Gustavo Henrique da Silva

Neutralization of the pharmacological effects of bothropstoxin-I from Bothrops jararacussu (jararacucu) venom by crotoxin antiserum and heparin

Bothropstoxin-I (BthTX-I), the principal myotoxin of Bothrops jararacussu venom, is devoid of phospholipase A(2) (PLA(2)) activity but capable of blocking neuromuscular transmission in mouse nerve-muscle preparations. In this study, the ability of crotoxin antiserum and heparin in preventing the neurotoxic and myotoxic effects of BthTX-I was investigated. Phrenic nerve-diaphragm preparations (PND) stimulated indirectly with supramaximal stimuli (0.2 ms, 0.1 Hz) were incubated with BthTX-I (20 microg/ml) alone or with BthTX-I preincubated with antiserum or heparin for 30 min at 37 degrees C prior to testing. Control preparations were incubated with Tyrode solution, antiserum or heparin alone. BthTX-I (20 microg/ml) produced 50% neuromuscular blockade in the PND preparations in 31+/-4min, with complete blockade occurring in 120 min. The antiserum and heparin significantly prevented the neuromuscular blockade caused by BthTX-I (84 +/- 4% and 100% protection, respectively). Light microscopy examination of the muscles at the end of the 120 min incubation showed that BthTX-I damaged 48 +/- 6% of the fibers. Preincubating the toxin with antivenom significantly reduced the extent of this damage (only 15 +/- 4% of fibers affected, corresponding to 69% protection, P<0.01) whereas heparin offered no protection (34 +/- 7% of fibers affected, not significantly different from that seen with toxin alone). These results show that the antivenom was more effective in neutralizing the myotoxic effects of BthTX-I than was heparin.

Acute blood-brain barrier permeabilization in rats after systemic Phoneutria nigriventer venom

A highly controlled transport of substances at the interface between blood and brain characterizes the blood-brain barrier (BBB), fundamental for maintenance of the homeostasis of the cerebral milieu. In this study, we investigated the time course (15 min, 1, 2, and 5 h) of BBB opening induced by intravenous (i.v.) injection of Phoneutria nigriventer spider venom (PNV) using quantitative and morphological approaches on cerebellum and hippocampus vessels for assessment of BBB permeability. The results showed vasogenic edema and tracer extravasation faster and severalfold higher in hippocampus than in cerebellum. Reactive astrocytes with swollen perivascular end-feet processes were found only in cerebellum. An immediate and total degradation of laminin in capillaries occurred resulting in the disappearance of the basement membrane. In medium-sized vessels, this effect was less prominent. The changes were transient, with cerebellum in general presenting a faster recovery. However, at 5 h laminin was overexpressed, principally in hippocampus. The rapid and abrupt shift of laminin expression in capillaries (at 15 min) coincided with the immediate and severe signs of intoxication shown by the animals, but not with the peak of leakage of vessels and vasogenic edema, which occurred later (1-2 h). The findings suggest a complex regulatory mechanism, since the extension of BBB impairment caused by PNV depends on the region of the SNC, and on the vessels types. It is suggested that the components of the BBB (gliovascular unit) have a critical role in these differences. P. nigriventer venom can be a useful tool to explore the mechanisms of BBB.

Natural lipids-based NLC containing lidocaine: from pre-formulation to in vivo studies

Abstract In a nanotechnological approach we have investigated the use of natural lipids in the preparation of nanostructured lipid carriers (NLC). Three different NLC composed of copaiba oil and beeswax, sweet almond oil and shea butter, and sesame oil and cocoa butter as structural matrices were optimized using factorial analysis; Pluronic® 68 and lidocaine (LDC) were used as the colloidal stabilizer and model encapsulated drug, respectively. The optimal formulations were characterized by different techniques (IR‐ATR, DSC, and TEM), and their safety and efficacy were also tested. These nanocarriers were able to upload high amounts of the anesthetic with a sustained in vitro release profile for 24 h. The physicochemical stability in terms of size (nm), PDI, zeta potential (mV), pH, nanoparticle concentration (particles/mL), and visual inspection was followed during 12 months of storage at 25 °C. The formulations exhibited excellent structural properties and stability. They proved to be nontoxic in vitro (cell viability tests with Balb/c 3T3 fibroblasts) and significantly improved the in vivo effects of LDC, over the heart rate of zebra fish larvae and in the blockage of sciatic nerve in mice. The results from this study support that the proper combination of natural excipients is promising in DDS, taking advantage of the biocompatibility, low cost, and diversity of lipids. Graphical abstract Illustrative representation of a NLC prepared with natural lipids. Three different optimized NLC systems for lidocaine delivery were prepared, using copaiba oil and beeswax, sweet almond oil and shea butter, or sesame oil and cocoa butter as the lipid matrix, plus Pluronic® 68 as surfactant. The systems were evaluated from pre‐formulation to in vivo studies and the most prominent results are highlighted. Figure. No Caption available.

Advances in Hybrid Polymer-Based Materials for Sustained Drug Release

The use of biomaterials composed of organic pristine components has been successfully described in several purposes, such as tissue engineering and drug delivery. Drug delivery systems (DDS) have shown several advantages over traditional drug therapy, such as greater therapeutic efficacy, prolonged delivery profile, and reduced drug toxicity, as evidenced by in vitro and in vivo studies as well as clinical trials. Despite that, there is no perfect delivery carrier, and issues such as undesirable viscosity and physicochemical stability or inability to efficiently encapsulate hydrophilic/hydrophobic molecules still persist, limiting DDS applications. To overcome that, biohybrid systems, originating from the synergistic assembly of polymers and other organic materials such as proteins and lipids, have recently been described, yielding molecularly planned biohybrid systems that are able to optimize structures to easily interact with the targets. This work revised the biohybrid DDS clarifying their advantages, limitations, and future perspectives in an attempt to contribute to further research of innovative and safe biohybrid polymer-based system as biomaterials for the sustained release of active molecules.

Optimised NLC: a nanotechnological approach to improve the anaesthetic effect of bupivacaine

The short time of action and systemic toxicity of local anaesthetics limit their clinical application. Bupivacaine is the most frequently used local anaesthetic in surgical procedures worldwide. The discovery that its S(-) enantiomeric form is less toxic than the R(+) form led to the introduction of products with enantiomeric excess (S75:R25 bupivacaine) in the market. Nevertheless, the time of action of bupivacaine is still short; to overcome that, bupivacaine S75:R25 (BVCS75) was encapsulated in nanostructured lipid carriers (NLC). In this work, we present the development of the formulation using chemometric tools of experimental design to study the formulation factors and Raman mapping associated with Classical Least Squares (CLS) to study the miscibility of the solid and the liquid lipids. The selected formulation of the nanostructured lipid carrier containing bupivacaine S75:R25 (NLCBVC) was observed to be stable for 12 months under room conditions regarding particle size, polydispersion, Zeta potential and encapsulation efficiency. The characterisation by DSC, XDR and TEM confirmed the encapsulation of BVCS75 in the lipid matrix, with no changes in the structure of the nanoparticles. The in vivo analgesic effect elicited by NLCBVC was twice that of free BVCS75. Besides improving the time of action, no statistical difference in the blockage of the sciatic nerve of rats was found between 0.125% NLCBVC and 0.5% free BVCS75. Therefore, the formulation allows a reduction in the required anaesthesia dose, decreasing the systemic toxicity of bupivacaine, and opening up new possibilities for different clinical applications.

Nanohybrid hydrogels designed for transbuccal anesthesia

Background Local anesthesia in dentistry is by far the most terrifying procedure for patients, causing treatment interruption. None of the commercially available topical formulations is effective in eliminating the pain and phobia associated to the needle insertion and injection. Materials and methods In this work we prepared a nanostructured lipid-biopolymer hydrogel for the sustained delivery of lidocaine–prilocaine (LDC-PLC) for transbuccal pre-anesthesia. The lipid was composed of optimized nanostructured lipid carriers (NLC) loaded with 5% LDC-PLC (NLC/LDC-PLC). The biopolymer counterpart was selected among alginate, xanthan (XAN), and chitosan matrices. The XAN-NLC hydrogel presented the most uniform aspect and pseudoplastic rheological profile, as required for topical use; therefore, it was selected for subsequent analyses. Accelerated stability tests under critical conditions (40°C; 75% relative humidity) were conducted for 6 months, in terms of drug content (mg/g), weight loss (%), and pH. Results In vitro LDC-PLC release profile through Franz diffusion cells revealed a bimodal kinetics with a burst effect followed by the sustained release of both anesthetics, for 24 hours. Structural analyses (fourier transform infrared spectroscopy, differential scanning calorimetry and scanning electron microscopy) gave details on the molecular organization of the hybrid hydrogel, confirming the synergic interaction between the components. Safety and efficacy were evaluated through in vitro cell viability (3T3, HaCat, and VERO cells) and in vivo antinociceptive (tail-flick, in mice) tests, respectively. In comparison to a control hydrogel and the eutectic mixture of 5% LDC-PLC cream (EMLA®), the XAN-NLC/LDC-PLC hybrid hydrogel doubled and quadrupled the anesthetic effect (8 hours), respectively. Conclusion Considering such exciting results, this multifaceted nanohybrid system is now ready to be further tested in clinical trials.

Bupivacaine (S75:R25) Loaded in Nanostructured Lipid Carriers: Factorial Design, HPLC Quantification Method and Physicochemical Stability Study

BACKGROUND Bupivacaine is the most used local anesthetic in surgical procedures, producing prolonged anesthesia. The major limiting factor for the clinical use of bupivacaine comes from its systemic toxicity. Nanostructured lipid carriers (NLC) are vehicles for sustained drug delivery that are able to minimize the toxicity and to increase the action time of lipophilic drugs. METHODS This work reports a 22 factorial design, which elucidates the role of the lipids mixture in the NLC, towards an optimized formulation. It also provides a new method for bupivacaine S75:R25 (BVCS75) quantification in NLC. Moreover, physicochemical stability studies on the prepared NLC formulations were carried out by monitoring particle size, polydispersity, Zeta potential and BVCS75 encapsulation efficiency for 90 days, at 25°C. RESULTS The factorial design showed that the liquid lipid Capryol 90® has a negative effect over particle size and PDI values while cetyl palmitate presented a positive effect in size. The analytical method was accurate, reproducible, specific and linear over the concentration range of 0.16-54.00 µg.mL-1 BVCS75 with limits of quantification and detection of 0.10 and 0.03 µg.mL-1, respectively. The validated method was used to quantify the BVCS75 encapsulation (55.5 ±2.8 %). Encapsulation did not affect the nanoparticles morphology (confirmed by Transmission Electron Microscopy), but increased their Zeta potential (from -15.7 to -37.0 mV). The NLC physical stability was maintained (particles: size < 170 nm, polydispersity <0.16, and number = 8.85 ±0.11 x 1013 particles.mL-1) during storage. CONCLUSION These results support further investigations on the use of BVCS75-in-NLC formulation for surgical anesthesia, aiming the development of a potent and less toxic nanostructured lipid carrier formulation for BVCS75.