US 9,283,194 B2 · Methods for protease assisted protein delivery

Abstract

A method for intracellular delivery of single proteins or other cargo molecules by encapsulation within nanocapsules formed by interfacial polymerization of one or more types of monomers and selected protease cleavable cross-linkers is provided. The thin positively charged capsules are readily brought into the cytosol of target cells by endocytosis. The capsules are degraded by the action of endogenous proteases or co-delivered proteases on the cross-linkers releasing the functional cargo unaltered. The cross-linkers can be adapted to be cleavable by specific enzymes selected from available intracellular enzymes within the target cell or co-delivered or self-cleaving when the cargo itself is a protease. The nanocapsules produced by the methods have been shown to have long-term stability, high cell penetration capability, low toxicity and efficient protease-modulated specific degradability without affecting cargo protein function.

Claims20

Dependent claims

A method for producing an enzymatically-degradable nanocapsule that encapsulates not more than one core cargo molecule, the method comprising:

(a) selecting a core cargo molecule for encapsulation;
(b) preparing a plurality of cross-linkers having at least one enzyme cleavage site;
(c) physically adsorbing a plurality of shell monomers and cross-linkers to said core cargo molecule, wherein said adsorbing is modulated by electrostatic forces between the monomers and the core cargo molecule;
and
(d) polymerizing a polymeric shell comprising the plurality of adsorbed shell monomers and cross-linkers around said core cargo molecule to provide a enzymatically-degradable nanocapsule;
wherein:
the core cargo molecules are not covalently coupled to the enzymatically-degradable nanocapsules:
said enzymatically-degradable nanocapsule encapsulates not more than one core cargo molecule;
and
said cross-linker enzyme cleavage sites of said cross-linkers are configured to be cleaved in the presence of site specific enzymes.

7 more elements

A method for selective intracellular delivery of cargo molecules to a cell or tissue, comprising:

selecting a target cellular system and a cargo molecule;
selecting an enzyme type and cleavable peptide sequence;
producing enzymatically-degradable nanocapsules that encapsulates not more than one core cargo molecule, comprising:
selecting a core cargo molecule for encapsulation;
preparing a plurality of cross-linkers having at least one selected enzyme cleavage site;
physically adsorbing a plurality of shell monomers and cross-linkers to the single core cargo molecule, wherein said adsorbing is modulated by electrostatic forces between the monomers and the core cargo molecule;
and polymerizing a polymeric shell comprising the plurality of adsorbed shell monomers and cross-linkers around said core cargo molecule to provide a enzymatically-degradable nanocapsule;
and placing said nanocapsules in proximity to target cells or tissues to allow for penetration of target cell membranes by said nanocapsules;
wherein:
the core cargo molecules are not covalently coupled to the enzymatically-degradable nanocapsules;
said enzymatically-degradable nanocapsule encapsulates not more than one core cargo molecule;
and
said cross-linker enzyme cleavage sites of said cross-linkers are configured to be cleaved by site specific enzymes to release the cargo molecule.

10 more elements

Dependencies are inferred from the claim text.

Methods for protease assisted protein delivery

Life of this patent

Methods for protease assisted protein delivery

Life of this patent

Abstract

Claims20