PEG Lipid Research Articles

The role of leukocyte activation in suspected Non-IgE excipient-related COVID-19 vaccine reactions: An exploratory hypothesis-driven study of pathogenesis

Background: Adverse allergic reactions due to the administration of vaccines developed for the protection of coronavirus disease 2019 (COVID-19) have been reported since the initiation of the vaccination campaigns in December 15, 2020. Current analyses provided by the Centers for Disease Control and Prevention and the U.S. Food and Drug Administration in the United States have estimated the rates of anaphylactic reactions in 2.5 and 11.1 per million of messenger RNA (mRNA) 1273 and BNT162b2 vaccines administered, respectively. The mechanisms by which these mRNA vaccines induce adverse vaccine reactions have been the subject of conflicting reports. Although skin testing with excipient components found in mRNA-1273 and BNT162b2 vaccines, such as polyethylene glycol (PEG) and related vaccine lipid products, were originally recommended to identify potential predictive biomarkers of adverse allergic reactions, more recent evidence has suggested that routine skin testing with these vaccine excipients have poor predictability and do not correlate with susceptibility to vaccine injury. Objective: The goal of this proof-of-concept (POC) exploratory study was to investigate the role of leukocyte activation (LA) induced by lipid excipients found in mRNA COVID-19 vaccines in the pathogenesis of COVID-19 mRNA vaccine‐associated adverse reactions by using an LA assay developed in our laboratory. Results: An LA assay was performed on blood samples obtained from 30 study subjects who were assigned to three study groups: group 1 consisted of 10 subjects who had received an mRNA COVID-19 vaccine and developed a serious vaccine adverse reaction; group 2 consisted of 10 subjects who had received a COVID-19 vaccine and developed a mild adverse reaction; and group 3 consisted of 10 subjects who had not received a COVID-19 vaccine and were asymptomatic. Five excipients were tested in each of the 10 subjects; hence, a potential of 50 reactions could be expressed in each of the three groups. In the subjects in group 1 who had shown clinically severe vaccine effects, 8 of 50 (16%) had severe LA index (LAI) responses (>144.83), 12 of 50 (24%) had moderate LAI responses (87.62 ‐144.82), and 30 of 50 (60%) had no reaction (0 ‐ 87.61). In the subjects in group 2 who had shown clinically mild vaccine effects, 4 of 50 (8%) had severe LAI responses (>144.83), 9 of 50 (18%) had moderate LAI responses (87.62 ‐144.82), and 37 of 50 (74%) had no reaction. In the subjects in group 3 who had not received the vaccine and, therefore, had no clinical vaccine effects, 2 of 50 (4%) had severe LAI responses (>144.83), 10 of 50 (20%) had moderate LAI responses (87.62 ‐144.82), and 38 of 50 (76%) had no reaction LA index (LAI) responses. Conclusion: The results of this exploratory POC study suggest that the measurement of LA induced by PEG and other vaccine-related lipid excipients found in mRNA COVID-19 vaccines may provide a novel and useful predictive biomarker associated with adverse non‐immunoglobulin E (IgE) related allergic reactions to these vaccines. The study results also underscore growing concerns related to these non-IgE hypersensitivity reactions and their potential for pathogenesis of adverse vaccine reactions. This is particularly noteworthy because, with the continuing emergence of novel and evolving variants of severe acute respiratory syndrome corona-virus 2 mutants, yearly immunization with mRNA vaccines will most likely be recommended. Although the study was not sufficiently powered to draw definitive conclusions with regard to associations between vaccine-associated COVID-19 reactions and LA, the trends of a more severe set of clinical reactions seen associated with LAI reactivity scores, particularly with ALC-0159 (2-[{polyethylene glycol} 2000]-N,N-ditetradecylacetamide), suggest a potential benefit worthy of exploration in future randomized controlled trials.

Microfluidic post-insertion of polyethylene glycol lipids and KK or RGD high functionality and quality lipids in milk-derived extracellular vesicles

To achieve the desired delivery effect, extracellular vesicles (EVs) must bypass rapid clearance from circulation and exhibit affinity for target cells; however, it is difficult to simultaneously incorporate two materials into EVs. Post-insertion is a general modification method that can be performed by simply mixing different solutions. Previously, we have developed a microfluidic post-insertion method that supported fast and upscaled modification of EVs using KK-modified high-functionality and -quality (HFQ) lipids. Here, we used microfluidic post-insertion to achieve simultaneous incorporation of polyethylene glycol (PEG) lipids and KK or RGD-modified HFQ lipids into milk-derived EVs to avoid uptake from the reticuloendothelial system and increase the uptake into target cells. PEG lipid and HFQ lipids were formulated to produce micelles and subsequently mixed with EV solution using a microfluidic device. Compared to bulk mixing, microfluidic post-insertion showed higher cellular association. Altered cellular association capacities and endocytic pathways indicated simultaneous incorporation. The cellular association of modified EVs can be adjusted by altering the ratio of (EK)4-KK in micelles with slight changes in physicochemical properties. Furthermore, microfluidic post-insertion is also suitable for (SG)5-RGD, which is insoluble in phosphate-buffered saline (PBS). Our results may be valuable for the development and manufacture of functional EVs as drug delivery systems for clinical applications.

Recent Advances in Lipid Nanoparticles and Their Safety Concerns for mRNA Delivery.

The advent of lipid nanoparticles (LNPs) as a delivery platform for mRNA therapeutics has revolutionized the biomedical field, particularly in treating infectious diseases, cancer, genetic disorders, and metabolic diseases. Recent Advances in Therapeutic LNPs: LNPs, composed of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids, facilitate efficient cellular uptake and cytosolic release of mRNA while mitigating degradation by nucleases. However, as synthetic entities, LNPs face challenges that alter their therapeutic efficacy and safety concerns. Toxicity/Reactogenicity/Immunogenicity: This review provides a comprehensive overview of the latest advancements in LNP research, focusing on preclinical safety assessments encompassing toxicity, reactogenicity, and immunogenicity. Summary and Outlook: Additionally, it outlines potential strategies for addressing these challenges and offers insights into future research directions for enhancing the application of LNPs in mRNA therapeutics.

Insights into the formulation of lipid nanoparticles for the optimization of mRNA therapeutics.

mRNA-based therapeutics increasingly demonstrate significant potential in treating various diseases, including infectious diseases, cancers, and genetic disorders. Effective delivery systems are crucial for advancing mRNA therapeutics. Lipid nanoparticles (LNPs) serve as an excellent carrier, widely validated for their safety and tolerability in commercially available mRNA vaccines. Standard LNPs typically consist of four components: ionizable lipids (ILs), helper lipids, cholesterol, and polyethylene glycol-lipids (PEG-lipids), with the structural design of ILs gradually becoming a focal point of research interest. The chemical structures and formulations of the other components also significantly affect the delivery efficiency, targeting specificity, and stability of LNPs. The complex formulations of LNPs may hinder the clinical transformation of mRNA therapeutics and have raised widespread concerns about their safety. This review aims to summarize the progress of LNPs-based mRNA therapeutics in clinical trials, focusing on adverse effects that occurred during these trials. It also discusses representative innovations in LNP components, highlighting challenges and potential ways in this research field. We firmly believe this review will promote further improvements and designs of LNP compositions to optimize mRNA therapeutics. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Lipid-Based Structures.

Acid-degradable lipid nanoparticles enhance the delivery of mRNA.

Lipid nanoparticle (LNP)-mRNA complexes are transforming medicine. However, the medical applications of LNPs are limited by their low endosomal disruption rates, high toxicity and long tissue persistence times. LNPs that rapidly hydrolyse in endosomes (RD-LNPs) could solve the problems limiting LNP-based therapeutics and dramatically expand their applications but have been challenging to synthesize. Here we present an acid-degradable linker termed 'azido-acetal' that hydrolyses in endosomes within minutes and enables the production of RD-LNPs. Acid-degradable lipids composed of polyethylene glycol lipids, anionic lipids and cationic lipids were synthesized with the azido-acetal linker and used to generate RD-LNPs, which significantly improved the performance of LNP-mRNA complexes in vitro and in vivo. Collectively, RD-LNPs delivered mRNA more efficiently to the liver, lung, spleen and brains of mice and to haematopoietic stem and progenitor cells in vitro than conventional LNPs. These experiments demonstrate that engineering LNP hydrolysis rates in vivo has great potential for expanding the medical applications of LNPs.

Trends in Serum Shifts: Unveiling Hyperglycaemia Links PostCOVID-19 Vaccination.

Clinical endocrinology has observed emerging endocrine complications following COVID-19 vaccination, amidst successful reductions in COVID-19 hospitalizations and deaths. The Pfizer-BioNTech and Moderna mRNA vaccines have demonstrated efficacy. Reports indicate a potential association between SARS-CoV-2 vaccination and diabetes, exploring interactions with ACE-2 receptors and molecular mimicry. Additionally, altered liver and kidney function tests post-vaccination prompt investigation into their role in predicting type 2 diabetes. This study aims to explore these biochemical abnormalities in a case-control, single-centre prospective study. This prospective study aimed to evaluate a total of five hundred healthy donors, out of which 203 qualified for final analysis. Participants were selected based on their vaccination status with a COVID-19 vaccine and prior exposure to the SARS-CoV-2 virus. Donors without prior SARS-CoV-2 infection were excluded from the study. Included participants were adults who had received three doses of the COVID-19 vaccine. A total of 203 individuals were included in the study, comprising 104 with type 2 diabetes mellitus (T2DM) and 99 without. Demographic characteristics including age, sex, nationality, Rh factors, ABO blood groups, liver function tests (LFT), kidney function tests (KFT), lactate dehydrogenase (LDH), and mineral ion levels were analysed. Among the participants, the distribution based on HbA1c levels showed 47.8% with HbA1c <7% classified as normal, 38.48% with HbA1c 8-10% classified as high, and 16.64% with HbA1c <10% classified as uncontrolled diabetes. Significant findings included a decrease in magnesium levels to 0.77±0.82 mmol/L (p<0.04*), an increase in LDH levels to 420.70±356.26 µL (p<0.01*), and elevated levels of alkaline phosphatase (143.22 ± 142.62 µL, p<0.001), gamma-glutamyl transferase (GGT) (55.70 ± 32.20 µL, p<0.001), and serum bilirubin (9.23 ± 4.87 µmol/L, p<0.001). Creatinine levels were significantly lower at 116.75 ± 101.94 µmol/L (p#60;0.001), while uric acid levels were significantly elevated at 305.92 ± 145.04 µmol/L (p<0.001) in individuals with uncontrolled HbA1c <10%. A majority of these individuals belonged to the O+ blood group. This study underscores significant shifts in serum biomarkers and their complex interplay with mRNA-based SARS-CoV-2 vaccination and diabetes, particularly in uncontrolled cases. The findings suggest potential autoimmune reactions triggered by the self-adjuvant properties of mRNA and polyethylene glycol lipid conjugates. Variations observed among different blood groups may correspond to racial disparities influencing molecular mimicry mechanisms. Despite these insights, the underlying pathophysiological mechanisms remain unclear, highlighting the critical need for further research to validate and expand upon these findings.

Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms “siHBV”) with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.

PEG-lipid-modified agonistic antibody against tumor necrosis factor receptor family elicits superior apoptosis-inducing activity against human carcinoma

We have recently developed a novel PEG-lipid-modified antibody to enhance the induction of apoptosis by the agonistic antibody. The chemically modified TRA-8 antibody [anti-death receptor 5 (DR5) antibody] with PEG-lipid (DSPE-PEG) demonstrated significant cytotoxic activity in vitro without the need for crosslinking with a secondary antibody, which is typically required. We investigated the correlation between the PEG-lipid structure and the cytotoxic activity of the modified antibodies by varying the PEG length or lipid structure. However, when the DSPE-PEG-modified TRA-8 antibody was incubated with plasma, it lost its cytotoxic activity, likely due to degradation in the DSPE-PEG component. Nevertheless, by designing new PEG-lipids that are intended to be resistant to enzymatic degradation, we were able to prevent this degradation and restore the cytotoxic activity of the modified antibody. These findings provide valuable insights for the design of PEG-lipid-modified antibodies and suggest their potential effectiveness in enhancing cancer therapy.

Influence of structural variations in polysarcosine functionalized lipids on lipid nanoparticle‐mediated mRNA delivery

AbstractLipid nanoparticles (LNPs) have been demonstrated to be potent and well‐tolerated vehicles for delivering mRNA in vaccination and therapeutics. However, the presence of anti‐poly(ethylene glycol) (PEG) antibodies in the body resulted in the problems of hypersensitivity reaction, accelerated blood clearance and high systemic reactogenicity after repeated dosing of PEG lipid‐contained LNPs, thus limiting the utility for in vivo messenger RNA (mRNA) delivery. Here, we synthesized well‐defined polysarcosine functionalized lipids (pSar‐lipids) with various hydrophobic tail lengths and molecular weights by the accelerated ring‐opening polymerization of sarcosine N‐carboxyanhydride (NCA). The obtained pSar‐lipids were utilized as PEG lipid alternatives to explore structure–activity relationships of pSar‐lipid‐based LNPs. The results demonstrated that pSar‐lipid‐based LNPs by intravenous administration represented higher mRNA delivery efficiency in the liver and spleen with the increased hydrophobic tail length of pSar‐lipids. Importantly, more significant preference for mRNA delivery into the liver was identified by increasing the molecular weight of pSar segments. As a result, this work elucidated the effect of structural variations in pSar‐lipids on LNP‐mediated in vivo mRNA delivery, providing clues to optimize pSar‐lipids as potential alternatives to PEG lipids for developing next‐generation of LNP delivery systems.

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core–shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150–200 nm sized particles with a small positive surface charge (+3–5 mV) and high pDNA encapsulation efficiencies (EE) of 75–90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80–100%), whereas polymer E100 demonstrated dose-dependent behaviour (20–90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26–42% at 48–72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography

The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thiophilic adsorption chromatography. The purification of IgY anti-MPT64 was achieved by initial PEG lipid precipitation, then an optimized purification by varying the gradient concentration of elution buffer into five steps gradient (0-20, 20-40, 40-60, 60-80, and 80-100%v/v) for three injection column volume (CV) each and two steps gradient (0-50 and 50-100%v/v) in eight CV for each concentration. The obtained IgY was characterized by SDS-PAGE and dot-blot then determined its content levels using the Lowry method. The results showed that the five steps gradient purification was found to provide a better purity level of IgY than the two steps gradient. However, the IgY content obtained in the two steps gradient purification (2.2632± 0.011 mg/mL) was higher than the five steps gradient purification (1.35482 ± 0.023 mg/mL). Nevertheless, both purified IgY results can recognize MPT64 protein through a dot blot test. Therefore, it can be summarized that thiophilic adsorption chromatography with five steps gradient of purification was an efficient process to obtain a higher purity of IgY anti-MPT64, especially to be targeted as a diagnostic kit component for MPT64 detection.

Engineering LNPs with polysarcosine lipids for mRNA delivery

Since the approval of the lipid nanoparticles (LNP)-mRNA vaccines against the SARS-CoV-2 virus, there has been an increased interest in the delivery of mRNA through LNPs. However, current LNP formulations contain PEG lipids, which can stimulate the generation of anti-PEG antibodies. The presence of these antibodies can potentially cause adverse reactions and reduce therapeutic efficacy after administration. Given the widespread deployment of the COVID-19 vaccines, the increased exposure to PEG may necessitate the evaluation of alternative LNP formulations without PEG components. In this study, we investigated a series of polysarcosine (pSar) lipids as alternatives to the PEG lipids to determine whether pSar lipids could still provide the functionality of the PEG lipids in the ALC-0315 and SM-102 LNP systems. We found that complete replacement of the PEG lipid with a pSar lipid can increase or maintain mRNA delivery efficiency and exhibit similar safety profiles in vivo.

Long-Term Stable Liposome Modified by PEG-Lipid in Natural Seawater.

This paper describes the stabilization of liposomes using a PEGylated lipid, N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (DSPE-PEGs), and the evaluation of the survival rate in natural seawater for future environmental applications. Liposomes in natural seawater were first monitored by confocal microscopy, and the stability was compared among different lengths and the introduction ratio of DSPE-PEGs. The survival rate increased with an increase in the PEG ratio. In addition, the survival rate in different cationic solutions (Na+, K+, Mg2+, and Ca2+ solutions) was studied to estimate the effects of the DSPE-PEG introduction. We propose that these variations in liposome stability are due to the cations, specifically the interaction between the poly(ethylene glycol) (PEG) chains and divalent ions, which contribute to making it difficult for cations to access the lipid membrane. Our studies provide insights into the use of PEG lipids and may offer a promising approach to the fabrication of liposomal molecular robots using different natural environments.

PEG-Lipid-PLGA Hybrid Particles for Targeted Delivery of Anti-Inflammatory Drugs.

Hybrid nanoparticles (HNPs) were designed by combining a PLGA core with a lipid shell that incorporated PEG-Lipid conjugates with various functionalities (-RGD, -cRGD, -NH2, and -COOH) to create targeted drug delivery systems. Loaded with a neutral lipid orange dye, the HNPs were extensively characterized using various techniques and investigated for their uptake in human monocyte-derived macrophages (MDMs) using FC and CLSM. Moreover, the best-performing HNPs (i.e., HNP-COOH and HNP-RGD as well as HNP-RGD/COOH mixed) were loaded with the anti-inflammatory drug BRP-201 and prepared in two size ranges (dH ~140 nm and dH ~250 nm). The HNPs were examined further for their stability, degradation, MDM uptake, and drug delivery efficiency by studying the inhibition of 5-lipoxygenase (5-LOX) product formation, whereby HNP-COOH and HNP-RGD both exhibited superior uptake, and the HNP-COOH/RGD (2:1) displayed the highest inhibition.

Networked Cluster Formation via Trigonal Lipid Modules for Augmented Ex Vivo NK Cell Priming.

Current cytokine-based natural killer (NK) cell priming techniques have exhibited limitations such as the deactivation of biological signaling molecules and subsequent insufficient maturation of the cell population during mass cultivation processes. In this study, we developed an amphiphilic trigonal 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) lipid-polyethylene glycol (PEG) material to assemble NK cell clusters via multiple hydrophobic lipid insertions into cellular membranes. Our lipid conjugate-mediated ex vivo NK cell priming sufficiently augmented the structural modulation of clusters, facilitated diffusional signal exchanges, and finally activated NK cell population with the clusters. Without any inhibition in diffusional signal exchanges and intrinsic proliferative efficacy of NK cells, effectively prime NK cell clusters produced increased interferon-gamma, especially in the early culture periods. In conclusion, the present study demonstrates that our novel lipid conjugates could serve as a promising alternative for future NK cell mass production.

Development of Lipid Nanoparticle Formulation for the Repeated Administration of mRNA Therapeutics.

During the COVID-19 pandemic, mRNA vaccines emerged as a rapid and effective solution for global immunization. The success of COVID-19 mRNA vaccines has increased interest in the use of lipid nanoparticles (LNPs) for the invivo delivery of mRNA therapeutics. Although mRNA exhibits robust expression profiles, transient protein expression is often observed, raising uncertainty regarding the frequency of its administration. Additionally, various RNA therapeutics may necessitate repeated dosing to achieve optimal therapeutic outcomes. Nevertheless, the impact of repeated administrations of mRNA/LNP on immune responses and protein expression efficacy remains unclear. In this study, we investigated the influence of the formulation parameters, specifically ionizable lipids and polyethylene glycol (PEG) lipids, on the repeat administration of mRNA/LNP. Our findings revealed that ionizable lipids had no discernible impact on the dose-responsive efficacy of repeat administrations, whereas the lipid structure and molar ratio of PEG lipids were primary factors that affected mRNA/LNP performance. The optimization of the LNP formulation with PEG lipid confirmed the sustained dose-responsive efficacy of mRNA after repeated administrations. This study highlights the critical importance of optimizing LNP formulations for mRNA therapeutics requiring repeated administrations.

PH-dependent structural transitions in cationic ionizable lipid mesophases are critical for lipid nanoparticle function.

Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal [Formula: see text] and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid [Formula: see text] phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal [Formula: see text] transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-[Formula: see text] transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.

Effective Gene Therapy for Hemophilia a: Novel Re-Dosable Non-Viral Formulation That Provides Stable, and Durable FVIII Expression with Improved Tolerability

Current first-line treatments for Hemophilia A patients are clotting factor replacement or bi-specific antibodies. These treatments require continuous, lifelong infusions, yet many patients have breakthrough bleeds. There remains a high unmet need for safe, effective, and durable therapies. We have developed a liver-directed non-viral in vivo gene insertion approach using the piggyBac® DNA insertion system. Unlike conventional AAV-based gene therapy, our platform enables delivery of large transgenes, the ability to stably and efficiently integrate the therapeutic transgene into the genome, and the potential for re-dosing to titrate to target FVIII activity levels. A key challenge for all gene insertion systems relates to the need to safely and efficiently deliver the transgene DNA. In the current study we evaluated a novel hepatocyte-targeted non-viral platform able to co-deliver both DNA and mRNA with superior safety and specificity relative to the traditional liver-directed LNP concept. We first evaluated a two nanoparticle system using conventional 4-component liver-directed LNPs, with one lipid nanoparticle (LNP) encapsulating the mRNA for the super piggyBac (SPB) transposase (LNP-SPB), and a second LNP encapsulating a plasmid containing the hFVIII transposon DNA (LNP-hFVIII). We subsequently developed a novel co-encapsulated LNP formulation, comprising both SPB mRNA and transposon plasmid DNA (LNP-SPB-hFVIII). In juvenile WT mice we observed a 50% increase in hFVIII antigen expression compared to the dual nanoparticle approach. This was further validated in a severe hemophilia A mouse model (FVIII knock-out). Following a single dose of the co-encapsulated LNP-SPB-hFVIII to adult hemophilia A mice tolerized to human FVIII, we observed ~30% of normal hFVIII expression sustained over the duration of the 7 month study. To further support the concept of re-dosing, we treated immunocompetent adult hemophilia A mice tolerized to human FVIII with repeated administrations every 3 weeks. We observed a dose-proportionate increase in FVIII activity after each administration, reaching an average hFVIII activity of 96% of normal, following 3 repeated doses. Traditional LNPs use several lipid components that typically include an ionizable lipid, cholesterol, a polyethylene glycol (PEG) lipid, and a structural lipid, each with a unique function to effectively encapsulate and deliver the DNA and mRNA required for the piggyBac platform. Cellular transfection of these traditional LNPs has been demonstrated to occur by passive mechanisms such as micropinocytosis or Apolipoprotein E (ApoE)-mediated endocytosis by the low-density lipoprotein receptor (LDLR). However, this can also result in delivery to unwanted cell-types, particularly tissue-resident immune cells. To address this potential failure mode, we explored the addition of a N-Acetylgalactosamine (GalNac)-based targeting ligand to our co-encapsulated LNP formulation (LNP-SPB-hFVIII-G). In immunocompetent animals, targeted LNPs yielded a ~2-3-log reduction in pro-inflammatory serum cytokines (IL-6, IFNɣ) while maintaining high hFVIII expression and no elevation in transaminases (ALT, AST). In conclusion, our results demonstrate the capabilities of the piggyBac DNA insertion system and non-viral approach in providing stable FVIII transgene expression through genomic integration, along with the potential for redosing. Additionally, we have highlighted the tolerability profile of our current generation of liver-targeted non-viral delivery platform. Altogether, these data provide proof-of-principle toward developing an effective and durable therapy for Hemophilia A.

Synthesis and Characterization of Paclitaxel-Loaded PEGylated Liposomes by the Microfluidics Method.

For cancer therapy, paclitaxel (PX) possesses several limitations, including limited solubility and untargeted effects. Loading PX into nanoliposomes to enhance PX solubility and target their delivery as a drug delivery system has the potential to overcome these limitations. Over the other conventional method to prepare liposomes, a microfluidic system is used to formulate PX-loaded PEGylated liposomes. The impact of changing the flow rate ratio (FRR) between the aqueous and lipid phases on the particle size and polydispersity index (PDI) is investigated. Moreover, the effect of changing the polyethylene glycol (PEG) lipid ratio on the particle size, PDI, stability, encapsulation efficiency % (EE %), and release profile is studied. The physicochemical characteristics of the obtained formulation were analyzed by dynamic light scattering, FTIR spectroscopy, and AFM. This work aims to use microfluidic technology to produce PEGylated PX-loaded liposomes with a diameter of <200 nm, low PDI < 0.25 high homogeneity, and viable 28 day stability. The results show a significant impact of FRR and PEG lipid ratio on the empty liposomes' physicochemical characteristics. Among the prepared formulations, two formulations produce size-controlled, low PDI, and stable liposomes, which make them preferable for PX encapsulation. The average EE % was >90% for both formulations, and the variation in the PEG lipid ratio affected the EE % slightly; a high packing for PX was reported at different drug concentrations. A variation in the release profiles was notified for the different PEG lipid ratios.

A Workflow for Lipid Nanoparticle (LNP) Formulation Optimization using Designed Mixture-Process Experiments and Self-Validated Ensemble Models (SVEM).

We present a Quality by Design (QbD) styled approach for optimizing lipid nanoparticle (LNP) formulations, aiming to offer scientists an accessible workflow. The inherent restriction in these studies, where the molar ratios of ionizable, helper, and PEG lipids must add up to 100%, requires specialized design and analysis methods to accommodate this mixture constraint. Focusing on lipid and process factors that are commonly used in LNP design optimization, we provide steps that avoid many of the difficulties that traditionally arise in the design and analysis of mixture-process experiments by employing space-filling designs and utilizing the recently developed statistical framework of self-validated ensemble models (SVEM). In addition to producing candidate optimal formulations, the workflow also builds graphical summaries of the fitted statistical models that simplify the interpretation of the results. The newly identified candidate formulations are assessed with confirmation runs and optionally can be conducted in the context of a more comprehensive second-phase study.