Mild preparation of hyaluronic acid/silk fibroin sponges by modified crosslinking method.

The composites composed of hyaluronic acid (HA) and silk fibroin (SF) exhibit great potential in diverse biomedical applications. However, the utilization of commercial crosslinkers such as 1,4-butanediol diglycidyl ether (BDDE) for crosslinking HA typically necessitates harsh conditions involving strong alkaline, which greatly limits its potential applications. In this study, a mild modified approach was developed to fabricate HA/SF blend sponges crosslinked by BDDE without alkaline conditions. The blend solutions were cryo-concentrated to induce crosslinking reactions. The mechanism of freezing crosslinking was elucidated by investigating the effects of ice crystal growth and HA molecular weight on the degree of crosslinking. The results revealed that HA achieved efficient crosslinking when its molecular weight exceeds 1000 kDa and freezing temperatures ranged from -40 °C to -20 °C. After introducing SF, multiple crosslinks were formed between SF and HA chains, producing water-stable porous sponges. The SEM results demonstrated that the introduction of SF effectively enhanced the interconnectivity between macropores through creating subordinate holes onto the pores wall. Raising the SF content significantly enhanced compression strength, resistance to enzymatic degradation and cell viability of blend sponges. This study provides a novel strategy for designing bioactive HA/SF blend sponges as substitutes for tissue repair and wound dressing.

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) and MMP2SSP (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 24 h. Conclusion: CASFMP is a promising compound for use in gene therapy.

A novel dual-function fluorescent probe for the rapid detection of bisulfite and hydrogen peroxide in aqueous solution and living cells.

In this work, Hcy-OB, a novel hemicyanine-based biocompatible dual-function fluorescence probe for bisulfite and H2O2 detection is designed and synthesized. Based on a 1,4-addition reaction, Hcy-OB can be used for bisulfite detection with fast response, high sensitivity and low detection limit (120 nM). In addition, the probe is successfully applied to the detection of bisulfite in aqueous solution. Furthermore, Hcy-OB shows excellent performance for hydrogen peroxide detection with the oxidation of phenylboronic acid. Hcy-OB shows excellent selectivity to H2O2 over other interfering substances with detection limit of H2O2 is calculated to be 70 nM. Most importantly, due to its good cell membrane permeability and low cytotoxicity, Hcy-OB has been applied to monitor and image H2O2 in living cells and mice.

Polyethylenimine-Modified Bombyx mori Silk Fibroin as a Delivery Carrier of the ING4-IL-24 Coexpression Plasmid.

One of the major challenges for lung cancer gene therapy is to find a gene delivery vector with high efficiency and low toxicity. In this study, low-molecular-weight polyethyleneimine (PEI, 1.8 kDa) was grafted onto the side chains of Bombyx mori silk fibroin (BSF) to prepare cationized BSF (CBSF), which was used to package the plasmid DNA (pDNA) encoded by the inhibitor of growth 4 (ING4) and interleukin-24 (IL-24). FTIR and 1H-NMR spectra demonstrated that PEI was effectively coupled to the side chains of BSF by amino bonds. The results of the trinitrobenzene sulfonic acid method and zeta potential showed that the free amino group content on BSF increased from 125.1 ± 1.2 µmol/mL to 153.5 ± 2.2 µmol/mL, the isoelectric point increased from 3.68 to 8.82, and the zeta potential reversed from - 11.8 ± 0.1 mV to + 12.4 ± 0.3 mV after PEI grafting. Positively charged CBSF could package pDNA to form spherical CBSF/pDNA complexes. In vitro, human lung adenocarcinoma A549 cells and human embryonic lung fibroblast WI-38 cells were transfected with CBSF/pDNA complexes. Confocal laser scanning microscopy analysis and flow cytometry tests showed that CBSF/pDNA complexes can effectively transfect A549 cells, and the transfection efficiency was higher than that of 25 kDa PEI/pDNA complexes. CCK-8 assay results showed that CBSF/pDNA complexes significantly inhibited the proliferation of A549 cells but had no significant effect on WI-38 cells and exhibited lower cytotoxicity to WI-38 cells than 25 kDa PEI. Therefore, a gene delivery system, constructed with the low-molecular-weight PEI-modified silk fibroin protein and the ING4-IL-24 double gene coexpression plasmid has potential applications in gene therapy for lung cancer.

High Molecular Weight Silk Fibroin Prepared by Papain Degumming.

A major challenge for the silk textile industry and for the process of silk-based biomaterials is to find a degumming method that can completely remove sericin while avoiding obvious hydrolysis damage to the silk fibroin. In this study, papain was used to degum Bombyx mori silk fibers under nearly neutral conditions based on the specificity of papain to sericin. The degumming efficiency was investigated, as well as the mechanical properties and molecular weight of the sericin-free silk fibroin. The results indicated that increasing the papain concentration aided in sericin removal, as the concentration increased to 3.0 g/L, the degummed fibers showed a clean, smooth surface morphology and exhibited a yellow color when stained by picric acid and carmine, confirming the complete removal of sericin from silk fibroin. Furthermore, an analysis of the amino acid composition indicated that the silk fibroin suffered less damage because papain specifically cleaved the binding sites between L-arginine or L-lysine residue and another amino acid residue in sericin, leading to a significantly higher molecular weight and improved tensile strength compared to traditional sodium carbonate degumming. This study provides a novel degumming method which cannot only completely remove sericin, but also maintain the original strong mechanical properties and high molecular weight of silk fibroin.

The Interactions of Quantum Dot-Labeled Silk Fibroin Micro/Nanoparticles with Cells.

When silk fibroin particles are used for controlled drug delivery, particle size plays a key role in the location of the carrier on the cells as well as the transport pathway, utilization efficiency, and therapeutic effect of the drugs. In this study, the interactions of different-sized silk fibroin particles and cell lines were investigated. Silk fibroin microparticles with dry size of 1.9 ± 0.4 µm (2.7 ± 0.3 µm in wet state) and silk fibroin nanoparticles with dry size of 51.5 ± 11.0 nm (174.8 ± 12.5 nm in wet state) were prepared by salting-out method and high-voltage electrospray method, respectively. CdSe/ZnS quantum dots were coupled to the surface of the micro/nanoparticles. Photostability observations indicated that the fluorescence stability of the quantum dots was much higher than that of fluorescein isothiocyanate. In vitro, microparticles and nanoparticles were co-cultured with human umbilical vein endothelial cells EA.hy 926 and cervical cancer cells HeLa, respectively. The fluorescence test and cell viability showed that the EA.hy926 cells tended to be adhered to the microparticle surfaces and the cell proliferation was significantly promoted, while the nanoparticles were more likely to be internalized in HeLa cells and the cell proliferation was notably inhibited. Our findings might provide useful information concerning effective drug delivery that microparticles may be preferred if the drugs need to be delivered to normal cell surface, while nanoparticles may be preferred if the drugs need to be transmitted in tumor cells.

Porous Poly(Hexamethylene Biguanide) Hydrochloride Loaded Silk Fibroin Sponges with Antibacterial Function.

In order to endue silk fibroin (SF) sponges with antibacterial function, positively charged poly(hexamethylene biguanide) hydrochloride (PHMB) was incorporated in SF through electrostatic interaction and by freeze-drying technique. The influence of PHMB on the structure and antibacterial activities of SF sponges was investigated. The zeta potential of SF was increased significantly when PHMB was incorporated in SF. The pores with size from 80 to 300 µm and the microscale holes in the pore walls within PHMB-loaded SF sponges provided the channels of PHMB release. The PHMB loaded in the porous sponges showed continuous and slow release for up to 20 days. Effective growth inhibition of both Escherichia coli and Staphylococcus aureus was achieved when the mass ratio of PHMB/SF was higher than 2/100. These results suggest that the porous PHMB/SF sponges have the potential to be used as a novel wound dressing for open skin wounds.

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

INTRODUCTION: Cancer 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 αvß3 and αvß5 integrin receptors, which are overexpressed in tumor vessels and most tumor cells. METHODS: In 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. RESULTS: Human 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. CONCLUSION: The 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.

Silk fibroin scaffolds loaded with angiogenic genes in adenovirus vectors for tissue regeneration.

Vascularization remains a critical challenge in dermal tissue regeneration. In this study, a vascular endothelial growth factor (VEGF165) and angiopoietin-1 (Ang-1) dual gene coexpression vector that encoded green fluorescent protein (GFP) was constructed from an arginine-glycine-aspartic acid-modified adenovirus. Silk fibroin (SF) scaffolds loaded with adenovirus vectors were fabricated by freeze-drying method. In vitro, the human endothelial-derived cell line EA.hy926 was infected with adenovirus vectors and then expressed GFP, secreted VEGF165 and Ang-1, and promoted cell proliferation effectively. The VEGF165 and Ang-1 genes loaded in the SF scaffolds significantly promoted the formation of abundant microvascular networks in the chick embryo chorioallantoic membrane. In vivo, angiogenic genes loaded in the scaffolds promoted vascularization and collagen deposition in scaffolds, thus effectively accelerating dermal tissue regeneration in a dorsal full-thickness skin defect wound model in Sprague-Dawley rats. In conclusion, SF scaffolds loaded with arginine-glycine-aspartic acid-modified adenovirus vectors encoding VEGF165 and Ang-1 could stimulate the formation of vascular networks through the effective expression of target genes in vascular endothelial cells, thereby accelerating the regeneration of dermal tissue.

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.

Porous Silk Fibroin Microspheres Sustainably Releasing Bioactive Basic Fibroblast Growth Factor.

Basic fibroblast growth factor (bFGF) plays a significant role in stimulating cell proliferation. It remains a challenge in the field of biomaterials to develop a carrier with the capacity of continuously releasing bioactive bFGF. In this study, porous bFGF-loaded silk fibroin (SF) microspheres, with inside-out channels, were fabricated by high-voltage electrostatic differentiation, and followed by lyophilization. The embedded bFGF exhibited a slow release mode for over 13 days without suffering burst release. SEM observations showed that incubated L929 cells could fully spread and produce collagen-like fibrous matrix on the surface of SF microspheres. CLSM observations and the results of cell viability assay indicated that bFGF-loaded microspheres could significantly promote cell proliferation during five to nine days of culture, compared to bFGF-unloaded microspheres. This reveals that the bFGF released from SF microspheres retained obvious bioactivity to stimulate cell growth. Such microspheres sustainably releasing bioactive bFGF might be applied to massive cell culture and tissue engineering as a matrix directly, or after being combined with three-dimensional scaffolds.