Protein charge regulator and protein-encapsulating polymer micelle complex

1. A polyion complex comprising a cationic polymer at least partially having a polycation moiety; and a protein whose overall charge is converted by a protein charge regulator;

wherein the protein charge regulator comprises a compound represented by the following formula (I):

wherein R a and R b each independently represent a hydrogen atom, or an optionally substituted alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group; alternatively, R a and R b may be linked to each other to form an aromatic ring or a cycloalkyl ring together with their adjacent carbon atoms; and a bond between the carbon atoms adjacent to R a and R b , respectively, may be either a single bond or a double bond, and

wherein the protein is not associated with an endosome membrane.

2. The polyion complex according to claim 1 , wherein the polycation moiety is a polypeptide having cationic groups in its side chains.

3. The polyion complex according to claim 1 , wherein the cationic polymer is represented by the following formula (1):

wherein R 1 and R 2 each independently represent a hydrogen atom or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms,

R 3 and R 4 each represent a residue derived from an amine compound having a primary amine,

L 1 represents NH, CO, or a group represented by the following formula (4):

—(CH 2 ) p1 —NH—  (4)

wherein p1 represents an integer of 1 to 6, or a group represented by the following formula (5):

-L 2a -(CH 2 ) q1 -L 3a -  (5)

wherein L 2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L 3a represents NH or CO, and q1 represents an integer of 1 to 6,

m1, m2 and m3 each independently represent an integer of 0 to 500 provided that the sum of m1, m2 and m3 represents an integer of 10 to 500, m4 represents an integer of 1 to 5, m5 represents an integer of 1 to 5, and n represents an integer of 0 to 500, and

the symbol “/” means that ml+m2+m3 units of the respective monomers shown in the left and right sides of this symbol are sequenced in any order.

4. The polyion complex according to claim 3 , wherein the group —R 3 and/or the group —R 4 in the polymer represents a group represented by the following formula (2):

—NH—(CH 2 ) r —X 1   (2)

wherein X 1 represents an amine compound residue derived from a primary, secondary or tertiary amine compound or a quaternary ammonium salt, and r represents an integer of 0 to 5, or a group represented by the following formula (3):

—[NH—(CH 2 ) s ] t —X 2   (3)

wherein X 2 represents an amine compound residue derived from a primary, secondary or tertiary amine compound or a quaternary ammonium salt, and s and t, which are independent of each other, represent an integer of 1 to 5 independently in each [NH—(CH 2 ) s ] unit and an integer of 2 to 5, respectively.

5. The polyion complex according to claim 4 , wherein R 3 and/or R 4 is —NH—NH 2 or —NH—(CH 2 ) 2 —NH—(CH 2 ) 2 —NH 2 .

6. The polyion complex according to claim 1 , wherein the polycation moiety in the cationic polymer is bound to the protein through an electrostatic interaction.

7. The polyion complex according to claim 1 , wherein the cationic polymer is a polymer further having a polyethylene glycol moiety,

wherein the protein and the polycation moiety in the cationic polymer form a core region, while the polyethylene glycol moiety in the cationic polymer forms a shell region around the core region.

8. The polyion complex according to claim 1 , wherein the compound represented by formula (1) is dissociated from the protein released from the polyion complex upon introduction into cells, whereby the overall charge of the protein returns to its inherent charge.

9. A device for intracellular protein delivery comprising the polyion complex according to claim 1 .

10. A kit for intracellular protein delivery comprising a cationic polymer at least partially having a polycation moiety and a protein charge regulator,

wherein the protein charge regulator comprises a compound represented by the following formula (I):

wherein R a and R b each independently represent a hydrogen atom, or an optionally substituted alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group; alternatively, R a and R b may be linked to each other to form an aromatic ring or a cycloalkyl ring together with their adjacent carbon atoms; and a bond between the carbon atoms adjacent to R a and R b , respectively, may be either a single bond or a double bond, and

wherein the protein is not associated with an endosome membrane.

11. The polyion complex according to claim 1 , wherein the compound represented by formula (I) is at least one of the compounds represented by the following formulae (Ia) to (Ig).

12. The polyion complex according to claim 1 , wherein the protein charge regulator converts the overall charge of a basic or neutral protein into that of an acidic protein.

13. The polyion complex according to claim 1 , wherein the protein charge regulator establishes binding between the compound represented by formula (I) and an amino group contained in the protein.

14. The polyion complex according to claim 1 , wherein a mixing ratio (N/C ratio) between a total number (N) of cationic groups in the cationic polymer and a total number (C) of carboxyl groups in the protein is 0.1 to 200.

15. The polyion complex according to claim 1 , wherein a particle size of the polyion complex is 5 to 200 nm.

16. The polyion complex according to claim 1 , wherein the protein is an enzyme.

17. The polyion complex according to claim 1 , wherein the protein is an antibody.

18. The polyion complex according to claim 1 , wherein the protein is an antibody fragment selected from the group consisting of Fab, Fab′, F(ab′) 2 , H chain, L chain, H chain V region, and L chain V region, scFv, scFv dimer, dsFv and fragments at least partially containing complementarity determining regions (CDRs).