RESUMEN

Microneedles (MNs) have attracted great interest as a drug delivery alternative to subcutaneous injections for treating diabetes mellitus. We report MNs prepared from polylysine-modified cationized silk fibroin (SF) for responsive transdermal insulin delivery. Scanning electron microscopy analysis of MNs' appearance and morphology revealed that the MNs were well arranged and formed an array with 0.5 mm pitch, and the length of single MNs is approximately 430 µm. The average breaking force of an MN is above 1.25 N, which guarantees that it can pierce the skin quickly and reach the dermis. Cationized SF MNs are pH-responsive. MNs dissolution rate increases as pH decreases and the rate of insulin release are accelerated. The swelling rate reached 223% at pH = 4, while only 172% at pH = 9. After adding glucose oxidase, cationized SF MNs are glucose-responsive. As the glucose concentration increases, the pH inside the MNs decreases, the MNs' pore size increases, and the insulin release rate accelerates. In vivo experiments demonstrated that in normal Sprague Dawley (SD) rats, the amount of insulin released within the SF MNs was significantly smaller than that in diabetic rats. Before feeding, the blood glucose (BG) of diabetic rats in the injection group decreased rapidly to 6.9 mmol/L, and the diabetic rats in the patch group gradually reduced to 11.7 mmol/L. After feeding, the BG of diabetic rats in the injection group increased rapidly to 33.1 mmol/L and decreased slowly, while the diabetic rats in the patch group increased first to 21.7 mmol/L and then decreased to 15.3 mmol/L at 6 h. This demonstrated that the insulin inside the microneedle was released as the blood glucose concentration increased. Cationized SF MNs are expected to replace subcutaneous injections of insulin as a new modality for diabetes treatment.

RESUMEN

Silk fibroin (SF) has attracted much attention due to its high, tunable mechanical strength and excellent biocompatibility. Imparting the ability to respond to external stimuli can further enhance its scope of application. In order to imbue stimuli-responsive behavior in silk fibroin, we propose a new conjugated material, namely cationic SF (CSF) obtained by chemical modification of silk fibroin with ε-Poly-(L-lysine) (ε-PLL). This pH-responsive CSF hydrogel was prepared by enzymatic crosslinking using horseradish peroxidase and H2O2. Zeta potential measurements and SDS-PAGE gel electrophoresis show successful synthesis, with an increase in isoelectric point from 4.1 to 8.6. Fourier transform infrared (FTIR) and X-ray diffraction (XRD) results show that the modification does not affect the crystalline structure of SF. Most importantly, the synthesized CSF hydrogel has an excellent pH response. At 10 wt.% ε-PLL, a significant change in swelling with pH is observed. We further demonstrate that the hydrogel can be glucose-responsive by the addition of glucose oxidase (GOx). At high glucose concentration (400 mg/dL), the swelling of CSF/GOx hydrogel is as high as 345 ± 16%, while swelling in 200 mg/dL, 100 mg/dL and 0 mg/dL glucose solutions is 237 ± 12%, 163 ± 12% and 98 ± 15%, respectively. This shows the responsive swelling of CSF/GOx hydrogels to glucose, thus providing sufficient conditions for rapid drug release. Together with the versatility and biological properties of fibroin, such stimuli-responsive silk hydrogels have great potential in intelligent drug delivery, as soft matter substrates for enzymatic reactions and in other biomedical applications.

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To reduce the pain caused by subcutaneous injections, microneedle patches as the new transdermal drug delivery method are gaining increased attention. In this study, we fabricated a composite insulin-loaded microneedle patch. Silk fibroin, a natural polymer material, was used as the raw material. The tip of the microneedle had good dissolving property and was able to dissolve rapidly to promote the release of insulin. The pedestal had the property of swelling without dissolving and was carrying insulin as a drug store. The insulin carried by the pedestal could release continuously through the micropore channels created by the microneedles. This kind of microneedle could achieve a sustained release effect. It was observed that the insulin had good storage stability in this kind of microneedle, and it maintained more than 90% of its biological activity after 30 days. The results of transdermal delivery to diabetic rats showed that the microneedle patches displayed an apparent hypoglycemic effect and indicated a sustained release effect. These drug-loaded silk microneedle patches may act as potential delivery systems for the treatment of diabetes.

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Silk fibroin is a multisegment natural protein composed of a heavy (H) chain, a light (L) chain and a P25 glycoprotein chain. Herein, we developed a dialysis separation technique under reducing conditions to break the disulfide bond between the H-chain and L-chain and remove the low-molecular-weight portions of the protein. Thus, a high-molecular-weight silk fibroin polypeptide (HSF) material was obtained. SDS-PAGE electrophoresis showed that the molecular weight of HSF was over 80 kDa, similar to the size of the silk fibroin H-chain. Amino acid analysis result demonstrated that the amino acid composition of HSF was almost identical to that of H-chain composition. Importantly, the HSF material obtained has a high surface activity, which can reduce the surface tension of water to below 20 mN/m; at high temperature and high concentration, it can also form a unique nanofibrous network with a lamellar crystalline structure. HSF can further form a rod-shaped structure in a strong polar environment and become a star-shaped fibrous network in a weak polar environment. When the pH value of HSF solution was adjusted from 6 to 8, a structural transition from a folded crank sheet-like structure with micellar beads to a ring-like fibrous structure was observed. During the conversion of HSF from colloidal particles to nanofibers, its molecular conformation also transformed from random coils to ß-sheets. These tunable properties indicate that HSF materials have a wide range of applications in biomedical and green chemistry fields.

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This work illustrates the feasibility of a microneedle based electrochemical biosensor for continuous glucose monitoring. The device consists of three silk/d-sorbitol pyramidal microneedles integrated with platinum (Pt) and silver (Ag) wires and immobilized glucose selective enzyme (glucose oxidase, GOD) during fabrication. The silk/d-sorbitol composite can provide a biocompatible environment for the enzyme molecules. The break strength can be controlled by the ratio of silk to d-sorbitol, which guarantees microneedle penetrate into skin. The enzymatic-amperometric responses and glucose concentration were linearly correlated, and cover physiological conditions. The microneedle displays high stability both in long-term monitoring and storage, even at 37 °C. Our results reveal that this new microneedle biosensor is a promising tool for wearable minimally invasive continuous glucose monitoring in practical applications.

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A photocurable silk fibroin hydrogel is prepared, for the first time, using natural silk protein fibroin and biophotosensitizer riboflavin. Riboflavin is excited by ultraviolet light to generate a triplet state which is transferred to produce active oxygen radicals with singlet oxygen as the main component. Active oxygen radicals can induce chemical cross-linking of amino-, phenol- and other groups in the silk fibroin macromolecules to form a photocurable hydrogel. The different biophysical characterizations of the gelation of this modified fibroin protein solution were studied by using Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, microplate reader and texture analyzer. The aggregate structures, surface morphologies, mechanical properties, light transmission and degradation properties of the gel were studied. The investigations showed that the silk fibroin/riboflavin hydrogels predominantly have random coils or alpha helix structures. These gels show resilience up to 90% after 80% compression and a light transmission of up to 97%. The cell culture experiment exhibits that the hydrogel has a satisfactory cytocompatibility.

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