Preparation and Characterization of Natural Silk Fibroin Hydrogel for Protein Drug Delivery

In recent years, hydrogels have been widely used as drug carriers, especially in the area of protein delivery. The natural silk fibroin produced from cocoons of the Bombyx mori silkworm possesses excellent biocompatibility, significant bioactivity, and biodegradability. Therefore, silk fibroin-based hydrogels are arousing widespread interest in biomedical research. In this study, a process for extracting natural silk fibroin from raw silk textile yarns was established, and three aqueous solutions of silk fibroin with different molecular weight distributions were successfully prepared by controlling the degumming time. Silk fibroin was dispersed in the aqueous solution as “spherical” aggregate particles, and the smaller particles continuously accumulated into large particles. Finally, a silk fibroin hydrogel network was formed. A rheological analysis showed that as the concentration of the silk fibroin hydrogel increased its storage modulus increased significantly. The degradation behavior of silk fibroin hydrogel in different media verified its excellent stability, and the prepared silk fibroin hydrogel had good biocompatibility and an excellent drug-loading capacity. After the protein model drug BSA was loaded, the cumulative drug release within 12 h reached 80%. We hope that these investigations will promote the potential utilities of silk fibroin hydrogels in clinical medicine.     

Effect of Silk Protein Processing on Drug Delivery from Silk Films

Sericin removal from the core fibroin protein of silkworm silk is a critical first step in the use of silk for biomaterial‐related applications, but degumming can affect silk biomaterial properties, including molecular weight, viscosity, diffusivity and degradation behavior. Increasing the degumming time (10, 30, 60, and 90 min) decreases the average molecular weight of silk protein in solution, silk solution viscosity, and silk film glass‐transition temperature, and increases the rate of degradation of a silk film by protease. Model compounds spanning a range of physical‐chemical properties generally show an inverse relationship between degumming time and release rate through a varied degumming time silk coating. Degumming provides a useful control point to manipulate silk's material properties.

     

Preparation and Characterization of a Novel Drug Delivery System: Biodegradable Nanoparticles in Thermosensitive Chitosan/Gelatin Blend Hydrogels

A novel injectable in situ gelling drug delivery system (DDS) consisting of biodegradable N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanoparticles and thermosensitive chitosan/gelatin blend hydrogels was developed for prolonged and sustained controlled drug release. Four different HTCC nanoparticles, prepared based on ionic process of HTCC and oppositely charged molecules such as sodium tripolyphosphate, sodium alginate and carboxymethyl chitosan, were incorporated physically into thermosensitive chitosan/gelatin blend solutions to form the novel DDSs. Resulting DDSs interior morphology was evaluated by scanning electron microscopy. The effect of nanoparticles composition on both the gel process and the gel strength was investigated from which possible hydrogel formation mechanisms were inferred. Finally, bovine serum albumin (BSA), used as a model protein drug, was loaded into four different HTCC nanoparticles to examine and compare the effects of controlled release of these novel DDSs. The results showed that BSA could be sustained and released from these novel DDSs and the release rate was affected by the properties of nanoparticle: the slower BSA release rate was observed from DDS containing nanoparticles with a positive charge than with a negative charge. The described injectable drug delivery systems might have great potential application for local and sustained delivery of protein drugs.     

Green Electrospun Silk Fibroin Nanofibers Loaded with Cationic Ethosomes for Transdermal Drug Delivery

Transdermal drug delivery system(TDDS) facilitates the controlled release of active ingredients penetrating through the skin, avoiding the liver first pass effect. Electrospinning is a simple process to fabricate ultrafine fibers with a higher specific surface area, making them excellent candidates for drug delivery. In current work, a novel silk fibroin(SF) nanofiber loaded with cationic ethosomes(CEs) was prepared via green electrospinning. The data of Fourier transform infrared spectroscopy(FTIR) and laser scanning confocal microscopy(LSCM) confirmed the existence of CEs in the SF nanofibers. The morphology of the nanofibers was not significantly affected by the incorporation of CEs as shown by scanning electron microscopy(SEM) images. The CEs-loaded SF nanofibrous patch (CEs-SFnP) showed good cytocompatibility as proved by both cell counting Kit-8(CCK-8) assay and SEM. Using doxorubicin hydrochloride(Dox) as a model drug, the transdermal performance of CEs-SFnP was evaluated through Franz diffusion cell against mouse skin. The results indicated that CEs-SFnP can effectively deliver drug into the skin, with a much higher permeation rate than the normal nanofibers without CEs. The as-spun CEs-SFnP in this study could find promising applications in TDDS.     

Functionalization of Silk Fibroin with NeutrAvidin and Biotin

New methods are needed to modify silk biomaterials with bioactive molecules for tissue engineering and drug delivery. In the present study, silk fibroin in solution or in microsphere format was coupled with NeutrAvidin via carbodiimide chemistry. Silk fibroin retained its self‐assembly features after reaction. It was found that more than four NeutrAvidin molecules bound to one silk molecule. Non‐specific binding of biotin or NeutrAvidin to silk microspheres could be reduced by pre‐treatment of the microspheres with BSA or post‐treatment with detergent. The NeutrAvidin‐coupled silk microspheres were coupled with biotinylated anti‐CD3 antibody and the functionalized microspheres were able to specifically bind to the CD3 positive T‐lymphocytic cell line Jurkat.

     

甲醇-水混合溶剂后处理制备丝素蛋白膜及其药物释放研究

采用溶液浇注法制备丝素蛋白薄膜, 应用傅里叶红外光谱(FTIR)和X 射线衍射(XRD)研究了浓度不同的甲醇-水混合溶剂处理后丝素蛋白薄膜的结构变化, 并以罗丹明B 为模型药物与丝素蛋白构建药物缓释体系, 考察了丝素蛋白膜的结晶结构对药物释放动力学的影响. 结果显示, 在甲醇体积比浓度Φ_MeOH＝50%～90%的范围内, 丝素蛋白材料中以β-折叠为主的silk Ⅱ 结晶含量随着混合溶剂中甲醇浓度的增加而先增加后下降, 在Φ_MeOH＝80%附近出现最大值. 罗丹明B 从丝素蛋白膜的释放属于Fickian 扩散机理, 其扩散指数n 随着丝素蛋白膜中β-折叠含量的增加而增加, silk Ⅱ结晶是丝素蛋白材料药物释放的天然调节器.     

Chitosan Cross-Linked Films for Drug Delivery Application

This work evaluated such a cross-linked chitosan based controlled release device to be later used for sustained drug release. Cross linking was required to control chitosan swelling/deswelling rate. Hexamethylene 1,6-Bis (aminocarboxysulfonate), a bisulfite blocked diisocyanate obtained by the reaction of 1,6 Hexamethylene Diisocyanate and Sodium bisulfite, was used as cross linking agent. Two films formulations were tested: 30 and 50% cross-linked, and they were prepared by solvent evaporation technique. Chitosan cross-linked films were characterized for cross linkage by FTIR, for hydrophilicity by Contact Angle and for swelling behavior by Gravimetric method. Cross linking reaction was confirmed by FTIR. Moreover, cross linking increased the hydrophilic character of cross-linked films and suppressed swelling. However, 30% cross-linked film swollen less than the 50% one, while 50% cross-linked film swollen less than chitosan film itself. This behavior was attributed to the hydrophilic character of the cross linking agent and to the polymeric network formation by cross linking.     

Crosslinked Silk Fibroin/Gelatin/Hyaluronan Blends as Scaffolds for Cell-Based Tissue Engineering

3D porous scaffolds fabricated from binary and ternary blends of silk fibroin (SF), gelatin (G), and hyaluronan (HA) and crosslinked by the carbodiimide coupling reaction were developed. Water-stable scaffolds can be obtained after crosslinking, and the SFG and SFGHA samples were stable in cell culture medium up to 10 days. The presence of HA in the scaffolds with appropriate crosslinking conditions greatly enhanced the swellability. The microarchitecture of the freeze-dried scaffolds showed high porosity and interconnectivity. In particular, the pore size was significantly larger with an addition of HA. Biological activities of NIH/3T3 fibroblasts seeded on SFG and SFGHA scaffolds revealed that both scaffolds were able to support cell adhesion and proliferation of a 7-day culture. Furthermore, cell penetration into the scaffolds can be observed due to the interconnected porous structure of the scaffolds and the presence of bioactive materials which could attract the cells and support cell functions. The higher cell number was noticed in the SFGHA samples, possibly due to the HA component and the larger pore size which could improve the microenvironment for fibroblast adhesion, proliferation, and motility. The developed scaffolds from ternary blends showed potential in their application as 3D cell culture substrates in fibroblast-based tissue engineering.     

Drug Delivery Systems Based on Porous Chitosan/Polyacrylic acid Microspheres

Porous microspheres have been prepared by suspension free radical polymerization of acrylic acid (AA) in the presence of chitosan (CHI). The microspheres were characterized by FTIR and environmental SEM. The PAA content of the microspheres was estimated to be in the range 45–50 wt.‐%. The swelling degree of these particles is almost constant in the range 2 < pH < 5, but it increases considerably as the pH is raised from 5 to 10. The release profiles of microspheres loaded with meclofenamic acid (MF) were determined at pH 2, 7.4, and 10. The in vitro release of MF at different pHs was modulated by the solubility of the drug. These microcapsules are biodegradable and presented good biocompatibility and biodegradability during in vivo experiments.

ESEM microphotograph of the porous PAA/CHI microspheres.     

纳米TiO2/丝素复合膜的制备

探讨了纳米TiO2在丝素溶液中的分散条件,在此基础上制备了纳米TiO2/丝素复合膜,并用原子力显微镜(AFM)和扫描电子能谱(EDS)对复合膜进行了表征。实验结果表明,制膜方法较为合理。当以有机物A为分散剂,纳米TiO2含量为1/1000时,其以粒径50 nm左右均匀地分散于复合膜中。     

Novel Polyelectrolyte Carboxymethyl Konjac Glucomannan–Chitosan Nanoparticles for Drug Delivery

Summary: Carboxymethyl Konjac Glucomannan–Chitosan (CKGM‐CS) nanoparticles, which are well dispersed and stable in aqueous solution, were spontaneously prepared under very mild conditions by polyelectrolyte complexation. Investigations of the physicochemical properties of these nanoparticles were undertaken. This study showed that the nanoparticulate system driven by complex formation has potential as an advanced drug‐delivery system for water‐soluble drugs.

Preparation mechanism of CS–CKGM nanoparticles.     

Stabilization and Release of Enzymes from Silk Films

A significant challenge remains to protect protein drugs from inactivation during production, storage, and use. In the present study, the stabilization and release of horseradish peroxidase (HRP) in silk films was investigated. Water‐insoluble silk films were prepared under mild aqueous conditions, maintaining the activity of the entrapped enzyme. Depending on film processing and post‐processing conditions, HRP retained more than 90% of the initial activity at 4 °C, room temperature and 37 °C over two months. The stability of protein drugs in silk films is attributed to intermolecular interactions between the silk and the enzymes, based on Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The unique structural feature of silk molecules, periodic hydrophobic‐hydrophilic domains, enabled strong interactions with proteins. The entrapped protein was present in two states, untrapped active and trapped inactive forms. The ratio between the two forms varied according to processing conditions. Proteolytic degradation and dissolution of the silk films resulted in the release of the bound enzyme which was otherwise not released by diffusion; enzyme recovered full activity upon release. There was a linear relationship between silk degradation/dissolution and the release of entrapped enzyme. Modifying the secondary structure of the silk matrix and the interactions with the non‐crystalline domains resulted in control of the film degradation or dissolution rate, and therefore the release rate of the entrapped enzyme. Based on the above results, silk materials are an intriguing carrier for proteins in terms of both retention of activity and controllable release kinetics from the films.

     

具有定向形貌的丝蛋白支架的制备

利用冰晶为模板并结合冷冻干燥技术, 成功地获得了具有一定力学强度的定向多孔丝蛋白支架. 采用扫描电镜、流变、拉曼光谱和压缩测试等方法, 考察了丝蛋白原液在不同pH值和初始浓度的情况下对定向支架的成因、内部形貌及力学性能的影响. 结果表明, 当溶液的pH值为4.4, 浓度由低到高时, 所制备的丝蛋白支架内部将分别出现纤维状结构、轴向片层状和梭状多孔结构. 特别是由初始浓度为15% (及以上)的丝蛋白溶液定向冷冻得到的支架, 在压缩试验中表现出较好的力学性能, 其轴向压缩模量和屈服应力分别达122.6和6.9 MPa, 满足了进一步应用的基本需求.     

Silk Fibroin-Coated Liposomes as Biomimetic Nanocarrier for Long-Term Release Delivery System in Cancer Therapy

Despite much progress in cancer therapy, conventional chemotherapy can cause poor biodistribution and adverse side-effects on healthy cells. Currently, various strategies are being developed for an effective chemotherapy delivery system. Silk fibroin (SF) is a natural protein used in a wide range of biomedical applications including cancer therapy due to its biocompatibility, biodegradability, and unique mechanical properties. In this study, SF-coated liposomes (SF-LPs) were prepared as a biomimetic drug carrier. Physicochemical properties of SF-LPs were characterized by Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering, zeta potential measurement, and transmission electron microscopy (TEM). In vitro release of SF-LPs loaded with doxorubicin (DOX-SF-LPs) was evaluated over 21 days. Anticancer activity of DOX-SF-LPs was determined against MCF-7 and MDA-MB231 cells using the MTT assay. SF-LPs containing 1% SF exhibited favorable characteristics as a drug carrier. SF coating modified the kinetics of drug release and reduced the cytotoxic effect against L929 fibroblasts as compared to the uncoated liposomes containing cationic lipid. DOX-SF-LPs showed anticancer activity against breast cancer cells after 48 h or 72 h at 20 μM of DOX. This approach provides a potential platform of long-term release that combines biocompatible SF and phospholipids for cancer therapy, achieving efficient drug delivery and reducing side-effects.     

Advanced Silk Fibroin Biomaterials-Based Microneedles for Healthcare

Microneedles are a promising transdermal drug delivery system that has the advantages of minimal invasiveness, painlessness, and on-demand drug delivery compared with commonly used medical techniques. Natural resources are developed as next-generation materials for microneedles with varying degrees of success. Among them, silk fibroin is a natural polymer obtained from silkworms with good biocompatibility, high hardness, and controllable biodegradability. These properties provide many opportunities for integrating silk fibroin with implantable microneedle systems. In this review, the research progress of silk fibroin microneedles in recent years is summarized, including their materials, processing technology, detection, drug release methods, and applications. Besides, the research and development of silk fibroin in a multidimensional way are analyzed. Finally, it is expected that silk fibroin microneedles will have excellent development prospects in various fields.     

柞蚕丝素／纳米TiO2复合膜的制备及其结构表征

为了获得良好性能的柞蚕丝素复合膜,本文采用复合法制备了不同配比柞蚕丝素/纳米TiO2复合膜,并与纯的丝素膜作了比较,用SEM、DSC、TG和IR进行了表征.SEM测试表明在分散剂聚乙烯醇作用下,适量的纳米TiO2能均匀分散丝素溶液中.DSC测试表明复合膜b、c和d的Tm均高于纯的柞蚕丝素膜a的Tm,然而随着纳米TiO2加入量的继续增加,对应复合膜的Tm有所降低.TG结果表明,随着纳米TiO2加入量的增加,复合膜的热稳定性得到提高.IR测试表明丝素复合膜的结晶结构从Silk I向SilkII转化.     

Silk Sericin Retards the Crystallization of Silk Fibroin

Summary: The thermal and structural analysis of silk fibroin (SF) and silk sericin (SS) blend films reveals that the crystallization of SF is retarded in the presence of SS. Although a phase separation was observed, there might be a strong interaction at the boundary of the SF and SS through intermolecular hydrogen bonding, which restricts the conformational transition of SF.

TEM image of the cross‐section of the SF/SS blend (75:25) film (magnification: ×15 000).     

Tramadol Release from a Delivery System Based on Alginate‐Chitosan Microcapsules

Alginate‐chitosan microcapsules to control the release of Tramadol‐HCl were prepared using two different methods. In the two‐stage procedure (Variant I) alginate was first pumped into a CaCl₂/NaCl solution and then transferred into a chitosan solution. In the one‐stage procedure (Variant II) alginate was directly pumped into a chitosan/CaCl₂ solution, and different behavior could be noted in each case. The microcapsules were spherical in both variants and they swelled to a greater extent in a basic medium as compared to an acid one. The drug release profile of Tramadol from microcapsules in simulated gastric fluid and simulated intestinal fluid was also studied. The maximum release of Tramadol at 24 h was 64% and 86% for Variant I and II, respectively, in simulated intestinal fluid. Release was adjusted using the power law of the semi‐empirical Peppas equation in order to gain information about the release mechanism. In both cases the values of the exponent were found to be between 0.53 and 0.84 for swellable microcapsules in simulated gastric and intestinal fluids, respectively, indicating anomalous drug transport for both variants. The good results obtained with alginate‐chitosan microcapsules are comparable to those of the best products so far described in the scientific bibliography and in addition, chitosan is useful in pharmacy.

Surface morphology of Tramadol‐loaded microcapsule.