In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.

From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases.

Vaccination represents the best line of defense against infectious diseases and is crucial in curtailing pandemic spread of emerging pathogens to which a population has limited immunity. In recent years, mRNA vaccines have been proposed as the new frontier in vaccination, owing to their facile and rapid development while providing a safer alternative to traditional vaccine technologies such as live or attenuated viruses. Recent breakthroughs in mRNA vaccination have been through formulation with lipid nanoparticles (LNPs), which provide both protection and enhanced delivery of mRNA vaccines in vivo. In this review, current paradigms and state-of-the-art in mRNA-LNP vaccine development are explored through first highlighting advantages posed by mRNA vaccines, establishing LNPs as a biocompatible delivery system, and finally exploring the use of mRNA-LNP vaccines in vivo against infectious disease towards translation to the clinic. Furthermore, we highlight the progress of mRNA-LNP vaccine candidates against COVID-19 currently in clinical trials, with the current status and approval timelines, before discussing their future outlook and challenges that need to be overcome towards establishing mRNA-LNPs as next-generation vaccines. STATEMENT OF SIGNIFICANCE: With the recent success of mRNA vaccines developed by Moderna and BioNTech/Pfizer against COVID-19, mRNA technology and lipid nanoparticles (LNP) have never received more attention. This manuscript timely reviews the most advanced mRNA-LNP vaccines that have just been approved for emergency use and are in clinical trials, with a focus on the remarkable development of several COVID-19 vaccines, faster than any other vaccine in history. We aim to give a comprehensive introduction of mRNA and LNP technology to the field of biomaterials science and increase accessibility to readers with a new interest in mRNA-LNP vaccines. We also highlight current limitations and future outlook of the mRNA vaccine technology that need further efforts of biomaterials scientists to address.

Lipid-Based Nanoparticles in the Clinic and Clinical Trials: From Cancer Nanomedicine to COVID-19 Vaccines.

COVID-19 vaccines have been developed with unprecedented speed which would not have been possible without decades of fundamental research on delivery nanotechnology. Lipid-based nanoparticles have played a pivotal role in the successes of COVID-19 vaccines and many other nanomedicines, such as Doxil® and Onpattro®, and have therefore been considered as the frontrunner in nanoscale drug delivery systems. In this review, we aim to highlight the progress in the development of these lipid nanoparticles for various applications, ranging from cancer nanomedicines to COVID-19 vaccines. The lipid-based nanoparticles discussed in this review are liposomes, niosomes, transfersomes, solid lipid nanoparticles, and nanostructured lipid carriers. We particularly focus on the innovations that have obtained regulatory approval or that are in clinical trials. We also discuss the physicochemical properties required for specific applications, highlight the differences in requirements for the delivery of different cargos, and introduce current challenges that need further development. This review serves as a useful guideline for designing new lipid nanoparticles for both preventative and therapeutic vaccines including immunotherapies.

Digestion of Lipid-Based Formulations Not Only Mediates Changes to Absorption of Poorly Soluble Drugs Due to Differences in Solubilization But Also Reflects Changes to Thermodynamic Activity and Permeability.

The aim of this study was to evaluate the effect of lipid digestion on the permeability and absorption of orally administered saquinavir (SQV), a biopharmaceutics classification system (BCS) class IV drug, in different lipid-based formulations. Three LBFs were prepared: a mixed short- and medium-chain lipid-based formulation (SMCF), a medium-chain lipid-based formulation (MCF), and a long-chain lipid-based formulation (LCF). SQV was loaded into these LBFs at 26.7 mg/g. To evaluate the pharmacokinetics of SQV in vivo, drug-loaded formulations were predispersed in purified water at 3% w/w and orally administered to rats. A low dose (0.8 mg/rat) was employed to limit confounding effects on drug solubilization, and consistent with this design, presolubilization of SQV in the LBFs did not increase in vivo exposure compared to a control suspension formulation. The areas under the plasma concentration-time curve were, however, significantly lower after administration of SQV as MCF and LCF compared to SMCF. To evaluate the key mechanisms underpinning absorption, each LBF containing SQV was digested, and the flux of SQV from the digests across a dialysis membrane was evaluated in in vitro permeation experiments. This study revealed that the absorption profiles were driven by the free concentration of SQV and that this varied due to differences in SQV solubilization in the digestion products generated by LBF digestion. The apparent first-order permeation rate constants of SQV (kapp,total) were estimated by dividing the flux of SQV in the dialysis membrane experiments by the concentration of total SQV on the donor side. kapp,total values strongly correlated with in vivo AUC. The data provide one of the first studies of the effect of digestion products on the free concentration of a drug in the GI fluid and oral absorption. This simple permeation model may be a useful tool for the evaluation of the impact of lipid digestion on apparent drug permeability from lipid-based formulations. These effects should be assessed alongside, and in addition to, the more well-known effects of lipids on enhancing intestinal solubilization of poorly water-soluble drugs.

Interaction with biliary and pancreatic fluids drives supersaturation and drug absorption from lipid-based formulations of low (saquinavir) and high (fenofibrate) permeability poorly soluble drugs.

Drug absorption from lipid-based formulations (LBFs) in the gastrointestinal (GI) tract is the result of a series of processes, including formulation dispersion, interaction with biliary and pancreatic secretions, drug solubilisation and supersaturation, and finally intestinal permeability. Optimal formulation design is dependent on a good understanding of the limitations to, and drivers of, absorption, but for LBFs the complexity of these processes makes data interpretation complex. The current study has re-examined a previous in vitro digestion-in situ perfusion model to increase physiological relevance and has used this model to examine drug absorption from LBFs. The composition of rat bile and jejunal fluid was also characterised to identify in vivo-relevant conditions. Digestion was initiated using rat bile/pancreatic fluid and the formulation and digestive enzymes mixed immediately prior to entry into the jejunum (allowing dilution/digestion to occur at the absorptive site). These conditions were employed to study drug absorption from LBFs of high (fenofibrate, FFB) and low (saquinavir, SQV) permeability compounds. The impact of polymeric precipitation inhibitors (PPIs) was also evaluated. For FFB, supersaturation, initiated by formulation interaction with biliary/pancreatic fluids, appeared to drive absorption and the addition of the PPIs poly(glycidyl methacrylate) (PPGAE) and hydroxypropylmethyl cellulose (HPMC), reduced drug precipitation, increased FFB supersaturation and increased absorption from a Type IV LBF of FFB. For a Type IIIB LBF however, PPIs were ineffective at increasing absorption. The impact of PPIs on the absorption of a less permeable drug, SQV, was similarly evaluated and again drug absorption appeared to be related to the extent of supersaturation, although in this case PPI were unable to promote absorption. For both FFB and SQV, drug absorption patterns obtained with the in vitro digestion-in situ perfusion mode, correlated well with in vitro supersaturation data and in vivo drug exposure data from oral bioavailability studies. The data are consistent with a mode of drug absorption where rapid dilution of LBFs with biliary and pancreatic secretions at the absorptive site in the upper small intestine drives transient supersaturation, that supersaturation is a significant driver of drug absorption for both low and high permeability drugs, and that PPIs delay drug precipitation, enhance supersaturation and promote drug absorption in a drug and formulation specific manner.

The Importance of Poly(ethylene glycol) and Lipid Structure in Targeted Gene Delivery to Lymph Nodes by Lipid Nanoparticles.

Targeted delivery of nucleic acids to lymph nodes is critical for the development of effective vaccines and immunotherapies. However, it remains challenging to achieve selective lymph node delivery. Current gene delivery systems target mainly to the liver and typically exhibit off-target transfection at various tissues. Here we report novel lipid nanoparticles (LNPs) that can deliver plasmid DNA (pDNA) to a draining lymph node, thereby significantly enhancing transfection at this target organ, and substantially reducing gene expression at the intramuscular injection site (muscle). In particular, we discovered that LNPs stabilized by 3% Tween 20, a surfactant with a branched poly(ethylene glycol) (PEG) chain linking to a short lipid tail, achieved highly specific transfection at the lymph node. This was in contrast to conventional LNPs stabilized with a linear PEG chain and two saturated lipid tails (PEG-DSPE) that predominately transfected at the injection site (muscle). Interestingly, replacing Tween 20 with Tween 80, which has a longer unsaturated lipid tail, led to a much lower transfection efficiency. Our work demonstrates the importance of PEGylation in selective organ targeting of nanoparticles, provides new insights into the structure-property relationship of LNPs, and offers a novel, simple, and practical PEGylation technology to prepare the next generation of safe and effective vaccines against viruses or tumours.

A Nonionic Polyethylene Oxide (PEO) Surfactant Model: Experimental and Molecular Dynamics Studies of Kolliphor EL.

Polyethoxylated, nonionic surfactants are important constituents of many drug formulations, including lipid-based formulations. In an effort to better understand the behavior of formulation excipients at the molecular level, we have developed molecular dynamics (MD) models for the widely used surfactant Kolliphor EL (KOL), a triricinoleate ester of ethoxylated glycerol. In this work, we have developed models based on a single, representative molecular component modeled with 2 force field variations based on the GROMOS 53A6DBW and 2016H66 force field parameters for polyethoxylate chains. To compare the computational models to experimental measurements, we investigated the phase behavior of KOL using nephelometry, dynamic light scattering, cross-polarized microscopy, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The potential for digestion of KOL was also evaluated using an in vitro digestion experiment. We found that the size and spherical morphology of the KOL colloids at low concentrations was reproduced by the MD models as well as the growing interactions between the aggregates to from rod-like structures at high concentrations. We believe that this model reproduces the phase behavior of KOL relevant to drug absorption and that it can be used in whole formulation simulations to accelerate the formulation development.

Polymeric Precipitation Inhibitors Promote Fenofibrate Supersaturation and Enhance Drug Absorption from a Type IV Lipid-Based Formulation.

The ability of lipid-based formulations (LBFs) to increase the solubilization, and prolong the supersaturation, of poorly water-soluble drugs (PWSDs) in the gastrointestinal (GI) fluids has generated significant interest in the past decade. One mechanism to enhance the utility of LBFs is to prolong supersaturation via the addition of polymers that inhibit drug precipitation (polymeric precipitation inhibitors or PPIs) to the formulation. In this work, we have evaluated the performance of a range of PPIs and have identified PPIs that are sufficiently soluble in LBF to allow the construction of single phase formulations. An in vitro model was first employed to assess drug (fenofibrate) solubilization and supersaturation on LBF dispersion and digestion. An in vitro-in situ model was subsequently employed to simultaneously evaluate the impact of PPI enhanced drug supersaturation on drug absorption in rats. The stabilizing effect of the polymers was polymer specific and most pronounced at higher drug loads. Polymers that were soluble in LBF allowed simple processing as single phase formulations, while formulations containing more hydrophilic polymers required polymer suspension in the formulation. The lipid-soluble polymers Eudragit (EU) RL100 and poly(propylene glycol) bis(2-aminopropyl ether) (PPGAE) and the water-soluble polymer hydroxypropylmethyl cellulose (HPMC) E4M were identified as the most effective PPIs in delaying fenofibrate precipitation in vitro. An in vitro model of lipid digestion was subsequently coupled directly to an in situ single pass intestinal perfusion assay to evaluate the influence of PPIs on fenofibrate absorption from LBFs in vivo. This coupled model allowed for real-time evaluation of the impact of supersaturation stabilization on absorptive drug flux and provided better discrimination between the different PPIs and formulations. In the presence of the in situ absorption sink, increased fenofibrate supersaturation resulted in increased drug exposure, and a good correlation was found between the degree of in vitro supersaturation and in vivo drug exposure. An improved in vitro-in vivo correlation was apparent when comparing the same formulation under different supersaturation conditions. These observations directly exemplify the potential utility of PPIs in promoting drug absorption from LBF, via stabilization of supersaturation, and further confirm that relatively brief periods of supersaturation may be sufficient to promote drug absorption, at least for highly permeable drugs such as fenofibrate.

Computational Models of the Intestinal Environment. 3. The Impact of Cholesterol Content and pH on Mixed Micelle Colloids.

In this study, we use molecular dynamics (MD) and experimental techniques (nephelometry and dynamic light scattering) to investigate the influence of cholesterol content and pH on the colloidal structures that form in the gastrointestinal (GI) tract upon lipid digestion. We demonstrate that the ionization state of the molecular species is a primary driver for the self-assembly of aggregates formed by model bile and therefore should be considered when performing in silico modeling of colloidal drug delivery systems. Additionally, the incorporation of physiological concentrations of cholesterol within the model systems does not affect size, number, shape, or dynamics of the aggregates to a significant degree. The MD data shows a reduction in aggregate size with increasing pH, a preference for glycodeoxycholate (GDX) to occupy the aggregate surface, and that the mixed micellar aggregates are oblate spheroids (disc-like). The results obtained assist in understanding the process by which pH and cholesterol influence self-assembly of mixed micelles within the GI tract. The MD approach provides a platform for investigation of interactions of drugs and formulation excipients with the endogenous contents of the GI tract.