Drug Reprofiling to Identify Potential HIV-1 Protease Inhibitors.

The use of protease inhibitors in human immunodeficiency virus type 1 (HIV-1) treatment is limited by adverse effects, including metabolic complications. To address these challenges, efforts are underway in the pursuit of more potent and less toxic HIV-1 protease inhibitors. Repurposing existing drugs offers a promising avenue to expedite the drug discovery process, saving both time and costs compared to conventional de novo drug development. This study screened FDA-approved and investigational drugs in the DrugBank database for their potential as HIV-1 protease inhibitors. Molecular docking studies and cell-based assays, including anti-HIV-1 in vitro assays and XTT cell viability tests, were conducted to evaluate their efficacy. The study findings revealed that CBR003PS, an antibiotic currently in clinical use, and CBR013PS, an investigational drug for treating endometriosis and uterine fibroids, exhibited significant binding affinity to the HIV-1 protease with high stability. Their EC50 values, measured at 100% cell viability, were 9.4 nM and 36.6 nM, respectively. Furthermore, cell-based assays demonstrated that these two compounds showed promising results, with therapeutic indexes higher than 32. In summary, based on their favorable therapeutic indexes, CBR003PS and CBR013PS show potential for repurposing as HIV-1 protease inhibitors.

Development of Mucoadhesive Electrospun Scaffolds for Intravaginal Delivery of Lactobacilli spp., a Tenside, and Metronidazole for the Management of Bacterial Vaginosis.

Bacterial vaginosis (BV) is an infection of the vagina associated with thriving anaerobes, such as Gardnerella vaginitis and other associated pathogens. These pathogens form a biofilm responsible for the recurrence of infection after antibiotic therapy. The aim of this study was to develop a novel mucoadhesive polyvinyl alcohol and polycaprolactone electrospun nanofibrous scaffolds for vaginal delivery, incorporating metronidazole, a tenside, and Lactobacilli. This approach to drug delivery sought to combine an antibiotic for bacterial clearance, a tenside biofilm disruptor, and a lactic acid producer to restore healthy vaginal flora and prevent the recurrence of bacterial vaginosis. F7 and F8 had the least ductility at 29.25% and 28.39%, respectively, and this could be attributed to the clustering of particles that prevented the mobility of the crazes. F2 had the highest at 93.83% due to the addition of a surfactant that increased the affinity of the components. The scaffolds exhibited mucoadhesion between 31.54 ± 0.83% and 57.86 ± 0.95%, where an increased sodium cocoamphoacetate concentration led to increased mucoadhesion. F6 showed the highest mucoadhesion at 57.86 ± 0.95%, as compared to 42.67 ± 1.22% and 50.89 ± 1.01% for the F8 and F7 scaffolds, respectively. The release of metronidazole via a non-Fickian diffusion-release mechanism indicated both swelling and diffusion. The anomalous transport within the drug-release profile pointed to a drug-discharge mechanism that combined both diffusion and erosion. The viability studies showed a growth of Lactobacilli fermentum in both the polymer blend and the nanofiber formulation that was retained post-storage at 25 °C for 30 days. The developed electrospun scaffolds for the intravaginal delivery of Lactobacilli spp., along with a tenside and metronidazole for the management of bacterial vaginosis, provide a novel tool for the treatment and management of recurrent vaginal infection.

Deep Eutectic Solvents for Subcutaneous Delivery of Protein Therapeutics.

Proteins are among the most common therapeutics for the treatment of diabetes, autoimmune diseases, cancer, and metabolic diseases, among others. Despite their common use, current protein therapies, most of which are injectables, have several limitations. Large proteins such as monoclonal antibodies (mAbs) suffer from poor absorption after subcutaneous injections, thus forcing their administration by intravenous injections. Even small proteins such as insulin suffer from slow pharmacokinetics which poses limitations in effective management of diabetes. Here, a deep eutectic-based delivery strategy is used to offer a generalized approach for improving protein absorption after subcutaneous injections. The lead formulation enhances absorption of mAbs after subcutaneous injections by ≈200%. The same composition also improves systemic absorption of subcutaneously injected insulin faster than Humalog, the current gold-standard of rapid acting insulin. Mechanistic studies reveal that the beneficial effect of deep eutectics on subcutaneous absorption is mediated by their ability to reduce the interactions of proteins with the subcutaneous matrix, especially collagen. Studies also confirm that these deep eutectics are safe for subcutaneous injections. Deep eutectic-based formulations described here open new possibilities for subcutaneous injections of therapeutic proteins.

SNAC for Enhanced Oral Bioavailability: An Updated Review.

The delivery of proteins and peptides via an oral route poses numerous challenges to improve the oral bioavailability and patient compliance. To overcome these challenges, as well as to improve the permeation of proteins and peptides via intestinal mucosa, several chemicals have been studied such as surfactants, fatty acids, bile salts, pH modifiers, and chelating agents, amongst these medium chain fatty acid like C10 (sodium caprate) and Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) and its derivatives that have been well studied from a clinical perspective. This current review enumerates the challenges involved in protein and peptide delivery via the oral route, i.e., non-invasive routes of protein and peptide administration. This review also covers the chemistry behind SNAC and toxicity as well as mechanisms to enhance the oral delivery of clinically proven molecules like simaglutide and other small molecules under clinical development, as well as other permeation enhancers for efficient delivery of proteins and peptides.

Freeze-drying for the preservation of immunoengineering products.

Immunoengineering technologies harness the power of immune system modulators such as monoclonal antibodies, cytokines, and vaccines to treat myriad diseases. Immunoengineering innovations have showed great promise in various practices including oncology, infectious disease, autoimmune diseases, and transplantation. Despite the countless successes, the majority of immunoengineering products contain active moieties that are prone to instability. The current review aims to feature freeze-drying as a robust and scalable solution to the inherent stability challenges in immunoengineering products by preventing the active moiety from degradation. Furthermore, this review describes the stability issues related to immunoengineering products and the utility of the lyophilization process to preserve the integrity and efficacy of immunoengineering tools ranging from biologics to nanoparticle-based vaccines. The concept of the freeze-drying process is described highlighting the quality by design (QbD) for robust process optimization. Case studies of lyophilized immunoengineering technologies and relevant clinical studies using immunoengineering products are discussed.

Discovery of Novel HIV Protease Inhibitors Using Modern Computational Techniques.

The human immunodeficiency virus type 1 (HIV-1) has continued to be a global concern. With the new HIV incidence, the emergence of multi-drug resistance and the untoward side effects of currently used anti-HIV drugs, there is an urgent need to discover more efficient anti-HIV drugs. Modern computational tools have played vital roles in facilitating the drug discovery process. This research focuses on a pharmacophore-based similarity search to screen 111,566,735 unique compounds in the PubChem database to discover novel HIV-1 protease inhibitors (PIs). We used an in silico approach involving a 3D-similarity search, physicochemical and ADMET evaluations, HIV protease-inhibitor prediction (IC50/percent inhibition), rigid receptor-molecular docking studies, binding free energy calculations and molecular dynamics (MD) simulations. The 10 FDA-approved HIV PIs (saquinavir, lopinavir, ritonavir, amprenavir, fosamprenavir, atazanavir, nelfinavir, darunavir, tipranavir and indinavir) were used as reference. The in silico analysis revealed that fourteen out of the twenty-eight selected optimized hit molecules were within the acceptable range of all the parameters investigated. The hit molecules demonstrated significant binding affinity to the HIV protease (PR) when compared to the reference drugs. The important amino acid residues involved in hydrogen bonding and п-п stacked interactions include ASP25, GLY27, ASP29, ASP30 and ILE50. These interactions help to stabilize the optimized hit molecules in the active binding site of the HIV-1 PR (PDB ID: 2Q5K). HPS/002 and HPS/004 have been found to be most promising in terms of IC50/percent inhibition (90.15%) of HIV-1 PR, in addition to their drug metabolism and safety profile. These hit candidates should be investigated further as possible HIV-1 PIs with improved efficacy and low toxicity through in vitro experiments and clinical trial investigations.

Biomimetic Targeted Theranostic Nanoparticles for Breast Cancer Treatment.

The development of biomimetic drug delivery systems for biomedical applications has attracted significant research attention. As the use of cell membrane as a surface coating has shown to be a promising platform for several disease treatments. Cell-membrane-coated nanoparticles exhibit enhanced immunocompatibility and prolonged circulation time. Herein, human red blood cell (RBC) membrane-cloaked nanoparticles with enhanced targeting functionality were designed as a targeted nanotheranostic against cancer. Naturally, derived human RBC membrane modified with targeting ligands coated onto polymeric nanoparticle cores containing both chemotherapy and imaging agent. Using epithelial cell adhesion molecule (EpCAM)-positive MCF-7 breast cancer cells as a disease model, the nature-inspired targeted theranostic human red blood cell membrane-coated polymeric nanoparticles (TT-RBC-NPs) platform was capable of not only specifically binding to targeted cancer cells, effectively delivering doxorubicin (DOX), but also visualizing the targeted cancer cells. The TT-RBC-NPs achieved an extended-release profile, with the majority of the drug release occurring within 5 days. The TT-RBC-NPs enabled enhanced cytotoxic efficacy against EpCAM positive MCF-7 breast cancer over the non-targeted NPs. Additionally, fluorescence images of the targeted cancer cells incubated with the TT-RBC-NPs visually indicated the increased cellular uptake of TT-RBC-NPs inside the breast cancer cells. Taken together, this TT-RBC-NP platform sets the foundation for the next-generation stealth theranostic platforms for systemic cargo delivery for treatment and diagnostic of cancer.

Modulation of Gastrointestinal Mucus Properties with Ionic Liquids for Drug Delivery.

The mucus barrier lining the gastrointestinal tract poses a significant barrier to the oral delivery of macromolecular drugs. Successful approaches to overcoming this barrier have primarily focused on reducing drug and carrier interactions with mucus or disrupting the mucus layer directly. Choline-based ionic liquids (ILs) such as choline geranate and choline glycolate (CGLY) have recently been shown to be effective in enhancing the intestinal absorption of macromolecules such as insulin and immunoglobulin (IgG), respectively. Herein, the use of choline-based ILs as mucus-modulating agents for safely improving drug penetration through mucus is described. Choline-based ILs significantly increase the diffusion rates of cationic dextrans through mucin solution. Choline-maleic acid (CMLC 2:1) enhances the diffusion of 4 kDa cationic dextran in mucin solution by more than fourfold when compared to phosphate-buffered saline control. Choline-based ILs also reduce mucus viscosity without significantly impacting the native mucus gel structure. In vitro studies in a mucus-secreting coculture model with Caco-2 and HT29MTX-E12 cells further demonstrate the effectiveness of ILs in improving transport of cationic molecules in the presence of secreted mucus. This work demonstrates the potential for choline-based ionic liquids to be used as nondestructive mucus-modulating agents for enabling enhanced oral delivery of macromolecular drugs.

Ionic-Liquid-Based Safe Adjuvants.

Adjuvants play a critical role in the design and development of novel vaccines. Despite extensive research, only a handful of vaccine adjuvants have been approved for human use. Currently used adjuvants are mostly composed of components that are non-native to the human body, such as aluminum salt, bacterial lipids, or foreign genomic material. Here, a new ionic-liquid-based adjuvant is explored, synthesized using two metabolites of the body, choline and lactic acid (ChoLa). ChoLa distributes the antigen efficiently upon injection, maintains antigen integrity, enhances immune infiltration at the injection site, and leads to a potent immune response against the antigen. Thus, it can serve as a promising safe adjuvant platform that can help to protect against pandemics and future infectious threats.

Amphiphilic Polyacrylamide Excipients Lead to a Record-Breaking Fast-Acting Insulin.

Fast-acting insulins are central to the regulation of prandial glucose in diabetic patients. Current fast-acting insulins require 20-30 min for the onset and longer for the peak blood concentrations. The recent work by Mann et al. used high-throughput synthesis and screening of polyacrylamide-based excipients to yield a formulation with pharmacokinetics that is faster than the currently available fast-acting insulins.

Inhibition of Pathogen Adhesion by Bacterial Outer Membrane-Coated Nanoparticles.

Anti-adhesion therapies interfere with the bacterial adhesion to the host and thus avoid direct disruption of bacterial cycles for killing, which may alleviate resistance development. Herein, an anti-adhesion nanomedicine platform is made by wrapping synthetic polymeric cores with bacterial outer membranes. The resulting bacterium-mimicking nanoparticles (denoted "OM-NPs") compete with source bacteria for binding to the host. The "top-down" fabrication of OM-NPs avoids the identification of the adhesins and bypasses the design of agonists targeting these adhesins. In this study, OM-NPs are made with the membrane of Helicobacter pylori and shown to bind with gastric epithelial cells (AGS cells). Treatment of AGS cells with OM-NPs reduces H. pylori adhesion and such anti-adhesion efficacy is dependent on OM-NP concentration and its dosing sequence.

Biomimetic Micromotor Enables Active Delivery of Antigens for Oral Vaccination.

Vaccination represents one of the most effective means of preventing infectious disease. In order to maximize the utility of vaccines, highly potent formulations that are easy to administer and promote high patient compliance are desired. In the present work, a biomimetic self-propelling micromotor formulation is developed for use as an oral antivirulence vaccine. The propulsion is provided by a magnesium-based core, and a biomimetic cell membrane coating is used to detain and neutralize a toxic antigenic payload. The resulting motor toxoids leverage their propulsion properties in order to more effectively elicit mucosal immune responses. After demonstrating the successful fabrication of the motor toxoids, their uptake properties are shown in vitro. When delivered to mice via an oral route, it is then confirmed that the propulsion greatly improves retention and uptake of the antigenic material in the small intestine in vivo. Ultimately, this translates into markedly elevated generation of antibody titers against a model toxin. This work provides a proof-of-concept highlighting the benefits of active oral delivery for vaccine development, opening the door for a new set of applications, in which biomimetic motor technology can provide significant benefits.

Composite thermoresponsive hydrogel with auranofin-loaded nanoparticles for topical treatment of vaginal trichomonad infection.

Trichomonas vaginalis is responsible for the most common non-viral sexually-transmitted disease worldwide. Standard treatment is with oral nitro-heterocyclic compounds, metronidazole or tinidazole, but resistance to these drugs is emerging and adverse effects can be problematic. Topical treatment offers potential benefits for increasing local drug concentrations and efficacy, while reducing systemic drug exposure, but no topical strategies are currently approved for trichomoniasis. The anti-rheumatic drug, auranofin (AF), was recently discovered to have significant trichomonacidal activity, but has a long plasma half-life and significant adverse effects. Here, we used this drug as a model to develop a novel topical formulation composed of AF-loaded nanoparticles (NP) embedded in a thermoresponsive hydrogel for intravaginal administration. The AF-NP composite gel showed sustained drug release for at least 12 h, and underwent sol-gel transition with increased viscoelasticity within a minute. Intravaginal administration in mice showed excellent NP retention for >6 h and markedly increased local AF levels, but reduced plasma and liver levels compared to oral treatment with a much higher dose. Furthermore, intravaginal AF-NP gel greatly outperformed oral AF in eliminating vaginal trichomonad infection in mice, while causing no systemic or local toxicity. These results show the potential of the AF-NP hydrogel formulation for effective topical therapy of vaginal infections.

Coating nanoparticles with gastric epithelial cell membrane for targeted antibiotic delivery against Helicobacter pylori infection.

Inspired by the natural pathogen-host interactions and adhesion, this study reports on the development of a novel targeted nanotherapeutics for the treatment of Helicobacter pylori (H. pylori) infection. Specifically, plasma membranes of gastric epithelial cells (e.g. AGS cells) are collected and coated onto antibiotic-loaded polymeric cores, the resulting biomimetic nanoparticles (denoted AGS-NPs) bear the same surface antigens as the source AGS cells and thus have inherent adhesion to H. pylori bacteria. When incubated with H. pylori bacteria in vitro, the AGS-NPs preferentially accumulate on the bacterial surfaces. Using clarithromycin (CLR) as a model antibiotic and a mouse model of H. pylori infection, the CLR-loaded AGS-NPs demonstrate superior therapeutic efficacy as compared the free drug counterpart as well as non-targeted nanoparticle control group. Overall, this work illustrates the promise and strength of using natural host cell membranes to functionalize drug nanocarriers for targeted drug delivery to pathogens that colonize on the host cells. As host-pathogen adhesion represents a common biological event for various types of pathogenic bacteria, the bioinspired nanotherapeutic strategy reported here represents a versatile delivery platform that may be applied to treat numerous infectious diseases.

Micromotor Pills as a Dynamic Oral Delivery Platform.

Tremendous progress has been made during the past decade toward the design of nano/micromotors with high biocompatibility, multifunctionality, and efficient propulsion in biological fluids, which collectively have led to the initial investigation of in vivo biomedical applications of these synthetic motors. Despite these recent advances in micromotor designs and mechanistic research, significant effort is needed to develop appropriate formulations of micromotors to facilitate their in vivo administration and thus to better test their in vivo applicability. Herein, we present a micromotor pill and demonstrate its attractive use as a platform for in vivo oral delivery of active micromotors. The micromotor pill is comprised of active Mg-based micromotors dispersed uniformly in the pill matrix, containing inactive (lactose/maltose) excipients and other disintegration-aiding (cellulose/starch) additives. Our in vivo studies using a mouse model show that the micromotor pill platform effectively protects and carries the active micromotors to the stomach, enabling their release in a concentrated manner. The micromotor encapsulation and the inactive excipient materials have no effects on the motion of the released micromotors. The released cargo-loaded micromotors propel in gastric fluid, retaining the high-performance characteristics of in vitro micromotors while providing higher cargo retention onto the stomach lining compared to orally administrated free micromotors and passive microparticles. Furthermore, the micromotor pills and the loaded micromotors retain the same characteristics and propulsion behavior after extended storage in harsh conditions. These results illustrate that combining the advantages of traditional pills with the efficient movement of micromotors offer an appealing route for administrating micromotors for potential in vivo biomedical applications.

Neutralization of cholera toxin with nanoparticle decoys for treatment of cholera.

Diarrheal diseases are a major cause of morbidity and mortality worldwide. In many cases, antibiotic therapy is either ineffective or not recommended due to concerns about emergence of resistance. The pathogenesis of several of the most prevalent infections, including cholera and enteroxigenic Escherichia coli, is dominated by enterotoxins produced by lumen-dwelling pathogens before clearance by intestinal defenses. Toxins gain access to the host through critical host receptors, making these receptors attractive targets for alternative antimicrobial strategies that do not rely on conventional antibiotics. Here, we developed a new nanotechnology strategy as a countermeasure against cholera, one of the most important and prevalent toxin-mediated enteric infections. The key host receptor for cholera toxin, monosialotetrahexosylganglioside (GM1), was coated onto the surface of polymeric nanoparticles. The resulting GM1-polymer hybrid nanoparticles were shown to function as toxin decoys by selectively and stably binding cholera toxin, and neutralizing its actions on epithelial cells in vitro and in vivo. Furthermore, the GM1-coated nanoparticle decoys attenuated epithelial 3',5'-cyclic adenosine monophosphate production and fluid responses to infection with live Vibrio cholera in cell culture and a murine infection model. Together, these studies illustrate that the new nanotechnology-based platform can be employed as a non-traditional antimicrobial strategy for the management of enteric infections with enterotoxin-producing pathogens.

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors.

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound-powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA-loaded nanomotors to directly penetrate through the plasma membrane of GFP-expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA-loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor-based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.

Hybrid biomembrane-functionalized nanorobots for concurrent removal of pathogenic bacteria and toxins.

With the rapid advancement of robotic research, it becomes increasingly interesting and important to develop biomimetic micro- or nanorobots that translate biological principles into robotic systems. We report the design, construction, and evaluation of a dual-cell membrane-functionalized nanorobot for multipurpose removal of biological threat agents, particularly concurrent targeting and neutralization of pathogenic bacteria and toxins. Specifically, we demonstrated ultrasound-propelled biomimetic nanorobots consisting of gold nanowires cloaked with a hybrid of red blood cell (RBC) membranes and platelet (PL) membranes. Such hybrid cell membranes have a variety of functional proteins associated with human RBCs and PLs, which give the nanorobots a number of attractive biological capabilities, including adhesion and binding to PL-adhering pathogens (e.g., Staphylococcus aureus bacteria) and neutralization of pore-forming toxins (e.g., α-toxin). In addition, the biomimetic nanorobots displayed rapid and efficient prolonged acoustic propulsion in whole blood, with no apparent biofouling, and mimicked the movement of natural motile cells. This propulsion enhanced the binding and detoxification efficiency of the robots against pathogens and toxins. Overall, coupling these diverse biological functions of hybrid cell membranes with the fuel-free propulsion of the nanorobots resulted in a dynamic robotic system for efficient isolation and simultaneous removal of different biological threats, an important step toward the creation of a broad-spectrum detoxification robotic platform.

Toxoid Vaccination against Bacterial Infection Using Cell Membrane-Coated Nanoparticles.

As nanoparticles exhibit unique properties attractive for vaccine development, they have been progressively implemented as antigen delivery platforms and immune potentiators. Recently, cell membrane-coated nanoparticles have provided a novel approach for intercepting and neutralizing bacterial toxins by leveraging their natural affinity to cellular membranes. Such toxin-nanoparticle assemblies, termed nanotoxoids, allow rapid loading of different types of toxins and have been investigated for their ability to effectively confer protection against bacterial infection. This topical review will cover the current progress in antibacterial vaccine nanoformulations and highlight the nanotoxoid platform as a novel class of nanoparticulate vaccine. We aim to provide insights into the potential of nanotoxoids as a platform that is facile to implement and can be broadly applied to help address the rising threat of super pathogens.

Biomimetic Platelet-Camouflaged Nanorobots for Binding and Isolation of Biological Threats.

One emerging and exciting topic in robotics research is the design of micro-/nanoscale robots for biomedical operations. Unlike industrial robots that are developed primarily to automate routine and dangerous tasks, biomedical nanorobots are designed for complex, physiologically relevant environments, and tasks that involve unanticipated biological events. Here, a biologically interfaced nanorobot is reported, made of magnetic helical nanomotors cloaked with the plasma membrane of human platelets. The resulting biomimetic nanorobots possess a biological membrane coating consisting of diverse functional proteins associated with human platelets. Compared to uncoated nanomotors which experience severe biofouling effects and hence hindered propulsion in whole blood, the platelet-membrane-cloaked nanomotors disguise as human platelets and display efficient propulsion in blood over long time periods. The biointerfaced nanorobots display platelet-mimicking properties, including adhesion and binding to toxins and platelet-adhering pathogens, such as Shiga toxin and Staphylococcus aureus bacteria. The locomotion capacity and platelet-mimicking biological function of the biomimetic nanomotors offer efficient binding and isolation of these biological threats. The dynamic biointerfacing platform enabled by platelet-membrane cloaked nanorobots thus holds considerable promise for diverse biomedical and biodefense applications.