Indian Journal of Experimental Biology Vol. 56, October 2018, pp. 707-715

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Nanotechnology in venom research—an overview

Antony Gomes*1, Sourav Ghosh1, Jayeeta Sengupta2, Kalyani Saha1 & Aparna Gomes3

1Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta, 92, APC Road, Kolkata-700 009, West Bengal, India 2Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Canada, T6G 1H9 3CSIR-Indian Institute of Chemical Biology (IICB), Kolkata-700 032, West Bengal, India

Received 02 June 2017; revised 12 July 2018

Nanotechnology has revolutionized the paradigm of today’s upcoming biological sciences through its applications in the field of biomedical research. One such promising aspect is by interfacing this modern technology with snake venom research. Snake venom is a valuable resource of bioactive molecules, which has shown efficient and promising contributions in biomedical research. The potentiality of merging these two unique fields lies in the approach of interfacing active bioactive molecules derived from snake venoms, which would yield better therapeutic molecules for future applications in terms of drug delivery, enhanced stability, reduced toxicity, bioavailability and targeted drug delivery. Available literature on nanoconjugation of snake venom bioactive molecules have suggest that these molecules have better therapeutic advantage in several fields of biomedical research viz., arthritis, cancer, etc. Another perspective in snake venom research could be green synthesis or herbal based synthesis of nanoparticles, which has shown enhanced effect in snake venom neutralizing capacity. Therefore, in terms of snake venom therapeutic potential and development of snake venom antidote, nanotechnology is a prodigious tool to be taken into serious consideration by the researchers. In this review, a comprehensive overview has been given on bridging nanoparticles with active biomolecules derived from snake venoms/herbs, current scientific evidences and records in this field, present trends and developments in nanotechnology in venom research along with future prospects in this arena. This may open new domains in snake venom research using nanotechnology in the near future.

Keywords: Antidote, Ayurveda, Drug delivery, Herbal, Nano applications, Nanoconjugation, Snake envenomation, Venoms-toxins

Nanotechnology and snake venom technology in almost every field bridging different Since the birth of its concept, nanotechnology has disciplines, such as biology, chemistry, physics, been employed in various sectors including electronics, mechanics, optics, mathematics and information and computing technology, energy, others. Due to its rapid growth since its basic origin, it sensors, biotechnology, biomedicine, food industry has climbed the steps of development and reached and even agriculture owing to its unique physico- almost every sector of applications. It is a big shot chemical properties which are completely different commercial market today which has billions of from their bulk form1. In fact, nanoscale materials, investments in global perspectives, and is expected to aggregates of atoms and molecules, gives rise to some be a giant market of future. Several nanoproducts are unforeseen properties which needs analysis and commercially available already and innumerable further detailing since these properties are what which products are under research and development. makes the nanotechnology— an unique technology. Nanotechnology can therefore be considered as the These unique yet highly potential properties of next technological revolution which has direct impact nanomaterials cannot be predicted by quantum on human health and environment. mechanics (individual atoms) or classical physics With wide array of applications of nanotechnology (bulk materials). The novel properties of nanomaterial in biomedical sciences, it is essential to know how has opened the door to diverse applications of this these nanoparticles actually work within the ______biological system. Owing to their miniature size, these particles can translocate from their entry portals *Correspondence: Phone: +91 33 23508386; Fax: +91 33 23519755, 22413288 (routes exposed) into the circulatory and immune E-mail: [email protected] systems, and ultimately to body tissues and organs. 708 INDIAN J EXP BIOL, OCTOBER 2018

Research in the field of cellular and interaction minerals for medicinal purposes8. In the Samhita of nanoparticles is vital owing to its molecular chess period (600-1000 BC) ayurvedic formulations used game, which it plays within the system. This opens a metals in powdered forms named Ayaskrati. The new door to the use of nanomaterials in drug delivery metals used in that period included mercury (Parada), and targeted therapy, and has been discussed in detail gold (Swarna), silver (Rajata), copper (Tamra), iron in this review. The way these particles behave greatly (Lauha), tin (Vanga), lead (Naga), zinc (Yasada), etc. depends on their size, morphology, shape, pH, charge, The metals were in micro forms and their internal use dose, route of exposure, crystalinity, etc.2. Hence, was limited because of their toxic effects. these particles become favorite candidates to be Development of Rasashastra of Ayurveda in manipulated for specific biomedical and research 7th century AD introduced many new pharmaceutical applications. techniques for metal formulations in Ayurvedic 9 The modern trend in snake venom research preparations including Shodana, Jarana and Marana , includes the utilization of nanotechnology3. Research which converted the metals into very fine, absorbable, in nanotechnology and snake venom/toxin has gained therapeutically most effective and least or non-toxic two different dimensions: (i) Use of nanotechnology form of medicine known as Bhasmas. Literature in the development of snake venom antidote; and shows that Ayurvedic processing of metallic formulations bring down its shape to nanometer (ii) Application of nanotechnology in the development 9 of drug clue against pathophysiology (arthritis, scale . Being too small, they are more powerful cancer, HIV, etc.). Till date, antisnake venom serum because the constituent metals and minerals do not (ASVS) has been the only clinically used antidote react with the tissues of the body. The metals could against snake envenomation developed in 1894 at combine with herbs which help in assimilation and delivery of the ingredients into the human Pasteur Institute, Paris, by Dr. Calmette. The 10 limitations (cost factor, self life, no protection against cell/tissues . There is a clear indication that metal- local effects, hemorrhage and organ toxicity) and side herb combination can work as medical wonders. effects (anaphylactic shock, pyrogenic reaction, serum Nanotechnology in the development of snake sickness, etc.) in patients are well known. Therefore, venom antidote worldwide, scientists are looking for a better Antisnake venom herbal compounds conjugated antisnake venom antidote. The herbal resources of the with nanoparticle have been a blooming area of world having antisnake venom potential have been 11 4 current day research. Alam et al. (1994) identified known for a long time . Folk and traditional medicinal 2-hydroxy-4-methoxy-benzoic acid (HMBA) from the system mentions about different herbs active against root extract of the Indian Sarsaparilla (Hemidesmus snake venom. The first scientific evaluation of herb as 5 indicus) having viper venom neutralizing effects in snake venom antidote was done by Knowles . He animal models. However, HMBA posses low efficacy screened several plants/plant constituents used by against viper venom and also had toxicity which was traditional practitioners, but failed to report their the major drawback of this herbal compound. But effectiveness against snake envenomation. Later, when conjugated with gold nanoparticle, it increased Mhaskar and Caius (1931) reported the the viper venom neutralizing efficacy of HMBA and ineffectiveness of herbs and herbal constituents 12,13 6 decreased the toxicity . Vitex negundo, a woody, against snake venom . It was contradicted later by aromatic shrub found in Afghanistan, India, Pakistan, many reports on the effectiveness of herbal antidotes Thailand, Sri Lanka, eastern Africa and Madagascar against snake envenomation. From the present has been used as antidote against venom for laboratory many herbs and herbal constituents have many years14. Saha et al. (2015)15 has synthesized been identified as an active antidote for snake 4 gold nanoparticle from gold salt using the reducing venom . Further studies with these herbs and herbal property of Vitex negundo and examined its potency constituents established their disadvantages such as 7 against viper venom induced acute stress and acute toxicities, low efficacy, etc , which confirmed the cytokine response in experimental animal model. need to improve the activity of the herb and herbal Characterization of gold nanoparticle was done using constituent against snake envenomation. dynamic light scattering (hydrodynamic diameter and India’s traditional medicinal system Ayurveda zeta potential), fourier transform infrared mentioned the utilization of herbs, metals and spectroscopy and transmission electron microscopy. It GOMES et al.: NANOTECHNOLOGY IN SNAKE VENOM RESEARCH 709

was found that the synthesized gold nanoparticles of these herbo-nano-conjugated biomolecules involve were of 25-40 nm size with nearly spherical shape. the cellular signaling molecules, the cytokines, Treatment with Vitex negundo-reduced gold enzymes and pro-inflammatory markers. However, nanoparticle significantly restored viper venom- more detail research is needed in this area to validate induced antioxidant parameters and cytokines the actions of hero-nano-conjugate as an effective responses. The particle was capable of reaching the alternative antidote against snake-bite. snake venom target sites thereby significantly Detection of snake venom type in victims is an neutralizing the deleterious venom effects on the essential step before snake venom therapeutics. Gold major organs (liver and kidney). Curcumin is an nanoparticle based lateral flow assay has been utilized active compound found in well known spice turmeric for detection of venom and Russell’s (Curcuma longa). The anti-inflammatory and viper venom in mice model21. It is based on paper antioxidant potential of curcumin has been well immunochromatography assay for detection of two established16. Curcumin was conjugated with gold snake venom species using polyvalent antisnake nanoparticle by adsorption method, and its antiviper venom antibodies. The immunoassay was rapid, venom activity was assessed in experimental selective, and sensitive to detect venom animals17. It was proposed that curcumin-conjugated concentrations upto 0.1 ng/mL. It has a potential to be gold nanoparticle may act by neutralizing viper a field diagnostic test to detect snake envenomation venom-induced local damages at the vascular bed by and assist in saving lives of snakebite victims. inhibiting the pro-oxidant activity of the venom, by direct inhibition at the enzymatic level, and by Nanoparticle snake venom/fraction against interfering with cellular markers including pathophysiology inflammatory markers and antioxidants17. Some Over the years, snake venom molecules have been nanoparticles have pharmacokinetic and protein- targeted as medical research tools or as binding properties that make them a low cost and therapeutic/diagnostic agent22,23. Calmette et al.24 easily available alternative for antivenom against (1933) proposed that the physiologically active systemically distributed snake venom toxins. Karain components of snake venom might have therapeutic et al. (2016)18 injected LD50 doses of southern potential. They showed that Cobra venom could kill Pacific rattlesnake (Crotalus oreganus helleri) venom cancer cells in animal model. Thereafter, many into crickets (Acheta domesticus) and found the reports have established the anticancer potential of ability of C60 fullerene to bind venom toxins. different species of , Viperidae and 25-29 Treatment with C60 fullerene increased the survival Crotalidae snake venoms . It was also noticed that rate of cricket after envenomation, compared to these compounds are very toxic, producing 30 control crickets. Survival analysis confirmed that C60 neurological, cardiac, respiratory adverse effects . fullerene protected crickets up to 60 h, which was Many attempts have been made to increase the significantly longer than controls. It may further therapeutic potential and to decrease the toxicity of considered as a simple, low cost treatment for venom-toxins in biological systems. One of such snakebite18. Oliveira et al. (2018)19 showed the approach in modern research arena has been neutralization of silver nanoparticles with 50 nm nanotechnology, which may provide increased diameter against Bothrops jararacussu snake venom efficacy and decreased toxicity (Table 1). A common induced neurotoxicity and myotoxicity in ex vivo and approach is to attach the protein toxin to the in vitro studies. Soares et al. (2018)20 has produced nanoparticle via non-covalent electrostatic adsorption antidote against Bothrops jararaca and process31. However this non-covalent interaction is B. erythromelas snake venom using chitosan polymer not at all stable at high salt concentrations and at pH nanoparticle as an adjuvant. They have showed that values more than the isoelectric point of the protein. chitosan polymer nanoparticle have potential A covalent bond between venom protein toxin and immunoadjuvant properties, without profound nanoparticle is more stable and its attachment site to inflammatory effect of the venom20. the nanoparticle may be controlled. Nanoparticle The molecular mechanism of action of these conjugation with snake venom/toxin may cause herbal-nanoparticle remain to be established and a big protein folding/unfolding32. It has been advised to challenge for the toxinologists. The molecular targets verify venom/toxin structure-function after 710 INDIAN J EXP BIOL, OCTOBER 2018

Table 1 — Snake venom/toxin-nanoparticle used against experimental pathophysiology Type of venom/toxin-nanoparticle Effective against Activity Naja kaouthia cytotoxin 1 (NKCT1) conjugated gold Cancer Significant inhibition of leukemic cell growth in dose and time nanoparticle (GNP-NKCT1) dependent manner36. GNP-NKCT1 Cancer Anticancer effect in vivo and in vitro in EAC cells and antitumor effect in EAC-induced mice37. GNP-NKCT1 Cancer Caspase dependent apoptotic pathway and autophagy inducing potential in human leukemic U936 and K562 cell lines38. GNP-NKCT1 Cancer Targeted delivery in cancer cells39. GNP-NKCT1 Cancer Down regulation of cyclin dependent kinase-4 and MAP kinase in human breast cancer cell line40 aegyptia venom combined with silica Cancer Combination with venom with nanoparticle strongly induced nanoparticle apoptosis in MDA-MB-231 and MCF-7 cancer cells41 Walterinnesia aegyptia venom combined with silica Cancer Inhibition of EGF-1 and IL-6-mediated multiple myoloma cell nanoparticle proliferation, change in cell cycle pattern and enhancement in induction of apoptosis in multiple myoloma cells42. Walterinnesia aegyptia venom combined with silica Cancer Decrease in the viability of multiple myoloma cells43. nanoparticle Walterinnesia aegyptia venom combined with silica Cancer Inhibition of multiple myoloma cell proliferation44. nanoparticle GNP-NKCT1 Arthritis Decrease in rheumatoid arthritis induced paw/ankle swelling, urinary markers, serum markers and cytokines47. GNP-NKCT1 Arthritis Change in physical parameter, urinary markers, serum and synovial membrane pro-inflammatory markers and metalloproteinase in collagenase induced osteoarthritic animals48 Echis carinatus venom-chitosan nanoparticles Drug delivery Noble drug delivery vehicle49 Agkistrodon halys venom-chitosan nanoparticles Drug delivery Noble drug delivery vehicle35 conjugation with nanoparticle. Co-functionalization of capacity of venom was achieved at 500 μg/mL nanoparticle with polyethylene glycol may help in concentration with chitosan at concentration 2 and protein unfolding, and can be used in snake 3 mg/mL. Zolfagharian and Mohammadpourdounighi venom/toxin loaded nanoparticle formation. (2009)34 established a small scale double emulsion Nanoparticles can be modified to carry drugs and technique for incorporation of Naja naja oxiana imaging probes both and designed to specifically venom into poly (lactide-co-glycolide) microspheres. target molecules of diseased tissues. Nanoparticles for They also showed that the antigenicity of venom was anticancer drug delivery had reached the first clinical retained after incorporation into poly (lactide-co- trial in the mid-1980s, and the first nanoparticles (e.g. glycolide) microspheres. Agkistrodon halys snake liposomal with encapsulated doxorubicin) had entered venom loaded chitosan nanoparticle was showed to be the pharmaceutical market in 1995. Since then, effective as adjuvant and antigen delivery system35. numerous new nanoparticles for cancer drug delivery Bhowmik et al. (2013)36 has reported the antileukemic have been approved and/or are currently under potential of PEGylated gold nanoparticle conjugated development due to their many advantages. Their with Naja kaouthia venom protein toxin NKCT1. advantages include enhancing solubility of Gold nanoparticle conjugated NKCT1 (GNP-NKCT1) hydrophobic drugs, prolonging circulation time, was prepared by borohydrate reduction method. It minimizing nonspecific uptake, preventing significantly inhibited leukemic cell growth in by undesirable off-target and side effects, improving dose or time dependent manner by 2-3 folds more intracellular penetration, and allowing for specific than NKCT1 alone. GNP-NKCT1 induced growth cancer targeting. Mohammadpourdounighi et al. arrest of leukemic cells at G1 phase. Cell line (2010)33 encapsulated Naja naja oxiana venom in migration was significantly low in GNP-NKCT1 biodegradable and nontoxic chitosan nanoparticle. treated cells. Bhowmik et al. (2014)37 also reported Chitosan nanoparticle was produced by ionic gelation the influence of GNP-NKCT1 on Ehrlich Ascites process of tripolyphosphate. Optimum loading Carcinoma (EAC) and EAC induced solid tumor GOMES et al.: NANOTECHNOLOGY IN SNAKE VENOM RESEARCH 711

bearing mice. The study established the MDA-MB-231 and MCF-7 cells. The same venom- characterization of GNP-NKCT1, where scanning nanoparticle combination showed decrease in surface electron microscopy showed the formation of gold expression of the chemokine receptors CXCR3, nanoparticle and energy dispersive X ray CXCR4 and CXCR6 to a greater extent than venom spectroscopy and X ray powder diffraction confirmed alone in breast cancer cell line. Venom-nanoparticle 60-90% gold nanoparticles in the solution. GNP- combination also reduced migration in response to the NKCT1 showed anticancer effect in vivo and in vitro cognate ligands CXCL10, CXCL12 and CXCL16. It in EAC cells and antitumor effect in EAC-induced strongly inhibited insulin-like growth factor 1 mice. It decreased the tumor volume and tumor (EGF-1)- and IL-6-mediated multiple myoloma cell weight in EAC induced tumor in male albino mice proliferation, changed the cell cycle pattern and and induced caspase dependent apoptosis pathway in enhanced the induction of apoptosis in multiple EAC cell. Bhowmik and Gomes has established the myoloma cells. The combination of venom with caspase dependent apoptotic pathway and autophagy nanoparticle robustly decreased the expression of inducing potential of GNP-NKCT1 in human cyclin D1, Bcl2 and the phosphorylation of AKT, leukemic U936 and K562 cell lines38. Induction of increased the expression of cyclin B1, altered the GNP-NKCT1 induced loss of mitochondrial mitochondrial membrane potential, increased the membrane potential and high reactive oxygen species activity of caspase-3, -8 and -9; and sensitized generation in both the leukemic cell lines. It multiple myoloma cells to growth arrest and downregulated PI3K/Akt and mTOR expression apoptosis42. Sayed et al. (2012)43 has demonstrated followed by autophagic cell death. Lysosomal the antiproliferative and apoptotic effect of staining confirmed lysosomal enzyme involvement in Walterinnesia aegyptia venom and Walterinnesia the autophagic response. Up regulation of Atg 3, aegyptia venom + silica nanoparticle on multiple Atg12, Beclin 1, LC3-II protein and BIF-1 and down myeloma cells. In this study, venom alone and regulation of Atg4B were also showed. Another study venom-nanoparticle both decreased the viability of established the increased uptake and targeted delivery multiple myoloma cells. Venom combined with of GNP-tagged NKCT1 in cancer cells39. GNP- nanoparticle strongly inhibited multiple myoloma cell NKCT1 down-regulated cyclin dependent kinase-4 proliferation. Cell cycle analysis of multiple myoloma and MAP kinase through estrogen receptor mediated cells indicated the altered cell cycle and increased cell cycle arrest in human breast cancer cell line40. apoptosis induction after venom-nanoparticle From the above findings, it is clear that tagging of treatment. Badr et al. (2013)44 has reported the gold nanoparticle with an anticancer snake venom therapeutic efficacy and molecular mechanisms of toxin NKCT1 increased the efficacy of the venom snake (Walterinnesia aegyptia) venom-loaded silica toxin to kill the cancer cells. nanoparticles in the treatment of breast cancer- and Al-Sadoon et al. (2012)41 has established the prostate cancer-bearing experimental mouse model. In potential antiproliferative property of Walterinnesia this study, xenograft breast and prostate tumor mice aegyptia venom alone or combined with silica models were established. Treatment with venom alone nanoparticle against mice immune cell and human and venom-loaded nanoparticle significantly breast carcinoma cell line (MDA-MB-231 and decreased both breast and prostate tumor volumes MCF-7). In this study, the IC50 value of venom and compared to treatment with nanoparticle or vehicle venom+silica nanoparticle were 50 and 20 ng/mL, alone. It was observed that venom-loaded respectively, which indicated the increase of anti- nanoparticle strongly inhibited insulin-like growth proliferative property of venom when combined with factor 1 and epidermal growth factor mediated silica nanoparticle. At these concentrations, the proliferation of breast and prostate cancer cells, venom did not affect the viability of normal breast respectively, and increased the apoptosis induction by epithelial cells (MCF-10). Combination with venom inducing the activity of caspase-3,–8, and -9 in with nanoparticle strongly induced apoptosis in both breast and prostate cancer cells. Combination MDA-MB-231 and MCF-7 cancer cells without of venom with nanoparticle decreased the significant effect on normal MCF-10 cells. Venom- phosphorylation of AKT, ERK, and IκBα, decreased nanoparticle combination reduced the expression of the expression of cyclin D1, surviving, and the Bcl2 and increased the activation of caspase 3 in antiapoptotic Bcl2 family members Bcl2, BclXL, and 712 INDIAN J EXP BIOL, OCTOBER 2018

52 Mcl1, markedly increased the expression of cyclin B1 biopotential. In a study by Barros et al. (2016) , Asp- and the proapoptotic Bcl2 family members Bak, Bax, PLA2 isolated from Bothrops jararacussu snake and Bim, changed the mitochondrial membrane venom was incorporated into liposome nanoparticles potential and sensitized tumor cells to growth arrest. and its efficacy against Leishmaniasis was shown Badr et al. (2013)45 also evaluated the impact of experimentally. Liposomes were formed combination of Walterinnesia aegyptia venom loaded homogeneously using the extrusion method and silica nanoparticle on the migration, invasion, physico-chemically characterized. The activity of proliferation and apoptosis of prostate cancer cells. Asp49-liposomes was evaluated in vitro against Snake venom/toxins have been identified as Leismaniasis and J774 macrophages. The results antiarthritic agent in animal models22. Gomes et al. indicated that Asp49-liposome is a promising tool to (2010)46 showed that Indian monocellate cobra (Naja enhance microbicidal activity of the infected kaouthia) venom had antiarthritic activity against macrophages in experimental leishmaniasis. Freund’s complete adjuvant (FCA) induced arthritis However, the major drawback of these bioactive in male albino rats. Antiarthritic potential of snake toxin molecules is their toxicity, which limits their venom/toxin conjugated nanoparticle has been entry in clinical trials. In order to overcome the evaluated in animal model. Saha et al. (2014)47 have toxicity, several attempts have been made such as conjugated NKCT1 with gold nanoparticle and liposome encapsulation, silica coating, examined its antiarthritic potential in animal model. nanoconjugation, etc. Application of nanotechnology Arthritis was induced in mice by FCA. In this study, in current biomedical research revealed that NKCT1 conjugated gold nanoparticle significantly nanoparticles have unusual properties to improve the changed arthritis induced paw/ankle swelling, urinary pharmacological and therapeutic properties of drugs. markers, serum markers and cytokines. Gomes et al. Nano conjugation of therapeutically potent toxin (2016)48 also reported the antiosteoarthritis potential molecules and pre-existing clinically used drugs, not of NKCT1 conjugated gold nanoparticle in only provides a media for better drug delivery but also collagenase induced experimental osteoarthritis. enhance stability, bioavailability and targeted drug Physical parameter (ankle diameter), urinary markers delivery. Saha et al. (2014)30 showed that GNP (hydroxyproline, glucosamine, pyridoline, deoxy- conjugation with protein NKCT1 significantly pyridoline), serum and synovial membrane pro- decreased the minimal lethal dose of NKCT1, thus inflammatory markers (TNF-α, IL-1β, IL-17, VEGF) making the molecule less toxic. Subchronic toxicity and metalloproteinase (MMP1) were significantly study of NKCT1/GNP-NKCT1 showed significant restored in osteoarthritic . reduction in food and water intake, which reflected in Use of snake venom along with nanoparticle is now body weight loss and decreased fecal consistency in an emerging area of drug delivery research49. The NKCT1 treated mice group. GNP conjugation with feasibility of hydrophilic nanoparticles has been protein NKCT1 significantly decreased (1 fold) the broadly investigated for use in drug delivery and minimal lethal dose of NKCT1, thus making the therapeutic systems. In a study, Agkistrodon halys molecule less toxic. snake venom was loaded in chitosan nanoparticles in order to be used as an advanced adjuvant and antigen Future directions of nanotechnology in snake delivery system in antidote production industry35. venom research Celen et al. (2018)50 has encapsulated Vipera Snake venom is a natural biological store of ammodytes transcaucasiana venom in PAMAM-G4 therapeutically active components including dendrimer nanoparticle by sol-gel method and showed neurotoxic, cardiotoxic, cytotoxic, nerve growth its efficacy in drug delivery systems. The surface factor, lectins, disintrigrins, haemorrhagins and many plasmon resonance property of nanoparticle has other different enzymes. These proteins not only been utilized in the detection of snake venom cause death to animals and humans, but can also be protein using integrated biosensor, which can be used for the treatment of cancer, arthritis, thrombosis used in detecting the concentration of venom and many other diseases22. Application of proteins in the blood of snake bitten patients in the nanotechnology along with therapeutically active 51 near future . Phospholipase A2 (PLA2) is an snake venom/toxins may open a new domain of important constituent of snake venom with enormous therapeutics. Possible snake venom/toxins that can be GOMES et al.: NANOTECHNOLOGY IN SNAKE VENOM RESEARCH 713

Table 2 — Possible snake venom/toxins that may be used in management of snake bite; (v) Nanoparticle nanotechnology research conjugated snake venom/toxins in the management of Snake Source Therapeutic Use communicable and non-communicable diseases; Venom/Toxins (vi) Nanoparticle conjugated snake venom/toxin as NN32 Naja naja venom Anticancer, immunomodulator in immunodeficiency diseases; and Antiarthritis, Anti- inflammatory, (vii) Detail toxicity studies with the herbo- Antioxidant53,54 nanoparticle and nanoparticle conjugated snake drCT-I Daboia russellii venom Anticancer55 venom/toxin are also required. Labein Macrovipera lebetina venom Anticancer56 What is more important at this stage is that CC5 and CC8 Cerastes cerastes venom Anticancer57 initiative should be taken by the Government and BnSP-6 Bothrops pauloensis venom Anticancer58 59 industries to take/support this emerging area of BF-F47 Bungarus fasciatus venom Antiarthritic research so that snake bite problem in tropical Cobratoxin Thailand cobra venom Antiarthritic60 Whole venom Naja kaouthia Antiarthritic61 countries may be tackled successfully. Further, ethical Viperatoxin-II Daboia russellii venom Antibacterial62 issues and biosafety measures in nanotechnology NN-XIb-PLA2 Naja naja venom Antibacterial63 research should be followed according to guidelines. Bothropstoxin-I Bothrops sp. Venom Antibacterial64 utilized in pathophysiology with the help of Conflict of interests nanotechnology are summarized in Table 253-64. No conflict of interest exists among the authors. The questions may arise from the above research Acknowledgement findings that (i) whether conjugate of venom/toxin The Indian Council of Medical Research, New with suitable nanoparticle will increase the efficacy Delhi, India is acknowledged for providing research for developing therapeutic agents against killer associate-1 fellowship to Sourav Ghosh (Ref. diseases (arthritis, cancer, HIV, etc.); (ii) whether 58/32/2012-BMS; Dt. 4.3.2015). conjugate of nanoparticle with venom/toxin shall decrease its toxicity to make it safe for drug trials; and References (iii) whether we can conjugate venom/toxins to more 1 Salata OV, Applications of nanoparticles in biology and than one nanoparticles, to make it more effective? medicine. J Nanobiotechnol, 2 (2004) 3. 2 Albanese A, Tang PS & Chan WC, The effect of Ultimately, the target for nanoparticle and snake nanoparticle size, shape, and surface chemistry on biological venom/toxin research should be the development of systems. Annu Rev Biomed Eng, 14 (2012) 1. drug clues against incurable diseases, such as cancer, 3 Utkin YN, Modern Trends in Animal Venom Research - arthritis, HIV, etc. Omics and Nanomaterials. World J Biol Chem, 8 (2017) 4. 4 Gomes A, Das R, Sarkhel S, Mishra R, Mukherjee S, The need for an efficient technology/tool for the Bhattacharya S & Gomes A, Herbs and Herbal Constituents study of snake venom was felt for a long time. Active Against Snake Bite. Indian J Exp Biol, 48 (2010) 865. Nanotechnology is the upcoming technology that has 5 Knowles R, The mechanism and treatment of snake bite in started its journey in the field of snake venom India. Trans R Soc Trop Med Hyg, 15 (1921) 72. 6 Mhaskar KS & Caius JF, Indian Plant remedies in snake bite. research. Until now, very few approaches have been Indian J Med Res, 19 (1931) 28. taken to explore nanotechnology in snake venom 7 Gomes A, Ghosh S, Sengupta J, Datta P & Gomes A, research. Not only in the field of supportive/alternate Herbonanoceuticals: A new step towards herbal therapeutics. antidote of snake venom is required but also the need Med Aromat Plants, 3 (2014) 162. 8 Madhavacharya (ed.), Sarvadarshanasamgraha. Umashankar of therapeutic agents against incurable diseases was Sharma Rishi, Choukhamba Vidya Bhavan, Varanasi (1964). started using snake venom/toxin. The upcoming 9 Chaudhary A, Ayurvedic bhasma: nanomedicine of ancient applications of nanotechnology in snake venom India—its global contemporary perspective. J Biomed research remain to be explored are (i) Nanoparticle Nanotechnol, 7 (2011) 68. 10 Paul S & Chugh A, Assessing the role of Ayurvedic aided detection, distribution and targeting of snake ‘Bhasmas’ as Ethnonanomedicine in the metal based venom/toxins; (ii) Nanoparticle aided interaction of nanomedicine patent regime. J Intellect Pro Rig, 16 (2011) snake venom/toxin in target organ; (iii) Nanoparticle 509. aided repair/healing of major organs (liver, kidney, 11 Alam MI, Auddy B & A Gomes, Isolation, purification and partial characterization of viper venom inhibiting factor from lung etc) affected in snake-bite; (iv) Nanoparticle the root extract of Indian medicinal plant sarsaparilla conjugated herbal antidote (supported therapy) in the (Hemidesmus indicus R. Br.). Toxicon, 32 (1994) 1551. 714 INDIAN J EXP BIOL, OCTOBER 2018

12 Gomes A, Sengupta J, Ghosh & Gomes A, Application of 27 Gomes A, Roy CS, Saha A & Mishra R, A heat stable gold nanoparticle conjugation with 2-hydroxy-4-methoxy protein toxin (drCT-I) from the Indian viper (Daboia russellii benzoic acid (HMBA) from Hemidesmus indicus root russellii) venom having antiproliferative, cytotoxic and enhancing neutralization of snake (viper) venom activity. apoptotic activities. Toxicon, 49 (2007) 46. J Nanosci Nanotechnol, 16 (2016) 8322. 28 Debnath A, Chatterjee U, Das M, Vedasiromoni JR & 13 Saha K & Gomes A, Russell’s viper venom induced Gomes A, Venom of Indian monocellate cobra and Russell’s nephrotoxicity, myotoxicity, hepatotoxicity: neutralization viper show anticancer activity in experimental models. J with gold nanoparticle conjugated 2-hydroxy-4-methoxy Ethnopharmacol, 111 (2007) 681. benzoic acid (GNP-HMBA) in experimental animal model. 29 Gomes A, Saha PP, Bhattacharya S, Ghosh S & Gomes A, Indian J Exp Biol, 55 (2017) 7. Therapeutic potential of krait venom. Toxicon, 131 (2017) 48. 14 Alam MI & A Gomes, Snake venom neutralization by Indian 30 Saha PP, Bhowmik T, Dasgupta AK & Gomes A, In vivo and medicinal plants (Vitex negundo and Emblica officinialis). in vitro toxicity of nanogold conjugated snake venom protein J Ethnopharmacol, 86 (2003) 75. toxin GNP-NKCT1. Toxicol Rep, 1 (2014) 74. 15 Saha K, Ghosh S, Ghosh S, Dasgupta SC, Gomes A & 31 Aubin-Tam ME, Conjugation of nanoparticles to proteins. Gomes A, Neutralization of Naja kaouthia venom induced Methods Mol Biol, 1025 (2013) 19. acute toxicity and stress response with herbal gold nano 32 Aubin-Tam ME & Hamad-Schifferli K, Structure and particle (VN-GNP) in experimental animal models. J Toxins, function of nanoparticle-protein conjugates. Biomed Mater, 3 2 (2015) 8. (2008) 034001. 16 Menon VP & Sudheer AR, Antioxidant and anti-inflammatory properties of curcumin. Adv Exp Med Biol, 595 (2007) 105. 33 Mohammadpourdounighi N, Behfar A, Ezabadi A, 17 Ghosh S & Gomes A, Russell’s viper (Daboia russelli Zolfagharian H & Heydari M, Preparation of chitosan russelli) venom toxicity neutralizing efficacy of curcumin- nanoparticles containing Naja naja oxiana snake venom. gold nanoparticle (C-GNP) in experimental animal model. Nanomedicine, 6 (2010) 137. J Toxins, 3 (2016) 6. 34 Zolfagharian H & Mohammadpourdounighi N, Encapsulation of Naja Naja Oxiana Snake venom into Poly 18 Karain BD, Lee MKY & Hayes WK, C60 Fullerenes as a (lactide-co-glycolide) microspheres. Archives of Razi Novel Treatment for Poisoning and Envenomation: A Proof- Institute, 64 (2009) 101. of-Concept Study for Snakebite. J Nanosci Nanotechnol, 16 35 Mohammadpourdounighi N, Javadi M, Zolfagharian H & (2016) 7764. Awadi MR, Preparation and Characterization of Agkistrodon 19 Oliveira ICF, de Paula MO, Lastra HCB, Alves BB, Moreno DAN, Halys Venom Entrapped Chitosan Nanoparticles: Novel and Yoshida EH, Amaral Filho J, Cogo JC, Varanda EA, Rai M, Advanced Antigen Delivery and Adjuvant System. Journal of Santos CAD & Oshima-Franco Y, Activity of silver Kerman University of Medical Sciences, 23 (2016) 618. nanoparticles on prokaryotic cells and Bothrops jararacussu 36 Bhowmik T, Saha PP, Dasgupta A & Antony Gomes. snake venom. Drug Chem Toxicol, 2 (2018) 1. Antileukemic potential of PEGylated gold nanoparticles 20 Soares KSR, Gláucia-Silva F, Daniele-Silva A, Torres-Rêgo M, conjugated with protein toxin (NKCT1) isolated from Indian Araújo NK, Menezes YAS, Damasceno IZ, Tambourgi DV, cobra (Naja kaouthia) venom. Cancer Nano, 4 (2013) 39. da Silva-Júnior AA & Fernandes-Pedrosa MF, Antivenom 37 Bhowmik T, Saha PP, DasGupta AK & Gomes A, Influence production against Bothrops jararaca and Bothrops of Gold Nanoparticle Tagged Snake Venom Protein Toxin erythromelas snake venoms using cross-linked chitosan NKCT1 on Ehrlich Ascites Carcinoma (EAC) and EAC nanoparticles as an immunoadjuvant. Toxins (Basel), 10 Induced Solid Tumor Bearing Male Albino Mice. Curr Drug (2018) doi: 10.3390/toxins10040158. Deliv, 11 (2014) 652. 21 Pawade BS, Salvi NC, Shaikh IK, Waghmare AB, Jadhav ND, 38 Bhowmik T & Gomes A, NKCT1 (purified Naja kaouthia Wagh VB, Pawade AS, Waykar IG & Potnis-Lele M, Rapid protein toxin) conjugated gold nanoparticles induced Akt/mTOR and selective detection of experimental snake envenomation inactivation mediated autophagic and caspase 3 activated - Use of gold nanoparticle based lateral flow assay. Toxicon, apoptotic cell death in leukemic cell. Toxicon, 121 (2016) 86. 119 (2016) 299. 39 Bhowmik T, Saha PP, Sarkar A & Gomes A, Evaluation of 22 Pal SK, Gomes A, Dasgupta SC & Gomes A, Snake venom cytotoxicity of a purified venom protein from Naja kaouthia as therapeutic agents: from toxin to drug development. (NKCT1) using gold nanoparticles for targeted delivery to Indian J Exp Biol, 40 (2002) 1353. cancer cell. Chem Biol Interact, 261 (2016) 35. 23 Biswas A, Gomes A, Sengupta J, Datta P, Singha S, 40 Bhowmik T & Gomes A, Down-regulation of cyclin- Dasgupta AK & Gomes A, Nanoparticle-conjugated animal dependent kinase-4 and MAPK through estrogen receptor venom-toxins and their possible therapeutic potential. mediated cell cycle arrest in human breast cancer induced by J Venom Res, 3 (2012) 15. gold nanoparticle tagged toxin protein NKCT1. Chem Biol 24 Calmette A, Saenz A & Costil L, Effects du venin de Cobra Interact, 268 (2017) 119. sur les greffes cancereuses et sur le cancer spontane 41 Al-Sadoon MK, Abdel-Maksoud MA, Rabah DM & Badr G, (adenocarcinoma) de la souris. CR Acad Sci, 197 (1933) 205. Induction of Apoptosis and Growth Arrest in Human Breast 25 Tu AT & Giltner JB, Cytotoxic effects of snake venoms on Carcinoma Cells by a Snake (Walterinnesia aegyptia) KB and Yoshida sarcoma cells. Res Commun Chem Pathol Venom Combined With Silica Nanoparticles: Crosstalk Pharmacol, 9 (1974) 783. Between Bcl2 and Caspase 3. Cell Physiol Biochem, 30 26 Iwaguchi T, Takhechi M & Hayashi K, Cytolytic activity of (2012) 653. cytotoxin isolated from Indian Cobra venom against 42 Al-Sadoon MK, Rabah DM & Badr G, Enhanced anticancer experimental tumor cell. Biochem Int, 10 (1985) 343. efficacy of snake venom combined with silica nanoparticles GOMES et al.: NANOTECHNOLOGY IN SNAKE VENOM RESEARCH 715

in a murine model of human multiple myeloma: molecular 54 Gomes A, Datta P, Das T, Biswas AK & Gomes A, Anti targets for cell cycle arrest and apoptosis induction. Cell arthritic and anti inflammatory activity of a cytotoxic protein Immunol, 284 (2013) 129. NN-32 from Indian spectacle cobra (Naja naja) venom in 43 Sayed D, Al-Sadoon MK & Badr G, Silica nanoparticles male albino rats. Toxicon, 90 (2014) 106. sensitize human multiple myeloma cells to snake 55 Gomes A, Choudhury SR, Saha A, Mishra R, Giri B, Biswas AK, (Walterinnesia aegyptia) venom-induced apoptosis and Debnath A & Gomes A, A heat stable protein toxin (drCT-I) growth arrest. Oxid Med Cell Longev, 2012 (2012) 386286. from the Indian Viper (Daboia russelli russelli) venom 44 Badr G, Al-Sadoon MK & Rabah DM, Therapeutic efficacy having antiproliferative, cytotoxic and apoptotic activities. and molecular mechanisms of snake (Walterinnesia Toxicon, 49 (2007) 46. aegyptia) venom-loaded silica nanoparticles in the treatment 56 Zakraoui O, Marcinkiewicz C, Aloui Z, Othman H, Grépin R, of breast cancer- and prostate cancer-bearing experimental Haoues M, Essafi M, Srairi-Abid N, Gasmi A, Karoui H, mouse models. Free Radic Biol Med, 65 (2013) 175. Pagès G & Essafi-Benkhadir K, Lebein, a snake venom 45 Badr G, Al-Sadoon MK, Rabah DM & Sayed Douaa, Snake disintegrin, suppresses human colon cancer cells (Walterinnesia aegyptia) venom-loaded silica proliferation and tumor-induced angiogenesis through cell nanoparticles induce apoptosis and growth arrest in cycle arrest, apoptosis induction and inhibition of VEGF human prostate cancer cells. Apoptosis, 18 (2013) 300. expression. Mol Carcinog, 56 (2017) 18. 46 Gomes A, Bhattacharya S, Chakraborty M, Bhattacharjee P, 57 Ben-Mabrouk H, Zouari-Kessentini R, Montassar F, Koubaa ZA, Mishra R & Gomes A, Anti-arthritic activity of Indian Messaadi E, Guillonneau X, ElAyeb M, Srairi-Abid N, Luis J, monocellate cobra (Naja kaouthia) venom on adjuvant Micheau O & Marrakchi N, CC5 and CC8, two homologous induced arthritis. Toxicon, 55 (2010) 670. disintegrins from Cerastes cerastes venom, inhibit in vitro 47 Saha PP, Bhowmik T, Dasgupta AK & Gomes A, Nano gold and ex vivo angiogenesis. Int J Biol Macromol, 86 (2016) conjugation, anti-arthritic potential and toxicity studies of snake 670. Naja kaouthia (Lesson, 1831) venom protein toxin NKCT1 in 58 Azevedo FV, Lopes DS, Cirilo Gimenes SN, Achê DC, male albino rats and mice. Indian J Exp Biol, 52 (2014) 763. Vecchi L, Alves PT, Guimarães Dde O, Rodrigues RS, 48 Gomes A, Saha PP, Bhowmik T, Dasgupta SC & Dasgupta AK, Goulart LR, Rodrigues Vde M & Yoneyama KA, Human Protection against osteoarthritis in experimental animals by breast cancer cell death induced by BnSP-6, a Lys-49 PLA₂ nanogold conjugated snake venom protein toxin GNP- homologue from Bothrops pauloensis venom. Int J Biol NKCT1. Indian J Med Res 144 (2016) 910. Macromol, 82 (2016) 671. 49 Mohammadpour DN, Mehrabi M, Avadi MR, Zolfagharian H & 59 Gomes A, Ghosh S, Ghosh S, Saha K, Saha PP, Dasgupta SC Rezayat M, Preparation, characterization and stability & Gomes A, Anti-osteoarthritic activity of Bungarus investigation of chitosan nanoparticles loaded with the Echis fasciatus venom fraction BF-F47 involving molecular carinatus snake venom as a novel delivery system. Arch Razi markers in the rats. Toxicon, 118 (2016) 43. Inst, 70 (2015) 269. 50 Çelen Ç, Keçeciler C, Karış M, Göçmen B, Yesil-Celiktas O, 60 Liu YL, Lin HM, Zou R, Wu JC, Han R, Raymond LN, Reid PF Nalbantsoy A. Cytotoxicity of Silica Nanoparticles with & Qin ZH, Suppression of complete Freund's adjuvant- Transcaucasian Nose Horned Viper, Vipera ammodytes- induced adjuvant arthritis by cobratoxin. Acta Pharmacol transcaucasiana, Venom on U87MG and SHSY5Y Neuronal Sin, 30 (2009) 219. Cancer Cells. Appl Biochem Biotechnol, (2018) doi: 61 Gomes A, Bhattacharya S, Chakraborty M, Bhattacharjee P, 10.1007/s12010-018-2742-2. Mishra R & Gomes A, Anti-arthritic activity of Indian 51 Choudhury SN, Konwar B, Kaur S, Doley R & Mondal B, monocellate cobra (Naja kaouthia) venom on adjuvant Study on Snake Venom Protein-Antibody Interaction by induced arthritis. Toxicon, 55 (2010) 670. Surface Plasmon Resonance Spectroscopy. Photonic Sens, 62 Samy RP, Stiles BG, Chinnathambi A, Zayed ME, Alharbi SA, 8 (2018) 193. Franco OL, Rowan EG, Kumar AP, Lim LH & Sethi G, 52 Barros NBD, Macedo SRA, Ferreira AS, Tagliari MP, Zanchi FB, Viperatoxin-II: A novel viper venom protein as an effective Kayano AM, Soares AM & Nicolete R, Liposomes containing bactericidal agent. FEBS Open Bio, 5 (2015) 928. an ASP49-phospholipase A 2 from Bothrops jararacussu snake 63 Sudarshan S & Dhananjaya BL, Antibacterial activity of an venom as experimental therapy against cutaneous leishmaniasis. acidic phospholipase A2 (NN-XIb-PLA2) from the venom of Int Immunopharmacol, 36 (2016) 225. Naja naja (Indian cobra). Springer plus, 5 (2016) 112. 53 Das T, Bhattacharya S, Halder B, Biswas A, Das Gupta S, 64 Santos-Filho NA, Lorenzon EN, Ramos MA, Santos CT, Gomes A & Gomes A, Cytotoxic and antioxidant property of Piccoli JP, Bauab TM, Fusco-Almeida AM & Cilli EM, a purified fraction (NN-32) of Indian Naja naja venom on Synthesis and characterization of an antibacterial and non- Ehrlich ascites carcinoma in BALB/c mice. Toxicon, 57 toxic dimeric peptide derived from the C-terminal region of (2011) 1065. Bothropstoxin-I. Toxicon, 103 (2015) 160.