Controlling silk fibroin particle features for drug delivery

Abstract

Silk proteins are a promising material for drug delivery due to their aqueous processability, biocompatibility, and biodegradability. A simple aqueous preparation method for silk fibroin particles with controllable size, secondary structure and zeta potential is reported. The particles were produced by salting out a silk fibroin solution with potassium phosphate. The effect of ionic strength and pH of potassium phosphate solution on the yield and morphology of the particles was determined. Secondary structure and zeta potential of the silk particles could be controlled by pH. Particles produced by salting out with 1.25 m potassium phosphate pH 6 showed a dominating silk II (crystalline) structure whereas particles produced at pH 9 were mainly composed of silk I (less crystalline). The results show that silk I-rich particles possess chemical and physical stability and secondary structure which remained unchanged during post treatments even upon exposure to 100% ethanol or methanol. A model is presented to explain the process of particle formation based on intra- and intermolecular interactions of the silk domains, influenced by pH and kosmotropic salts. The reported silk fibroin particles can be loaded with small molecule model drugs, such as alcian blue, rhodamine B, and crystal violet, by simple absorption based on electrostatic interactions. In vitro release of these compounds from the silk particles depends on charge-charge interactions between the compounds and the silk. With crystal violet we demonstrated that the release kinetics are dependent on the secondary structure of the particles.

Figure 1

Influence of process parameters on silk particle formation by salting out. a) Salting out efficiency as a function of ionic strength of potassium phosphate at pH 8. b) Salting out efficiency as a function of pH employing 1.25 M potassium phosphate. c–e) Scanning electron micrographs of particles produced by salting out with 1.25 M potassium phosphate at c) pH 4, d) pH 5 and e) pH 6.

Figure 2

FTIR spectra of silk particles produced by salting out with potassium phosphate (1.25 M, pH 8) followed by treatment with ethanol, methanol or sonication. a) EtOH (incubated for 24h). b) MeOH (incubated for 24h). c) Sonication with 20% maximum amplitude for exposure times indicated.

Figure 3

Size analysis of silk particles produced from different concentrations of protein by salting out with potassium phosphate (1.25 M, pH 8). a) Size distributions of silk particles as a function of protein concentration. b) Average size of silk particles as a function of protein concentration. Error bars indicate the width of the size distribution. c)–e) Scanning electron micrographs of silk particles produced by salting out with potassium phosphate (1.25 M, pH 8) from silk fibroin solution of c) 0.25 mg/ml, d) 2 mg/ml e) 20 mg/ml.

Figure 4

Control of secondary structure and zeta potential of silk fibroin particles. a) FTIR spectra of particles produced by salting out with 1.25 M potassium phosphate at different pH values. b) Zeta potential of particles produced by salting out with 1.25 M potassium phosphate at different pH values. Error bars indicate the width of the zeta potential distribution of the particles. c–d) Fourier self deconvolution of FTIR spectra for particles produced by salting out with 1.25 M potassium phosphate at c) pH 6 and d) pH 8.

Figure 5

Silk fibroin characteristics and particle formation. a) Characteristics of silk fibroin considering the charge distribution along the amino acid chain. b) Model for silk fibroin particle formation. Top: Assumed configuration of amino acid chains at different pHs. Middle: Particle nucleation of micellar like structures. Bottom: Particle formation and stabilization of secondary structures through clustering of micellar like structures in the presence of potassium phosphate (>1.0 M).

Figure 6

Loading of silk fibroin particles. a) Loading efficiencies of silk fibroin particles incubated in aqueous solutions containing the model drugs (MD) crystal violet, alcian blue and rhodamin B of different concentrations yielding molar ratios of model drug (MD) : silk fibroin (SF) as indicated. b) Loading of particles (i.e. model drug content). c) Zeta potential of crystal violet, alcian blue and rhodamine B loaded particles as a function of loading. d) Confocal microscopy of RhB loaded particles.

Figure 7

Release from silk fibroin particles. a) Release of rhodamine B, crystal violet and alcian blue from particles produced by salting with potassium phosphate (1.25 M, pH 8) b) Crystal violet release from particles produced at different pH values.