Silk particles for controlled and sustained delivery of compounds
The present invention relates to a method of producing and loading silk particles, preferably spider silk particles, with a compound. In particular, the present invention provides a novel two step method for loading silk particles, preferably spider silk particles, with small and water-soluble compounds. Also disclosed are silk particles, preferably spider silk particles, loaded with at least one compound which are eminently suited as carriers for controlled and sustained delivery applications. Furthermore, the invention relates to pharmaceutical or cosmetic compositions comprising said silk particles, preferably spider silk particles, and a pharmaceutically active compound or cosmetic compound for controlled and sustained release. The present invention is also directed to silk particles, preferably spider silk particles, loaded with a compound obtainable by the method according to the invention.
1. A method of producing spider silk particles loaded with a compound comprising the steps of:
i) providing spider silk particles that consist of an inner solid matrix with an outer surface, both the inner solid matrix and the outer surface homogenously comprising one or more spider silk polypeptides, wherein the one or more spider silk polypeptides comprise at least two identical repetitive units, and wherein the spider silk particles are produced by protein aggregation,
and
ii) incubating said spider silk particles with at least one compound.
2. The method of claim 1 , wherein the spider silk particles provided in step i) are produced by the steps of:
a) providing an aqueous solution comprising one or more spider silk polypeptides comprising at least two identical repetitive units,
b) triggering aggregation of the spider silk polypeptides to form spider silk particles, and
c) separating the spider silk particles by phase separation.
3. The method of claim 1 , wherein the compound is able to permeate into the spider silk particles.
4. The method of claim 1 , wherein at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the loaded compound is located within the spider silk particles.
5. The method of claim 1 , wherein the at least two identical repetitive units each comprise at least one consensus sequence selected from the group consisting of:
i) GPGXX (SEQ ID NO:3), wherein X is any amino acid, preferably in each case independently selected from the group consisting of A, S, G, Y, P and Q;
ii) GGX, wherein X is any amino acid, preferably in each case independently selected from the group consisting of Y, P, R, S, A, T, N and Q; and
iii) A x , wherein x is an integer from 5 to 10.
6. The method of claim 5 , wherein the repetitive unit of the respective spider silk polypeptide is independently selected from module A (SEQ ID NO:20) or variants thereof, module C (SEQ ID NO:21) or variants thereof, module Q (SEQ ID NO:22) or variants thereof, module A C (SEQ ID NO:29), module A K (SEQ ID NO:30), module C C (SEQ ID NO:31), module C K1 (SEQ ID NO:32), module C K2 (SEQ ID NO:33) or module C KC (SEQ ID NO:34).
7. The method of claim 6 , wherein the spider silk polypeptide further comprises at least one non-repetitive (NR) unit.
8. The method of claim 7 , wherein the non-repetitive (NR) unit is independently selected from the group consisting of NR3 (SEQ ID NO:41 and SEQ ID NO:45) or variants thereof and NR4 (SEQ ID NO:42 and SEQ ID NO:46) or variants thereof.
9. The method of claim 1 , wherein the compound is a pharmaceutically active compound, a cosmetic substance, an agricultural substance, a chemoattractant, a chemorepellent, an anti-fungal substance, an anti-bacterial substance, a nutrient, a dietary supplement, a dye, a fragrance or an agent selected from the group consisting of hemostatic agents, growth stimulating agents, inflammatory agents, anti-fouling agents, antimicrobial agents and UV protecting agents.
10. The method of claim 1 , wherein the compound has an overall positive net charge.
11. The method of claim 1 , wherein the compound is able to permeate into the spider silk particles by electrostatic interaction and/or diffusion.
12. The method of claim 1 , wherein the compound has a neutral or alkaline nature.
13. Spider silk particles produced from the method of claim 1 , which consist of an inner solid matrix with an outer surface, both the inner solid matrix and the outer surface homogenously comprising at least one spider silk polypeptide comprising at least two identical repetitive units, wherein the spider silk particles are loaded with at least one compound.
14. The spider silk particles of claim 13 , wherein, at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the loaded compound is located within the spider silk particles.
15. The spider silk particles of claim 13 , wherein the spider silk polypeptide comprises at least two identical repetitive units each comprise at least one consensus sequence selected from the group consisting of:
i) GPGXX (SEQ ID NO:3), wherein X is any amino acid, preferably in each case independently selected from the group consisting of A, S, G, Y, P and Q;
ii) GGX, wherein X is any amino acid, preferably in each case independently selected from the group consisting of Y, P, R, S, A, T, N and Q; and
iii) A x , wherein x is an integer from 5 to 10.
16. The spider silk particles of claim 15 , wherein the repetitive unit of the spider silk polypeptide is independently selected from module A (SEQ ID NO:20) or variants thereof, module C (SEQ ID NO:21) or variants thereof, module Q (SEQ ID NO:22) or variants thereof, module A C (SEQ ID NO:29), module A K (SEQ ID NO:30), module C C (SEQ ID NO:31), module C K1 (SEQ ID NO:32), module C K2 (SEQ ID NO:33) or module C KC (SEQ ID NO:34).
17. The spider silk particles of claim 16 , wherein the spider silk polypeptide further comprises one or more non-repetitive (NR) units.
18. The spider silk particles of claim 17 , wherein the NR unit is independently selected from the group consisting of NR3 (SEQ ID NO:41 and SEQ ID NO:45) or variants thereof and NR4 (SEQ ID NO:42 and SEQ ID NO:46) or variants thereof.
19. The spider silk particles of claim 13 , wherein the compound is a pharmaceutically active compound, a cosmetic substance, an agricultural substance, a chemoattractant, a chemorepellent, an anti-fungal substance, an anti-bacterial substance, a nutrient, a dietary supplement, a dye, a fragrance or an agent selected from the group consisting of hemostatic agents, growth stimulating agents, inflammatory agents, anti-fouling agents, antimicrobial agents and UV protecting agents.
20. The spider silk particles of claim 13 , wherein the compound has an overall positive net charge.
21. The spider silk particles of claim 13 , wherein the compound is able to permeate into the spider silk particles by electrostatic interaction and/or diffusion.
22. The spider silk particles of claim 13 , wherein the compound has a neutral or alkaline nature.
23. The spider silk particles of claim 13 , wherein the compound is released from the spider silk particles by diffusion upon exposure to physiological conditions.
24. The spider silk particles of claim 23 , wherein less than 20%, preferably less than 15%, and most preferably less than 10% of the compound is released within the first 24 hours.
25. A pharmaceutical composition comprising the spider silk particles according to claim 19 , and additionally a pharmaceutically acceptable buffer, diluent and/or excipient, the pharmaceutical composition being useful for controlled and sustained delivery, wherein the compound is a pharmaceutically active compound.
26. A cosmetic composition comprising the spider silk particles according to claim 19 for controlled and sustained delivery, wherein the compound is a cosmetic compound.