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Current Nanomedicine


ISSN (Print): 2468-1873
ISSN (Online): 2468-1881

Research Article

Non-Clinical Investigation of Tuberculosis Drugs: Conjugated Norbornene- Based Nanocarriers Toxic Impacts on Zebrafish

Author(s): Thangammal Anju*, Radhakrishnan Preetha, Raja Shunmugam, Shivshankar R. Mane, Jesu Arockiaraj and Shivasekar Ganapathy

Volume 11, Issue 4, 2021

Page: [224 - 236] Pages: 13

DOI: 10.2174/2468187312666211221130125

Price: $65


Introduction: Rifampicin conjugated (R-CP) and rifampicin-isoniazid dual conjugated (RI-CP) norbornene-derived nanocarriers are newly designed for pH stimuli-responsive delivery of tuberculosis (TB) drugs. Its biosafety level is yet to be well established.

Objectives: This study aimed to assess the impacts of the nanocarriers on liver cells using the zebrafish animal model and human liver cell line model (HepG2).

Methods: Initially, lethal dose concentration for the norbornene-derived nanocarrier systems in zebrafish was determined. The toxic effects were analysed at the sub-lethal drug concentration by histopathological study, total GSH level, gene expression, and DNA damage in zebrafish liver cells. Fish erythrocyte nuclear abnormalities were also evaluated. Cell viability and oxidative stress level (ROS generation) after exposure to the nanoconjugates were determined using HepG2 cells in the in vitro study.

Results: In vivo studies of both R-CP and RI-CP showed 100% mortality at 96 hours for exposure concentration >100mg/l and showed toxic changes in zebrafish liver histology, GSH, and DNA damage levels. Noticeably upregulated PXR, CYP3A, and cyp2p6 genes were observed in RI-CP exposure than in RIF or R-CP molecules. The in vitro study revealed a dose-dependent effect on cell viability and ROS generation for RIF, R-CP, and RI-CP exposures in HepG2 cells.

Conclusion: The current study reports that the rifampicin conjugated (R-CP) and rifampicin-isoniazid conjugated (RI-CP) norbornene derived nanocarriers exhibit enhanced toxic responses in both adult zebrafish and HepG2 cells. The pH-sensitive norbornene-derived nanocarriers on conjugation with different drugs exhibited varied impacts on hepatic cells. Hence the present investigation recommends a complete metabolomics analysis and norbornene carrier-drug interaction study to be performed for each drug conjugated norbornene nanocarrier to ensure its biosafety.

Keywords: Rifampicin, drug-carrier interaction, nanocarrier toxicity, norbornene, pH-sensitive, biosafety.

Graphical Abstract
Deng Y, Zhang X, Shen H, He Q, Wu Z, Liao W. Application of the nano-drug delivery system in treatment of cardiovascular diseases. Front Bioeng Biotechnol 2020; 7: 1-18.
Tapia-Hernández JA, Rodríguez-Felix F, Juárez-Onofre JE, et al. Zein-polysaccharide nanoparticles as matrices for antioxidant compounds: A strategy for prevention of chronic degenerative diseases. Food Res Int 2018; 111: 451-71.
[] [PMID: 30007708]
Moreno-v MJ, Rodríguez-f F, L AG, Del-toro-s CL. Heliyon Sustainable-green synthesis of silver nanoparticles using saf flower (Carthamus tinctorius L.) waste extract and its antibacterial activity. 2021.
Rodríguez-Félix F, Del-Toro-Sánchez CL, Javier Cinco-Moroyoqui F, et al. Preparation and characterization of quercetin-loaded zein nanoparticles by electrospraying and study of in vitro Bioavailability. J Food Sci 2019; 84(10): 2883-97.
[] [PMID: 31553062]
Mitchell MJ, Billingsley MM, Haley RM, Wechsler ME, Peppas NA, Langer R. Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov 2021; 20(2): 101-24.
[] [PMID: 33277608]
Pradhan D, Biswasroy P, Goyal A, Ghosh G, Rath G. Recent advancement in nanotechnology-based drug delivery system against viral infections. 2021.
He B, Sui X, Yu B, Wang S, Shen Y, Cong H. Recent advances in drug delivery systems for enhancing drug penetration into tumors. Drug Deliv 2020; 27(1): 1474-90.
[] [PMID: 33100061]
Jong WH De, Borm P JA. Drug delivery and nanoparticles : Applications and hazards. Int J Nanomedicine 2008; 3(2): 133-49.
Mudshinge SR, Deore AB, Patil S, Bhalgat CM. Nanoparticles: Emerging carriers for drug delivery. Saudi Pharm J 2011; 19(3): 129-41.
[] [PMID: 23960751]
Gringolts ML, Denisova YI, Finkelshtein ES, Kudryavtsev YV. Olefin metathesis in multiblock copolymer synthesis. Beilstein J Org Chem 2019; 15: 218-35.
[] [PMID: 30745996]
Gringolts ML, Denisova YI, Shandryuk GA, et al. Synthesis of norbornene-cyclooctene copolymers by the cross-metathesis of polynorbornene with polyoctenamer. RSC Advances 2015; 5(1): 316-9.
Zhang K, Tew GN. Cyclic polymers as a building block for cyclic brush polymers and gels. React Funct Polym 2014; 80(1): 40-7.
Walker CN, Sarapas JM, Kung V, Hall AL, Tew GN. Multiblock copolymers by thiol addition across norbornene. ACS Macro Lett 2014; 3(5): 453-7.
Mukherjee S, Dinda H, Chakraborty I, Bhattacharyya R, Das Sarma J, Shunmugam R. Engineering camptothecin-derived norbornene polymers for theranostic application. ACS Omega 2017; 2(6): 2848-57.
[] [PMID: 30023678]
Verduzco R, Li X, Pesek SL, Stein GE. Structure, function, self-assembly, and applications of bottlebrush copolymers. In: Chem Soc Rev. 2015; pp. 2405-20.
Rao NV, Dinda H, Venu P, Sarma J Das, Shunmugam R. Smart nanocarrier from norbornene based triblock copolymers for the sustained release of multi-cancer drugs’. RSC Advances 2014; 4: 45625-34.
Mane SR, Rao N. Amphiphilic homopolymer vesicles as unique nano-carriers for cancer therapy. Macromolecules 2012; 45(19): 8037-42.
Mane SR, Sathyan A, Shunmugam R. Synthesis of norbornene derived helical copolymer by simple molecular marriage approach to produce smart nanocarrier. Sci Rep 2017; 7: 44857.
[] [PMID: 28327656]
Mane SR, Sathyan A, Shunmugam R. Biomedical applications of pH-responsive amphiphilic polymer nanoassemblies. ACS Appl Nano Mater 2020; 2104-17.
Rao NV, Mane SR, Kishore A, Das Sarma J, Shunmugam R. Norbornene derived doxorubicin copolymers as drug carriers with pH responsive hydrazone linker. Biomacromolecules 2012; 13(1): 221-30.
[] [PMID: 22107051]
Gautam RK, Chattopadhyaya MC. Advanced Nanomaterials for Wastewater Remediation. 1st ed. CRC PRESS 2016.
Tina L, Id M, Hella J, et al. High burden of tuberculosis infection and disease among people receiving medication- assisted treatment for substance use disorder in Tanzania. 2021.
Pai M, Behr MA, Dowdy D, et al. Tuberculosis Nat Rev | Dis Prim 2016; 2(1): 1-23.
Chakaya J, Khan M, Ntoumi F, et al. Global tuberculosis report 2020 – reflections on the global tb burden , treatment and prevention efforts. Inter J Infec Dis 2020; 4-9.
Mane SR, Chatterjee K, Dinda H, Das Sarma J, Shunmugam R. Stimuli responsive nanocarrier for an effective delivery of multi-frontline tuberculosis drugs. Polym Chem 2014; 5(8): 2725-35.
Clark JRA, Dobson PJ, Mahapatra I, Owen R, Lead JR, Lynch I. Expert perspectives on potential environmental risks from nanomedicines and adequacy of the current guideline on environmental risk assessment. Environ Sci Nano 2018; 5(8): 1873-89.
Anju T, Preetha R, Shunmugam R, Mane SR, Arockiaraj J, Kumaresan V. Norbornene derived nanocarrier reduces isoniazid mediated liver toxicity: assessment in HepG2 cell line and zebrafish model. RSC Advances 2016; 6(115): 114927-36.
Krishnaraj C, Harper SL, Yun SI. In vivo toxicological assessment of biologically synthesized silver nanoparticles in adult Zebrafish (Danio rerio). J Hazard Mater 2016; 301: 480-91.
[] [PMID: 26414925]
Westerfield M. The zebrafish book a guide for the laboratory use of zebrafish (danio rerio). 4th ed. Eugene: University of Oregon Press 2000.
Fako VE, Furgeson DY. Zebrafish as a correlative and predictive model for assessing biomaterial nanotoxicity. Adv Drug Deliv Rev 2009; 61(6): 478-86.
[] [PMID: 19389433]
Chakraborty C, Sharma AR, Sharma G, Lee SS. Zebrafish: A complete animal model to enumerate the nanoparticle toxicity. J Nanobiotechnology 2016; 14(1): 65.
[] [PMID: 27544212]
Braunbeck T, Boettcher M, Hollert H, et al. Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species -- an update. Altern Anim Exp 2005; 22(2): 87-102.
[PMID: 15953964]
Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C Toxicol Pharmacol 2009; 149(2): 196-209.
[] [PMID: 19095081]
Saad M, Cavanaugh K, Verbueken E, Pype C, Casteleyn C. Xenobiotic metabolism in the zebra fish : a review of the spatiotemporal distribution, modulation and activity of Cytochrome P450 families 1 to 3. 2016.
Bresolin T, de Freitas Rebelo M, Celso Dias Bainy A. Expression of PXR, CYP3A and MDR1 genes in liver of zebrafish. Comp Biochem Physiol C Toxicol Pharmacol 2005; 140(3-4): 403-7.
[] [PMID: 15914091]
Tseng HP, Hseu TH, Buhler DR, Wang WD, Hu CH. Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva. Toxicol Appl Pharmacol 2005; 205(3): 247-58.
[] [PMID: 15922010]
Wei Y, Tang C, Sant V, Li S, Poloyac SM, Xie W. A molecular aspect in the regulation of drug metabolism: does PXR-induced enzyme expression always lead to functional changes in drug metabolism? Curr Pharmacol Rep 2017; 2(4): 187-92.
[] [PMID: 27795941]
Chen J, Raymond K. Roles of rifampicin in drug-drug interactions: underlying molecular mechanisms involving the nuclear pregnane X receptor. Ann Clin Microbiol Antimicrob 2006; 5: 3.
[] [PMID: 16480505]
Wang Y, Xiang X, Huang WW, et al. Association of PXR and CAR polymorphisms and antituberculosis drug-induced hepatotoxicity. Sci Rep 2019; 9: 2217.
Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther 2013; 138(1): 103-41.
[] [PMID: 23333322]
Bainya Afonso C D, Kubotaa Akira, Goldstone Jared V, et al. Functional characterization of a full length pregnane X receptor, expression in Vivo, and identification of PXR alleles, in zebrafish (Danio rerio). Aquat Toxicol 2013; 15: 447-57.
Schroeder I. A mechanistic study of organochlorine hepatotoxicity. 2011.
Gerets HHJ, Hanon E, Cornet M, et al. Selection of cytotoxicity markers for the screening of new chemical entities in a pharmaceutical context: a preliminary study using a multiplexing approach. Toxicol in vitro 2009; 23(2): 319-32.
[] [PMID: 19110050]
Gerets HHJ, Tilmant K, Gerin B, et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol Toxicol 2012; 28(2): 69-87.
[] [PMID: 22258563]
Van Rie A, Dow A. Neurodevelopmental trajectory of HIV-infected children accessing care in Kinshasa, democratic republic of congo. Test Chem 2011; 72(2): 181-204.
Preetha R, Jayaprakash NS, Singh ISB. Synechocystis MCCB 114 and 115 as putative probionts for Penaeus monodon post-larvae. 2007; 74: 243-7.
Culling CFA. Histopathological and histochemical techniques (including museum techniques). 1974.
Rahman I, Kode A, Biswas SK. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 2006; 1(6): 3159-65.
[] [PMID: 17406579]
Tsedensodnom O, Vacaru AM, Howarth DL, Yin C, Sadler KC. Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease. Dis Model Mech 2013; 6(5): 1213-26.
[PMID: 23798569]
Tice RR, Agurell E, Anderson D, et al. Single cell gel / comet assay : Guidelines for in vitro and in vivo genetic toxicology testing. 2000; 221: 206-21.
Babaei F, Ramalingam R, Tavendale A, et al. Novel blood collection method allows plasma proteome analysis from single zebrafish. J Proteome Res 2013; 12(4): 1580-90.
[] [PMID: 23413775]
Agrawal Karoon. Cleft palate repair and variations. Indian J Plast Surgery 2009; 42(3): 102-9.
Ateeq B, Abul farah M, Niamat Ali M, Ahmad W. Induction of micronuclei and erythrocyte alterations in the catfish Clarias batrachus by 2,4-dichlorophenoxyacetic acid and butachlor. Mutat Res 2002; 518(2): 135-44.
[] [PMID: 12113764]
Hoffman DJ, Rattner BA, Burton GAJ, Cairns JJ. Handbook of Ecotoxicology. 2nd ed. Lewis Publisher 2002.
Sajid M, Ilyas M, Basheer C, et al. Impact of nanoparticles on human and environment: review of toxicity factors, exposures, control strategies, and future prospects. Environ Sci Pollut Res Int 2015; 22(6): 4122-43.
[] [PMID: 25548015]
Nel A, Xia T, Mädler L, Li N. Toxic potential of materials at the nanolevel. Science (80- ) 2006; 311(5761): 622-7.
Larsson Å, Bengtsson BE. Disturbed ion balance in flounder. Aquat Toxicol 1981; 1(1): 19-35.
Weis JS, Smith G, Zhou T, Santiago-bass C, Weis P. Effects of contaminants on behavior: Biochemical mechanisms and ecological consequences. 2001; 51(3): 209-17.
Figueiredo-fernandes A, Ferreira-cardoso J V, Garcia-santos S, et al. Histopathological changes in liver and gill epithelium of Nile tilapia , Or eochromis niloticus , exposed to waterborne copper 1 Oreochromis. 2007; 27(3): 103-9.
Abar M, Akbulut C, Yön ND, et al. Biosciences histological changes in the liver of the Zebrafish, (Danio Rerio) after exposure to poly (2-ethyl-2-oxazoline). Biosciences 2015; 80: 30891-4.
Yön Nd, Akbulut Cansu AM, Kayhan FE, Kaymak G. Histological changes in the liver of the Swordtail fish , Xiphophorus helleri (Pisces : Poecilidae) after exposure to deltamethrin author’s names and affiliations histological changes in the liver of the Swordtail fish. xiphophorus helleri. 2014.
Eroglu A, Dogan Z, Kanak EG, Atli G, Canli M. Effects of heavy metals (Cd, Cu, Cr, Pb, Zn) on fish glutathione metabolism. Environ Sci Pollut Res Int 2015; 22(5): 3229-37.
[] [PMID: 24793073]
Oberdörster E. Manufactured nanomaterials (fullerenes, C60) induce oxidative stress in the brain of juvenile largemouth bass. Environ Health Perspect 2004; 112(10): 1058-62.
[] [PMID: 15238277]
Zhang DL, Liu SY, Zhang J, Hu CX, Li DH, Liu YD. Antioxidative responses in zebrafish liver exposed to sublethal doses Aphanizomenon flos-aquae DC-1 aphantoxins. Ecotoxicol Environ Saf 2015; 113(DECEMBER): 425-32.
[] [PMID: 25544652]
Timme-Laragy AR, Van Tiem LA, Linney EA, Di Giulio RT. Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish. Toxicol Sci 2009; 109(2): 217-27.
[] [PMID: 19233942]
Watkins RE, Wisely GB, Moore LB, et al. The human nuclear xenobiotic receptor PXR: Structural determinants of directed promiscuity. Science (80- ) 2001; 292(5525): 2329-33.
Maglich JM, Caravella JA, Lambert MH, Willson TM, Moore JT, Ramamurthy L. The first completed genome sequence from a teleost fish (Fugu rubripes) adds significant diversity to the nuclear receptor superfamily. Nucleic Acids Res 2003; 31(14): 4051-8.
[] [PMID: 12853622]
Kubota A, Goldstone JV, Lemaire B, Takata M, Woodin BR, Stegeman JJ. Role of pregnane X receptor and aryl hydrocarbon receptor in transcriptional regulation of pxr, CYP2, and CYP3 genes in developing zebrafish. Toxicol Sci 2015; 143(2): 398-407.
[] [PMID: 25424564]
Cheng J, Ma X, Krausz KW, Idle JR, Gonzalez FJ. Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity. Drug Metab Dispos 2009; 37(8): 1611-21.
[] [PMID: 19460945]
Wassmur B. Detoxification Mechanisms in Fish. University of Gothenburg 2012.
Li F, Lu J, Cheng J, et al. Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid co-therapy. Nat Med 2013; 19(4): 418-20.
[] [PMID: 23475203]
Jatav SK, Kulshrestha A, Zacharia A, et al. Spirulina maxima protects liver from isoniazid and rifampicin drug toxicity. J Evid Based Complementary Altern Med 2014; 19(3): 189-94.
[] [PMID: 24742608]
Rocco L, Frenzilli G, Fusco D, Peluso C, Stingo V. Evaluation of zebrafish DNA integrity after exposure to pharmacological agents present in aquatic environments. Ecotoxicol Environ Saf 2010; 73(7): 1530-6.
[] [PMID: 20696478]
Mekkawy IA, Mahmoud UM, Sayed Ael-D. Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822). Tissue Cell 2011; 43(4): 223-9.
[] [PMID: 21501852]
Sayed AEH, Mahmoud UM, Mekkawy IA. Erythrocytes alterations of monosex tilapia (Oreochromis niloticus, Linnaeus, 1758) produced using methyltestosterone. Egypt J Aquat Res 2016; 42(1): 83-90.
Siu WHL, Cao J, Jack RW, et al. Application of the comet and micronucleus assays to the detection of B[a]P genotoxicity in haemocytes of the green-lipped mussel (Perna viridis). Aquat Toxicol 2004; 66(4): 381-92.
[] [PMID: 15168946]
Ahmed MK, Habibullah-Al-Mamun M, Hossain MA, et al. Assessing the genotoxic potentials of arsenic in tilapia (Oreochromis mossambicus) using alkaline comet assay and micronucleus test. Chemosphere 2011; 84(1): 143-9.
[] [PMID: 21382637]
Faßbender C, Braunbeck T. Assessment of genotoxicity in gonads, liver and gills of zebrafish (Danio rerio) by use of the comet assay and micronucleus test after In Vivo exposure to methyl methanesulfonate. Bull Environ Contam Toxicol 2013; 91(1): 89-95.
[] [PMID: 23620131]
Mitchelmore C, Chipman JK. Detection of DNA strand breaks in brown trout (Salmo trutta) hepatocytes and blood cells using the single cell gel electrophoresis (comet) assay. Aquat Toxicol 1998.
Cotelle S, Férard JF. Comet assay in genetic ecotoxicology: a review. Environ Mol Mutagen 1999; 34(4): 246-55.
[<246::AID-EM4>3.0.CO;2-V] [PMID: 10618172]
Deventer K. Detection of genotoxic effects on cells of liver and gills of B. rerio by means of single cell gel electrophoresis. Bull Environ Contam Toxicol 1996; 56(6): 911-8.
[] [PMID: 8661880]
Miyawaki I. Application of zebrafish to safety evaluation in drug discovery. J Toxicol Pathol 2020; 33: 197-210.
Profile SEE. A molecular toolbox for genetic manipulation of zebrafish. Adv Genom Genet 2015.
Horzmann KA, Freeman JL. Making Waves: New developments in toxicology with the zebrafish. 2018; 163(1): 5-12.
Zakaria ZZ, Benslimane FM, Nasrallah GK, et al. Using zebrafish for investigating the molecular mechanisms of drug-induced cardiotoxicity. 2018.
Pamela J, Tal T, Yaghoobi B, Lein PJ. Sciencedirect toxicology translational toxicology in zebrafish. Curr Opin Toxicol 2020; 23–24: 56-66.
Vahdati-Mashhadian N, Jafari MR, Sharghi N, Sanati T. Protective effects of vitamin C and NAC on the toxicity of rifampin on HepG2 cells. Iran J Pharm Res 2013; 12(1): 141-6.
[PMID: 24250582]
Kushwaha A, Rani Radha, A V. Environmental fate and ecotoxicity of engineered nanoparticles. 1st ed. CRC PRESS 2016.
Kanwal Z, Raza MA, Manzoor F, et al. A comparative assessment of nanotoxicity induced by metal (silver, nickel) and metal oxide (cobalt, chromium) nanoparticles in Labeo rohita. Nanomaterials (Basel) 2019; 9(2): 309.
[] [PMID: 30823536]
Arbex MA, Varella MdeC, Siqueira HR, Mello FA. Antituberculosis drugs: drug interactions, adverse effects, and use in special situations. Part 1: first-line drugs. J Bras Pneumol 2010; 36(5): 626-40.
[] [PMID: 21085830]
Sridhar A, Sandeep Y, Krishnakishore C, Sriramnaveen P, Manjusha Y, Sivakumar V. Fatal poisoning by isoniazid and rifampicin. Indian J Nephrol 2012; 22(5): 385-7.
[] [PMID: 23326053]

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