Lipid Nanoparticles For Delivery Research Articles

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Entrapment of lipid nanoparticles in peripheral endosomes but not lysosomes impairs intracellular trafficking and endosomal escape

The uptake and intracellular trafficking of lipid nanoparticles (LNPs) along the endolysosomal pathway leading to releasing compartments is critical for delivery efficiency. How the players of the processes interact with each other to affect LNP delivery remains unclear. Here, we employed a recently developed, highly sensitive LNP labeling platform in combination with known-state of endolysosomal activity of cells to address this outstanding question with spatiotemporal analysis. We found the endolysosomal activity (endolyosomal pH, endolysosomal protease activation), which was regulated by nutrients, determines the active endocytosis activity of cells. Elevated internalization of DNA and LNP alike by cells correlated with increased endolysosomal activity. Similar to naked DNA, elevated internalization of LNP resulted in entrapment of LNPs in peripheral endosomes, which significantly impaired the intracellular trafficking of LNP to the perinuclear lysosome region and cytosolic release of LNP cargo. On the other hand, we found the extent of perinuclear lysosomal LNP accumulation positively correlated with the level of transgene expression. Moreover, we found continuous internalization of LNP was necessary not only to saturate the degradation compartments to overcome rapid degradation of LNP but also to maintain a necessary pool of releasing compartments, shuttling between peripheral endosomes and lysosomes via anterograde transport and retrograde transport along microtubules respectively, for meaningful endosomal release. Our results suggest the balance between endocytosis and intracellular trafficking needs to be fine-tuned according to endolysosomal activity of target cell to achieve optimal cytosol release.

Personalized Membrane Protein Vaccine Based on a Lipid Nanoparticle Delivery System Prevents Postoperative Recurrence in Colorectal Cancer Models

While accessing tumor neoantigens and developing effective delivery systems have posed significant challenges in therapeutic oncology vaccines, this study introduces a cost- and time-efficient personalized tumor vaccine demonstrating potent anti-tumor effects in a mouse xenograft model. This vaccine utilizes a lipid nanoparticle (C5 LNP) system loaded with membrane protein antigens (mAg) derived from surgically excised tumor tissue. Its safety and efficacy were validated in a B16-OVA murine model. C5/OVA exhibited significant uptake by dendritic cells (DCs), leading to cross-presentation, maturation, and subsequent initiation of robust cell-mediated and humoral immune responses. This resulted in clear tumor growth suppression and extended survival in the B16-OVA model. Furthermore, the personalized C5/mAg vaccine effectively inhibited tumor growth in a colorectal carcinoma model. When combined with anti-PD-1 therapy, it notably increased complete remission (CR) rates in the CT26 xenograft model. Vaccinated mice demonstrated 100% resistance to tumor rechallenge, underscoring the vaccine's ability to induce long-term immune memory. This study presents a promising personalized cancer vaccine delivery system with potential for both treatment and prevention of carcinoma in clinical applications. Statement of significanceIn this paper, we present a scheme for manufacturing personalized tumor vaccines. The vaccine component consists of lipid nanoparticles (LNPs) designated C5 and membrane protein antigens (mAg) derived from autologous tumor tissue surgically resected from the patient. Our study demonstrates that the personalized mAg-C5 vaccine for colorectal carcinoma significantly inhibits tumor growth. Furthermore, conjugation with anti-PD-1 therapy demonstrably increased the complete remission (CR) rate in the murine CT26 xenograft tumor model. Additionally, mice treated with the C5/mAg vaccine exhibited 100% resistance to tumor growth in a colon carcinoma rechallenge model, indicating the induction of immune memory by the vaccine.Our research results suggest that the mAg-LNP vaccine is a simple, cost-effective treatment that clinicians can easily and quickly integrate into routine practice.

EXTH-68. EPIGENETIC EDITING BY MRNA-BASED CRISPROFF ENABLES FLEXIBLE AND DURABLE TARGETING OF GLIOBLASTOMA DRIVERS MGMT AND TERT

Abstract Alterations in cis-regulatory elements, such as hypomethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter and activating mutations within the telomerase reverse transcriptase (TERT) promoter, are pervasive drivers of glioblastoma (GBM) tumorigenesis that are challenging to target. CRISPRoff is an engineered CRISPR/Cas9 system that can potently and heritably silence cis-regulatory elements through DNA methylation. Here, we develop an mRNA-based CRISPRoff platform to target the MGMT and TERT promoters in GBM, and we establish proof-of-principal in vivo targeting through lipid nanoparticles (LNP). Epigenetic editing in GBM cells and primary GBM organoids was performed using either electroporation or LNP encapsulation of CRISPRoff mRNA and sgRNAs. Target gene silencing and functional phenotypes were assessed by bisulfite sequencing, RT-qPCR, RNA-seq, western blot, cell viability/apoptosis, and telomere restriction fragment assays. In vivo efficacy was demonstrated using intracranial GBM xenografts, first through transplantation of cells with CRISPRoff delivered ex vivo, then with direct delivery of LNPs using convection enhanced delivery (CED) coupled with immunofluorescence/ immunohistochemistry. CRISPRoff targeting of MGMT promoter resulted in >99% reduction in MGMT expression in MGMT unmethylated primary GBM organoids, inducing up to 150-fold sensitization to temozolomide. Temozolomide sensitization was retained after intracrania transplantation, and silencing was durable in clonally isolated GBM cells continuously passaged for over 8 months after transient delivery of CRISPRoff. CRISPRoff targeting of the TERT promoter in primary GBM cultures harboring the G228A TERT promoter mutation silenced TERT expression by up to 99%, overcoming constitutive activation of TERT, and induced telomere shortening sufficient for complete replicative senescence. CED of mRNAs encapsulated by LNPs based on cKK-E12 or Lipid A9 resulted in tumor-selective uptake of mRNA transcripts, establishing a foundation for direct delivery of CRISPRoff in vivo bypassing the blood-brain-barrier. In summary, we establish a flexible and durable epigenetic editing system against multiple regulatory elements driving GBM, leveraging LNPs with potential for in vivo application.

Modified PEG-Lipids Enhance the Nasal Mucosal Immune Capacity of Lipid Nanoparticle mRNA Vaccines.

Omicron, the predominant variant of SARS-CoV-2, exhibits strong immune-evasive properties, leading to the reduced efficacy of existing vaccines. Consequently, the development of versatile vaccines is imperative. Intranasal mRNA vaccines offer convenient administration and have the potential to enhance mucosal immunity. However, delivering vaccines via the nasal mucosa requires overcoming complex physiological barriers. The aim of this study is to modify PEGylated lipids to enhance the mucosal immune efficacy of the vaccine. The PEGylated lipid component of lipid nanoparticle (LNP) delivery vectors was modified with chitosan or mannose to generate novel LNPs that enhance vaccine adhesion or targeting on mucosal surfaces. The impact of the mRNA encoding the receptor-binding domain of Omicron BA.4/BA.5 on the immune response was examined. Compared to the unmodified LNP group, the IgG and IgA titers in the chitosan or mannose-modified LNP groups showed an increasing trend. The chitosan-modified group showed better effects. Notably, the PEGylated lipid with 1.5 mol% of chitosan modification produced high levels of IgG1 and IgG2a antibodies, promoting Th1/Th2 responses while also generating high levels of IgA, which can induce stronger cellular immunity, humoral immunity, and mucosal immunity. The 1.5 mol% of chitosan-modified LNPs (mRNA-LNP-1.5CS) can serve as a safe and effective carrier for intranasal mRNA vaccines, offering a promising strategy for combating the Omicron variant.

Advances in mRNA LNP-Based Cancer Vaccines: Mechanisms, Formulation Aspects, Challenges, and Future Directions.

After the COVID-19 pandemic, mRNA-based vaccines have emerged as a revolutionary technology in immunization and vaccination. These vaccines have shown remarkable efficacy against the virus and opened up avenues for their possible application in other diseases. This has renewed interest and investment in mRNA vaccine research and development, attracting the scientific community to explore all its other applications beyond infectious diseases. Recently, researchers have focused on the possibility of adapting this vaccination approach to cancer immunotherapy. While there is a huge potential, challenges still remain in the design and optimization of the synthetic mRNA molecules and the lipid nanoparticle delivery system required to ensure the adequate elicitation of the immune response and the successful eradication of tumors. This review points out the basic mechanisms of mRNA-LNP vaccines in cancer immunotherapy and recent approaches in mRNA vaccine design. This review displays the current mRNA modifications and lipid nanoparticle components and how these factors affect vaccine efficacy. Furthermore, this review discusses the future directions and clinical applications of mRNA-LNP vaccines in cancer treatment.

Lipid nanoparticle delivery of TALEN mRNA targeting LPA causes gene disruption and plasma lipoprotein(a) reduction in transgenic mice

Lipoprotein(a), or Lp(a), is encoded by the LPA gene and is a causal genetic risk factor for cardiovascular disease. Individuals with high Lp(a) are at risk for cardiovascular morbidity and are refractory to standard lipid-lowering agents. Lp(a)-lowering therapies currently in clinical development require repetitive dosing, while a gene editing approach presents an opportunity for a single-dose treatment. In this study, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALENs) were designed to target human LPA for gene disruption and permanent Lp(a) reduction. TALEN mRNAs were screened in vitro and found to cause on-target gene editing and target protein reduction with minimal off-target editing. TALEN mRNAs were then encapsulated with LUNAR®, a proprietary lipid nanoparticle (LNP), and administered to transgenic mice that expressed a human LPA transgene. A single dose of TALEN mRNA-LNPs reduced plasma Lp(a) levels in mice by over 80%, which was sustained for at least 5 weeks. Moreover, both standard and long-read next generation sequencing confirmed the presence of gene-inactivating deletions at LPA transgene loci. Overall, this study serves as a proof-of-concept for using TALEN-mediated gene editing to disrupt LPA in vivo, paving the way for the development of a feasible gene editing therapy for patients with high Lp(a).

Alpha-Pinene-encapsulated lipid nanoparticles diminished inflammatory responses in THP-1 cells and imiquimod-induced psoriasis-like skin injury and splenomegaly in mice.

Psoriasis, a persistent skin condition caused by the disorder of the immune system, impacts approximately 1.25 million individuals globally. Nevertheless, the presence of adverse effects in conventional clinical drugs necessitates further exploration of novel medications or combination therapies to mitigate these reactions and enhance their effectiveness. Hence, our intention here in this paper is to utilize the lipid nanoparticle delivery system for overcoming the volatility and hydrophobic properties of α-pinene, a naturally occurring compound renowned for its anti-inflammatory and antiviral effects, and further explore its potential pharmacological applications both in vitro and in vivo. The production of α-pinene lipid nanoparticles (APLNs) was achieved through the utilization of high pressure homogenization methods. APLNs was successfully fabricated with enhanced stability and water solubility. Meanwhile, the application of APLNs could drastically reduce the expression of lipopolysaccharide (LPS)-induced inflammation-related factors in THP-1 cells. Administration of APLNs to a mouse model of auricular swelling could effectively reduce redness and swelling in the auricles of mice as well. Furthermore, APLNs were also found to alleviate skin damage in mice with Imiquimod (IMQ)-induced psoriasis model, as well as decrease the levels of psoriasis-related protein nuclear factor kappa-B (NF-κB) and interleukin-17 (IL-17), interleukin-23 (IL-23), and other inflammation-related cytokines. More importantly, utilization of APLNs successfully mitigated the systemic inflammatory reactions in mice, resulting in the reduction of spleen-to-body ratio (wt%) and of inflammatory cytokines' expression in the serum. Overall, our results suggest that with the help of lipid nanoparticle encapsulation, APLNs possess a better pharmacological effect in anti-inflammation and could potentially serve as an anti-psoriasis drug.

Macrophage-specific lipid nanoparticle therapy blocks the lung's mechanosensitive immunity due to macrophage-epithelial interactions.

The lung's mechanosensitive immune response, which occurs when pulmonary alveoli are overstretched, is a major impediment to ventilation therapy for hypoxemic respiratory failure. The cause is not known. We tested the hypothesis that alveolar stretch causes stretch of alveolar macrophages (AMs), leading to the immune response. In lungs viewed by optical imaging, sessile AMs expressed gap junctional protein connexin-43 (Cx43), and they communicated with the alveolar epithelium through gap junctions. Alveolar hyperinflation increased Ca2+ in the AMs but did not stretch the AMs. The Ca2+ response, and concomitant TNFα secretion by AMs were blocked in mice with AM-specific deletion of Cx43. The AM responses, as also lung injury due to mechanical ventilation at high tidal volume, were inhibited by AM-specific delivery of lipid nanoparticles containing Xestospongin C, which blocked the induced Ca2+ increases. We conclude, Cx43- and Ca2+-dependent AM-epithelial interactions determine the lung's mechanosensitive immunity, providing a basis for therapy for ventilator-induced lung injury.

Composition of lipid nanoparticles for targeted delivery: application to mRNA therapeutics.

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.

Hepatocyte-Targeted Lipid Nanoparticle Delivery of HERC2 Plasmid Controls Drug-Induced Hepatotoxicity by Limiting β-Catenin-Regulated CYP2E1 Expression.

Understanding the molecular mechanisms that bridge hepatic inflammation and liver injury is crucial for developing effective therapeutic strategies for drug-induced liver injury (DILI) management. HECT domain and RCC1-like domain 2(HERC2) belongsto the large Herc family of ubiquitin E3 ligases, which are implicated in tissue development and inflammation. The observation reveals a pronounced HERC2 expression in specific hepatocyte subsets that proliferate in response to DILI in humans, prompting an investigation into the role of HERC2 in distinct DILI progression. Under the APAP challenge, liver-specific HERC2-deficient mice suffer more severe liver damage. Integrated single-cell RNA sequencing analysis unveils a negative correlation between HERC2 and CYP2E1, a vital metabolic enzyme for xenobiotics, in hepatocytes from APAP-challenged mice. Mechanistically, HERC2 interacts with β-catenin to promote its ubiquitination, thereby governing CYP2E1 transcriptional regulation. Targeted hepatic delivery of lipid nanoparticle-encapsulated HERC2-overexpressing plasmid markedly reduces liver damage caused by APAP overdose. Collectively, these findings elucidate a previously unrecognized protective role of HERC2 in protecting against acute liver injury associated with drug metabolism disorders, highlighting its potential as a therapeutic target in treating DILI.

Paracyclophane-based ionizable lipids for efficient mRNA delivery in vivo

mRNA therapeutics utilizing lipid nanoparticle (LNP) delivery technology represent a medical innovation for the treatment of various diseases. Amine-derived ionizable cationic lipids have been regarded as the pivotal component of LNPs, which often utilize commercially available small amine molecules as their cores. Given that even minor changes in the structure of ionizable lipids can result in significant differences in the delivery performance, there is a growing need to redesign the lipid amine-cores to optimize mRNA therapy. Here, we rationally design and synthesize a library of 198 paracyclophane-based ionizable lipids (PILs), which are then formulated into LNPs for mRNA delivery in vitro and in vivo. The resulting PIL LNPs display favorable characteristics, including appropriate particle sizes, zeta potentials, mRNA binding capability, efficacious endosomal escape, and robust mRNA delivery in vitro. Tailoring the PIL structures further enables mRNA expression specifically in the liver or simultaneously across multi-organs in vivo. Notably, the optimized PIL LNPs demonstrate superior efficacy compared to the U.S. Food and Drug Administration (FDA) approved DLin-MC3-DMA LNPs following intravenous administration. Additionally, when administered intramuscularly, our PIL LNPs exhibit higher efficacy than the SM-102 and ALC-0315 LNPs that are employed in the coronavirus disease 2019 (COVID-19) mRNA vaccines. These findings demonstrate the potential of paracyclophane-based ionizable lipids in advancing mRNA therapeutics, particularly for liver-targeted drugs and vaccines.

Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.

Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.

Impact of Protein Coronas on Lipid Nanoparticle Uptake and Endocytic Pathways in Cells.

Lipid nanoparticles (LNPs), widely used in disease diagnosis and drug delivery, face the challenge of being surrounded by biological macromolecules such as proteins upon entering the human body. These molecules compete for binding sites on the nanoparticle surfaces, forming a protein corona. The impact of different types of protein coronas on LNP delivery remains unclear. In this study, we employed a newly developed, highly sensitive LNP labeling platform and analyzed the endocytosis of HeLa cells under different nutritional conditions using proteomics to address this critical issue. Our research found that under conditions of complete medium and amino acid starvation, most DNA-FITC vesicles in HeLa cells were located in the perinuclear region 4 h after transfection. In contrast, under serum starvation conditions, only a small portion of DNA-FITC vesicles were in the perinuclear region. On the other hand, through proteomics, we discovered that cells that were enriched in amino acids and complete medium contained more proteins, whereas those under serum starvation had relatively fewer enriched proteins. Through KEGG pathway enrichment analysis, we identified the phagosome and endocytosis pathways as particularly important. Lastly, differential analysis of proteins in these pathways revealed that proteins such as F-actin, Coronin, vATPase, TUBA, TUBB, MHCII, and TSP may have significant impacts on cellular endocytosis. Our research findings indicate that it is necessary to regulate cellular endocytosis based on different protein coronas to achieve optimal cytoplasmic release.

Discovery of Ketal-Ester Ionizable Lipid Nanoparticle with Reduced Hepatotoxicity, Enhanced Spleen Tropism for mRNA Vaccine Delivery.

The safety and efficacy of the lipid nanoparticle (LNP) delivery system are crucial for the successful development of messenger RNA vaccines. We designed and synthesized a series of ketal ester lipids (KELs), featuring a biodegradable ketal moiety in the linker and ester segments in the tail. Through iterative optimization of the head and tail groups of KELs, we tuned the pKa and molecular shapes, and identified (4S)-KEL12 as a safe and efficient ionizable lipid for mRNA delivery. (4S)-KEL12 LNP showed significantly higher delivery efficacy and lower toxicity than the DLin-MC3-DMA LNP. In comparison to SM-102 LNP, (4S)-KEL12 LNP exhibited better spleen tropism, reduced liver tropism, and hepatotoxicity. Additionally, (4S)-KEL12 demonstrated good biodegradability following intramuscular or intravenous injection. Notably, (4S)-KEL12 LNP encapsulated with a therapeutic mRNA cancer vaccine elicited robust cellular immune responses leading to substantial tumor regression along with prolonged survival in tumor-bearing mice. Our results suggest that (4S)-KEL12 LNP holds great promise for mRNA vaccine delivery. The comprehensive analysis of the structure-activity relationship, toxicity, biodegradability, distribution, expression, efficacy, and stereochemistry of these LNPs will greatly contribute to the rational design and discovery of novel lipid-based delivery systems.

Immune Implications of Cholesterol-Containing Lipid Nanoparticles.

The majority of clinically approved nanoparticle-mediated therapeutics are lipid nanoparticles (LNPs), and most of these LNPs are liposomes containing cholesterol. LNP formulations significantly alter the drug pharmacokinetics (PK) due to the propensity of nanoparticles for uptake by macrophages. In addition to readily engulfing LNPs, the high expression of cholesterol hydroxylases and reactive oxygen species (ROS) in macrophages suggests that they will readily produce oxysterols from LNP-associated cholesterol. Oxysterols are a heterogeneous group of cholesterol oxidation products that have potent immune modulatory effects. Oxysterols are implicated in the pathogenesis of atherosclerosis and certain malignancies; they have also been found in commercial liposome preparations. Yet, the in vivo metabolic fate of LNP-associated cholesterol remains unclear. We review herein the mechanisms of cellular uptake, trafficking, metabolism, and immune modulation of endogenous nanometer-sized cholesterol particles (i.e., lipoproteins) that are also relevant for cholesterol-containing nanoparticles. We believe that it would be imperative to better understand the in vivo metabolic fate of LNP-associated cholesterol and the immune implications for LNP-therapeutics. We highlight critical knowledge gaps that we believe need to be addressed in order to develop safer and more efficacious lipid nanoparticle delivery systems.

A biodegradable lipid nanoparticle delivers a Cas9 ribonucleoprotein for efficient and safe in situ genome editing in melanoma

The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo. Statement of significanceCRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.

Development of mRNA Lipid Nanoparticles: Targeting and Therapeutic Aspects.

Lipid nanoparticles (LNPs) have emerged as leading non-viral carriers for messenger RNA (mRNA) delivery in clinical applications. Overcoming challenges in safe and effective mRNA delivery to target tissues and cells, along with controlling release from the delivery vehicle, remains pivotal in mRNA-based therapies. This review elucidates the structure of LNPs, the mechanism for mRNA delivery, and the targeted delivery of LNPs to various cells and tissues, including leukocytes, T-cells, dendritic cells, Kupffer cells, hepatic endothelial cells, and hepatic and extrahepatic tissues. Here, we discuss the applications of mRNA-LNP vaccines for the prevention of infectious diseases and for the treatment of cancer and various genetic diseases. Although challenges remain in terms of delivery efficiency, specific tissue targeting, toxicity, and storage stability, mRNA-LNP technology holds extensive potential for the treatment of diseases.

Principles of lipid nanoparticle design for mRNA delivery

AbstractmRNA therapeutics have significantly evolved within the life sciences, particularly in applications such as vaccines, tumor immunotherapy, protein replacement, gene editing, and monoclonal antibody therapy. To fully realize the potential of mRNA drugs and mitigate the adverse effects, substantial vector materials have been developed for delivery of these pharmaceutical agents. Lipid nanoparticles (LNPs) represent the most clinically advanced mRNA carriers, recognized by U.S. Food and Drug Administration approved mRNA vaccines and numerous clinical trials. Diverse therapeutic applications necessitate tailored design of LNPs. Herein, we outline the principles of LNP design for mRNA delivery, focusing specifically on their effectiveness, targeting capabilities, safety profiles, and nanoparticle stability. Additionally, we present the latest advancements in mRNA‐LNP technology. This review aims to elucidate the benefits and design principles of LNP delivery systems for mRNA therapeutics, providing insights into breakthroughs and innovative ideas for further enhancing these advantages. These summaries are dedicated to promoting the broader applications of LNP‐mRNA drugs, aiming to advance the treatment of serious diseases in an effective and safe manner.

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge. Upon administration to human embryonic kidney (HEK 293) cells, plain and PEGylated LNPs were internalized through different endocytic routes, as revealed by spatiotemporal correlation spectroscopy. An imaging-derived mean square displacement (iMSD) analysis shows that PEGylated LNPs exhibit a significantly stronger preference for caveolae-mediated endocytosis (CAV) and clathrin-mediated endocytosis (CME) pathways compared to plain LNPs, with these latter being better tailored to MCR-dependent internalization and trafficking. This suggests that PEG plays a crucial role in directing LNPs toward specific cellular uptake routes. Further studies should explore how PEG-mediated endocytosis impacts intracellular trafficking and ultimately translates to therapeutic efficacy, guiding the design of next-generation LNP delivery systems.