Silk fibroin as a vehicle for drug delivery applications.

Silk fibroin (SF), a naturally occurring protein polymer, has several unique properties making it a favorable matrix for the incorporation and delivery of a range of therapeutic agents. SF is biocompatible, slowly biodegradable, and endowed with excellent mechanical properties and processability. Novel manufacturing techniques including mild all-aqueous processes have expanded its range of application even to sensitive protein and nucleic acid therapeutics. SF matrices were demonstrated to successfully deliver protein drugs and preserve their potency. Adjustments in SF crystallinity, concentration and structure, the design of the delivery systems as well as the molecular weight and structure of the embedded agents represent important variables when it comes to precisely tailor the release kinetics of SF matrices. Other strategies to fine-tune the release from SF matrices comprise the embedment of drug loaded micro- or nanoparticles or the coating of micro- or nanoparticles with SF films. So far, the main focus of SF drug delivery systems has been on tissue regeneration applications. For instance, growth factor loaded SF scaffolds were suggested for the tissue engineering of bone and cartilage, as well as for vascular and nerve regeneration devices and wound healing products. Moreover, SF matrices were proposed for oral, transmucosal and ocular drug delivery. This article reviews SF properties and fabrication processes that affect the release from SF drug delivery systems. For illustration, we discuss a variety of examples for the incorporation of drugs into SF systems and their release.

The use of sulfonated silk fibroin derivatives to control binding, delivery and potency of FGF-2 in tissue regeneration.

The development of biomaterials that mimic the physiological binding of growth factors to the extracellular matrix (ECM) is an appealing strategy for advanced growth factor delivery systems. In vivo, fibroblast growth factor 2 (FGF-2) binds to the sulfated glycosaminoglycan heparan sulfate, which is a major component of the ECM. Therefore, we tested whether silk fibroin (SF) decorated with a sulfonated moiety could mimic the natural ECM environment and lead to advanced delivery of this heparin-binding growth factor. Using a diazonium coupling reaction, modified SF derivatives containing approximately 20, 40, 55 and 70 sulfonic acid groups per SF molecule were obtained. Films of the SF derivative decorated with 70 sulfonic acid groups per SF molecule resulted in a 2-fold increase in FGF-2 binding as compared to native SF. More than 99% of bound FGF-2 could be retained on all SF derivatives. However, protection of FGF-2 potency was only achieved with at least 40 sulfonic acid groups per SF molecule, as observed by reduced metabolic activity and enhanced levels of phosphorylated extracellular signal-regulated kinases (pERK1/2) in cultured human mesenchymal stem cells (hMSCs). This study introduces a first step towards the development of an ECM-mimicking biomaterial for sustained, non-covalent binding, controlled delivery and preserved potency of biomolecules.

Development of silk fibroin-based beads for immobilized cell fermentations.

Silk fibroin was evaluated as a new matrix for immobilized cell fermentation. Silk fibroin was extracted from Bombyx mori cocoon, purified, concentrated in polyethylene glycol solution and diluted to 3 wt% with distilled water. This fibroin solution was used to encapsulate sensitive cells of the probiotic strain, Bifidobacterium longum ATCC 15707. Polymer droplets produced with an encapsulator were collected in liquid nitrogen and lyophilized. A low overall survival of 0.2% was measured after lyophilization. Lyophilized beads were hardened for 24 h under vacuum with an atmosphere of 89% relative humidity. The inoculated beads were colonized in two successive batch fermentations. Structure of silk fibroin beads and colonization of cells were examined with scanning electron microscopy. Colonized beads were tested in continuous fermentations for cell production. A biomass productivity of 1.7 x 10(9) CFU ml(-1) h(-1) was achieved, which was limited by loss of bead structure. This instability might be due to bead degradation by proteolytic activity of cells and/or limited mechanical stability during continuous fermentation in the stirred tank reactor.

Microporous silk fibroin scaffolds embedding PLGA microparticles for controlled growth factor delivery in tissue engineering.

The development of prototype scaffolds for either direct implantation or tissue engineering purposes and featuring spatiotemporal control of growth factor release is highly desirable. Silk fibroin (SF) scaffolds with interconnective pores, carrying embedded microparticles that were loaded with insulin-like growth factor I (IGF-I), were prepared by a porogen leaching protocol. Treatments with methanol or water vapor induced water insolubility of SF based on an increase in beta-sheet content as analyzed by FTIR. Pore interconnectivity was demonstrated by SEM. Porosities were in the range of 70-90%, depending on the treatment applied, and were better preserved when methanol or water vapor treatments were prior to porogen leaching. IGF-I was encapsulated into two different types of poly(lactide-co-glycolide) microparticles (PLGA MP) using uncapped PLGA (50:50) with molecular weights of either 14 or 35 kDa to control IGF-I release kinetics from the SF scaffold. Embedded PLGA MP were located in the walls or intersections of the SF scaffold. Embedment of the PLGA MP into the scaffolds led to more sustained release rates as compared to the free PLGA MP, whereas the hydrolytic degradation of the two PLGA MP types was not affected. The PLGA types used had distinct effects on IGF-I release kinetics. Particularly the supernatants of the lower molecular weight PLGA formulations turned out to release bioactive IGF-I. Our studies justify future investigations of the developed constructs for tissue engineering applications.

Growth factor gradients via microsphere delivery in biopolymer scaffolds for osteochondral tissue engineering.

Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.

Silk fibroin spheres as a platform for controlled drug delivery.

The goal of this proof-of-concept study was the fabrication of drug-loaded silk fibroin (SF) spheres under very mild processing conditions. The spheres were fabricated using the laminar jet break-up of an aqueous SF solution, which was induced by a nozzle vibrating at controlled frequency and amplitude. SF particles were spherical in shape as determined by SEM with diameters in the range of 101 microm to 440 microm, depending on the diameter of the nozzle and the treatment to induce water insolubility of SF. Both treatments, either methanol or exposure to water vapor, resulted in an increase in beta-sheet content as analyzed by FTIR. High encapsulation efficiencies, close to 100%, were obtained when salicylic acid and propranolol hydrochloride-loaded SF spheres were left untreated or exposed to water vapor. Methanol treatment resulted in drug leaching and lowered the overall encapsulation efficiency. When 9% SF solutions were used for SF sphere preparation, release rates were more sustained than from spheres made with 3% SF solutions, and propranolol hydrochloride release was more sustained than salicylic acid release. However, no difference in the release profiles was observed between methanol and water vapor treated SF spheres. Because of its very mild conditions, which are potentially advantageous for the encapsulation of sensitive drugs, we also tested this method for the encapsulation of insulin-like growth factor I (IGF-I). Again encapsulation efficiencies were close to 100%, even after treatment with methanol. IGF-I was continuously released over 7 weeks in bioactive form, as analyzed by the proliferation of MG-63 cells. These results favor further investigation of SF spheres as a platform for the controlled release of sensitive biologicals.

Silk coatings on PLGA and alginate microspheres for protein delivery.

Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, poly(lactic-co-glycolic acid) (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres, the coating was approximately 1microm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was approximately 10microm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca(2+)-cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamine-conjugated bovine serum albumin (Rh-BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.

Silk microspheres for encapsulation and controlled release.

A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load protein drugs in active form for controlled release. The lipid was subsequently removed by methanol or sodium chloride treatments, resulting in silk microspheres consisting of beta-sheet structure and about 2 mum in diameter. NaCl treated microspheres had smoother surfaces compared to the methanol treatments based on SEM analysis, and both types of microspheres had a mixture of multilamellar and unilamellar structures. A model protein drug, horseradish peroxidase, was encapsulated in the microspheres. Freeze-thaw cycles during preparation led to higher loading of the peroxidase due to improved mixing between the silk and drug, while without this process the drug and silk remained in separate layers or domains in microspheres. This partitioning was determined with fluorescein-labeled silk and rhodamine-labeled dextran. Small molecules such as the enzyme substrate 3,3',5,5'-tetramethylbenzidine, Mw=240 Da, and its oxidized product freely diffused through the MeOH- and NaCl-processed silk microspheres so that enzyme loading and activity could be determined. Enzyme activity was retained during processing and in the final microspheres. The enzyme release profile depended on the NaCl-process used in microsphere preparation. The physically cross-linked beta-sheet structure of silk fibroin and the residual lipids in the microspheres played important roles in controlling enzyme release profiles. The silk microspheres have the potential for diverse applications where controlled protein release from biocompatible, mechanically tough, and slowly biodegradable carriers is desirable.

Insulin-like growth factor I-releasing alginate-tricalciumphosphate composites for bone regeneration.

PURPOSE: Development and characterization of an in situ-forming, osteoconductive, and growth factor-releasing bone implant. METHODS: Injectable in situ-forming scaffolds were prepared from a 2% (m/v) alginate solution, tricalciumphosphate (TCP) granules, and poly(lactide-co-glycolide) microspheres (MS), loaded with the osteoinductive growth factor insulin-like growth factor I (IGF-I). Scaffolds were prepared by mixing the components followed by hydrogel formation through calcium carbonate-induced physical cross-linking of the alginate at slightly acidic pH. Physical-chemical properties and cell biocompatibility using osteoblast-like cells (MG-63 and Saos-2) of these scaffolds were investigated. RESULTS: The addition of TCP to the alginate resulted in reduced swelling and gelation time and an increase in stiffness. Osteoblast-like cells (MG-63 and Saos-2) did not show toxic reactions and adhered circumferentially to the TCP granules surface. The addition of the IGF-I MS resulted in an up to sevenfold increased proliferation rate of MG-63 cells as compared to scaffold preparations without IGF-I MS. The alkaline phosphate (ALP) activity-a parameter for osteblastic activity-increased with increasing amounts of TCP in Saos-2 loaded composite scaffolds. CONCLUSIONS: A prototype in situ-hardening composite system for conformal filling of bone defects supporting osteoblastic activity for further clinical testing in relevant fracture models was developed and characterized.