Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications in C-C bond formation and tandem catalysis

The straightforward synthesis of novel enzyme-metalNP nanobiohybrids in aqueous medium was developed. These new nanobiohybrids were excellent multivalent catalysts combining both activities in various sets of synthetic reactions even at ultra-low concentrations (ppb amount). © 2013 The Royal Society of Chemistry.This research was supported by The Spanish National Research Council (CSIC). Authors thank European Community (FP7-MULTIFUN) for the contract of M.M.Peer Reviewe

Parvalbumin-expressing ependymal cells in rostral lateral ventricle wall adhesions contribute to aging-related ventricle stenosis in mice

Aging-associated ependymal-cell pathologies can manifest as ventricular gliosis, ventricle enlargement, or ventricle stenosis. Ventricle stenosis and fusion of the lateral ventricle (LV) walls is associated with a massive decline of the proliferative capacities of the stem cell niche in the affected subventricular zone (SVZ) in aging mice. We examined the brains of adult C57BL/6 mice and found that ependymal cells located in the adhesions of the medial and lateral walls of the rostral LVs upregulated parvalbumin (PV) and displayed reactive phenotype, similarly to injury-reactive ependymal cells. However, PV+ ependymal cells in the LV-wall adhesions, unlike injury-reactive ones, did not express glial fibrillary acidic protein. S100B+/PV+ ependymal cells found in younger mice diminished in the LV-wall adhesions throughout aging. We found that periventricular PV-immunofluorescence showed positive correlation to the grade of LV stenosis in nonaged mice (10-month-old) PV-knock out (PV-KO) mice. This suggests an involvement of PV+ ependymal cells in aging-associated ventricle stenosis. Additionally, we observed a time-shift in microglial activation in the LV-wall adhesions between age-grouped PV- KO and wild-type mice, suggesting a delay in microglial activation when PV is absent from ependymal cells. Our findings implicate that compromised ependymal cells of the adhering ependymal layers upregulate PV and display phenotype shift to “reactive” ependymal cells in aging-related ventricle stenosis; moreover, they also contribute to the progression of LV-wall fusion associated with a decline of the affected SVZ-stem cell niche in aged mice

Percutaneous Alcohol Sclerotherapy of Simple Hepatic Cysts. Results From a Multicentre Survey in Italy

The increased use of Ultrasonography (US) has led to increased detection of simple hepatic cysts. For symptomatic cysts treatment is necessary. Until some years ago surgery was the only therapy. We have treated a large number of patients with Percutaneous Alcohol Sclerotherapy (PAS) and evaluated retrospectively the efficacy of this approach

Methods for preparation and administration of lutetium-177 oxodotreotide 3.7 GBq: proceedings from an Italian advisory board

n/

Applications of Nanomaterials Based on Magnetite and Mesoporous Silica on the Selective Detection of Zinc Ion in Live Cell Imaging

Functionalized magnetite nanoparticles (FMNPs) and functionalized mesoporous silica nanoparticles (FMSNs) were synthesized by the conjugation of magnetite and mesoporous silica with the small and fluorogenic benzothiazole ligand, that is, 2(2-hydroxyphenyl)benzothiazole (hpbtz). The synthesized fluorescent nanoparticles were characterized by FTIR, XRD, XRF, 13C CP MAS NMR, BET, and TEM. The photophysical behavior of FMNPs and FMSNs in ethanol was studied using fluorescence spectroscopy. The modification of magnetite and silica scaffolds with the highly fluorescent benzothiazole ligand enabled the nanoparticles to be used as selective and sensitive optical probes for zinc ion detection. Moreover, the presence of hpbtz in FMNPs and FMSNs induced efficient cell viability and zinc ion uptake, with desirable signaling in the normal human kidney epithelial (Hek293) cell line. The significant viability of FMNPs and FMSNs (80% and 92%, respectively) indicates a potential applicability of these nanoparticles as in vitro imaging agents. The calculated limit of detections (LODs) were found to be 2.53 X 10-6 and 2.55 X 10-6 M for Fe3O4-H@hpbtz and MSN-Et3N-IPTMS-hpbtz-f1, respectively. FMSNs showed more pronounced zinc signaling relative to FMNPs, as a result of the more efficient penetration into the cells.This research was funded by several sources. The URJC authors thank the financial support of theMinisterio de Economía y Competitividad and FEDER (Grants nos. CTQ2015-66164-R and CTQ2017-90802-REDT) and Universidad Rey Juan Carlos-Banco de Santander for supporting our excellence group QUINANOAP. The partial support of this work by the Isfahan University of Technology Research Council (grant number 500/95/24305 and the Iran National Science Foundation through INSF grant number 95828071 is also acknowledged. The CNIC is supported by the Spanish Ministerio de Ciencia, Innovación y Universidades and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). M.F. would like to thank MEyC for the research grant no. SAF2014-59118-JIN, co-funded by Fondo Europeo de Desarrollo Regional (FEDER) and COST Action CA1520: ‘European Network on NMR Relaxometry-EURELAX’. M.F. would also like to thank the Community of Madrid for research contract num. 2017-T1/BIO-4992 (‘Atraccion de Talento’ Action) cofunded by Universidad Complutense de Madrid

Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide

[EN] Production of citrus flavonoids prunin and naringenin was performed selectively through the enzyme hydrolysis of naringin, a flavonoid glycoside abundant in grapefruit wastes. To produce the monoglycoside flavonoid, prunin, crude naringinase from Penicillium decumbens was purified by a single purification step resulting in an enzyme with high -rhamnosidase activity. Both crude and purified enzymes were covalently immobilized on graphene oxide. The activity of the immobilized enzymes at different pH levels and temperatures, and the thermal stability were determined and compared with those exhibited by the free naringinases using specific substrates: p-nitrophenyl--d-glucoside (Glc-pNP) and p-nitrophenyl-alpha-l-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on GO were tested in the hydrolysis of naringin, giving naringenin and prunin, respectively, in excellent yields. The supported enzymes can be reused many times without loss of activity. The naringinase stabilized on GO has high potential to produce the valuable citrus flavonoids prunin and naringenin.Authors acknowledge the financial support from MICINN Project CTQ-2015-67592-P and Program Severo Ochoa (SEV-2016-0683). JVC thanks Universitat Politecnica de Valencia for predoctoral fellowships. JY and AC thank the support from the National Natural Science Foundation of China (Grant No. 21320102001) and the 111 Project (Grant No. B17020).Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Iborra Chornet, S.; Yu, J.... (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry. 21(4):839-849. https://doi.org/10.1039/c8gc03661fS839849214Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. J., Rosa, M. E., Calado, A. R., & Ribeiro, M. H. L. (2010). An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis. Process Biochemistry, 45(6), 841-850. doi:10.1016/j.procbio.2010.02.004C. Grassin and P.Fauquembergue , in Industrial Enzymology , ed. S. West and T. Godfrey , Nature Publishing Group , New York , 2nd edn, 1996 , p. 225Tsen, H.-Y., & Tsai, S.-Y. (1988). Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources. Journal of Fermentation Technology, 66(2), 193-198. doi:10.1016/0385-6380(88)90047-7SOARES, N. F. F., & HOTCHKISS, J. H. (1998). Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice. Journal of Food Science, 63(1), 61-65. doi:10.1111/j.1365-2621.1998.tb15676.xPuri, M., Kaur, H., & Kennedy, J. F. (2005). Covalent immobilization of naringinase for the transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80(10), 1160-1165. doi:10.1002/jctb.1303Norouzian, D., Hosseinzadeh, A., Inanlou, D. N., & Moazami, N. (1999). World Journal of Microbiology and Biotechnology, 15(4), 501-502. doi:10.1023/a:1008980018481Nishita, M., Park, S.-Y., Nishio, T., Kamizaki, K., Wang, Z., Tamada, K., … Minami, Y. (2017). Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness. Scientific Reports, 7(1). doi:10.1038/s41598-016-0028-xZhang, Y., Wu, C., Guo, S., & Zhang, J. (2013). Interactions of graphene and graphene oxide with proteins and peptides. Nanotechnology Reviews, 2(1), 27-45. doi:10.1515/ntrev-2012-0078Mathesh, M., Luan, B., Akanbi, T. O., Weber, J. K., Liu, J., Barrow, C. J., … Yang, W. (2016). Opening Lids: Modulation of Lipase Immobilization by Graphene Oxides. ACS Catalysis, 6(7), 4760-4768. doi:10.1021/acscatal.6b00942Li, W., Wen, H., Shi, Q., & Zheng, G. (2016). Study on immobilization of (+) γ-lactamase using a new type of epoxy graphene oxide carrier. Process Biochemistry, 51(2), 270-276. doi:10.1016/j.procbio.2015.11.030Hong, S.-G., Kim, J. H., Kim, R. E., Kwon, S.-J., Kim, D. W., Jung, H.-T., … Kim, J. (2016). Immobilization of glucose oxidase on graphene oxide for highly sensitive biosensors. Biotechnology and Bioprocess Engineering, 21(4), 573-579. doi:10.1007/s12257-016-0373-4Liu, F., Piao, Y., Choi, K. S., & Seo, T. S. (2012). Fabrication of free-standing graphene composite films as electrochemical biosensors. Carbon, 50(1), 123-133. doi:10.1016/j.carbon.2011.07.061Wang, Z., Zhou, X., Zhang, J., Boey, F., & Zhang, H. (2009). Direct Electrochemical Reduction of Single-Layer Graphene Oxide and Subsequent Functionalization with Glucose Oxidase. The Journal of Physical Chemistry C, 113(32), 14071-14075. doi:10.1021/jp906348xSingh, R. K., Kumar, R., & Singh, D. P. (2016). Graphene oxide: strategies for synthesis, reduction and frontier applications. RSC Advances, 6(69), 64993-65011. doi:10.1039/c6ra07626bVila-Real, H., Alfaia, A. J., Bronze, M. R., Calado, A. R. T., & Ribeiro, M. H. L. (2011). Enzymatic Synthesis of the Flavone Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase. Enzyme Research, 2011, 1-11. doi:10.4061/2011/692618Mamma, D., Kalogeris, E., Hatzinikolaou, D. G., Lekanidou, A., Kekos, D., Macris, B. J., & Christakopoulos, P. (2004). Biochemical Characterization of the Multi-enzyme System Produced byPenicillium decumbensGrown on Rutin. Food Biotechnology, 18(1), 1-18. doi:10.1081/fbt-120030382Chang, H.-Y., Lee, Y.-B., Bae, H.-A., Huh, J.-Y., Nam, S.-H., Sohn, H.-S., … Lee, S.-B. (2011). Purification and characterisation of Aspergillus sojae naringinase: The production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG-CoA reductase. Food Chemistry, 124(1), 234-241. doi:10.1016/j.foodchem.2010.06.024Yadav, S., Yadava, S., & Yadav, K. D. S. (2013). Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011. Process Biochemistry, 48(9), 1348-1354. doi:10.1016/j.procbio.2013.05.001Zhu, Y., Jia, H., Xi, M., Xu, L., Wu, S., & Li, X. (2017). Purification and characterization of a naringinase from a newly isolated strain of Bacillus amyloliquefaciens 11568 suitable for the transformation of flavonoids. Food Chemistry, 214, 39-46. doi:10.1016/j.foodchem.2016.06.108Zhang, T., Yuan, W., Li, M., Miao, M., & Mu, W. (2018). Purification and characterization of an intracellular α-l-rhamnosidase from a newly isolated strain, Alternaria alternata SK37.001. Food Chemistry, 269, 63-69. doi:10.1016/j.foodchem.2018.06.134Vila-Real, H., Alfaia, A. J., Rosa, J. N., Gois, P. M. P., Rosa, M. E., Calado, A. R. T., & Ribeiro, M. H. (2011). α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol–gel matrices: Activity and stability studies. Journal of Biotechnology, 152(4), 147-158. doi:10.1016/j.jbiotec.2010.08.005Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., … Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85. doi:10.1016/0003-2697(85)90442-7Erickson, H. P. (2009). Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy. Biological Procedures Online, 11(1), 32-51. doi:10.1007/s12575-009-9008-xZhang, J., Zhang, F., Yang, H., Huang, X., Liu, H., Zhang, J., & Guo, S. (2010). Graphene Oxide as a Matrix for Enzyme Immobilization. Langmuir, 26(9), 6083-6085. doi:10.1021/la904014zMarolewski, A. (1996). Fundamentals of Enzyme Kinetics. Revised Edition By Athel Cornish-Bowden. Portland Press, London. 1995. xiii + 343 pp. 17.5 cm × 24.5 cm. ISBN 1-85578-072-0. $29.00. Journal of Medicinal Chemistry, 39(4), 1010-1011. doi:10.1021/jm9508447Romero, C., Manjón, A., Bastida, J., & Iborra, J. (1985). A method for assaying the rhamnosidase activity of naringinase. Analytical Biochemistry, 149(2), 566-571. doi:10.1016/0003-2697(85)90614-1Fox, D. W., Savage, W. L., & Wender, S. H. (1953). Hydrolysis of Some Flavonoid Rhamnoglucosides to Flavonoid Glucosides. Journal of the American Chemical Society, 75(10), 2504-2505. doi:10.1021/ja01106a507Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426-428. doi:10.1021/ac60147a030LAEMMLI, U. K. (1970). Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227(5259), 680-685. doi:10.1038/227680a0Heukeshoven, J., & Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6(3), 103-112. doi:10.1002/elps.1150060302Sheldon, R. A., & van Pelt, S. (2013). Enzyme immobilisation in biocatalysis: why, what and how. Chem. Soc. Rev., 42(15), 6223-6235. doi:10.1039/c3cs60075

A Novel Halophilic Lipase, LipBL, Showing High Efficiency in the Production of Eicosapentaenoic Acid (EPA)

Background: Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity. Methods and Findings: A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C b-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80uC and the pH optimum determined at 25uC was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested. Conclusions: In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs

Synthesis of a theranostic platform based on fibrous silica nanoparticles for the enhanced treatment of triple-negative breast cancer promoted by a combination of chemotherapeutic agents.

A new series of theranostic silica materials based on fibrous silica particles acting as nanocarriers of two different cytotoxic agents, namely, chlorambucil and an organotin metallodrug have been prepared and structurally characterized. Besides the combined therapeutic activity, these platforms have been decorated with a targeting molecule (folic acid, to selectively target triple negative breast cancer) and a molecular imaging agent (Alexa Fluor 647, to enable their tracking both in vitro and in vivo). The in vitro behaviour of the multifunctional silica systems showed a synergistic activity of the two chemotherapeutic agents in the form of an enhanced cytotoxicity against MDA-MB-231 cells (triple negative breast cancer) as well as by a higher cell migration inhibition. Subsequently, the in vivo applicability of the siliceous nanotheranostics was successfully assessed by observing with in vivo optical imaging techniques a selective tumour accumulation (targeting ability), a marked inhibition of tumour growth paired to a marked antiangiogenic ability after 13 days of systemic administration, thus, confirming the enhanced theranostic activity. The systemic nanotoxicity was also evaluated by analyzing specific biochemical markers. The results showed a positive effect in form of reduced cytotoxicity when both chemotherapeutics are administered in combination thanks to the fibrous silica nanoparticles. Overall, our results confirm the promising applicability of these novel silica-based nanoplatforms as advanced drug-delivery systems for the synergistic theranosis of triple negative breast cancer.We would like to thank the funding of the Ministerio de Ciencia e Innovación of Spain (former Ministerio de Ciencia Innovación y Universidades of Spain) and FEDER, Una manera de hacer Europa for the grant number RTI2018-094322-B-I00. We would also like to thank Comunidad de Madrid for the predoctoral grant PEJD-2017-PRE/BMD3512 (I.M.-P.). M.M, Y.L.M., and M.F. are grateful to the Comunidad Autónoma de Madrid and FEDER for the I + D collaborative Programme in Biomedicine NIETO-CM (Project reference B2017-BMD3731). M.F. and K.O.P. thank the Comunidad Autonoma ´ de Madrid for research project No. 2017-T1/BIO-4992 (“Atraccion ´ de Talento” Action) cofunded by Universidad Complutense de Madrid. M.F is grateful to Instituto de Salud Carlos III (ISCIII) for project No DTS20/00109 (AES-ISCIII). M. M., M.F. and L.L.C would also like to thank Comunidad de Madrid for the predoctoral grant IND2020/BIO-17523. M.F. and K.O.P. acknowledge the support of Microscopy & Dynamic Imaging Unit of CNIC, Madrid, Spain. The Unit is part of the ReDiB-ICTS and has the support of FEDER, “Una manera de hacer Europa.” The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovacion ´ (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/ 501100011033).S

Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices

This is the peer reviewed version of the following article: J. M. Carceller, J. P. Martínez Galán, R. Monti, J. C. Bassan, M. Filice, J. Yu, M. J. Climent, S. Iborra, A. Corma, ChemCatChem 2020, 12, 4502, which has been published in final form at https://doi.org/10.1002/cctc.202000320. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] The crude naringinase from Penicillium decumbens and a purified naringinase with high a-L-rhamnosidase activity could be covalently immobilized on two-dimensional zeolite ITQ-2 after surface modification with glutaraldehyde. The influence of pH and temp. on the enzyme activity (in free and immobilized forms) as well as the thermal stability were detd. using the specific substrate: p-nitrophenyl-alpha-L-rhamnopyranoside (Rha-pNP). The crude and purified naringinase supported on ITQ-2 were applied in the hydrolysis of naringin, giving the flavonoids naringenin and prunin resp. with a conversion >90% and excellent selectivity. The supported enzymes showed long term stability, being possible to perform up to 25 consecutive cycles without loss of activity, showing its high potential to produce the valuable citrus flavonoids prunin and naringenin. We have also succeeded in the application of the immobilized crude naringinase on ITQ-2 for debittering grapefruit juices in a continuous process that was maintained operating for 300 h, with excellent results.The authors acknowledge financial support from PGC2018-097277-B-100 (MCIU/AEI/FEDER,UE) project and Program Severo Ochoa (SEV-2016-0683). Jilin agreement 111 Project (Grant No. B17020). JMC thanks to Universitat Politecnica de Valencia for predoctoral fellowships.Carceller-Carceller, JM.; Martínez Galán, JP.; Monti, R.; Bassan, JC.; Filice, M.; Yu, J.; Climent Olmedo, MJ.... (2020). Covalent Immobilization of Naringinase over Two-Dimensional 2D Zeolites and its Applications in a Continuous Process to Produce Citrus Flavonoids and for Debittering of Juices. ChemCatChem. 12(18):4502-4511. https://doi.org/10.1002/cctc.202000320S450245111218Puri, M., & Banerjee, U. C. (2000). Production, purification, and characterization of the debittering enzyme naringinase. Biotechnology Advances, 18(3), 207-217. doi:10.1016/s0734-9750(00)00034-3Vila-Real, H., Alfaia, A. J., Rosa, M. E., Calado, A. R., & Ribeiro, M. H. L. (2010). An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis. Process Biochemistry, 45(6), 841-850. doi:10.1016/j.procbio.2010.02.004RoitNer, M., Schalkhammer, T., & Pittner, F. (1984). Preparation of prunin with the help of immobilized naringinase pretreated with alkaline buffer. Applied Biochemistry and Biotechnology, 9(5-6), 483-488. doi:10.1007/bf02798402Ribeiro, I. A., Rocha, J., Sepodes, B., Mota-Filipe, H., & Ribeiro, M. H. (2008). Effect of naringin enzymatic hydrolysis towards naringenin on the anti-inflammatory activity of both compounds. Journal of Molecular Catalysis B: Enzymatic, 52-53, 13-18. doi:10.1016/j.molcatb.2007.10.011Puri, M., Marwaha, S. S., Kothari, R. M., & Kennedy, J. F. (1996). Biochemical Basis of Bitterness in Citrus Fruit Juices and Biotech Approaches for Debittering. Critical Reviews in Biotechnology, 16(2), 145-155. doi:10.3109/07388559609147419Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2015). Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts. Biotechnology Advances, 33(5), 435-456. doi:10.1016/j.biotechadv.2015.03.006Garcia-Galan, C., Berenguer-Murcia, Á., Fernandez-Lafuente, R., & Rodrigues, R. C. (2011). Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance. Advanced Synthesis & Catalysis, 353(16), 2885-2904. doi:10.1002/adsc.201100534ONO, M., TOSA, T., & CHIBATA, I. (1978). Preparation and properties of immobilized naringinase using tannin-aminohexyl cellulose. Agricultural and Biological Chemistry, 42(10), 1847-1853. doi:10.1271/bbb1961.42.1847Tsen, H.-Y., & Tsai, S.-Y. (1988). Comparison of the kinetics and factors affecting the stabilities of chitin-immobilized naringinases from two fungal sources. Journal of Fermentation Technology, 66(2), 193-198. doi:10.1016/0385-6380(88)90047-7SOARES, N. F. F., & HOTCHKISS, J. H. (1998). Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice. Journal of Food Science, 63(1), 61-65. doi:10.1111/j.1365-2621.1998.tb15676.xPuri, M., Kaur, H., & Kennedy, J. F. (2005). Covalent immobilization of naringinase for the transformation of a flavonoid. Journal of Chemical Technology & Biotechnology, 80(10), 1160-1165. doi:10.1002/jctb.1303Lei, S., Xu, Y., Fan, G., Xiao, M., & Pan, S. (2011). Immobilization of naringinase on mesoporous molecular sieve MCM-41 and its application to debittering of white grapefruit. Applied Surface Science, 257(9), 4096-4099. doi:10.1016/j.apsusc.2010.12.003Luo, J., Li, Q., Sun, X., Tian, J., Fei, X., Shi, F., … Liu, X. (2019). The study of the characteristics and hydrolysis properties of naringinase immobilized by porous silica material. RSC Advances, 9(8), 4514-4520. doi:10.1039/c9ra00075eNunes, M. A. P., Vila-Real, H., Fernandes, P. C. B., & Ribeiro, M. H. L. (2009). Immobilization of Naringinase in PVA–Alginate Matrix Using an Innovative Technique. Applied Biochemistry and Biotechnology, 160(7), 2129-2147. doi:10.1007/s12010-009-8733-6Busto, M. D., Meza, V., Ortega, N., & Perez-Mateos, M. (2007). Immobilization of naringinase from Aspergillus niger CECT 2088 in poly(vinyl alcohol) cryogels for the debittering of juices. Food Chemistry, 104(3), 1177-1182. doi:10.1016/j.foodchem.2007.01.033Huang, W., Zhan, Y., Shi, X., Chen, J., Deng, H., & Du, Y. (2017). Controllable immobilization of naringinase on electrospun cellulose acetate nanofibers and their application to juice debittering. International Journal of Biological Macromolecules, 98, 630-636. doi:10.1016/j.ijbiomac.2017.02.018Gong, X., Xie, W., Wang, B., Gu, L., Wang, F., Ren, X., … Yang, L. (2017). Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage. Scientific Reports, 7(1). doi:10.1038/s41598-017-17172-wCarceller, J. M., Martínez Galán, J. P., Monti, R., Bassan, J. C., Filice, M., Iborra, S., … Corma, A. (2019). Selective synthesis of citrus flavonoids prunin and naringenin using heterogeneized biocatalyst on graphene oxide. Green Chemistry, 21(4), 839-849. doi:10.1039/c8gc03661fPuri, M., Marwaha, S. S., & Kothari, R. M. (1996). Studies on the applicability of alginate-entrapped naringiase for the debittering of kinnow juice. Enzyme and Microbial Technology, 18(4), 281-285. doi:10.1016/0141-0229(95)00100-xNorouzian, D., Hosseinzadeh, A., Inanlou, D. N., & Moazami, N. (1999). World Journal of Microbiology and Biotechnology, 15(4), 501-502. doi:10.1023/a:1008980018481Saallah, S., Naim, M. N., Lenggoro, I. W., Mokhtar, M. N., Abu Bakar, N. F., & Gen, M. (2016). Immobilisation of cyclodextrin glucanotransferase into polyvinyl alcohol (PVA) nanofibres via electrospinning. Biotechnology Reports, 10, 44-48. doi:10.1016/j.btre.2016.03.003Cipolatti, E. P., Valério, A., Henriques, R. O., Moritz, D. E., Ninow, J. L., Freire, D. M. G., … de Oliveira, D. (2016). Nanomaterials for biocatalyst immobilization – state of the art and future trends. RSC Advances, 6(106), 104675-104692. doi:10.1039/c6ra22047aCorma, A., Fornes, V., & Rey, F. (2002). Delaminated Zeolites: An Efficient Support for Enzymes. Advanced Materials, 14(1), 71-74. doi:10.1002/1521-4095(20020104)14:13.0.co;2-wGallego, E. M., Portilla, M. T., Paris, C., León-Escamilla, A., Boronat, M., Moliner, M., & Corma, A. (2017). «Ab initio» synthesis of zeolites for preestablished catalytic reactions. Science, 355(6329), 1051-1054. doi:10.1126/science.aal0121Margarit, V. J., Díaz-Rey, M. R., Navarro, M. T., Martínez, C., & Corma, A. (2018). Direct Synthesis of Nano-Ferrierite along the 10-Ring-Channel Direction Boosts Their Catalytic Behavior. Angewandte Chemie, 130(13), 3517-3521. doi:10.1002/ange.201711418Barbosa, O., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Rodrigues, R. C., & Fernandez-Lafuente, R. (2014). Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization. RSC Adv., 4(4), 1583-1600. doi:10.1039/c3ra45991hSmith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., … Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85. doi:10.1016/0003-2697(85)90442-7Marolewski, A. (1996). Fundamentals of Enzyme Kinetics. Revised Edition By Athel Cornish-Bowden. Portland Press, London. 1995. xiii + 343 pp. 17.5 cm × 24.5 cm. ISBN 1-85578-072-0. $29.00. Journal of Medicinal Chemistry, 39(4), 1010-1011. doi:10.1021/jm9508447Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426-428. doi:10.1021/ac60147a030Cheong, M. W., Liu, S. Q., Zhou, W., Curran, P., & Yu, B. (2012). Chemical composition and sensory profile of pomelo (Citrus grandis (L.) Osbeck) juice. Food Chemistry, 135(4), 2505-2513. doi:10.1016/j.foodchem.2012.07.012Fox, D. W., Savage, W. L., & Wender, S. H. (1953). Hydrolysis of Some Flavonoid Rhamnoglucosides to Flavonoid Glucosides. Journal of the American Chemical Society, 75(10), 2504-2505. doi:10.1021/ja01106a507Corma, A., Fornes, V., Pergher, S. B., Maesen, T. L. M., & Buglass, J. G. (1998). Delaminated zeolite precursors as selective acidic catalysts. Nature, 396(6709), 353-356. doi:10.1038/24592Camblor, M. A., Corma, A., & Valencia, S. (1998). Characterization of nanocrystalline zeolite Beta. Microporous and Mesoporous Materials, 25(1-3), 59-74. doi:10.1016/s1387-1811(98)00172-3Beck, J. S., Vartuli, J. C., Roth, W. J., Leonowicz, M. E., Kresge, C. T., Schmitt, K. D., … Schlenker, J. L. (1992). A new family of mesoporous molecular sieves prepared with liquid crystal templates. Journal of the American Chemical Society, 114(27), 10834-10843. doi:10.1021/ja00053a02

Role of Folic Acid in the Therapeutic Action of Nanostructured Porous Silica Functionalized with Organotin(IV) Compounds against Different Cancer Cell Lines

The synthesis, characterization and cytotoxic activity against different cancer cell lines of various mesoporous silica-based materials containing folate targeting moieties and a cytotoxic fragment based on a triphenyltin(IV) derivative have been studied. Two different mesoporous nanostructured silica systems have been used: firstly, micronic silica particles of the MSU-2 type and, secondly, mesoporous silica nanoparticles (MSNs) of about 80 nm. Both series of materials have been characterized by different methods, such as powder X-ray diffraction, X-ray fluorescence, absorption spectroscopy and microscopy. In addition, these systems have been tested against four different cancer cell lines, namely, OVCAR-3, DLD-1, A2780 and A431, in order to observe if the size of the silica-based systems and the quantity of incorporated folic acid influence their cytotoxic action. The results show that the materials are more active when the quantity of folic acid is higher, especially in those cells that overexpress folate receptors such as OVCAR-3 and DLD-1. In addition, the study of the potential modulation of the soluble folate receptor alpha (FOLR1) by treatment with the synthesized materials has been carried out using OVCAR-3, DLD-1, A2780 and A431 tumour cell lines. The results show that a relatively high concentration of folic acid functionalization of the nanostructured silica together with the incorporation of the cytotoxic tin fragment leads to an increase in the quantity of the soluble FOLR1 secreted by the tumour cells. In addition, the studies reported here show that this increase of the soluble FOLR1 occurs presumably by cutting the glycosyl-phosphatidylinositol anchor of membrane FR-α and by the release of intracellular FR-α. This study validates the potential use of a combination of mesoporous silica materials co-functionalized with folate targeting molecules and an organotin(IV) drug as a strategy for the therapeutic treatment of several cancer cells overexpressing folate receptors.Spanish Government RTI2018-094322-B-I00 CTQ2017-90802-REDTMinistry of Research and Innovation, CNCS-UEFISCDI within PNCDI III PN-III-P4-ID-PCCF-2016-014