Preparation of Silk Fibroin Nanoparticles and Enzyme-Entrapped Silk Fibroin Nanoparticles

Materials and Reagents

1. Qualitative Filter Paper, 0.22 μm, Round, 15 cm (Sangon Biotech (Shanghai) Co., Ltd., catalog number: F503312)
2. Dialysis membrane (Standard, RC, 5 KD, 18 mm Width) (BBI, catalog number: F128058)
3. The cocoons of silkworm Bombyx mori, provided by Sericultural Institute, Soochow University, Suzhou, P. R. China 
4. Na₂CO₃ (Sangon Biotech (Shanghai) Co., Ltd., catalog number: A500840)
5. CaCl₂ (Sangon Biotech (Shanghai) Co., Ltd., catalog number: A501330)
   
6. Ethanol (Sangon Biotech Shanghai Co., Ltd., catalog number: A500737) 
7. Acetone (Sigma-Aldrich, catalog number: 320110)
8. Polyethylene glycol-2000 (Sangon Biotech (Shanghai) Co., Ltd., catalog number: A601785)
9. 50 mmol/L phosphate buffer (pH 7.0) (see Recipes)
10. Ajisawa's reagent (CaCl₂/ethanol/H₂O [1:2:8 molar ratio]) (see Recipes)
    

Equipment

1. Glass Injector, 20 ml
2. Glass beaker, 50 ml
3. High-speed Refrigerated Centrifuge (Beckman Coulter, model: Avanti^® J-30I)
4. Scanning Electron Microscope (Hitachi, model: S-570)
5. Ultra-sonic Instrument (Sonics Vibra cell) (Sonics & Materials, model: VCX 130 PB)
   

Procedure

Figure 1 shows a schematic of silk fibroin nanoparticles (SFNs) or enzyme-entrapped SFNs preparation using acetone.


Figure 1. Schematic of silk fibroin nanoparticles (SFNs) or enzyme-entrapped SFNs preparation using acetone

1. Degum the clean cocoon shell twice in boiling 0.2% (w/v) Na₂CO₃ for 0.5 h each time. 
2. After the degumming treatment, wash the resulting silk materials repeatedly in deionized water and then air dry.
3. Immerse the degummed silk fibers into 20-fold volume (w/v) of Ajisawa's reagent (CaCl₂/ethanol/H₂O, 1:2:8 molar ratio) (Ajisawa, 1998) and stir at 70 °C (water bath) for 4 h.
4. Centrifuge the silk fibroin-salt solution at 7,000 x g for 10 min to remove impurities and insoluble substances. 
5. Dialyze the filtered solution or supernatant continuously for 48 h with running deionized water to remove CaCl₂, smaller molecules and some impurities using a cellulose semi-permeable membrane (cutoff 5 kDa).
6. Concentrate the aqueous solutions of regenerated silk fibroin to 1%-5% of silk fibroin solution at 4 °C by using PEG and finally store it at 4 °C for SFN preparation.
7. If preparing enzyme-entrapped silk fibroin nanoparticles (SFNs), add a given amount of enzyme (liquid or powder) to the above silk fibroin solution and homogenize the mixture mildly via shaking by hand. 
8. Rapidly Inject the silk fibroin solution or a mixture with enzyme into acetone (acetone is more than 70% of the final mixture) by using a 20 ml injector at room temperature; while injection proceeded, rapidly shake the 50 ml beaker or container containing the protein/enzyme by hand. 
9. The milk-like silk protein particles or enzyme-entrapped silk protein particles form immediately and suspend in the mixture composed of water and acetone. 
10. These particles are water-insoluble and can precipitate slowly due to nanoparticle aggregating. Filtrate and collect the precipitates of these nanoparticles through 0.22 μm filter paper, or purify from the mixture by centrifugation at 108,000 x g for 0.5 h to separate the particles from the solvent. 
11. After subject these filtered retentates or centrifuged precipitates to ultrasonic treatment (Output Watts: 16) five times for 2-min each, the nanoparticles or enzyme-entrapped nanoparticles disperse evenly in 50 mM phosphate buffer or water and can be stored at 4 °C for a long time.
    

Data analysis

Our previous experiments showed that the solubilization of the degummed silk fibroin fiber in the CaCl₂ ternary system at 70 °C for 4 h resulted in amide-bond breakages in the silk fibroin molecular chains. SDS-PAGE showed that the molecular masses and distributions of liquid silk fibroin are affected by the dissolving conditions (Zhang et al., 2007). In fact, the regenerated liquid silk is a mixture of gradually degraded polypeptides of silk fibroin, with a size range of about 10-70 kDa. When a given amount of silk fibroin solution without or with containing L-asparaginase is rapidly injected into an excess of acetone, a milky suspension of silk protein forms at once, and simultaneously L-asparaginase is tightly entrapped in the silk fibroin nanoparticles (SFNs). Scanning Electron Microscope (SEM) observations showed that the silk nanoparticles are globular granules whether it contains enzymes or no enzymes (Figure 2). All these granules are about 50-120 nm in diameter.


Figure 2. SEM images (10⁴x) in the left (SFNs) and right (L-asparaginase-entrapped SFNs) by using a Hitachi S-570 Scanning Electron Microscope

Recipes

1. 50 mmol/L phosphate buffer (pH 7.0)
   Dissolve 7.800 g NaH₂PO₄•2H₂O to 500 ml dH₂O, and adjust the salt solution to pH 7.00 with 0.1 N NaOH solution, finally adjust the volume of the phosphate solution to 1,000 ml with dH₂O
2. Ajisawa's reagent (CaCl₂/ethanol/H₂O [1:2:8 molar ratio])
   Add 55.5 g CaCl₂ to 72 ml dH₂O, after the mixture solution cool to room temperature, add 57.5 ml ethanol the mixture. The total volume of the ternary solvent is about 165 ml
   

Acknowledgments

This work was supported by the earmarked fund for the China Agriculture Research System (CARS-18-ZJ0502) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, P. R. China. This modified protocol is based on our previously published work (Zhang et al., 2007 and 2011).

Competing interests

The author declares no conflicts of interest or competing interests.

References

1. Ajisawa, A. (1998). Dissolution of silk fibroin with calcium chloride/ethanol aqueous solution. J Seric Sci Jpn 67(2): 91-94. 
2. Wang, H. Y. and Zhang, Y. Q. (2013). Effect of regeneration of liquid silk fibroin on its structure and characterization. Soft Matter 9(1): 138-145.
3. Zhang, Y. Q., Shen, W. D., Xiang, R. L., Zhuge, L. J., Gao, W. J. and Wang, W. B. (2007). Formation of silk fibroin nanoparticles in water-miscible organic solvent and their characterization. J Nanopart Res 9(5): 885-900.
4. Zhang, Y. Q., Wang, Y. J., Wang, H. Y., Zhu, L. and Zhou, Z. Z. (2011). Highly efficient processing of silk fibroin nanoparticle-L-asparaginase bioconjugates and their characterization as a drug delivery system. Soft Matter 7(20): 9728-9736.