Enhanced Transfection Efficiency Research Articles

Spermine Significantly Increases the Transfection Efficiency of Cationic Polymeric Gene Vectors.

Background/Objectives: Non-viral vectors have gained recognition for their ability to enhance the safety of gene delivery processes. Among these, polyethyleneimine (PEI) stands out as the most widely utilized cationic polymer due to its accessibility. Traditional methods of modifying PEI, such as ligand conjugation, chemical derivatization, and cross-linking, are associated with intricate preparation procedures, limited transfection efficiency, and suboptimal biocompatibility. Methods: In this investigation, enhanced transfection efficiency was achieved through the straightforward physical blending of PEI carriers with spermine. Results: Transfection assays explored the maximal enhancement potential conferred by spermine, alongside further methodological refinements aimed at optimizing transfection efficacy, showcasing a potential increase of up to 40.7%. Through the comparison of different addition sequences of spermine, the optimal complex PEI/Spermine/DNA for transfection efficiency was selected. Characterization of PEI/Spermine/DNA revealed that, compared to PEI/DNA, its particle size increased to approximately 150 nm. Molecular dynamics simulation results revealed that spermine can enhance the interaction between PEI and DNA, thereby forming a system with lower energy and greater stability. Mechanistic inquiries studies also disclosed that spermine augments the endosomal escape capability of PEI carriers without altering pathways involved in the cellular uptake of gene nanoparticles, thereby facilitating heightened gene expression. Conclusions: PEI-Sper emerges as a promising non-viral vector for gene delivery, distinguished by its simplicity in preparation, cost-effectiveness, and superior transfection efficiency.

Impact of Acetylation, Succinylation, and pH on DNA Packaging in PEI-Based Polyplexes.

Polyethylenimine (PEI) is a widely used cationic polymer for nonviral gene delivery, often modified to enhance transfection efficiency and reduce cytotoxicity. This study investigates how acetylation, succinylation (acPEI and zPEI), and pH influence the internal DNA packaging of polyplexes. Both modifications alter physicochemical properties, leading to complexes that decondense more readily with increasing modification. X-ray scattering reveals that high acetylation produces loosely packed DNA, while succinylation unexpectedly tightens DNA packing at higher modification levels. Polyplexes formed at low pH (pH 4) are more stable and tightly packed than those formed at pH 7.5. Acidifying polyplexes initially formed at pH 7.5 induces structural rearrangement to tighter DNA packing accompanied by significant PEI release, providing direct evidence for models where free PEI aids endosomal escape. These findings challenge conventional assumptions about PEI behavior and offer new insights into DNA packaging, emphasizing tailored polymer modifications and pH conditions to optimize gene delivery.

Novel electroporation microchip with field constriction enhances transfection efficiency and survival rates of feline embryos

This study introduces a low-voltage electroporation microchip designed for transfection in cat embryos, featuring real-time impedance monitoring. The microchip uses a field constriction strategy, which localises the electric field to the membrane region in contact with the micro-orifice, enhancing electroporation efficiency while minimising damage. Embryos were positioned on the orifice, and a series of voltage pulses (10, 15, and 20 V) were applied. Electroporation efficacy was assessed using fluorescent dyes, followed by real-time impedance measurements to monitor the membrane resealing time. It provided valuable insights into membrane recovery times, essential for optimizing gene editing conditions to ensure efficient delivery and maintain cell integrity. The results demonstrated that the microchip with 15 V achieved a 69.5% higher electroporation rate and 100% of survival compared to conventional devices (p < 0.05). Additionally, the microchip successfully facilitated the transfer of green fluorescent protein genes into embryos, achieving a 78.5% success rate significantly greater compared to 53.6% with the conventional device (p < 0.05). This innovative microchip provides transformative transfection technology for safer and more efficient genomic modifications in embryos. It holds promising applications across species and therapeutic interventions, paving the way for future studies in advanced genomic research.

Spleen‐Targeted mRNA Nanoparticles for Modulating B Cell Hyperactivation in Rheumatoid Arthritis Therapy

AbstractMessenger RNA (mRNA)‐based therapies have emerged as a revolutionary strategy for treating various diseases. In autoimmune diseases like rheumatoid arthritis (RA), targeted mRNA delivery provides a potential intervention to modulate immune responses. However, achieving specific and efficient in vivo modulation of immune regulators, such as the inhibitory Fc gamma receptor, FcγRIIB, on B cells remains challenging. In this study, lipid polymer nanoparticles (LPNs) formulated with AMB‐POC18 lipidoid and poly(ethylene glycol)‐block‐polylactide (PEG‐PLA) are engineered to deliver FcγRIIB mRNA (mFcγRIIB) specifically to splenic B cells for RA treatment. Protein corona analysis indicated that selective adsorption of complement C3 on the LPNs' surface facilitated their targeted delivery to the spleen, enhancing transfection efficiency in B cells following intravenous administration. In a collagen‐induced arthritis mouse model, mFcγRIIB/LPNs effectively upregulated FcγRIIB expression in splenic B cells, significantly reducing autoimmune responses and alleviating RA symptoms. Further mechanistic studies elucidated that increased FcγRIIB expression suppressed B cell activation via the FcγRIIB/Lyn/SHP‐1 signaling pathway. This work underscored the potential of the spleen‐targeted mRNA delivery system for RA therapy, providing a precise and targeted approach to modulate B cell activity and mitigate autoimmune diseases.

Fine-Tuned "Click" Functionalization of PAMAM Dendrimers with a Linear Fluorinated Guanidino Linker: Synthesis, Characterization, and Applications.

This study presents the synthesis, characterization, and application of multifunctional PAMAM G2 and G4 dendrimers decorated with a linear fluorinated guanidino linker designed to improve gene delivery efficiency while minimizing cytotoxicity. For the first time, we were able to fine-tune the degree of grafting (DG) during the functionalization process through efficient "click" Michael addition, achieving the synthesis of a collection of six PAMAM conjugates that showed a significant enhancement in transfection efficiency (TE), surpassing the performance of traditional nonviral vectors. The incorporation of fluorinated moieties not only facilitated better deoxyribonucleic acid (DNA) condensation and TE but also introduced potential applications in 19F magnetic resonance imaging thanks to the sharp and intense fluorine nuclear magnetic resonance signals and favorable relaxation parameters. The new dendrimer conjugates demonstrated a promising balance between low cytotoxicity and high TE, with the low-generation PAMAM G2 with lower DG being the best-performing conjugate, making them strong candidates for further development in gene therapy. These findings highlight the potential of these multifunctional PAMAM dendrimers as efficient, nontoxic, and trackable gene delivery vectors.

Brief Comparison of the Efficacy of Cationic and Anionic Liposomes as Nonviral Delivery Systems.

In recent decades, the development and application of nonviral vectors, such as liposomes and lipidic nanoparticles, for gene therapy and drug delivery have seen substantial progress. The interest in the physicochemical properties and structures of the complexes liposome/DNA and liposome/RNA is due to their potential to substitute viruses as carriers of drugs or genetic material into cells with minimal cytotoxicity, which could lead to their use in gene therapy. Initially, cationic liposomes were utilized as nonviral DNA delivery vectors; subsequently, different molecules, such as polymers, were incorporated to enhance transfection efficiency. Additionally, liposome/protein complexes have been developed as nonviral vectors for the treatment of diseases. The most relevant internalization pathways of these vectors and the few transfection results obtained using targeted and nontargeted liposomes are discussed below. The high cytotoxicity of cationic liposomes represents a significant challenge for the development of gene therapy and drug delivery. Anionic liposomes offer a promising alternative to address the limitations of conventional cationic liposomes, including immune response, short circulation time, and low toxicity. This review will discuss the advantages of cationic liposomes and the novel anionic liposome-based systems that have emerged as a result. The advent of novel designs and manufacturing techniques has facilitated the development of innovative systems, designated as lipid nanoparticles (LNPs), which serve as highly efficacious regulators of the immune system.

High-Efficiency Targeted Gene Transfection of Cells Using Temporal and Spatial Shaping Femtosecond Laser Irradiation.

Laser-irradiation-assisted cell gene transfection is sterile and nontoxic, but the low transfection efficiency cannot meet the application requirements. To improve the efficiency, a temporal and spatial shaping method of a femtosecond laser is proposed. Using the time shaping method, we can segment the pulse into subpulses of varying energies and with a defined delay, thereby influencing the interaction between electrons and photons, ultimately enhancing transfection efficiency. The transfection efficiency is further improved by spatially shaping the laser pulse to extend the focusing beam's working distance and reduce the cell's sensitivity to the focal position. Through the characterization of the viability and transfection efficiency of HEK-293T cells, the method achieved efficient and active transfection, with a maximum transfection efficiency of 45.1% and a cell survival rate of 93.6%. This method provides key technical support for femtosecond laser transfection and promotes its further application in clinical practice.

Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Role of size, surface charge, and PEGylated lipids of lipid nanoparticles (LNPs) on intramuscular delivery of mRNA

Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs.Graphical

Tuning extracellular fluid viscosity to enhance transfection efficiency

Tuning extracellular fluid viscosity to enhance transfection efficiency

Swarms of Enzymatic Nanobots for Efficient Gene Delivery.

This study investigates the synthesis and optimization of nanobots (NBs) loaded with pDNA using the layer-by-layer (LBL) method and explores the impact of their collective motion on the transfection efficiency. NBs consist of biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and are powered by the urease enzyme, enabling autonomous movement and collective swarming behavior. In vitro experiments were conducted to validate the delivery efficiency of fluorescently labeled NBs, using two-dimensional (2D) and three-dimensional (3D) cell models: murine urothelial carcinoma cell line (MB49) and spheroids from human urothelial bladder cancer cells (RT4). Swarms of pDNA-loaded NBs showed enhancements of 2.2- to 2.6-fold in delivery efficiency and 6.8- to 8.1-fold in material delivered compared to inhibited particles (inhibited enzyme) and the absence of fuel in a 2D cell culture. Additionally, efficient intracellular delivery of pDNA was demonstrated in both cell models by quantifying and visualizing the expression of eGFP. Swarms of NBs exhibited a >5-fold enhancement in transfection efficiency compared to the absence of fuel in a 2D culture, even surpassing the Lipofectamine 3000 commercial transfection agent (cationic lipid-mediated transfection). Swarms also demonstrated up to a 3.2-fold enhancement in the amount of material delivered in 3D spheroids compared to the absence of fuel. The successful transfection of 2D and 3D cell cultures using swarms of LBL PLGA NBs holds great potential for nucleic acid delivery in the context of bladder treatments.

A Photothermal Polymeric Platform for Efficient and Safe Gene Transfection: When Polyethylenimine Collaborates with Indocyanine Green.

Gene transfection, defined by the delivery of nucleic acids into cellular compartments, stands as a crucial procedure in gene therapy. While branched polyethylenimine (PEI) is widely regarded as the "gold standard" for nonviral vectors, its cationic nature presents several issues, including nonspecific protein adsorption and notable cytotoxicity. Additionally, it often fails to achieve high transfection efficiency, particularly with hard-to-transfect cell types. To overcome these challenges associated with PEI as a vector for plasmid DNA (pDNA), the photothermal agent indocyanine green (ICG) is integrated with PEI and pDNA to form the PEI/ICG/pDNA (PI/pDNA) complex for more efficient and safer gene transfection. The negatively charged ICG serves a dual purpose: neutralizing PEI's excessive positive charges to reduce cytotoxicity and, under near-infrared irradiation, inducing local heating that enhances cell membrane permeability, thus facilitating the uptake of PI/pDNA complexes to boost transfection efficiency. Using pDNA encoding vascular endothelial growth factor as a model, our system shows enhanced transfection efficiency in vitro for hard-to-transfect endothelial cells, leading to improved cell proliferation and migration. Furthermore, in vivo studies reveal the therapeutic potential of this system in accelerating the healing of infected wounds by promoting angiogenesis and reducing inflammation. This approach offers a straightforward and effective method for gene transfection, showing potentials for tissue engineering and cell-based therapies.

Harnessing Guanidinium and Imidazole Functional Groups: A Dual-Charged Polymer Strategy for Enhanced Gene Delivery.

Histidine and arginine are two amino acids that exhibit beneficial properties for gene delivery. In particular, the imidazole group of histidine facilitates endosomal release, while the guanidinium group of arginine promotes cellular entry. Consequently, a dual-charged copolymer library based on these amino acids was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The content of the N-acryloyl-l-histidine (His) monomer was systematically increased, while maintaining consistent levels of methyl N-acryloyl-l-argininate hydrochloride (ArgOMe) or N-(4-guanidinobutyl)acrylamide hydrochloride (GBAm). The resulting polymers formed stable, nanosized polyplexes when complexed with nucleic acids. Remarkably, candidates with increased His content exhibited reduced cytotoxicity profiles and enhanced transfection efficiency, particularly retaining this performance level at lower pDNA concentrations. Furthermore, endosomal release studies revealed that increased His content improved endosomal release, while ArgOMe improved cellular entry. These findings underscore the potential of customized dual-charged copolymers and the synergistic effects of His and ArgOMe/GBAm in enhancing gene delivery.

Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Optimizing Biocompatibility and Gene Delivery with DMAEA and DMAEAm: A Niacin-Derived Copolymer Approach.

Gene therapy is pivotal in nanomedicine, offering a versatile approach to disease treatment. This study aims to achieve an optimal balance between biocompatibility and efficacy, which is a common challenge in the field. A copolymer library is synthesized, incorporating niacin-derived monomers 2-acrylamidoethyl nicotinate (AAEN) or 2-(acryloyloxy)ethyl nicotinate (AEN) with N,N-(dimethylamino)ethyl acrylamide (DMAEAm) or hydrolysis-labile N,N-(dimethylamino)ethyl acrylate (DMAEA). Evaluation of the polymers' cytotoxicity profiles reveals that an increase in AAEN or DMAEA molar ratios correlates with improved biocompatibility. Remarkably, an increase in AAEN in both DMAEA and DMAEAm copolymers demonstrated enhanced transfection efficiencies of plasmid DNA in HEK293T cells. Additionally, the top-performing polymers demonstrate promising gene expression in challenging-to-transfect cells (THP-1 and Jurkat cells) and show no significant effect on modulating immune response induction in ex vivo treated murine monocytes. Overall, the best performing candidates exhibit an optimal balance between biocompatibility and efficacy, showcasing potential advancements in gene therapy.

Optimizing Transfection Efficiency in CAR-T Cell Manufacturing through Multiple Administrations of Lipid-Based Nanoparticles.

The existing manufacturing protocols for CAR-T cell therapies pose notable challenges, particularly in attaining a transient transfection that endures for a significant duration. To address this gap, this study aims to formulate a transfection protocol utilizing multiple lipid-based nanoparticles (LNPs) administrations to enhance transfection efficiency (TE) to clinically relevant levels. By systematically fine-tuning and optimizing our transfection protocol through a series of iterative refinements, we have accomplished a remarkable one-order-of-magnitude augmentation in TE within the immortalized T-lymphocyte Jurkat cell line. This enhancement has been consistently observed over 2 weeks, and importantly, it has been achieved without any detrimental impact on cell viability. In the subsequent phase of our study, we aimed to optimize the gene delivery system by evaluating three lipid-based formulations tailored for DNA encapsulation using our refined protocol. These formulations encompassed two LNPs constructed from ionizable lipids and featuring systematic variations in lipid composition (iLNPs) and a cationic lipoplex (cLNP). Our findings showcased a notable standout among the three formulations, with cLNP emerging as a frontrunner for further refinement and integration into the production pipeline of CAR-T therapies. Consequently, cLNP was scrutinized for its potential to deliver CAR-encoding plasmid DNA to the HEK-293 cell line. Confocal microscopy experiments demonstrated its efficiency, revealing substantial internalization compared to iLNPs. By employing a recently developed confocal image analysis method, we substantiated that cellular entry of cLNP predominantly occurs through macropinocytosis. This mechanism leads to heightened intracellular endosomal escape and mitigates lysosomal accumulation. The successful expression of anti-CD19-CD28-CD3z, a CAR engineered to target CD19, a protein often expressed on the surface of B cells, was confirmed using a fluorescence-based assay. Overall, our results indicated the effectiveness of cLNP in gene delivery and suggested the potential of multiple administration transfection as a practical approach for refining T-cell engineering protocols in CAR therapies. Future investigations may focus on refining outcomes by adjusting transfection parameters like nucleic acid concentration, lipid-to-DNA ratio, and incubation time to achieve improved TE and increased gene expression levels.

Microfluidic Mechanoporation: Current Progress and Applications in Stem Cells.

Intracellular delivery, the process of transporting substances into cells, is crucial for various applications, such as drug delivery, gene therapy, cell imaging, and regenerative medicine. Among the different approaches of intracellular delivery, mechanoporation stands out by utilizing mechanical forces to create temporary pores on cell membranes, enabling the entry of substances into cells. This method is promising due to its minimal contamination and is especially vital for stem cells intended for clinical therapy. In this review, we explore various mechanoporation technologies, including microinjection, micro-nano needle arrays, cell squeezing through physical confinement, and cell squeezing using hydrodynamic forces. Additionally, we highlight recent research efforts utilizing mechanoporation for stem cell studies. Furthermore, we discuss the integration of mechanoporation techniques into microfluidic platforms for high-throughput intracellular delivery with enhanced transfection efficiency. This advancement holds potential in addressing the challenge of low transfection efficiency, benefiting both basic research and clinical applications of stem cells. Ultimately, the combination of microfluidics and mechanoporation presents new opportunities for creating comprehensive systems for stem cell processing.

Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.

Leveraging the extensive surface area of the lungs for gene therapy, the inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Our results demonstrated the superiority of the MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of the nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful lung-specific mRNA transfection without observable signs of toxicity. This MAP may represent an advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells.

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines' culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

Abstract 7249: Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy

Abstract Cancer poses a global health challenge, driving the quest for innovative therapies. Messenger ribonucleic acid (mRNA) lipid nanoparticles (LNPs) are a promising strategy in cancer immunotherapy. However, a major obstacle lies in efficiently transfecting immune cells like macrophages and dendritic cells (DCs) with LNPs. This study seeks to improve cancer immunotherapy by optimizing mRNA-LNP formulations. Therefore, we systematically evaluated three components of LNPs, namely ionizable lipids, phospholipids, and sterols, and fine-tuned the components and their molar ratios. A comprehensive screening process was conducted to determine the efficiency of LNPs, involving in vitro experiments on macrophages and DCs, as well as in vivo assessments. Among six ionizable lipids assessed in different cell lines (RAW264.7 and DC2.4), SM-102 significantly enhanced the transfection efficiency in macrophages and DCs. Subsequently, we evaluated these LNPs in an in vivo setting. We quantified the bioluminescence signal after administering LNPs loaded with luciferase mRNA. The results demonstrated a significant increase in luciferase expression when using SM-102 and ALC-0315 lipids. To validate this finding, we conducted a T cell response study, which revealed an increased percentage of CD8+ T cells with SM-102-based LNPs compared to other formulations. Having identified SM-102 as a potent ionizable lipid, we proceeded to evaluate three phospholipids in vitro. The results indicated a significant enhancement in transfection efficiency with DOPE lipids compared to DSPC. Consequently, we selected DOPE as the phospholipid for subsequent screening. Lastly, we explored different types of sterols with varying molar ratios in macrophages and DC cells. Our findings unveiled a notable improvement in mRNA delivery, particularly in DCs, when the molar ratio of β-sitosterol was increased to 19.5%, while concurrently decreasing the molar ratio of cholesterol to 19% within the same formulation. It is noteworthy that the addition of β-sitosterol alone led to reduced transfection efficiency in DCs but resulted in a significant enhancement in macrophages. Lastly, we evaluated the top four formulations in vivo, and the results demonstrated a significant increase when β-sitosterol was added to the formulation with cholesterol, at molar ratios of 19.5% and 19%, respectively. Overall, altering lipid composition and their molar ratios profoundly impact mRNA-LNP transfection. The optimized formulations, comprising SM-102, DOPE, and cholesterol with or without β-sitosterol, outperformed the alternatives. These refined formulations hold promise for clinical applications, potentially enhancing passive therapeutic agent delivery into macrophages and DCs, offering promising prospects for advancing cancer immunotherapy. Citation Format: Yasir Alshehry, Matthew Fernandez, Raneem Aldaqqa, Asma Al-Terawi, Douglas Sweet, Sandro da Rocha. Enhancing mRNA- lipid nanoparticles formulations to improve transfection efficiency in macrophages and dendritic cells in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7249.