DNA aptamer-conjugated lipid nanoparticle for targeted PTEN mRNA delivery to prostate cancer cells.

The use of messenger RNA (mRNA) as a cancer vaccine and gene therapy requires targeted vehicle delivery to the site of disease. Here, we designed a mRNA-encapsulating lipid nanoparticle (LNP) conjugated with anti-programmed death-ligand 1 (PD-L1) DNA aptamer that delivers mRNA encoding a tumor suppressor gene, namely phosphatase and tensin homolog (PTEN), to castration-resistant prostate cancer (CRPC) cells expressing PD-L1 on the cell surface. The DNA aptamer-conjugated LNP-based mRNA delivery system (Apt-LNP[PTEN mRNA]) mediated efficient mRNA delivery and transfection in CRPC cells than LNPs without targeting ligands. Cancer-targeted PTEN mRNA delivery using Apt-LNPs achieved significantly higher PTEN expression via aptamer-mediated endocytosis in target cancer cells compared with non-targeted LNP delivery, resulting in significant downregulation of AKT phosphorylation. This enhanced PI3K/AKT pathway regulation, and in turn reduced cell migration after two days along with a 70 % decrease in cell viability, leading to effective apoptotic cell death. In a CRPC xenograft model, Apt-LNP[PTEN mRNA] led to an approximate 60 % reduction in tumor growth, which was attributable to the effective PTEN restoration and PI3K/AKT signaling pathway regulation. PTEN expression was significantly enhanced in CRPC tumor tissues, which abolished cancer cell tumorigenicity. These findings demonstrated the potential of Apt-LNPs for targeted mRNA delivery to cancer cells, thus providing a promising tool for targeted mRNA delivery to a range of cancers and tissues using a conventional LNP systems.

Advancing mRNA Therapeutics: The Role and Future of Nanoparticle Delivery Systems.

The coronavirus (COVID-19) pandemic has underscored the critical role of mRNA-based vaccines as powerful, adaptable, readily manufacturable, and safe methodologies for prophylaxis. mRNA-based treatments are emerging as a hopeful avenue for a plethora of conditions, encompassing infectious diseases, cancer, autoimmune diseases, genetic diseases, and rare disorders. Nonetheless, the in vivo delivery of mRNA faces challenges due to its instability, suboptimal delivery, and potential for triggering undesired immune reactions. In this context, the development of effective drug delivery systems, particularly nanoparticles (NPs), is paramount. Tailored with biophysical and chemical properties and susceptible to surface customization, these NPs have demonstrated enhanced mRNA delivery in vivo and led to the approval of several NPs-based formulations for clinical use. Despite these advancements, the necessity for developing a refined, targeted NP delivery system remains imperative. This review comprehensively surveys the biological, translational, and clinical progress in NPs-mediated mRNA therapeutics for both the prevention and treatment of diverse diseases. By addressing critical factors for enhancing existing methodologies, it aims to inform the future development of precise and efficacious mRNA-based therapeutic interventions.

An overview of lipid constituents in lipid nanoparticle mRNA delivery systems.

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 Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Delivery of mRNA Using Biomimetic Vectors: Progress and Challenges.

Messenger RNA (mRNA) is an emerging class of therapeutic agents for treating a wide range of diseases. However, due to the instability and low cell transfection rate of naked mRNA, the expression of delivered mRNA in target cells or tissues in vivo requires delivery strategies. Biomimetic vectors hold advantages such as high biocompatibility, tissue specific targeting ability and efficient delivery mechanisms, potentially overcoming challenges faced by other delivery vectors. In this review, biomimetic vector-based mRNA delivery systems are summarized and discuss the possible challenges and prospects of such delivery systems, which may contribute to the progress and application of mRNA-based therapy in the biomedical field.

Branching in poly(amine-co-ester) polyplexes impacts mRNA transfection.

Branching is a key structural parameter of polymers, which can have profound impacts on physicochemical properties. It has been demonstrated that branching is a modulating factor for mRNA delivery and transfection using delivery vehicles built from cationic polymers, but the influence of polymer branching on mRNA delivery remains relatively underexplored compared to other polymer features such as monomer composition, hydrophobicity, pKa, or the type of terminal group. In this study, we examined the impact of branching on the physicochemical properties of poly(amine-co-esters) (PACE) and their efficiency in mRNA transfection in vivo and in vitro under various conditions. PACE polymers were synthesized with various degrees of branching ranging from 0 to 0.66, and their transfection efficiency was systemically evaluated. We observed that branching improves the stability of polyplexes but reduces the pH buffering capacity. Therefore, the degree of branching (DB) must be optimized in a delivery route specific manner due to differences in challenges faced by polyplexes in different physiological compartments. Through a systematic analysis of physicochemical properties and mRNA transfection in vivo and in vitro, this study highlights the influence of polymer branching on nucleic acid delivery.

Rational design of lipid nanoparticles: overcoming physiological barriers for selective intracellular mRNA delivery.

This review introduces the typical delivery process of messenger RNA (mRNA) nanomedicines and concludes that the delivery involves a at least four-step SCER cascade and that high efficiency at every step is critical to guarantee high overall therapeutic outcomes. This SCER cascade process includes selective organ-targeting delivery, cellular uptake, endosomal escape, and cytosolic mRNA release. Lipid nanoparticles (LNPs) have emerged as a state-of-the-art vehicle for in vivo mRNA delivery. The review emphasizes the importance of LNPs in achieving selective, efficient, and safe mRNA delivery. The discussion then extends to the technical and clinical considerations of LNPs, detailing the roles of individual components in the SCER cascade process, especially ionizable lipids and helper phospholipids. The review aims to provide an updated overview of LNP-based mRNA delivery, outlining recent innovations and addressing challenges while exploring future developments for clinical translation over the next decade.

Unraveling mRNA delivery bottlenecks of ineffective delivery vectors by co-transfection with effective carriers.

The messenger RNA (mRNA) SARS-CoV-2 vaccines have demonstrated the therapeutic potential of this novel drug modality. Protein expression is the consequence of a multistep delivery process that relies on proper packaging into nanoparticle carriers to protect the mRNA against degradation enabling effective cellular uptake and endosomal release and liberating the mRNA in the cytosol. Bottlenecks along this route remain challenging to pinpoint. Although methods to assess endosomal escape of carriers have been developed, versatile strategies to identify bottlenecks along the delivery trajectory are missing. Here, it is shown that co-incubating an inefficient nanoparticle formulation with an efficient one solves this problem. Cells were co-incubated with mRNA nanoparticles formed with either the efficient cell-penetrating peptide (CPP) PepFect14 or the inefficient CPP nona-arginine (R9). Co-transfection enhanced cellular uptake and endosomal escape of R9-formulated mRNA, resulting in protein expression, demonstrating that both vectors enter cells along the same route. In addition, cells were transfected with a galectin-9-mCherry fusion protein to detect endosomal rupture. Remarkably, despite endosomal release, mRNA remained confined to punctate structures, identifying mRNA liberation as a further bottleneck. In summary, co-transfection offers a rapid means to identify bottlenecks in cytosolic mRNA delivery, supporting the rational design and optimization of intracellular mRNA delivery systems.

Full-cycle study on developing a novel structured micromixer and evaluating the nanoparticle products as mRNA delivery carriers.

Achieving precise control of nanoparticle size while maintaining consistency and high uniformity is of paramount importance for improving the efficacy of nanoparticle-based therapies and minimizing potential side effects. Although microfluidic technologies are widely used for reliable nanoparticle synthesis, they face challenges in meeting critical homogeneity requirements, mainly due to imperfect mixing efficiency. Furthermore, channel clogging during continuous operation presents a significant obstacle in terms of quality control, as it progressively impedes the mixing behavior necessary for consistent nanoparticle production for therapeutic delivery and complicates the scaling-up process. This study entailed the development of a 3D-printed novel micromixer embedded with hemispherical baffle microstructures, a dual vortex mixer (DVM), which integrates Dean vortices to generate two symmetrical counter-rotating intensified secondary flows. The DVM with a relatively large mixer volume showed rapid mixing characteristics even at a flow rate of several mL min-1 and produced highly uniform lipids, liposomes, and polymer nanoparticles in a size range (50-130 nm) and polydispersity index (PDI) values below 0.15. For the evaluation of products, SARS-CoV-2 Spike mRNA-loaded lipid nanoparticles were examined to verify protein expression in vitro and in vivo using firefly luciferase (FLuc) mRNA. This showed that the performance of the system is comparable to that of a commercial toroidal mixer. Moreover, the vigorous in-situ dispersion of nanoparticles by harnessing the power of vortex physically minimizes the occurrence of aggregation, ensuring consistent production performance without internal clogging of a half-day operation and facilitating quality control of the nanoparticles at desired scales.

Tailoring Highly Branched Poly(ß-amino ester)s for Efficient and Organ-Selective mRNA Delivery.

Development of mRNA therapeutics necessitates targeted delivery technology, while the clinically advanced lipid nanoparticles face difficulty for extrahepatic delivery. Herein, we design highly branched poly(ß-amino ester)s (HPAEs) for efficacious organ-selective mRNA delivery through tailoring their chemical compositions and topological structures. Using an "A2+B3+C2" Michael addition platform, a combinatorial library of 219 HPAEs with varied backbone structures, terminal groups, and branching degrees are synthesized. The branched topological structures of HPAEs provide enhanced serum resistance and significantly higher mRNA expression in vivo. The terminal amine structures of HPAEs determine the organ-selectivity of mRNA delivery following systemic administration: morpholine facilitates liver targeting, ethylenediamine favors spleen delivery, while methylpentane enables mRNA delivery to the liver, spleen, and lungs simultaneously. This study represents a comprehensive exploration of the structure-activity relationship governing both the efficiency and organ-selectivity of mRNA delivery by HPAEs, suggesting promising candidates for treating various organ-related diseases.

Evaluation of mRNA lipoplexes prepared using modified ethanol injection method as a tumour vaccine.

We have previously demonstrated that messenger RNA (mRNA) lipoplexes composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and polyethylene glycol-cholesteryl ether (PEG-Chol) exhibited high protein expression in the lungs and spleen of mice after intravenous injection and induced high levels of antigen-specific IgG1 upon immunisation. In this study, we optimised PEG modification in mRNA lipoplexes to reduce mRNA accumulation in the lungs and evaluated the suppression of tumour growth in mice bearing mouse lymphoma E.G7-ovalbumin (OVA) tumours by immunising them with an intravenous injection of OVA mRNA lipoplexes. PEGylation of mRNA lipoplexes with 3 mol% PEG-Chol (LP-DC-1-16-3PCL) prevented agglutination of erythrocytes and reduced accumulation in the lungs. Intravenous injection of LP-DC-1-16-3PCL lipoplexes containing OVA mRNA into mice induced high levels of anti-OVA IgG1 (83,000 mU/mL) in serum, and exhibited a high cytotoxic activity (97%) against E.G7-OVA cells by the splenocytes of mice. Furthermore, immunisation with LP-DC-1-16-3PCL lipoplexes containing OVA mRNA suppressed E.G7-OVA tumour growth compared to control mRNA. Based on these results, LP-DC-1-16-3PCL lipoplexes may be an effective mRNA vaccine for inducing antibody- and cytotoxic cell-mediated immune responses to tumours through intravenous injection.

Recent Advances in Biomaterials for mRNA Delivery to Immune Cells.

Modulating the function of immune cells by targeting the cells themselves has become a key strategy in immunotherapy for combating various diseases such as cancer, autoimmune disorders, and infectious ailments. The use of mRNA (mRNA) is a powerful tool for transiently inducing protein expression, which is often used for genetic manipulation. However, its inherent instability and inability to precisely reach target cells necessitate the use of biomaterials for safe and effective delivery. Additionally, transfecting immune cells is difficult and complex due to their resistance mechanisms, signaling pathways, and cellular interactions. This review focuses on the recent development of biomaterials for mRNA delivery to immune cells, including lipid nanoparticles and polymeric carriers. It also outlines the challenges of targeting and delivering therapeutic payloads to immune cells, providing commentary and outlook on the design of next-generation materials. Finally, this approach has the potential to significantly enhance the precision and effectiveness of therapeutic interventions for various diseases, shaping the future of mRNA delivery for immune conditions.

Engineering CaP-Pickering emulsion for enhanced mRNA cancer vaccines via dual DC and NK activations.

mRNA delivery systems, such as lipid nanoparticle (LNP), have made remarkable strides in improving mRNA expression, whereas immune system activation operates on a threshold. Maintaining a delicate balance between antigen expression and dendritic cell (DC) activation is vital for effective immune recognition. Here, a water-in-oil-in-water (w/o/w) Pickering emulsion stabilized with calcium phosphate nanoparticles (CaP-PME) is developed for mRNA delivery in cancer vaccination. CaP-PME efficiently transports mRNA into the cytoplasm, induces pro-inflammatory responses and activates DCs by disrupting intracellular calcium/potassium ions balance. Unlike LNP, CaP-PME demonstrates a preference for DCs, enhancing their activation and migration to lymph nodes. It elicits interferon-γ-mediated CD8+ T cell responses and promotes NK cell proliferation and activation, leading to evident NK cells infiltration and ameliorated tumor microenvironment. The prepared w/o/w Pickering emulsion demonstrates superior anti-tumor effects in E.G7 and B16-OVA tumor models, offering promising prospects as an enhanced mRNA delivery vehicle for cancer vaccinations.

Bioinspired cellular membrane-derived vesicles for mRNA delivery.

The rapid advancement of mRNA as vaccines and therapeutic agents in the biomedical field has sparked hope in the fight against untreatable diseases. Successful clinical application of mRNA therapeutics largely depends on the carriers. Recently, a new and exciting focus has emerged on natural cell-derived vesicles. These nanovesicles offer many functions, including enhanced drug delivery capabilities and immune evasion, thereby presenting a unique and promising platform for the effective and safe delivery of mRNA therapeutics. In this study, we summarize the characteristics and properties of biomimetic delivery systems for mRNA therapeutics. In particular, we discuss the unique features of cellular membrane-derived vesicles (CDVs) and the combination of synthetic nanovesicles with CDVs.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

In Silico Screening Accelerates Nanocarrier Design for Efficient mRNA Delivery.

Lipidic nanocarriers are a broad class of lipid-based vectors with proven potential for packaging and delivering emerging nucleic acid therapeutics. An important early step in the clinical development cycle is large-scale screening of diverse formulation libraries to assess particle quality and payload delivery efficiency. Due to the size of the screening space, this process can be both costly and time-consuming. To address this, computational models capable of predicting clinically relevant physio-chemical properties of dendrimer-lipid nanocarriers, along with their mRNA payload delivery efficiency in human cells are developed. The models are then deployed on a large theoretical nanocarrier pool consisting of over 4.5 million formulations. Top predictions are synthesised for validation using cell-based assays, leading to the discovery of a high quality, high performing, candidate. The methods reported here enable rapid, high-throughput, in silico pre-screening for high-quality candidates, and have great potential to reduce the cost and time required to bring mRNA therapies to the clinic.

Optimizing mRNA-Loaded Lipid Nanoparticles as a Potential Tool for Protein-Replacement Therapy.

Lipid nanoparticles (LNPs) tailored for mRNA delivery were optimized to serve as a platform for treating metabolic diseases. Four distinct lipid mixes (LMs) were formulated by modifying various components: LM1 (ALC-0315/DSPC/Cholesterol/ALC-0159), LM2 (ALC-0315/DOPE/Cholesterol/ALC-0159), LM3 (ALC-0315/DSPC/Cholesterol/DMG-PEG2k), and LM4 (DLin-MC3-DMA/DSPC/Cholesterol/ALC-0159). LNPs exhibited stability and homogeneity with a mean size of 75 to 90 nm, confirmed by cryo-TEM and SAXS studies. High mRNA encapsulation (95-100%) was achieved. LNPs effectively delivered EGFP-encoding mRNA to HepG2 and DC2.4 cell lines. LNPs induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs), revealing that LM1, LM2, and LM4 induced 1.5- to 4-fold increases in IL-8, TNF-α, and MCP-1 levels, while LM3 showed minimal changes. Reporter mRNA expression was observed in LNP-treated PBMCs. Hemotoxicity studies confirmed formulation biocompatibility with values below 2%. In vivo biodistribution in mice post intramuscular injection showed significant mRNA expression, mainly in the liver. The modification of LNP components influenced reactogenicity, inflammatory response, and mRNA expression, offering a promising platform for selecting less reactogenic carriers suitable for repetitive dosing in metabolic disease treatment.

Effect of vorinostat on protein expression in vitro and in vivo following mRNA lipoplex administration.

Previously, we demonstrated that cationic liposomes comprised of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and poly(ethylene glycol) cholesteryl ether induced substantial protein expression both in vitro and in vivo following the administration of mRNA/cationic liposome complexes (mRNA lipoplexes). The present study evaluated the effect of vorinostat, a histone deacetylase inhibitor, on protein expression levels in vitro and in vivo following the administration of mRNA lipoplexes. The half-maximal inhibitory concentration (IC50) values of vorinostat for human cervical carcinoma HeLa and human liver cancer HepG2 cells were determined to be 7.8 and 2.6 µM, respectively, following a 24 h incubation period. Treatment with 1 µM vorinostat resulted in a 2.7-fold increase in luciferase (Luc) activity for HeLa cells and a 1.6-fold increase for HepG2 cells at 24 h post-transfection with firefly Luc (FLuc) mRNA lipoplexes compared with untreated cells. However, treatment with 10 µM vorinostat decreased Luc activity compared with treatment with 1 µM vorinostat. Intravenous injection of Cy5-labeled mRNA lipoplexes into mice resulted in mRNA accumulation primarily in the lungs; however, co-injection with vorinostat at doses of 5 or 25 mg/kg resulted in mRNA accumulation in both the lungs and liver. Furthermore, intravenous injection of FLuc mRNA lipoplexes resulted in high Luc activity in both the lungs and spleen. Nevertheless, co-injection with vorinostat slightly decreased Luc activity in the lungs but not in the spleen. These findings indicated that vorinostat enhances in vitro protein expression from transfected mRNA after treatment with a lower concentration of IC50; however, it does not largely affect in vivo protein expression from the transfected mRNA.

Lung-Specific mRNA Delivery Enabled by Sulfonium Lipid Nanoparticles.

Among various mRNA carrier systems, lipid nanoparticles (LNPs) stand out as the most clinically advanced. While current clinical trials of mRNA/LNP therapeutics mainly address liver diseases, the potential of mRNA therapy extends far beyond─yet to be unraveled. To fully unlock the promises of mRNA therapy, there is an urgent need to develop safe and effective LNP systems that can target extrahepatic organs. Here, we report on the development of sulfonium lipid nanoparticles (sLNPs) for systemic mRNA delivery to the lungs. sLNP effectively and specifically delivered mRNA to the lungs following intravenous administration in mice. No evidence of lung and systemic inflammation or toxicity in major organs was induced by sLNP. Our findings demonstrated that the newly developed lung-specific sLNP platform is both safe and efficacious. It holds great promise for advancing the development of new mRNA-based therapies for the treatment of lung-associated diseases and conditions.

Branched-Tail Lipid Nanoparticles for Intravenous mRNA Delivery to Lung Immune, Endothelial, and Alveolar Cells in Mice.

Lipid nanoparticles (LNPs) are proven safe and effective delivery systems on a global scale. However, their efficacy has been limited primarily to liver and immune cell targets. To extend the applicability of mRNA drugs, 580 ionizable lipidoids are synthesized and tested for delivery to extrahepatocellular targets. Of these, over 40 enabled protein expression in mice, with the majority transfecting the liver. Beyond the liver, several LNPs containing new, branched-tail ionizable lipidoids potently delivered mRNA to the lungs, with cell-level specificity depending on helper lipid chemistry. Incorporation of the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 16 mol% enabled highly specific delivery to natural killer and dendritic cells within the lung. Although inclusion of the cationic lipid 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP) improved lung tropism, it decreased cell specificity, resulting in equal transfection of endothelial and lymphoid cells. DOTAP formulations are also less favorable than DOPE formulations because they elevated liver enzyme and cytokine levels. Together, these data identify a new branched-tailed LNP with a unique ability to selectively transfect lung immune cell populations without the use of toxicity-prone cationic helper lipids. This novel vehicle may unlock RNA therapies for lung diseases associated with immune cell dysregulation, including cancer, viral infections, and autoimmune disorders.

Steering the course of CAR T cell therapy with lipid nanoparticles.

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.