Microfluidic nanodevices for drug sensing and screening applications.

The outbreak of pandemics (e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 in 2019), influenza A viruses (H1N1 in 2009), etc.), and worldwide spike in the aging population have created unprecedented urgency for developing new drugs to improve disease treatment. As a result, extensive efforts have been made to design novel techniques for efficient drug monitoring and screening, which form the backbone of drug development. Compared to traditional techniques, microfluidics-based platforms have emerged as promising alternatives for high-throughput drug screening due to their inherent miniaturization characteristics, low sample consumption, integration, and compatibility with diverse analytical strategies. Moreover, the microfluidic-based models utilizing human cells to produce in-vitro biomimetics of the human body pave new ways to predict more accurate drug effects in humans. This review provides a comprehensive summary of different microfluidics-based drug sensing and screening strategies and briefly discusses their advantages. Most importantly, an in-depth outlook of the commonly used detection techniques integrated with microfluidic chips for highly sensitive drug screening is provided. Then, the influence of critical parameters such as sensing materials and microfluidic platform geometries on screening performance is summarized. This review also outlines the recent applications of microfluidic approaches for screening therapeutic and illicit drugs. Moreover, the current challenges and the future perspective of this research field is elaborately highlighted, which we believe will contribute immensely towards significant achievements in all aspects of drug development.

Effects of Sodium Silicate Complex against Hemorrhagic Activities Induced by Protobothrops mucrosquamatus Venom.

Protobothrops mucrosquamatus poses a serious medical threat to humans in Southern and Southeastern Asia. Hemorrhage is one of the conspicuous toxicities related to the pathology of P. mucrosquamatus envenoming. Previous in vitro and in vivo studies showed that a silica-derived reagent, sodium silicate complex (SSC), was able to neutralize hemorrhagic and proteolytic activities induced by pit viper venoms, including Crotalus atrox, Agkistrodoncontortrix contortrix and Agkistrodon piscivorus leucostoma. In this study, we validated that SSC could neutralize enzymatic and toxic effects caused by the venom of P. mucrosquamatus. We found that SSC inhibited the hemolytic and proteolytic activities induced by P. mucrosquamatus venom in vitro. In addition, we demonstrated that SSC could block intradermal hemorrhage caused by P. mucrosquamatus venom in a mouse model. Finally, SSC could neutralize lethal effects of P. mucrosquamatus venom in the mice. Therefore, SSC is a candidate for further development as a potential onsite first-aid treatment for P. mucrosquamatus envenoming.

Snake venom proteome of Protobothrops mucrosquamatus in Taiwan: Delaying venom-induced lethality in a rodent model by inhibition of phospholipase A2 activity with varespladib.

Protobothrops mucrosquamatus, also known as the brown spotted pit viper or Taiwanese habu, is a medically significant venomous snake in Taiwan, especially in the northern area. To more fully understand the proteome profile of P. mucrosquamatus, we characterized its venom composition using a bottom-up proteomic approach. Whole venom components were fractionated by RP-HPLC and then analyzed by SDS-PAGE. Each protein band in gels was excised and subjected to protein identification by LC-MS/MS. A subsequent proteomic analysis revealed the presence of 61 distinct proteins belonging to 19 families in P. mucrosquamatus venom. Snake venom metalloproteinase (SVMP; 29.4%), C-type lectin (CLEC; 21.1%), snake venom serine protease (SVSP; 17.6%) and phospholipase A2 (PLA2; 15.9%) were the most abundant protein families, whereas several low-abundance proteins, categorized into eight protein families, were demonstrated in P. mucrosquamatus venom for the first time. Because PLA2 is known to make a major contribution to venom lethality, we evaluated whether the known PLA2 inhibitor, varespladib, was capable of preventing the toxic effects of P. mucrosquamatus venom. This small-molecule drug demonstrated the ability to inhibit PLA2 activity in vitro (IC50 = 101.3 nM). It also blunted lethality in vivo, prolonging survival following venom injection in a mouse model, but it showed limited potency against venom-induced local hemorrhage in this model. Our findings provide essential biological and pathophysiological insights into the composition of P. mucrosquamatus venom and suggest PLA2 inhibition as an adjunctive or alternative therapeutic strategy in the clinical management of P. mucrosquamatus envenoming in emergency medicine. SIGNIFICANCE: P. mucrosquamatus envenomation is a significant medical concern in Taiwan, especially in the northern region. Although antivenom is commonly used for rescuing P. mucrosquamatus envenoming, severe clinical events still occur, with more than 20% of cases requiring surgical intervention. Small-molecule therapy offers several advantages as a potential adjunctive, or even alternative, to antivenom treatment, such as heat stability, low antigenicity and ease of administration, among others. A deeper understanding of the venom proteome of P. mucrosquamatus would aid in the discovery of small-molecule drugs that could be repurposed to target specific venom proteins. Here, we applied a bottom-up proteomic approach to characterize the protein profile of P. mucrosquamatus venom. Varespladib, a small-molecule drug used to treat inflammatory disease, was repurposed to inhibit the toxicity of P. mucrosquamatus venom, and was shown to reduce the lethal effects of P. mucrosquamatus envenomation in a rodent model. Varespladib might be used as a first-aid therapeutic against P. mucrosquamatus envenoming in the pre-referral period and/or as an adjunctive agent administered together with anti-P. mucrosquamatus antivenom.

Pathogenesis of local necrosis induced by Naja atra venom: Assessment of the neutralization ability of Taiwanese freeze-dried neurotoxic antivenom in animal models.

Naja atra envenomation is one of the most significant clinical snakebite concerns in Taiwan. Taiwanese freeze-dried neurotoxic antivenom (FNAV) is currently used clinically for the treatment of cobra snakebite, and has been shown to limit the mortality of cobra envenomation to less than 1%. However, more than half of victims (60%) require surgery because of local tissue necrosis, a major problem in patients with cobra envenomation. Although the importance of evaluating the neutralizing effect of FNAV on this pathology is recognized, whether FNAV is able to prevent the local necrosis extension induced by N. atra venom has not been investigated in detail. Cytotoxins (CTXs) are considered as the major components of N. atra venom that cause necrosis. In the current study, we isolated CTXs from whole cobra venom and used both whole venom and purified CTXs to develop animal models for assessing the neutralization potential of FNAV against venom necrotizing activity. Local necrotic lesions were successfully produced in mice using CTXs in place of whole N. atra venom. FNAV was able to rescue mice from a subcutaneously injected lethal dose of cobra venom; however, it was unable to prevent CTX-induced dermo-necrosis. Furthermore, using the minimal necrosis dose (MND) of CTXs and venom proteome data, we found a dose of whole N. atra venom suitable for FNAV and developed a workable protocol for inducing local necrosis in rodent models that successfully imitated the clinical circumstance of cobra envenoming. This information provides a more comprehensive understanding of the pathophysiology of N. atra envenomation, and serves as a guide for improving current antivenom strategies and advancing clinical snakebite management in Taiwan.