Sequentially decomposable polypeptide-based nanocarriers with protective shell and preparation thereof

1. A sequentially decomposable polypeptide-based nanocarrier with a protective shell, comprising:

a plurality of first polypeptide-based copolymers, each comprising a first hydrophilic polymer and a first polypeptide, wherein the first hydrophilic polymer is conjugated to the N-terminus of the first polypeptide via an acid-labile linkage; and

a plurality of second polypeptide-based copolymers, each comprising a second hydrophilic polymer, a second polypeptide, and an active targeting molecule, wherein the second hydrophilic polymer is conjugated at one end to the N-terminus of the second polypeptide and conjugated at the other end to the active targeting molecule;

wherein the plurality of first polypeptide-based copolymers and the plurality of second polypeptide-based copolymers are assembled into a polypeptide core and a hydrophilic polymer outer layer;

wherein the first polypeptide-based copolymers and the second polypeptide-based copolymers are mixed and aligned radially in the polypeptide-based nanocarrier with the protective shell;

wherein the first hydrophilic polymer has a molecular weight of 3400 Da, and the second hydrophilic polymer has a molecular weight of 1100 Da;

wherein both the first polypeptide and the second polypeptide have a first amino acid sequence, and each sequentially comprises an acidic amino acid segment, an acid-responsive amino acid segment, and a hydrophobic amino acid segment from the N-terminus to the C-terminus;

wherein the acid-responsive amino acid segment consists of an amino acid having a side chain with a pKa value of about 6; and

wherein the protective shell is an acid-soluble mineralized protective shell comprising an acid-soluble mineral deposited on the acidic amino acid segment.

2. The polypeptide-based nanocarrier with the protective shell of claim 1 , wherein the first polypeptide-based copolymers and the second polypeptide-based copolymers are in a molar ratio of about 1:2 to 2:1, and the polypeptide-based nanocarrier with the protective shell is at a size of about 100-200 nm.

3. The polypeptide-based nanocarrier with the protective shell of claim 1 , wherein the acidic amino acid segment consists of 10-20 acidic amino acid residues, the acid-responsive amino acid segment consists of 5-15 acid-responsive amino acid residues, and the hydrophobic amino acid segment consists of 5-15 hydrophobic amino acid residues.

4. The polypeptide-based nanocarrier with the protective shell of claim 1 , wherein the acidic amino acid segment consists of glutamic acid, aspartic acid, or combinations thereof.

5. The polypeptide-based nanocarrier with the protective shell of claim 1 , wherein the acid-labile linkage is hydrolyzed at pH 6.5-7.

6. The polypeptide-based nanocarrier with the protective shell of claim 1 , wherein the acid-soluble mineral is calcium phosphate or calcium carbonate.

7. The polypeptide-based nanocarrier with the protective shell of claim 1 , which further encapsulates a hydrophobic agent.

8. A sequentially decomposable polypeptide-based nanocarrier with a protective shell, comprising:

a plurality of first polypeptide-based copolymers, each comprising a first hydrophilic polymer and a first polypeptide, wherein the first hydrophilic polymer is conjugated to the N-terminus of the first polypeptide via an acid-labile linkage; and

a plurality of second polypeptide-based copolymers, each comprising a second hydrophilic polymer, a second polypeptide, and an active targeting molecule, wherein the second hydrophilic polymer is conjugated at one end to the N-terminus of the second polypeptide and conjugated at the other end to the active targeting molecule;

wherein the plurality of first polypeptide-based copolymers and the plurality of second polypeptide-based copolymers are assembled into a polypeptide core and a hydrophilic polymer outer layer;

wherein the first polypeptide-based copolymers and the second polypeptide-based copolymers are mixed and aligned radially in the polypeptide-based nanocarrier with protective shell;

wherein the first hydrophilic polymer has a molecular weight of 3400 Da, and the second hydrophilic polymer has a molecular weight of 1100 Da;

wherein both the first polypeptide and the second polypeptide have a first amino acid sequence, and each sequentially comprises a cysteine segment, an acid-responsive amino acid segment, and a hydrophobic amino acid segment from the N-terminus to the C-terminus; wherein the acid-responsive amino acid segment consists of an amino acid having a side chain with a pKa value of about 6; and

wherein the protective shell is a redox-responsive protective shell comprising a plurality of disulfide bonds between the cysteine segments of the first polypeptide-based copolymers and the second polypeptide-based copolymers in proximity to each other.

9. The polypeptide-based nanocarrier with the protective shell of claim 8 , wherein the first polypeptide-based copolymers and the second polypeptide-based-copolymers are in a molar ratio of about 1:2 to 2:1, and the polypeptide-based nanocarrier with the protective shell is at a size of about 100-200 nm.

10. The polypeptide-based nanocarrier with the protective shell of claim 8 , wherein the acid-labile linkage is hydrolyzed at pH 6.5-7.

11. The polypeptide-based nanocarrier with the protective shell of claim 8 , which further encapsulates a hydrophobic agent.

12. A method of preparing the sequentially decomposable polypeptide-based nanocarrier with the protective shell of claim 1 , comprising the steps of:

(a) preparing separately a first polypeptide-based copolymer and a second polypeptide-based copolymer by chemical grafting, wherein the first polypeptide-based copolymer comprises a first hydrophilic polymer and a first polypeptide, and the second polypeptide-based copolymer comprises a second hydrophilic polymer and a second polypeptide, wherein both the first polypeptide and the second polypeptide have a first amino acid sequence, and each sequentially comprises an acidic amino acid segment, an acid-responsive amino acid segment, and a hydrophobic amino acid segment from the N-terminus to the C-terminus, wherein the first hydrophilic polymer and the acidic amino acid segment of the first polypeptide are conjugated by an acid-labile linkage, and an end of the second hydrophilic polymer is conjugated with an active targeting molecule, wherein the first hydrophilic polymer is conjugated to the N-terminus of the first polypeptide via the acid-labile linkage, and the second hydrophilic polymer is conjugated at one end to the N-terminus of the second polypeptide and conjugated at the other end to the active targeting molecule; wherein the first hydrophilic polymer has a molecular weight of 3400 Da, and the second hydrophilic polymer has a molecular weight of 1100 Da;

(b) mixing the first polypeptide-based copolymer and the second polypeptide-based copolymer in a polar solvent to allow self-assembly into a polypeptide-based nanocarrier; and

(c) adding a cation aqueous solution and an anion aqueous solution into the polar solvent containing the polypeptide-based nanocarrier to form a first layer of an acid-soluble mineralized protective shell on the acidic amino acid segment of the polypeptide-based nanocarrier, and repeating the addition step multiple times for formation of multiple layers of the acid-soluble mineralized protective shell to obtain the polypeptide-based nanocarrier with the protective shell.

13. The method of claim 12 , wherein in step (c) the cation aqueous solution is a calcium ion aqueous solution, the anion aqueous solution is a phosphate aqueous solution or a carbonate aqueous solution, and the addition step is repeated at least 5 times.

14. The method of claim 12 , wherein in step (b) the first polypeptide-based-copolymer and the second polypeptide-based copolymer are mixed in a molar ratio of about 1:2 to 2:1.

15. The method of claim 12 , wherein in step (a) the acidic amino acid segment consists of 10-20 acidic amino acid residues, the acid-responsive amino acid segment consists of 5-15 acid-responsive amino acid residues, and the hydrophobic amino acid segment consists of 5-15 hydrophobic amino acid residues.

16. The method of claim 12 , wherein in step (a) the acidic amino acid segment consists of glutamic acid, aspartic acid, or combinations thereof.

17. The method of claim 12 , wherein in step (a) the acid-labile linkage is hydrolyzed at pH 6.5-7.