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Lipid nanoparticles encapsulating mRNA (LNP‐mRNA) revolutionized medicine over the past several years. While clinically approved indications currently focus on infectious disease vaccination, LNP‐mRNA based treatments also hold promise for cancer immunotherapy. However, the route of dosing may impact treatment efficacy, safety, and dose. To minimize adverse effects, it is hypothesized that LNP‐mRNA can be used to activate and engineer dendritic cells (DC) ex vivo before re‐administration of these cells. Here, it is shown that LNP‐mRNA engineered DCs can indeed vaccinate recipient mice. Vaccinated mice showed strong anti‐tumor T cell responses, rejected tumor challenge, and displayed no evidence of toxicity. Further, it is found that DC specific ablation of the immune activating kinase NFkB inducing kinase (NIK) abrogated vaccination efficacy, demonstrating that adoptively transferred DCs can be functionally modified in addition to their antigen presentation capacity. Collectively, these studies show that ex vivo LNP‐mRNA engineering of DCs is a feasible and robust therapeutic strategy for cancer. [ABSTRACT FROM AUTHOR]

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations. [ABSTRACT FROM AUTHOR]

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy. [ABSTRACT FROM AUTHOR]

Breast cancer is a diverse female malignancy; its classification is based on clinical evidence and pathological elucidation. Large public drug screening data databases combined with transcriptome measures have helped develop predictive computational models. Breast cancer is frequent among women worldwide. Several genes increase breast cancer risk. The Mammalian Target of Rapamycin (popularly known as mTOR) is a risk factor mutated in numerous breast carcinoma types. This has caught the scientific community's focus, which is attempting to generate creative, potent, and bio-available ligands for future anti-cancer treatments to establish a practical therapeutic approach. mTOR is a protein kinase involved in cell proliferation, survival, metabolism, and immune response. Activating mTOR promotes cancer growth and spread. To generate a bioavailable and effective mTOR inhibitor, we used computer-aided drug design to study chromones and flavonoids, two naturally occurring chemicals with many biological activities. We used Curcuma longaderived tiny nano-molecules, which can be coated using liposomes to target mTOR to prevent breast cancer growth. The significant interactions of Curcumin were anticipated using molecular docking. It had the highest binding affinity at -12.26 kcal/mol. 100 nanoseconds of molecular dynamic modelling confirmed Curcumin and mTOR receptor interaction. Liposomes are a form of medicine carrier. To improve healthcare, more liposome-like nanostructures are being made. Nanostructures' interactions with living creatures are being studied. Half-life, tissue accumulation, and toxicity have been studied. Future medication distribution may use nanocarriers having a liposome-like form, enabling targeted nano-delivery. Curcumin's interaction with the active site increased the complex's structural stability during its expansion. Our results may help future investigations of Curcumin's efficacy as a possible lead treatment targeting mTOR receptors in breast cancer. Using Curcumin as a potential anti-cancer drug with lipid-coated nano-particles allows for tailored administration. [ABSTRACT FROM AUTHOR]

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field. [ABSTRACT FROM AUTHOR]

The full potential of ionizable lipid nanoparticles (LNPs) as an in vivo nucleic acid delivery platform has not yet been realized given that LNPs primarily accumulate in the liver following systemic administration, limiting their success to liver-centric conditions. The engineering of LNPs with antibody targeting moieties can enable extrahepatic tropism by facilitating site-specific LNP tethering and driving preferential LNP uptake into receptor-expressing cell types via receptor-mediated endocytosis. Obstetric conditions stemming from placental dysfunction, such as preeclampsia, are characterized by overexpression of cellular receptors, including the epidermal growth factor receptor (EGFR), making targeted LNP platforms an exciting potential treatment strategy for placental dysfunction during pregnancy. Herein, an EGFR antibody-conjugated LNP (aEGFR-LNP) platform was developed by engineering LNPs with increasing densities of antibody functionalization. aEGFR-LNPs were screened in vitro in immortalized placental trophoblasts and in vivo in non-pregnant and pregnant mice and compared to non-targeted formulations for extrahepatic, antibody-targeted mRNA LNP delivery to the placenta. Our top performing LNP with an intermediate density of antibody functionalization (1:5 aEGFR-LNP) mediated a ∼twofold increase in mRNA delivery in murine placentas and a ∼twofold increase in LNP uptake in EGFR-expressing trophoblasts compared to non-targeted counterparts. These results demonstrate the potential of antibody-conjugated LNPs for achieving extrahepatic tropism, and the ability of aEGFR-LNPs in promoting mRNA delivery to EGFR-expressing cell types in the placenta. [Display omitted] • Strain-promoted azide-DBCO cycloaddition produces stable antibody-conjugated LNPs • Pregnancy alters EGFR antibody-conjugated LNP biodistribution in vivo • Intermediate EGFR antibody density enhances mRNA LNP delivery to the placenta • EGFR antibody-conjugated LNPs enhance uptake in EGFR-expressing trophoblasts [ABSTRACT FROM AUTHOR]

Polymer vesicles and lipid nanoparticles are supramolecular structures with similar physicochemical properties that are self-assembled from different amphiphilic molecules. Because of their efficient drug encapsulation capability, they are good candidates for drug delivery systems. In recent years, nanoparticles with different compositions, sizes, and morphologies have been applied to the delivery of a wide variety of different therapeutic molecules, such as nucleic acids, proteins, and enzymes; their remarkable chemical versatility allows for customization to specific biological applications. In this review, design approaches for polymer vesicles and lipid nanoparticles are summarized with representative examples in terms of their physicochemical properties (size, shape, and mechanical features), preparation strategies (film rehydration, solvent switch, and nanoprecipitation), and applications (with a focus on diagnosis, imaging, and RNA-based therapy). Finally, the challenges limiting the transition from laboratory to clinical application and future perspectives are discussed. [ABSTRACT FROM AUTHOR]

The objective of this investigation was to formulate an ideal nanostructured lipid carrier (NLC) for glib-enclamide in oral capsules. Employing factorial design, the NLC formulation was achieved through high-pressure homogenization using Design-Expert® software, optimized using a 32-factor experimental design and response surface analysis. Comprehensive analyses were conducted to characterize NLCs and determine the optimal formulation. Rigorous evaluations, including drug content, in vitro diffusion and stability studies, were performed. Safety assessments, efficacy studies and optimization led to the identification of an optimal NLC formulation with specific lipid and surfactant percentages, resulting in a desirable particle size (nm) and a robust zeta potential indicative of strong physical stability. [ABSTRACT FROM AUTHOR]

Schematic illustration of the effects of inhibiting miR-17 in microglia of 5XFAD mouse brains in vivo. Anti-17 MLNPs injected through the cisterna magna in 5XFAD mice are attached to mannose receptors on the surface of microglia and internalized. This leads to a significant and specific downregulation of miR-17 in microglia. Notably, Amyloid-beta loads in the brain are significantly reduced. MiR-17 inhibition in turn, decreases immune cell activation and infiltration, dampens neuroinflammation and reduces microglial ASC expression. Subsequently, the expression of synaptic genes is improved in 5XFAD mouse brains. Importantly, the behavior and memory of 5XFAD mice are improved. [Display omitted] • Mannose lipid nanoparticles selectively deliver miR-17 inhibitor, antagomir, to the microglia of 5XFAD mice when injected via intracisternal magna. • Inhibition of miR-17 in microglia decreases microglia and immune cell activation in the brains of 5XFAD mice. • Inhibition of miR-17 in microglia dampens neuroinflammation and decreases amyloid beta deposition. • Microglial miR-17 inhibition in 5XFAD mice improved synaptic function and resulted in better anxiety and spatial memory behavioral tests performance. Neuroinflammation and accumulation of Amyloid Beta (Aβ) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aβ accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aβ burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs. [ABSTRACT FROM AUTHOR]

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 μL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100–200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents. [ABSTRACT FROM AUTHOR]

Controlling the end‐groups of biocompatible polymers is crucial for enabling polymer‐based therapeutics and nanomedicine. Typically, end‐group diversification is a challenging and time‐consuming endeavor, especially for polymers prepared via ionic polymerization mechanisms with limited functional group tolerance. In this study, we present a facile end‐group diversification approach for poly(2‐oxazoline)s (POx), enabling quick and reliable production of heterotelechelic polymers to facilitate POxylation. The approach relies on the careful tuning of reaction parameters to establish differential reactivity of a pentafluorobenzyl initiator fragment and the living oxazolinium chain‐end, allowing the selective introduction of N‐, S‐, O‐nucleophiles via the termination of the polymerization, and a consecutive nucleophilic para‐fluoro substitution. The value of this approach for the accelerated development of nanomedicine is demonstrated through the synthesis of well‐defined lipid‐polymer conjugates and POx‐polypeptide block‐copolymers, which are well‐suited for drug and gene delivery. Furthermore, we investigated the application of a lipid‐POx conjugate for the formulation and delivery of mRNA‐loaded lipid nanoparticles for immunization against the SARS‐COV‐2 virus, underscoring the value of POx as a biocompatible polymer platform. [ABSTRACT FROM AUTHOR]

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA‐targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR‐associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD. [ABSTRACT FROM AUTHOR]

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mRNA therapeutics have shown great potential for a broad spectrum of disease treatment. However, the challenges of mRNA's inherent instability and difficulty in cellular entry have hindered its progress in the biomedical field. To address the cellular barriers and deliver mRNA to cells of interest, various delivery systems are designed. Among these, lipid nanoparticles (LNPs) stand out as the most extensively used mRNA delivery systems, particularly following the clinical approvals of corona virus disease 2019 (COVID‐19) mRNA vaccines. LNPs are comprised of ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol derived lipids (PEG‐lipids). In this review, we primarily summarize the recent advancements of the LNP mRNA delivery technology, focusing on the structures of four lipid constituents and their biomedical applications. We delve into structure–activity relationships of the lipids, while also exploring the future prospects and challenges in developing more efficacious mRNA delivery systems. This article is categorized under:Therapeutic Approaches and Drug Discovery > Emerging TechnologiesBiology‐Inspired Nanomaterials > Lipid‐Based StructuresNanotechnology Approaches to Biology > Nanoscale Systems in Biology [ABSTRACT FROM AUTHOR]

Abstract Over the last two decades, lipid nanoparticles (LNPs) have evolved as an effective biocompatible and biodegradable RNA delivery platform in the fields of nanomedicine, biotechnology, and drug delivery. They are novel bionanomaterials that can be used to encapsulate a wide range of biomolecules, such as mRNA, as demonstrated by the current successes of COVID‐19 mRNA vaccines. Therefore, it is important to provide a perspective on LNPs for RNA delivery, which further offers useful guidance for researchers who want to work in the RNA‐based LNP field. This perspective first summarizes the approaches for the preparation of LNPs, followed by the introduction of the key characterization parameters. Then, the in vitro cell experiments to study LNP performance, including cell selection, cell viability, cellular association/uptake, endosomal escape, and their efficacy, were summarized. Finally, the in vivo animal experiments in the aspects of animal selection, administration, dosing and safety, and their therapeutic efficacy were discussed. The authors hope this perspective can offer valuable guidance to researchers who enter the field of RNA‐based LNPs and help them understand the crucial parameters that RNA‐based LNPs demand.

IntroductionThe approval of chimeric antigen receptor (CAR) T cell therapies for the treatment of B cell malignancies has fueled the development of numerous ex vivo cell therapies. However, these cell therapies are complex and costly, and unlike in hematological malignancies, outcomes with most T cell therapies in solid tumors have been disappointing. Here, we present a novel approach to directly program myeloid cells in vivo by administering novel TROP2 CAR mRNA encapsulated in lipid nanoparticles (LNPs).MethodsThe CAR comprises a TROP2 specific single-chain variable fragment (scFv) fused to a truncated CD89 which requires association with the FcRγ signal adapter to trigger myeloid-specific cell activation. The mRNA encoding the TROP2 CAR was encapsulated in LNPs. Co-immunoprecipitation, flow cytometry and enzyme-linked immunosorbent assay (ELISA) were used to measure CAR expression and functional activity in vitro. Anti-tumor efficacy of the TROP2 CAR mRNA/LNP was evaluated after intravenous administration in various murine tumor models.ResultsIn vitro, transient expression of the TROP2 CAR on monocytes triggers antigen-dependent cytotoxicity and cytokine release. In tumor bearing mice and cynomolgus monkeys, the TROP2 CAR mRNA/LNP are primarily expressed by myeloid cells. In a mouse xenograft model, intravenous administration of TROP2 CAR mRNA/LNP results in tumor growth inhibition and in a B16/F10-OVA immunocompetent melanoma mouse model, anti-tumor efficacy of a gp75-specific CAR correlates with increased number of activated T cells, activation of dendritic cells and a humoral response against B16/F10-OVA melanoma tumors.DiscussionsThese findings demonstrate that myeloid cells can be directly engineered in vivo to kill tumor cells and orchestrate an adaptive immune response and guide clinical studies for the treatment of solid tumors.

Acute respiratory distress syndrome (ARDS) is a complex and life-threatening disease characterized by severe respiratory failure. The lethality of ARDS remains alarmingly high, especially with the persistent ravages of coronavirus disease 2019 (COVID-19) in recent years. ARDS is one of the major complications of neocoronavirus pneumonia and the leading cause of death in infected patients. The large-scale outbreak of COVID-19 has greatly increased the incidence and mortality of ARDS. Despite advancements in our understanding of the causes and mechanisms of ARDS, the current clinical practice is still limited to the use of supportive medications to alleviate its progression. However, there remains a pressing need for effective therapeutic drugs to combat this devastating disease. In this comprehensive review, we discuss the commonly used therapeutic drugs for ARDS, including steroids, vitamin C, targeted inhibitors, and heparin. While these medications have shown some promise in managing ARDS, there is still a significant gap in the availability of definitive treatments. Moreover, we highlight the potential of nanocarrier delivery systems, such as liposomes, lipid nanoparticles, polymer nanoparticles, and inorganic nanoparticles, as promising therapeutic approaches for ARDS in the future. These innovative delivery systems have demonstrated encouraging results in early clinical trials and offer the potential for more targeted and effective treatment options. Despite the promising early results, further clinical trials are necessary to fully assess the efficacy and safety of nanotherapies for ARDS. Additionally, more in-depth research should be conducted to focus on the continuous development of precision therapies targeting different stages of ARDS development or different triggers. This will provide more ideas and rationale for the treatment of ARDS and ultimately lead to better patient outcomes.

Despite the wide range of applications of mRNA therapies, major difficulties exist in the efficient delivery of mRNA into oligodendrocytes, a type of glial cell in the brain. Commonly used viral vectors are not efficient in transforming oligodendrocytes. In this study, we introduced mRNAs into oligodendrocytes with high efficiency and specificity using LUNAR lipid nanoparticles. The uptake of LUNAR lipid nanoparticles occurred via low-density lipoprotein receptors in the presence of apoprotein E. A single dose of LUNAR-human galactosylceramidase mRNA significantly improved phenotypes and survival of twitcher mice, a mouse model of Krabbe disease wherein oligodendrocytes are damaged by galactosylceramidase deficiency. This approach to mRNA therapeutics, combined with cell-specific nanocarriers, demonstrates remarkable potential for the treatment of neurological disorders associated with oligodendrocytes.

RNA science has experienced a surge, particularly its application to vaccine and therapeutic development. Delivery of RNA molecules into the brain remains one of the biggest therapeutic challenges with the blood-brain barrier excluding most drug-delivery modalities such as lipid nanoparticles. As RNA molecules are predisposed to degradation, harnessing its potential as a therapeutic agent requires protection during delivery into target cells. Several solutions have been offered to protect RNA molecules from degradation, with extracellular vesicles (EVs) garnering attention as naturally occurring nanoparticles released by cells, holding promise as carriers for RNA molecules. These EVs, which offer inherent protection to their cargo and have shown promise in efficiently and safely delivering RNA therapeutics present a promising avenue for advancing RNA-based therapeutics in combating various diseases. Here, we review this fast-developing field looking at the range of delivery strategies developed for RNA/EV brain delivery. We summarize the major issues with conventional RNA delivery techniques while emphasizing the potential benefits of EV-based treatments and suggest promising ways forward to progress the field so that we may realize the promise of delivering therapeutic RNA molecules in EVs as potential treatments for neurological disorders.

For cystic fibrosis patients, a lung-targeted gene therapy would significantly alleviate pulmonary complications associated with morbidity and mortality. However, mucus in the airways and cell entry pose huge delivery barriers for local gene therapy. Here, we used phage display technology to select for and identify mucus- and cell-penetrating peptides against primary human bronchial epithelial cells from cystic fibrosis patients cultured at the air-liquid interface. At the air-liquid interface, primary human bronchial epithelial cells produce mucus and reflect cystic fibrosis disease pathology, making it a clinically relevant model. Using this model, we discovered a lead candidate peptide and incorporated it into lipid nanoparticles to deliver mRNA to primary human bronchial epithelia in vitro and mouse lungs in vivo. Compared to lipid nanoparticles without our peptide, peptide-lipid nanoparticles demonstrated up to 7.8-fold and 3.4-fold higher reporter luciferase bioactivity in vitro and in vivo, respectively. Importantly, these peptides facilitated higher specific uptake of nanoparticles into lung epithelia relative to other cell types. Since gene delivery to primary human bronchial epithelia is a significant challenge, we are encouraged by these results and anticipate that our peptide could be used to successfully deliver cystic fibrosis gene therapies in future work.

ABSTRACTThe research and development of messenger RNA (mRNA) cancer vaccines have gradually overcome numerous challenges through the application of personalized cancer antigens, structural optimization of mRNA, and the development of alternative RNA-based vectors and efficient targeted delivery vectors. Clinical trials are currently underway for various cancer vaccines that encode tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), or immunomodulators. In this paper, we summarize the optimization of mRNA and the emergence of RNA-based expression vectors in cancer vaccines. We begin by reviewing the advancement and utilization of state-of-the-art targeted lipid nanoparticles (LNPs), followed by presenting the primary classifications and clinical applications of mRNA cancer vaccines. Collectively, mRNA vaccines are emerging as a central focus in cancer immunotherapy, offering the potential to address multiple challenges in cancer treatment, either as standalone therapies or in combination with current cancer treatments.

Pronounced T cell exhaustion characterizes immunosuppressive tumors, with the tumor microenvironment (TME) employing multiple mechanisms to elicit this suppression. Traditional immunotherapies, such as immune checkpoint blockade, often fail due to their focus primarily on T cells. To overcome this, we utilized a proinflammatory cytokine, interleukin (IL)-12, that re-wires the immunosuppressive TME by inducing T cell effector function while also repolarizing immunosuppressive myeloid cells. Due to toxicities observed with systemic administration of this cytokine, we utilized lipid nanoparticles encapsulating mRNA encoding IL-12 for intratumoral injection. This strategy has been proven safe and tolerable in early clinical trials for solid malignancies. We report an unprecedented loss of exhausted T cells and the emergence of an activated phenotype in murine pancreatic ductal adenocarcinoma (PDAC) treated with stereotactic body radiation therapy (SBRT) and IL-12mRNA. Our mechanistic findings reveal that each treatment modality contributes to the T cell response differently, with SBRT expanding the T cell receptor repertoire and IL-12mRNA promoting robust T cell proliferation and effector status. This distinctive T cell signature mediated marked growth reductions and long-term survival in local and metastatic PDAC models. This is the first study of its kind combining SBRT with IL-12mRNA and provides a promising new approach for treating this aggressive malignancy.

Lipid nanoparticles (LNPs) have emerged as a prominent delivery system for nucleic acid drugs, attracting significant attention, especially through the successful development of several commercial products. As a key component in LNPs, cationic lipids have long served as a key technical barrier to block competitors by building up a complex patent thicket. However, there have been few studies as yet that have comprehensively analyzed the patented compounds in LNP formulations, despite a large number of technical reviews and original articles. In this context, this study focuses on analyzing the macroscopic landscapes and microscopic molecular characteristics of LNP patents, aiming to provide a valuable reference for researchers and developers in making informed technological and commercial decisions. By mining 2,994 patents, 265 formulas, 7,674 compounds, and 28,789 fragments, this work sketches the empirical golden ratio of lipid materials in LNP formulation, discloses the advanced technology in the formulation, characterizes high-frequency fragments of heads, linkers and tails in both novel cationic lipids as well as targeting lipids, and establishes a virtual focus library of LNP materials.

Today, lipid nanoparticles (LNPs) are some of the main delivery systems for mRNA-based therapeutics. The scope of LNP applications in terms of RNA is not limited to antiviral vaccines but encompasses anticancer drugs and therapeutics for genetic (including rare) diseases. Such widespread use implies high customizability of targeted delivery of LNPs to specific organs and tissues. This review addresses vector-free options for targeted delivery of LNPs, namely the influence of lipid composition of these nanoparticles on their biodistribution. In the review, experimental studies are examined that are focused on the biodistribution of mRNA or of the encoded protein after mRNA administration via LNPs in mammals. We also performed a comprehensive analysis of individual lipids’ functional groups that ensure biodistribution to desired organs. These data will allow us to outline prospects for further optimization of lipid compositions of nanoparticles for targeted delivery of mRNA therapeutics.

While oral desensitization is capable of alleviating peanut allergen anaphylaxis, long-term immune tolerance is the sought-after goal. We developed a liver-targeting lipid nanoparticle (LNP) platform to deliver mRNA-encoded peanut allergen epitopes to liver sinusoidal endothelial cells (LSECs), which function as robust tolerogenic antigen-presenting cells that induce FoxP3+ regulatory T-cells (Tregs). The mRNA strand was constructed by including nucleotide sequences encoding for nonallergenic MHC-II binding T-cell epitopes, identified in the dominant peanut allergen, Ara h2. These epitopes were inserted in the mRNA strand downstream of an MHC-II targeting sequence, further endowed in vitro with 5 and 3 capping sequences, a PolyA tail, and uridine substitution. Codon-optimized mRNA was used for microfluidics synthesis of LNPs with an ionizable cationic lipid, also decorated with a lipid-anchored mannose ligand for LSEC targeting. Biodistribution to the liver was confirmed by in vivo imaging, while ELISpot assays demonstrated an increase in IL-10-producing Tregs in the spleen. Prophylactic administration of tandem-repeat or a combination of encapsulated Ara h2 epitopes induced robust tolerogenic effects in C3H/HeJ mice, sensitized to and subsequently challenged with crude peanut allergen extract. In addition to alleviating physical manifestations of anaphylaxis, there was suppression of Th2-mediated cytokine production, IgE synthesis, and mast cell release, accompanied by increased IL-10 and TGF-β production in the peritoneum. Similar efficacy was demonstrated during LNP administration postsensitization. While nondecorated particles had lesser but significant effects, PolyA/LNP-Man lacked protective effects. These results demonstrate an exciting application of mRNA/LNP for treatment of food allergen anaphylaxis, with the promise to be widely applicable to the allergy field.

Abstract Lipid nanoparticles (LNPs) have proven themselves as transformative actors in chimeric antigen receptor (CAR) T cell therapy, surpassing traditional methods and addressing challenges like immunogenicity, reduced toxicity, and improved safety. Promising preclinical results signal a shift toward safer and more effective CAR T cell treatments. Ongoing research aims to validate these findings in clinical trials, marking a new era guided by LNPs utility in CAR therapy. Herein, we explore the preference for LNPs over traditional methods, highlighting the versatility of LNPs and their effective delivery of nucleic acids. Additionally, we address key challenges in clinical considerations, heralding a new era in CAR T cell therapy. Graphical Abstract

Ilaria Ottonelli,1,* Elisa Adani,2,* Andrea Bighinati,2 Sabrina Cuoghi,1 Giovanni Tosi,1,3 Maria Angela Vandelli,1 Barbara Ruozi,1 Valeria Marigo,2,3 Jason Thomas Duskey1 1Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Center for Neuroscience and Neurotechnology, Modena, Italy*These authors contributed equally to this workCorrespondence: Jason Thomas Duskey, Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 103, Modena, MO, Italy, 41125, Tel +390592058573, Email jtduskey@unimore.it Valeria Marigo, Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 287, Modena, MO, 41125, Italy, Tel +390592055392, Email vmarigo@unimore.itPurpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity.Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical “post-formulation” DNA addition vs “pre” addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake.Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 μg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 μg/mL of pDNA with the “post” method, while maintaining their characteristics up to 100 μg/mL of pDNA with the “pre” addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 μg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes.Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.Keywords: gene therapy, lipid nanoparticles, microfluidics, lipoplexes, DNA delivery

Abstract Tyrosine kinase inhibitors have been the standard treatment for patients with Philadelphia chromosome-positive (Ph+) leukemia. However, a series of issues, including drug resistance, relapse and intolerance, are still an unmet medical need. Here, we report the targeted siRNA-based lipid nanoparticles in Ph+ leukemic cell lines for gene therapy of Ph+ leukemia, which specifically targets a recently identified NEDD8 E3 ligase RAPSYN in Ph+ leukemic cells to disrupt the neddylation of oncogenic BCR-ABL. To achieve the specificity for Ph+ leukemia therapy, a single-chain fragment variable region (scFv) of anti-CD79B monoclonal antibody was covalently conjugated on the surface of OA2-siRAPSYN lipid nanoparticles to generate the targeted lipid nanoparticles (scFv-OA2-siRAPSYN). Through effectively silencing RAPSYN gene in leukemic cell lines by the nanoparticles, BCR-ABL was remarkably degraded accompanied by the inhibition of proliferation and the promotion of apoptosis. The specific targeting, therapeutic effects and systemic safety were further evaluated and demonstrated in cell line-derived mouse models. The present study has not only addressed the clinical need of Ph+ leukemia, but also enabled gene therapy against a less druggable target. Graphical Abstract

We developed innovative self-amplifying mRNA (sa-mRNA) vaccine based on the derivative of S and Nsp3 proteins, which are considered crucial adhering to human host cells. We performed B-cell, Major histocompatibility complex (MHC) I, and II epitope which were merged with the KK and GPGPG linker. We also incorporated 5ʹ cap sequence, Kozak sequence, replicase sequence, 3ʹ/5ʹ UTR, and poly A tail within the vaccine structure. The vaccine structure was subsequently docked and run the molecular dynamic simulation with TLR7 molecules. As the results of immune response simulation, the immune response was accelerated drastically up to >10-fold for immunoglobulin, interferon-γ, interleukin-2, immunoglobulin M (IgM) + immunoglobulin G (IgG) isotype, IgM isotype, and IgG1 isotype in secondary and tertiary dose, whereas natural killer cells, macrophages, and dendritic cells showed relatively high concentrations after the first dose. As our finding, the IgM + IgG, IgG1 + IgG2, and IgM level (induced by sa-mRNA vaccine) ensued three times with two-fold increase in days 25, and 50, then decreased after days 70–150. However, 150–350 days demonstrated constantly in the range of 20,000–21,000.

Abstract Nonviral delivery of the CRISPR/Cas9 system provides great benefits for in vivo gene therapy due to the low risk of side effects. However, in vivo gene editing by delivering the Cas9 ribonucleoprotein (RNP) is challenging due to the poor delivery into target tissues and cells. Here, we introduce an effective delivery method for the CRISPR/Cas9 RNPs by finely tuning the formulation of ionizable lipid nanoparticles. The LNPs delivering CRISPR/Cas9 RNPs (CrLNPs) are demonstrated to induce gene editing with high efficiencies in various cancer cell lines in vitro. Furthermore, we show that CrLNPs can be delivered into tumor tissues with high efficiency, as well as induce significant gene editing in vivo. The current study presents an effective platform for nonviral delivery of the CRISPR/Cas9 system that can be applied as an in vivo gene editing therapeutic for treating various diseases such as cancer and genetic disorders.

Understanding and meticulously engineering the biomolecular corona on the surface of lipid nanoparticles can accelerate their successful clinical applications beyond mRNA vaccines. We outline the major hurdles of clinical development faced by lipid nanoparticles and discuss how considering and modifying the biomolecular corona could mitigate these challenges. [ABSTRACT FROM AUTHOR]

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer.

Nowadays, skin is one of the organs most commonly affected by diseases (infections, inflammations, and injuries) due to exposure to the external environment. Although topical treatment represents the most suitable administration route, it is poorly effective due to the low permeability of the drug through the skin. Skin drug delivery by lipid nanocarriers (LNs) appears to be a suitable therapeutic strategy to overcome these issues, allowing it to reach a topical or systemic effect. Several LN-based products have been developed to enhance the permeation of bioactive compounds through the skin, obtaining interesting results in both pharmaceutical and cosmetic fields. Therefore, this review aims to analyze the scientific literature regarding the use of LNs to treat major skin diseases (psoriasis, wound healing, atopic dermatitis, and acne) and esthetic skin defects (wrinkles and cellulite). Furthermore, attention has been paid to the transdermal application of LNs (topical formulations, transdermal patches, and microneedles), being a new topic in recent years.

Microfluidic devices are greatly affected by the materials used. The materials used in previous studies had problems in various aspects, such as processing, adsorption, and price. This study will investigate the materials needed to overcome such problems. Various microfluidic devices based on the perfluorinated compound perfluoropolyether (PFPE) were fabricated and mixed with hydrophilic and amphiphilic monomers, including poly(ethylene glycol) diacrylate, polyethylene glycol monomethacrylate, poly(ethylene glycol) methyl ether methacrylate, acrylic acid, and 2-hydroxyethyl methacrylate. A PFPE-based sheet with a repeating structure of hydrophobic and hydrophilic groups was fabricated. Thus, the hydrophilicity of highly hydrophobic PFPE was enhanced. The fluidic channel was engraved on a PFPE-based sheet using laser cutting and a fabricated microfluidic device. The channels of microfluidic devices are micro-scale (100 µm~300 µm). The lipid nanoparticles and droplets generated through the microfluidic device demonstrated uniform particles continuously.

Background/Objectives: Oxidative stress significantly impacts skin health, contributing to conditions like aging, pigmentation, and inflammatory disorders. Curcumin, with its potent antioxidant properties, faces challenges of low solubility, stability, and bioavailability. This study aimed to encapsulate curcumin in three lipid nanocarriers—solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and nanoemulsions (NEs)—to enhance its stability, bioavailability, and antioxidant efficacy for potential therapeutic applications in oxidative-stress-related skin disorders. Methods: The lipid nanocarriers were characterized for size, polydispersity index, ζ-potential, and encapsulation efficiency. Stability tests under various conditions and antioxidant activity assays (DPPH and FRAP methods) were conducted. Cytotoxicity in human dermal fibroblasts was assessed using MTT assays, while the expression of key antioxidant genes was evaluated in human dermal fibroblasts under oxidative stress. Skin penetration studies were performed to analyze curcumin’s distribution across the stratum corneum layers. Results: All nanocarriers demonstrated high encapsulation efficiency and stability over 90 days. NLCs exhibited superior long-term stability and enhanced skin penetration, while NE formulations facilitated rapid antioxidant effects. Antioxidant assays confirmed that curcumin encapsulation preserved and enhanced its bioactivity, particularly in NLCs. Gene expression analysis revealed upregulation of key antioxidant markers (GPX1, GPX4, SOD1, KEAP1, and NRF2) with curcumin-loaded nanocarriers under oxidative and non-oxidative conditions. Cytotoxicity studies confirmed biocompatibility across all formulations. Conclusions: Lipid nanocarriers effectively enhance curcumin’s stability, antioxidant activity, and skin penetration, presenting a targeted strategy for managing oxidative stress in skin applications. Their versatility offers opportunities for tailored therapeutic formulations addressing specific skin conditions, from chronic disorders like psoriasis to acute stress responses such as sunburn.

Lipid nanoparticles (LNPs) are versatile delivery systems with high interest because they allow the release of hydrophobic and hydrophilic molecules, such as essential oils (EOs) and plant extracts. This review covers published works between 2019 and 2024 that have reported the use of essential EO-based LNPs with antimicrobial properties and applications in human and animal health, as well as biopesticides. In the human healthcare field, reports have addressed the effect of encapsulating EOs in lipid nanosystems with antiviral, antibacterial, antiprotozoal and antifungal activities. In animal care, this still needs to be more deeply explored while looking for more sustainable alternatives against different types of parasites that affect animal health. Overall, the antibacterial activities of LNPs carrying EOs are described as alternatives to the use of synthetic antibiotics. In the field of agriculture, studies showed that these approaches in the control of phytopathogens and other pests that affect food production. There is a growing demand for innovative and more sustainable technologies. However, there are still some challenges to be overcome in order to allow these innovations to reach the market.

Malignant growth is expected to surpass other significant causes of death as one of the top reasons for dismalness and mortality worldwide. According to a World Health Organization (WHO) study, this illness causes approximately between 9 and 10 million instances of deaths annually. Chemotherapy, radiation, and surgery are the three main methods of treating cancer. These methods seek to completely eradicate all cancer cells while having the fewest possible unintended impacts on healthy cell types. Owing to the lack of target selectivity, the majority of medications have substantial side effects. On the other hand, nanomaterials have transformed the identification, diagnosis, and management of cancer. Nanostructures with biomimetic properties have been grown as of late, fully intent on observing and treating the sickness. These nanostructures are expected to be consumed by growth in areas with profound disease. Furthermore, because of their extraordinary physicochemical properties, which incorporate nanoscale aspects, a more prominent surface region, explicit geometrical features, and the ability to embody different substances within or on their outside surfaces, nanostructures are remarkable nano-vehicles for conveying restorative specialists to their designated regions. This review discusses recent developments in nanostructured materials such as graphene, dendrimers, cell-penetrating peptide nanoparticles, nanoliposomes, lipid nanoparticles, magnetic nanoparticles, and nano-omics in the diagnosis and management of cancer.

Background/Objectives: Following tooth extraction, resveratrol (RSV) can support healing by reducing inflammation and microbial risks, though its poor solubility limits its effectiveness. This study aims to develop a solid nanocomposite by embedding RSV in lipid nanoparticles (mLNP) within a hydrophilic matrix, to the scope of improving local delivery and enhancing healing. Hydroxyapatite (HXA), often used as a bone substitute, was added to prevent post-extraction alveolus volume reduction. Methods: The mLNP-RSV dispersion was mixed with seven different polymers in various mLNP/polymer ratios. Following freeze-drying, the powders were redispersed, and the resulting dispersions were tested by DLS experiments. Then, the best two nanocomposites underwent extensive characterization by SEM, XRD, FTIR, Raman spectroscopy, and thermal analysis as well as in vitro partitioning studies aimed at verifying their ability to yield the mLNP-RSV from the hydrophilic matrix to a lipophilic tissue. The characterizations led to identify the best nanocomposite, which was further combined with HXA to obtain hybrid nanocomposites, further evaluated as pharmaceutical powders or in form of mini-tablets. Results: PEG-based nanocomposites emerged as optimal and, following HXA insertion, the resulting powders revealed adequate bulk properties, making them useful as a pharmaceutical intermediate to produce ≈59 mm3 mini-tablets, compliant with the post-extraction socket. Moreover, they were proven ex vivo to be able to promote RSV and GA accumulation into the buccal tissue over time. Conclusions: The here-proposed mini-tablet offers an innovative therapeutic approach for alveolar wound healing promotion as they led to a standardized dose administration, while being handy and stable in terms of physical solid identity as long as it takes to suture the wound.

Background: Acute liver injury (ALI) is a prevalent and potentially lethal condition globally, where pharmacotherapy plays a vital role. However, challenges such as rapid drug excretion and insufficient concentration at hepatic lesions often impede the treatment’s effectiveness. Methods: We successfully prepared glycyrrhizinate monoammonium cysteine (GMC)-loaded lipid nanoparticles (LNPs) using high-pressure homogenization. The characterization and safety of the LNPs were measured using electrophoretic light scattering (ELS), transmission electron microscopy (TEM), dynamic light scattering (DLS), cytotoxicity assays, and hemolysis tests. The distribution of LNPs in mice was explored using fluorescence labeling methods. The encapsulation efficiency of LNP-GMC was detected using High-Performance Liquid Chromatography (HPLC), and its slow-release effect on GMC was assessed through dialysis. The therapeutic effects of LNP-GMC and pure GMC on the ALI model were evaluated using fibroblast activation protein inhibitor (FAPI) PET imaging, blood biochemical indicators, and liver pathology slices. Results: The encapsulation of GMC in LNPs enhances drug stability and prolongs its hepatic retention, significantly improving its bioavailability and sustained release within the liver. This study also explores the expression of fibroblast activation protein (FAP) in ALI, employing 68Ga-FAPI PET/CT imaging for effective differentiation and assessment of liver injury. Conclusions: Our results suggest that LNPs offer an enhanced therapeutic approach for ALI treatment, reducing the required drug dosage, and 68Ga-FAPI PET/CT imaging provides a novel method for diagnosis and treatment assessment. This study contributes valuable insights into the utilization of LNPs in liver disease treatment, presenting a promising direction for future clinical applications.

Background/Objectives: This study aims to evaluate the efficacy of curcumin (CUR), a natural polyphenol with potent antimicrobial and anti-inflammatory properties, when formulated as solid lipid nanoparticles (CUR-loaded SLN) against Enterococcus faecalis. Methods: Solid lipid nanoparticles (SLNs) were prepared as a carrier for CUR, which significantly improved its solubility. SLNs made with cetyl palmitate and Tween 80 were obtained via the hot ultrasonication method. The physicochemical properties of CUR-loaded SLNs were evaluated, including their size, stability, and release profile. Antimicrobial testing was conducted against both sessile and planktonic E. faecalis populations. Cytotoxicity was assessed on human gingival fibroblasts. Results: The CUR-loaded SLNs exhibited about 200 nm and a −25 mV surface potential, and the encapsulation of CUR did not affect the physicochemical properties of SLNs. CURs were released from SLNs in a controlled and sustained manner over 100 h. The nanoparticles remained stable for at least two months when stored at 4 °C or 25 °C, making them suitable for clinical use. Antioxidant activity was confirmed through DPPH and ABTS assays. Free CUR significantly reduced the planktonic E. faecalis CFU counts by approximately 65% after 24 h of exposure. However, this inhibitory effect diminished with longer exposure times (48 and 72 h). Antimicrobial activity studies of CUR-loaded SLNs showed dose- and time-dependent effects, in the 2.5–10 µg/mL range, against both sessile and planktonic E. faecalis populations, over 24 to 72 h. The CUR-loaded SLNs showed good cytocompatibility with human fibroblasts up to 2.5 μg/mL, suggesting low toxicity. Conclusions: CUR-loaded SLNs demonstrate significant antimicrobial activity against E. faecalis, along with good cytocompatibility, indicating their potential as an effective adjunct therapy in endodontic treatments.

Background: Nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) have emerged as a promising vaccine strategy, especially for COVID-19. While the LNPs protect mRNA from degradation and efficiently deliver the mRNA to antigen-presenting cells, the effect of lipid composition on the immunogenicity and protective efficacy of mRNA/LNP vaccines is not well characterized. Studies on using the mRNA/LNP platform for vaccines have largely focused on the nucleic acid cargo with less attention paid to the LNP vehicle. Whether the composition and biophysical properties of LNPs impact vaccine performance remains to be fully elucidated. Methods: In the present study, we used SARS-CoV-2 Spike-mRNA as a prototype vaccine to study the effect of four different LNPs with various lipid compositions. Results: We demonstrate that when the same Spike-mRNA was delivered in the LNP4 formulation based on phospholipid 1,2-dioleoyl-sn-glycero-3-Phosphoethanolamine, it outperformed other LNPs (LNP1, LNP2, and LNP3) that are based on different lipids. Compared to the other three LNPs, LNP4 (i) enhanced the phenotypic and functional maturation of dendritic cells; (ii) induced strong T-cell responses; (iii) increased the secretion of proinflammatory cytokines and pro-follicular T helper (Tfh) cell cytokines; (iv) induced higher neutralization IgG titers; and (v) provided better protection against SARS-CoV-2 infection and COVID-19-like symptoms in the hamster model. Furthermore, we compared LNP-4 with the commercially available LNPs and found it to provide better T-cell immunity against COVID-19 in hamsters. Conclusion: This study suggests mRNA vaccines encapsulated in Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine containing LNPs induced Potent B- and T cell immunity. The mechanisms by which Phospholipid 1,2-Dioleoyl-sn-Glycero-3-PhosphoEthanolamine-based LNPs may activate protective B and T cells are discussed.

Background: Although mRNA vaccines encapsulated in lipid nanoparticles (LNPs) have demonstrated a safety profile with minimal serious adverse events in clinical trials, there is opportunity to further reduce mRNA reactogenicity. The development of naked mRNA vaccines could improve vaccine tolerability. Naked nucleic acid delivery using the jet injection method may be a solution. Methods: In the first part of the study, the optimal conditions providing low traumatization and high expression of the model mRNA-GFP molecule in the tissues of laboratory animals were determined. Then, we used the selected protocol to immunize BALB/c mice with mRNA-RBD encoding the SARS-CoV-2 receptor-binding domain (RBD). It was demonstrated that mice vaccinated with naked mRNA-RBD developed a high level of specific antibodies with virus-neutralizing activity. The vaccine also induced a strong RBD-specific T-cell response and reduced the viral load in the lungs of the animals after infection with the SARS-CoV-2 virus. The level of immune response in mice immunized with mRNA-RBD using a spring-loaded jet injector was comparable to that in animals immunized with mRNA-RBD encapsulated in LNPs. Results: In this study, the efficacy of an inexpensive, simple, and safe method of mRNA delivery using a spring-loaded jet injector was evaluated and validated. Conclusions: Our findings suggest that the jet injection method may be a possible alternative to LNPs for delivering mRNA vaccines against SARS-CoV-2 infection.