Biocatalytic virus capsid as nanovehicle for enzymatic ...

Biotechnology Journal

Biotechnol. J. 2017, 12, 1600706

DOI 10.1002/biot.201600706

www.biotechnology-journal.com

Research Article

Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells Alejandro Tapia-Moreno, Karla Juarez-Moreno, Oscar Gonzalez-Davis, Ruben D. Cadena-Nava and Rafael Vazquez-Duhalt Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Ensenada, Baja California, Mexico

Most of the drugs used in chemotherapy should be activated by a transformation catalyzed by cytochrome P450 (CYP) enzymes. In this work, bacteriophage P22 virus-like particles (VLPs) containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed. The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P22. CYP and coat protein were both heterologously expressed in E. coli. The VLPs with enzymatic activity were covered with polyethylene glycol that was functionalized in its distal end with folic acid in order to be recognized by folate receptors exhibited on tumor cells. The capacity of biocatalytic VLPs to be recognized and internalized into tumor cells is demonstrated. The VLP-treated cells showed enhanced capacity for the transformation of the pro-drug tamoxifen, which resulted in an increase of the cell sensitivity to this oncological drug. In this work, the potential use of biocatalytic VLPs vehicles as a delivery system of medical relevant enzymes is clearly demonstrated. In addition to cancer treatment, this technology also offers an interesting platform as nano-bioreactors for intracellular delivery of enzymatic activity for other diseases originated by the lack of enzymatic activity.

Received Revised Accepted Accepted article online

29 NOV 2016 28 MAR 2017 31 MAR 2017 03 APR 2017

Keywords: Chemotherapy · Cytochrome P450 · Enzyme delivery · Nanoparticles · Virus-like particles

1 Introduction Nanotechnology is making huge progress in the industrial, biomedical and environmental fields. New materials, with unexpected properties, are rapidly developing from nanotechnological techniques. One of the most promising areas is the design and production of biocatalytic nanoreactors based on the combination of the catalytic properties of enzymes and the unique characteristics of nanosized materials [1–5]. This is, certainly, an opportunity to

Correspondence: Prof. Rafael Vazquez-Duhalt, Centro de Nanociencias y Nanotecnología UNAM, Km 107 carretera Tijuana-Ensenada, Ensenada, Baja California, 22860 Mexico E-mail: [email protected] Abbreviations: BFC, 7-benzyloxy-4-(trifluoromethyl)-coumarin; CYP, cyto chrome P450; 2,6-DMP, 2,6-Dimethoxyphenol; EPT, Enzyme Prodrug Therapy; FA, folic acid; 4HT, 4-hydroxy-tamoxifen; NHS, N-hydroxysuccinimide; PEG, polyethylene glycol; TEM, transmission electron microscopy; VLPs, virus-like particles

© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

solve different challenges in industrial, environmental processes, and especially in biomedical treatments. Nanomedicine is the use of nanomaterials for medical purposes. This strategy is making promising advances for anti-cancer schemes [6–8]. Abundant research efforts are currently performed to optimize nanovehicles for oncological drug delivery [9–11]. The main goal of nanocarriers is to design target therapeutics to improve the pharmaceutical and therapeutic benefits of conventional drugs. More than 14 million new cancer cases and eight million cancer deaths occurred in 2012 worldwide [12], and so far the most used strategy to treat cancer tumors is chemotherapy [13]. Conventional chemotherapeutic strategies imply the fact that cytotoxic drugs are not able to specifically recognize cancer cells. Thus, the administered substances and their active metabolites accumulate in most normal tissues as well as in tumors in which their concentrations are significantly higher than in serum. [14–16]. Nowadays, many studies using nanomaterials for oncological treatments focused precisely on drug delivery to specific tissues in the body to reduce the doses, minimize

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Scheme 1. Schematic representation of functionalized P22-CYP VLPs. (A) Assembly of VLP nanoparticles. This is due to non-covalent interaction between the scaffold protein and the interior of the coat protein. These biocatalytic VLPs are able to transform prodrugs into their active form. (B) Chemical functionalization. The VLPs are covered with polyethylene glycol containing folic acid at the polymer distal end. The PEG could mitigate the immune response. (C) Specific VLP delivery. The aim of VLPs functionalized with PEG-folic acid is to be targeted to the over-expressed receptors in tumor cells and once internalized be able to deliver the therapeutic enzyme activity.

side effects and improve its function compared to conventional treatments [17–20]. Among diverse approaches, Enzyme Prodrug Therapy (EPT) has been recently proposed for improving the efficiency and then minimizing the side effects of chemotherapeutic treatments [21, 22]. Also, emerging strategies involving the use of virus-like particles (VLPs) have been proposed. VLPs have demonstrated their potential use as nanobioreactors, which are suitable for the encapsulation of significant quantities of therapeutic enzymes used for cancer treatment along with their intrinsic properties of protease resistance and stability [23–26]. Thus, VLPs, as biological nanostructures, seem to be attractive vehicles for biocatalytic activity delivery. The presence of amine and carboxylic groups on the capsid surface allows the strategy to attach specific compounds, as ligands, to be recognized by the targeted cells [27], making these nanovehicles able to deliver therapeutic enzymes. Folate receptors in the tumor cells have been used for nanoparticle targeting [28, 29] The aim of this work is to demonstrate that VLPs bionanoreactors containing cytochrome P450 (CYP) can be specifically targeted to the over-expressed receptors in cancer cells. The functionalization of biocatalytic VLPs with polyethylene glycol (PEG), containing folic acid as receptor ligand, could improve the efficacy of the anti-carcinogenic pro-drug tamoxifen (Scheme 1). First, the coat protein from the P22 bacteriophage and a fusion protein containing CYP and the viral scaffold peptide were heterologously produced. Then, the biocatalytic VLPs were purified and chemically covered with PEG-containing folic acid at the polymer end as a ligand for cell receptors.

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2 Materials and methods 2.1 Materials Electrocompetent cells of E. coli BL21(DE3) were obtained from Lucigen (Middleton, WI). 2,6-Dimethoxyphenol (2,6-DMP), 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and hydrogen peroxide, were purchased from SigmaAldrich (St. Louis, MO). Heterobifunctionalized polyethylene glycol (PEG) linker (5000 Da) with folic acid (FA) and N-hydroxysuccinimide (NHS) at each respective end (FA-PEG-NHS) was purchased from Nanocs Inc. (New York, NY, USA). TOX1 in vitro toxicology assay kit was obtained from Sigma-Aldrich (St. Louis, MO). Copper grids (400-mesh) coated with formvar/carbon support film for TEM were purchased from TedPella (USA).

2.2 Plasmid constructs The pBAD CYP-SP plasmid (AmpR) containing the truncated scaffold protein SP141-303 fused to the cytochrome P450 variant “21B3” of CYPBM3 from Bacillus megaterium and the pRSF P22 plasmid (KamR) containing the P22 coat protein from the bacteriophage P22, CPP22, were obtained as previously described [26].

2.3 Differential protein expression and purification of the P22-CYP VLPs The protein expression strategy and the purification procedure were performed as previously described [26]. The first expression of CYP-SP gene was induced with 0.125%



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of l-arabinose, and subsequently, the coat protein (CPP22) was induced with 0.3 mM IPTG. The bacterial culture was centrifuged and the cell pellet resuspended in the lysis buffer and sonicated. Then, the lysed mixture was centrifuged (12 000 × g) and the supernatant was ultracentrifuged over a 35% w/v sucrose cushion at 215 000 × g for 50 min at 4°C in an OptimaXPN-100 ultracentrifuge (Beckman Coulter®) equipped with fixed angle rotor type 50.1 Ti (Beckman Coulter, Inc). The pellet was resuspended in PBS buffer and then purified through a size exclusion column (Sephacryl S-500, 60 × 1.6 cm GE Healthcare) in FPLC (AKTA Pharmacia). Fractions containing wellformed VLPs were pooled and ultracentrifuged. The purity of VLPs was confirmed by both gel electrophoresis and transmission electron microscopy (TEM). The VLPs concentration was estimated by UV absorbance at 280 nm according to Patterson et al. [30] using the Protein Calculator v3.3 (Chris Putnam, Scripps) and with an extinction coefficient of 44 920 M–1 cm–1 for coat protein and 52 830 M–1 cm–1 for CYP-SP.

2.4 Chemical functionalization of P22-CYP with NHS-PEG-FA Heterofunctionalized PEG with FA and succinimide moieties was covalently attached to solvent exposed free amino groups of the VLPs using the succinimide-activated esters. The reaction was performed with a NHS-PEGFA molar excess of 5 to CPP22 (15 mg mL–1) free amino groups on the capsid surface. The reaction was carried out in 50 mM sodium phosphate buffer containing 25 mM sodium chloride (pH 8.0) for 2 h at room temperature. Samples were purified by dialysis for 24 hr at 4°C against 2 L of 50 mM Tris-HCl buffer, pH 8.0.

2.5 Transmission electron microscopy TEM analyses were carried out as previously described [26]. A sample (10 µL) of a P22-CYP or P22-CYP-PEG-FA nanoparticles (0.1 mg mL–1) were applied to copper grids with carbon and Formvar coating, incubated (1 min) and then stained with 5 μL 1% uranyl acetate for 1 min. Samples were analyzed by TEM with an electron microscope JEOL JEM-2010 (JEOL Ltd., Peabody, MA, USA) operated at 100 kV. The electron microscopy images were processed using ImageJ software (NIH).

2.6 Dynamic light scattering Size and Z potential measurements of P22-CYP and P22CYP-PEG-FA samples were carried out with a Zetasizer NanoZS (Malvern, UK), which uses back scatter and phase analysis light scattering for size and Z potential determinations. VLPs samples were dialyzed in borate buffer (pH 9) at 4°C for more than 12 h, using a membrane cutoff of 8–12 kDa.



2.7 Enzymatic assays The enzymatic activities of free CYP, P22-CYP, and P22CYP-PEG-FA nanoparticles were determined in 1 mL reaction mixture containing 500 µM 2,6-dimetoxyphenol (2,6-DMP) in 50 mM Tris–HCl buffer (pH 8) at 25°C. The reactions were initiated by adding 5 mM H2O2 and monitored at 468 nm (e468 = 14 800 M–1 cm–1) using an Agilent 8453 UV-Vis spectrophotometer.

2.8 Cell lines and cell culture Human cervix carcinoma HeLa cells (ATCC- CCL-2) and human breast adenocarcinoma MCF7 cells (ATCC- HTB22) were purchased from the American Type Culture Collection (ATCC). HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (Gemini Bio Products), 1.5 g L–1 sodium bicarbonate, 1% l-glutamine and 1% Penicillinstreptomycin (Sigma-Aldrich). On the other hand, breast cancer MCF7 cells were cultured in Eagle’s Minimal Essential Medium (EMEM) enriched with 10% fetal bovine serum, 1.5 g L–1 sodium bicarbonate and 1% l-glutamine, and supplemented with 1% penicillin-streptomycin and 0.01 mg mL–1 of human recombinant insulin (Sigma-Aldrich). Cells were maintained and propagated in growth medium at 37°C and 5% CO2.

2.9 CYP enzymatic activity in tumor cells with or without treatment with biocatalytic VLPs The endogenous CYP activity of untreated cells and enhanced CYP activity of CYP-PEG-FA-treated cells were assayed in both HeLa and MCF7 cell lines. The transformation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to the fluorescent product 7-hydroxy-4-(trifluoromethyl)coumarin (HFC) was used to estimate the CYP activity according to Arora et al. [31] with some modifications previously described [26]. CYP activity was quantified spectrofluorimetrically by the transformation of BFC reagent to the fluorescent product HFC. As the control, endogenous CYP enzyme activity was evaluated in untreated HeLa and MCF7 cells. Briefly, 400 000 cells were seeded in the cell culture Petri dishes and incubated in maintenance cell culture medium for 12 h at 37°C and 5% CO2. The culture medium was then discarded, and the specific CYP substrate BFC (15 µL of 20 mM BFC in 150 µL of DMEM medium) was added and maintained in darkness for 10 min at room temperature. Then, 1.5 mL of DMEM medium was added and incubated for 30 min at 37°C and 5% CO2. Cells were rinsed three times with 1x PBS, and the CYP reaction was started by adding 4.5 µL of 1 M hydrogen peroxide. After 10 min reaction, cells were rinsed and harvested with trypsin/EDTA treatment. Cells were counted and their number adjusted at 250 000 cells. The fluorescence inten-

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sity of cell suspension was measured in a spectrofluoro meter (Hitachi F-7000), using an excitation wavelength at 254 nm and emission measurement at 510 nm.

2.10 Confocal microscopy cell imaging Treated and untreated HeLa and MCF7 cell cultures were incubated in the presence of BFC reagent for 10 min as described above and then fixed for 15 min with 4% formaldehyde-PBS solution at 4°C. After fixation, cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at 4°C in darkness. Cell nuclei were stained by incubating the cells with DAPI at 0.5 ng/mL in darkness for 10 min and then washed five times with 1x PBS. Stained cell nuclei were visualized with an inverted laser-scanning microscope (OlympusFluoview, FV-100) with excitation at 405 nm and emission through DAPI filters at 455 nm. The CYP activity was visualized by the BFC transformation into the fluorescent product HFC using an excitation wavelength of 488 nm and emission detection at 515–530 nm through a GFP filter. A Plan achromatic 60 X/1.48 N.A oil immersion objective was used. Laser intensity was kept at 20% to reduce photo-bleaching. A photomultiplier module allows providing a simultaneous view of the fluorescence in the entire cell. Confocal images were captured using the FV-10 ASW software and were analyzed with the FV10ASW viewer version 4.1 from Olympus.

2.11 Tamoxifen susceptibility assay Tamoxifen susceptibility was assayed in both human cervix adenocarcinoma (HeLa) cells or human breast adenocarcinoma (MCF7) cells as follows: A 96-well plate was used to seed 10 000 cells of HeLa or MCF7 cells per well and the cells were incubated for 24 h in their specific cell culture medium at 37°C and 5% CO2. Then, cell medium was discarded and 3.14 × 1011 of P22-CYP-PEGFA nanoparticles in 100 µL of DMEM medium were added to each well and then incubated for 12 h at 37°C and 5% CO2. This incubation time was selected after preliminary experiments at 4, 8 and 12 h, being the last that showed the highest nanoparticle internalization. After the incubation time, the medium was discarded, and 50 µL of DMEM medium containing 3 mM of hydrogen peroxide (final concentration) was added to each well and left reacting for 10 min at 37°C and 5% CO2 followed by a cell rinse with 1x PBS. Then, tamoxifen at different concentrations was added to each well. For HeLa cells, two-fold serial dilutions of tamoxifen ranging from 7.81; 15.62; 31.25 and 62.50 μM were added to each well in a final volume of 100 µL of DMEM medium. In the case of MCF7 cells, tamoxifen was added at 0.781, 1.56, 3.125, 6.25 and 12.5 μM to each well in a final volume of 100 µL of MCF7 cell medium. Both cell cultures were incubated for 18 h at 37°C and 5% CO2. After this time, the medium was removed and cells were washed out with 300 µL of 1x PBS

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and MTT cytotoxic determination assay was achieved. Tamoxifen sensitivity was compared to those from the control experiments without VLPs treatment. The experiments were performed in three independent replicates.

2.12 MTT cytotoxicity assay Both, HeLa and MCF7 cells with or without incubation with P22-CYP-PEG-FA were exposed to different concentrations of tamoxifen as described above. After drug incubation, the viability of cells was tested by a colorimetric assay based on the reduction of the MTT reagent (methy134-thiazolyltetrazolium) by using the TOX1 in vitro toxicology assay kit (Sigma-Aldrich). MTT reagent was added to the plate following the instructions of the manufacturer. The negative control of cell viability was obtained adding 100% DMSO (dimethyl sulfoxide), which induces total cell death. Cell survival positive control was achieved by incubating the cells with either DMEM or EMEM medium, simulating cell behavior under ideal conditions. Experiments were conducted in three independent replicates. The absorbance measurement of MTT reduction was achieved with a 96-well plate reader (Thermo Scientific) at 570 and 690 nm. The absorbance results from positive survival control (cell medium) were used to establish 100% of cell survival, then the direct comparison of the experimental groups was done and depicted as survival percentage related to the different tamoxifen concentrations.

3 Results 3.1 Biocatalytic nanoparticles production and characterization The virus-like nanoparticles (VLPs) containing CYP were produced by differential protein expression in E. coli cells, first induced by l-arabinose for the gene of the CYPBM3scaffold fusion protein (CYP-SP) and then with IPTG to induce the P22 coat protein (CP). The protein expression reached 120 mg of capsid proteins per liter of culture. The biocatalytic virus capsids of P22-CYP were purified by ultracentrifugation and gel permeation chromatography. The VLP preparations showed to be monodisperse with an average diameter of 53.8 ± 5 nm. The TEM analyzes showed that the packaging of the enzyme did not affect the assembly of the P22 capsid nanoparticles maintaining their quasispherical and well-structured form (Fig 1A). The effective expression of coat protein P22 and the fusion protein of scaffold peptide-cytochrome P450 (SPCYP) was demonstrated by gel electrophoresis (Fig. 2). Electrophoresis gel showed only two proteins after assembling of the viral capsids, one of the 71.5 kDa that corresponds to the SP-CYP and the P22 coat protein of 46.6 kDa. Images were taken on a Major Science Gel Documentation System, and the proportions of each pro-



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Figure 1. Transmission electron micrographs. (A) P22-CYP virus-like nanoparticles. (B) Functionalized P22-CYP-PEG-FA virus-like nanoparticles. VLPs were applied to copper grids with carbon and Formvar coating and stained with 1% uranyl acetate. Samples were analyzed by TEM with an electron microscope operated at 100 kV. Scale bar represents100 nm. Inserts, unmodified and pegylated VLPs. (C) Size distribution and Zeta potential of unmodified and pegylated VLPs.

tein were estimated by densitometry measurements with the software ImageJ (NHI) to determine the ratio of CP to SP-CYP fusion protein. It is known that the P22 capsid is formed by 420 coat proteins (60 hexamers and 12 pentamers) with the aid of 60 to 300 scaffold proteins [32, 33]. From the gel image analysis and considering the capsid internal volume of 5.8 × 104 nm3 [34] an internal CYP con-


centration of 3.14 mM has been estimated, representing 109.7 (± 2.8) CYP molecules per capsid.

3.2 Chemical functionalization of VLPs The surface of biocatalytic VLPs was covered with polyethylene glycol containing a folic acid moiety at the poly-

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Figure 2. Electrophoresis SDS-PAGE of purified VLPs. The 12% electrophoresis gel was stained with InstantBlue (Expedeon, Cambridge, UK). The VLPs contain only two proteins: the P22 coat protein (46.6 kDa) and the SP-CYP fusion protein (71.5 kDa). MWM, molecular weight markers. After pegylation different CPPEG conjugates could be detected. Well 1, molecular weight markers (MWM); well 2, non-pegylated VLPs (P22-CYP); and well 3, and pegylated VLPs (P22-CYP-PEG-FA).

mer distal end (P22-CYP-PEG-FA). The main goal of pegylation is to mitigate immune responses or adverse immunologically related responses associated with capsid proteins that may affect their safety and efficacy. The spherical shape is conserved after functionalization, and a covering layer of PEG could be observed (Fig. 1B). The effective pegylation was confirmed by SDS electrophoresis detected by Coomassie (Fig. 2). Coomassie blue is not a preferred detection system for pegylated proteins, since this dye interacts with free amino groups [35], which are bonded with PEG molecules, decreasing the sensitivity to this dye [36]. The CP protein has 20 sites susceptible to be pegylated and as expected, coat proteins showed different degrees of pegylation. In addition, the average nanoparticle diameter of pegylated and non-pegylated nanoparticles were analyzed by dynamic light scattering. The P22-CYP-PEG-FA showed an average diameter of 73.7 nm, which is 10 nm higher than these from nonpegylated VLPs (Fig. 1C). As expected, the Zeta potential of two preparations are different. P22-CYP showed –42.55 mV, while P22-CYP-PEG-FA showed a value of –12.6 mV. The VLPs pegylation was also confirmed by gel permeation chromatography.

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3.3 Catalytic characterization The catalytic activity of VLP preparations was evaluated with 2,6-dimetoxyphenol (Table 1). The non-functionalized VLPs (P22-CYP) showed 50% of the catalytic activity found in the free enzyme, based on its CYP content, while the catalytic activity of P22-CYP-PEG-FA nanoparticles is slightly lower than the uncovered VLPs showing the transformation of 1290 molecules of substrate per minute per biocatalytic capsid (Table 1). Table 1. Catalytic activity of free CYPBM3, and functionalized and nonfunctionalized CYP containing VLPs

Free CYP P22-CYP P22-CYP-PEG-FA

Specific activity (mU mg CYP–1)

VLPs activity (Substrate molecules min–1 capsid–1)

888 (± 3)b) 466 (± 24) 334 (± 39)

– 1746 (± 93) 1292 (± 149)

a) The reaction mixture contained a catalytic saturating concentration of 500 µM 2,6-DMP. b) Average values and standard deviation from three independent replicates.



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3.4 Tumor cell recognition and internalization The cell internalization capacity of P22-CYP-PEG-FA nanoparticles via folate receptor-mediated endocytosis was assayed in two cell lines (Figs. 3 and 4). Human cervix carcinoma HeLa and breast adenocarcinoma MCF7 cells were incubated with P22-CYP-PEG-FA nanoparticles. To corroborate the VLP uptake into both cancer cell lines, the intracellular activity of CYP was evaluated by the transformation of its specific substrate 7-benzyloxy-4-trifluoromethylcoumarin (BFC) into the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). The fluorescence of HFC was observed by confocal microscopy, and the cell internalization of P22-CYP-PEG-FA in both human cervix carcinoma (HeLa) cells and human breast adenocarcinoma (MCF7) cells was clearly demonstrated (Figs. 3 and 4). Cell nuclei were stained with DAPI labeled as “n”. The endogenous activity of CYP inside the cells was


observed in panels 3B and 4B. As expected, both the endogenous and the internalized P22-CYP-PEG-FA produced the fluorescence product HFC that is restricted to the cell cytoplasm as it is pointed out by arrows (panels 3B, 4B, 3H and 4H). The cytoplasmic fluorescence intensity of treated cells with P22-CYP-PEG-FA nanoparticles was considerably higher when compared with that observed in untreated cells (endogenous CYP, Figs. 3B and 4B) and treated with non-functionalized nanoparticles (P22-CYP) (Figs. 3E and 4E). Interestingly, control experiments with non-functionalized biocatalytic VLPs showed some CYP activity in the outer membrane of the tumor cells and not nanoparticle internalization (Figs. 3E and 4E) that could be attributed to unspecific adsorption of VLPs into cells.

Figure 3. Cytochrome P450 enzyme activity in human cervix carcinoma HeLa cells targeted with P22-CYP-PEG-FA nanoparticles. Staining with DAPI shows nuclei of HeLa cells labeled as “n” in panels (A), (D) and (G). Endogenous enzyme activity was measured by the transformation of BFC into the HFC fluorescent reagent as observed in panel (B). CYP activity of cells treated with non-functionalized VLPs is shown in panel (E). CYP activity of functionalized P22-CYP-PEG-FA in HeLa cells is shown in panel (H). Merge of DAPI and HFC localize the CYP activity in the cytoplasm of cells (yellow arrows) as observed in panels (C), (F) and (I). Scale bar represents 40 μm. As shown in histogram, panel (J), cytochrome P450 activity was measured by the fluorescence intensity of the transformation rate of BFC to HFC in 250 000 HeLa cells untreated and incubated with P22-CYP-PEG-FA. Fluorescence was detected with an excitation/emission spectra at 254/510 nm, respectively. Statistical significance was analyzed by the Student’s t test (**p