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Improved Stability, Antitumor Effect, and Controlled Release of Recombinant Soluble TRAIL by Combining Genetic Engineering with Coaxial Electrospinning Bin Yu,†,‡,⊥ Xueping Zhang,§,⊥ Jingyi Yan,‡ Dong Liu,† Libo Li,† Renjun Pei,∥ Xianghui Yu,*,‡ and Tianyan You*,†
Downloaded by UNIV AUTONOMA DE COAHUILA at 04:07:03:666 on June 05, 2019 from https://pubs.acs.org/doi/10.1021/acsabm.9b00119.
†
School of Agricultural Equipment Engineering Institute of Agricultural Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, China ‡ National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun 130012, China § Department of Materials Science and Engineering, National University of Singapore, Singapore 117575, Singapore ∥ Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences, Suzhou, Jiangsu 215123, China S Supporting Information *
ABSTRACT: Soluble tumor necrosis factor related apoptosis-inducing ligand (sTRAIL) is a promising candidate for antitumor protein drugs. However, the low stability and poor pharmacokinetic profile significantly limit its clinical application. In this work, a novel strategy was used to improve sTRAIL performance by combining genetic engineering with coaxial electrospinning. We first obtained a stable homotrimer via genetic engineering using the adenovirus knobless fiber motif to modify sTRAIL. In vitro studies showed that the engineered sTRAIL was more stable and had higher antitumor activity than wild sTRAIL. This new recombinant sTRAIL was then encapsulated into poly(lacticco-glycolic acid) (PLGA) nanofibers by coaxial electrospinning to further enhance the stability and achieve the controlled release of sTRAIL. Surprisingly, the recombinant sTRAIL maintained its biological activity during the electrospinning process and exhibited a good cumulative release. The establishment of incorporation treatment of breast cancer mouse model demonstrated that the PLGA/sTRAIL nanofiber mats can effectively inhibit the growth of breast cancer tumor cells. KEYWORDS: sTRAIL, genetic engineering, coaxial electrospinning, controlled release, tumor treatment a portion of dimers, which would have hepatocyte toxicity.10,11 Chemical modification methods such as PEGylation and longcirculating TRAIL liposomes were also widely used to enhance the pharmaceutical characteristics and biological activities of recombinant sTRAIL.12−15 Besides, in vivo safety analyses are imperative before these studies are ready for clinical application. Therefore, more ideal modification strategies are still required with safety being taken into primary consideration. Recently, our group has designed and expressed a highly stable and active recombinant sTRAIL homotrimer (FA1FT) similar to natural TRAIL by using the adenovirus knobless fiber motif.16 Unfortunately, this new recombinant protein did not show better pharmacokinetic characteristics in vivo than wild TRAIL did, since it appeared to be concentrated in the liver. In order to further enhance the stability and prolong the half-life of sTRAIL in vivo, construction of a controlled drug
1. INTRODUCTION Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential candidate for anticancer drugs due to its selective cytotoxicity in many cancer cells.1,2 TRAIL induces apoptosis by recognizing death receptors (DR4 and DR5) and subsequently recruiting Fas-associated death domains.3 Native and recombinant soluble TRAIL (sTRAIL) must form a homotrimer to become biologically active.4 However, noncovalently associated TRAIL is sensitive to degradation, since the homotrimer is formed by chelation of one zinc atom with the Cys230 of each monomer.5 A large number of preclinical studies showed that recombinant sTRAIL can safely and effectively inhibit tumor cell growth.6,7 However, early clinical studies have shown that recombinant sTRAIL has instability in vivo, short half-life, and low sensitivity in many tumor cells.1,8 Various approaches have been developed to improve the stability and selective antitumor activity of recombinant sTRAIL. Genetic engineering is one of the most important strategies by redesigning its structure with different trimerization motifs.9 Unfortunately, most engineering approaches altered the native trimeric structure of TRAIL and produced © XXXX American Chemical Society
Received: February 12, 2019 Accepted: May 22, 2019 Published: May 22, 2019 A
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials
Scheme 1. Schematic Illustration of the Combined Modification Strategy for Enhancing the Stability, Antitumor Effect, and Controlled Release of sTRAIL
GCTAGCATGGGTGCCATTACAGTAGGAAAC-3′ (S-sTRAIL); 5′-GCTAGCATGAAGCGCGCAAGACCGTCTGA-3′ (TS9sTRAIL), and the reverse primer for all constructs was 5′AAGCTTTTAGCCAACTAAAAAGGCCC-3′. The fusion fragments were cloned into the pET-28a (+) prokaryotic expression vector (Novagen, Madison, WI) at the Nhe I and Hind III restriction sites. Then the recombinant proteins were expressed in Escherichia coli and were purified by Ni-affinity chromatography as described previously.16 SDS-PAGE and GDS-PAGE were used to analyze the purified recombinant proteins has been described.25 2.2. Cell Viability Assay. The cell viability was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay according to the previous report.16 Experimental cells (1 × 104/100 μL) in a 96-well plate were treated with different concentrations of recombinant protein (0.01, 0.1, 1, 10, and 100 nM) at 37 °C for 16 h. Then the cells were incubated for another 4 h with 20 μL of MTT (5 mg/mL) added in each well. The absorbance was measured at 570 nm. Cell survival ratio (%) was calculated according to the following formula: proliferation = [(average A value of experimental group − average A value of control group)/(average A value of positive group − average A value of control group)] × 100%. 2.3. Flow Cytometry Analysis of Apoptosis. Cancer cell apoptosis induced by recombinant proteins was analyzed by using the Annexin-V(FITC)/PI Apoptosis Kit (Biosea, Beijing, China). Cells were incubated with 10 nM recombinant proteins for 6 h, which were then digested and resuspended in binding buffer. After dark staining with Annexin V (0.6 mg/mL) and PI (5 mg/mL) at room temperature for 15 min, all cells were washed once with PBS and analyzed by flow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, NJ). 2.4. Analysis of the Stability of Recombinant Proteins. The in vitro stability of sTRAIL, S-sTRAIL, and TS9-sTRAIL in PBS was investigated by the change in activity over time at physiological temperature. Recombinant proteins (100 μg/mL) were incubated in PBS (pH = 7.4) at 37 °C for 0, 15, and 30 min and 1, 2, 6, 12, and 24 h, and then the killing activity of proteins to ZR-75-30 (human breast cancer cell line) cells was analyzed using MTT assays. For the pH sensitivity analysis, 100 μg/mL protein solutions in PBS at various pH values (pH = 1, 3, 5, 7, 9, or 11) were incubated at 4 °C for 2 h, and then the killing activity of proteins was also analyzed using MTT assays. The biological stability of recombinant proteins was evaluated
delivery system for sTRAIL would be the desired strategy since it could maintain effective drug concentration in vivo release time, thus eliminating the need for frequent administration and reducing the side effect and resistance. Electrospinning is a widely used technique to prepare nanofibers.17 Due to its unique nanostructure and the ability to incorporating electronic, magnetic, or biological functionalities, electrospun nanofibers show great potential in nanodevices, tissue engineering, and drug delivery.18−21 For drug delivery, electrospun nanofibers feature high encapsulation efficiency, large loading capacity, and different controlled drug release. Studies have demonstrated that small molecule drugs, biomacromolecules such as proteins, DNA and RNA can be incorporated into electrospun nanofibers for the treatment of diseases such as tumors.22,23 A recent study showed that smallmolecule-drug-loaded electrospun mats implanted to cover the solid tumor locally could effectively suppress cancer growth in vivo.24 Herein, a novel strategy for improving the stability and antitumor activity of sTRAIL was developed by combining genetic engineering with coaxial electrospinning. As shown in Scheme 1, we first created a novel engineered sTRAIL by an optimized fusion method with adenovirus fiber shaft to the amino terminus. Then the more stable recombinant sTRAIL proteins were encapsulated into biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanofibers by coaxial electrospinning to obtain controlled drug release. Finally, the treatment of a solid tumor was investigated with the recombinant sTRAIL-loaded nanofiber mats.
2. EXPERIMENTAL SECTION 2.1. Construction, Expression, and Purification of Recombinant Proteins. The N-terminal tail and the shaft region of human adenovirus type 5 (Ad5) fiber were obtained from a rAd5 vector. The sTRAIL (aa 114−281) fragments were derived from a recombinant sTRAIL expression vector pET-28a-TRAIL (96−281). The fusion gene was constructed by overlap PCR. The forward primers were 5′GCTAGCATGGTGAGAGAAAGAGGTCCTCA-3′ (sTRAIL); 5′B
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 1. Preparation of recombinant sTRAIL proteins and analysis of its biological activities. (A) Schematic representation of the design of engineered sTRAIL used in this study. (B) SDS-PAGE and GDS-PAGE analysis of sTRAIL, S-sTRAIL, and TS9-sTRAIL. The arrows indicate the monomer, dimer, and trimer forms of recombinant sTRAIL proteins, respectively. (C) Cytotoxicity of sTRAIL, S-sTRAIL, and TS9-sTRAIL in ZR75-30 and normal human liver cells QSG-7701 was evaluated by the MTT assay. Results are displayed as mean ± SD from three independent experiments (n = 3). (D) Apoptosis-inducing ability of sTRAIL, S-sTRAIL, and TS9-sTRAIL was evaluated through flow cytometry. QSG-7701 and ZR-75-30 cells were treated with 10 nM recombinant proteins for 6 h and then examined using a PI/Annexin V staining kit and flow cytometry. by incubation with 50% rat plasma. The samples were also incubated at 37 °C for different times, and then the cytotoxicity of recombinant proteins was tested by MTT assays. 2.5. In Vivo Pharmacokinetics of Recombinant Proteins. Female BALB/c mice (3 mice per group), 6−7 weeks old, were injected intravenously (i.v.) with 1 nM of recombinant protein. Blood samples were taken from the tail at 5, 10, 30, 60, 120, and 240 min intervals. After centrifugation at 3000 rpm for 20 min at 4 °C, the concentration of recombinant protein was detected by the enzymelinked immunosorbent assay (ELISA).16 2.6. Preparation of Coaxial Electrospinning Nanofibers. PLGA [lactide:glycolide (75:25), Mw = 66 000−107 000] and hexafluoro-2-isopropanol (HFIP) were obtained from Sigma-Aldrich. The PLGA shell solution was prepared by dissolving 340 mg of PLGA in 1 mL of HFIP and stirring for 2 h. PBS containing 5 mg/mL sTRAIL or S-sTRAIL or 10 mg/mL TS9-sTRAIL was put into the inner coaxial needle by using a small syringe pump. The flow rate of the shell PLGA solution was 1 mL/h, while that of the core solution ranged from 0.1 to 0.3 mL/h. A grounded fixed indium tin oxide (ITO) glass (15 × 15 cm2) was used as the fiber collector. The distance of tip-to-collector was 15 cm, and the applied voltage was 20 kV. The nanofiber morphology was examined by scanning electron microscopy (SEM) using a PHILIPS XL-30 field-emission scanning electron microscope with an accelerating voltage of 20 kV. 2.7. Protein Release from the Nanofibers. The fibrous membrane (5 mg) was suspended in 1.0 mL of PBS (pH = 6.0 or 7.4) in 1.5 mL EP tubes (n = 3), which was then placed in a shaking bath at 37 °C and 40 rpm for 30 days. Then 0.2 mL of the supernatant was taken at predetermined intervals and replaced with fresh buffer accordingly. The protein concentrations were determined by a bicinchoninic acid (BCA) quantitative method. The morphology change of fibers under the release condition was inspected using SEM
at different time points. Cytotoxicity of proteins released from nanofibers was assessed by the MTT assay. 2.8. In Vivo Antitumor Activities of Recombinant Proteins. Female BALB/c nude mice, 6 weeks old, were obtained from HFK BIOSCIENCE Co., LTD (Beijing, China). Here, 1 ×106 ZR-75-30 cells were implanted subcutaneously (s.c.) into the right flank of each mouse. The volume of the tumor was calculated according to the following equation: tumor volume (mm3) = length × width2 × 0.5. In systemic administration treatment, mice were given daily intraperitoneal (i.p.) injections of recombinant proteins (0.1 nM/mice/ day) or PBS (100 μL/mice/day) for 7 days when tumor volume reached about 100 mm3. The growth of the tumor was tracked for 16 days in the treatment groups. For subcutaneous implantation treatment, mice (tumor volume ≈ 200 mm3) were also divided into four groups (n = 9 per group). The fibrous membrane weight was controlled as 10 mg (10 nM protein included) in each sample. The protein-loaded fibrous membrane was implanted according to procedures described previously.24 The tumor growth rate was calculated with the following formula: the tumor growth rate (%) = [volume of tumor from sacrificed mice (Vt)/initial volume of the tumor (V0)] × 100%. 2.9. Statistical Analysis. Statistical calculations were carried out by using the GraphPad Prism 5.0 statistical program.
3. RESULTS AND DISCUSSION 3.1. Design and Expression Analysis of Engineered Trimeric sTRAIL. Our prior studies showed that FA1FT with the fowl Ad1 full-length knobless fiber has a more stable trimer structure compared to HA5FT and HA5ST which contain the shortened shaft repeats (1 or 2) of human Ad5 fiber. However, FA1FT was also found to be concentrated in the liver, which C
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 2. Improved characteristics of engineered sTRAIL proteins in vitro and in vivo. Cytotoxicity of sTRAIL, S-sTRAIL and TS9-sTRAIL were evaluated by using the ZR-75-30 cells. Recombinant proteins were incubated at 37 °C in PBS (A), various pH values and at 4 °C in PBS (B) or 37 °C in plasma for the indicated times (C). The samples were collected and kept on ice until use in the MTT assay (mean ± SEM, n = 3). (D) Pharmacokinetic profiles of sTRAIL, S-sTRAIL, and TS9-sTRAIL. Mice were administered an i.v. injection of proteins (1 nM per mouse). The concentrations of recombinant proteins in blood were monitored by the ELISA. The results are displayed as the mean ± SD from three different mice (n = 3).
led to reduced antitumor activity and poor pharmacokinetic characteristics.16 Therefore, in order to obtain a highly stable and active trimeric sTRAIL with avoiding the hepatocyte tropism, two kinds of trimerization elements based on the shaft of human adenovirus 5 (Ad5) fiber were selected to engineer the sTRAIL first. Fusion sTRAIL (114−281) containing the N-terminal tail and nine shaft repeats (TS9-sTRAIL) or containing the C-terminal last shaft repeat (S-sTRAIL) were expressed in E. coli as described previously (Figures 1A and S1), separated, and characterized by SDS-PAGE, gradually denatured SDS-PAGE (GDS-PAGE), and circular dichroism (CD) spectroscopy (Figures 1B and S2). Analysis by GDSPAGE indicated that the preparation of TS9-sTRAIL contained more stable trimeric proteins than that of sTRAIL. 3.2. Biological Activities Analysis of Engineered sTRAIL. Since previous studies have shown that polyhistidine tags cause sTRAIL to form dimers which are cytotoxic to normal hepatocytes and keratinocytes,10 all the purified proteins were digested by thrombin (Figure S3), and zinc was added to the recombinant protein sample to increase the formation of homotrimers. And we also showed that these untagged proteins were all trimeric form as analyzed by size exclusion chromatography (Figure S4). Then the in vitro cytotoxicity of engineered sTRAIL proteins was evaluated on a human liver normal cell line QSG7701 and a human breast tumor cell line ZR-75-30. All cell lines were obtained from the
American Type Culture Collection. As a result, both of SsTRAIL and TS9-sTRAIL showed remarkable higher cytotoxicity on ZR-75-30 compared with sTRAIL in 1−100 nM but no cytotoxicity to normal cell QSG7701 even in 100 nM (Figure 1C). To further confirm the apoptotic activity of recombinant proteins, FITC-conjugated Annexin V was used to detect phosphatidylserine exposed to apoptotic cell membranes and to quantify apoptotic cells by flow cytometry. Results showed that 57.4% and 67.5% of the ZR-75-30 cells were apoptotic after being treated with S-sTRAIL and TS9sTRAIL (10 nM), respectively, compared to 41.7% upon exposure to sTRAIL at the same concentration (Figure 1D). These results indicated that the modified protein, particularly TS9-sTRAIL, has a more stable trimer structure and corresponding stronger specific tumor cell apoptosis activity. 3.3. Stability of the Engineered sTRAIL in Vitro and in Vivo. To evaluate the physicochemical properties of the recombinant sTRAIL proteins, their stability at 37 °C incubation and at different pH values was investigated. When the sTRAIL was kept in PBS for 24 h, sTRAIL readily lost its toxicity to ZR-75-30 cancer cells. In contrast, S-sTRAIL and TS9-sTRAIL retained nearly 80% of their activity (Figure 2A). No clear pH sensitivity effect was also observed for engineered sTRAIL proteins (Figure 2B). In order to mimic the physiological environment, the biological stability of recombinant proteins in rat plasma at 37 °C was studied. While D
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 3. Modulation of recombinant protein release from biodegradable nanofibers prepared by coaxial electrospinning. (A) SEM images of coaxially electrospun PLGA/sTRAIL nanofibers (PLGA as shell and recombinant proteins as core) prepared with the inner flow rates of 0.1, 0.2, 0.25, and 0.3 mL/h, respectively. The outer flow rate was set at 1.0 mL/h in all cases. The concentration of the protein solution was 5 or 10 mg/mL (TS9-sTRAIL). (B) Recombinant proteins release profiles of the fibrous membrane in 0.02 M, PBS (pH = 6.0 or 7.4) at 37 °C. (C) Cytotoxicity to ZR-75-30 cells of released proteins from nanofibers incubated in PBS for a period. For MTT analysis, the concentration of the released sTRAIL was 50 nM while that of S-sTRAIL and TS9-sTRAIL was 10 nM.
core were fabricated through coaxial electrospinning. We chose PLGA for encapsulating these proteins mainly based on its good biocompatibility, biodegradability, and release behavior.20 As shown in Figure 3A, the inner flow rates have a significant effect on the morphology of PLGA/sTRAIL nanofibers. When the inner flow rate was as low as 0.1 or 0.2 mL/h, smooth and well-defined PLGA/sTRAIL nanofibers with a diameter between 200 and 700 nm were obtained. However, with the inner flow rate being increased to 0.25 mL/ h, the electrospinning process became unstable. With a much higher inner flow rate (0.3 mL/h), no continuous and uniform nanofibers can be produced. In order to increase the loading amount of proteins in the composite nanofibers, we used 0.2 mL/h as the inner flow rate to prepare nanofibers for subsequent experiments. In this condition, about 1 nmol of recombinant proteins was encapsulated in 1 mg of PLGA nanofibers. The protein released from the nanofibers in 0.02 M, pH 7.4 or 6.0 phosphate buffer solution (PBS) at 37 °C was analyzed by BCA protein quantitative method. As shown in Figure 3B, about 10%−15% of the protein was released on the first day, followed by a relatively stable release of about 3 weeks. Results also suggested that all proteins were encapsulated in the nanofibers and nearly 90% of proteins could be released. Cytotoxicity of released recombinant proteins was analyzed by MTT at different time points. Results showed that the killing activity of S-sTRAIL and TS9-sTRAIL was not affected following a 14 day incubation period in fibers, while sTRAIL lost its partial activity even after 1 day incubation (Figure 3C). During the release study, the morphological changes of the nanofibers upon autocatalytic degradation in PBS (pH 7.4) and in vivo were also monitored by SEM, and a gradual
sTRAIL lost its cytotoxicity after 1 h incubation, S-sTRAIL and TS9-sTRAIL retained 80% and 50% cytotoxicity following a 12 h incubation period, respectively (Figure 2C). To assess the pharmacokinetic profile of S-sTRAIL and TS9-sTRAIL compared to sTRAIL, the serum samples were collected at different time points (5, 10, 30, 60, 120, and 240 min) after a single intravenous (i.v.) injection of 1 nM protein into nude mice, and then the protein concentrations were tested by ELISA. Unexpectedly, results showed that the concentration of TS9-sTRAIL in the serum was tremendously reduced only 5 min after administration. In comparison, the pharmacokinetic profile of S-sTRAIL was much plainer than that of sTRAIL or TS9-sTRAIL (Figure 2D). This promotion in half-life is very beneficial for its application considering that its half-life is only a few minutes reported earlier for recombinant sTRAIL. These results also demonstrated that genetic modification may have different effects on the in vitro stability and in vivo half-life of recombinant sTRAIL. 3.4. Encapsulation of Recombinant sTRAIL Proteins in PLGA Nanofibers. To further enhance the pharmaceutical characteristics of engineered sTRAIL proteins, we then designed them to combine electrospinning technology for the controlled release of bioactive recombinant proteins. Protein drugs are commonly admixed with organic solvents in the traditional fabrication process of a sustained release system, which denatures the proteins.23 The coaxial electrospinning method can reduce the contact between proteins and organic solvents, and it is expected to improve the bioactivity of proteins and become a new type drug release system.26 To investigate the feasibility of encapsulating engineered sTRAIL protein into the core−shell nanofibers for sustained release, the nanofibers with PLGA as a shell and recombinant proteins as a E
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 4. Antitumor activity of recombinant proteins in ZR-75-30 tumor-bearing mice. (A) Schematic diagram of the systemic administration experiment design. (B) Tumor growth was suppressed by recombinant proteins (0.1 nM per mouse, n = 10) after 7 day i.p. injection. (C) Representative mouse with implantation treatment was imaged in each group on day 10 after inoculation. Treatment was initiated when the tumors reached 200 mm3. (D) Changes of the tumor volume on the 1st, 7th, and 14th day after the fibrous membrane was implanted under the skin of tumor-bearing mice. (E) Tumor tissues were imaged in each group (n = 3) at the end of the experiment (day 14). Statistical analysis was performed by using the unpaired t test, two-tailed test, *P < 0.05, **P < 0.01.
group after 14 days (Figure 4E). These results indicated that, by implanting the engineered sTRAIL proteins loaded nanofibers on tumor tissue, the rapid growth of tumor cells can be effectively suppressed in less than 1 week. And this effect could be maintained even after 21 days (data not shown), with the continuous release of the protein.
degradation process was observed (Figure S5). These studies demonstrated that the coaxial electrospinning membrane could control the release of engineered recombinant sTRAIL proteins and help maintain its stability. 3.5. Antitumor Activity of Engineered sTRAIL and PLGA/sTRAIL Nanofibers in Vivo. In order to analyze the antitumor ability of the engineered sTRAIL proteins in vivo and evaluate the therapeutic potential of PLGA/sTRAIL nanofibers, both systemic administrations of recombinant proteins and implantation treatment of nanofibers were performed. The tumor transplant and treatment schedule of the systemic administration experiment showed in Figure 4A. The recombinant proteins were injected i.p. with a low concentration (0.1 nmol) of sTRAIL, S-sTRAIL or TS9sTRAIL daily for 7 days. As shown in Figure 4B, tumor growth was effectively inhibited during protein injection in both sTRAIL and S-sTRAIL treated mice. Especially in the SsTRAIL treated group, the tumor volume was significantly smaller than that of all other three groups on day 16. This is consistent with its good stability and pharmacokinetic characteristics. However, compared with the PBS group, TS9-sTRAIL treatment did not remarkably inhibit tumor growth. Considering its poor pharmacokinetic properties, we think that TS9-sTRAIL may also be concentrated in the liver; it remarkably reduced its antitumor activity in vivo, which is similar to one of our previous designs, FA1FT.16 Therefore, we then performed a subcutaneous implantation experiment with PLGA/sTRAIL nanofibers (Figure 4C). The inhibition on the growth of tumor was assessed on the 7th and 14th days separately. The volume of tumor increased by 6 and 14 times in the PBS group, while it increased by 2.5 and 8 times in the sTRAIL group on the 7th and 14th days, respectively. More importantly, in the S-sTRAIL and TS9-sTRAIL groups, the tumor volume decreased by 20−60% at these time points (Figure 4D). The pictures also clearly revealed an obvious decline in tumor volume in the S-sTRAIL and TS9-sTRAIL
4. CONCLUSIONS In this study, a novel strategy that could improve the stability, antitumor activity, and pharmaceutical characteristics of sTRAIL was presented by combining genetic engineering with coaxial electrospinning. We demonstrated that engineered sTRAIL encapsulated in nanofibers for implantation is a conceptually viable therapeutic strategy with reduced dosing frequency and hepatotoxicity and improved antitumor activity. As sTRAIL has been shown to enhance the effects of other therapies such as chemical drugs, it is believed that the PLGA/ sTRAIL nanofibers will further improve its antitumor activity by the encapsulated toxic small molecule in the shell or core of a nanofiber. Additionally, our strategy could be a potential approach for some other unstable bioactive recombinant protein drugs.
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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/acsabm.9b00119. DNA sequences of sTRAIL (114−281) and the trimerization domain used; circular dichroic spectroscopy of sTRAIL, S-sTRAIL, and TS9-sTRAIL; thrombin cleavage site of purified sTRAIL proteins and identification of sTRAIL, S-sTRAIL, and TS9-sTRAIL after removing His tag by Western blotting; size exclusion chromatography of sTRAIL, S-sTRAIL, and TS9sTRAIL; morphology of nanofibers incubated in release F
DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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(14) Jiang, T. Y.; Mo, R.; Bellotti, A.; et al. Gel-liposome-mediated co-delivery of anticancer membrane-associated proteins and smallmolecule drugs for enhanced therapeutic efficacy. Adv. Funct. Mater. 2014, 24, 2295−2304. (15) Pan, L. Q.; Wang, H. B.; Lai, J.; et al. Site-specific PEGylation of a mutated-cysteine residue and its effect on tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Biomaterials 2013, 34, 9115−9123. (16) Yan, J.; Wang, L.; Wang, Z.; et al. Engineered adenovirus fiber shaft fusion homotrimer of soluble TRAIL with enhanced stability and antitumor activity. Cell Death Dis. 2016, 7, No. e2274. (17) Chou, S. F.; Carson, D.; Woodrow, K. A. Current strategies for sustaining drug release from electrospun nanofibers. J. Controlled Release 2015, 220, 584−591. (18) Hong, Y. L.; Chen, X. S.; Jing, X. B.; et al. Preparation, bioactivity, and drug release of hierarchical nanoporous bioactive glass ultrathin fibers. Adv. Mater. 2010, 22, 754−758. (19) Hong, Y. L.; Chen, X. S.; Jing, X. B.; et al. Fabrication and drug delivery of ultrathin mesoporous bioactive glass hollow fibers. Adv. Funct. Mater. 2010, 20, 1503−1510. (20) Chen, M. L.; Gao, S.; Dong, M. D.; et al. Chitosan/siRNA nanoparticles encapsulated in PLGA nanofibers for siRNA delivery. ACS Nano 2012, 6, 4835−4844. (21) Miao, Y.; Zhu, H.; Chen, D.; et al. Electrospun fibers of layered double hydroxide/biopolymer nanocomposites as effective drug delivery systems. Mater. Chem. Phys. 2012, 134, 623−630. (22) Zamani, M.; Prabhakaran, M. P.; Ramakrishna, S. Advances in drug delivery via electrospun and electrosprayed nanomaterials. Int. J. Nanomed. 2013, 8, 2997−3017. (23) Hu, X.; Liu, S.; Zhou, G.; et al. Electrospinning of polymeric nanofibers for drug delivery applications. J. Controlled Release 2014, 185, 12−21. (24) Liu, D.; Liu, S.; Jing, X.; et al. Necrosis of cervical carcinoma by dichloroacetate released from electrospun polylactide mats. Biomaterials 2012, 33, 4362−4369. (25) Yu, B.; Wang, C.; Dong, J. N.; et al. Chimeric hexon HVRs protein reflects partial function of adenovirus. Biochem. Biophys. Res. Commun. 2012, 421, 170−176. (26) Jiang, H.; Hu, Y.; Li, Y.; et al. A facile technique to prepare biodegradable coaxial electrospun nanofibers for controlled release of bioactive agents. J. Controlled Release 2005, 108, 237−243.
buffer (PBS) and under the skin of mice (in vivo) (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (X.H.Y). *E-mail:
[email protected] (T.Y.Y). ORCID
Renjun Pei: 0000-0002-9353-3935 Tianyan You: 0000-0003-1579-7078 Author Contributions ⊥
B.Y. and X.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by funding from the National Natural Science Foundation of China (No. 21675065), the Science & Technology Development Plan of Jilin Province (No. 20140520007JH), and the Project funded by China Postdoctoral Science Foundation (No. 2014T70301). Animal experiments were approved by and performed in strict accordance with the guidelines of the Animal Experiment Committee of Jilin University.
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DOI: 10.1021/acsabm.9b00119 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
