Lipid Nanoparticle System Research Articles

SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants

The continual emergence of SARS-CoV-2 variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Towards enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle system (SMART-LNPs) that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric-S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5’&3’ UTRs. In a mouse model, SMART-LNPs-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced IFNγ producing T-cells against SARS-CoV-2 virus compared to non-targeted LNPs-TS1 vaccine. Further, T-cells analysis revealed that SMART-LNPs-TS1 vaccine induced long-lived memory T-cell subsets, Th1-dominant and cytotoxic T-cells immune responses against SARS-CoV-2 virus. Importantly, SMART-LNPs-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-gen delivery system for mRNA-based immune therapies.

PH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles.

Self-assembled lipid nanoparticles (LNPs), serving as essential nanocarriers in recent COVID-19 mRNA vaccines, provide a stable and versatile platform for delivering a wide range of biological materials. Notably, LNPs with unique inverse mesostructures, such as cubosomes and hexosomes, are recognized as fusogenic nanocarriers in the drug delivery field. This study delves into the physicochemical properties, including size, lyotropic liquid crystalline mesophase, and apparent pKa of LNPs with various lipid components, consisting of two ionizable lipids (ALC-0315 and SM-102) used in commercial COVID-19 mRNA vaccines and a well-known inverse mesophase structure-forming helper lipid, phytantriol (PT). Two partial mesophase diagrams are generated for both ALC-0315/PT LNPs and SM-102/PT LNPs as a function of two factors, ionizable lipid ratio (α, 0-100 mol%) and pH condition (pH 3-11). Furthermore, the impact of different LNP stabilizers (Pluronic F127, Pluronic F108, and Tween 80) on their pH-dependent phase behavior is evaluated. The findings offer insights into the self-assembled mesostructure and ionization state of the studied LNPs with potentially enhanced endosomal escape ability. This research is relevant to developing innovative next-generation LNP systems for delivering various therapeutics.

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Selection of GalNAc-Conjugated siKeap1 as Disease-Specific Delivery System for Chemotherapy-Induced Liver Injury and Chronic Liver Disease.

Chemotherapy-induced liver injury (CILI) is a pressing concern in cancer patients. One promising approach involves activating nuclear factor erythroid 2-related factor 2 (Nrf2) to mitigate CILI. However, selectively activating liver Nrf2 without compromising chemotherapy's efficacy has remained elusive. Herein, two RNAi delivery strategies were explored: lipid nanoparticle (LNP) and N-acetylgalactosamine (GalNAc) delivery systems loaded with siRNA designed to silence Kelch-like-ECH associated protein 1 (Keap1) by aiming for liver-specific Nrf2 activation. Remarkably, siKeap1-LNP exhibited unintended tumor targeting alongside liver effects, thereby potentially promoting tumor progression. Conversely, siKeap1-GalNAc did not compromise chemotherapy efficacy and outperformed the conventional Nrf2 activator, bardoxolone, in mitigating CILI. This study proposes siKeap1-GalNAc as a promising therapeutic avenue for liver injury. Importantly, our study bridges a crucial gap concerning the delivery system for liver targeting but not tumor targeting and underscores the importance of selecting nucleic acid delivery systems tailored to specific diseases, not just to specific organs.

Dual-targeted lipid nanoparticles system for synergistic anti-inflammation and cartilage repair in the treatment of temporomandibular joint osteoarthritis

Synovial inflammation and cartilage degeneration are two crucial pathologic features in temporomandibular joint osteoarthritis (TMJ OA). Cartilage repair can be very thorny in inflammatory environment, which is highly associated with the activated macrophages. Thus, simultaneous inflammation suppression and cartilage repair are strongly required. However, the clinically used intra-articular injection agents face the problems of insufficient inflammation suppression, deficient cartilage repair and non-targeted therapy. Hence, we developed the novel dual-targeted lipid nanoparticles (LNPs) loaded with miR-330-3p, an important inflammation inhibitor, and kartogenin (KGN), a pro-chondrogenic small molecular, for synergistic anti-inflammation and cartilage repair of TMJ OA. The folic acid (FA) and collagen II-targeting peptide (WYRGRL) were modified on the surface of LNPs for precise delivery to macrophages or chondrocytes, respectively. The dual-targeted miR-330-3p@FA-LNP and KGN@WYRGRL-LNP (KGN@W-LNP) both manifested high bioavailability and selectively active cellular uptake. Furthermore, it functioned synergistically to alleviate synovium inflammation and cartilage degeneration via modulating the M1 to M2 repolarization of macrophages and maintaining the homeostasis of chondrocytes, with a low intra-articular injection dose of miR-330-3p@FA-LNP (0.0125 nmol) and KGN@W-LNP (0.025 nmol) in vivo. RNA-seq and further validation demonstrated that miR-330-3p functioned by inhibiting SPARC and KGN functioned by upregulating EPHB4. In summary, this dual-targeted LNPs system provides a promising therapeutic strategy for TMJ OA treatment.

Base-modified factor VIII mRNA delivery with galactosylated lipid nanoparticles as a protein replacement therapy for haemophilia A.

The bleeding disorder hemophilia A (HemA) requires systemic functional factor VIII protein infusions on prophylactic schedules. Recently, chemically modified mRNAs have emerged as promising protein replacement therapies to reduce repeated infusions and improve safety profiles. However, the influence of base modifications on mRNA translation kinetics to specific cell types remains unexplored. In this study, towards developing mRNA therapeutics for haemophilia A, we synthesized chemically modified mRNAs with commercially available base modifications of adenine, guanine, uridine, and cytidine, and evaluated in vitro transcription yield and translation kinetics in hepatic cell lines using reporter eGFP mRNA. Our findings demonstrated that mRNA with N1-methyl pseudouridine (m1Ψ) showed a 5-12-fold increase in translation efficiency in both hepatic and endothelial cell lines. As a proof of concept for developing mRNA therapy for HemA, where FVIII is deficient, we developed a m1Ψ modified functional FVIII mRNA with our liver-targeting lipid nanoparticle (Gal-LNP) system. We evaluated its delivery efficiencies in both hepatic cell lines and the HemA mouse model. The m1Ψ-FVIII mRNA showed high therapeutic efficacy up to 15 days in vivo in the HemA mouse model. Gal-LNPs were found to be safe for systemic administration. Our study reveals that incorporating m1Ψ base modifications on mRNAs could improve therapeutic efficacy in liver- and endothelial-based therapeutics. Optimized mRNA synthesis for superior expression kinetics in hepatic cells and its delivery with liver-targeted nanoparticles may emerge as protein replacement therapies for monogenic liver disorders.

TET1-Lipid Nanoparticle Encapsulating Morphine for Specific Targeting of Peripheral Nerve for Pain Alleviation.

Opioids are irreplaceable analgesics owing to the lack of alternative analgesics that offer opioid-like pain relief. However, opioids have many undesirable central side effects. Restricting opioids to peripheral opioid receptors could reduce those effects while maintaining analgesia. To achieve this goal, we developed Tet1-LNP (morphine), a neural-targeting lipid nanoparticle encapsulating morphine that could specifically activate the peripheral opioid receptor in the dorsal root ganglion (DRG) and significantly reduce the side effects caused by the activation of opioid receptors in the brain. Tet1-LNP (morphine) were successfully prepared using the thin-film hydration method. In vitro, Tet1-LNP (morphine) uptake was assessed in differentiated neuron-like PC-12 cells and dorsal root ganglion (DRG) primary cells. The uptake of Tet1-LNP (morphine) in the DRGs and the brain was assessed in vivo. Von Frey filament and Hargreaves tests were used to assess the antinociception of Tet1-LNP (morphine) in the chronic constriction injury (CCI) neuropathic pain model. Morphine concentration in blood and brain were evaluated using ELISA. Tet1-LNP (morphine) had an average size of 131 nm. Tet1-LNP (morphine) showed high cellular uptake and targeted DRG in vitro. CCI mice treated with Tet1-LNP (morphine) experienced prolonged analgesia for nearly 32 h compared with 3 h with free morphine (p < 0.0001). Notably, the brain morphine concentration in the Tet1-LNP (morphine) group was eight-fold lower than that in the morphine group (p < 0.0001). Our study presents a targeted lipid nanoparticle system for peripheral neural delivery of morphine. We anticipate Tet1-LNP (morphine) will offer a safe formulation for chronic neuropathic pain treatment, and promise further development for clinical applications.

Antigen‐specific mRNA lipid nanoparticle platforms for the prevention and treatment of allergy and autoimmune diseases

AbstractWhile most nanomedicines primarily aim to stimulate the immune system against infections or tumors, there is a growing demand for inducing immune tolerance under certain conditions, such as allergic and autoimmune diseases. Researchers have explored nanotechnology‐based strategies to induce immune tolerance in a targeted and specific manner. One approach involves the use of nanoparticles (NPs) to encapsulate immunosuppressive drugs and/or antigens to educate naïve T cells and promote the generation of antigen‐specific regulatory T cells that inhibit immune responses. However, this approach has certain limitations. The hydrophobicity of proteins or peptides restricts the degree to which they can be encapsulated in NPs, which in turn, affects their loading efficiency and treatment efficacy. With the emergence of mRNA lipid nanoparticle (LNP) platforms, there is the possibility of overcoming the limitations of protein and peptide encapsulation. To date, mRNA LNP systems have been shown to provide organ, cellular, and subcellular targeting for the induction of immune tolerance. This method of drug delivery is flexible and scalable that can be customized for a specific patient, resulting in an effective means of administering relevant proteins or epitopes to induce antigen‐specific immune tolerance. With continued research and development, this technology could offer a safer and more effective alternative to current therapies, ultimately improving the quality of life of patients worldwide.

In Vivo Delivery of RNA By Lipid Nanoparticles (LNP) to Hematopoietic Stem Cells and T Cells in Humanized Mice and Non-Human Primates

Genomic medicines have significant therapeutic potential to cure rare genetic diseases and cancer, but currently, most treatments rely on ex vivo cell editing, which is time consuming, costly, and suffers from logistic and manufacturing challenges. In contrast, in vivo delivery of genomic medicines would unlock new therapeutic areas and provide broader patient access. However, the success of in vivo delivery is dependent on the development of safe delivery vehicles capable of transporting the genomic medicines to their intended target tissues and cells. In recent years, lipid nanoparticles (LNPs) have emerged as versatile delivery solutions for RNA therapeutics but have seldom been used fo r in vivo delivery beyond liver and muscle tissue. Challenges in extrahepatic delivery of LNPs include adequate tissue-specific targeting to achieve therapeutic levels of genome editing while avoiding anti-drug immune responses. We have developed novel LNPs designed to deliver RNA payloads to either T cells or hematopoietic stem cells (HSCs) in vivo. Using our novel T-cell LNPs, we have demonstrated single-dose in vivo delivery of GFP mRNA in two humanized mouse models. In the spleen of humanized NSG mice (pre-engrafted with human HSCs 12-weeks prior to study), we achieved GFP expression in 24% of human CD3 + T cells. In another model, NSG mice infused with human T cells 14-days prior to study, GFP expression was observed in 76% of T cells in the spleen. Single-dose administration of this T-cell LNP in rhesus macaque resulted in 40% GFP expression in peripheral blood T cells. Furthermore, we did not observe any detectable GFP expression in human or rhesus B cells, suggesting this LNP system may be suitable for the development of in vivo CAR-T therapies for B cell malignancies. Separately, we treated humanized NBSGW mice (pre-engrafted with human HSCs for 12 weeks) with a single dose of our HSC LNPs delivering GFP mRNA. Expression of GFP was observed in approximately 90% of bone marrow hematopoietic stem and progenitor cells (HSPCs defined as Lin -CD34 +CD38 - cells) and approximately 95% of long-term bone marrow HSC-enriched population (HSCs defined as Lin -CD34 +CD38 -CD90 +CD45RA - cells). Single-dose intravenous administration of the same LNP in cynomolgus macaques led to GFP expression in 61% of HSPCs and 62% of HSCs in the bone marrow. Gene editing presents a greater challenge than reporter mRNA delivery due to the increased size and complexity of the RNA cargos. Toward this goal, we went on to demonstrate that our HSC LNP platform can deliver Cas9 mRNA, with a beta-2 microglobulin (B2M) sgRNA, to human HSCs in humanized NBSGW mice resulting in the knockout (KO) of the B2M gene through the introduction of insertion or deletion mutations. Likewise, following administration of a B2M editing T-cell LNP in humanized NSG mice, we demonstrated B2M KO in splenic human CD3 + T cells. Together, these in vivo delivery results indicate our LNP delivery platform is capable of efficiently delivering RNA payloads to different hematopoietic cell types in blood and tissues, providing a potential delivery platform for in vivo genome editing.

Telomerase mRNA Enhances Human Skin Engraftment for Wound Healing.

Deep skin wounds represent a serious condition and frequently require split-thickness skin grafts (STSG) to heal. The application of autologous human-skin-cell-suspension (hSCS) requires less donor skin than STSG without compromising the healing capacity. Impaired function and replicative ability of senescent cutaneous cells in the aging skin affects healing with autologous hSCS. Major determinants of senescence are telomere erosion and DNA damage. Human telomerase reverse transcriptase (hTERT) adds telomeric repeats to the DNA and can protect against DNA damage. Herein, hTERT mRNA lipid nanoparticles (LNP) are proposed and evaluated for enhancing cellular engraftment and proliferation of hSCS. Transfection with optimized hTERT mRNA LNP system enables delivery and expression of mRNA in vitro in keratinocytes, fibroblasts, and in hSCS prepared from donors' skin. Telomerase activity in hSCS is significantly increased. hTERT mRNA LNP enhance the generation of a partial-thickness human skin equivalent in the mouse model, increasing hSCS engraftment (Lamin) and proliferation (Ki67), while reducing cellular senescence (p21) and DNA damage (53BP1).

Novel Ionizable Lipid Nanoparticles for SARS‐CoV‐2 Omicron mRNA Delivery (Adv. Healthcare Mater. 13/2023)

Ionizable Lipid Nanoparticles Lipid nanoparticle (LNP) systems are currently the leading nonviral delivery systems for enabling the clinical potential of mRNA drugs. Ionizable lipid is the critical component of lipid nanoparticles. In article 2202590 by Gengshen Song, Jing Yang, Shengqi Wang, and co-workers, a novel ionizable lipid YK009 is designed and developed for LNP formulation. The YK009-LNP shows high mRNA delivery efficacy and good safety. An mRNA vaccine against the SARS-CoV-2 Omicron variant delivered by YK009-LNP can trigger a robust immune response against this viral infection.

Liver-derived extracellular vesicles from patients with hepatitis B virus-related acute-on-chronic liver failure impair hepatic regeneration by inhibiting on FGFR2 signaling via miR-218-5p.

Impaired liver regeneration in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients is closely related to prognosis; however, the mechanisms are not yet defined. Liver-derived extracellular vesicles (EVs) may be involved in the dysregulation of liver regeneration. Clarifying the underlying mechanisms will improve the treatments for HBV-ACLF. EVs were isolated by ultracentrifugation from liver tissues of HBV-ACLF patients (ACLF_EVs) after liver transplantation, and their function was investigated in acute liver injury (ALI) mice and AML12 cells. Differentially expressed miRNAs (DE-miRNAs) were screened by deep miRNA sequencing. The lipid nanoparticle (LNP) system was applied as a carrier for the targeted delivery of miRNA inhibitors to improve its effect on liver regeneration. ACLF_EVs inhibited hepatocyte proliferation and liver regeneration, with a critical role of miR-218-5p. Mechanistically, ACLF_EVs fused directly with target hepatocytes and transferred miR-218-5p into hepatocytes, acting by suppressing FGFR2 mRNA and inhibiting the activation of ERK1/2 signaling pathway. Reducing the level of miR-218-5p expression in the liver of ACLF mice partially restored liver regeneration ability. The current data reveal the mechanism underlying impaired liver regeneration in HBV-ACLF that promotes the discovery of new therapeutic approaches.

Abstract P4-07-50: mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation

Abstract During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr® Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T-47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3®, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (&amp;gt;94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferase-mRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research. Citation Format: Karem A. Court, Anjana Tiwari, Eric Chau, John Cooke, Biana Godin. mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-50.

Lipid Nanoparticle and Liposome Reference Materials: Assessment of Size Homogeneity and Long-Term -70 °C and 4 °C Storage Stability.

With recent advances and anticipated proliferation of lipid nanoparticle (LNP)-delivered vaccines and therapeutics, there is a need for the availability of internationally recognized reference materials of LNP systems. Accordingly, we developed six LNP and liposome (anionic, neutral, and cationic each) candidate reference material formulations and thoroughly characterized by dynamic light scattering their particle hydrodynamic size (Z-avr) and polydispersity. We also evaluated the particle size homogeneity and long-term -70 °C and 4 °C storage stability using multiple large sets of randomly selected vials for each formulation. The formulations stored at -70 °C remained stable and homogeneous for a minimum of 9 months. The Z-avr relative combined uncertainty and the long-term variability were both <1.3% for liposome formulations and anionic LNPs, (3.9% and 1.7%) for neutral LNPs, and (6.7% and 4.4%) for cationic LNPs. An inadvertent few-hour-long storage temperature increase to -35 °C due to a freezer malfunction resulted in a small change of the size and size distribution of anionic liposomes and LNPs but, unexpectedly, a larger size increase of the neutral and cationic liposomes (≤5%) and LNPs (≤25%). The mean Z-avr values of the LNPs stored at 4 °C appeared to slowly increase with t1/3, where t is the storage time, and the Z-avr between-vial heterogeneity and mean polydispersity index values appeared to decrease; no change was observed for liposomes. The size and size distribution evolution of LNPs stored at 4 °C was attributed to an incomplete equilibration of the formulations following the addition of sucrose prior to the initial freezing. Such a process of size increase and size distribution narrowing has not been previously discussed nor observed in the context of LNPs.

Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.

Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.

Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery.

A new methodology termed selective organ targeting (SORT) was recently developed that enables controllable delivery of nucleic acids to target tissues. SORT lipid nanoparticles (LNPs) involve the inclusion of SORT molecules that accurately tune delivery to the liver, lungs and spleen of mice after intravenous administration. Nanoparticles can be engineered to target specific cells and organs in the body by passive, active and endogenous targeting mechanisms that require distinct design criteria. SORT LNPs are modular and can be prepared using scalable, synthetic chemistry and established engineering formulation methods. This protocol provides detailed procedures, including the synthesis of a representative ionizable cationic lipid, preparation of multiple classes of SORT LNPs by pipette, vortex and microfluidic mixing methods, physical characterization, and in vitro/in vivo mRNA delivery evaluation. Depending on the scale of the experiments, the synthesis of the ionizable lipid requires 4-6 d; LNPs can be formulated within several hours; LNP characterization can be completed in 2-4 h; and in vitro/in vivo evaluation studies require 1-14 d, depending on the design and application. Our strategy offers a versatile and practical method for rationally designing nanoparticles that accurately target specific organs. The SORT LNPs generated as described in this protocol can therefore be applied to multiple classes of LNP systems for therapeutic nucleic acid delivery and facilitate the development of protein replacement and genetic medicines in target tissues. This protocol does not require specific expertise, is modular to various lipids within defined physicochemical classes, and should be accomplishable by researchers from various backgrounds.

Novel pH-Responsive Cubosome and Hexosome Lipid Nanocarriers of SN-38 Are Prospective for Cancer Therapy.

Highlights1) What are the main findings? Lipid nanoparticles containing novel synthetic aminolipids were formulated. Their internal nanostructures are sensitive to pH, being an inverse hexagonal phase at pH 7.4 and a bicontinuous cubic phase at pH 4.0–pH 7.0.Poorly soluble and highly potent anticancer drug SN-38 was encapsulated successfully in the nanoparticles with a ~100-fold increase in solubility.SN-38 was released faster at a tumour-relevant acidic pH compared to neutral pH. 2) What is the implication of the main finding? Aminolipids can be incorporated into self assembled lipid nanoparticles to achieve desirable pH responsiveness.The SN-38-loaded pH-sensitive lipid nanoparticles represent a promising candidate for delivering the potent drug SN-38 to tumour sites.The pH-sensitive lipid nanoparticles in this study can be a viable alternative to the currently used irinotecan. pH-responsive nanoparticles enable the selective delivery of a chemotherapeutic agent to tumours while reducing adverse effects. Herein we synthesised four novel aminolipids and developed pH-responsive nanostructured lipid nanoparticles (LNP), which exhibited a slow-releasing hexagonal structure (H2) at physiological pH and quick release bicontinuous cubic phase (Q2) at the acidic tumour pH. The nanoparticles were used to encapsulate and control the release of the chemotherapeutic agent SN-38. High-throughput formulation techniques were employed to fabricate LNP by mixing various amounts of aminolipid with monoolein (MO). The effect of aminolipids on MO self-assembled structures was studied using small-angle X-ray scattering (SAXS) at various pH values. Out of the four studied aminolipid-MO LNP systems, the nanoparticles containing N-(Pyridin-4-ylmethyl) oleamide (OAPy-4) or N-(2(piperidin-1yl)ethyl) oleamide (OAPi-1) exhibited a pH-induced H2 to Q2 phase transition in a tumour-relevant pH range (pH 5.5–7.0). SN-38 is 1000 times more efficacious than the commercially available prodrug irinotecan. However, low solubility in water and instability at physiological pH makes it unsuitable for clinical use. SN-38 was loaded into LNP containing MO and aminolipid OAPy-4. The drug loading and entrapment efficiency were determined, and the results indicated that the aqueous solubility of SN-38 loaded in LNP dispersions was ~100 times higher compared to the solubility of the pure drug in aqueous solution. Furthermore, we demonstrated that the in vitro SN-38 release rate from LNPs was faster at lower pH (pH 5) than at neutral pH. Therefore, pH-responsive LNPs developed in this study can potentially be employed in delivering and controlling the release of the potent drug SN-38 to tumour sites.

Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo.

Chimeric receptor T cells (CAR-T) can effectively cure leukemia; however, there are two limitations: a complicated preparation process ex vivo and cytokine release syndrome (CRS). In this study, we constructed a lipid nanoparticle system modified by CD3 antibody on the surface, loading with the plasmid containing the combination gene of interleukin 6 short hairpin RNA (IL-6 shRNA) and CD19-CAR (AntiCD3-LNP/CAR19+shIL6). The system targeted T cells by the mediation of CD3 antibody and stably transfected T cells to transform them into CAR-T cells with IL-6 knockdown, thus killing CD19-highly expressed leukemia tumor cells and reducing CRS caused by IL-6. In vivo experiments showed that AntiCD3-LNP/CAR19+shIL6 could stably transfect T cells and produce CAR-T within 90days to kill the tumor. This significantly prolonged the survival time of leukemia model mice and demonstrated the prepared LNP exhibited the same anti-tumor effect as the traditional CAR-T cells prepared ex vivo. In this study, CAR-T cells were directly produced in vivo after intravenous injection of the lipid nanoparticles, without the need of using the current complex process ex vivo. Additionally, IL-6 expression was silenced, which would be helpful to reduce the CRS and improve the safety of CAR-T therapy. This method improves the convenience of using CAR-T technology and is helpful in further promoting the clinical application of CAR-T.

Solid Lipid Nanoparticles of Curcumin Designed for Enhanced Bioavailability and Anticancer Efficiency.

Curcumin (Cur) has anticancer properties but exhibits poor aqueous solubility, permeability, and photostability. In this study, we aimed to develop a solid lipid nanoparticle (SLN) system to enhance Cur bioavailability. The characteristics of Cur-loaded SLNs prepared by sonication were evaluated using UV–vis and Fourier transform infrared spectroscopy. The mean particle size of the stearic acid-based, lauric acid-based, and palmitic acid-based SLNs was 14.70–149.30, 502.83, and 469.53 nm, respectively. The chemical interactions between Cur and lipids involved hydrogen bonding and van der Waals forces. The formulations with high van der Waals forces might produce a neat arrangement between Cur and lipids, leading to a decrease in particle size. The Cur formulations showed enhanced cytotoxicity in HeLa, A549, and CT-26 cells compared with pure Cur. Additionally, the anticancer effect is dependent on particle size and the type of cell line. Therefore, Cur-loaded SLNs have the potential for use in anticancer therapy.

Solid lipid nanoparticles and nanostructured lipid carriers of dual functionality at emulsion interfaces. Part I: Pickering stabilisation functionality

Solid lipid nanoparticles and nanostructured lipid carriers are two types of lipid nanoparticulate systems, that have been primarily studied for their capability to function as active carriers, and only more recently utilised in Pickering emulsion stabilisation. Unveiling the factors that impact upon the lipid particle characteristics related to their Pickering functionality could enable the development of a liquid formulation with tailored microstructure and potentially the capacity to display a two-fold performance. In part I, this work investigates how certain formulation characteristics, namely solid-to-liquid lipid mass ratio and presence of unadsorbed surfactant in the aqueous carrier phase, affect the structural properties of the lipid particles, and in turn how these influence their Pickering stabilisation capacity. The effect of the formulation parameters was assessed in terms of the wettability and physicochemical properties of the lipid particles, including particle size, crystallinity and interfacial behaviour. Lipid particles fabricated with higher liquid lipid content (70% w/w) were shown to be more hydrophilic and have lower surfactant decoration at their surface compared to particles containing lower or no liquid lipid in their crystalline matrix. The emulsion stabilisation ability through a Pickering mechanism was confirmed for all types of lipid particles using polarised microscopy. Increasing liquid lipid content and removal of excess surfactant did not compromise the particle stabilisation capacity, though emulsion droplets of larger sizes were initially acquired in the latter case. The particle-stabilised emulsions maintained their physical integrity, with particles retaining close association with the emulsion interface over a storage period of 12 weeks.