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Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs. [ABSTRACT FROM AUTHOR]

Abstract Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs. Graphical Abstract

mRNA vaccines have been demonstrated as a powerful alternative to traditional conventional vaccines because of their high potency, safety and efficacy, capacity for rapid clinical development, and potential for rapid, low-cost manufacturing. These vaccines have progressed from being a mere curiosity to emerging as COVID-19 pandemic vaccine front-runners. The advancements in the field of nanotechnology for developing delivery vehicles for mRNA vaccines are highly significant. In this review we have summarized each and every aspect of the mRNA vaccine. The article describes the mRNA structure, its pharmacological function of immunity induction, lipid nanoparticles (LNPs), and the upstream, downstream, and formulation process of mRNA vaccine manufacturing. Additionally, mRNA vaccines in clinical trials are also described. A deep dive into the future perspectives of mRNA vaccines, such as its freeze-drying, delivery systems, and LNPs targeting antigen-presenting cells and dendritic cells, are also summarized. [ABSTRACT FROM AUTHOR]

The messenger RNA (mRNA) vaccines for COVID-19, Pfizer-BioNTech and Moderna, were authorized in the US on an emergency basis in December of 2020. The rapid distribution of these therapeutics around the country and the world led to millions of people being vaccinated in a short time span, an action that decreased hospitalization and death but also heightened the concerns about adverse effects and drug-vaccine interactions. The COVID-19 mRNA vaccines are of particular interest as they form the vanguard of a range of other mRNA therapeutics that are currently in the development pipeline, focusing both on infectious diseases as well as oncological applications. The Vaccine Adverse Event Reporting System (VAERS) has gained additional attention during the COVID-19 pandemic, specifically regarding the rollout of mRNA therapeutics. However, for VAERS, absence of a reporting platform for drugvaccine interactions left these events poorly defined. For example, chemotherapy, anticonvulsants, and antimalarials were documented to interfere with the mRNA vaccines, but much less is known about the other drugs that could interact with these therapeutics, causing adverse events or decreased efficacy. In addition, SARS-CoV-2 exploitation of host cytochrome P450 enzymes, reported in COVID-19 critical illness, highlights viral interference with drug metabolism. For example, patients with severe psychiatric illness (SPI) in treatment with clozapine often displayed elevated drug levels, emphasizing drug-vaccine interaction. [ABSTRACT FROM AUTHOR]

Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The messenger RNA (mRNA) vaccines for COVID-19, Pfizer-BioNTech and Moderna, were authorized in the US on an emergency basis in December of 2020. The rapid distribution of these therapeutics around the country and the world led to millions of people being vaccinated in a short time span, an action that decreased hospitalization and death but also heightened the concerns about adverse effects and drug-vaccine interactions. The COVID-19 mRNA vaccines are of particular interest as they form the vanguard of a range of other mRNA therapeutics that are currently in the development pipeline, focusing both on infectious diseases as well as oncological applications. The Vaccine Adverse Event Reporting System (VAERS) has gained additional attention during the COVID-19 pandemic, specifically regarding the rollout of mRNA therapeutics. However, for VAERS, absence of a reporting platform for drug-vaccine interactions left these events poorly defined. For example, chemotherapy, anticonvulsants, and antimalarials were documented to interfere with the mRNA vaccines, but much less is known about the other drugs that could interact with these therapeutics, causing adverse events or decreased efficacy. In addition, SARS-CoV-2 exploitation of host cytochrome P450 enzymes, reported in COVID-19 critical illness, highlights viral interference with drug metabolism. For example, patients with severe psychiatric illness (SPI) in treatment with clozapine often displayed elevated drug levels, emphasizing drug-vaccine interaction.

The effects of polyethylene glycol- (PEG) modified lipids and gangliosides on the Ca 2+ induced interaction between liposomes composed of palmitoyl-oleoyl phosphatidylethanolamine (POPE) and palmitoyl-oleoyl phosphatidylserine (POPS) was investigated at physiological ionic strength. Förster resonance energy transfer (FRET) studies complemented with dynamic light scattering (DLS) and cryo-transmission electron microscopy (Cryo-EM) show that naked liposomes tend to adhere, rupture, and collapse on each other's surfaces upon addition of Ca 2+ , eventually resulting in the formation of large multilamellar aggregates and bilayer sheets. Noteworthy, the presence of gangliosides or PEGylated lipids does not prevent the adhesion-rupture process, but leads to the formation of small, long-lived bilayer fragments/disks. PEGylated lipids seem to be more effective than gangliosides at stabilizing these structures. Attractive interactions arising from ion correlation are proposed to be a driving force for the liposome-liposome adhesion and rupture processes. The results suggest that, in contrast with the conclusions drawn from previous solely FRET-based studies, direct liposome-liposome fusion is not the dominating process triggered by Ca 2+ in the systems studied., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Long-circulating liposomes are typically prepared with poly(ethylene glycol)- (PEG-) modified lipids, where the lipid portion is inserted in the lipid bilayers as an anchor and the hydrophilic PEG coats the surface to prevent liposome aggregation and rapid clearance in vivo . However, these steric protection effects are compromised upon systemic administration due to low retention of PEGylated lipids within liposome membranes upon dilution. Hence, a series of PEGylated bolaamphiphiles (PEG-bolas) were for the first time developed to increase retention in the lipid bilayer, presumably leading to enhanced integrity of the PEG protective layer upon dilution. We hypothesized that PEG-bolas with a sufficiently long hydrophobic domain and rigid central group could predominantly adopt a membrane-spanning configuration, taking full advantage of steric protection offered by PEG and enhanced retention in liposomes enabled by the bola geometry. In this paper, liposomes stabilized by PEG-bolas comprised of a biphenyl core and twelve-carbon alkyl chain not only exhibited similar storage and biological stability compared to conventional PEGylated lipid stabilized liposomes, but also significantly improved retention upon dilution. Our findings facilitate new designs of liposome-stabilizing agents and can be applied to improve the delivery efficiency of liposomal delivery vehicles in vivo . [ABSTRACT FROM AUTHOR]

Nanosized formulations of poorly water-soluble drugs show great potential due to improved bioavailability. In order to retain colloidal stability, the nanocrystals need to be stabilized. Here we explore the use of the poly(ethylene glycol) (PEG) conjugated phospholipids DSPE-PEG2000 and DSPE-PEG5000 as stabilizers of felodipine and griseofulvin nanocrystals. Nanocrystal stability and physicochemical properties were examined and the interaction between the PEGylated lipids and the nanocrystal surface as well as a macroscopic model surface was investigated. Using quartz crystal microbalance with dissipation monitoring both mass adsorption and the thickness of the adsorbed layer were estimated. The results indicate that the PEGylated lipids are adsorbed as flat layers of around 1–3 nm, and that DSPE-PEG5000 forms a thicker layer compared with DSPE-PEG2000. In addition, the mass adsorption to the drug crystals and the model surface are seemingly comparable. Furthermore, both DSPE-PEG2000 and DSPE-PEG5000 rendered stable drug nanocrystals, with a somewhat higher surface binding and stability seen for DSPE-PEG2000. These results suggest DSPE-PEG2000 and DSPE-PEG5000 as efficient nanocrystal stabilizers, with DSPE-PEG2000 giving a somewhat higher surface coverage and superior colloidal stability, whereas DSPE-PEG5000 shows a more extended structure that may have advantages for prolongation of circulation time in vivo and facilitation for targeting modifications. [ABSTRACT FROM AUTHOR]

Abstract: The thermotropic phase behavior of a suite of newly developed self-forming synthetic biopolymers has been investigated by variable-temperature Fourier transform infrared (FT-IR) absorption spectroscopy. The temperature-induced infrared spectra of these artificial biopolymers (lipids) composed of 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12), 1,2-dioleoyl-rac-glycerol-3-dodecaethylene glycol (GDO-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) in the spectral range of 4000–500cm−1 have been acquired by using a thin layered FT-IR spectrometer in conjunction with a custom built temperature-controlled demountable liquid cell having a pathlength of ∼15μm. The lipids under consideration have long hydrophobic acyl chains and contain various units of hydrophilic polyethylene glycol (PEG) headgroups. In contrast to conventional phospholipids, this new kind of lipids forms liposomes or nanovesicles spontaneously upon hydration, without requiring external activation energy. We have found that the thermal stability of the PEGylated lipids differs greatly depending upon the acyl chain-lengths as well as the nature of the associated bonds and the number of PEG headgroup units. In particular, GDM-12 (saturated 14 hydrocarbon chains with 12units of PEG headgroup) exhibits one sharp order–disorder phase transition over a temperature range increasing from 3°C to 5°C. Similarly, GDS-23 (saturated 18 hydrocarbon chains with 23units of PEG headgroup) displays comparatively broad order–disorder phase transition profiles between temperature 17°C and 22°C. In contrast, GDO-12 (monounsaturated 18 hydrocarbon chains with 12units of PEG headgroup) does not reveal any order–disorder transition phenomena demonstrating a highly disordered behavior for the entire temperature range. To confirm these observations, differential scanning calorimetry (DSC) was applied to the samples and revealed good agreement with the infrared spectroscopy results. Finally, the investigation of thermal properties of lipids is extremely critical for numerous purposes and the result obtained in this work may find application in various studies including the development of PEGylated lipid based novel drug and substances delivery vehicles. [Copyright &y& Elsevier]

Abstract: 4-amino-2-phenyl, 6(p-fluor-phenyl)-5-carbonitrile-pyrimidine (APCP) is a new derivative of pyrimidine with low solubility in water and anti-inflammatory properties. We compared the interfacial behaviors of spread films of poly(ethylene glycol)-grafted phospholipid (DSPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and APCP and a mixture of these molecules. The surface pressure–area (Π–A) isotherm showed that APCP and DSPE-PEG2000 molecules were stable at the air/water interface and could be evenly inserted into a DPPC floating monolayer. The introduction of APCP into the DPPC/(DSPE-PEG2000) binary monolayer generally causes an overall increase in surface potential. Analyses of distance variation between the grafted sites are associated with a change of mushroom to brush conformation and this behavior is observed for the DPPC/(DSPE-PEG2000) and DPPC/(DSPE-PEG2000)/APCP monolayers. Langmuir–Blodgett (LB) films of molecules of biological interest were transferred onto mica in order to investigate their interaction. AFM images do not show any regular shape or size and are randomly distributed. [ABSTRACT FROM AUTHOR]

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e.g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin- and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K16 nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-µm particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved ~2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we have utilized the advantages of biophotonics and nanotechnology in conjunction with vibrational spectroscopic techniques to understand the various properties of biological macromolecules known as lipid bilayers or liposomes and nanovesicles. Liposomes are spherical particles that can encapsulate a fraction of the solvent into their interior. Liposomes are constructed of polar lipids which are characterized by having a lipophilic and hydrophilic group on the same molecules. Liposomes are nowadays used extensively as a model for biological membranes as well as drug and gene delivery vehicles. Here, we have studied newly developed novel self-forming synthetic PEGylated lipids (trademarked as QuSomes) and nanovesicles by means of Raman vibrational spectroscopic techniques. In contrast to conventional phospholipids, these new kinds of lipids spontaneously form liposomes or lipid vesicles upon hydration, without the supply of external activation energy. In addition, scanning electron microscopy (SEM) has been used for the imaging of single lipid vesicle and fluorescence correlation spectroscopy (FCS) has been utilized to measure the size distribution of such nanoparticles in suspensions. Furthermore, we have obtained an excellent signal-to-noise ratio in the Raman spectra for single particle of ∼100nm dimension. The cross-sections of associated Raman signal for the lipid vesicles in PBS suspension have been estimated to be of the order of 10−28 cm2/(nanoparticles sr). In this work, we have found that the Raman spectra of PEGylated lipids are dominated by vibrational bands arising mainly from C–H and C–C stretching modes that have been used to identify important fingerprint regions. These spectra have been studied extensively as a function of temperature, pH value and size distribution of such nanoparticles to unravel the structural changes and conformational order and disorder phenomena. [Copyright &y& Elsevier]

In this work, we have aimed to merge the advantages of nanotechnology and biophotonics in conjunction with vibrational spectroscopic techniques in order to understand the various aspects of new kinds of synthetic bionanoparticles originating from self-forming synthetic biopolymers known as polyethylene glycol (PEG)ylated lipids. In particular, two complementary molecular spectroscopic techniques based on thin-layered Fourier transform infrared and confocal laser tweezers. Raman spectroscopy has been employed for the investigations of newly developed artificial PEGylated lipids trademarked as QuSomes. These novel types of synthetic lipids are composed of 1,2-dimyristoyl- rac-glycerol-3-dodecaethylene glycol (GDM-12), 1,2-dioleoyl- rac-glycerol-3-dodecaethylene glycol (GDO-12), and 1,2-distearoyl- rac-glycerol-3-triicosaethylene glycol (GDS-23). The lipid labeled GDM-12 has saturated 14 acyl chains whereas GDO-12 is characterized by monounsaturated 18 acyl chains, and GDS-23 is composed of saturated 18 acyl chains in their hydrophobic chain. Similarly, GDM-12 and GDO-12 contain 12 units, and GDS-23 contains 23 units of hydrophilic PEG head groups. In contrast to conventional phospholipids, this novel kind of lipid can form liposomes spontaneously upon hydration, without the input of external activation energy. In addition, fluorescence correlation spectroscopy has been utilized to measure the size distribution of such nanoparticles in suspension as well as scanning electron microscopy has been applied for the imaging purposes. Although such PEGylated lipids show a common spectral pattern, important differences in the spectra have been observed, enabling us to distinguish these different lipids on the basis of characteristic features calculated from the spectroscopic band component analysis. Finally, in this study, detailed spectroscopic results due to the vibrational band assignments and band component analysis corresponding to various functional groups for individual nanoparticles have been analyzed and discussed. [ABSTRACT FROM AUTHOR]

Abstract: Near-infrared (NIR) spectroscopy has been used to analyze a suite of synthesized PEGylated lipids (1–3) trademarked as QuSomes™. The three amphiphiles used in this study, differ in their hydrophobic chain length and contain various units of polyethylene glycol (PEG) head groups. Whilst the spectra of QuSomes™ show a common pattern, differences in the spectra are observed which enable the lipids to be distinguished. NIR absorption spectra of these new artificial lipids have been recorded in the spectral range of 4800–9000cm−1 (∼2100–1100nm) by using a new miniaturized spectrometer based on micro-optical-electro-mechanical systems (MOEMS) technology. Three NIR spectral regions are identified, (a) the high wavenumber region between 6500 and 9000cm−1 attributed to the first overtone of the hydroxyl stretching and second overtone of the C–H stretching mode; (b) the 5350–5900cm−1 region attributed to first overtone of the C–H stretching mode; and (c) the 4800–5300cm−1 region attributed to the combination O–H stretching and second overtone of the Cing mode. For each of these regions, the lipids show distinctive spectra which allow their identification and characterization. NIR spectroscopy is a less used technique which does have great potential for the study of lipids, particularly to examine the behaviour of nanovesicles (liposomes) formed from lipids in aqueous suspensions. The study of such lipids is important since they are used as membrane models and prominent candidate for substance and drug delivery systems. [Copyright &y& Elsevier]

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers. [ABSTRACT FROM AUTHOR]