Multidrug-resistant bacterial infections are a threat to public health worldwide, which boosts the urgent need for pharmacological research for new drugs. Although the peptides without disulfide bridges from scorpions have shown antimicrobial action, usually their toxicity hamper their pharmacological application. Stigmurin is a non-hemolytic cationic peptide from Tityus stigmurus venom with antibacterial effect and toxicity on normal cells. In this approach, the conformational changes and stability of two Stigmurin analog peptides, named StigA8 and StigA18, were evaluated by circular dichroism, as well as the mechanism of interaction with bacterial membranes in silico. In addition, the in vitro and in vivo antibacterial activity and the action against the biofilm formed by multidrug-resistant Staphylococcus aureus were investigated. StigA8 (+4) and StigA18 (+5) revealed the ability to change their structural conformation depending on the medium composition, and high stability at different temperatures and pH conditions. Both analog peptides showed greater ability to interact with bacterial membranes in silico when compared to the native one. StigA8 and StigA18 demonstrated low hemolytic action, with non-toxic effect on G. mellonella larvae up to 120 mg/kg. StigA8 and StigA18 presented a broad spectrum of antibacterial action in vitro, especially against multidrug-resistant clinical isolates. The analog peptides (7.5 µM) also reduced the biofilm biomass of multidrug-resistant S. aureus, as well as increased the larval survival of the Galleria mellonella infected larvae. Therefore, StigA8 and StigA18 showed a beneficial potential in the treatment of bacterial infections, constituting promising bioactive components for the development of new antimicrobial agents.
Methicillin-Resistant Staphylococcus aureus , Scorpion Venoms , Animals , Anti-Bacterial Agents/pharmacology , Bacteria , Biofilms , Microbial Sensitivity Tests , Peptides/chemistry , Peptides/pharmacology , Scorpion Venoms/chemistry , Scorpion Venoms/pharmacology , Scorpions/chemistry
Trypanosoma cruzi is a protozoan parasite responsible for Chagas disease, which affects millions around the world and is not treatable in its chronic stage. Sodium diethyldithiocarbamate is a compound belonging to the carbamate class and, in a previous study, demonstrated high efficacy against T. cruzi, showing itself to be a promising compound for the treatment of Chagas disease. This study investigates the encapsulation of sodium diethyldithiocarbamate by poly-lactic acid in nanoparticles, a system of biodegradable nanoparticles that is capable of reducing the toxicity caused by free DETC against cells and maintaining the antiparasitic activity. The nanosystem PLA-DETC was fabricated using nanoprecipitation, and its physical characterization was measured via DLS, SEM, and AFM, demonstrating a small size around 168 nm and a zeta potential of around -19 mv. Furthermore, the toxicity was determined by MTT reduction against three cell lines (VERO, 3T3, and RAW), and when compared to free DETC, we observed a reduction in cell mortality, demonstrating the importance of DETC nanoencapsulation. In addition, the nanoparticles were stained with FITC and put in contact with cells for 24 h, followed by confirmation of whether the nanosystem was inside the cells. Lastly, the antiparasitic activity against different strains of T. cruzi in trypomastigote forms was determined by resazurin reduction and ROS production, which demonstrated high efficacy towards T. cruzi equal to that of free DETC.
Chagas disease is caused by Trypanosoma cruzi and affects thousands of people. Drugs currently used in therapy are toxic and have therapeutic limitations. In addition, the genetic diversity of T. cruzi represents an important variable and challenge in treatment. Sodium diethyldithiocarbamate (DETC) is a compound with pharmacological versatility acting as metal chelators and ROS generation. Thus, the objective was to characterize the antiparasitic action of DETC against different strains and forms of T. cruzi and their mechanism. The different strains of T. cruzi were grown in LIT medium. To evaluate the antiparasitic activity of DETC, epimastigote and trypomastigote forms of T. cruzi were used by resazurin reduction methods and by counting. Different response patterns were obtained between the strains and an IC50 of DETC ranging from 9.44 ± 3,181 to 60.49 ± 7.62 µM. Cell cytotoxicity against 3T3 and RAW cell lines and evaluated by MTT, demonstrated that DETC in high concentration (2222.00 µM) presents low toxicity. Yet, DETC causes mitochondrial damage in T. cruzi, as well as disruption in parasite membrane. DETC has antiparasitic activity against different genotypes and forms of T. cruzi, therefore, representing a promising molecule as a drug for the treatment of Chagas disease.
Chagas Disease/parasitology , Ditiocarb/pharmacology , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects
Among several bioactive peptides identified from the venom glands of the Tityus stigmurus scorpion, one peptide with hypotensive action (TistH, Tityus stigmurus Hypotensin) showed multifunctional and biotechnological applications. The maximum efficacy of this class of compounds can be achieved by immobilizing it in specific and suitable biomaterials or suitable carriers. In this study, distinct entrapment methods of TistH in chitosan nanoparticles was tested using its incorporation (CN-TistH-Inc) or adsorption (CN-TistH-Ads) methods by ionotropic gelification. Physico-chemical properties as well as biocompatibility and antifungal efficacy were assessed for different samples. Atomic force microscopy and field emission gun scanning electronic microscopy images associated with particle size measurements demonstrated that the two methods induced cationic spherical, small (< 160â¯nm), and narrow-sized (PdI about 0.3) nanoparticles, even after peptide loading greater than 96.5%, which was confirmed using Fourier transform infrared spectroscopy. The colloidal suspensions showed to be stable for 8â¯weeks and were able to induce the desired slow in vitro peptide release. Cytotoxicity assays performed in normal cells originated from murine macrophages (RAW 264.7) and kidneys of African green monkeys (Vero E6) suggested biocompatibility of samples. The CN-TistH-Inc and CN-TistH-Ads showed a minimal inhibitory concentration of 89.2⯵g.mL-1 against Candida albicans, 11.1⯵g.mL-1 for C. parapsilosis and C. tropicalis, confirmed by minimum fungicidal concentrations assay. Moreover, the TistH-loaded cross-linked chitosan nanoparticles significantly reduced the biofilm formation of clinical yeast sepsis of C. tropicalis and C. krusei, as well as clinical yeasts of vulvovaginal candidiasis of C. albicans. In this approach, biodegradable nanocarriers prepared using simple and reproducible methods demonstrated the ability to deliver the TistH peptide from T. stigmurus and improve its antifungal efficacy.
Antifungal Agents , Arthropod Proteins , Candida/growth & development , Chitosan , Nanoparticles/chemistry , Peptides , Scorpion Venoms/chemistry , Scorpions/chemistry , Animals , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Arthropod Proteins/chemistry , Arthropod Proteins/pharmacology , Chitosan/chemistry , Chitosan/pharmacology , Chlorocebus aethiops , Mice , Peptides/chemistry , Peptides/pharmacology , RAW 264.7 Cells , Vero Cells
Snakebite envenoming is a tropical disease neglected worldwide. In Brazil, the Crotalus durissus cascavella (CDC) snake belongs to a genus with venom of highest lethality. A search for new immunoadjuvants aimed to expand the therapeutic alternatives to improve vaccines and antivenom. This approach proposed to produce small and narrow-sized cationic CDC venom-loaded chitosan nanoparticles (CHNP) able to induce antibody response against the CDC venom. The ionic gelation method induced the formation of stable and slightly smooth spherical nanoparticles (<160â¯nm) with protein loading efficiency superior to 90%. The interactions between venom proteins and CHNP assessed using FT-IR spectroscopy corroborated with the in vitro release behavior of proteins from nanoparticles. Finally, the immunization animal model using BALB/c mice demonstrated the higher effectiveness of CDC venom-loaded CHNP compared to aluminum hydroxide, a conventional immunoadjuvant. Thus, CHNPs loaded with CDC venom exhibited a promising biotechnological approach to immunotherapy.
Antivenins/chemistry , Antivenins/immunology , Biotechnology , Crotalus , Immunotherapy , Nanoparticles , Animals , Antivenins/metabolism , Female , Male , Mice , Nanoparticles/chemistry , Particle Size , Safety
In Brazil, envenomation by snakes of the genus Bothrops is clinically relevant, particularly for the species Bothrops jararaca and B. erythromelas. The most effective treatment for envenomation by snakes is the administration of antivenoms associated with adjuvants. Novel adjuvants are required to reduce side effects and maximize the efficiency of conventional serum and vaccine formulations. The polymer chitosan has been shown to have immunoadjuvant properties, and it has been used as a platform for delivery systems. In this context, we evaluated the potential immunoadjuvant properties of chitosan nanoparticles (CNPs) loaded with B. jararaca and B. erythromelas venoms in the production of sera against these venoms. Stable CNPs were obtained by ionic gelation, and mice were immunized subcutaneously for 6 weeks with 100 µL of each snake venom at concentrations of 5.0 or 10.0% (w/w), encapsulated in CNPs or associated with aluminium hydroxide (AH). The evaluation of protein interactions with the CNPs revealed their ability to induce antibody levels equivalent to those of AH, even with smaller doses of antigen. In addition, the CNPs were less inflammatory due to their modified release of proteins. CNPs provide a promising approach for peptide/protein delivery from snake venom and will be useful for new vaccines.
Adjuvants, Immunologic/administration & dosage , Antivenins/blood , Bothrops , Chitosan/administration & dosage , Crotalid Venoms/administration & dosage , Nanoparticles/administration & dosage , Adjuvants, Immunologic/chemistry , Animals , Chitosan/chemistry , Crotalid Venoms/chemistry , Female , Male , Mice, Inbred BALB C , Nanoparticles/chemistry
Snakebite envenoming is a tropical disease neglected worldwide. In Brazil, the Crotalus durissus cascavella (CDC) snake belongs to a genus with venom of highest lethality. A search for new immunoadjuvants aimed to expand the therapeutic alternatives to improve vaccines and antivenom. This approach proposed to produce small and narrow-sized cationic CDC venom-loaded chitosan nanoparticles (CHNP) able to induce antibody response against the CDC venom. The ionic gelation method induced the formation of stable and slightly smooth spherical nanoparticles (<160?nm) with protein loading efficiency superior to 90%. The interactions between venom proteins and CHNP assessed using FT-IR spectroscopy corroborated with the in vitro release behavior of proteins from nanoparticles. Finally, the immunization animal model using BALB/c mice demonstrated the higher effectiveness of CDC venom-loaded CHNP compared to aluminum hydroxide, a conventional immunoadjuvant. Thus, CHNPs loaded with CDC venom exhibited a promising biotechnological approach to immunotherapy.
In Brazil, envenomation by snakes of the genus Bothrops is clinically relevant, particularly for the species Bothrops jararaca and B. erythromelas. The most effective treatment for envenomation by snakes is the administration of antivenoms associated with adjuvants. Novel adjuvants are required to reduce side effects and maximize the efficiency of conventional serum and vaccine formulations. The polymer chitosan has been shown to have immunoadjuvant properties, and it has been used as a platform for delivery systems. In this context, we evaluated the potential immunoadjuvant properties of chitosan nanoparticles (CNPs) loaded with B. jararaca and B. erythromelas venoms in the production of sera against these venoms. Stable CNPs were obtained by ionic gelation, and mice were immunized subcutaneously for 6 weeks with 100 mu L of each snake venom at concentrations of 5.0 or 10.0% (w/w), encapsulated in CNPs or associated with aluminium hydroxide (AH). The evaluation of protein interactions with the CNPs revealed their ability to induce antibody levels equivalent to those of AH, even with smaller doses of antigen. In addition, the CNPs were less inflammatory due to their modified release of proteins. CNPs provide a promising approach for peptide/protein delivery from snake venom and will be useful for new vaccines.
Snakebite envenoming is a tropical disease neglected worldwide. In Brazil, the Crotalus durissus cascavella (CDC) snake belongs to a genus with venom of highest lethality. A search for new immunoadjuvants aimed to expand the therapeutic alternatives to improve vaccines and antivenom. This approach proposed to produce small and narrow-sized cationic CDC venom-loaded chitosan nanoparticles (CHNP) able to induce antibody response against the CDC venom. The ionic gelation method induced the formation of stable and slightly smooth spherical nanoparticles (<160?nm) with protein loading efficiency superior to 90%. The interactions between venom proteins and CHNP assessed using FT-IR spectroscopy corroborated with the in vitro release behavior of proteins from nanoparticles. Finally, the immunization animal model using BALB/c mice demonstrated the higher effectiveness of CDC venom-loaded CHNP compared to aluminum hydroxide, a conventional immunoadjuvant. Thus, CHNPs loaded with CDC venom exhibited a promising biotechnological approach to immunotherapy.
In Brazil, envenomation by snakes of the genus Bothrops is clinically relevant, particularly for the species Bothrops jararaca and B. erythromelas. The most effective treatment for envenomation by snakes is the administration of antivenoms associated with adjuvants. Novel adjuvants are required to reduce side effects and maximize the efficiency of conventional serum and vaccine formulations. The polymer chitosan has been shown to have immunoadjuvant properties, and it has been used as a platform for delivery systems. In this context, we evaluated the potential immunoadjuvant properties of chitosan nanoparticles (CNPs) loaded with B. jararaca and B. erythromelas venoms in the production of sera against these venoms. Stable CNPs were obtained by ionic gelation, and mice were immunized subcutaneously for 6 weeks with 100 mu L of each snake venom at concentrations of 5.0 or 10.0% (w/w), encapsulated in CNPs or associated with aluminium hydroxide (AH). The evaluation of protein interactions with the CNPs revealed their ability to induce antibody levels equivalent to those of AH, even with smaller doses of antigen. In addition, the CNPs were less inflammatory due to their modified release of proteins. CNPs provide a promising approach for peptide/protein delivery from snake venom and will be useful for new vaccines.
