Understanding structure activity relationships of Good HEPES lipids for lipid nanoparticle mRNA vaccine applications.

Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.

High affinity protein surface binding through co-engineering of nanoparticles and proteins.

Control over supramolecular recognition between proteins and nanoparticles (NPs) is of fundamental importance in therapeutic applications and sensor development. Most NP-protein binding approaches use 'tags' such as biotin or His-tags to provide high affinity; protein surface recognition provides a versatile alternative strategy. Generating high affinity NP-protein interactions is challenging however, due to dielectric screening at physiological ionic strengths. We report here the co-engineering of nanoparticles and protein to provide high affinity binding. In this strategy, 'supercharged' proteins provide enhanced interfacial electrostatic interactions with complementarily charged nanoparticles, generating high affinity complexes. Significantly, the co-engineered protein-nanoparticle assemblies feature high binding affinity even at physiologically relevant ionic strength conditions. Computational studies identify both hydrophobic and electrostatic interactions as drivers for these high affinity NP-protein complexes.

Impact of COVID-19 on the Intestinal Microbiome.

PURPOSE OF REVIEW: This review article aims to explore the GI changes induced by SARS-CoV-2 and how gut microbial homeostasis can influence these changes and affect the lung-gut axis and its relationship with the induction of the cytokine release syndrome in severe COVID-19 patients. RECENT FINDINGS: Coronavirus disease 2019 (COVID-19) affects not only the respiratory system but can produce multi-systemic damage. The expression of angiotensin-converting enzyme 2 (ACE-2) receptors in the gastrointestinal (GI) tract, the high prevalence of GI symptoms in severely ill COVID-19 patients, and the abnormalities described in the gut microbiome in these patients have raised concerns about the influence of GI tract as a risk factor or as a potential modulator to reduce the severity of COVID-19. Understanding the mechanisms by which gut dysbiosis may influence viral transmission and disease progression in COVID-19 may help in shaping how accessible therapies, like diet modulation, can potentially help beat the devastating consequences of COVID-19.

Nanotherapeutics using all-natural materials. Effective treatment of wound biofilm infections using crosslinked nanoemulsions.

Bacterial wound infections are a threat to public health. Although antibiotics currently provide front-line treatments for bacterial infections, the development of drug resistance coupled with the defenses provided through biofilm formation render these infections difficult, if not impossible, to cure. Antimicrobials from natural resources provide unique antimicrobial mechanisms and are generally recognized as safe and sustainable. Herein, an all-natural antimicrobial platform is reported. It is active against bacterial biofilms and accelerates healing of wound biofilm infections in vivo. This antimicrobial platform uses gelatin stabilized by photocrosslinking using riboflavin (vitamin B2) as a photocatalyst, and carvacrol (the primary constituent of oregano oil) as the active antimicrobial. The engineered nanoemulsions demonstrate broad-spectrum antimicrobial activity towards drug-resistant bacterial biofilms and significantly expedite wound healing in an in vivo murine wound biofilm model. The antimicrobial activity, wound healing promotion, and biosafety of these nanoemulsions provide a readily translatable and sustainable strategy for managing wound infections.

Polymeric Nanoparticles Active against Dual-Species Bacterial Biofilms.

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.

Intracellular Activation of Anticancer Therapeutics Using Polymeric Bioorthogonal Nanocatalysts.

Bioorthogonal catalysis provides a promising strategy for imaging and therapeutic applications, providing controlled in situ activation of pro-dyes and prodrugs. In this work, the use of a polymeric scaffold to encapsulate transition metal catalysts (TMCs), generating bioorthogonal "polyzymes," is presented. These polyzymes enhance the stability of TMCs, protecting the catalytic centers from deactivation in biological media. The therapeutic potential of these polyzymes is demonstrated by the transformation of a nontoxic prodrug to an anticancer drug (mitoxantrone), leading to the cancer cell death in vitro.

High-content and high-throughput identification of macrophage polarization phenotypes.

Macrophages are plastic cells of the innate immune system that perform a wide range of immune- and homeostasis-related functions. Due to their plasticity, macrophages can polarize into a spectrum of activated phenotypes. Rapid identification of macrophage polarization states provides valuable information for drug discovery, toxicological screening, and immunotherapy evaluation. The complexity associated with macrophage activation limits the ability of current biomarker-based methods to rapidly identify unique activation states. In this study, we demonstrate the ability of a 2-element sensor array that provides an information-rich 5-channel output to successfully determine macrophage polarization phenotypes in a matter of minutes. The simple and robust sensor generates a high dimensional data array which enables accurate macrophage evaluations in standard cell lines and primary cells after cytokine treatment, as well as following exposure to a model disease environment.

Functionalized Polymers Enhance Permeability of Antibiotics in Gram-negative MDR Bacteria and Biofilms for Synergistic Antimicrobial Therapy.

The emergence of multi-drug resistant pathogenic bacteria constitutes a key threat to global health. Infections caused by multi-drug resistant Gram-negative bacteria are particularly challenging to treat due to the ability of pathogens to prevent antibiotic penetration inside the bacterial membrane. Antibiotic therapy is further rendered ineffective due to biofilm formation where the protective Extracellular Polymeric Substance (EPS) matrix limits the diffusion of antibiotics inside the biofilm. We hypothesized that careful engineering of chemical groups on polymer scaffolds could enable polymers to penetrate the barriers of Gram-negative bacterial membrane and biofilm matrix. Here, we present the use of engineered polymeric nanoparticles in combination with antibiotics for synergistic antimicrobial therapy. These polymeric nanoparticles enhance the accumulation of antibiotics inside Gram-negative bacteria and biofilm matrix, resulting in increased potency of antibiotics in combination therapy. Sub-lethal concentrations of engineered polymeric nanoparticles reduce the antibiotic dosage by 32-fold to treat MDR bacteria and biofilms. Tailoring of chemical groups on polymers demonstrate a strong-structure activity relationship in generating additive and synergistic combinations with antibiotics. This study demonstrates the ability of polymeric nanoparticles to 'rejuvenate' antibiotics rendered ineffective by resistant bacteria and provides a rationale to design novel compounds to achieve effective antimicrobial combination therapies.

Phytochemical-Based Nanocomposites for the Treatment of Bacterial Biofilms.

Biofilm infections are responsible for at least 65% of human bacterial infections. These biofilms are refractory to conventional antibiotics, leading to chronic infections and nonhealing wounds. Plant-derived antibiotics (phytochemicals) are promising alternative antimicrobial treatments featuring antimicrobial properties. However, their poor solubility in aqueous media limits their application in treating biofilm infections. Phytochemicals were incorporated into cross-linked polymer nanocomposite "sponges" for the treatment of bacterial biofilms. The results indicated encapsulating low log P phytochemicals effectively eliminated biofilms while demonstrating low cytotoxicity against mammalian fibroblast cells.

Rapid Identification of Biofilms Using a Robust Multichannel Polymer Sensor Array.

Infections caused by bacterial biofilms are challenging to diagnose because of the complexity of both the bacteria and the heterogeneous biofilm matrix. We report here a robust polymer-based sensor array that uses selective interactions between polymer sensor elements and the biofilm matrix to identify bacteria species. In this array, an appropriate choice of fluorophore enabled excimer formation and interpolymer FRET, generating six output channels from three polymers. Selective multivalent interactions of these polymers with the biofilm matrices caused differential changes in fluorescent patterns, providing a species-based signature of the biofilm. The real-world potential of the platform was further validated through identification of mixed-species bacterial biofilms and discrimination of biofilms in a mammalian cell-biofilm co-culture wound model.

Water-Dispersible and Biocompatible Iron Carbide Nanoparticles with High Specific Absorption Rate.

Magnetic nanoparticles are important tools for biomedicine, where they serve as versatile multifunctional instruments for a wide range of applications. Among these applications, magnetic hyperthermia is of special interest for the destruction of tumors and triggering of drug delivery. However, many applications of magnetic nanoparticles require high-quality magnetic nanoparticles displaying high specific absorption rates (SARs), which remains a challenge today. We report here the functionalization and stabilization in aqueous media of highly magnetic 15 nm iron carbide nanoparticles featuring excellent heating power through magnetic induction. The challenge of achieving water solubility and colloidal stability was addressed by designing and using specific dopamine-based ligands. The resulting nanoparticles were completely stable for several months in water, phosphate, phosphate-buffered saline, and serum-containing media. Iron carbide nanoparticles displayed high SARs in water and viscous media (water/glycerol mixtures), even after extended exposition to water and oxygen (SAR up to 1000 W·g-1 in water at 100 kHz, 47 mT). The cytotoxicity and cellular uptake of iron carbide nanoparticles could be easily tuned and were highly dependent on the chemical structure of the ligands used.

Control of Intra- versus Extracellular Bioorthogonal Catalysis Using Surface-Engineered Nanozymes.

Bioorthogonal transformation of prodrugs and profluorophores using transition metal catalysts (TMCs) offers a promising strategy for therapeutic and imaging applications. Here, we report the surface engineering of nanoparticles to specifically localize gold nanoparticles (AuNPs) with encapsulated TMCs (nanozymes) to either the inside or outside of cells. The ability to control nanozyme localization and hence activity was demonstrated by the activation of pro-fluorophores and prodrugs intra- and extracellularly, establishing the potential of engineered nanozyme platforms for both diagnostic and therapeutic purposes.

Dual Functionalization of Nanoparticles for Generating Corona-Free and Noncytotoxic Silica Nanoparticles.

Protein coronas form on the surfaces of nanomaterials in biological fluids. This layer of proteins affects the properties of nanomaterials, altering their behavior and masking engineered functionality. The use of nonfouling moieties reduces or prevents corona formation; however, these ligands typically complicate functionalization. We present here a surface modification strategy for silica nanoparticles using specific molar ratios of zwitterionic and amine moieties. Through proper balance of ligands, we were able to generate particles that featured reactive "handles", while retaining nonfouling properties, high hemocompatibility, and low cytotoxicity.

Matrix-Incorporated Polydopamine Layer as a Simple, Efficient, and Universal Coating for Laser Desorption/Ionization Time-of-Flight Mass Spectrometric Analysis.

Self-oxidative copolymerization of dopamine with α-cyano-4-hydroxycinnamic acid (CHCA) provides an efficient and multifunctional platform for laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) analysis. The polydopamine coating layer embedded with the CHCA matrix can be readily formed on nanomaterials and solid substrates without additional surface treatments to generate an efficient LDI-TOF-MS platform for the analysis of small molecules as well as synthetic polymers. This coating can be further functionalized with specific ligands for target enrichment from complex biological media, providing analyte capture for subsequent LDI-TOF-MS analysis.

Dynamically crosslinked polymer nanocomposites to treat multidrug-resistant bacterial biofilms.

Multidrug-resistant biofilms are highly resistant to current antimicrobial therapies. We have developed an antimicrobial platform that integrates the bacteria-killing phytochemical carvacrol into dynamically crosslinked polymer nanocomposites (DCPNs). Taking advantage of a reversibly crosslinked Schiff-base scaffold throughout the engineered emulsions, DCPNs exhibited long-term shelf-life and good stability in serum, while readily disassembling in acidic microenvironments. Furthermore, we demonstrated that DCPNs efficiently penetrate the biofilm matrix, eradicating both Gram-negative/positive bacteria enclosed within. Moreover, DCPNs showed no observable toxicity to fibroblast mammalian cells with the same antimicrobial concentrations necessary to eradicate MDR biofilms. Given their potent antimicrobial and stimuli-responsive dissociation characteristics in a biofilm setting, DCPNs are a suitable therapeutic platform for combating MDR bacterial infections.

Engineered Polymer Nanoparticles with Unprecedented Antimicrobial Efficacy and Therapeutic Indices against Multidrug-Resistant Bacteria and Biofilms.

The rapid emergence of antibiotic-resistant bacterial "superbugs" with concomitant treatment failure and high mortality rates presents a severe threat to global health. The superbug risk is further exacerbated by chronic infections generated from antibiotic-resistant biofilms that render them refractory to available treatments. We hypothesized that efficient antimicrobial agents could be generated through careful engineering of hydrophobic and cationic domains in a synthetic semirigid polymer scaffold, mirroring and amplifying attributes of antimicrobial peptides. We report the creation of polymeric nanoparticles with highly efficient antimicrobial properties. These nanoparticles eradicate biofilms with low toxicity to mammalian cells and feature unprecedented therapeutic indices against red blood cells. Most notably, bacterial resistance toward these nanoparticles was not observed after 20 serial passages, in stark contrast to clinically relevant antibiotics where significant resistance occurred after only a few passages.

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system.

Biodegradable Nanocomposite Antimicrobials for the Eradication of Multidrug-Resistant Bacterial Biofilms without Accumulated Resistance.

Infections caused by multidrug-resistant (MDR) bacteria are a rapidly growing threat to human health, in many cases exacerbated by their presence in biofilms. We report here a biocompatible oil-in-water cross-linked polymeric nanocomposite that degrades in the presence of physiologically relevant biomolecules. These degradable nanocomposites demonstrated broad-spectrum penetration and elimination of MDR bacteria, eliminating biofilms with no toxicity to cocultured mammalian fibroblast cells. Notably, serial passaging revealed that bacteria were unable to develop resistance toward these nanocomposites, highlighting the therapeutic promise of this platform.