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Fullerenol-Based Intracellular Delivery of Methotrexate: A Water-Soluble Nanoconjugate for Enhanced Cytotoxicity and Improved Pharmacokinetics

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Abstract

Derivatization of fullerenes to polyhydroxylated fullerenes, i.e., fullerenols (FLU), dramatically decreases their toxicity and has been reported to enhance the solubility as well as cellular permeability. In this paper, we report synthesis of FLU as nanocarrier and subsequent chemical conjugation of Methotrexate (MTX) to FLU with a serum-stable and intracellularly hydrolysable ester bond between FLU and MTX. The conjugate was characterized for physiochemical attributes, micromeritics, drug-loading, and drug-release and evaluated for cancer cell-toxicity, cellular-uptake, hemocompatibility, protein binding, and pharmacokinetics. The developed hemocompatible FL-MTX offered lower protein binding vis-à-vis naïve drug and substantially higher drug loading. The conjugate offered pH-dependent release of 38.20 ± 1.19% at systemic pH and 85.67 ± 3.39% at the cancer cell pH. FLU-MTX-treated cells showed significant reduction in IC50 value vis-à-vis the cells treated with pure MTX. Analogously, the results from confocal scanning laser microscopy also confirmed the easy access of the dye-tagged FLU-MTX conjugate to the cell interiors. In pharmacokinetics, the AUC of MTX was enhanced by approx. 6.15 times and plasma half-life was enhanced by 2.45 times, after parenteral administration of single equivalent dose in rodents. FLU-MTX offered enhanced availability of drug to the biological system, meanwhile improved the cancer-cell cytotoxicity, sustained the effective plasma drug concentrations, and offered substantial compatibility to erythrocytes.

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References

  1. Kokubo K, Matsubayashi K, Tategaki H, Takada H, Oshima T. Facile synthesis of highly water-soluble fullerenes more than half-covered by hydroxyl groups. ACS Nano. 2008;2(2):327–33. https://doi.org/10.1021/nn700151z.

    Article  CAS  PubMed  Google Scholar 

  2. Maney V, Singh M. An in vitro assessment of novel chitosan/bimetallic PtAu nanocomposites as delivery vehicles for doxorubicin. Nanomedicine (Lond). 2017;12(21):2625–40. https://doi.org/10.2217/nnm-2017-0228.

    Article  CAS  Google Scholar 

  3. Pujara N, Siddharth J, Wong KY, McGuckin M, Popat A. Enhanced colloidal stability, solubility and rapid dissolution of resveratrol by nanocomplexation with soy protein isolate. J Colloid Interface Sci. 2017;488:303–8. https://doi.org/10.1016/j.jcis.2016.11.015.

    Article  CAS  PubMed  Google Scholar 

  4. Soo E, Thakur S, Qu Z, Jambhrunkar S, Parekh HS, Popat A. Enhancing delivery and cytotoxicity of resveratrol through a dual nanoencapsulation approach. J Colloid Interface Sci. 2016;462:368–74. https://doi.org/10.1016/j.jcis.2015.10.022.

    Article  CAS  PubMed  Google Scholar 

  5. Kevin A, Udofot O, Singh M, Krishnan S, Reams R, Rosenberg J, et al. Smart thermosensitive liposomes for effective solid tumor therapy and in vivo imaging. PLoS One. 2017;12:1–22.

    Google Scholar 

  6. Guan J, Zhou ZQ, Chen MH, Li HY, Tong DN, Yang J, et al. Folate-conjugated and pH-responsive polymeric micelles for target-cell-specific anticancer drug delivery. Acta Biomater. 2017;60:244–55. https://doi.org/10.1016/j.actbio.2017.07.018.

    Article  CAS  PubMed  Google Scholar 

  7. Xu C, Niu Y, Popat A, Jambhrunkar S, Karmakar S, Yu C. Rod-like mesoporous silica nanoparticles with rough surfaces for enhanced cellular delivery. J Mater Chem B. 2014;2(3):253–6. https://doi.org/10.1039/C3TB21431A.

    Article  CAS  Google Scholar 

  8. Mody KT, Mahony D, Zhang J, Cavallaro AS, Zhang B, Popat A, et al. Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to bovine viral diarrhoea virus E2 protein. Biomaterials. 2014;35:9972–83.

    Article  CAS  PubMed  Google Scholar 

  9. Abbaraju PL, Meka AK, Jambhrunkar S, Zhang J, Xu C, Popat A, et al. Floating tablets from mesoporous silica nanoparticles. J Mater Chem B. 2014;2(47):8298–302. https://doi.org/10.1039/C4TB01337A.

    Article  CAS  Google Scholar 

  10. Raza K, Thotakura N, Kumar P, Joshi M, Bhushan S. C60-fullerenes for delivery of docetaxel to breast cancer cells : a promising approach for enhanced efficacy and better pharmacokinetic profile. Int J Pharm. 2015;495(1):551–9. https://doi.org/10.1016/j.ijpharm.2015.09.016.

    Article  CAS  PubMed  Google Scholar 

  11. Sen T, Bruce I. Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations. Langmuir. 2005;21:7029–35.

    Article  PubMed  Google Scholar 

  12. Foley S, Crowley C, Smaihi M, Bonfils C, Erlanger BF, Seta P, et al. Cellular localisation of a water-soluble fullerene derivative. Biochem Biophys Res Commun. 2002;294(1):116–9. https://doi.org/10.1016/S0006-291X(02)00445-X.

    Article  CAS  PubMed  Google Scholar 

  13. Petrovic D, Seke M, Srdjenovic B, Djordjevic A. Applications of anti/Prooxidant fullerenes in nanomedicine along with fullerenes influence on the immune system. J Nanomater. 2015;2015:1–11. https://doi.org/10.1155/2015/565638.

    Article  Google Scholar 

  14. Lens M. Recent progresses in application of fullerenes in cosmetics. Recent Pat Biotechnol. 2011;5(2):67–73. https://doi.org/10.2174/187220811796365707.

    Article  CAS  PubMed  Google Scholar 

  15. Vraneš M, Borišev I, Tot A, Armaković S, Armaković S, Jović D, et al. Self-assembling, reactivity and molecular dynamics of fullerenol nanoparticles. Phys Chem Chem Phys. 2016;19(1):135–44. https://doi.org/10.1039/c6cp06847b.

    Article  PubMed  Google Scholar 

  16. Troshin PA, Kolesnikov D, Burtsev AV, Lubovskaya RN, Denisenko NI, Popov AA, et al. Bromination of [60] fullerene. I. High-yield synthesis of C60Br x (x= 6, 8, 24). Fullerenes, Nanotubes and Carbon Nanostruct. 2003;11(1):47–60. https://doi.org/10.1081/FST-120018666.

    Article  CAS  Google Scholar 

  17. Wu CY, Benet LZ. Predicting drug disposition via application of BCS: transport/absorption/elimination interplay and development of a biopharmaceutics drug disposition classification system. Pharm Res. 2005;22(1):11–23.

    Article  CAS  PubMed  Google Scholar 

  18. Radic S, Nedumpully-Govindan P, Chen R, Salonen E, Brown JM, Ke PC, et al. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding. Nano. 2014;6(14):8340–9.

    CAS  Google Scholar 

  19. Jia M, Li Y, Yang X, Huang Y, Wu H, Huang Y, et al. Development of both methotrexate and mitomycin C loaded PEGylated chitosan nanoparticles for targeted drug codelivery and synergistic anticancer effect. ACS Appl Mater Interfaces. 2014;6(14):11413–23.

    Article  CAS  PubMed  Google Scholar 

  20. Das M, Datir SR, Singh RP, Jain S. Augmented anticancer activity of a targeted, intracellularly activatable, theranostic nanomedicine based on fluorescent and radiolabeled, methotrexate-folic acid-multiwalled carbon nanotube conjugate. Mol Pharm. 2013;10(7):2543–57. https://doi.org/10.1021/mp300701e.

    Article  CAS  PubMed  Google Scholar 

  21. Joshi M, Kumar P, Kumar R, Sharma G, Singh B, Katare OP, et al. Aminated carbon-based “cargo vehicles” for improved delivery of methotrexate to breast cancer cells. Mater Sci Eng C. 2017;75:1376–88. https://doi.org/10.1016/j.msec.2017.03.057.

    Article  CAS  Google Scholar 

  22. Thotakura N, Sharma G, Singh B, Kumar V, Raza K. Aspartic acid derivatized hydroxylated fullerenes as drug delivery vehicles for docetaxel: an explorative study. Artif Cells Nanomed Biotechnol. 2017. https://doi.org/10.1080/21691401.2017.1392314.

  23. Lokerse WJ, Kneepkens EC, ten Hagen TL, Eggermont AM, Grüll H, Koning GA. Depth study on thermosensitive liposomes: optimizing formulations for tumor specific therapy and in vitro to in vivo relations. Biomaterials. 2016;82:138–50. https://doi.org/10.1016/j.biomaterials.2015.12.023.

    Article  CAS  PubMed  Google Scholar 

  24. Djordjević A, Vojinović-Miloradov M, Petranović N, Devečerski A, Lazar D, Ribar B. Catalytic preparation and characterization of C60Br24. Fuller Sci Technol. 1998;6(4):689–94. https://doi.org/10.1080/10641229809350229.

    Article  Google Scholar 

  25. Kokubo K, Shirakawa S, Kobayashi N, Aoshima H, Oshima T. Facile and scalable synthesis of a highly hydroxylated water-soluble fullerenol as a single nanoparticle. Nano Res. 2011;4(2):204–15. https://doi.org/10.1007/s12274-010-0071-z.

    Article  CAS  Google Scholar 

  26. Lin YS, Tungpradit R, Sinchaikul S, An FM, Liu DZ, Phutrakul S, et al. Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters. J Med Chem. 2008;51(23):7428–41. https://doi.org/10.1021/jm8006257.

    Article  CAS  PubMed  Google Scholar 

  27. Bhatia A, Singh B, Raza K, Wadhwa S, Katare OP. Tamoxifen-loaded lecithin organogel (LO) for topical application: development, optimization and characterization. Int J Pharm. 2013;444(1):47–59. https://doi.org/10.1016/j.ijpharm.2013.01.029.

    Article  CAS  PubMed  Google Scholar 

  28. Koch-Weser J, Sellers EM. Binding of drugs to serum albumin. N Engl J Med. 1976;294(6):311–6. https://doi.org/10.1056/NEJM197602052940605.

    Article  CAS  PubMed  Google Scholar 

  29. Semenov KN, Charykov NA, Murin IV, Pukharenko YV. Physico-chemical properties of the fullerenol-70 water solutions. J Mol Liq. 2015;202:1–8. https://doi.org/10.1016/j.molliq.2014.12.002.

    Article  CAS  Google Scholar 

  30. Saxena S, Saxena U. Development of bimetal oxide doped multifunctional polymer nanocomposite for water treatment. Int Nano Lett. 2016;6(4):223–34. https://doi.org/10.1007/s40089-016-0188-5.

    Article  CAS  Google Scholar 

  31. Raza K, Kumar D, Kiran C, Kumar M, Guru SK, Kumar P, et al. Conjugation of docetaxel with multiwalled carbon nanotubes and codelivery with piperine: implications on pharmacokinetic profile and anticancer activity. Mol Pharm. 2016;13(7):2423–32. https://doi.org/10.1021/acs.molpharmaceut.6b00183.

    Article  CAS  PubMed  Google Scholar 

  32. Chen Y, Sha X, Zhang W, Zhong W, Fan Z, Ren Q, et al. Pluronic mixed micelles overcoming methotrexate multidrug resistance: in vitro and in vivo evaluation. Int J Nanomedicine. 2013;8:1463–6. https://doi.org/10.2147/IJN.S42368.

    PubMed  PubMed Central  Google Scholar 

  33. Thakur CK, Thotakura N, Kumar R, Kumar P, Singh B, Chitkara D, et al. Polymeric nanocarriers with better delivery potential for tamoxifen. Int J Biol Macromol. 2016;93(Pt A):381–9. https://doi.org/10.1016/j.ijbiomac.2016.08.080.

  34. Anselmo AC, Mitragotri S. An overview of clinical and commercial impact of drug delivery systems. J Control Release. 2014;190:15–28. https://doi.org/10.1016/j.jconrel.2014.03.053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Singh A, Thotakura N, Kumar R, Singh B, Sharma G, Katare OP, et al. PLGA-soya lecithin based micelles for enhanced delivery of methotrexate: cellular uptake, cytotoxic and pharmacokinetic evidences. Int J Biol Macromol. 2017;95:750–6. https://doi.org/10.1016/j.ijbiomac.2016.11.111.

    Article  CAS  PubMed  Google Scholar 

  36. Misra C, Kumar M, Sharma G, Kumar R, Singh B, Katare OP, et al. Glycinated fullerenes for tamoxifen intracellular delivery with improved anticancer activity and pharmacokinetics. Nanomedicine. 2017;12(9):1011–23. https://doi.org/10.2217/nnm-2016-0432.

    Article  CAS  PubMed  Google Scholar 

  37. Zhang J, Chen XG, Huang L, Han JT, Zhang XF. Self-assembled polymeric nanoparticles based on oleic acid-grafted chitosan oligosaccharide: biocompatibility, protein adsorption and cellular uptake. J Mater Sci Mater Med. 2012;23(7):1775–83. https://doi.org/10.1007/s10856-012-4651-1.

    Article  CAS  PubMed  Google Scholar 

  38. Kumar P, Sharma G, Kumar R, Malik R, Singh B, Katare OP, et al. Vitamin-derived Nanolipoidal carriers for brain delivery of dimethyl fumarate: a novel approach with preclinical evidence. ACS Chem Neurosci. 2017;8(6):1390–6. https://doi.org/10.1021/acschemneuro.7b00041.

    Article  CAS  PubMed  Google Scholar 

  39. Raza K, Kumar N, Misra C, Kaushik L, Guru SK, Kumar P, et al. Dextran-PLGA-loaded docetaxel micelles with enhanced cytotoxicity and better pharmacokinetic profile. Int J Biol Macromol. 2016;88:206–12. https://doi.org/10.1016/j.ijbiomac.2016.03.064.

    Article  CAS  PubMed  Google Scholar 

  40. Lampman GM, Pavia DL, Kriz GS, Vyvyan JR. Introduction to spectroscopy, 4th ed.; 2010. p. 119–136.

  41. Indian Pharmacopoeia. The Indian Pharmacopoeia Commission, Ministry of Health and Family Welfare. Ghaziabad: Government of India; 2007.

  42. Troshin PA, Kemnitz E, Troyanov SI. Characterization of reactions of fullerene C 60 with bromine. Crystal structures of bromofullerenes C60 Br6, C60 Br6· CS2, C60 Br8· CHBr3·2Br2, and C60 Br24· C6 H4 Cl2· Br 2. Russ Chem Bull. 2004;53(12):2787–92. https://doi.org/10.1007/s11172-005-0191-x.

    Article  CAS  Google Scholar 

  43. Injac R, Prijatelj M, Strukelj B. Fullerenol nanoparticles: toxicity and antioxidant activity. Oxidative Stress Nanotechnol: Methods Protoc. 2013;1028:75–100. https://doi.org/10.1007/978-1-62703-475-3_5.

    Article  CAS  Google Scholar 

  44. Frohlich E. Hemocompatibility of inhaled environmental nano- particles: potential use of in vitro testing. J Hazard Mater. 2017;336:158–67.

  45. Narayanan D, Nair S, Menon DA. Systematic evaluation of hydroxyethyl starch as a potential nanocarrier for parenteral drug delivery. Int J Biol Macromol. 2015;74:575–84. https://doi.org/10.1016/j.ijbiomac.2014.12.012.

    Article  CAS  PubMed  Google Scholar 

  46. Raza K, Kumar P, Kumar N, Malik R. Pharmacokinetics and biodistribution of nanoparticles. In: Nimesh S, Chandra R, Gupta N, editors. Advances in nanomedicine for the delivery of therapeutic nucleic acids. Sawston: Woodhead publishing; 2017. p. 165–81. https://doi.org/10.1016/B978-0-08-100557-6.00009-2.

    Chapter  Google Scholar 

  47. Kumar M, Raza K. C60-fullerenes as drug delivery carriers for anticancer agents: Promises and hurdles. Pharm Nanotechnol. 2017;05:1–1. https://doi.org/10.2174/2211738505666170301142232.

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Acknowledgements

University Grant Commission, New Delhi, India, is highly acknowledged for Rajeev Gandhi National Fellowship to Mr. Manish Kumar (F1-17.1/2014-15/RGNF-2014-15 ST-RAJ-73879/(SA-III/Website)). This work would have been impossible without the gift sample of MTX from M/s Ipca Laboratories, Mumbai, India. Their support for the academic cause is highly appreciated.

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Authors and Affiliations

  1. Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Bandar Sindri, Dist., Ajmer, Rajasthan, 305817, India

    Shradha Bahuguna, Manish Kumar & Kaisar Raza

  2. Division of Pharmaceutics, University Institute of Pharmaceutical Sciences, Centre of Advanced Studies, Panjab University, Chandigarh, 160014, India

    Gajanand Sharma & Bhupinder Singh

  3. UGC-Centre of Excellence in Applications of Nanomaterials, Nanoparticles and Nanocomposites, Panjab University, Chandigarh, 160014, India

    Rajendra Kumar & Bhupinder Singh

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  1. Shradha Bahuguna
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Correspondence to Kaisar Raza.

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Bahuguna, S., Kumar, M., Sharma, G. et al. Fullerenol-Based Intracellular Delivery of Methotrexate: A Water-Soluble Nanoconjugate for Enhanced Cytotoxicity and Improved Pharmacokinetics. AAPS PharmSciTech 19, 1084–1092 (2018). https://doi.org/10.1208/s12249-017-0920-0

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