Cyp-p22 biocatalytic nanoparticles with cytochrome p450 activity for prodrug activation
AssigneeUniversidad Nacional Autonoma De Mexico
InventorsRafael Vazquez Duhalt, Lorena Paulina Sanchez Sanchez
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Abstract
Hybrid proteins with cytochrome P450 activity and which are encapsulated in a nanocapsid (nanoparticles charged with cytochrome P450 activity) are designed and synthesized, these hybrid proteins being immunologically inert and recognized by breast cancer cells.
Claims33
An immunologically inert and functionalized CYP-P22 biocatalytic nanoparticle that activates prodrugs in a target cell, comprising a gene encoding CYPBM3 fused to SP, a fragment of P22 scaffold protein, and a gene encoding a coat protein of P22 bacteriophage.
An immunologically inert and functionalized CYP-P22 biocatalytic nanoparticle that activates prodrugs in a target cell, for the manufacture of a medicine useful for contributing in the treatment of cancer of a mammalian or human patient.
A method for producing immunologically inert and functionalized CYP-P22 biocatalytic nanoparticles that activate prodrugs in a target cell, comprising the following steps:
- A. Cloning of CYP gene is carried out in pETDuet+SP+CP P22 vector;
- B. Ligation between PCR product encoding for the CYP gene and pETDuet+SP+CP P22 linearized vector is made and the cytochrome gene fused to the scaffolding protein gene (CYP-SP) is obtained;
- C. Plasmid is purified by alkaline lysis and the plasmids will be sequenced to verify the correct incorporation of CYP gene into the pETDuet vector in phase with the scaffold protein that results in the pETDuet CYP-SP+CP P22 plasmid;
- D. CYPBM3-SP plasmid and CP P22 are expressed;
- E. pETDuet CYP-SP+CP P22 plasmid is transformed into electrocompetent cells;
- F. CYP-P22 biocatalytic nanoparticles are purified by supernatant ultracentrifugation from cell lysis;
- G. A gel filtration is then carried out and the fractions corresponding to elution of the correctly assembled capsids are collected and then concentrated by ultracentrifugation
- H. Capsid pellet is resuspended and its structure analyzed by transmission electron microscopy (TEM);
- I. Pegylation of the CYP-P22 biocatalytic nanoparticles is performed using bifunctional polyethylene glycol;
- J. CYP-P22 biocatalytic nanoparticles are functionalized with a cyclic peptide or ligand related to the receptor that is present in tumor cells;
- K. CYP-P22 biocatalytic nanoparticles are subjected to 5 mM H2O2 or glucose oxidase+glucose and reaction progress is monitored by HPLC equipped with a C18 reverse phase column to determine the enzymatic activity
- L. Determine the affinity of functionalized nanoparticles in tumor cells in vitro using human MCF7 breast cancer cells in Dulbecco's Modified Eagle's Medium (DMEM medium) supplemented with 10% fetal bovine serum.
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A cytochrome P450 encapsulated load in an immunologically inert and functionalized P22 bacteriophage nanocapsid that activates prodrugs in a target cell, characterized in that comprises from 90 to 150 molecules of CY/capsid.
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Dependencies are inferred from the claim text.