Microporous Drug‐Eluting Large Silk Particles Through Cryo‐Granulation

Abstract

A facile method for the preparation of large, microporous, drug-loaded particles is presented. High shear bollus injections of silk with cross-linker and drug colloids into super-cooled hexane were utilized to trigger phase separation of silk droplets, followed by immediate freezing at-60 o C. A subsequent-20 o C freeze-thaw of the frozen droplets resulted in self-assembly (crystallization) of the silk. The silk particles developed an internal interconnected microporous morphology with 0.1-10 µm in diameter pores. The silk particles ranged in diameter from 100 to 1,300 µm, with particle mean diameter and polydispersity controlled by the starting concentration of the cross-linking agent and silk, the rheology of the reaction mixture, and the injection pressure (80-300kPa). Cryogranulation provided a one-step process to produce microporous meso-scale silk particles with encapsulated drugs, such as doxorubicin chloride (DoxR), tobramycin sulfate (TS), kanamycin sulfate (KS) or gentamicin sulfate (GS). Almost 100% drug encapsulation efficiency was achieved in the process, and subsequent release profiles depended on the starting concentration of both the drug, silk, and pH of the elution medium. Kirby-Bauer tests and bioluminescent imaging confirmed the retention of anti-bacterial potency of the antibiotics pre-encapsulated in the cryo-particles, and macroparticles cytocompatibility towards human fibroblast and kidney cells.

Controlled Cryogelation and Catalytic Cross-Linking Yields Highly Elastic and Robust Silk Fibroin Scaffolds

“…Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy spectra in Figure A enables an assessment of the conformation structure of the SF and the trace of epoxy groups from EGDE in the SF scaffolds. The fresh SF scaffolds after cryogelation reactions, termed prerinse CC-SF, showed characteristic peaks (854, 910, and 1103 cm –1 ) corresponding to the EGDE cross-linker . After rinsing, these peaks disappeared for the CC-SF scaffolds, indicating that EGDE or TEMED molecules can be removed.…”

“…Many techniques, such as freeze-drying, salt-leaching, electrospinning, 3D-printing, hydrogels, and gas foaming, have been explored to prepare SF scaffolds. To construct the cross-linking structure in SF, chemical cross-linking is often introduced through enzymatic catalysis, photocrosslinking of dityrosine bond formation, or reacting with other agents such as genipin. − Although these cross-linkers have been reported with low cytotoxicity, , some of them, e.g ., horseradish peroxidase, are expensive to use. Ethylene glycol diglycidyl ether (EGDE) is a commonly used chemical cross-linker in biology and clinical medicine. , Recently, cryogelation, a subambient gelation process, was used to introduce both physical and chemical cross-links in SF hydrogels by Okay et al − The cryogelled SF possessed a homogeneous morphology and a dense cross-linking structure.…”

“…To construct the cross-linking structure in SF, chemical cross-linking is often introduced through enzymatic catalysis, photocrosslinking of dityrosine bond formation, or reacting with other agents such as genipin. − Although these cross-linkers have been reported with low cytotoxicity, , some of them, e.g ., horseradish peroxidase, are expensive to use. Ethylene glycol diglycidyl ether (EGDE) is a commonly used chemical cross-linker in biology and clinical medicine. , Recently, cryogelation, a subambient gelation process, was used to introduce both physical and chemical cross-links in SF hydrogels by Okay et al − The cryogelled SF possessed a homogeneous morphology and a dense cross-linking structure. The underlying mechanism is that at freezing temperature the polymer solution will be phase-separated into ice and concentrated polymer solution, where the cross-linking agent and the catalyst help polymers accomplish a higher degree of chemical cross-linking.…”

Human Adipose-Derived Mesenchymal Stem Cells-Incorporated Silk Fibroin as a Potential Bio-Scaffold in Guiding Bone Regeneration

“…After lyophilization, the SF scaffold displayed bands at 1619 cm −1 (amide I), 1521 cm −1 with a shoulder at 1538 cm −1 (amide II), and 1231 cm −1 (amide III). The band at 1642 cm −1 in the SF solution was remarkably observed to shift to 1619 cm −1 in the SF scaffold, representing the transition of random coil to β-sheet formation, as the lyophilization process and ethanol treatment increase the crystallinity and diminish the water solubility of the treated samples [27,28]. In addition to favorable mechanical properties, this transition makes SF more attractive as a biomaterial for BTE because the β-sheet structure can also act as a nucleating site to promote mineralization and cell adhesion capabilities [14,29].…”