification of the protein drug, and the modified protein binds to the extracellular matrix at the tumor site and induces apoptosis. In this study, the Kd values of IFNA2b and IFNA2b-CBD for collagen were measured by an ELISA-based assay and Scatchard analysis as reported,15 which was easy to perform without special equipment. However, methods such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can provide more accurate data to quantitatively determine the protein−protein interaction.57 Despite the limitations of method applied, the measured Kd values of IFNA2b and IFNA2b-CBD for collagen has only a 1.65-fold difference. This difference is indeed lower than other published data about the difference of Kd values between native growth factor and CBD modified growth factor, which can be as low as 2.57-fold.31 However, CH-IFNA2b-CBD did show different behaviors compared with CH-IFNA2b. It is possibly because that in the assay used to measure, the Kd values collagens were first immobilized onto the plate surface, while collagens and IFNA2b or IFNA2b-CBD were all in solution in the assay of preparing the collagen hydrogels. As indicated by the results of rheology (Figure 2), gelation turbidity (Figure 3), SEM (Figure 4A), and the early stage of degradation by collagenase (Figure S2), IFNA2b-CBD may interact with the collagen molecules or even participate in collagen fibril assembly in hydrogel formation. Nevertheless, the underlying mechanism of different behaviors between CH-IFNA2b-CBD and CH-IFNA2b remains to be explored. It should be noted that, while IFNA2b has both direct and indirect apoptotic activity,3 we only exploited the former for targeting the tumor cells. Considering that IFNA2b can directly induce apoptosis in a p53-dependent manner,18 IFNA2b-CBD was tested against MCF-7 cells which have fully functional p53. Indeed, IFNA2b or IFNA2b-CBD may be effective against a number of solid tumor types, as long as p53 activity is intact.
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CONCLUSIONS In this study, we have generated a fusion protein consisting of collagen-binding domain and IFNA2b, and we constructed a collagen hydrogel material containing it. The fusion protein showed an apoptotic activity similar to IFNA2b but with relatively greater affinity for collagen. Incorporation into collagen hydrogel enables controlled release of the fusion protein without loss of activity in vitro. In an in vivo xenografted tumor model, such a hydrogel exhibits a moderate degree of apoptotic activity, reduces tumor volumes, and extends survival.
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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/acsbiomaterials.8b00490.
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Construction of plasmids and induced expression of proteins; degradation properties of CH, CH-IFNA2b, and CH-IFNA2b-CBD in vitro (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hui-Qi Xie: 0000-0003-0760-0853 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. Mei Gong and Dong-Ze Li for their critical comments. This work was supported by National Key R&D Program of China (Grant No. 2017YFC1104702) and National Natural Science Foundation of China (Grant No. 81473446 and 31771065).
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REFERENCES
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