Lipid Nanoparticle Delivery Systems Research Articles

Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics.

Several studies have utilized a lipid nanoparticle delivery system to enhance the effectiveness of mRNA therapeutics and vaccines. However, these nanoparticles are recognized as foreign materials by the body and stimulate innate immunity, which in turn impacts adaptive immunity. Therefore, it is crucial to understand the specific type of innate immune response triggered by lipid nanoparticles. This article provides an overview of the immunological response in the body, explores how lipid nanoparticles activate the innate immune system, and examines the adverse effects and immunogenicity-related development pathways associated with these nanoparticles. Finally, we highlight and explore strategies for regulating the immunogenicity of lipid nanoparticles.

Site-Specific Bioorthogonal Activation of DNAzymes for On-Demand Gene Therapy.

RNA-cleaving DNAzymes hold great promise as gene silencers, and spatiotemporal control of their activity through site-specific reactions is crucial but challenging for on-demand therapy. We herein report a novel design of a bioorthogonally inducible DNAzyme that is deactivated by site-specific installation of bioorthogonal caging groups on the designated backbone sites but restores the activity via a phosphine-triggered Staudinger reduction. We perform a systematical screening for installing the caging groups on each backbone site in the catalytic core of 10-23 DNAzyme and identify an inducible DNAzyme with very low leakage activity. This design is demonstrated to achieve bioorthogonally controlled cleavage of exogenous and endogenous mRNA in live cells. It is further extended to photoactivation and endogenous stimuli activation for spatiotemporal or targeted control of gene silencing. The bioorthogonally inducible DNAzyme is applied to a triple-negative breast cancer mouse model using a lipid nanoparticle delivery system, demonstrating high efficiency in knockdown of Lcn2 oncogenes and substantial suppression of tumor growth, thus highlighting the potential of precisely controlling the DNAzyme functions for on-demand gene therapy.

The impact of nucleoside base modification in mRNA vaccine is influenced by the chemistry of its lipid nanoparticle delivery system

The use of modified nucleosides is an important approach to mitigate the intrinsic immunostimulatory activity of exogenous mRNA and to increase its translation for mRNA therapeutic applications. However, for vaccine applications, the intrinsic immunostimulatory nature of unmodified mRNA could help induce productive immunity. Additionally, the ionizable lipid nanoparticles (LNPs) used to deliver mRNA vaccines can possess immunostimulatory properties that may influence the impact of nucleoside modification. Here we show that uridine replacement with N1-methylpseudouridine in an mRNA vaccine encoding influenza hemagglutinin had a significant impact on the induction of innate chemokines/cytokines and a positive impact on the induction of functional antibody titers in mice and macaques when MC3 or KC2 LNPs were used as delivery systems, while it impacted only minimally the titers obtained with L319 LNPs, indicating that the impact of nucleoside modification on mRNA vaccine efficacy varies with LNP composition. In line with previous observations, we noticed an inverse correlation between the induction of high innate IFN-α titers in the macaques and antigen-specific immune responses. Furthermore, and consistent with the species specificity of pathogen recognition receptors, we found that the effect of uridine replacement did not strictly translate from mice to non-human primates.

Analysis of patent development status of lipid nanoparticle delivery system for mRNA vaccines

Analysis of patent development status of lipid nanoparticle delivery system for mRNA vaccines

Self-Assembly of Tunable Intrinsically Disordered Peptide Amphiphiles.

Intrinsically disordered peptide amphiphiles (IDPAs) present a novel class of synthetic conjugates that consist of short hydrophilic polypeptides anchored to hydrocarbon chains. These hybrid polymer-lipid block constructs spontaneously self-assemble into dispersed nanoscopic aggregates or ordered mesophases in aqueous solution due to hydrophobic interactions. Yet, the possible sequence variations and their influence on the self-assembly structures are vast and have hardly been explored. Here, we measure the nanoscopic self-assembled structures of four IDPA systems that differ by their amino acid sequence. We show that permutations in the charge pattern along the sequence remarkably alter the headgroup conformation and consequently alter the pH-triggered phase transitions between spherical, cylindrical micelles and hexagonal condensed phases. We demonstrate that even a single amino acid mutation is sufficient to tune structural transitions in the condensed IDPA mesophases, while peptide conformations remain unfolded and disordered. Furthermore, alteration of the peptide sequence can render IDPAs to become susceptible to enzymatic cleavage and induce enzymatically activated phase transitions. These results hold great potential for embedding multiple functionalities into lipid nanoparticle delivery systems by incorporating IDPAs with the desired properties.

Developing the solid lipid nanoparticles of Losartan using the stearic acid and glyceryl monostearate as the carrier matrices

This research work deals with the design and development of the solid lipid nanoparticles of Losartan to improve solubility of poorly soluble drug. Losartan solid lipid nanoparticles are formed using hot homogenization technique followed by ultrasonication technique. Experiment trend is predicted using the standard calibration curve. The parameters studied are the percent drug release, entrapment efficiency, particle size of SLN and the in-vitro drug release of prepared SLN. The results obtained in this research work clearly indicated that the developed solid lipid nanoparticle delivery system for the highly lipophilic drug, Losartan using stearic acid, glyceryl monostearate as carrier matrices can be used effectively for hypertension.

Pharmacotechnological Advances for Clinical Translation of Essential Oils for the Treatment of Pain and Agitation in Severe Dementia

The demand for natural products is steadily increasing, and pharmacotechnological engineering is needed to allow rigorous investigation of their efficacy and safety in clinical conditions representing still unmet needs. Among aged patients affected by dementia, up to 80% of residents in nursing homes suffer from chronic pain and 97% from fluctuant neuropsychiatric symptoms (NPS), of which the most challenging is agitation. It is, at least in part, due to undertreated pain and treated with antipsychotics almost doubling the risk of death. In the frame of a scoping review assessing the existence of essential oils undergoing engineering pharmacotechnological processes using solid lipid nanoparticle delivery systems for clinical translation in pain and/or neuropsychiatric symptoms of dementia following the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR), here we identified that the sole essential oil engineered to overcome the criticisms of aromatherapy clinical trials in pain and dementia is the essential oil of bergamot (BEO). Therefore, we present the process leading to the actually ongoing randomized, double-blind, placebo-controlled NCT04321889 clinical trial to assess the efficacy and safety of intervention with bergamot in the management of agitation and pain in severe dementia to be followed also for the proof of concept of efficacy and safety of other essential oils.

A flexible, thermostable nanostructured lipid carrier platform for RNA vaccine delivery

Current RNA vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are limited by instability of both the RNA and the lipid nanoparticle delivery system, requiring storage at −20°C or −70°C and compromising universally accessible vaccine distribution. This study demonstrates the thermostability and adaptability of a nanostructured lipid carrier (NLC) delivery system for RNA vaccines that has the potential to address these concerns. Liquid NLC alone is stable at refrigerated temperatures for ≥1 year, enabling stockpiling and rapid deployment by point-of-care mixing with any vaccine RNA. Alternatively, NLC complexed with RNA may be readily lyophilized and stored at room temperature for ≥8 months or refrigerated temperature for ≥21 months while still retaining the ability to express protein in vivo. The thermostability of this NLC/RNA vaccine delivery platform could significantly improve distribution of current and future pandemic response vaccines, particularly in low-resource settings.

Cardiovascular Complications of SARS-CoV-2 Vaccines: An Overview.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the deadly disease known as coronavirus disease 2019 (COVID-19) that has reached pandemic proportions. Currently, there is no definitive treatment for COVID-19, although many vaccines have been developed. The World Health Organization has approved the safety and efficacy of the AstraZeneca/Oxford, Johnson and Johnson/Janssen (JnJ), Moderna, Pfizer/BioNTech, Sinopharm, and Sinovac vaccines so far. The approved formulations of AstraZeneca, JnJ, and Gam-COVID-vac (Sputnik V) contain DNA delivered within non-replicating recombinant adenovirus vector-based systems, while the Pfizer and Moderna vaccines utilize mRNA technology and lipid nanoparticle delivery systems. All of these vaccines encode production of the SARS-CoV-2 spike (S) protein, ultimately triggering immunity in the human body. COVID-19 causes several cardiovascular complications, such as arrhythmias, myocarditis, pericarditis, and venous thromboembolism. SARS-CoV-2 vaccines have been associated with rare, but sometimes fatal, cardiovascular side effects, which are the topics of this review. SARS-CoV-2 vaccines in general may cause thromboembolic events, such as cerebral vein thrombosis, and mRNA-based vaccines in particular may cause myocarditis/pericarditis, with the latter more likely to occur in younger adults after the second vaccination dose. Nevertheless, the advantages of these vaccines for ending the pandemic and/or decreasing the mortality rate outweigh any risk for the rare cardiovascular complications.

A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

From the Literature

From the Literature

Development of next generation nanomedicine-based approaches for the treatment of cancer: we've barely scratched the surface.

Interest in nanomedicines has grown rapidly over the past two decades, owing to the promising therapeutic applications they may provide, particularly for the treatment of cancer. Personalised medicine and ‘smart’ actively targeted nanoparticles represent an opportunity to deliver therapies directly to cancer cells and provide sustained drug release, in turn providing overall lower off-target toxicity and increased therapeutic efficacy. However, the successful translation of nanomedicines from encouraging pre-clinical findings to the clinic has, to date, proven arduous. In this review, we will discuss the use of nanomedicines for the treatment of cancer, with a specific focus on the use of polymeric and lipid nanoparticle delivery systems. In particular, we examine approaches exploring the surface functionalisation of nanomedicines to elicit active targeting and therapeutic effects as well as challenges and future directions for nanoparticles in cancer treatment.

Biomedicine in the COVID Age: Opportunities, Responses, and Challenges

According to one of the earliest definitions, biomedicine means “clinical medicine based on the principles of physiology and biochemistry.” Clinicians for quite some time preferred the use of the term medical research to describe what they considered the clinical findings pertaining to various issues related to clinical studies. Since the time when the basic molecules of life, deoxyribonucleic acids, were characterized and the genetic code elucidated, there has been great excitement, anticipation, and promise for the development of precision and personalized medicine. However, the progress has been considerably slow and at times disappointing. The unprecedented coronavirus disease created a worldwide panic and exposed all our weaknesses and unpreparedness. It also demonstrated a global demand for better public health infrastructure and preparedness to combat future pandemics. This unprecedented public health crisis acted as a great stimulus for putting together a concerted effort to develop vaccines. According to the experts, the time was right and within 48 hours after the information on the SARS-CoV-2 genome was posted, Moderna scientists had on paper a workable mRNA, which would code for the spike protein. The immune engineers at Moderna as well as BioNTech were able to put together a lipid nanoparticle delivery system for safe delivery of this precious cargo to the appropriate cells. Professor Cody Meissner at Tufts University School of Medicine in Boston says, “It is absolutely astonishing that this happened [COVID Vaccine development] in such a short time—to me, it is equivalent to putting a person on the Moon.” It is indeed a great achievement, and it demonstrated the power of basic science and emerging technologies. The extraordinary success of mRNA vaccines has opened new avenues for mRNA-based therapies. mRNAs, siRNAs, and non-coding miRNAs will play a very important role as novel therapeutics soon. Furthermore, this success has acted as a catalyst for ongoing work on the use of small RNAs for therapeutic purposes. Having said that, I must say that there are a great many challenges that need to be addressed.

DNAzyme-Catalyzed Cellular Oxidative Stress Amplification for Pro-protein Activation in Living Cells.

The conditional control of protein function in response to the physiological change of cells is of great interest for studying protein function in biological settings and developing protein therapeutics. We report herein that catalase (CAT) DNAzyme can potentiate the generation of reactive oxygen species (ROS) in living cells by knocking down catalase expression, which could further activate a reactive oxygen species (ROS)-responsive pro-protein, RNase A-NBC, in situ. Using an optimized lipid nanoparticle delivery system to simultaneously introduce CAT DNAzyme and RNase A-NBC into cells, we show that the pro-protein, RNase A-NBC, could be activated in a significantly enhanced manner to prohibit tumor cell growth in different types of cancer cells. We believe the methodology of regulating pro-protein activity using DNAzyme biocatalysis to differentiate intracellular environment could further be extended to other functional proteins, and even fundamental investigations in living systems to develop pro-protein therapeutics.

Preclinical Assessment of a Gene Editing Approach in a Mouse Model of MNGIE

Background: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare and progressive disease caused by mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). This results in a systemic accumulation of nucleosides and mitochondrial toxicity. AAV-based liver-directed gene therapy has shown efficacy in preclinical studies, but it has limitations such as a decline of transgene expression long-term. We propose that gene editing can circumvent some of these problems. Methods: We performed a preclinical study in the murine model of MNGIE to assess that the coordinated delivery of CRISPR/Cas9 and a TYMP cDNA caused the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci in hepatocytes. CRISPR/Cas9 was delivered either as mRNA using nanoparticles or in AAV2/8 viral vectors; the latter were also used to package the TYMP cDNA. We assessed the efficient integration of the templates into the liver cells, and monitored the nucleoside levels in plasma to detect the biochemical correction. Findings: The best results were obtained using lipid nanoparticles. The consistent and stable nucleoside reduction observed correlated with the presence of TYMP mRNA and functional enzyme in the liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with high levels of TP activity in plasma. Interpretation: Liver-directed gene editing is a feasible approach to achieve the long-term biochemical correction of the disease, with several advantages over previous methods. Funding Information: This project was made possible with financial support from the Instituto de Salud Carlos III/FEDER (grant PI15/00172) and from MINECO/FEDER (grant RTI2018-094734-B-C22). The support of the Agencia de Gestio d’Ajuts Universitaris i de Recerca (AGAUR) of the Generalitat de Catalunya, through SGR 2017 1559 grant, is also acknowledged. Declaration of Interests: S.H.Y.F. and Y.K.T. are employees of Acuitas Therapeutics, a company focused on the development of lipid nanoparticle delivery systems for nucleic-acid-based drugs. RM was the recipient of personal fees associated with advisory tasks and of financial support from Modis Therapeutics for research outside the submitted work. RM also holds a patent “Deoxynucleoside therapy for diseases caused by unbalanced nucleotide pools including mitochondrial DNA depletion syndromes” (PCT/US16/038110), with royalties paid to Modis Therapeutics. INCOMPLETE Ethics Approval Statement: All animal procedures were performed in accordance with European recommendations and were approved by our institutional ethics committee (Comite Etic d’Experimentacio Animal).

Nanomaterial Delivery Systems for mRNA Vaccines.

The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines.

Recent developments in solid lipid nanoparticle and surface-modified solid lipid nanoparticle delivery systems for oral delivery of phyto-bioactive compounds in various chronic diseases.

Solid lipid nanoparticle (SLN) delivery systems have a wide applicability in the delivery of phyto-bioactive compounds to treat various chronic diseases, including diabetes, cancer, obesity and neurodegenerative diseases. The multiple benefits of SLN delivery include improved stability, smaller particle size, leaching prevention and enhanced lymphatic uptake of the bioactive compounds through oral delivery. However, the burst release makes the SLN delivery systems inadequate for the oral delivery of various phyto-bioactive compounds that can treat such chronic diseases. Recently, the surface-modified SLN (SMSLN) was observed to overcome this limitation for oral delivery of phyto-bioactive compounds, and there is growing evidence of an enhanced uptake of curcumin delivered orally via SMSLNs in the brain. This review focuses on different SLN and SMSLN systems that are useful for oral delivery of phyto-bioactive compounds to treat various chronic diseases.

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of invivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of invivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

Controlling the gastrointestinal fate of nutraceutical and pharmaceutical-enriched lipid nanoparticles: From mixed micelles to chylomicrons

The oral bioavailability of lipophilic bioactive compounds such as many pharmaceuticals and nutraceuticals can be enhanced using triacylglycerol-based lipid nanoparticle delivery systems. These digestible lipid nanoparticles are dissembled in the gastrointestinal tract to form mixed micelles that solubilize and transport the lipophilic bioactives to the intestinal epithelium cells where they are absorbed. In these cells, the lipid digestion products and bioactive agents contained within the mixed micelles are then packaged into biological lipid protein nanoparticles (e.g., chylomicrons) that are secreted into the lymph. In this study, we examined the influence of fatty acid type (i.e., oleic acid, linoleic acid, and linolenic acid) on the properties of mixed micelles, cellular lipid droplets, and lipoprotein nanoparticles, and on the bioavailability of a highly lipophilic nutraceutical: 5-demethylnobiletin (5DN). There were distinct differences in the structural properties of lipoprotein nanoparticles formed depending on fatty acid unsaturation. Oleic acid (C18:1) was most effective in enhancing intestinal uptake of 5DN and led to the formation of the largest chylomicrons. Linoleic acid (C18:2) and linolenic acid (C18:3) also promoted intestinal uptake of 5DN and formation of chylomicrons, but they were less efficient than oleic acid. The metabolism of 5DN within the intestinal epithelium cells was greatly reduced when 5DN was incorporated into chylomicrons, presumably because they were isolated from metabolic enzymes in the cytoplasm. These results have important implications for the rational design of lipid nanoparticle-based delivery systems for lipophilic nutraceuticals and pharmaceuticals by targeting them to the lymphatic circulation.

Physicochemical characterization by AFM, FT-IR and DSC and biological assays of a promising antileishmania delivery system loaded with a natural Brazilian product

The control and treatment of Leishmaniasis, a neglected and infectious disease affecting approximately 12 million people worldwide, are challenging. Leishmania parasites multiply intracellularly within macrophages located in deep skin and in visceral tissues, and the currently employed treatments for this disease are subject to significant drawbacks, such as resistance and toxicity. Thus, the search for new Leishmaniasis treatments is compulsory, and Ocotea duckei Vattimo, a plant-derived product from the biodiverse Brazilian flora, may be a promising new treatment for this disease. In this regard, the aim of this work was to develop and characterize a delivery system based on solid lipid nanoparticles (SLN) that contain the liposoluble lignan fraction (LF) of Ocotea duckei Vattimo, which targets the Leishmania phagolysosome of infected macrophages. LF-loaded SLNs were obtained via the hot microemulsion method, and their physical and chemical properties were comprehensively assessed using PCS, AFM, SEM, FT-IR, DSC, HPLC, kinetic drug release studies, and biological assays. The size of the developed delivery system was 218.85±14.2nm, its zeta potential was −30mV and its entrapment efficiency (EE%) was high (the EEs% of YAN [yangambin] and EPI-YAN [epi-yangambin] markers were 94.21±0.40% and 94.20±0.00%, respectively). Microscopy, FT-IR and DSC assays confirmed that the delivery system was nanosized and indicated a core–shell encapsulation model, which corroborated the measured kinetics of drug release. The total in vitro release rates of YAN and EPI-YAN in buffer (with sink conditions attained) were 29.6±8.3% and 34.3±8.9%, respectively, via diffusion through the cellulose acetate membrane of the SLN over a period of 4h. After 24h, the release rates of both markers reached approximately 45%, suggesting a sustained pattern of release. Mathematical modeling indicated that both markers, YAN and EPI-YAN, followed matrix diffusion-based release kinetics (Higuchi’s model) with an estimated diffusion coefficient (D) of 1.3.10−6cm2/s. The LF-loaded SLNs were non-toxic to murine macrophages (20–80μgmL−1 range) and exerted a prominent anti-leishmanial effect (20μgmL−1). These data suggest this new and well-characterized lipid nanoparticle delivery system safely and effectively kills Leishmania and warrants further clinical investigation.