Antheraea Pernyi Silk Fibroin Research Articles

Targeting Antheraea pernyi silk fibroin modified dual-gene coexpressing vector enhances gene transport and promotes lung tumor suppression

Poor systemic administration capability, a natural tendency to target CAR-positive cells, nonspecific shedding to normal organs, and poor viral persistence in tumor tissues are major hindrances to the therapeutic benefit of adenovirus (Ad) gene vectors in the clinical setting. Antheraea pernyi silk fibroin (ASF) grafted with targeted peptides was used to coat ING4-IL-24 dual-gene coexpressing adenovirus for targeted gene therapy of lung carcinoma. The dual-gene vector with a diameter of 390 nm could target and infect H460 lung tumor cells, internalize into cells, express the ING4 and IL-24 genes at a high level, effectively inhibit the proliferation of lung tumor cells, and induce their apoptosis. The in vivo treatment of H460 human lung carcinoma xenograft tumors showed that the dual-gene coexpressing vector suppressed the proliferation of lung tumor cells by downregulating the expression of Ki67 and Bcl-2, promoted apoptosis by upregulating the expression of C Caspase-3 and Bax, and blocked tumor angiogenesis by downregulating the expression of VEGF and CD31, thus exerting a multichannel tumor inhibition effect. Surface modification of Ad with targeted cationic silk fibroin is an effective way to solve the natural tendencies and in vivo instability of adenovirus vectors, and such vectors have potential for clinical application.

Silk Fibroin-Modified Liposome/Gene Editing System Knocks out the PLK1 Gene to Suppress the Growth of Lung Cancer Cells.

Polo-like protein kinase 1 (PLK1) plays a key role in lung cancer cell mitosis. The knockout of PLK1 gene by the CRISPR-Cas9 system can effectively inhibit the proliferation of tumor cells, but there is no suitable vector for in vivo delivery. In this study, CRISPR-Cas9 gene knockout plasmids encoding sgRNA, Cas9 and green fluorescent protein were constructed. Then, the plasmids were packaged with liposome (Lip) and cholesterol-modified Antheraea pernyi silk fibroin (CASF) to obtain the CASF/Lip/pDNA ternary complex. The CASF/Lip/pDNA complex was transfected into lung cancer cells A549 to investigate the transfection efficiency, the PLK1 gene knockout effect and the inhibitory effect on lung cancer cells. The results showed that the transfection efficiency of the CASF/Lip/pDNA complex was significantly higher than that of the Lip/pDNA binary complex, and the expression of PLK1 in cells transfected with CASF/Lip/pDNA complexes was significantly lower than that in cells transfected with Lip/pDNA complexes. The CASF/Lip/pDNA complex significantly increased the apoptosis rate and decreased the proliferation activity of lung cancer cells compared with Lip/pDNA complexes. The cytotoxicity of the complexes was evaluated by coculture with the human bronchial epithelial cells BEAS2B. The results showed that CASF/Lip/pDNA complexes exhibited lower cytotoxicity than Lip/pDNA complexes. The fibroin-modified liposome/PLK1 gene knockout system not only effectively inhibited the growth of lung cancer cells but also showed no obvious toxicity to normal cells, showing potential for clinical application in lung cancer therapy.

Self-assembled mesoporous particles of Antheraea pernyi silk fibroin for encapsulation and sustained release of 5-fluorouracil

Mesoporous particles have attracted considerable attention in drug delivery system, because they can offer fast mass transport and overcome the diffusion restriction as occurred in micro/nano- porous particles. Conventional mesoporous particles are generally dominated by inorganic compounds, little concern has been raised over biodegradable organic polymers, especially silk fibroin. Here, we show that Antheraea pernyi (A. pernyi) silk fibroin, rich in Arg-Gly-Asp (RGD) moiety, can be developed into mesoporous particles via self-assembly in a direct and eco-friendly (without any organic solvents) manner for sustained release of 5-fluorouracil (5-FU). By cultivating a mild condition at 40 ℃, a mesoporous structure was successfully achieved without any foreign organic solvents. The self-assembled mesoporous particles were quasi-spherical in shape with an average diameter of 1.58 µm. Mesopores ran through the entire particles to construct a divergent structure with external pore diameters ranging from 30 to 60 nm and internal pore width of 6.225 nm. The mesoporous particles were predominated by β-sheet conformation (∼45%), which effectively reduced the weight-loss ratio to 2.6%. 5-FU was encapsulated into the mesoporous particles by hydrogen bonding and van der Waals force as well as ionic interactions. Drug loading efficiency and encapsulation efficiency were found to be 26.24% and 50.36%, respectively. The mesoporous particles enabled sustained drug release, with approximately 90% release after 110 h. After a small burst release (20%) of the surface-bound 5-FU, a slower linear release dominated by Fickian-diffusion occurred. These features together with the excellent biocompatibility of A. pernyi silk indicated that the self-assembled mesoporous particles would be an ideal carrier for sustained drug release to meet critical requirements of biomaterials. Furthermore, without any foreign materials, this one-step strategy may offer new green chemistry approaches as well as options to load bioactive drugs.

Antheraea pernyi silk fibroin bioinks for digital light processing 3D printing.

The application of three-dimensional (3D) bioprinting has increased in the biomedical field. The lack of bioinks with both biocompatibility and printability is still a problem to be solved. Silk fibroin materials have good biocompatibility and have a broad application prospect in the field of biomedical materials. At present, most research usually involves Bombyx mori silk fibroin (BSF). However, BSF has low cell adhesion. Compared with BSF, Antheraea pernyi silk fibroin (ASF) isolated from typical non-mulberry silk exhibits a unique arginine-glycine-aspartate (RGD) sequence with good cell adhesion enhancement. In this study, we developed a bioink based on ASF for digital light processing (DLP) 3D bioprinting. The ASF-based bioinks (ASF-MA) were produced by a methacryloylation process using methacrylic anhydride (MA) to achieve the properties of photopolymerization reaction. The ASF-MA hydrogel has mechanical properties, biocompatibility, and especially cell adhesion. Meanwhile, we found that the ASF-MA hydrogels promoted the adhesion, migration, and proliferation of S16 cells. Hence, the ASF-MA hydrogels had the potential applications in biomedical fields.

Feasibility and Safety of Regenerated Wild Antheraea pernyi Silk Fibroin/Polyvinyl Alcohol Scaffold in Repair of Calcaneal Tendon Defects

This research discussed the value of regenerated wild Antheraea pernyi silk fibroin (RWSF)/polyvinyl alcohol (PVA) nanofiber scaffold (NFS) in repairing the calcaneal tendon defect (CTD). RWSF was prepared by saturated salt solution (SSS) method, and then RWSF/PVA NFS was prepared by electrospinning using RWSF and PVA as raw materials. Fourier transform infrared spectroscopy (FTIS) was applied to detect the characteristic absorption spectra of WSF, RWSF, and RWSF/PVA. The ultimate tensile strength (UTS) and elongation at break (BE) of RWSF/PVA NFS were analyzed by mechanical tester. The cytotoxicity of RWSF/PVA NFS was determined by MTT assay. 18 SD rats were randomly rolled into an operation group, control group, and experimental group, with 6 rats in each. Meanwhile, 27 rats were randomly grouped into three: blank group, model group, and experimental group. HE staining, Masson staining, and biomechanical properties of the regenerated fibers were analyzed in the calcaneal tendon tissues (CTTs) of rats in different groups. Expressions of tendon-related genes and inflammatory factors in CTTs in various groups were compared by RT-PCR. The results revealed that the UTS and BE of PVA and RWSF/PVA were much higher than those of natural acellular tendon (P <0.01). On day 15 after operation, the hair in the incision area of rats in the Ope, Con, and Exp groups grew normally. The implanted RWSF/PVA NFS in the Exp group adhered closely to the surrounding muscle tissue and degraded gradually, and there were still trace inflammatory cells at the junction. The tendon cross sectional area (CSA) in the Model group and RWSF/PVA group was greatly higher based on that in the Blank group (P <0.05), and the UTS in of Model group was much higher than that in the Blank group but lower to the Model group, showing great differences with P <0.05. The Collagen I, Collagen III, TGF-β1, BGN, and TNMD in CTTs in the RWSF/PVA group were higher to the Model group 2 months ago (P <0.05); while Collagen I, TGF-β1, BGN, and TNMD were still much higher 3 months later (P <0.01) but Collagen III was lower with an obvious difference (P <0.05). At 5 months, IL-1β and TNF-α in the RWSF/PVA group were greatly lower in contrast to the model group, presenting extremely obvious differences (P <0.001). The results indicated that the RWSF/PVA NFS exhibited a good biocompatibility, can accelerate the collagen secretion, promote TGF-β1, inhibit IL-1β and TNF-α factors, thus being conductive to repair of CTD. In conclusion, RWSF/PVA NFS possessed a good biocompatibility, can promote collagen secretion, elevate the TGF-β1, and inhibit IL-1β and TNF-α factors to participate in calcaneal CTD repair, showing a high value in repair of CTD.

Effect of Degumming Methods on Structure and Properties of Regenerated Antheraea Pernyi Silk Fibroin Films

ABSTRACT The extraction of fibroin (degumming) from the silk fibers impacts the properties of silk fibroin. However, the effect of the degumming method on structures and properties of regenerated Antheraea pernyi silk fibroin (ASF) biomaterials has not been investigated in detail. In this study, Na2CO3 and NaHCO3 are selected to remove sericin of Antheraea pernyi silks. The results show that the silk fibroin prepared by NaHCO3 degumming possessed a higher molecular weight, demonstrated by SDS-PAGE and viscosity testing. Na2CO3 degumming produced more low molecular weight silk fibroin protein below 40 kDa, whereas NaHCO3 processing produced more silk fibroin protein above 70 kDa. The increased molecular weight significantly improved the mechanical properties of ASF film. The enzyme degradation results indicated that the degradation rate of ASF film prepared by Na2CO3 degumming was faster in the early stage due to the presence of low molecular weight silk fibroin. After 21 days, the two silk fibroin films showed approximate degradation ratios. These results showed that the degumming conditions have a significant effect on the structure and properties of ASF biomaterials, and provide options for regulating the mechanical properties and degradation rate of ASF biomaterials.

Near-Infrared-Enpowered Nanomotor-Mediated Targeted Chemotherapy and Mitochondrial Phototherapy to Boost Systematic Antitumor Immunity.

Phototherapy is an important strategy to inhibit tumor growth and activate antitumor immunity. However, the effect of photothermal/photodynamic therapy (PTT/PDT) is restricted by limited tumor penetration depth and unsatisfactory potentiation of antitumor immunity. Here, a near-infrared (NIR)-driven nanomotor is constructed with a mesoporous silicon nanoparticle (MSN) as the core, end-capped with Antheraea pernyi silk fibroin (ApSF) comprising arginine-glycine-aspartate (RGD) tripeptides. Upon NIR irradiation, the resulting ApSF-coated MSNs (DIMs) loading with photosensitizers (ICG derivatives, IDs) and chemotherapeutic drugs (doxorubicin, Dox) can efficiently penetrate into the internal tumor tissues and achieve effective phototherapy. Combined with chemotherapy, a triple-modal treatment (PTT, PDT, and chemotherapy) approach is developed to induce the immunogenic cell death of tumor cells and to accelerate the release of damage-associated molecular patterns. In vivo results suggest that DIMs can promote the maturation of dendritic cells and surge the number of infiltrated immune cells. Meanwhile, DIMs can polarize macrophages from M2 to M1 phenotypes and reduce the percentages of immunosuppressive Tregs, which reverse the immunosuppressive tumor microenvironment and activate systemic antitumor immunity. By achieving synergistic effects on the tumor inhibition and the antitumor immunity activation, DIMs show great promise as new nanoplatforms to treat metastatic breast cancer.

Antheraea pernyi silk fibroin-coated adenovirus as a VEGF165-Ang-1 dual gene delivery vector

Vascularization is a key challenge in the regeneration of tissues containing blood vessels. In this study, spermine was used for cationic modification of Antheraea pernyi silk fibroin (ASF) to synthesize cationized ASF (CASF). CASF/Ad complexes prepared by coating adenovirus (Ad) with CASF were used as delivery vectors for vascular endothelial growth factor 165 and angiopoietin-1 dual genes. The results showed that the zeta potential of the Ad was reversed from −7.75 mV to approximately +8.40 mV after CASF coating, and the sizes of the CASF/Ad complexes were 200 to 290 nm. Furthermore, human umbilical vein endothelial cells HUVECs were cocultured and infected with CASF/Ad in vitro. The results of confocal laser scanning microscopy, flow cytometry and CCK-8 assay showed that coating Ad with CASF at concentration of 20 and 50 µg/mL not only reduced the cytotoxicity of naked Ad, but also significantly promoted cell proliferation. Therefore, the CASF/Ad complexes could be beneficial to reduce the dosage of Ad and the potential toxicity risk of high doses of Ad in vivo, which has the potential of application to promote vascular network regeneration.

3D Printed Gelatin Scaffold with Improved Shape Fidelity and Cytocompatibility by Using Antheraea pernyi Silk Fibroin Nanofibers

3D Printed Gelatin Scaffold with Improved Shape Fidelity and Cytocompatibility by Using Antheraea pernyi Silk Fibroin Nanofibers

Oral nanotherapeutics based on Antheraea pernyi silk fibroin for synergistic treatment of ulcerative colitis

Ulcerative colitis (UC) with its rapidly increasing incidence has become an emerging challenge for public health. Therapeutic agents are required to be specifically delivered to colon epithelial cells and macrophages with controlled release in the cytoplasm for wound healing, inflammation alleviation, and microbiota rebalance. As a promising biomaterial for accomplishing this, Antheraea pernyi silk fibroin (ApSF) was selected and engineered to form nanoparticles (NPs) loaded with the anti-inflammatory drug, resveratrol (Res). The intrinsic features of these fabricated Res-ApNPs included targeting of colonic epithelial cells and macrophages, lysosomal escape capacity, and responsiveness to pH, reactive oxygen species (ROS), and glutathione, which were pertinent to their functional units such as arginine-glycine-aspartate tripeptides, α-helixes, β-sheets, and disulfide bonds, enabling on-demand release of Res molecules in the cytoplasm of target cells. The Res-ApNP treatment restored damaged colonic epithelial barriers, polarized macrophages to type M2, alleviated inflammatory reactions, and reduced the level of intracellular ROS. Oral treatment with chitosan-alginate hydrogels embedded with Res-ApNPs substantially relieved UC symptoms, as evidenced by decreased colonic inflammation, increased synthesis of tight-junction proteins, and rebalanced intestinal microbiota. Our findings suggest that these high-performance ApSF-based NPs can be developed as effective drug carriers for oral UC treatment.

Fabrication of Antheraea pernyi Silk Fibroin-Based Thermoresponsive Hydrogel Nanofibers for Colon Cancer Cell Culture.

Antheraea pernyi silk fibroin (ASF)-based nanofibers have wide potential for biomaterial applications due to superior biocompatibility. It is not clear whether the ASF-based nanofibers scaffold can be used as an in vitro cancer cell culture platform. In the current study, we fabricated novel ASF-based thermoresponsive hydrogel nanofibers by aqueous electrospinning for colon cancer (LoVo) cells culture. ASF was reacted with allyl glycidyl ether (AGE) for the preparation of allyl silk fibroin (ASF-AGE), which provided the possibility of copolymerization with allyl monomer. The investigation of ASF-AGE structure by 1H NMR revealed that reactive allyl groups were successfully linked with ASF. ASF-based thermoresponsive hydrogel nanofibers (p (ASF-AGE-NIPAAm)) were successfully manufactured by aqueous electrospinning with the polymerization of ASF and N-isopropylacrylamide (NIPAAm). The p (ASF-AGE-NIPAAm) spinning solution showed good spinnability with the increase of polymerization time, and uniform nanofibers were formed at the polymerization time of 360 min. The obtained hydrogel nanofibers exhibited good thermoresponsive that the LCST was similar with PNIPAAm at about 32 °C, and good degradability in protease XIV PBS solution. In addition, the cytocompatibility of colon cancer (LoVo) cells cultured in hydrogel nanofibers was assessed. It was demonstrated that LoVo cells grown on hydrogel nanofibers showed improved cell adhesion, proliferation, and viability than those on hydrogel. The results suggest that the p (ASF-AGE-NIPAAm) hydrogel nanofibers have potential application in LoVo cells culture in vitro. This study demonstrates the feasibility of fabricating ASF-based nanofibers to culture LoVo cancer cells that can potentially be used as an in vitro cancer cell culture platform.

Cleavable poly(ethylene glycol) branched chain-modified Antheraea pernyi silk fibroin as a gene delivery carrier.

Aim: To obtain a gene carrier that can effectively deliver loaded therapeutic genes to tumor cells, avoid toxic effects on normal cells and reduce nonspecific adsorption of plasma proteins. Methods:The conjugate of poly(ethylene glycol) (PEG) andMMP2SSP (PEG-MMP2SSP) was covalently coupled to cationized Antheraea pernyi silk fibroin (CASF) through disulfide bond exchange reaction to obtain a PEG-MMP2SSP-modified CASF (CASFMP). Results: The PEG chains were effectively cleaved from the CASFMP by MMP2. CASFMP/pDNA complexes inhibited human fibrosarcoma cell proliferation, and its cytotoxicity to human normal embryonic kidney cells was significantly lower than that of poly(ethylenimine)/pDNA after coculturing with cells for 24h. Conclusion: CASFMP is a promising compound for use in gene therapy.

Comparison of Native and Regenerated Antheraea Pernyi Silk Fibroin

The regenerated Antheraea pernyi silk fibroin (RASF) was harvested by dissolving the silk fibers in a calcium nitrate solution. XRD result demonstrated that both native and regenerated Antheraea pernyi silk fibroin involved the α-helix conformation, and DTG curves showed that their thermal decompositions were quite similar and proceeded three steps. However, rheological measurements figured out that the molecule weight of RASF decreased from 246 kDa to 199 kDa, comparing with native Antheraea pernyi silk fibroin. Also, the tensile properties of the RASF film, which were the same to those of regenerated Bombyx mori silk fibroin film, were observed.

Degradable allyl Antheraea pernyi silk fibroin thermoresponsive hydrogels to support cell adhesion and growth.

At present, Antheraea pernyi silk fibroin (ASF) based hydrogels have wide potential applications as biomaterials because of their superior cytocompatibility. Herein, ASF is used as a nucleophilic reagent, reacted with allyl glycidyl ether (AGE) for the preparation of allyl silk fibroin (ASF-AGE). The investigation of ASF-AGE structure by 1H NMR and FTIR are revealed that reactive allyl groups were obtained on ASF by nucleophilic substitution. A series of ASF based hydrogels are manufactured by N-isopropylacrylamide (NIPAAm) copolymerization bridged with ASF-AGE. By the silk fibroin self-assembly process, stably physical cross-linked hydrogels are formed without any crosslinking agent. These hydrogels exhibit good thermoresponsive and degradability, for which the LCST was about 32 °C, and these hydrogels can be degraded in protease XIV solution. Excellent cell proliferation, viability and morphology is demonstrated for b End.3 cells on the hydrogels by the characteristic MTT assay, CLSM and SEM. The cytocompatibility of b End.3 cells was demonstrated with excellent cell adhesion and growth on these ASF based hydrogels in vitro. These degradable and thermoresponsive ASF based hydrogels may find potential applications for cells delivery devices and tissue engineering.

Biomimetic and hierarchical nerve conduits from multifunctional nanofibers for guided peripheral nerve regeneration

Development of a functional nerve conduit to replace autografts remains a significant challenge particularly considering the compositional complexity and structural hierarchy of native peripheral nerves. In the present study, a multiscale strategy was adopted to fabricate 3D biomimetic nerve conduit from Antheraea pernyi silk fibroin (ApF)/(Poly(L-lactic acid-co-caprolactone)) (PLCL)/graphene oxide (GO) (ApF/PLCL/GO) nanofibers via nanofiber dispersion, template-molding, freeze-drying and crosslinking. The resultant conduits exhibit parallel multichannels (ϕ = 125 µm) surrounded by biomimetic fibrous fragments with tailored degradation rate and improved mechanical properties in comparison with the scaffold without GO. In vitro studies showed that such 3D biomimetic nerve scaffolds had the ability to offer an effective guiding interface for neuronal cell growth. Furthermore, these conduits showed a similarity to autografts in vivo repairing sciatic nerve defects based on a series of analysis (walking track, triceps weight, morphogenesis, vascularization, axonal regrowth and myelination). The conduits almost completely degraded within 12 weeks. These findings demonstrate that the 3D hierarchical nerve guidance conduit (NGC) with fascicle-like structure have great potential for peripheral nerve repair.

Silk as templates for hydroxyapatite biomineralization: A comparative study of Bombyx mori and Antheraea pernyi silkworm silks

Silk is extensively investigated in bone tissue engineering due to its extraordinary mechanical properties and ability to regulate biomineralization. Protein templates regulate biomineralization process through chemical interaction with ions. However, the effect of structural differences in silk fibroin on biomineralization has not been studied in detail. In this study, Antheraea pernyi silk fibroin (ASF) and Bombyx mori silk fibroin (BSF) fibers were used as templates to study the effect of silk species on biomineralization. The results showed that silk fibroin could induce the formation of calcium-deficient hydroxyapatite in simulated body fluid (SBF), and the SBF treatment resulted in the formation of silk I crystals. Compared with BSF, ASF exhibited a higher ability to induce mineralization, which may depend on the differences in hydrophilic amorphous fractions between ASF and BSF. The amorphous fractions of ASF contain more acidic amino acids, which can provide more nucleation sites in the initial stage of mineralization, resulting in faster mineralization process and more mineral deposits. This study decodes the key role of silk fibroin fractions on biomineralization, and provides deeper insights for the study of silk fibroin as biomineralization template and bone repair materials.

Cationic Antheraea pernyi Silk Fibroin-Modified Adenovirus-Mediated ING4 and IL-24 Dual Gene Coexpression Vector Suppresses the Growth of Hepatoma Carcinoma Cells.

IntroductionCancer gene therapy requires both effective tumor suppressor genes and safe vectors that express target genes efficiently. Inhibitor of growth 4 (ING4) inhibits tumor growth via multiple pathways. Interleukin-24 (IL-24) also has tumor-suppressive activity against a broad spectrum of human cancers. Adenovirus (Ad) vectors exhibit high infection efficiency, but potential toxicity related to high doses of adenovirus has led to careful reconsideration of their use in human clinical trials. Antheraea pernyi silk fibroin (ASF) is a cytocompatible and biodegradable natural polymer, and it possesses Arg–Gly–Asp sequences exhibiting a high binding affinity and selectivity for αvβ3 and αvβ5 integrin receptors, which are overexpressed in tumor vessels and most tumor cells.MethodsIn this study, an Arg-Gly-Asp peptide-modified Ad vector coexpressing ING4 and IL-24 was constructed by homologous recombination of the dual gene coexpression transfer plasmid and RGD-modified pAdEasy-1 adenoviral backbone plasmid. The cationic ASF (CASF) was prepared by modifying ASF with low-molecular-weight PEI. The negatively charged Ad vector was modified with CASF to form a CASF/Ad complex.ResultsHuman hepatoma carcinoma SMMC-7721 cells and normal hepatic L-02 cells were infected with the CASF/Ad complex, which showed significantly higher infection efficiency than the naked Ad. The CASF/Ad complex could effectively mediate the expression of the target gene ING4 in SMMC-7721 cells and the secretion of the target gene IL-24 from SMMC-7721 cells, thus inducing apoptosis of hepatoma carcinoma SMMC-7721 cells. The viability of SMMC-7721 and L-02 cells infected with the CASF/Ad complex was further assessed, and it was found that the growth of SMMC-7721 cells was significantly inhibited but that the growth and proliferation of L-02 cells were not affected.ConclusionThe CASF/Ad complex constructed in this study, showing improved infection efficiency and enhanced suppressive effects on human hepatoma carcinoma SMMC-7721 cells, has the potential to reduce the dose of adenovirus and still maintain high infection efficiency and tumor inhibition.

Hepatoma Cell-Targeted Cationized Silk Fibroin as a Carrier for the Inhibitor of Growth 4-Interleukin-24 Double Gene Plasmid.

The construction of a targeted gene delivery system with low cytotoxicity to normal tissues is an urgent need for the clinical treatment of liver cancer. In this study, Antheraea pernyi silk fibroin (ASF) was cationized with low-molecular-weight polyethylenimine (PEI, 1.8 kDa) to synthesize a cationized Antheraea pernyi silk fibroin (CASF). The highly cancer-selective hepatoma targeted peptide, HCBP1 (sequence FQHPSFI), was coupled to the side chains of CASF to synthesize a hepatoma-targeted CASF (CASFP). CASFP relied on the positive charges of CASF could package the pDNA encoded the inhibitor of growth 4 (ING4) and interleukin-24 (IL-24) to form CASFP/pDNA complexes. The results showed that the zeta potential of ASF was reversed from -9.08 ± 0.20 to +11.33 ± 0.38 mV, and its isoelectric point significantly increased from 4.31 to 9.38 after PEI modification. The Fourier transform infrared spectroscopy results and the 1Hydrogen-nuclear magnetic resonance spectra demonstrated that HCBP1 could be coupled to the side chains of CASF under the action of the bifunctional reagent N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). In vitro, human hepatocellular carcinoma HepG2 cells and human normal hepatic L-02 cells were transfected with the CASFP/pDNA complexes. The results of confocal laser scanning microscope analysis and cell viability assays showed that the complexes were able to transfect HepG2 cells and effectively inhibit their proliferation but had no obvious cytotoxicity to L-02 cells. In this study, a new gene delivery system, constructed by using HCBP1-modified CASF and the ING4-IL-24 dual-gene co-expression plasmid, was able to inhibit the proliferation of hepatocellular carcinoma cells but had no obvious cytotoxicity to normal hepatic cells. Therefore, the gene delivery system has the potential for application as a gene therapy in liver cancer.

Fabrication and characterization of regenerated Antheraea pernyi silk fibroin scaffolds for Schwann cell culturing

Antheraea pernyi silk, which is from a kind of non-mulberry silkworm produced in China, has better mechanical properties and cytocompatibility than the extensively studied silk from mulberry silkworm (Bombyx mori). In this work, the regenerated Antheraea pernyi silk fibroin (RASF) scaffolds were prepared from RASF aqueous solutions with different concentrations by a freeze-drying method. The morphologies, structures and properties of the obtained RASF scaffolds were investigated. It was found that with the decrease of RASF solution concentration and the increase of freezing temperature, the porosity of the scaffold increased while its apparent density decreased. The micro-morphology of the scaffold from the 1 wt% RASF solution was irregular. When the solution concentration was increased to the range of 4–13 wt%, the micro-morphologies of the obtained scaffolds mainly showed lamellar-like structure. And with the increase of solution concentration, the lamellar gap of the scaffold decreased while the lamellar thickness of the scaffold increased. However, with the further increase of solution concentration, the morphology of scaffold could gradually transfer into porous structure. In addition, the scaffold micro-morphology could be also affected by freezing temperature. Moreover, with the increase of RASF solution concentration or (and) the decrease of freezing temperature, the obtained scaffold had higher content of β-sheet conformation and higher crystallinity, which could improve its mechanical properties. It was further found that the swelling ratio and degradation rate of RASF scaffold prepared for tissue engineering increased with the decrease of solution concentration. The results of MTT indicated that the obtained RASF scaffold had no cytotoxicity. Moreover, the results of field emission scanning electron microscopy and laser confocal scanning microscopy revealed that the viability, cytocompatibility and migration ability of Schwann cells cultured on RASF scaffold were much better than those cultured on regenerated Bombyx mori silk fibroin (RBSF) scaffold. The RASF scaffold prepared in this work may have great potential applications in the area of nerve tissue engineering.

Regenerated Antheraea pernyi Silk Fibroin/Poly(N-isopropylacrylamide) Thermosensitive Composite Hydrogel with Improved Mechanical Strength.

At present, Antheraea pernyi silk fibroin (ASF) has attracted research efforts to investigate it as a raw material for fabrication of biomedical devices because of its superior cytocompatibility. Nevertheless, native ASF is not easily processed into a hydrogel without any crosslinking agent, and a single hydrogel shows poor mechanical properties. In this paper, a series of ASF/poly (N-isopropylacrylamide) (PNIPAAm) composite hydrogels with different ASF contents were manufactured by a simple in situ polymerization method without any crosslinking agent. Meanwhile, the structures, morphologies and thermal properties of composite hydrogels were investigated by XRD, FTIR, SEM, DSC and TGA, respectively. The results indicate that the secondary structure of silk in the composite hydrogel can be controlled by changing the ASF content and the thermal stability of composite hydrogels is enhanced with an increase in crystalline structure. The composite hydrogels showed similar lower critical solution temperatures (LCST) at about 32 °C, which matched well with the LCST of PNIPAAm. Finally, the obtained thermosensitive composite hydrogels exhibited enhanced mechanical properties, which can be tuned by varying the content of ASF. This strategy to prepare an ASF-based responsive composite hydrogel with enhanced mechanical properties represents a valuable route for developing the fields of ASF, and, furthermore, their attractive applications can meet the needs of different biomaterial fields.