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Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus serrulatus Scorpion Venom.

ABSTRACT: This study assesses the efficacy of different nanoemulsion formulations as new and innovative adjuvants for improving the in vivo immunization against the Tityus serrulatus scorpion venom. Nanoemulsions were designed testing key-variables such as surfactants, co-solvents, and the influence of the temperature, which would be able to induce the phase transition from a liquid crystal to a stable nanoemulsion, assessed for four months. Additionally, cationic-covered nanoemulsion with hyper-branched poly(ethyleneimine) was prepared and its performance was compared to the non-cationic ones. The physicochemical properties of the selected nanoemulsions and the interactions among their involved formulation compounds were carefully monitored. The cytotoxicity studies in murine macrophages (RAW 264.7) and red blood cells were used to compare different formulations. Moreover, the performance of the nanoemulsion systems as biocompatible adjuvants was evaluated using mice immunization protocol. The FTIR shifts and the zeta potential changes (from -18.3 ± 1.0 to + 8.4 ± 1.4) corroborated with the expected supramolecular anchoring of venom proteins on the surface of the nanoemulsion droplets. Cell culture assays demonstrated the non-toxicity of the formulations at concentrations less than 1.0 mg/mL, which were able to inhibit the hemolytic effect of the scorpion venom. The cationic-covered nanoemulsion has shown superior adjuvant activity, revealing the highest IgG titer in the immunized animals compared to both the non-cationic counterpart and the traditional aluminum adjuvant. In this approach, we demonstrate the incredible potential application of nanoemulsions as adjuvants, using a nanotechnology platform for antigen delivery system on immune cells. Additionally, the functionalization with hyper-branched poly(ethyleneimine) enhances this recognition and improves its action in immunization.

SUBMITTER: Medeiros ASA

PROVIDER: S-EPMC7599857 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against <i>Tityus serrulatus</i> Scorpion Venom.

Medeiros Arthur Sérgio Avelino de ASA Torres-Rêgo Manoela M Lacerda Ariane Ferreira AF Rocha Hugo Alexandre Oliveira HAO Egito Eryvaldo Sócrates Tabosa do ESTD Cornélio Alianda Maira AM Tambourgi Denise V DV Fernandes-Pedrosa Matheus de Freitas MF da Silva-Júnior Arnóbio Antônio AA

Pharmaceutics 20200929 10

This study assesses the efficacy of different nanoemulsion formulations as new and innovative adjuvants for improving the in vivo immunization against the <i>Tityus serrulatus</i> scorpion venom. Nanoemulsions were designed testing key-variables such as surfactants, co-solvents, and the influence of the temperature, which would be able to induce the phase transition from a liquid crystal to a stable nanoemulsion, assessed for four months. Additionally, cationic-covered nanoemulsion with hyper-br ...[more]

PMID: 33003322

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Project description:Antimicrobial peptides (AMPs) are ubiquitous and multipotent components of the innate immune defense arsenal used by both prokaryotic and eukaryotic organisms. The search for new AMPs has increased in recent years, due to the growing development of microbial resistance to therapeutical drugs. In this work, we evaluate the effects of Tityus serrulatus venom (Tsv), its fractions and its major toxin Ts1, a beta-neurotoxin, on fungi growth. The fractions were obtained by ion-exchange chromatography of Tsv. The growth inhibition of 11 pathogenic and non-pathogenic filamentous fungi (Aspergillus fumigatus, A. nidulans, A. niger, A. terreus, Neurospora crassa, Penicillium corylophilum, P. ochrochloron, P. verrucosum, P. viridicatum, P. waksmanii, and Talaromyces flavus) was evaluated by quantitative microplate reader assay. Tsv (100 and 500 ?g/well, which correspond to 1 and 5 mg/mL, respectively, of total soluble protein) was active in inhibiting growth of A. nidulans, A. terreus, P. corylophilum, and P. verrucosum, especially in the higher concentration used and at the first 30 h. After this period, fungi might have used Tsv components as alternative sources of nutrients, and therefore, increased their growth tax. Only fractions IX, X, XI, XIIA, XIIB (3 and 7.5 ?g/well, which correspond to 30 and 75 ?g/mL, respectively, of total soluble protein) and Ts1 (1.5, 3, and 6 ?g/well, which correspond to 2.18, 4.36, and 8.72 ?M, respectively) showed antifungal activity. Ts1 showed to be a non-morphogenic toxin with dose-dependent activity against A. nidulans, inhibiting 100% of fungal growth from 3 ?g/well (4.36 ?M). The inhibitory effect of Ts1 against A. nidulans growth was accompanied by fungistatic effects and was not amended by 1 mM CaCl2 or tetrodotoxin (46.98 and 93.96 ?M). The structural differences between Ts1 and drosomycin, a potent cysteine-rich antifungal peptide, are discussed here. Our results highlight the antifungal potential of the first cysteine-containing scorpion toxin. Since Ts1 is a multifunctional toxin, we suggest that it could be used as a template in the design of engineered scorpion AMPs and in the search for new mechanisms of action of antifungal drugs.

Project description:BackgroundThe hyaluronidase enzyme is generally known as a spreading factor in animal venoms. Although its activity has been demonstrated in several organisms, a deeper knowledge about hyaluronidase and the venom spreading process from the bite/sting site until its elimination from the victim's body is still in need. Herein, we further pursued the goal of demonstrating the effects of inhibition of T. serrulatus venom (TsV) hyaluronidase on venom biodistribution.Methods and principal findingsWe used technetium-99m radiolabeled Tityus serrulatus venom (99mTc-TsV) to evaluate the venom distribution kinetics in mice. To understand the hyaluronidase's role in the venom's biodistribution, 99mTc-TsV was immunoneutralized with specific anti-T.serrulatus hyaluronidase serum. Venom biodistribution was monitored by scintigraphic images of treated animals and by measuring radioactivity levels in tissues as heart, liver, lungs, spleen, thyroid, and kidneys. In general, results revealed that hyaluronidase inhibition delays venom components distribution, when compared to the non-neutralized 99mTc-TsV control group. Scintigraphic images showed that the majority of the immunoneutralized venom is retained at the injection site, whereas non-treated venom is quickly biodistributed throughout the animal's body. At the first 30 min, concentration peaks are observed in the heart, liver, lungs, spleen, and thyroid, which gradually decreases over time. On the other hand, immunoneutralized 99mTc-TsV takes 240 min to reach high concentrations in the organs. A higher concentration of immunoneutralized 99mTc-TsV was observed in the kidneys in comparison with the non-treated venom. Further, in situ neutralization of 99mTc-TsV by anti-T.serrulatus hyaluronidase serum at zero, ten, and 30 min post venom injection showed that late inhibition of hyaluronidase can still affect venom biodistribution. In this assay, immunoneutralized 99mTc-TsV was accumulated in the bloodstream until 120 or 240 min after TsV injection, depending on anti-hyaluronidase administration time. Altogether, our data show that immunoneutralization of hyaluronidase prevents venom spreading from the injection site.ConclusionsBy comparing TsV biodistribution in the absence or presence of anti-hyaluronidase serum, the results obtained in the present work show that hyaluronidase has a key role not only in the venom spreading from the inoculation point to the bloodstream, but also in venom biodistribution from the bloodstream to target organs. Our findings demonstrate that hyaluronidase is indeed an important spreading factor of TsV and its inhibition can be used as a novel first-aid strategy in envenoming.

Project description:BACKGROUND:The yellow scorpion Tityus serrulatus (Ts) is responsible for the highest number of accidents and the most severe scorpion envenoming in Brazil. Although its venom has been studied since the 1950s, it presents a number of orphan peptides that have not been studied so far. The objective of our research was to isolate and identify the components present in the fractions VIIIA and VIIIB of Ts venom, in order to search for a novel toxin. The major isolated toxins were further investigated for macrophage modulation. METHODS:The fractions VIIIA and VIIIB, obtained from Ts venom cation exchange chromatography, were rechromatographed on a C18 column (4.6?×?250 mm) followed by a reversed-phase chromatography using another C18 column (2.1?×?250 mm). The main eluted peaks were analyzed by MALDI-TOF and Edman's degradation and tested on macrophages. RESULTS:The previously described toxins Ts2, Ts3-KS, Ts4, Ts8, Ts8 propeptide, Ts19 Frag-II and the novel peptide Ts19 Frag-I were isolated from the fractions VIIIA and VIIIB. Ts19 Frag-I, presenting 58 amino acid residues, a mass of 6,575 Da and a theoretical pI of 8.57, shares high sequence identity with potassium channel toxins (KTx). The toxins Ts4, Ts3-KS and the partially purified Ts19 Frag-I did not produce cytotoxic effects on macrophage murine cells line (J774.1). On the other hand, Ts19 Frag-I induced the release of nitric oxide (NO) by macrophages, while Ts4 and Ts3-KS did not affect the NO production at the tested concentration (50 ?g/mL). At the same concentration, Ts19 Frag-I and Ts3-KS increased the production of interleukin-6 (IL-6). Ts19 Frag-I and Ts4 did not induce the release of IL-10, IL-1? or tumor necrosis factor-? by macrophage cells using the tested concentration (50 ?g/mL). CONCLUSIONS:We partially purified and determined the complete sequence and chemical/physical parameters of a new ?-KTx, denominated Ts19 Frag-I. The toxins Ts4, Ts3-KS and Ts19 Frag-I showed no cytotoxicity toward macrophages and induced IL-6 release. Ts19 Frag-I also induced the release of NO, suggesting a pro-inflammatory activity.

Dataset Information

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against Tityus serrulatus Scorpion Venom.

Publications

Self-Assembled Cationic-Covered Nanoemulsion as A Novel Biocompatible Immunoadjuvant for Antiserum Production Against <i>Tityus serrulatus</i> Scorpion Venom.

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