Nanoarchitectonics of an acetogenin-enriched nanosystem mediated by an aqueous extract ofAnnona cherimolaMill with anti-inflammatory and proapoptotic activity against HepG2 cell line.

For the first time, this study shows the nanoarchitectonic process to obtain an acetogenin-enriched nanosystem (AuNPs-Ac) using an aqueous extract fromAnnona cherimolaMill (ACM) composed of gold nanoparticles embedded in an organic matrix that acts as stabilizing agent and presents anti-inflammatory activity and cytotoxical effect against HepG2 cell line, promoting apoptosis. The synthesis of AuNPs-Ac was confirmed by x-ray diffraction analysis, showing metallic gold as the only phase, and the scanning transmission microscope showed an organic cap covering the AuNPs-Ac. Fourier-transformed infrared suggests that the organic cap comprises a combination of different annonaceous acetogenins, alkaloids, and phenols by the presence of bands corresponding to aromatic rings and hydroxyl groups. High-Performance Liquid Chromatography has demonstrated the presence of annonacin, a potent acetogenin, in the extract of ACM. Anin vitroanti-inflammatory activity of the extract of ACM and the AuNPs-Ac was performed using the albumin denaturation method, showing a nonlinear response, which is better than sodium diclofenac salt in a wide range of concentrations that goes from 200 to 400µg ml-1with both samples. The viability assay was studied using trypan blue, treating IMR90 and HepG2 at different concentrations of AuNPs-Ac. The results defined a median lethal dose of 800µg ml-1against HepG2 through apoptosis according to the ratio of caspase-cleaved 9/alpha-tubulin evaluated. It was also demonstrated that the nanosystem presents a higher cytotoxic effect on the HepG2 cell line than in IMR90, suggesting a targeted mechanism. In addition, the nanosystem performs better than using only the extract of ACM in the anti-inflammatory or antiproliferative test, attributed to their higher surface area.

Enhancing antioxidant properties of CeO2 nanoparticles with Nd3+ doping: structural, biological, and machine learning insights.

The antioxidant capabilities of nanoparticles are contingent upon various factors, including their shape, size, and chemical composition. Herein, novel Nd-doped CeO2 nanoparticles were synthesized and the neodymium content was varied to investigate the synergistic impact on the antioxidant properties of CeO2 nanoparticles. Incorporating Nd3+ induced changes in lattice parameters and significantly altered the morphology from nanoparticles to nanorods. The biological activity of Nd-doped CeO2 was examined against pathogenic bacterial strains, breast cancer cell lines, and antioxidant models. The antibacterial and anticancer activities of nanoparticles were not observed, which could be associated with the Ce3+/Ce4+ ratio. Notably, the incorporation of neodymium improved the antioxidant capacity of CeO2. Machine learning techniques were employed to forecast the antioxidant activity to enhance understanding and predictive capabilities. Among these models, the random forest model exhibited the highest accuracy at 96.35%, establishing it as a robust computational tool for elucidating the biological behavior of Nd-doped CeO2 nanoparticles. This study presents the first exploration of the influence of Nd3+ on the structural, optical, and biological attributes of CeO2, contributing valuable insights and extending the application of machine learning in predicting the therapeutic efficacy of inorganic nanomaterials.

Potential application of a fungal co-culture crude extract for the conservation of post-harvest fruits.

Fungal plant pathogens are responsible for serious losses in many economically important crop species worldwide. Due to the use of fungicides and the fungi genome plasticity, multi-drug resistant strains are emerging as a new generation of pathogens, causing an expansive range of superficial and systemic plant infections, or new opportunistic fungal pathogens for humans. The group of antagonistic fungi Trichoderma spp. has been widely used to enhance plant growth and for the control of different pathogens affecting crops. Although Neurospora crassa is not a mycoparasitic fungus, its secretion of secondary metabolites with antimicrobial activity has been described. In this work, the effect of crude extract of the monoculture of Trichoderma asperellum T8a or the co-culture with N. crassa as an inhibitory treatment against the fungal pathogens Botrytis cinerea and Fusarium solani was evaluated. The findings demonstrate that the secondary metabolites contained in the T. asperellum crude extract have a clear fungistatic activity against B. cinerea and F. solani. Interestingly, this fungistatic activity highly increases when T. asperellum is co-cultivated with the non-pathogenic fungus N. crassa. Moreover, the co-culture crude extract also showed antifungal activity on post-harvest fruits, and no toxic effects on Murine fibroblast L929 (CCL-1) and murine macrophages RAW 264.7 (TIB-71) were observed. All these results together are solid evidence of the potential of the co-culture crude extract of T. asperellum and N. crassa, as an antifungal agent against phytopathogenic fungi, or post-harvest fruits during the transportation or commercialization time.

Lanthanide-Doped ZnO Nanoparticles: Unraveling Their Role in Cytotoxicity, Antioxidant Capacity, and Nanotoxicology.

This study used a sonochemical synthesis method to prepare (La, Sm)-doped ZnO nanoparticles (NPs). The effect of incorporating these lanthanide elements on the structural, optical, and morphological properties of ZnO-NPs was analyzed. The cytotoxicity and the reactive oxygen species (ROS) generation capacity of ZnO-NPs were evaluated against breast (MCF7) and colon (HT29) cancer cell lines. Their antioxidant activity was analyzed using a DPPH assay, and their toxicity towards Artemia salina nauplii was also evaluated. The results revealed that treatment with NPs resulted in the death of 10.559-42.546% and 18.230-38.643% of MCF7 and HT29 cells, respectively. This effect was attributed to the ability of NPs to downregulate ROS formation within the two cell lines in a dose-dependent manner. In the DPPH assay, treatment with (La, Sm)-doped ZnO-NPs inhibited the generation of free radicals at IC50 values ranging from 3.898 to 126.948 µg/mL. Against A. salina nauplii, the synthesized NPs did not cause death nor induce morphological changes at the tested concentrations. A series of machine learning (ML) models were used to predict the biological performance of (La, Sm)-doped ZnO-NPs. Among the designed ML models, the gradient boosting model resulted in the greatest mean absolute error (MAE) (MAE 9.027, R2 = 0.86). The data generated in this work provide innovative insights into the influence of La and Sm on the structural arrangement and chemical features of ZnO-NPs, together with their cytotoxicity, antioxidant activity, and in vivo toxicity.

Antibacterial Activity of Biosynthesized Copper Oxide Nanoparticles (CuONPs) Using Ganoderma sessile.

Copper oxide nanoparticles (CuONPs) were synthesized using an eco-friendly method and their antimicrobial and biocompatibility properties were determined. The supernatant and extract of the fungus Ganoderma sessile yielded small, quasi-spherical NPs with an average size of 4.5 ± 1.9 nm and 5.2 ± 2.1 nm, respectively. Nanoparticles were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and zeta potential analysis. CuONPs showed antimicrobial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa). The half-maximal inhibitory concentration (IC50) for E. coli was 8.5 µg/mL, for P. aeruginosa was 4.1 µg/mL, and for S. aureus was 10.2 µg/mL. The ultrastructural analysis of bacteria exposed to CuONPs revealed the presence of small CuONPs all through the bacterial cells. Finally, the toxicity of CuONPs was analyzed in three mammalian cell lines: hepatocytes (AML-12), macrophages (RAW 264.7), and kidney (MDCK). Low concentrations (<15 µg/mL) of CuONPs-E were non-toxic to kidney cells and macrophages, and the hepatocytes were the most susceptible to CuONPs-S. The results obtained suggest that the CuONPs synthesized using the extract of the fungus G. sessile could be further evaluated for the treatment of superficial infectious diseases.

Green nanoarchitectonics of carbon quantum dots from Cinchona Pubescens Vahl as targeted and controlled drug cancer nanocarrier.

Carbon quantum dots (CQDs) are a new carbon-based nanomaterial that has attracted tremendous attention due to their excellent fluorescent properties, chemical stability, water solubility, and biocompatibility features. Here, fluorescent CQDs synthesized by a green nanoarchitectonic method using Cinchona Pubescens Vahl extract were evaluated as drug nanocarriers for carboplatin (CBP) delivery. The characterization methods showed CQDs with semispherical shapes and sizes around 5 nm, temperature- and pH-dependent functional groups that interact with the CBP molecule adding specificity to the drug-delivery system. Based on the load efficiency results, it seems that the CQDs can carry almost 100 µg of carboplatin for every 1 mg of CQDs. This is possible due to the self-assembly process that takes place through the interaction between the protonation/deprotonation functional groups of CQDs and the hydrolyzed CBP molecule. Through this process, it is created spherical nanoparticles with an average size of 77.44 nm. The CQDs-CBP nanoparticles release the drug through a diffusion-controlled release mechanism where the acidic media is preferred, and the EPR effect also plays a helpful role. Besides, the viability test shows that the CQDs have almost null cytotoxicity suggesting that they could be used as a promising cancer treatment, improving the efficiency of cell internalization and significantly increasing their drug delivery.

Highly Antifungal Activity of Biosynthesized Copper Oxide Nanoparticles against Candida albicans.

Candida albicans (ATCC SC5314) was exposed to biosynthesized copper oxide nanoparticles (CuONPs) to determine their inhibitory capacity. Nanoparticles were polydisperse of small size (5.8 ± 3.5 nm) with irregular shape. The minimum inhibitory concentration (MIC) against C. albicans was 35.5 µg/mL. The production of reactive oxygen species (ROS) of C. albicans was verified when exposed to different concentrations of CuONPs. Ultrastructural analysis of C. albicans revealed a high concentration of CuONPs in the cytoplasm and outside the cell; also, nanoparticles were detected within the cell wall. Cytotoxic analyses using fibroblasts (L929), macrophages (RAW 264.7), and breast (MCF-12) cell lines show good results of cell viability when exposed at the MIC. Additionally, a hemocompatibility analysis was carried out and was found to be below 5%, considered the threshold for biocompatibility. Therefore, it is concluded that the biosynthesized CuONPs have a high potential for developing a topical antifungal treatment.

Targeted Enzymatic VLP-Nanoreactors with ß-Glucocerebrosidase Activity as Potential Enzyme Replacement Therapy for Gaucher's Disease.

Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29 mM) and the functionalized nanoreactors (KM =0.32 mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pH 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.

Optimized Synthesis of Small and Stable Silver Nanoparticles Using Intracellular and Extracellular Components of Fungi: An Alternative for Bacterial Inhibition.

Silver nanoparticles (AgNPs) represent an excellent option to solve microbial resistance problems to traditionally used antibiotics. In this work, we report optimized protocols for the production of AgNPs using extracts and supernatants of Trichoderma harzianum and Ganoderma sessile. AgNPs were characterized using UV-Vis spectroscopy and transmission electron microscopy, and the hydrodynamic diameter and Z potential were also determined. The obtained AgNPs were slightly larger using the fungal extract, and in all cases, a quasi-spherical shape was obtained. The mean sizes of AgNPs were 9.6 and 19.1 nm for T. harzianum and 5.4 and 8.9 nm for G. sessile using supernatant and extract, respectively. The AgNPs were evaluated to determine their in vitro antibacterial effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentration (MIC) was determined, and in all cases the AgNPs showed an antimicrobial effect, with a MIC varying from 1.26-5.0 µg/mL, depending on the bacterial strain and type of nanoparticle used. Cytotoxicity analyses of AgNPs were carried out using macrophages and fibroblast cell lines. It was determined that the cell viability of fibroblasts exposed for 24 h to different concentrations of AgNPs was more than 50%, even at concentrations of up to 20 µg/mL of silver. However, macrophages were more susceptible to exposure at higher concentrations of AgNPs as their viability decreased at concentrations of 10 µg/mL. The results presented here demonstrate that small AgNPs are obtained using either supernatants or extracts of both fungal strains. A remarkable result is that very low concentrations of AgNPs were necessary for bacterial inhibition. Furthermore, AgNPs were stable for more than a year, preserving their antibacterial properties. Therefore, the reported optimized protocol using fungal supernatants or extracts may be used as a fast method for synthesizing small AgNPs with high potential to use in the clinic.

Exploring In Vitro Biological Cellular Responses of Pegylated ß-Cyclodextrins.

ßCDPEG5 and ßCDPEG2 are two derivatives comprising seven PEG linear chains of 5 and 2 kDa, respectively, conjugated to ßCD. As ßCDPEGs display different physicochemical properties than their precursors, they could also trigger distinct cellular responses. To investigate the biological behavior of ßCDPEGs in comparison to their parent compounds, we performed broad toxicological assays on RAW 264.7 macrophages, MC3T3-E1 osteoblasts, and MDCK cells. By analyzing ROS and NO2- overproduction in macrophages, we found that ßCDPEGs induced a moderate stress response without affecting cell viability. Although MC3T3-E1 osteoblasts were more sensitive than MDCK cells to ßCDPEGs and the parent compounds, a similar pattern was observed: the effect of ßCDPEG5 on cell viability and cell cycle progression was larger than that of ßCDPEG2; PEG2 affected cell viability and cell cycle more than ßCDPEG2; cell post-treatment recovery was favorable in all cases, and the compounds had similar behaviors regarding ROS generation. The effect on MDCK cell migration followed a similar pattern. In contrast, for osteoblasts, the interference of ßCDPEG5 with cell migration was smaller than that of ßCDPEG2; likewise, the effect of PEG2 was shorter than its conjugate. Overall, the covalent conjugation of ßCD and PEGs, particularly to yield ßCDPEG2, improved the biocompatibility profile, evidencing that a favorable biological response can be tuned through a thoughtful combination of materials. Moreover, this is the first time that an in vitro evaluation of ßCD and PEG has been presented for MC3T3-E1 and MDCK cells, thus providing valuable knowledge for designing biocompatible nanomaterials constructed from ßCD and PEGs.

Boosting cell proliferation in three-dimensional polyacrylates/nanohydroxyapatite scaffolds synthesized by deep eutectic solvent-based emulsion templating.

Among three-dimensional (3D) scaffold fabrication methods, porous polymers templated using high internal phase emulsions (HIPEs) have emerged as an attractive method due to the facile generation of interconnected porosity through a variety of synthetic routes. These include a bottom-up approach to selectively incorporate nanomaterials onto the inner walls in a nonaqueous environment. In this work, novel nonaqueous HIPEs made of different (meth)acrylate monomers and a deep eutectic solvent (DES) were formulated with nonfunctionalized nanohydroxyapatite (NHA), which also played the role of cosurfactant. Free radical polymerization of HIPEs yielded free-standing nanocomposites with 3D interconnected macroporosity and nonfunctionalized NHA selectively decorating the scaffolds' inner surface. The influence of different polymer functionalities, acrylate or methacrylate, their alkyl tail length, and the presence of NHA on MC3T3-E1 preosteoblast cell proliferation in vitro, reactive oxygen species (ROS) production and alkaline phosphatase (ALP) activity were evaluated. All materials presented promising biocompatibility, non-hemolytic activity, negligible inflammatory response along to remarkably enhanced cell proliferation (e.g., up to 160-fold cell proliferation increase compared with polystyrene plate) in vitro, which open the path for the development of scaffolds in regenerative medicine. It is noteworthy that polyHIPEs studied here were obtained using a green synthetic protocol where nonfunctionalized nanoparticles can be selectively incorporated into a scaffolds' inner walls. This versatile technique allows for the simple construction of 3D bioactive nanocomposite scaffolds with varied compositions for cell culture.

Virus-Based Nanoreactors with GALT Activity for Classic Galactosemia Therapy.

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia.

Nanotoxicological study of downconversion Y2 O3 :Eu3+ luminescent nanoparticles functionalized with folic acid for cancer cells bioimaging.

Luminescent lanthanide downconversion nanoparticles (DCNPs) provide a combination of high luminescence intensity, sharp emission peaks with narrow bandwidth and a large Stokes' shift, leading to high-performance biomedical applications mainly for imaging. The purpose of this study is to present a nanotoxicological study of DCNPs Y2 O3 codoped with Eu3+ and functionalized with folic acid (FA). These assessments include cytotoxicity, genotoxicity, hemocompatibility, and in vitro inflammatory studies. We demonstrated by flow cytometry and confocal microscope the internalization of FA-DCNPs in breast cancer and melanoma cells. They were synthesized by sol-gel method and coated with a thin silica shell to make them biocompatible; also they were functionalized with amino groups and FA ligands that bind to the folate receptors (FR) located on the surface of the cancer cells studied. This functionalization enables the DCNPs to be internalized into the cancer cells via endocytosis by the conjugation FA-FR. The DCNPs were characterized with transmission electron microscope, Fourier transform infrared spectroscopy and photoluminescence. The nanotoxicological assessments demonstrated that both nanoparticles (bare and functionalized) are no cytotoxic and no genotoxic at the tested concentrations (0.01-20 µg/mL) in three cell lines (breast, skin cancer, and osteoblasts). Also they are hemocompatible and do not exert nitric oxide production in vitro by macrophages. The FA-DCNPs were clearly localized into the cell cytoplasm with bright red luminescence. Thus, herein we present a complete nanotoxicological study of FA-DCNPs Y2 O3 codoped with Eu3+ and we conclude that these nanoparticles are biocompatible and can be further used for cancer cells bioimaging.

Dual-photosensitizer coupled nanoscintillator capable of producing type I and type II ROS for next generation photodynamic therapy.

The current photodynamic therapy (PDT) is majorly hindered by the shallow penetration depth and oxygen dependency, limiting its application to deep-seated solid hypoxic tumors. Thus, it is meaningful to develop efficient X-ray mediated PDT system capable of generating reactive oxygen species (ROS) under both the normoxic and hypoxic conditions. Herein, we report the synthesis and characterization of nanocomposite, YAG:Pr@ZnO@PpIX with an amalgamation of UV-emitting Y2.99Pr0.01Al5O12 (YAG:Pr) nanoscintillator, and zinc oxide (ZnO) and protoporphyrin IX (PpIX) as photosensitizers. YAG:Pr surface was coated with a ZnO layer (∼10 nm) by atomic layer deposition, and then PpIX was covalently conjugated via a linker to give YAG:Pr@ZnO@PpIX. The photo- and cathodoluminescence analyses gave the evidences of efficient energy transfer from YAG:Pr to ZnO at ∼320 nm, and YAG:Pr@ZnO to PpIX at Soret region (350-450 nm). The nanohybrid was able to produce both, Type I and Type II ROS upon direct and indirect photoactivation with UV365nm and UV290nm, respectively. In vitro cytotoxicity of non-activated YAG:Pr@ZnO@PpIX in mouse melanoma cells revealed low toxicity, which significantly enhanced upon photoactivation with UV365nm indicating the photokilling property of the nanohybrid. Overall, our preliminary studies successfully demonstrate the potential of YAG:Pr@ZnO@PpIX to overcome the limited penetration and oxygen-dependency of traditional PDT.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.

Light sheet microscopy and SrAl2 O4 nanoparticles codoped with Eu2+ /Dy3+ ions for cancer cell tagging.

Light sheet optical microscopy on strontium aluminate nanoparticles (SrAl2 O4 NPs)1 codoped with Eu2+ and Dy3+ was used for cancer cell tagging and tracking. The nanoparticles were synthesized by urea-assisted combustion with optimized percentage values of the 2 codoping rare-earth ions for cell viability and for lower cytotoxic effects. The optical properties of these materials showed an excitation wide range of wavelengths (λexc = 254-460 nm), a broad emission band (λem = 475-575 nm) with the maximum centered wavelength at 525 nm and a half lifetime within the seconds regime. The feasibility to measure the nanoparticle luminescence under the selective plane illumination configuration was studied by immersing the nanoparticles in 1% Agarose. The potential applicability of the synthesized nanophosphors for cancer cell tagging was demonstrated by using in vitro experiments with human breast adenocarcinoma MCF-7 cells. A single MCF-7 cell observed by the use of light sheet microscopy with UV excitation. The cell has been bio-labeled with FA-SrAl2 04 : Eu2+ , Dy3+ NPs and 4',6-diamidino-2-phenylindole, dihydrochloride for nucleus identification.

Biocatalytic virus capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor cells.

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.

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery.

BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

Antioxidant Capacity of Poly(Ethylene Glycol) (PEG) as Protection Mechanism Against Hydrogen Peroxide Inactivation of Peroxidases.

The ability of poly(ethylene glycol) (PEG) to protect enzymatic peroxidase activity was determined for horseradish peroxidase (HRP), versatile peroxidase (VP), commercial Coprinus peroxidase (BP), and chloroperoxidase (CPO). The operational stability measured as the total turnover number was determined for the four peroxidases. The presence of PEG significantly increased the operational stability of VP and HRP up to 123 and 195%, respectively, and dramatically increased the total turnover number of BP up to 597%. Chloroperoxidase was not protected by PEG, which may be due to the different oxidation mechanism, in which the oxidation is mediated by hypochlorous ion instead of free radicals as in the other peroxidases. The presence of PEG does not protect the enzyme when incubated only in the presence of H2O2 without reducing substrate. The catalytic constants (k cat) are insensitive to the presence of PEG, suggesting that the protection mechanism is not due to a competition between the PEG and the substrate as electron donors. On the other hand, PEG showed to have a significant antioxidant capacity. Thus, we conclude that the protection mechanism for peroxidases of PEG is based in its antioxidant capacity with which it is able scavenge or drain radicals that are harmful to the protein.