Development Of Lipid Nanoparticles Research Articles

Effectiveness and Safety of mRNA Vaccines in the Therapy of Glioblastoma.

Glioblastoma (GBM) is the most common and most malignant primary brain tumor, presenting significant treatment challenges due to its heterogeneity, invasiveness, and resistance to conventional therapies. Despite aggressive treatment protocols, the prognosis remains poor, with a median survival time of approximately 15 months. Recent advancements in mRNA vaccine technology, particularly the development of lipid nanoparticles (LNPs), have revitalized interest in mRNA-based therapies. These vaccines offer unique advantages, including rapid production, personalization based on tumor-specific mutations, and a strong induction of both humoral and cellular immune responses. mRNA vaccines have demonstrated potential in preclinical models, showing significant tumor regression and improved survival rates. Early-phase clinical trials have indicated that mRNA vaccines are safe and can induce robust immune responses in GBM patients. Combining mRNA vaccines with other immunotherapeutic approaches, such as checkpoint inhibitors, has shown synergistic effects, further enhancing their efficacy. However, challenges such as optimizing delivery systems and overcoming the immunosuppressive tumor microenvironment remain. Future research should focus on addressing these challenges and exploring combination therapies to maximize therapeutic benefits. Large-scale, randomized clinical trials are essential to validate the efficacy and safety of mRNA vaccines in GBM therapy. The potential to reshape the tumor microenvironment and establish long-term immunological memory underscores the transformative potential of mRNA vaccines in cancer immunotherapy.

Intranasal Delivery of Lipid Nanoparticles: A Ground-breaking Approach for Brain Targeting

Abstract: In the present scenario, various novel delivery systems are available for drug delivery to systemic circulation. So, to accomplish a greater therapeutic effect, the nature of the drug delivery is very important. This delivery is one of the innovative approaches where the drug is targeted to the brain through the nasal cavity. As we know, the human brain is the most crucial part of the body that controls various functions of our system. So, safely reaching the targeted site of the brain is necessary to achieve brain specificity. This delivery system helps us to tackle the problems that may arise in the other delivery system and helps the drug reach the brain without any difficulties. The major obstacles we faced during this delivery were the blood-brain barrier (BBB) and the brain-cerebrospinal fluid barrier. So, if we target the drug to the brain, then we have to overcome these challenges, and before that, we must have a clear understanding of the targeted site and the mechanism behind the drug targeting. Advancements in science and technology have helped discover many recent strategies and formulations available for intranasal delivery. The development of lipid nanoparticles is one of the primitive approaches for targeting any type of drug(hydrophilic/lipophilic) in the brain. So, the aim of this review mainly focused on the mechanism of intranasal delivery, the devices used, and some recent strategies like the development of lipid nanoparticles, surface-modified lipid nanocarriers, and noseto-brain patches. This review article also includes a few FDA-approved formulations for nose-to-brain delivery and their regulatory aspects related to clinical trials and future perspectives.

The 60-year evolution oflipid nanoparticles fornucleic acid delivery.

Delivery of genetic information to the interior of target cells in vivo has been a major challenge facing gene therapies. This barrier is now being overcome, owing in part to dramatic advances made by lipid-based systems that have led to lipid nanoparticles (LNPs) that enable delivery ofnucleic acid-based vaccines and therapeutics. Examples include the clinically approved COVID-19 LNP mRNA vaccines and Onpattro (patisiran), an LNP small interfering RNA therapeutic to treat transthyretin-induced amyloidosis (hATTR). In addition, a host of promising LNP-enabled vaccines and gene therapies are in clinical development. Here, we trace this success to two streams of research conducted over the past 60 years: the discovery of the transfection properties of lipoplexes composed of positively charged cationic lipids complexed with nucleic acid cargos and the development of lipid nanoparticles using ionizable cationic lipids. The fundamental insights gained from these two streams of research offer potential delivery solutions for most forms of gene therapies.

Evolution of the structure of lipid nanoparticles for nucleic acid delivery: From in situ studies of formulation to colloidal stability

The development of lipid nanoparticle (LNP) based therapeutics for delivery of RNA has triggered the advance of new strategies for formulation, such as high throughput microfluidics for precise mixing of components into well-defined particles. In this study, we have characterised the structure of LNPs throughout the formulation process using in situ small angle x-ray scattering in the microfluidic chip, then by sampling in the subsequent dialysis process. The final formulation was investigated with small angle x-ray (SAXS) and neutron (SANS) scattering, dynamic light scattering (DLS) and cryo-TEM. The effect on structure was investigated for LNPs with a benchmark lipid composition and containing different cargos: calf thymus DNA (DNA) and two model mRNAs, polyadenylic acid (polyA) and polyuridylic acid (polyU). The LNP structure evolved during mixing in the microfluidic channel, however was only fully developed during the dialysis. The colloidal stability of the final formulation was affected by the type of incorporated nucleic acids (NAs) and decreased with the degree of base-pairing, as polyU induced extensive particle aggregation. The main NA LNP peak in the SAXS data for the final formulation were similar, with the repeat distance increasing from polyU<polyA<DNA, following the expected extent of base-pairing.

An Ionizable Lipid Material with a Vitamin E Scaffold as an mRNA Vaccine Platform for Efficient Cytotoxic T Cell Responses.

RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.

A Multidimensional Approach to Modulating Ionizable Lipids for High‐Performing and Organ‐Selective mRNA Delivery

AbstractThe development of lipid nanoparticles (LNPs) has enabled a successful clinical application of mRNA vaccines. However, disclosure of design principles for the core component‐ionizable lipids (ILs), improving the delivery efficacy and organ targeting of LNPs, remains a formidable challenge. Herein, we report a powerful strategy to modulate ILs in one‐step chemistry using the Ugi four‐component reaction (Ugi‐4CR) under mild conditions. A large IL library of new structures was established simply and efficiently through a multidimensional approach, allowing us to identify the top‐performing ILs in delivering mRNA via the formulated LNPs. Adjusting the skeleton of ILs has transformed the organ‐specific and robust transfection in mRNA delivery from the liver to the spleen following different administration routes. Of note, a series of isomeric ILs were prepared and we found that the isomers mattered greatly in the performance of LNPs for mRNA delivery. Furthermore, owing to the bis‐amide bonds formed in the Ugi‐4CR reaction, the ILs within LNPs may form hydrogen bonding intermolecularly, facilitating the colloidal stabilization of LNPs. This work provides clues to the rapid discovery and rational design of IL candidates, assisting the application of mRNA therapeutics.

A Multidimensional Approach to Modulating Ionizable Lipids for High-Performing and Organ-Selective mRNA Delivery.

The development of lipid nanoparticles (LNPs) has enabled a successful clinical application of mRNA vaccines. However, disclosure of design principles for the core component-ionizable lipids (ILs), improving the delivery efficacy and organ targeting of LNPs, remains a formidable challenge. Herein, we report a powerful strategy to modulate ILs in one-step chemistry using the Ugi four-component reaction (Ugi-4CR) under mild conditions. A large IL library of new structures was established simply and efficiently through a multidimensional approach, allowing us to identify the top-performing ILs in delivering mRNA via the formulated LNPs. Adjusting the skeleton of ILs has transformed the organ-specific and robust transfection in mRNA delivery from the liver to the spleen following different administration routes. Of note, a series of isomeric ILs were prepared and we found that the isomers mattered greatly in the performance of LNPs for mRNA delivery. Furthermore, owing to the bis-amide bonds formed in the Ugi-4CR reaction, the ILs within LNPs may form hydrogen bonding intermolecularly, facilitating the colloidal stabilization of LNPs. This work provides clues to the rapid discovery and rational design of IL candidates, assisting the application of mRNA therapeutics.

Development of lipid nanoparticles(LNP)- RNA vaccine against Streptococcus pneumonae gene cluster facilitating the synthesis of the capsular polysaccharide in Egypt.

Development of lipid nanoparticles(LNP)- RNA vaccine against Streptococcus pneumonae gene cluster facilitating the synthesis of the capsular polysaccharide in Egypt.

Development of lipid nanoparticles with nystatin for an antifungal action

Fungal diseases currently affect about a quarter of the population worldwide. Fungi infections caused by Candida albicans have been described as a significant concern to public health. The spectrum of clinical diseases caused by this fungi species range between vulvovaginal candidiasis, oral candidiasis, candidemia and mucositis. The emergence of resistance mechanisms towards antifungal therapy greatly hampers successful management of illness and patient outcome. Nystatin, an antifungal drug, is categorized as a class IV of Biopharmaceutical Classification System, presenting low aqueous solubility and low intestinal permeability. Nowadays, the emerging platform of nanotechnology and lipid nanoparticles, notably solid lipid nanoparticles (SLN), has been subject to growing attention over recent past, owing to the promising properties of vectorization among a substantial variety of pharmaceutical drugs. Due to its hydrophobic proprieties, nystatin was encapsulated in SLN. Thus aiming to understand the relationship between the use of nanosystems and the improvement of the therapeutic effect. The aim of this work was to formulate SLN with nystatin by different methods (high speed homogenization and ultrasonication) with optimization of several parameters and formulation of 2 gels (one of them containing nanoparticles).&#x0D; Initially, 3 lipids were used: Compritol® 888 ATO, cetyl palmitate and Precirol® ATO 5 and, after the study of several parameters (size, encapsulation efficiency (EE) and polymorphic behaviour of the lipids), Precirol® ATO 5 was chosen as the lipid with the most satisfactory results. The results of the present work showed that the assay method of nystatin was linear, specific and presented repeatability. The average diameter of empty nanoparticles (NPs) and with drug (Precirol-NYS NPs) was, respectively, 306 nm and 260 nm and an EE of 67.8%. Regarding stability, SLN with drug proved to be more stable than SLN without drug. The polymer used for formulation of gels was the polymer commonly known by the trade name Carbopol® 940. The yield of 0.5% Carbopol® gel preparations and 0.5% Carbopol® gel + 10% Precirol-NYS NPs were 87.2% and 91.39%, respectively.

Development of Lipid Nanoparticles [LNP]-RNA Vaccine Against Neisseria Meningitidis CPS Gene Locus Facilitating the Synthesis of The Capsular Polysaccharide in Egypt

Development of Lipid Nanoparticles [LNP]-RNA Vaccine Against Neisseria Meningitidis CPS Gene Locus Facilitating the Synthesis of The Capsular Polysaccharide in Egypt

Microfluidic synthesis of nanomaterials for biomedical applications.

The field of nanomaterials has progressed dramatically over the past decades with important contributions to the biomedical area. The physicochemical properties of nanomaterials, such as the size and structure, can be controlled through manipulation of mass and heat transfer conditions during synthesis. In particular, microfluidic systems with rapid mixing and precise fluid control are ideal platforms for creating appropriate synthesis conditions. One notable example of microfluidics-based synthesis is the development of lipid nanoparticle (LNP)-based mRNA vaccines with accelerated clinical translation and robust efficacy during the COVID-19 pandemic. In addition to LNPs, microfluidic systems have been adopted for the controlled synthesis of a broad range of nanomaterials. In this review, we introduce the fundamental principles of microfluidic technologies including flow field- and multiple field-based methods for fabricating nanoparticles, and discuss their applications in the biomedical field. We conclude this review by outlining several major challenges and future directions in the implementation of microfluidic synthesis of nanomaterials.

SiRNA delivery to lymphatic endothelial cells via ApoE-mediated uptake by lipid nanoparticles.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.

PH-Responsive Lipid Nanoparticles Achieve Efficient mRNA Transfection in Brain Capillary Endothelial Cells.

The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.

Adoptive Cell Transfer and Vaccines in Melanoma: The Horizon Comes Into View.

Over the past four decades, cancer immunotherapy for melanoma has evolved from single-agent, type-I cytokine therapy to combination immune checkpoint inhibition. Along the way, breakthroughs in the fields of cell therapy and cancer vaccination have been made as well. The early data from adoptive cell therapy, involving the delivery of tumor infiltrating lymphocytes harvested from resected tumors, was generated at the National Cancer Institute. Subsequently, a limited number of centers across the globe have developed programs to deliver these therapies. Recently, more widespread availability of this therapy has been made possible by centralizing the growth and expansion of tumor infiltrating lymphocyte products, then distributing the products for delivery of therapy at numerous academic medical centers. Work is ongoing to optimize these treatments with additional cell types and/or modified cell products, and to determine the best ways of combining these treatments with immune checkpoint inhibition. Similarly, tumor vaccination strategies are undergoing dramatic changes, transitioning the field from peptide-based vaccines to next-generation sequencing and T-cell receptor sequencing. These changes help improve the selection of targeted antigens by finding more immunogenic options, and they help with the development of lipid nanoparticles and mRNA delivery. In short, evolution of the approaches that are revolutionizing infectious disease vaccination has been ongoing, and there are promising preliminary data in patients with melanoma.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.

Improvement of mRNA Delivery Efficiency to a T Cell Line by Modulating PEG-Lipid Content and Phospholipid Components of Lipid Nanoparticles.

(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.

Chimeric liposomes decorated with P407: an alternative biomaterial for producing stealth nano-therapeutics

The aim of the present study is the development and evaluation of the physicochemical properties of chimeric hydrogenated soya phosphatidylcholine (HSPC) and egg phosphatidylcholine (EggPC) liposomes with incorporated triblock copolymer Poloxamer P407 (P407). The physicochemical assay was held in water HPLC-grade and Foetal Bovine Serum (FBS), in order to determine whether these systems can be used as drug or antigen delivery nanosystems. Dynamic and electrophoretic light scattering (DLS/ELS) techniques were used for the measurement of the hydrodynamic diameter, the polydispersity index, and the ζ-potential of the prepared nanosystems. The incorporation of the P407 resulted in a size reduction of all systems. A decrease in the hydrodynamic diameter and polydispersity index were also found as a result of increasing the storage temperature from 4 °C to 25 °C, attributed to P407. The experiments that were carried out in FBS, showed that the addition of P407 improved systems stealth properties. Concluding, we propose P407 as a promising alternative to PEG in the development of lipid nanoparticles with optimized bio- and shelf-stability.

Development of lipid nanoparticles for delivery of siRNA to neural cells

Lipid nanoparticles (LNPs) are a clinically approved platform to deliver siRNA to the liver. We are optimizing this platform to deliver siRNA for the treatment of pain. siRNA against TRPV1 (siTRPV1) was chosen as a proof of concept. siTRPV1-LNPs were formulated using C12-200, an ionizable cationic lipid and helper lipids. Their particle size and surface charge was characterized using dynamic light scattering and siRNA encapsulation efficiency was determined using a Ribogreen assay. A panel of breast cancer cell lines (MCF-7, MDA-MB-231 and BT-549) that express TRPV1 were selected for in vitro screening of LNPs to determine their cytocompatibility and gene knockdown effects using an ATP assay, qPCR and western blotting. The effects of mixing speeds during LNP formulation, slow vs. fast, were compared for determining their effects on %siRNA encapsulation. The values increased from 74.4% at slow mixing to 83.8% at faster mixing speeds. siRNA-LNPs showed a particle diameter of ca. 251 nm compared to 434 nm for empty LNPs. A lower polydispersity index of 0.15 was observed for siRNA-LNPs compared to 0.41 for empty LNPs, suggesting narrower particle size distribution upon siRNA loading. Zeta potential of siRNA-LNPs (+0.15 mV) was slightly lower compared to empty LNPs (+0.41 mV). siRNA-LNPs had a negligible impact on cell viability, which was >95% in all three cell lines and primary rat dorsal root ganglion neurons four hours post-exposure. We are currently optimizing transfection parameters to determine TRPV1 knockdown in primary DRG cultures. LNPs were well-tolerated by TRPV1-expressing cell lines and primary DRG cultures, allowing their further exploration for TRPV1 silencing.

Lipid Nanoparticles as Delivery Systems for RNA-Based Vaccines.

There has been increased interest in the development of RNA-based vaccines for protection against various infectious diseases and also for cancer immunotherapies. Rapid and cost-effective manufacturing methods in addition to potent immune responses observed in preclinical and clinical studies have made mRNA-based vaccines promising alternatives to conventional vaccine technologies. However, efficient delivery of these vaccines requires that the mRNA be protected against extracellular degradation. Lipid nanoparticles (LNPs) have been extensively studied as non-viral vectors for the delivery of mRNA to target cells because of their relatively easy and scalable manufacturing processes. This review highlights key advances in the development of LNPs and reviews the application of mRNA-based vaccines formulated in LNPs for use against infectious diseases and cancer.

Development of lipid nanoparticles containing the xanthone LEM2 for topical treatment of melanoma

Melanoma is the most aggressive form of skin cancer. Thus, it is important to improve skin cancer treatment efficacy. Topical treatment of skin diseases is a worthy strategy since avoids systemic side effects commonly associated with oral and parenteral drugs administration. The 1-carbaldehyde-3,4-dimethoxyxanthone (LEM2), previously reported as a TAp73 activator, has potent antiproliferative effect on melanoma A375 cell lines. However, it presents poor aqueous solubility and poor bioavailability. Thus, the use of lipid nanoparticles to encapsulate LEM2 seems to be a promising strategy for topical delivery of this drug. In this work, unloaded nanostructured lipid carriers (NLCs) were obtained by hot high-pressure homogenization (HPH) and ultrasonication. These lipid nanoparticles were characterized, and stability tests were performed for 60 days. NLCs were stable and ultrasonication showed to be an easier method to prepare these nanoparticles when compared to hot HPH. Thus, LEM2 was encapsulated in NLCs, using the ultrasonication method, and the final loaded NLCs had a mean particle size suitable for topical application and a high encapsulation efficiency. This formulation seemed to be more cytotoxic against melanoma A375 cell line than unloaded NLCs, which indicates the potential use of NLCs as a carrier to LEM2, improving its efficacy.