CYP-P22 biocatalytic nanoparticles with cytochrome P450 activity for prodrug activation

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.

Claims

1. An immunologically inert and functionalized CYP-P22 biocatalytic nanoparticle that activates prodrugs in a target cell, comprising a) a polypeptide sequence of SEQ ID NO:4 which is a fusion of a scaffold protein SP and a CYPBM3 protein, b) a fragment of a P22 scaffold protein, c) a coat protein of P22 bacteriophage, d) a bifunctional dendritic polyethylene glycol, and e) a cyclic polypeptide of SEQ ID NO: 5.

2. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the CYP-P22 biocatalytic nanoparticle measures 53.62 nm in diameter.

3. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the CYP-P22 biocatalytic nanoparticle comprises from 90 to 150 molecules of CYP/capsid.

4. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the prodrug is selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, and loratadine.

5. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the target cell comprises mammalian or human patient tissues.

6. The CYP-P22 biocatalytic nanoparticle according to claim 5, wherein the tissue presents a tumor mass.

7. The CYP-P22 biocatalytic nanoparticle according to claim 6, wherein the tumor mass is a cancer selected from: breast cancer, and colon cancer.

8. 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, comprising: a) a polypeptide sequence of SEQ ID NO:4 which is a fusion of a scaffold protein SP and a CYPBM3 protein, b) a fragment of a P22 scaffold protein, c) a coat protein of P22 bacteriophage, d) a bifunctional dendritic polyethylene glycol, and e) a cyclic polypeptide of SEQ ID NO: 5.

9. The CYP-P22 biocatalytic nanoparticle according to claim 8, wherein the CYP-P22 biocatalytic nanoparticle is 53.62 nm in diameter.

10. The CYP-P22 biocatalytic nanoparticle according to claim 8, wherein the CYP-P22 biocatalytic nanoparticle comprises from 90 to 150 molecules of CYP/capsid.

11. The CYP-P22 biocatalytic nanoparticle according to claim 8, wherein the prodrug is selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, and loratadine.

12. The CYP-P22 biocatalytic nanoparticle according to claim 8, wherein the target cell comprises tissues from a mammalian or human patient.

13. The CYP-P22 biocatalytic nanoparticle according to claim 12, wherein the tissue presents a tumor mass.

14. The CYP-P22 biocatalytic nanoparticle according to claim 13, wherein the tumor mass is a cancer selected from: breast cancer, and colon cancer.

15. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the CYP-P22 is in combination with a prodrug.

16. The CYP-P22 biocatalytic nanoparticle according to claim 15, wherein the prodrug is selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, and loratadine.

17. The CYP-P22 biocatalytic nanoparticle according to claim 1, wherein the prodrug is selected from: tamoxifen, resveratrol, tegafur, isosfamide, clopidogrel, nabumetone, pafuramidine, and loratadine.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. General procedure scheme of cloning, expression and in vivo encapsulation of CYP.sub.BM3 in bacteriophage P22 capsids.

(2) FIGS. 2A-2D. Analysis of P22-CYP nanostructure formation by gel filtration chromatography. The correctly assembled structures elute around 65 min; the aberrant structures elute around 45 min. Protein elution was monitored at =280 nm. FIG. 2A: pET 15 h; 2B: pBAD+pRSF 1; 2C: pET 5 h; 2D: pBAD+pRSF 2.

(3) FIGS. 3A and 3B. Microphotography of P22 viral capsids where load or content of 90 to 150 molecules of cytochrome P450 per capsid is observed. Nanoparticle diameter is 53.62 nm. 3A) Analysis of molecular weight and P22-CYP encapsulation diameter by gel filtration chromatography (HPLC) coupled to MALS-QELS-RI detectors. 3B) P22-CYP particles seen at TEM. Negative staining

(4) FIG. 4. Catalytic activity of loaded nanoparticles with cytochrome P450 activity (CYP-P22 biocatalytic nanoparticle) compared to free-form enzyme: free CYP vs. CYP-P22 (5 mM H.sub.2O.sub.2).

(5) FIG. 5. Tamoxifen transformation products catalyzed by P22 nanoparticles containing cytochrome P450 (CYP-P22 biocatalytic nanoparticle). Transformation products are: 4-hydroxy tamoxifen; N-demethyltamoxifen; 4-hydroxy-N-dimethyltamoxifen (endoxifen); 3,4-hydroxytamoxifen.

(6) FIGS. 6A-6F. Cytochrome P450 activity in human cervical carcinoma cells treated with VLPs-CYP. Staining with DAPI shows cell nuclei marked with n, panels 6A and 6D. Endogenous CYP activity on BFC reagent in untreated cells, panel 6B. Increase in CYP activity in cells treated with VLPs-CYP, panel 6E. BFC reagent is transformed into a fluorescent product {HFC) and is located in cell cytoplasm (white arrows). Overlapping of images stained with DAPI and with BFC, panels 6C and 6F. Cells were observed with a 63 objective (DIC), 1.4 NA of planar-apochromatic oil immersion.

(7) FIG. 7. Tamoxifen transformation (a prodrug used for treatment of breast cancer) by CYP-P22 (biocatalytic nanoparticles) containing cytochrome P450.

(8) FIG. 8. Number of enzymes per capsid and proportion of active CYP for each expression system: co-expression and differential expression.

(9) FIG. 9. Plasmid pETDuet CYP-SP+CP P22.

(10) FIG. 10. Activity vs PH of CYP-P22 vs. free CYP

(11) FIG. 11. Protease activity on CYP-P22 and free CYP

(12) FIG. 12. Plasmid pCWori CYPBM3 21B3

(13) FIG. 13. Plasmid pBAD CYP-SP

(14) FIG. 14. Plasmid pRSF CP P22

(15) FIGS. 15A and 15B. Stability of free and encapsulated CYP in P22 at 40 C. (15A) and 50 C. (15B).

DETAILED DESCRIPTION OF THE INVENTION

(16) Terminology used in present invention is intended to describe particular embodiments and is not intended to be limiting of the invention. As used herein, CYP-P22 or CYP-P22 biocatalytic nanoparticle refers to a nanoparticle, nanostructure or biocatalytic nanocapsid loaded with cytochrome P450 activity (nanoparticles or nanostructures or nanocapsids containing cytochrome P450). More particularly, CYP-P22 is a cytochrome P450 encapsulated in a bacteriophage P22 nanocapsid.

(17) The term CYP-P22 should also be understood in its inverse form P22-CYP, both referring to CYP-P22 nanoparticle, the nanoparticle loaded with cytochrome P450.

(18) In turn, CYP-P22 (containing the cytochrome P450 nanoparticle or nanostructure) has enzymatic activity on prodrugs, such as, for example, prodrugs selected from: anticancer agents (Tamoxifen, Tegafur, Ifosphamide, Resveratrol, and the like), antithrombotic agents (e.g., Clopidogrel and the like), analgesics (such as nabumetone, and the like), antiparasitic agents (such as Pafuramidine and the like), antihistaminics (such as Loratadine, and the like), and others; and said CYP-P22 is immunologically inert and capable of being recognized by tumor cells, tumor tissues or other tissues of interest. Furthermore, prodrugs will be activated in a target cell or tissue of interest. Thus, in the present invention the CYP enzymatic activity is carried to the tissue or target cell that will activate the prodrugs in said targets selected from a mammalian or human patient suffering from a tumor mass or cancer selected from breast cancer or colon cancer or for other treatments, where the prodrug is activated by cytochrome P450, achieving a better treatment efficiency, a decrease in required medication doses and a decrease in side effects. Thus, the local concentration of active drug in tumor cells, tumor tissues or other tissues is increased, increasing drug efficiency in the tumor or tissue and reducing toxicity produced by the drug in the rest of the host cells.

(19) The invention consists of nanoparticles loaded with cytochrome P450 (enzymes of the cytochrome P450 group encapsulated within nanoparticles), where a CYP-SP protein is located inside a P22 nanocapsid. These biocatalytic nanoparticles are functionalized to be recognized by tumor cells, tumor tissues or other tissues in a treatment wherein the prodrug is activated by cytochrome P450, and are immunologically inert, and catalytically active. The present invention consists in cytochrome P450 molecule encapsulation within viral capsids. Enzymatic nanoparticles or nanoparticles loaded with cytochrome P450 activity will be coated with bifunctional dendritic polyethylene glycol and finally functionalized with some cyclic peptide or other specific ligand to be recognized by tumor cells or other tissues of interest of a mammal or human being. CYP-P22 biocatalytic nanoparticles are capable of transforming prodrugs used in chemotherapy or in other therapies, including treatments requiring cytochrome P450 enzymatic activity on selected prodrugs such as: anticancer agents (Tamoxifen, Tegafur, Ifosphamide, Resveratrol, and the like), antithrombotic agents (e.g., Clopidogrel and the like), analgesics (such as nabumetone, and the like), antiparasitic agents (such as Pafuramidine and the like), antihistaminics (such as Loratadine, and the like), and others. Due to their coating nature, biocatalytic nanoparticles are immunologically inert and are recognized by receptors located on the surface of tumor cells or cells of other tissues of interest. Nanoparticles containing cytochrome P450 (CYP-P22) increase enzymatic activity on the surface of the tumor tissues, tissues with a tumor mass or tissues of interest where prodrugs are activated more efficiently and at the required site, such as, for example, prodrugs used in chemotherapy or other therapies and are selected from antithrombotic, analgesic, antiparasitic, antihistaminic agents, and any other therapy or treatment wherein a prodrug is activated by P450.

(20) The method to produce or synthesize these CYP-P22 biocatalytic and immunologically inert vehicles (nanoparticles loaded with cytochrome P450 activity), that activate prodrugs in a target cell, for example to increase CYP activity in tumors or other tissues of interest and in a treatment wherein the prodrug is activated by cytochrome P450, includes the following procedure:

(21) A) Cloning of CYP gene is performed in the pETDuet+SP+CP P22 vector. For this end, oligonucleotides are designed to amplify the gene encoding for CYP with insertion of specific restriction sites, to allow subsequent gene ligation in pETDuet vector.

(22) B) Ligation is carried out between the PCR product encoding for CYP gene and the linearized vector pETDuet+SP+CP P22 and the cytochrome gene fused to scaffolding protein gene (CYP-SP) is obtained. Ligation is used to transform electrocompetent cells. The presence of the insert is checked by inoculating transformed cells in LB boxes with the specific selection antibiotic. The plasmid is purified by alkaline lysis and plasmids will be sequenced to verify the correct inclusion of CYP gene into pETDuet vector in phase with the scaffold protein that results in plasmid pETDuet CYP-SP+CP P22.

(23) C) Plasmid CYPBM3-SP and CP P22 is expressed. Plasmid pETDuet CYP-SP+CP P22 is transformed into electrocompetent BL21 cells. At the end of recovery, cells are cultured in boxes with the selection antibiotic and are grown. Induction of transformed strain is carried out in antibiotic cultures (for selection), the inducer and the aminolevulinic acid as a precursor in heme synthesis.

(24) D) P22 capsids containing cytochrome P450 are purified by supernatant ultracentrifugation from cell lysis. Subsequently the sample is subjected to gel filtration and the corresponding fractions to the elution of the correctly assembled capsids are collected and then concentrated by means of ultracentrifugation. The capsid pellet is resuspended and its structure is analyzed by transmission electron microscopy (TEM).

(25) E) Subsequently catalytic nanoparticle pegylation is performed. Modification with polyethylene glycol of viral capsid surface is carried out with bi-functional polyethylene glycol. In this stage, the safety of nanoparticles can be evaluated on activation of different lymphoid cell subpopulations. Likewise, nanoparticle toxicity is measured on these same linfoid cells.

(26) F) Nanoparticles loaded with cytochrome P450 activity are functionalized for targeting tumor cells such as breast tumor cells, or other tissues of interest, with some cyclic peptide or other ligand related to receptors that are present in tumor cells, tumor tissues or other tissues.

(27) G) Determination of enzymatic activity of loaded biocatalytic nanoparticles is carried out, for example by means of the transformation of Tamoxifen or another prodrug selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, loratadine. Reactions will be initiated by adding H.sub.2O.sub.2 5 mM or glucose oxidase+glucose and reaction progress is monitored by HPLC equipped with a C18 reverse phase column.

(28) H) Evaluation of affinity of the nanoparticles loaded with cytochrome P450 activity functionalized in tumor cells in vitro can be carried out for example in human MCF7 breast cancer cells maintained in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum. Cells are cultured at 37 C. in 5% CO.sub.2 and with nanoparticles before treatment, the cells are washed twice with serum-free medium and then 1 mL of serum-free medium will be added. Subsequently, the functionalized loaded nanoparticles are added to each well and incubated for 4 hours. After incubation, the supernatant is removed and 1 mL of fresh medium containing 10% fetal bovine serum is incubated for further 48 hours.

(29) I) The presence of biocatalytic nanoparticles (loaded nanoparticles) with CYP activity is evaluated in transformation of 7-benzyloxy-4-trifluorometyl-coumarin (BFC) monitored by fluorescence according to the method of Donato et al. [58]. The fluorescence assay for determining CYP activity is performed with a direct incubation of cultured tumor cells in a 12-well plate in the presence of BFC 100 M. BFC is added dissolved in acetonitrile, ensuring that final acetonitrile concentration in the wells does not exceed 0.5% (v/v). After 60 min incubation at 37 C. the incubation medium is removed and the conjugated products of CYP transformation will be hydrolyzed with a mixture of 3-giucoronidase/arylsulfatase for 2 hours at 37 C. Finally, samples are diluted with the corresponding solution and product fluorescence (7-hydroxy-4-trifluoromethylcoumarin, HFC) is quantified in a spectrofluorimeter with 410 nm excitation and 510 nm emission.

(30) J) CYP-P22 or biocatalytic nanoparticle efficiency in prodrug activation is determined by determining the tumor cell viability previously treated with nanoparticles in the presence of the prodrug or in combination with the prodrug selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, loratadine.

(31) The embodiments of the invention will become apparent from the following examples, since CYP-P22 specifically carry to tumor cells, tumor tissues or other tissues the cytochrome P450 activity that activates prodrugs used in chemotherapy more efficiently and at the required site, or in other treatments where the prodrug is activated by cytochrome P450, without limiting the scope to some type of cancer or condition; such as, a cancer selected from breast or colon cancer.

EXAMPLES

Example 1. CYP Expression and Encapsulation

(32) A) Cloning of CYP gene is carried out in pETDuet+SP+CP P22 vector. For such end, oligonucleotides (SEQ ID NO: 2 and 3) are designed to amplify the gene coding for CYP with insertion of specific restriction sites, NcoI and BamHI, to allow subsequent gene ligation in pETDuet vector (FIG. 1).

(33) TABLE-US-00003 CYPNcolfw SEQ.ID.NO.2 5AAAAATCATGCCATGGCAATTAAAGAAATGCCT3 CYPBamHIReverse SEQ.ID.NO.3 5AAAAAAGCGGGATCCAGTGCTAGGTGAAGGAA3

(34) A PCR reaction is carried out using the plasmid pCWori CYPBM3 (ampicillin resistance, double ptac promoter, IPTG inducible, gene coding for heme domain of CYPBM3 21B3 mutant) (FIG. 12), 10 picomoles of each oligo and Pfu Ultra DNA polymerase. Gene amplification (1400 bp) is checked by means of a 1% agarose gel electrophoresis using TAE as a run buffer. A voltage of 100 V is used for 25 min. PCR reaction is digested with 1 l of DpnI for 2 h at 37 C. to remove the parental plasmid. The reaction is then cleaned and DNA is resuspended in 40 uL of mQ grade water. PCR product is digested with 40 units of NcoI and BamHI restriction enzymes). Total reaction volume is brought to 50 uL and incubated at 37 C. for 16 h, the pETDuet plasmid containing the scaffold protein fragment gene, SP141-303, and the bacteriophage P22 coat protein is digested in the same way as the PCR product explained above. Digestion is run on a 1% agarose gel in TAE buffer. The band corresponding to the linearized vector is cut and DNA is extracted from agarose gel.

(35) B) Ligation is performed between the PCR product coding for the CYP gene and the pETDuet+SP+CP P22 linearized vector using the T4-DNA ligase enzyme incubating at room temperature for 6 h. 1 l of ligation was taken to transform 25 l of electrocompetent 10 G cells. 250 l of transformed cells were plated in a LB box with ampicillin and allowed growing for 16 h at 37 C. The presence of insert was verified (CYPBM3 gene) for 16 colonies by a colony PCR reaction with CYPNcolfw and CYPBamHIReverse oligonucleotides. Thus, cytochrome gene fused to the scaffold protein gene is obtained, while ligation is used to transform electrocompetent cells. The plasmid is purified by alkaline lysis and plasmids will be sequenced to check the correct incorporation of CYP gene into pETDuet vector in phase with the scaffold protein that results in pETDuet CYP-SP+CP P22 plasmid {FIG. 9), which comprises a gene encoding CYPBM3 21B3 fused to SP, a P22 scaffold protein fragment, plus the P22 bacteriophage coat protein gene.

(36) SEQ. ID. NO. 4 corresponds to CYPBM3 2163 amino acid sequence fused to P22 bacteriophage scaffold protein:

(37) TABLE-US-00004 MAIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVT RYLSSQRLVKEACDESRFDKNLSQALKFVRDFAGDGLATSWTHEKNWKKA RNILLPSLSQQAMKGYHAMMVDIAVQLVQKWERLNSDEKIEVPEDVTRLT LDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYD ENKRQFQEDIKVMDLVDKIIADRKASGEQSDDLLTHMLHGKDPETGEPLD DENIRYQIITFLIAGHETTSGLLTFALYFLVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKG DELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRAC IGQQFALHEATLVLGMMLKHFDFEDHTNYELDIEETLTLKPEGFVIKAKS KKIPLGGIPSPSTGSLVPRGSCRSNAVAEQGRKTQEFTQQSAQYVEAARK HYDAAEKLNIPDYQEKEDAFMQLVPPAVGADIMRLFPEKSAALMYHLGAN PEKARQLLAMDGQSALIELTRLSERLTLKPRGKQISSAPHADQPITGDVS AANKDAIRKQMDAAASKGDVETYRKLKAKLKGIR

(38) C) CYPBM3-SP and CP P22 plasmid is expressed. pETDuet CYP-SP+CP P22 plasmid is transformed into electrocompetent BL21 cells. At the end of the recovery, the cells are cultured at least 1 h in LB boxes with the selection antibiotic, ampicillin and grown for 16 h at 37 C.

(39) Induction of the transformed strain is carried out in cultures with the antibiotic (for selection), the inducer and aminolevulinic acid as a precursor in the heme synthesis.

(40) CYPBM3-SP and CP P22 Simultaneous Expression (Co-Expression)

(41) Two induction schemes were followed. For the first scheme, 2 mL of a transformed strain pre-culture was taken to inoculate a 250 mL culture of TB medium with Amp added with 0.5 m of Thiamine and trace elements. It was allowed to grow at 37 C. at 180 rpm until reaching OD.sub.600=0.8. At this point, it was induced with IPTG 0.5 mM and aminolevulinic acid 1 mM was added. The culture was allowed to grow for additional 5 h at 30 C. at 135 rpm. For the second scheme, 2 mL of a transformed strain pre-culture was taken to inoculate a 250 mL culture of TB medium with Amp added with Thiamine 0.5 mM and trace elements. It was allowed to grow at 37 C. for 7 h at 150 rpm. At this point, it was induced with IPTG 0.5 nM and aminolevulinic acid 1 mM was added. The culture was allowed to grow for additional 15 h at 30 C. at 135 rpm. At the end of the induction, the cultures were centrifuged cold at 3840g for 10 min, cells were resuspended in lysis buffer (Na2HPO4 50 mM, NaCl 100 mM, pH 7.6) and the sample was sonicated. It was centrifuged at 12,000g for 30 min at 4 C. and the supernatant was recovered. {FIG. 8)

(42) CYPBM3-SP and CP P22 Differential Expression

(43) Cloning of CYPBM3-SP Gene into pBAD Vector

(44) To clone the cytochrome gene fused to the scaffold protein gene (CYP-SP) in pBAD vector, pETDuet CYP-SP-CP P22 plasmid was digested with 30 units of NcoI and SacI restriction enzymes at 37 C. for 3 h. The reaction was cleaned using MinElute Reaction Cleanup Kit and DNA was resuspended in 30 l of mQ grade water. Subsequently the sample was incubated with 2.5 units of Antarctic phosphatase for 16 h at 37 C. in a final volume of 30 uL in order to dephosphonate pETDuet vector still present in the mixture thus avoiding its recirculation. Enzyme inactivation at 65 C. for 30 min was carried out.

(45) pBAD plasmid was also digested with 30 units of NcoI and SacI restriction enzymes at 37 C. for 3 h. Digestion was run on a 1% agarose gel in TAE buffer. The band corresponding to the linearized vector was cut and DNA extraction of agarose gel was done using the QIAquick gel extraction kit.

(46) Then ligation between the CYP-SP gene and the linearized vector pBAD was carried out using the 4-DNA ligase enzyme, incubating at room temperature for 6 h. 1 L of ligation was taken to transform 25 L of electrocompetent 10G cells. 250 l of transformed cells were plated in a LB box with ampicillin and allowed to grow for 16 h at 37 C. The presence of a CYP-SP construct for 8 colonies was verified by a colony PCR reaction with CYPNcolfw and CYPBamHIReverse oligonucleotides, following the same previously reported PCR program. Finally, 2 clones were randomly grown (with a previously verified insert) to purify plasmid by alkaline lysis using Qiagen solutions and columns (QIAprep Spin Miniprep kit).

(47) The correct incorporation of CYP-SP gene into pBAD vector was verified, resulting in plasmid pBAD CYP-SP.

(48) Plasmids pBAD CYP-SP (ampicillin resistance, araBAD promoter, arabinose-inducible, gene coding for CYPBM3 21B3 fused to SP141-303, a protein fragment of P22 scaffold) and pRSF CP P22 (kanamycin resistance, T7 promoter, IPTG inducible, gene encoding the bacteriophage P22 coat protein) (30 ng each) in 25 L of BL21 competent cells. At the end of 1 hour of recovery, 20 cells were plated in LB boxes with ampicillin and kanamycin and were grown for 16 h at 37 C. CYP-SP protein was firstly expressed to subsequently express the CP coat protein. Two schemes with different inducer concentrations were followed. 1 L of culture was carried out distributed in 4 flasks with 250 mL each. 2.5 mL of a transformed strain preculture was taken to inoculate each 250 mL culture of TB medium with Amp and Km added with Thiamine 0.5 mM and trace elements. It was allowed to grow at 35 C. at 150 rpm for 7 h. At this point CYP-SF expression with 0.2% L-arabinose was induced and aminolevulinic acid 1 mM was added. Cultures were allowed to grow for 16 h more at 30 C. at 120 rpm. Subsequently, CP expression was induced by adding 0.5 mM of IPTG and cultures were grown for 3 h more at 30 C. and 150 rpm. For the second induction scheme, differential expression was performed as explained above, but 0.125% L-arabinose and 0.3 Mm IPTG were used to induce cultures. At the end of induction cultures were centrifuged cold at 3840g for 10 min, cells were resuspended in lysis buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.6) and the sample was sonicated. Centrifuged at 12,000g for 30 min at 4 C. and the supernatant was recovered (FIG. 8)

Example 2. CYP-VLPs Purification and Characterization

(49) D) At the end of induction, cultures were centrifuged cold at 4000g for 10 min, cells were resuspended in lysis buffer and the sample was sonicated. It was centrifuged at 12,000g for 30 min at 4 C. and the supernatant was recovered. P22 capsids containing (encapsulating) cytochrome P450 (CYP-P22) are purified by supernatant ultracentrifugation from cell lysis using a 35% sucrose cushion.

(50) CYP-SP Amount and Concentration Per P22 Capsid

(51) The number of enzymes per capsid was calculated using the following equation:

(52) $\mathrm{CYP},{\mathrm{SP}}_{\mathrm{per}\mathrm{capsid}}=\frac{M_{\mathrm{caspid}+\mathrm{CYP},\mathrm{SP}}-M_{\mathrm{capsid}}}{M_{\mathrm{CYP},\mathrm{SP}}}$

(53) where M.sub.capsid-CYP.SP=Absolute capsid mass with encapsulated enzyme (experimentally determined value by HPLC-MALS-RI). M.sub.capsid=46.6 kDa420 subunits=19572 kDa. MCYP. SP=71.5 kDa (theoretically calculated with Serial Cloner 2.6 program, Franck Prez, SerialBasics).

(54) Using the number of enzymes per capsid, the CYP-SP concentration in the sample was calculated as follows:
A.sub.T=A.sub.CP+A.sub.CYP.SP

(55) where AT is the total sample absorbance at 280 nm, ACP is the absorbance contribution of the coat protein and ACYP-SP is the absorbance contribution of CYPBM3 fused to the scaffolding protein. To measure the total sample absorbance, a P22-CYP aliquot encapsulated in PBS buffer was denatured with 6M guanidine chloride and -mercaptoethanol 1 mM, the Abs280 was recorded after 5 min of incubation. Above equation can be rewritten according to the Lambert-Beer Law as:
A.sub.T=C.sub.CP.sub.CPl+C.sub.CYP.SP.sub.CYP.SPl

(56) where CCP and CCYP.SP are protein concentrations for CP and CYP-SP respectively; refers to the extinction coefficients for each protein, .sub.280 CP-44920 M.sup.1 cm.sup.1 and .sub.280 CYP.SP-52830 M.sup.1 cm.sup.1 (theoretically calculated with the ProtParam program, Gasteiger, 2005), and is the distance traveled by light through the cell (in this case=1 cm).

(57) The equation was then put in terms of a single variable, CCYP-SP, using the relationship between the CP number and the CYP-SP number per capsid. Example (assuming that 109.7 CYP-SP per capsid was encapsulated):
420CP:109.7CYP-SP 420/109.7=3.8
C.sub.CP=3.8(C.sub.CYP.SP)
A.sub.T=3.8C.sub.CYP.SP.sub.CPl+C.sub.CYP.SP.sub.CYP.SPl

(58) Finally, CYP-SP concentration in the sample is calculated by substituting the value of and l constants in the equation, as well as the experimentally calculated value for total absorbance.

(59) Determination of Kinetic Parameters for P22-CYP Pseudoviral Particles

(60) kCat and KM catalytic (apparent) constants were determined for the CYP encapsulated in P22 and the free CYP using as substrate both 2,6-DMP and H.sub.2O2. To calculate the kinetic parameters using 2,6-DMP as a substrate, two curves were constructed with two different concentrations of hydrogen peroxide, 5 mM and 60 mM. Reactions were carried out in 0.1 mL (50 mM Tris-HCl pH 8) with the following phenol concentrations: 10, 25, 50, 125, 250 and 500 M. The reaction was started by adding 5 mM or 60 mM of H.sub.2O.sub.2. The amount of encapsulated enzyme used was 42.9 picomoles (assays with 5 mM H.sub.2O.sub.2) and 21.45 picomoles (assays with 60 mM H.sub.2O.sub.2); the amount of protein was determined by a concentration assay, specific for CYP450, for binding to CO. The catalytic activity was spectrophotometrically monitored at 468 nm (.sub.468=14800 M.sup.1 cm.sup.1 using an Agilent 8453 UV-vis spectrophotometer. Catalytic constants were obtained through the GraphPad Prism 6 program.

(61) For determination of kinetic parameters using free CYP, an amount of enzyme equal to 27.3 picomoles (assays with 5 mM H.sub.2O.sub.2) and 15 picomoles (assays with 60 mM H.sub.2O.sub.2) was used.

(62) Regarding the determination of kinetic parameters using H.sub.2O.sub.2 as a substrate, a fixed concentration of 2,6-DMP equal to 500 M was used for activity assays. Reactions were carried out in 0.1 mL (50 mM Tris-HCl pH 8) with the following peroxide concentrations: 1, 2, 5, 10, 20, 40 and 60 mM. The reaction was started by adding the corresponding amount of H.sub.2O.sub.2. The amount of encapsulated enzyme used was 42.9 and 21.45 picomoles; the amount of protein was determined by the concentration test, specific for CYP450, for binding to CO. Catalytic activity was spectrophotometrically monitored at 468 nm (.sub.468=14800 M.sup.1 cm.sup.1) using an Agilent 8453 UV-vis spectrophotometer. Catalytic constants were obtained through the GraphPad Prism 6 program. For determination of kinetic parameters using free CYP, an amount of enzyme equal to 30 and 15 picomoles was used.

(63) To determine the integrity of P22-CYP pseudo-viral particles in presence of 5 and 60 mM H.sub.2O.sub.2, 115 g of dissolved particles were incubated in 100 L (100 mM Tris-HCl pH 8) with the aforementioned hydrogen peroxide concentrations for 5 min. Subsequently, the capsid diameter was monitored by dynamic light scattering for 4 min.

(64) Temperature Stability of P22-CYP Pseudo-Viral Particles

(65) Stability of encapsulated CYP and the free enzyme was determined by measuring the percentage of activity retention at different times when the protein was incubated at 40 and 50 C. in a water bath. Incubation times for each of the temperatures were 0, 5, 10, 15 and 30 min. At the end of each time, an aliquot was removed from the sample, centrifuged for 1 min at 16,000g and left to rest for 10 min to temper the sample before measuring activity. Catalytic activity was measured in a final volume of 0.1 mL in 100 mM Tris-HCl buffer pH 8 using 500 M 2,6-DMP as substrate and initiating the reaction with 5 mM H.sub.2O.sub.2.

(66) pH Activity Profile and Acid pH Stability of P22-CYP Pseudoviral Particles

(67) To determine the activity profile at different pH values for the encapsulated and free enzyme, catalytic activity was measured at the following pHs: 5 (100 mM sodium acetate), 6 (100 mM potassium phosphate}, 7, 8 and 9 (100 mM Tris-HCl), 10 (100 mM borates). Catalytic activity was measured in a final volume of 0.1 mL using as substrate 500 M 2,6-DMP and initiating the reaction with 5 mM H.sub.2O.sub.2.

(68) The stability of encapsulated CYP and the free enzyme at acidic pH (pH 5 and 6) was determined by measuring the percentage of activity retention upon incubation of the protein at pH 5 and pH 6. The sample was incubated (at room temperature) for 1 and 16 h in 100 mM sodium acetate buffer for pH 5 and 100 mM potassium phosphate buffer for pH 6. At the end of each time, an aliquot was removed from the sample and centrifuged for 3 min at 16,000g. Catalytic activity was measured in a final volume of 0.1 mL in 100 mM Tris-HCl buffer pH 8 using as substrate 500 M 2,6-DMP and initiating the reaction with 5 mM H.sub.2O.sub.2.

(69) Stability of P22-CYP Pseudoviral Particles to Protease Degradation

(70) For proteolysis assays, the encapsulated and free enzyme was treated with 10 U of trypsin per mg protein, incubating for 1 and 20 h at room temperature. At the end of each time, an aliquot was taken out of the sample and the residual activity was measured in 100 mM Tris-HCl buffer pH 8 using 500 M 2,6-DMP as substrate and initiating the reaction with 5 mM H.sub.2O.sub.2.

(71) Determination of Iron Atoms in CYB-SP by ICP-MS

(72) The amount of iron and sulfur in a sample of P22-CYP pseudo-viral particles is determined by mass spectrometry with inductively coupled plasma source (ICPMS), in order to calculate the number of CYPBM3 with heme incorporated in the structure. Sulfur is used as a reference to calculate the number of capsids per liter in the sample. The number of sulfur atoms per capsid is equal to 8401 (23 S for each CYP-SP and 14 S for each CP).

(73) Taking into account that the limit of detection for Fe is 0.03 mg L.sup.1 and that of S is 0.1 mg L.sup.1, 21.6 mg of P22-CYP encapsulation were used to be above the detection limit for both atoms. The sample was incubated in concentrated nitric acid for 16 h at 70 C., once solubilized, the sample was taken to a final volume of 50 mL reaching a final concentration of 5% HNO.sub.3 in mQ water. A sample with the same amount of buffer in which the protein was dissolved was also prepared as a control, brought to a final volume of 50 mL at a final concentration of 5% HNO.sub.3 in mQ water.

(74) Confinement Molarity and Capsid Occupation Percentage (CCMV and P22)

(75) Enzyme concentration within the capsid, confinement molarity, was calculated by applying the following equation:

(76) $M_{\mathrm{conf}}=\frac{\left({\mathrm{Enzymes}}_{\mathrm{per}\mathrm{capsid}}\right)\left(\frac{1\mathrm{mol}}{6.022{10}^{23}\mathrm{enzymes}}\right)}{\mathrm{Internal}{\mathrm{volume}}_{\mathrm{capsid}}}$

(77) Internal volume of P22 capsid is 5.810-20 L (58000 nm.sup.3) with an r.sub.internal=24 nm

(78) Percentage of capsid occupation by the enzyme was determined as follows:

(79) $\%\mathrm{Ocupaci}\stackrel{}{o}n=\frac{\left({\mathrm{Enzymes}}_{\mathrm{per}\mathrm{capsid}}\right)\left({\mathrm{Volume}}_{\mathrm{CYP}}\right)}{\mathrm{Internal}{\mathrm{volume}}_{\mathrm{capsid}}}100$

(80) CYPBM3 volume, 150.5 nm.sup.3, was calculated by obtaining the average protein radius (3.3 nm) with support of the Maestro 9.6 software (Schrdinger, Inc.).

(81) VLPs Analysis by Transmission Electron Microscope

(82) 6 L of sample (about 100 g mL.sup.1) were deposited on a copper grid covered with Formvar (Electron Microscopy Science). After 1 min, the remaining liquid was removed with a Whatman filter paper. 6 L of 2% uranyl acetate was added to the grid, after 1 min the excess contrast agent was removed with filter paper. Samples were viewed with a JEOL JEM-2010 transmission electron microscope operated at 200 keV and equipped with a BioScan 600-W llK digital camera mounted on the upper part.

(83) In Vivo CYPBM3 Encapsulation Results in P22-CYP

(84) In order to perform CYPBM3 encapsulation within the bacteriophage P22 capsid, the gene coding for the enzyme was fused with the nucleotide sequence of a truncated version of the scaffold protein (SP). This fragment comprising the C-terminal domain of the scaffold protein, and including amino acids 141 to 303, interacts with the coat proteins to catalyze, stabilize and direct the procapsid formation geometry. Specific oligonucleotides were designed to amplify 21B3 mutant CYPBM3 gene, adding NcoI and BamHI sites at the ends thereof, and then cloning it into the desired vector in phase with the la SP truncated gene.

(85) Two different strategies were used for capsid production in vivo, CYP3M3-SP simultaneous expression and bacteriophage P22 coat protein (CP P22), and expression at different times of the two proteins. For the first strategy, the pETDuet plasmid was used in which both genes are under control of the same promoter. For the second strategy, genes were cloned in different vectors (pBAD-CYPBM3-SP and pRSF-CP P22) in order to induce genes differentially. For this case, the gene encoding the SP-enzyme was firstly expressed to subsequently carry out the induction of the coat protein gene.

(86) The ease in the purification method involving only two steps (ultracentrifugation and gel filtration chromatography), results in high purity, which is an important advantage in VLPs production derived from P22 bacteriophage. For both protocols, differential coexpression and expression correctly assembled capsids were found with a gel filtration chromatography retention time, of around 65 min; however, proportion of correctly assembled capsids to aberrant species which elute from the column at 45 min, is different for each case (FIGS. 2A-2D).

(87) These differences are due to differences in the different protocols implemented in expression, and therefore CYP-SP and CP P22 protein concentration as well as in the SP/CP P22 ratio, these two factors have an important influence on P22 capsid assembly.

(88) Obtained viral capsids (CYP-P22 nanoparticles) reached values of 120 mg/L of culture. The number of CYP molecules that can be loaded or contained in each capsid is from 90 to 150 molecules of CYP/capsid; more preferably 109.72.8 molecules of CYP/capsid, which results in a local enzyme concentration (confinement molarity=M.sub.conf) of 3.14 mM. Obtained capsids are quasi-spherical and nanoparticle (CYP-P22) diameter was 53.62 determined by HPLC gel filtration chromatography coupled to multi-angle laser light scattering (MALS) detectors, quasi-elastic light scattering (QELS) and refractive index (RI). The presence of quasi-spherical capsids correctly assembled with CYP inside was verified by TEM (FIGS. 3A and 3B).

Example 3. Stability of P22-CYP Nanoparticles

(89) E) The stability of encapsulated CYP and the free enzyme was evaluated at two different temperatures: 40 C. and 50 C. The stability at 40 C. for the free and the encapsulated enzyme is practically the same, while at 50 C., the inactivation of the encapsulated enzyme was even faster than that found for free CYP (FIGS. 15A and 15B).

(90) Activity profile was determined at different pH. As seen in the pH profile generated for the encapsulated and free enzyme, both graphs are very similar each other, with the exception of the optimum activity retention by one more pH unit (pH 8 and 9) for the P22-CYP case (FIG. 10). Due to virus intrinsic capacity to protect the material stored inside, stability of the encapsulated and free CYP in the presence of a protease was evaluated. After an hour of incubation with trypsin, the encapsulated enzyme retains practically all the activity (96%); while the free CYP loses 40%. After 20 hours of incubation the enzyme within the viral capsid retained 38% of the activity and the free CYP retained only 18% of its capacity to transform the substrate (FIG. 11).

Example 4. Immunogenicity of Biocatalytic Nanoparticles

(91) F) Pegylation of the catalytic loaded nanoparticles is subsequently carried out. Modification with polyethylene glycol of the viral capsid surface is carried out with bifunctional polyethylene glycol. In this stage, nanoparticle safety was evaluated on the activation of different subpopulations of lymphoid cells. Likewise, the toxicity of the nanoparticles on these same lymphoid cells is measured.

(92) Modification with polyethylene glycol of the viral capsid surface is carried out with maleimide polyethylene glycol ester of succinimide ester (Mal-PEG5000-NHS). The reaction is carried out at pH 8 with an excess of 5 on a molar basis to the capsid free amino acids.

(93) Immunoassays are performed by ELISA technique, following the endpoint titration method. To this end, 96-well plates covered with nanoparticles suspended in cover buffer are used. After three successive washings using wash buffer (i.e., Concentrated Wash Buffer: sodium chloride 1.4 mol/l in buffer phosphates 100 mmol/l and nonionic surfactant 0.1 g/l), blocking buffer is applied into the wells (i.e., 3% Bovine Serum Albumin} for 1 h at 37 C. After three washes, 100 L/rabbit serum well obtained at days 0, 10, 40, 70 is applied, as well as at the end of the immunization protocol (day 90), diluted serially with factor 2 from 1/1000 in the blocking buffer, and incubated for 1 h at 37 C. Plates are washed again and bound antibodies are detected using anti-rabbit IgG conjugated with alkaline phosphatase followed by the addition of the respective substrate (100 L/well of p-nitrophenyl phosphate dissolved in Tris buffer). Serum titre is estimated as the inverse of the dilution thereof that produces 50% of the maximum absorbance recorded and data are compared with the titres obtained with CYP without modification.

(94) Safety of CYP-P22 (nanoparticles loaded with cytochrome P450 activity) on activation of different lymphoid cell subpopulations is evaluated. For such end, peripheral blood mononuclear cells isolated from healthy blood bank donors are incubated in the presence of different amounts of nanoparticles for 24 or 48 hours and cytosine or chemokine secretion is quantified from culture supernatant by a multiplex assay with beads coupled to a panel of specific antibodies against these analytes.

Example 5. Targeting of Biocatalytic CYP-P22 (Nanoparticles Loaded with Cytochrome P450 Activity)

(95) G) Loaded nanoparticles are functionalized for targeting breast tumor cells, tumor tissues or other tissue of interest with a cyclic peptide or another ligand.

(96) To synthesize CYP-P22 (loaded nanoparticles) functionalized with Arg-Ala-Asp-D-Phe-Cys cyclic peptide (SEQ ID NO: 5) to be recognized by the integrin v3/5 of the breast tumor cells. Two peptide equivalents are added to the reaction mixture under gentle stirring for 12 h. The reaction product is expressed against a phosphate buffer.

Example 6. Catalytic Activity

(97) H) Catalytic capacity of P22 capsids with CYP inside is evaluated. Reactions were carried out in 0.1 mL (50 mM Tris-HCl pH 8) with the following phenol concentrations: 10, 25, 50, 125, 250 and 500 M. The reaction was started by adding 5 mM or 60 mM of H.sub.2O.sub.2. The amount of encapsulated enzyme used was 42.9 picomoles, the amount of protein was determined by a CO binding assay specific for CYP450. Catalytic activity was spectrophotometrically monitored at 468 nm (468=14800 M.sup.1 cm.sup.1). The enzyme encapsulated in this VLP is catalytically active using 2,6-DMP and H.sub.2O.sub.2 as substrates (FIG. 4). For calculations, the CYP concentration determined by the CO (catalytically active protein) assay is taken into account. The encapsulated enzyme follows a Michaelis-Menten kinetics as does free CYP. The CYP 450 loaded nanoparticle-bacteriophage P22 nanocapsid has the following constants with respect to free CYP.

(98) TABLE-US-00005 TABLE 3 CYP-P22 and free CYP Kinetics. 2,6-DMP (5 mM H.sub.2O.sub.2) k.sub.cat app K.sub.M app k.sub.cat/K.sub.M (min.sup.1) (mM) (min.sup.1 M.sup.1) CYP-P22 127.2 (2.3) 51.1 (3.2) 2.5 CYP free 143.6 (4.6) 24.9 (3.3) 5.8

Example 7. Transformation of Prodrugs with CYP

(99) A determination of expressed and purified CYP enzymatic activity, for example by means of tamoxifen transformation, is shown in FIG. 5. Reactions will be initiated by adding 5 mM of H.sub.2O.sub.2 or glucose oxidase+glucose and reaction progress is monitored by HPLC equipped with a C18 reverse phase column. The reactions are carried out in a final volume of 0.5 mL (100 mM potassium phosphate buffer pH 7.4, 2 mM ascorbic acid) with the following substrate concentrations: 20, 40, 80, 140 and 200 M. Methanol concentration in the reaction (the solvent in which tamoxifen is dissolved) was always the same (1%). The enzyme amount to be used per test was between 90 and 225 picomoles. Reactions are initiated by adding 5 mM H.sub.2O.sub.2, or glucose oxidase, and carried out at 25 C. for 5 min. Reactions are terminated by adding 50 uL of acetic acid to be subsequently centrifuged (3 min at 13,000 rpm) and analyzed by HPLC following the elution gradient below: 0 to 10 min solvent B 40%, 10 to 20 min solvent B up to 65% with a flow of 0.75 mL min-1. Mobile phase A consists of a 10 mM ammonium acetate buffer (pH 3) and mobile phase B, 100% acetonitrile. Decrease in tamoxifen peak is monitored at 280 nm.

(100) Bacterial CYP is capable of transforming the drug into four products (FIG. 5), which are detected by liquid nanochromatography coupled to tandem mass spectrometry (nanoLC/MSMS). Identified compounds correspond to 4-hydroxytamoxifen, 4-hydroxy-N-demethyltamoxifen (endoxifen), Ndemethyltamoxyphen and dihydroxy tamoxifen. It is important to mention that control of tamoxifen with hydrogen peroxide (without enzyme) does not generate any product profile.

Example 8. Affinity and Efficiency of CYP-P22 Biocatalytic Nanoparticles

(101) J) Affinity evaluation of functionalized loaded nanoparticles in tumor cells in vitro is carried out in human MCF7 breast cancer cells that are maintained in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum. Cells are cultured at 37 C. in 5% CO.sub.2 and before treatment with nanoparticles (CYP-P22), cells are washed twice with serum-free medium and then 1 mL of serum-free medium will be added. Subsequently, functionalized nanoparticles are added to each well and incubated by 4 hours. After incubation, supernatant is removed and 1 mL of fresh medium containing 10% fetal bovine serum is incubated for further 48 hours. The presence of biocatalytic nanoparticles (CYP-P22) with CYP activity is evaluated in the transformation of 7-benzyloxy-4-trifluoromethyl-coumarin (BFC) monitored by fluorescence according to the method of Donato et al. [58]. Fluorescence assay for determining CYP activity is performed with direct incubation of cultured tumor cells in a 12-well plate in the presence of 100 M of BFC (FIG. 6A-6F). BFC is added dissolved in acetonitrile, ensuring that final acetonitrile concentration in the wells does not exceed 0.5% (v/v). After 60 min of incubation at 37 C. the incubation medium is removed and conjugated products of CYP transformation will be hydrolyzed with a mixture of 3-glucuronidase/arylsulfatase for 2 hours at 37 C. Finally, samples are diluted with the corresponding solution and product (7-hydroxy-4-trifluoromethylcoumarin, HFC) fluorescence is quantified in a spectrofluorimeter with 410 nm excitation and 510 nm emission. DAPI staining shows n-labeled cell nuclei, panels 6A and 6D. Endogenous CYP activity on BFC reagent in untreated cells, panel 6B. Increased CYP activity in cells treated with CYP-P22, panel 6E. BFC reagent is transformed into a fluorescent product (HFC) and is located in cell cytoplasm (white arrows). Overlapping of DAPI and BFC stained images, panels 6C and 6F. Cells were observed with a 63X objective (DIC), 1.4 NA of plan-apochromatic oil immersion.

(102) Images clearly show that cytochrome P450 activity in tumor cells treated with biocatalytic nanoparticles (CYP-P22) is much greater than that found endogenously in untreated cells.

(103) Nanoparticle efficiency in prodrug activation is carried out by determining the viability of previously treated tumor cells with nanoparticles in the presence of the prodrug or in combination with a prodrug selected from: tamoxifen, resveratrol, tegafur, ifosfamide, clopidogrel, nabumetone, pafuramidine, loratadine.

INDUSTRIAL APPLICABILITY

(104) The present invention relates to a therapeutic strategy based on nanotechnology that incorporates or loads the cytochrome P450 in a nanoparticle or nanocapsid, which in turn carries this cytochrome P450 enzymatic activity to a tissue of interest; for example, in tumor cells, tumor tissues or others allowing a greater efficiency in the prodrug activation through CYP. The matter above, contributes in the treatment of cancer, provides greater efficiency of cancer treatment with chemotherapy or in other treatments where the prodrug is activated by cytochrome P450, a decrease in the medication doses required and a decrease in side effects. With this, increase the local concentration of active drug in the vicinity of tumor cells or tissues of interest, increasing the concentration of active drug and therefore the drug efficiency in tumor or target tissue and reducing the toxicity produced by the drug in the rest of the body cells.

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