Javascript is currently disabled in your browser. When javascript is disabled, some functions of this website will not work.

Open access for scientific and medical research

From submission to the first editing decision.

From editor acceptance to publication.

The above percentage of manuscripts have been rejected in the past 12 months.

Open access to peer-reviewed scientific and medical journals.

Dove Medical Press is a member of OAI.

Batch reprints for the pharmaceutical industry.

We provide authors with real benefits, including fast processing of papers.

Register your specific details and specific drugs of interest, and we will match the information you provide with articles in our extensive database and send you a PDF copy via email in a timely manner.

Back to Journal »International Journal of Nanomedicine» Volume 16

Influence of silver nanoparticles on the toxic effect of Philodryas olfersii venom

Author Proença-Assunção JC, Farias-de-França AP, Tribuiani N, Cogo JC, Collaço RC, Randazzo-Moura P, Consonni SR, Chaud MV, Santos CA, Oshima-Franco Y 

Published on May 25, 2021, the 2021 volume: 16 pages 3555-3564

DOI https://doi.org/10.2147/IJN.S293366

Single anonymous peer review

Editor approved for publication: Prof. Dr. Anderson Oliveira Lobo

Jaqueline de Cássia Proença-Assunção,1 Anna Paula Farias-de-França,2 Natalia Tribuiani,1 Jose Carlos Cogo,3 Rita de Cássia Collaço,4 Priscila Randazzo-Moura,5 Sílvio Roberto Consonni,6 Marco1 Vinicius Chaud Santos, 6 Marco1 Vinicius Chaud dos Santos, Yoko Oshima-Franco1 1 University of Sorocaba (Uniso) Pharmacy Graduate Course; 2 Pharmacy Graduate Course, University of Sorocaba (Uniso), Sorocaba, SP, Brazil; 3 Bioengineering, Institute of Technology and Science, University of Sao Paulo, Brazil And biomedical engineering projects; 4 Department of Pharmacology, Faculty of Medical Sciences, Campinas State University (Unicamp), Campinas, SP, Brazil; 5 Surgery, Catholic University of Sao Paulo (PUCSP), Sorocaba, SP, Brazil; 6 Department of Biochemistry and Tissue Biology, Institute of Biology, Campinas State University, Campinas, Brazil Mailing address: Yoko Oshima-Franco University of Sorocaba (UNISO), Rodovia Raposo Tavares, Km 92.5, Sorocaba, 18023- 000, SP, Brazil Tel 55 15 2101-7197 Fax 55 15-2101-7112 Email yok[email protected] Purpose: Use curcumin solid dispersion (130 nm, 0.081 mg mL−1) to obtain silver nanoparticles by reducing salt To combat the toxicity of Philodryas olfersii venom-causing edema, myotoxicity and neurotoxicity. Methods: Inject 50 μg of venom individually or pre-incubate with 1, 10, and 100 μL AgNPs in each site, and evaluate the edema-inducing effect in the back skin of rats by plasma extravasation; by measuring before treatment and at the end of each experiment The creatine kinase released into the organ bath at the time is used to evaluate the muscle toxicity; and the neurotoxicity is evaluated in the chicken double bra using the traditional electromyography technique, facing the external body added to the bath before treatment and after each experiment. Source acetylcholine (ACh) and potassium chloride (KCl). A preliminary concentration-response curve of AgNPs was performed to select the concentration used for the neutralization test, which included neutralizing neuromuscular paralysis and edema caused by the venom by pre-incubating AgNPs with the venom for 30 minutes. Results: P. olfersii venom caused edema (n=6) and complete neuromuscular block (n=4), including complete and unrecovered ACh and KCl contracture block. After pre-incubation with 10 and 100 μL AgNPs, AgNPs produced a concentration-dependent reduction in venom-induced edema (n = 6) from 223.3% to 134.4% and 100.5%. The pre-incubation of venom and AgNPs (1 μL; n=6) can maintain 46.5 ± 10.9% of neuromuscular response under indirect stimulation, and maintain 39.2 ± 9.7% of exogenous nicotinic receptors and nicotinic receptors without electrical stimulation. 28.3 ± 8.1% of potassium on the reactive muscle membrane. CK release is not affected by any experimental protocol similar to the control. Conclusion: AgNPs interact with the components of P. olfersii venom and are responsible for the formation of edema activity and neuromuscular blockade, but they do not interact with the active components of the sarcolemma. The protective effect of AgNPs on avian preparations studied indicates that the molecular targets are internal and external nicotinic receptors. Keywords: Chick Shuangwen head-necked snake, dorsal snake, Philodryas olfersii, neuromuscular blocker, silver nanoparticles

Modern technologies such as nanotechnology have been revealed as a potential treatment for snake venom poisoning-a global and neglected disease whose official treatment is serum therapy1-because these materials are believed to prevent the venom from spreading through the body. 2,3 This is in line with the World Health Organization’s global strategy for the prevention and control of snakebite poisoning4,5

Considering that serum therapy is effective for systemic effects, but not for local effects, and must be administered as soon as possible after the venom to increase the chance of successful treatment. This is a disadvantage for residents in rural areas in many countries. Nanoparticles are used as a countermeasure. Promising options for snakebite 6 and have used Naja nigricollis (Elapidae), 2 Doboia russellie (Viperidae), 7 Bothrops jararaca and Bothrops erythromelas (Viperidae), 8 Bothrops jararacussu (Viperidae), 9 Daboia russelii (Viperidae) Shows their value and Naja kaouthia (Elapidae). 10,11

The reasons for using nanoparticles include venom toxin bridging as an interface between drug delivery and targeted therapy, 6 as shown by the antigen delivery of Naja naja oxiana venom encapsulated in chitosan nanoparticles targeting tumor cells. 12 In addition, the herbal Vitex negundo gold nanoparticles neutralize the acute toxicity, acute stress and cytokine response of the locust venom, 10 and titanium dioxide nanoparticles are used as an antidote against the lethal activity of Roche locust and locust venom. And B. erythromelas venom was tested as an immune adjuvant for the production of antivenom. 8

Nanoparticles are also used for more specific goals. Silver nanoparticles can promote the protection of neuromuscular block 9 induced by dimale borer venom and proteolysis of Russell tree venom. 7 Non-biological hydrogel nanoparticles can alleviate the many kinds found in black-necked calamus Progression of local tissue damage induced by phospholipase A2 and 3FTX isoforms, Venom 2

Colubridae snakes Philodryas olfersii (P. olfersii) and Philodryas patagoniensis (commonly called green snakes) are anti-drake snakes and are considered non-venomous, 13 and are restricted to South America. The poisoning of these species produces similar effects as the species of the genus Bicolor, which can lead to misidentification, which leads to the use of bicolor antivenom for treatment. 14-16 Although the incidence is low, bites from a twill snake can cause mild to severe symptoms and need to be reconsidered despite their medical importance.

Especially for P. olfersii, there are few snake bites reported in the literature. A retrospective analysis indicated that between 1982 and 199017, Brazil's Vital Hospital (Institute of Butantin, São Paulo, Brazil) treated 43 cases, and more cases were reported in subsequent years. 14 In addition, Recife (Pernambuco, Brazil) also reported deaths. 18 Symptoms after these accidents may include severe local pain, swelling, erythema, and ecchymosis at the bite site17,19 but the clotting time is normal. 19 Other studies have also reported venom bleeding, fibrinogen dissolution, and edema formation activities, 20 in addition to muscle toxicity, 21 neurotoxicity, 22 and the ability to trigger inflammatory cell infiltration. 23 Strangely, the venom increased the levels of creatine kinase in mice, but it did not affect the isolated mammalian preparation. 24 On the contrary, it can cause head drooping and paralysis in chickens, as well as irreversible neuromuscular blockade in isolated poultry preparations. 24,25 may be the result of its arboreal niche. 26

In this case, considering the activity of forming edema and the known effect of P. olfersii venom on avian preparations, a solid dispersion of curcumin (Curcuma longa Linn.) was used to reduce the salt and Pluronic F68 polymer 27 was used. To counteract the toxic effects of this snake venom (edema, neurotoxicity and myotoxicity), because strategies to minimize the progression caused by snake venom, especially local effects, do not exist and should have significant public health benefits.

The integrated nanoparticles in this study consisted of silver nitrate obtained by reducing the salt with a solid dispersion of curcumin (Curcuma longa Linn.). All reagents are from Sigma-Aldrich® (St. Louis, Missouri, USA), as described by Alves et al. 27 In this mechanism, it is believed that the polymeric compounds pluronic F68 and curcumin will affect the reduction and subsequent conversion of these salts. 28 In short, a jacketed glass reactor (250 ml) with a temperature of 80-90°C is used. Connect the bath and stir on an orbital shaker, add 45 mg of silver nitrate and 135 mg of solid curcumin dispersion. After 40 minutes, the resulting solution was taken out of the system and cooled at room temperature. Afterwards, the obtained nanoparticles were characterized by dynamic light scattering (DLS, Brookhaven-NanoBrook-90 Plus, New York, USA), showing an average size of 130 nm. According to other places, the concentration of AgNPs is called 0.081 mg mL-1,29,30 but additional experiments are needed to clarify the actual number. Since AgNPs, curcumin and pluronic F68 are a complex process, the process of obtaining nanoparticle AgNPs has not been purified, but this does not abolish the importance of the preliminary data described here.

For this analysis, we used silver nanoparticles obtained by dos Santos et al. in the absence of curcumin as a reference. Therefore, put 20 uL of each sample (with or without curcumin) on a piece of paraffin film. Next, a Formvar carbon-coated EM grid (G200H-Cu, Electron Microscopy Sciences©, Hatfield, USA) was placed on top of the droplet for 20 minutes. Then, carefully let each grid dry for 2 minutes, and then wash it twice with mili-Q water. Finally, the grid was air-dried for 10 minutes and stored in a LEO 906-Zeiss transmission electron microscope (Carl Zeiss Microscopy GmbH, Germany) at an acceleration voltage of 60 kV until inspection. TEM was performed in the electron microscope laboratory of the Institute of Biology, Campinas State University.

The freeze-dried venom of P. olfersii comes from the adult specimen pool kept in the snake tank of the Center for Natural Studies (CEN) donated by Dr. José Carlos Cogo of the University of Brazil (SP, Brazil). This work has been registered in the National Genetic Resources and Related Traditional Knowledge Management System (SISGEN no. AE18BF2).

Chicks HY-line W36 (4-8 days) were provided by Santa Bárbara Poultry Farming (Sorocaba, SP, Brazil) and were raised in metal cages with a sawdust substrate, where they could get food and water at will. The project was approved by the Animal Use Ethics Committee-Sorocaba University CEUA-SP Protocol No. 159/2019.

Male Wistar rats (400-500 grams; 3-6 months old) from the animal house of the School of Medical Science and Health (PuC-SP, Sorocaba, Brazil) were treated according to the following guidelines until the ethics committee The result of the experiment. The experimental protocol was approved by the laboratory animal care and use committee of the institution according to the protocol number. 2019/117. Animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals31 and the Guidelines for Animal Research: Reports of In vivo Experiments (ARRIVE). 32

Rats were anesthetized by inhalation of isoflurane (BioChimico®, Rio de Janeiro, RJ, Brazil) and thiopental sodium (ip 50 mg mL-1), using maintenance doses if necessary. The skin on the back was shaved, and the animal was injected with Evan blue dye (1 mL kg-1) through the bulbar penile vein according to other methods. 33 Then intradermal injection (fixed volume: 100 μL) carrier (saline control), silver nanoparticles (AgNPs control) and P. olfersii (50 μg of venom per site) or previously combined with 1, 10 and 100 μL of AgNPs Incubate (30 minutes), using a random sequence and balanced site pattern. Thirty minutes later, according to Rocha & Furtado, there was enough time for the venom to exert its maximum effect. 16 The rats were euthanized with an excess of isoflurane, the back skin was removed, and the diameter of the injection site was measured (Evans Blue Halo) Use calipers. Plasma extravasation is expressed as a percentage of the control. The results are compared with the saline control. Figure 1 shows the experiments conducted on the back of rats using 8 different experimental protocols (n = 6 per group), namely in 8 random application areas: 1. Saline (control); 2. Individual venom (50 per site) Micrograms); 3. Venom (50 μg per site) AgNPs (1 μL or 81 ng); 4. Venom (50 μg per site) AgNPs (10 μL or 810 ng); 5. Venom (50 μg per site) ) AgNPs (100 μL or 8100 ng); 6, AgNPs (1 µL); 7, AgNPs (10 µL); 8, AgNPs (100 µL). Figure 1 Random application of different protocols (1 to 8) on the depilatory area on the back of rats, n = 6 for each experimental group.

Figure 1 Random application of different protocols (1 to 8) on the depilatory area on the back of rats, n = 6 for each experimental group.

The chicks were euthanized by inhalation of an overdose of isoflurane (BioChimico®, Rio de Janeiro, RJ, Brazil) and the double abdominal muscles were removed according to Ginsborg and Warriner 34 as described elsewhere 35. In short, place the preparation under a tension of 1 g/g 0.5 cm in a 5 mL organ bath (Panlab® four-chamber organ bath) maintained at 37°C with carbogen (95% O2 and 5% CO2) Inflate and store in Krebs solution with the following composition (mM, pH 7.5)): sodium chloride, 118.1; potassium chloride, 4.8; CaCl2, 2.5; magnesium sulfate, 1.2; sodium bicarbonate, 25; and glucose 11.1 . Insert the bipolar platinum ring electrode around the tendon for the nerve trunk of the muscle to run inside the tendon. Field stimulation is done using a pulse generator and a host, and can be used for up to 4 units (LE12406TC, Panlab®) stimulators (0.1 Hz, 0.2 ms, 5-12 V). Muscle contractions transmitted from the "internal" receptors respond to neurotransmitters released from nerve endings, and contractures (depolarizing activity) transmitted from the "external" receptors respond to exogenously added acetylcholine34,36 The MLT0201 (ADInstruments) is recorded in the isometric length of the force displacement sensor model and coupled to the four-bridge amplifier model FE224 (ADInstruments). A PowerLab 4/35 system model 3504/P connected to a computer unit containing LabChart and LabChart Pro Modules software (ADInstruments) was used for data acquisition. Stabilize the BC formulation for at least 10 minutes before adding 40 μM acetylcholine (ACh) for 60 seconds (s) or 100 mM potassium chloride (KCl) for 60 seconds. Before or after the start of each experiment, the contracture of exogenous ACh or KCl was recorded without field stimulation, as a test to evaluate the pre-synaptic or post-synaptic effect and the integrity of the muscle membrane at the same time. Concentration-response curves were performed using 1, 5, and 25 µL of AgNP. When added to a 5 mL bath, they corresponded to 0.0162, 0.081, and 0.405 ng mL-1, respectively.

To quantify CK activity, a sample of BC bath solution (100 μL) was taken from the organ bath at 0 (control, after the addition of exogenous KCl and ACh, but before any treatment) and 120 minutes after each treatment. The volume taken out is replaced with an equal volume of Krebs solution. The collected samples were stored at 4°C for 2 hours until a commercial kit (CK-NAC REF 11.002.00, BioClin®, Belo Horizonte, MG, Brazil) was used.

All parameters are expressed as mean ± SEM, using Student's t test (the contraction response of acetylcholine and potassium chloride in the pre-incubation experiment) or one-way analysis of variance, and then the Tukey test (edema formation activity, CK release, pharmacological AgNPs) The concentration-response curve of ACh and KCl determination and contracture response), p <0.05 indicates significance. All data analysis was done using Origin© v.9.5 (OriginLab Corporation, Northampton, MA, USA).

The estimated concentration of the innovative AgNPs obtained with curcumin is 0.081 mg mL-1, which is equivalent to adding about 60% silver nitrate in the process, as described previously for other AgNPs obtained without curcumin. Figure 2 shows a photo panel obtained by standardized TEM of these nanoparticles to illustrate the success of nanoparticle production, using silver nanoparticles obtained in a similar process as a reference, but without curcumin. Figure 2 The image obtained from a transmission electron microscope (TEM, 60 kV, 100.000X). (A) Milli-Q water. (B) AgNPs 0.081 mg mL-1 was used as a reference in the absence of curcumin. 29 (C) AgNPs 0.081 mg mL-1 is synthesized with curcumin. The following figure shows the successful production process of curcumin-silver nanoparticles. Bar = 200nm.

Figure 2 The image obtained from a transmission electron microscope (TEM, 60 kV, 100.000X). (A) Milli-Q water. (B) AgNPs 0.081 mg mL-1 was used as a reference in the absence of curcumin. 29 (C) AgNPs 0.081 mg mL-1 is synthesized with curcumin. The following figure shows the successful production process of curcumin-silver nanoparticles. Bar = 200nm.

Since edema is an event that occurs quickly after a snake bite by P. olfersii, we hypothesized that AgNPs can minimize this effect. Therefore, we evaluated the protection of AgNPs produced by intradermal venom on vascular permeability of rat skin (Figure 3, and Tables S1 and S2). Please note that P. olfersii venom increased plasma leakage to 223.3% of the control, and the inhibition of venom-induced edema by AgNPs was effective at concentrations higher than 10 µL-not statistically significant compared to the saline control. Figure 3 Extravasation of plasma in the skin of the rat's back. The rats were anesthetized and the back skin was depilated. The animals received Evan blue dye (100 µL per 100 g) through the penile vein of the bulbar urethra. Then, they were injected intracutaneously (fixed volume: 100 μL) with vehicle (saline control), AgNPs (1, 10 and 100 μL) and P. olfersii venom alone (50 μg per site) or pre-incubated (30 minutes) Use 1, 10, and 100 µL of AgNP, using a random order in the balance and standardization stations. Thirty minutes later, measure the diameter of the injection site (Evan’s blue halo). Plasma leakage is expressed as a percentage of the control (% control). The column represents the mean ± SEM (n = 6). *Compared with the control, p<0.05. #p <0.05 compared with poison.

Figure 3 Extravasation of plasma in the skin of the rat's back. The rats were anesthetized and the back skin was depilated. The animals received Evan blue dye (100 µL per 100 g) through the penile vein of the bulbar urethra. Then, they were injected intracutaneously (fixed volume: 100 μL) with vehicle (saline control), AgNPs (1, 10 and 100 μL) and P. olfersii venom alone (50 μg per site) or pre-incubated (30 minutes) Use 1, 10, and 100 µL of AgNP, using a random order in the balance and standardization stations. Thirty minutes later, measure the diameter of the injection site (Evan’s blue halo). Plasma leakage is expressed as a percentage of the control (% control). The column represents the mean ± SEM (n = 6). *Compared with the control, p<0.05. #p <0.05 compared with poison.

The in vitro test was performed with a functional chicken Biventer cervicis (BC) preparation. Figure 4A shows the concentration-response curves of AgNPs added to the organ bath (volume 5 mL) in 1, 5, and 25 µL (volume). Please note that 1 µL of AgNPs (approximately 0.0162 ng mL-1 in 5 mL) selected for further pre-incubation analysis with P. olfersii venom will cause mild but significant (compared to Krebs control) nerves The muscle block is approximately 24 ± 12% at the end of 120 minutes. The blocking induced by AgNPs is concentration-dependent under indirect stimulation. Figure 4 Biventer's cervix preparation. (A) The concentration-response curve of AgNPs under indirect stimulation shows a concentration-dependent effect. (B) and (C) show the contracture response to ACh and KCl, respectively: Krebs control (n=7) venom (n=4) AgNPs 0.081 mg mL−1 (1 µL, n=6) pre-incubation (n = 6). Each point represents the mean ± SEM. * p <0.05 at all subsequent points and all concentrations compared to the control. #p<0.05 Compare between AgNP.

Figure 4 Biventer's cervix preparation. (A) The concentration-response curve of AgNPs under indirect stimulation shows a concentration-dependent effect. (B) and (C) show the contracture response to ACh and KCl, respectively: Krebs control (n=7) venom (n=4) AgNPs 0.081 mg mL−1 (1 µL, n=6) pre-incubation (n = 6). Each point represents the mean ± SEM. * p <0.05 at all subsequent points and all concentrations compared to the control. #p<0.05 Compare between AgNP.

The BC formulation allows the interpretation of the indirect evoked stimulus shown above (Figure 4A) as a contracture caused by exogenous acetylcholine and potassium chloride in the absence of electrical stimulation (Figure 4B and C, respectively). This is possible because, unlike mammalian tissues, avian products have multiple innervated fibers with a large number of endplates distributed along their length. In this analysis, in addition to this, silver nanoparticles alone only affected the amplitude of ACh or KCl contracture, because the results were not statistically different from the Krebs control. According to the myodynamic criteria, select a small amount of AgNPs for further pre-incubation analysis.

In the sequence, we tested the ability of 1 µL of AgNPs to inhibit the neuromuscular activity of P. olfersii venom. Figure 5A shows an experiment of indirect initiation that resulted in the release of ACh at the end of the endplate area. Note the rapid installation of neuromuscular paralysis caused by snake venom. AgNPs 1 µL and 50 µg mL-1 venom were pre-incubated for 30 minutes before being added to the organ bath, which significantly protected the preparation from neuromuscular block induced by venom alone and maintained a neuromuscular response of about 46.7±10.9%. The experiment is over, under indirect stimulation. Figure 5B shows the characteristic EMG recording of the BC preparation by P. olfersii venom, which induces a rapid increase in the amplitude of convulsions, followed by complete and irreversible neuromuscular blockade. Figure 5 Biventer's cervix preparation. (A) The protection of AgNPs is shown in the pre-incubation experiment. (B) The motor record shows an irreversible neuromuscular block caused by 50 µg mL-1 P. olfersii venom. (C) and (D) show contracture responses to ACh and KCl, respectively, which are blocked by venom. (E) CK activities of the experimental group. Please note that in this experimental model, venom alone does not induce the release of CK. In the pre-culture experiment, the comparison between (A) and (C) and (D) showed a positive correlation between the indirect stimuli added by exogenous ACh, but there was no positive correlation between exogenous KCl and CK measurements. Each point represents the mean ± SEM. *Compared to the control, p<0.05 at all subsequent points and concentrations. #p<0.05 compared with venom. The arrow in D indicates the blockade of venom.

Figure 5 Biventer's cervix preparation. (A) The protection of AgNPs is shown in the pre-incubation experiment. (B) The motor record shows an irreversible neuromuscular block caused by 50 µg mL-1 P. olfersii venom. (C) and (D) show contracture responses to ACh and KCl, respectively, which are blocked by venom. (E) CK activities of the experimental group. Please note that in this experimental model, venom alone does not induce the release of CK. In the pre-culture experiment, the comparison between (A) and (C) and (D) showed a positive correlation between the indirect stimuli added by exogenous ACh, but there was no positive correlation between exogenous KCl and CK measurements. Each point represents the mean ± SEM. *Compared to the control, p<0.05 at all subsequent points and concentrations. #p<0.05 compared with venom. The arrow in D indicates the blockade of venom.

The venom alone completely eliminated the contracture in response to both (Figure 5C and D, respectively). Therefore, the pre-incubation of AgNPs and venom showed a significant protective effect of AgNPs, retaining about 39.2±9.7% of the nicotine ACh response and 28.3±8.1% of the sensitivity of KCl to the sarcolemma.

Figure 5E shows the results obtained for creatine kinase (CK) activity in an aliquot collected from the bath 2 hours after each protocol. Note that compared to the control, no statistical changes were observed in preparations treated with AgNPs, venom, or pre-incubated samples (AgNPs venom).

The use of curcumin solid dispersion as a reducing agent to produce silver nanoparticles is an innovative method, in which both curcumin substances and pluronic reagents can promote the production of AgNPs through unclear mechanisms. Here, the protective effect of AgNPs on the toxic effects of Philodryas olfersii snake venom on mammals (in vivo) and birds (in vitro) was evaluated.

Although P. olfersii is considered a non-venomous snake, 13 a bite by these dorsal snakes may lead to misidentification with those snakes caused by the genus Diosaurus. 14-16 Then, determining the differences between them is important to resolve the appropriate treatment. Unlike Bothrops snakes, P. olfersii venom does not cause laboratory abnormalities or coagulation disorders,18 but can cause other common similar symptoms, such as edema. 16,20

In this study, a rat back skin model was selected to evaluate the ability of AgNPs to prevent the formation of venom edema, because this is a rapid event that was experimentally installed after P. olfersii injection. Plasma extravasation may have multiple causes37, as seen in some snake venoms. 38 The interaction between platelets and mast cells is one of the mechanisms by which snake venom induces plasma extravasation; this effect seems to also apply to P. olfersii venom, 23 can cause hypovolemic shock or hypothermia, and non-specific tissues Injury and inflammation. 37,39

Considering that nanoparticles have passive (extravasation achieved through increased vascular system permeability) and active (achieved through internalization that expresses their cytotoxic potential) targeting effects in cancer treatment, 40 believe that silver nanoparticles can avoid the effects of P The edema caused by olfersii venom is mainly due to their passive contact with the venom's compounds and reduce its toxicity.

The pharmacological results we show in Figures 4 and 5 were performed in a functional isolated chicken BC preparation, with the exception of the additional ability to directly stimulate the unknown function independent of muscle fibers. 41

The concentration-response curve of AgNPs showed that compared with the Krebs control, 1, 5 and 25 µL did not interfere with the contracture response of ACh or KCl (Figure 4B and C). These results are interesting for evaluating the ability of AgNPs to counteract the total blockade induced by P. olfersii venom on contraction (Figure 5A), nicotinic receptors, and sarcolemma.

The venom of P. olfersii lacks the enzymatic activities associated with phospholipase A2, fibrinogenase, platelet aggregates, nucleotidase, and DNase. 20,42 However, the venom expresses serine protease, metalloprotease, C-type lectin, and high alkaline phosphatase activity. 43,44 Future research needs to determine which venom component is responsible for the neuromuscular blockade caused by indirect stimulation; nevertheless, we have observed that the venom cannot induce this blockade at lower temperatures below 37°C (data not shown) ).

The venom completely prevented the contracture caused by the addition of exogenous acetylcholine (Figure 5C) and potassium chloride (Figure 5D) without stimulation. The silver nanoparticles pre-incubated with the venom partially eliminated (approximately 50%) the blockade induced indirectly by the venom alone (Figure 5B), showing some efficacy on the target responsible for paralytic activity. O'Brien et al. 2 described the successful use of polymer nanoparticles to isolate the main protein toxins in snakeskin snakes, but silver nanoparticles certainly showed different interactions with this venom component.

In the absence of stimulation, ACh-induced contracture (48.6 ± 8.4%) had a higher level of protection of AgNPs than KCl contracture (28.3 ± 8.1%), indicating that the protective effect was to protect nicotinic receptors rather than sarcomere. It can also be explained that the venom has very little access to internal/connection (caused by indirect stimulation) and external/extra connection (caused when facing exogenous acetylcholine without electrical stimulation) postsynaptic nicotine receptors. 35,36,45, 46 The addition of KCl directly depolarizes the tissues and leads to muscle contractures, and is widely used in Biventer cervicis preparations to prove the presence of compounds that affect muscle membrane potential; 47 KCl-induced contracture reduction indicates myotoxic damage 48 This study The results are consistent with the findings of myotoxins in the gland secretions of Philodryas olfersii Duvernoy, 49 and the myotoxic effects observed in experimental models, 21,23,25,50 including human accidents (edema, erythema, ecchymosis), area Lymphadenopathy, neurotoxicity and myotoxic effects, but no coagulation disorders) 18.

However, these results of the addition of exogenous KCl are not related to the amount of CK released into the bath. The reason why this biomarker does not provide a good measure of the damage caused by this venom may involve delayed release as shown by Philodryas patagoniensis 51 and P. olfersii. 16 In the latter venom, the maximum CK released in the blood is 180 minutes, and our experimental protocol is measured in 2 hours.

A key point of this work is to address the presence of curcumin in the colloidal dispersion of AgNPs. Curcumin has been used in histopathology,52 but there is no protection against muscle membrane damage caused by venom. On the other hand, it is speculated that curcumin can inhibit phospholipases, metalloproteinases and protein kinases, 53-57 This is consistent with this work and may be a positive feature of these nanoparticles. Finally, this study also revealed the local effects of using AgNPs as co-adjuvants to treat snakebites, which cannot be effectively neutralized by conventional serum therapy.

AgNPs interacted with the components of P. olfersii venom, resulting in edema and neuromuscular blockade, but the effect on the active components of the sarcolemma was less than that of ACh. The protective effect of AgNPs on avian preparations studied indicates that the molecular targets are internal and external nicotinic receptors.

JCPA was partially supported by Uniso (Sorocaba, SP, Brazil)'s Pharmaceutical Science Graduate Program; APFF was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp 2019/05891-7); NT was supported by higher education personnel Improved Coordination Organization (CAPES, Brazil) for PhD student funding. RCC is supported by Brazil's CAPES Postdoctoral Fellowship. This work was supported by Fapesp (2012/08271-0). We are grateful to the electron microscope laboratory of Campinas State University (UNICAMP) for equipment and technical assistance.

The authors report no conflicts of interest in this work.

1. World Health Organization. Health topic: snake bite. Available from: https://www.who.int/snakebites/en/. Visited April 23, 2020:

2. O'Brien J, Lee SH, Gutiérrez JM, Shea KJ. Engineered nanoparticles bind elastic snake venom toxins and inhibit skin necrosis caused by the venom. PLoS Negl Trop Dis. 2018;12(10):e0006736. doi:10.1371/journal.pntd.0006736

3. Science Daily. Nanoparticles used to treat snakebites. Science Daily; October 4, 2018. Available from: www.sciencedaily.com/releases/2018/10/181004143920.htm. Visited April 23, 2020.

4. WHO launches a global strategy for the prevention and control of snakebites. WHO department news. May 23, 2019. Available from: https://www.who.int/news-room/detail/23-05-2019-who-launches-global-strategy-for-prevention-and-control-of-snanebite-envenoming. May 2020 Visited on 23rd.

5. Williams DJ, Faiz MA, Abela-Ridder B, etc. A globally coordinated strategy for addressing priority neglected tropical diseases: snake bites. PLoS Negl Trop Dis. 2019;13(2):e0007059. doi:10.1371/journal.pntd.0007059

6. Biswas A, Gomes A, Sengupta J, etc. Nanoparticle-bound animal venom toxins and their possible therapeutic potential. J Venom Research. 2012; 3:15-21.

7. Hingane VC, Pangam D, Dongre PM. Inhibition of silver nanoparticles on crude snake venom: a biophysical and biochemical study. Biophysics Physicobiol. 2018; 15:204-213. doi:10.2142/biophysico.15.0_204

8. Soares K, Gláucia-Silva F, Daniele-Silva A, etc. Use cross-linked chitosan nanoparticles as an immune adjuvant to produce antivenom against Bothrops jararaca and Bothrops erythromelas snake venom. Toxin (Basel). 2018;10(4):158. doi:10.3390/toxins10040158

9. Oliveira ICF, de Paula MO, Lastra HCB, etc. The activity of silver nanoparticles on prokaryotic cells and bothrops jararacussu snake venom. Pharmaceutical Chemistry Toxicology. 2019;42(1):60–64. doi:10.1080/01480545.2018.1478850

10. Saha K, Ghosh S, Ghosh S, Dasgupta SC, Gomes A, Gomes A. In experimental animal models, Vitex negundo-gold nanoparticles (VN-GNP) neutralize the toxicity and stress induced by Naja kaouthia venom. J toxin. 2015;2(1):8.

11. Chakrabartty S, Alam MI, Bhagat S, etc. Titanium dioxide nanoparticles inhibit snake venom-induced aseptic inflammation and PLA2 activity in experimental animals. Scientific Reports 2019;9(1):11175. doi:10.1038/s41598-019-47557-y

12. Mohammadpourdounighi N, Behfar A, Ezabadi A, Zolfagharian H, Heydari M. Preparation of chitosan nanoparticles containing Naja naja oxiana snake venom. Nanomedicine. 2010;6(1):137–143. doi:10.1016/j.nano.2009.06.002

13. de Carvalho MA, Nogueira FN. Serpentes da área Urbana de Cuiabá, State of Mato Grosso: aspects ecológicos e acidentes ofídicos associados [Snakes from the urban area of ​​Cuiaba, State of Mato Grosso: ecological aspects and related snake bites]. Cad Saude Publica. 1998;14(4):753–763.

14. de Araújo ME, dos Santos AC. Cases of human poisoning caused by Philodryas olfersii and Philodryas patagoniensis (Serpentes: Colubridae). Rev Soc Bras Med Trop. 1997; 30(6): 517-519. doi:10.1590/S0037-86821997000600013

15. Rocha MM, Paixão-Cavalcante D, Tambourgi DV, Furtado MF. Duvernoy gland secretions of Philodryas olfersii and Philodryas patagoniensis (Colubridae): the neutralization of local and systemic effects of commercial bisexual antivenom (Bothrops). poison. 2006;47(1):95–103. doi:10.1016/j.toxicon.2005.10.005

16. Rocha MMT, Furtado MFD. Analysis of the biological activity of Philodryas olfersii (Lichtenstein) and P. patagoniensis (Girard) venom (Serpents, Colubridae). The priest bra zoo. 2007;24(2):410–418. doi:10.1590/S0101-81752007000200019

17. Ribeiro LA, Puorto G, Jorge MT. Bitten by a salamander snake Philodryas olfersii: a clinical and epidemiological study of 43 cases. poison. 1999;37(6):943-948. doi:10.1016/S0041-0101(98)00191-3

18. Correia JM, Santana Neto PL, Pinho MS, Silva JA, Amorim ML, Escobar JA. Poisoned by Philodryas olfersii (Liechtenstein, 1823) at Restauração Hospital in Recife, Pernambuco, Brazil: case report. Rev Soc Bras Med Trop. 2010;43(3):336-338. doi:10.1590/S0037-86822010000300025

19. Silveira PV, Nishioka SA. Brazilian teaching hospitals are non-venomous snake bites and snake bites are non-venomous. Analysis of 91 cases. Rev Inst Med Trop Sao Paulo. 1992;34(6):499-503. doi:10.1590/S0036-46651992000600002

20. Assakura MT, Salomão MG, Puorto G, Mandelbaum FR. Colubrid snake Philodryas olfersii (green snake) venom hemorrhage, fibrinogen dissolution and edema formation activity. poison. 1992;30(4):427–438. doi:10.1016/0041-0101(92)90539-H

21. Acosta de Pérez O, Leiva de Vila L, Peichoto ME, etc. Edema and myotoxic activity of Duvernoy gland secretions from Philodryas olfersii in northeastern Argentina. Biological cells. 2003;27(3):363–370. doi:10.32604/biocell.2003.27.363

22. Rodríguez-Acosta A, Lemoine K, Navarrete L, Girón ME, Aguilar I. Experimental oxygen toxemia produced by the venom of the opisthoglyphous lora snake (Philodryas olfersii). Rev Soc Bras Med Trop. 2006;39(2):193-197. doi:10.1590/S0037-86822006000200012

23. Oliveira JS, Sant'Anna LB, Oliveira Junior MC, etc. Local and hematological changes of Philodryas olfersii snake venom in mice. poison. 2017; 132: 9-17. doi:10.1016/j.toxicon.2017.03.013

24. Prado-Franceschi J, Hyslop S, Cogo JC, etc. The effect of Duvernoy gland secretions of Philodryas olfersii on striated muscle and neuromuscular junction: Some characteristics of neuromuscular part. poison. 1996;34(4):459–466. doi:10.1016/0041-0101(95)00146-8

25. Collaço RC, Cogo JC, Rodrigues-Simioni L, Rocha T, Oshima-Franco Y, Randazzo-Moura P. Protection of Mikania (guaco) extract against the toxicity of Philodryas olfersii snake venom. poison. 2012;60(4):614–622. doi:10.1016/j.toxicon.2012.05.014

26. Hauzman E, Bonci DM, Grotzner SR, etc. A comparative study on the morphology and spatial resolution of Dipsadidae snake photoreceptors and retinal ganglion cells. Brain behavior evolution. 2014;84(3):197-213. doi:10.1159/000365275

27. Alves TFR, Das Neves Lopes FCC, Rebelo MA, etc. Crystalline ethylene oxide and propylene oxide triblock copolymer solid dispersions can enhance solubility, stability and promote the controlled release of curcumin over time. The most recent Pat Drug Deliv formula. 2018;12(1):65–74. doi:10.2174/1872211312666180118104920

28. Rachmawati H, Al Shaal L, Müller RH, Keck CM. Development of curcumin nanocrystals: physical aspects. J Pharm Sci. 2013;102(1):204-214. doi:10.1002/jps.23335

29. Dos Santos CA, Seckler MM, Ingle A, Rai M. Comparison of the antibacterial activity of silver nanoparticles synthesized through biological and chemical pathways, using pluronic F68 as a stabilizer. IET Nano Biotechnology. 2016;10(4):200–205. doi:10.1049/iet-nbt.2015.0055

30. Alves TF, Chaud MV, Grotto D, etc. The combination of silver nanoparticles and curcumin solid dispersion: antibacterial and antioxidant properties. AAPS Pharmaceutical Technology. 2018;19(1):225–231. doi:10.1208/s12249-017-0832-z

31. National Research Council of the National Academy of Sciences. Guide to the care and use of laboratory animals. 8th edition. Washington, DC: National Academy of Sciences Press. year 2011. Available from https://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-use-of-laboratory-animals.pdf. Visited on September 14, 2019.

32. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving Bioscience Research Reports: ARRIVE Guidelines for Reporting Animal Research. Public Science Library Biology. 2010;8(6):e1000412. doi:10.1371/journal.pbio.1000412

33. Brain SD, Williams TJ. Inflammatory edema caused by the synergy between calcitonin gene-related peptide (CGRP) and mediators of increasing vascular permeability. Br J Pharmacol. 1985;86(4):855-860. doi:10.1111/j.1476-5381.1985.tb11107.x

34. Ginsborg BL, Warriner J. Cervical neuromuscular preparation of the isolated chick double temperature section. Br J Pharmacol Chemother. 1960; 15(3): 410–411. doi:10.1111/j.1476-5381.1960.tb01264.x

35. Tribuiani N, Tavares MO, Santana MN, etc. The ability of Terminalia fagifolia extract (Combretaceae) to neutralize the in vitro neuromuscular effects of Bothrops jararacussu venom. Nat Prod Res. 2017;31(23):2783–2787. doi:10.1080/14786419.2017.1292506

36. Chang CC, Tang SS. The difference between the internal and external acetylcholine receptors of the chicken double bra muscle. Naunyn Schmiedebergs Arch Pharmacol. 1974;282(4):379-388. doi:10.1007/BF00500986

37. Wick MJ, Harral JW, Loomis ZL, Dempsey EC. The optimized Evans Blue protocol for evaluating vascular leakage in mice. J Vis Exp. 2018; 139: 57037. doi:10.3791/57037

38. Nakamura Y, Sasaki T, Mochizuki C, etc. Snake erythromycin induces plasma extravasation through toxin-mediated interaction between platelets and mast cells. Scientific Reports 2019; 9(1): 15958. doi:10.1038/s41598-019-52449-2

39. Tsai M, Starkl P, Marihal T, Galli SJ. Test the "anaphylactoxin hypothesis": mast cells, IgE, and innate and acquired immune responses to venom. Curr Opin Immunol. 2015; 36:80-87. doi:10.1016/j.coi.2015.07.001

40. Cordani M, Somoza Á. Using metal nanoparticles to target autophagy: a promising cancer treatment strategy. Cell and molecular life sciences. 2019;76(7):1215–1242. doi:10.1007/s00018-018-2973-y

41. Cisterna BA, Vargas AA, Puebla C, etc. Active acetylcholine receptors can prevent skeletal muscle atrophy and facilitate the reinnervation of nerves. Nat community. 2020;11(1):1073. doi:10.1038/s41467-019-14063-8

42. Assakura MT, Reichl AP, Mandelbaum FR. Isolation and characterization of five fibrin (proto)lytic enzymes from the venom of Philodryas olfersii (green snake). poison. 1994;32(7):819-831. doi:10.1016/0041-0101(94)90007-8

43. Ching AT, Rocha MMT, Paes Leme AF, etc. Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii are revealed from Duvernoy's (venom) gland transcriptome. FEBS let. 2006;580(18):4417–4422. doi:10.1016/j.febslet.2006.07.010

44. Sales of PB, Santoro ML. The nuclease and DNase activities in Brazilian snake venom. Comp Biochem Physiol C Toxicol Pharmacol. 2008;174(1):85-95. doi:10.1016/j.cbpc.2007.08.003

45. Kingsburg BL. Spontaneous activity of muscle fibers in chicks. J Physiology. (London). 1960; 150(3): 707-717. doi:10.1113/jphysiol.1960.sp006413

46. ​​Ginsberg BL. Some characteristics of avian skeletal muscle fibers with multiple neuromuscular junctions. J Physiology. (London). 1960;154(3):581-598. doi:10.1113/jphysiol.1960.sp006597

47. Shieh BH, Pezzementi L, Schmidt J. Regulation of extracellular potassium and acetylcholine receptor synthesis in chicken muscle cells. Brain research. 1983;263(2):259-265. doi:10.1016/0006-8993(83)90318-9

48. Fernandez S, Hodgson W, Chaisakul J, etc. The in vitro toxic effect of puff adder (Bitis arietans) venom and the neutralization effect of antivenom. Toxin (Basel). 2014;6(5):1586–1597. doi:10.3390/toxins6051586

49. Prado-Franceschi J, Hyslop S, Cogo JC, etc. Characterization of myotoxin from Duvernoy gland secretions of allodontic dragon fish Philodryas olfersii (green snake): effects on striated muscle and neuromuscular junction. poison. 1998;36(10):1407–1421. doi:10.1016/S0041-0101(98)00075-0

50. Schezaro-Ramos R, Collaço RC, Cogo JC, etc. Cordia salicifolia and Lafoensia pacari plant extracts can counteract the local effects of the snake venom of Bothrops jararacussu and Philodryas olfersii. J Venom Research. 2020; 10:32-37.

51. Peichoto ME, Leiva LC, Guaimás Moya LE, Rey L, Acosta O. Duvernoy’s glands secrete Philodryas patagoniensis from northeastern Argentina: its effect on blood coagulation. poison. 2005;45(4):527–534. doi:10.1016/j.toxicon.2004.12.016

52. Dos Santos CA, Rai M, de Oliveira Júnior JM, etc. Curcumin and related antioxidants: applications in histopathology. pathology. First edition. Elsevier. roll. 1; 2020:197-204.

53. Yamamoto H, Hanada K, Kawasaki K, Nishijima M. Curcumin's inhibitory effect on mammalian phospholipase D activity. FEBS let. 1997;417(2):196-198. doi:10.1016/S0014-5793(97)01280-5

54. Hong J, Bose M, Ju J, etc. Regulation of arachidonic acid metabolism by curcumin and related diketone derivatives: effects on cytosolic phospholipase A2, cyclooxygenase and 5-lipoxygenase. Carcinogenicity. 2004;25(9):1671-1679. doi:10.1093/carcin/bgh165

55. Lin SS, Lai KC, Hsu SC, etc. Curcumin inhibits the migration and invasion of human A549 lung cancer cells by inhibiting matrix metalloproteinase-2 and -9 and vascular endothelial growth factor (VEGF). Cancer Letters. 2009;285(2):127–133. doi:10.1016/j.canlet.2009.04.037

56. Lin Jiankang, Chen YC, Huang Yutang, Lin Shao SY. Apigenin and curcumin inhibit protein kinase C and nuclear cancer gene expression as possible molecular mechanisms for cancer chemoprevention. J Cell Biochemistry. 1997; Supplement 28-29: 39-48.

57. Ghosh S, Gomes A. Russell's viper (Daboia russelli russelli) Curcumin gold nanoparticles (C-GNP) neutralizing venom toxicity in experimental animal models. J toxin. 2016;3(2):6.

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and include the Creative Commons Attribution-Non-commercial (unported, v3.0) license. By accessing the work, you hereby accept the terms. The use of the work for non-commercial purposes is permitted without any further permission from Dove Medical Press Limited, provided that the work has an appropriate attribution. For permission to use this work for commercial purposes, please refer to paragraphs 4.2 and 5 of our terms.

Contact Us• Privacy Policy• Associations and Partners• Testimonials• Terms and Conditions• Recommend this site• Top

Contact Us• Privacy Policy

© Copyright 2021 • Dove Press Ltd • Software development of maffey.com • Web design of Adhesion

The views expressed in all articles published here are those of specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.

Dove Medical Press is part of Taylor & Francis Group, the academic publishing department of Informa PLC. Copyright 2017 Informa PLC. all rights reserved. This website is owned and operated by Informa PLC ("Informa"), and its registered office address is 5 Howick Place, London SW1P 1WG. Registered in England and Wales. Number 3099067. UK VAT group: GB 365 4626 36

In order to provide our website visitors and registered users with services that suit their personal preferences, we use cookies to analyze visitor traffic and personalize content. You can understand our use of cookies by reading our privacy policy. We also retain data about visitors and registered users for internal purposes and to share information with our business partners. By reading our privacy policy, you can understand what data we retain, how we process it, who we share it with, and your right to delete data.

If you agree to our use of cookies and the content of our privacy policy, please click "Accept".