Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.

Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

pH-Responsive Polymeric Micelle Dynamic Complexes for Selective Killing of Helicobacter pylori.

Helicobacter pylori, the world's most common chronic infection-causing pathogen, is responsible for causing gastric ulcers, the fourth-leading cause of cancer-related death globally in 2020. In recent years, the effectiveness of the current treatment regimen (two antibiotics and one proton pump inhibitor) has often been plagued with problems such as resistance and the undesired elimination of commensal bacteria. Herein, we report the synthesis of block and random copolycarbonates, functionalized with cationic guanidinium and anionic acetate functional groups, aimed at selectively killing H. pylori in the acidic environment of the stomach, while remaining nontoxic to the commensal bacteria in the gut. The compositions of the polymers were fine-tuned so that the polymers were readily dispersed in water without any difficulty at both pH 3.0 and 7.4. The self-assembly behavior of the polymers at different pH values by dynamic light scattering showed that the random and block copolymers formed stable micelles in a simulated gastric environment (pH 3.0) while aggregated at pH 7.4. Both polymers demonstrated stronger antibacterial activity against H. pylori than the guanidinium-functionalized homopolymer without any acetate functional group at pH 3.0. The block copolymer was significantly more bactericidal at pH 3.0 across the concentrations tested, as compared to the random copolymer, while it did not show significant toxicity toward rat red blood cells (rRBCs) and HK-2 cells or bactericidal effect toward E. coli (a common gut bacterium) and nor caused aggregation of rRBCs at its effective concentration and at physiological pH of 7.4. Additionally, both the block and random copolymers were much more stable against hydrolysis at pH 3.0 than at pH 7.4. This study provides insight into the influence of both polymer architecture and dynamic assembly on the bioactivities of antimicrobial polymers, where the disassembly of coacervates into narrowly dispersed micelles at pH 3 make them potent antimicrobials aided by the protonated carboxylic acid block.

Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels.

The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T cells' immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory. Here we demonstrate an approach to safely anchor aPD1-formed nanogels onto T cell surface via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18 % of the group treated by equivalent dose of free aPD1 and T cells. The technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T cells.

Dental environment and practitioner preferences of southeast Asian youths with dental fear/anxiety.

OBJECTIVES: This study estimated the prevalence of dental fear/anxiety (DFA) in Southeast Asian youths and established their dental environment and practitioner preferences. METHODS: A convenience sample of youths were enrolled from a local polytechnic. The Index of Dental Anxiety and Fear (IDAF-4C) was used to ascertain the presence and severity of DFA. Socio-demographic and IDAF-4C data, along with participants' dental environment and practitioner partialities were gathered electronically. Statistical evaluations were performed with Kruskal-Wallis and Chi-square tests (α = 0.05). RESULTS: Data from a total of 215 participants were appraised. The mean age of the study sample was 18.9 ± 2.0 years (87.4% women). Of these, 12.6% had moderate-to-high DFA and 6.0% had high-to-extreme DFA. As a group, Southeast Asian youths generally liked dental clinics with adorned walls, cooler temperatures, magazines/books, background music and audio-visual devices. In addition, they favoured female practitioners who are younger (≤45 years old), friendly, talkative and maintain a professional relationship. However, those with high-to-extreme DFA preferred a warmer clinic environment and to have an informal relationship with their dental practitioners (p = 0.01). CONCLUSIONS: The prevalence of moderate-to-extreme DFA in Southeast Asian youths was 18.6%. Individuals with high-to-extreme DFA may have disparate dental environment and practitioner preferences compared to those with no-to-moderate DFA.

Fight bacteria with bacteria: Bacterial membrane vesicles as vaccines and delivery nanocarriers against bacterial infections.

Bacterial membrane vesicles (MVs) are particles secreted by bacteria with diameter of 20-400 nm. The pathogen-associated molecular patterns (PAMPs) present on the surface of MVs are capable of activating human immune system, leading to non-specific immune response and specific immune response. Due to the immunostimulatory properties and proteoliposome nanostructures, MVs have been increasingly explored as vaccines or delivery systems for the prevention and treatment of bacterial infections. Herein, the recent progresses of MVs for antibacterial applications are reviewed to provide an overview of MVs vaccines and MVs-related delivery systems. In addition, the safety issues of bacterial MVs are discussed to demonstrate their potential for clinical translation. In the end of this review, the challenges of bacterial MVs as vaccines and delivery systems for clinical applications are highlighted with the purpose of predicting future research directions in this field.

Accurate and Real-Time Temperature Monitoring during MR Imaging Guided PTT.

Photothermal therapy (PTT) is an efficient approach for cancer treatment. However, accurately monitoring the spatial distribution of photothermal transducing agents (PTAs) and mapping the real-time temperature change in tumor and peritumoral normal tissue remain a huge challenge. Here, we propose an innovative strategy to integrate T1-MRI for precisely tracking PTAs with magnetic resonance temperature imaging (MRTI) for real-time monitoring temperature change in vivo during PTT. NaBiF4: Gd@PDA@PEG nanomaterials were synthesized with favorable T1-weighted performance to target tumor and localize PTAs. The extremely weak susceptibility (1.04 × 10-6 emu g-1 Oe1-) of NaBiF4: Gd@PDA@PEG interferes with the local phase marginally, which maintains the capability of MRTI to dynamically record real-time temperature change in tumor and peritumoral normal tissue. The time resolution is 19 s per frame, and the detection precision of temperature change is approximately 0.1 K. The approach achieving PTT guided by multimode MRI holds significant potential for the clinical application.

Identification of Structural Attributes Contributing to the Potency and Selectivity of Antimicrobial Polyionenes: Amides Are Better Than Esters.

Polyionenes are a unique class of materials in which the charges reside along the polymer backbone and have emerged as an important class of antimicrobials. In this study, we have synthesized polyionenes based on quaternary ammonium salts consisting of amides or esters or amide/ester combinations. These materials have a broad spectrum of antimicrobial activity against various types of pathogenic microbes and exhibit a low minimum inhibitor concentration. Importantly, polyionenes with amides outperformed esters in terms of their antimicrobial activity, selectivity, and killing kinetics. Our findings offer insights into the macromolecular design to access selective and potent antimicrobial agents.

Subcutaneous vaccination using injectable biodegradable hydrogels for long-term immune response.

Prolonged vaccine release enables gradual immunostimulation, providing long-term immunity. Herein, Vitamin E-PEG-Vitamin E triblock 'ABA' hydrogel, which is formed through physical cross-linking of flower-shaped micelles and can reside in vivo for >17 weeks, was employed for delivery of cancer preventive vaccines to provide sustained anticancer immunity. Mice vaccinated with hydrogel formulations produced a significantly higher quantity of antibodies compared to solution formulations. OVA was used to study EG.7-OVA tumor rejection in vaccinated mice. Among all formulations, OVA-loaded hydrogel containing aluminum-based adjuvant had the best therapeutic outcome, and only 2/10 mice developed solid tumors with significantly smaller tumor size. Moreover, no adverse effect on liver and kidney was detected with the hydrogel formulation. In a lymphoma metastasis mouse model, vaccination with the OVA-loaded hydrogel and adjuvant resulted in increased survival (66.7%) compared to other formulations (12.5-50%) over 100 days. This hydrogel is a promising formulation for sustained delivery of vaccines.

Effective encapsulation of apomorphine into biodegradable polymeric nanoparticles through a reversible chemical bond for delivery across the blood-brain barrier.

Apomorphine (AMP, used for treatment of Parkinson's disease) is susceptible to oxidation. Its oxidized products are toxic. To overcome these issues, AMP was conjugated to phenylboronic acid-functionalized polycarbonate through pH-sensitive covalent boronate ester bond between phenylboronic acid and catechol in AMP. Various conditions (use of base as catalyst, reaction time and initial drug loading) were optimized to achieve high AMP conjugation degree and mitigate polymer degradation caused by amine in AMP. Pyridine accelerated AMP conjugation and yielded ~74% conjugation within 5 min. Tertiary amine groups were incorporated to polycarbonate, and served as efficient catalyst (~80% conjugation within 5 min). AMP-conjugated polymer self-assembled into nanoparticles. AMP release from the nanoparticles was minimal at pH 7.4, while in acidic environment (endolysosomes) rapid release was observed. Encapsulation protected AMP from oxidization. The nanoparticles were significantly accumulated in the brain tissue after intranasal delivery. These AMP-loaded nanoparticles have potential use for treatment of Parkinson's disease.

Delivery of NF-κB shRNA using carbamate-mannose modified PEI for eliminating cancer stem cells.

The presence of cancer stem cells (CSCs) is one of the main reasons that cause cancer relapse and metastasis. In this study, NF-κB shRNA was delivered to target CSCs using carbamate-mannose modified PEI (CMP) as a non-viral gene vector. The polymer was synthesized by blocking primary amine groups of branched PEI (10kDa) through nucleophilic addition between PEI and protected mannose-functionalized cyclic carbonate, followed by mannose deprotection. CMP/control shRNA nanocomplexes showed lower cytotoxicity and higher transfection efficiency in 4T1 murine breast cancer cells than unmodified PEI/control shRNA nanocomplexes. Importantly, CMP/NF-κB shRNA nanocomplexes (CMPN) were capable of inhibiting migration and invasion, decreasing mammosphere and colony formation and lowering ALDH+ CSC population. Furthermore, CMPN not only induced apoptosis and inhibited cell proliferation, but also sensitized the cells to the treatment with doxorubicin-loaded micellar nanoparticles. Therefore, CMPN may provide a promising approach for eliminating CSCs to prevent cancer relapse and metastasis.

Supramolecular nanofibers self-assembled from cationic small molecules derived from repurposed poly(ethylene teraphthalate) for antibiotic delivery.

Low molecular weight cationic compounds were synthesized from re-purposed poly(ethylene teraphthalate) (PET) and used to self-assemble into high aspect ratio supramolecular nanofibers for encapsulation and delivery of anionic antibiotics. The antibiotic piperacillin/tazobactam (PT) was successfully loaded into the nanofibers through ionic interaction between anionic PT and the cationic nanofibers without loss of the nanofiber features. These PT-loaded nanofibers demonstrated high loading efficiency and sustained delivery for PT. The antimicrobial activity of PT-loaded nanofibers remained potent towards both Gram-positive and Gram-negative bacteria. Importantly, in a P. aeruginosa-infected mouse skin wound model, the treatment with the PT-loaded nanofibers was more effective than free PT for wound healing as evidenced by the significantly lower P. aeruginosa counts at the wound sites and histological analysis. This strategy can be applied to deliver a variety of anionic antibiotics for improved treatment efficacy of various infections.

Disease-directed design of biodegradable polymers: Reactive oxygen species and pH-responsive micellar nanoparticles for anticancer drug delivery.

Herein, we report reactive oxygen species (ROS)- and pH-responsive biodegradable polyethylene glycol (PEG)-block-polycarbonate by installing thioether groups onto the polycarbonate and its self-assembled core/shell structured micelles for anticancer drug delivery. Oxidation of thioethers to sulfoxide and subsequently sulfone induces an increase in hydrophilicity, resulting in more hydrophilic micellar core. This phase-change caused the micelles to swell and enhance cargo release. Carboxylic acid groups have also been installed onto thioether-containing polycarbonate to promote loading of amine-containing anticancer doxorubicin through electrostatic interaction. Urea-functionalized thioether-containing PEG-block-polycarbonates were synthesized to mix with the acid-functionalized PEG-block-polycarbonate for stabilizing micelle structure through hydrogen-bonding interaction. The mixed micelles were 50 nm in diameter and had a 25 wt% loading capacity for doxorubicin. Enhanced drug release from the micelles was triggered by low pH and high content of ROS. Drug-encapsulated micelles accumulated in tumors through leaky tumor vasculature in PC-3 human prostate cancer xenograft mouse model.

Biodegradable Strain-Promoted Click Hydrogels for Encapsulation of Drug-Loaded Nanoparticles and Sustained Release of Therapeutics.

Biodegradable polycarbonate-based ABA triblock copolymers were synthesized via organocatalyzed ring-opening polymerization and successfully formulated into chemically cross-linked hydrogels by strain-promoted alkyne-azide cycloaddition (SPAAC). The synthesis and cross-linking of these polymers are copper-free, thereby eliminating the concern over metallic contaminants for biomedical applications. Gelation occurs rapidly within a span of 60 s by simple mixing of the azide- and cyclooctyne-functionalized polymer solutions. The resultant hydrogels exhibited pronounced shear-thinning behavior and could be easily dispensed through a 22G hypodermic needle. To demonstrate the usefulness of these gels as a drug delivery matrix, doxorubicin (DOX)-loaded micelles prepared using catechol-functionalized polycarbonate copolymers were incorporated into the polymer solutions to eventually form micelle/hydrogel composites. Notably, the drug release rate from the hydrogels was significantly more gradual compared to the solution formulation. DOX release from the micelle/hydrogel composites could be sustained for 1 week, while the release from the micelle solution was completed rapidly within 6 h of incubation. Cellular uptake of the released DOX from the micelle/hydrogel composites was observed at 3 h of incubation of human breast cancer MDA-MB-231 cells. A blank hydrogel containing PEG-(Cat)12 micelles showed almost negligible toxicity on MDA-MB-231cells where cell viability remained high at >80% after treatment. When the cells were treated with the DOX-loaded micelle/hydrogel composites, there was a drastic reduction in cell viability with only 25% of cells surviving the treatment. In all, this study introduces a simple method of formulating hydrogel materials with incorporated micelles for drug delivery applications.

Amphiphilic and Hydrophilic Block Copolymers from Aliphatic N-Substituted 8-Membered Cyclic Carbonates: A Versatile Macromolecular Platform for Biomedical Applications.

Introduction of hydrophilic components, particularly amines and zwitterions, onto a degradable polymer platform, while maintaining precise control over the polymer composition, has been a challenge. Recognizing the importance of these hydrophilic residues in multiple aspects of the nanobiomedicine field, herein, a straightforward synthetic route to access well-defined amphiphilic and hydrophilic degradable block copolymers from diethanolamine-derived functional eight-membered N-substituted aliphatic cyclic carbonates is reported. By this route, tertiary amine, secondary amine, and zwitterion residues can be incorporated across the polymer backbone. Demonstration of pH-responsiveness of these hydrophilic residues and their utility in the development of drug-delivery vehicles, catered for the specific requirements of respective model drugs (doxorubicin and diclofenac sodium salt) are shown. As hydrophilic components in degradable polymers play crucial roles in the biological interactions, these materials offers opportunities to expand the scope and applicability of aliphatic cyclic carbonates. Our approach to these functional polycarbonates will expand the range of biocompatible and biodegradable synthetic materials available for nanobiomedicine, including drug and gene delivery, antimicrobials, and hydrophilic polymers as poly(ethylene glycol) (PEG) alternatives.

pH and redox dual-responsive biodegradable polymeric micelles with high drug loading for effective anticancer drug delivery.

Diblock copolymers of poly(ethylene glycol) (PEG) and biodegradable polycarbonate functionalized with GSH-sensitive disulfide bonds and pH-responsive carboxylic acid groups were synthesized via organocatalytic ring-opening polymerization of functional cyclic carbonates with PEG having different molecular weights as macroinitiators. These narrowly-dispersed polymers had predictable molecular weights, and were used to load doxorubicin (DOX) into micelles primarily through ionic interactions. The DOX-loaded micelles exhibited the requisite small particle size (<100 nm), narrow size distribution and high drug loading capacity. When exposed to endolysosomal pH of 5.0, drug release was accelerated by at least two-fold. The introduction of GSH further expedited DOX release. Effective DOX release enhanced cytotoxicity against cancer cells. More importantly, the DOX-loaded micelles with the optimized composition showed excellent antitumor efficacy in nude mice bearing BT-474 xenografts without inducing toxicity. These pH and redox dual-responsive micelles have the potential as delivery carriers to maximize the therapeutic effect of anticancer drugs.

Broad-Spectrum Antimicrobial/Antifouling Soft Material Coatings Using Poly(ethylenimine) as a Tailorable Scaffold.

Microbial colonization and biofilm formation is the leading cause of contact lens-related keratitis. Treatment of the condition remains a challenge because of the need for prolonged therapeutic course and high doses of antimicrobial agents especially for biofilm eradication. The development of strategies to prepare nonfouling contact lens surfaces is a more practical way to ensure users' safety and relieve the excessive public healthcare burden. In this study, we report a series of polymers that were modified to introduce functionality designed to facilitate coating adhesion, antimicrobial and antifouling properties. Cyclic carbonate monomers having different functional groups including adhesive catechol, antifouling poly(ethylene glycol) (PEG), and hydrophobic urea/ethyl were conjugated onto branched poly(ethylenimine) (bPEI, 25 kDa) at various degrees in a facile and well-controlled manner using a simple one step, atom economical approach. Immersion of contact lenses into an aqueous solution of the catechol-functionalized polymers at room temperature resulted in robust and stable coating on the lens surfaces, which survived the harsh condition of autoclaving and remained on the surface for a typical device application lifetime (7 days). The deposition of the polymer was unambiguously confirmed by static contact angle measurement and X-ray photoelectron spectroscopy (XPS). Polymer coating did not change light transmission significantly. Combinatorial optimization demonstrated that lenses coated with bPEI functionalized with catechol, PEG (5 kDa) and urea groups at 1:12:3:23 molar ratio for 18 h provided the highest antifouling effect against four types of keratitis-causing pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and Fusarium solani, after 7 days of incubation. The polymer coating also inhibited protein adsorption onto the contact lens surfaces after exposure to bovine serum albumin solution for up to 24 h, owing to the flexible and large PEG constituent. Notably, all the polymer coatings used in this study were biocompatible, achieving ≥90% cell viability following direct contact with human corneal epithelial cells for 24 h. Hence, these polymer coatings are envisaged to be promising for the prevention of contact lens-related keratitis.

Injectable biodegradable hydrogels from vitamin D-functionalized polycarbonates for the delivery of avastin with enhanced therapeutic efficiency against metastatic colorectal cancer.

Humanized vascular endothelial growth factor (VEGF) antibody (bevacizumab; Avastin) is a highly effective monoclonal antibody against metastatic colorectal cancer and several other advanced late stage cancers. However, limited aqueous solubility and short circulation half-life of the antibody result in long infusion time (30-90 min) and frequent injections. Such direful medical procedures often cause considerable patient inconvenience and prolonged pharmacy preparation. Subcutaneous delivery of Avastin using injectable hydrogels can continuously provide Avastin to treat the malignancy and mitigate antibody degradation. In this study, ABA triblock copolymers of vitamin D-functionalized polycarbonate and poly(ethylene glycol), that is, VDm-PEG-VDm were synthesized and employed to form physically cross-linked injectable hydrogels for encapsulation and subcutaneous delivery of Avastin in a sustained fashion. Antitumor studies were performed using two different HCT116 xenograft mouse models: a subcutaneous and an intraperitoneal metastatic tumor models. The therapeutic efficacy of Avastin-loaded hydrogel injected subcutaneously (s.c.) was compared to an Avastin solution injected via either intravenous (i.v.) or intraperitoneal (i.p.) route. In the subcutaneous tumor model, the Avastin-loaded hydrogel resulted in greater tumor suppression as compared to i.v. and i.p. administration of Avastin solution. The biodistribution pattern of the hydrogel delivery system was also different from the other formulations as there was significantly higher accumulation in the tumor tissue and lesser accumulation within the liver and kidneys as compared to Avastin delivered through i.v. and i.p. administration. Furthermore, in vivo studies carried out on mice with peritoneal metastasis demonstrated that Avastin-loaded hydrogel and weekly administration of Avastin solution resulted in higher survival (87 and 77% over 62 days, respectively) when compared to the control, blank hydrogel and bolus Avastin solution (i.v.; 50-60%). The antimetastatic activity of Avastin delivered using a one-time injection of the hydrogel was as effective as that of 4× weekly injections (i.v.) of Avastin. The reduced injection frequency provided by the subcutaneous formulation may enhance patient convenience and compliance for metastatic cancer therapy.

Broad-spectrum antimicrobial polycarbonate hydrogels with fast degradability.

In this study, a new family of broad-spectrum antimicrobial polycarbonate hydrogels has been successfully synthesized and characterized. Tertiary amine-containing eight-membered monofunctional and difunctional cyclic carbonates were synthesized, and chemically cross-linked polycarbonate hydrogels were obtained by copolymerizing these monomers with a poly(ethylene glycol)-based bifunctional initiator via organocatalyzed ring-opening polymerization using 1,8-diazabicyclo[5.4.0]undec-7-ene catalyst. The gels were quaternized using methyl iodide to confer antimicrobial properties. Stable hydrogels were obtained only when the bifunctional monomer concentration was equal to or higher than 12 mol %. In vitro antimicrobial studies revealed that all quaternized hydrogels exhibited broad-spectrum antimicrobial activity against Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), Pseudomonas aeruginosa (Gram-negative), and Candida albicans (fungus), while the antimicrobial activity of the nonquaternized hydrogels was negligible. Moreover, the gels showed fast degradation at room temperature (4-6 days), which makes them ideal candidates for wound healing and implantable biomaterials.

Thermoresponsive Random Poly(ether urethanes) with Tailorable LCSTs for Anticancer Drug Delivery.

A new class of thermoresponsive random polyurethanes is successfully synthesized and characterized. Poly(ethylene glycol) diol (Mn = 1500 Da) and 2,2-dimethylolpropionic acid are reacted with isophorone diisocyanate in the presence of methane sulfonic acid catalyst. It is found that these polyurethanes are thermoresponsive in aqueous media and manifest a lower critical solution temperature (LCST) that can be easily tuned from 30 °C to 70 °C by increasing the poly(ethylene glycol) content. Their sharp LCST transitions make these random polyurethanes ideal candidates for stimuli-responsive drug delivery applications. To that end, the ability of these systems to efficiently sequester doxorubicin (up to 36 wt%) by means of a sonication/dialysis method is successfully demonstrated. Additionally, it is also demonstrated that accelerated doxorubicin release kinetics from the nanoparticles can be attained above the LCST.

Overcoming multidrug resistance in microbials using nanostructures self-assembled from cationic bent-core oligomers.

Novel cationic molecules based on rigid terephthalamide-bisurea cores flanked by imidazolium moieties are described. In aqueous media, these compounds self-assemble into supramolecular nanostructures with distinct morphologies. The compound with optimal hydrophilic/hydrophobic balance displays potent antimicrobial activity and high selectivity towards clinically-isolated MRSA without inducing drug-resistance. These self-assembled cationic antimicrobial nanostructures show promise for the prevention and treatment of multidrug-resistant infections.