Opportunities for innovation: Building on the success of lipid nanoparticle vaccines

Lipid nanoparticle (LNP) formulations of messenger RNA (mRNA) have demonstrated high efficacy as vaccines against SARS-CoV-2. The success of these nanoformulations underscores the potential of LNPs as a delivery system for next-generation biological therapies. In this article, we highlight the key considerations necessary for engineering LNPs as a vaccine delivery system and explore areas for further optimisation. There remain opportunities to improve the protection of mRNA, optimise cytosolic delivery, target specific cells, minimise adverse side-effects and control the release of RNA from the particle. The modular nature of LNP formulations and the flexibility of mRNA as a payload provide many pathways to implement these strategies. Innovation in LNP vaccines is likely to accelerate with increased enthusiasm following recent successes; however, any advances will have implications for a broad range of therapeutic applications beyond vaccination such as gene therapy.     

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles

In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of −13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.     

Real-Time pH-Dependent Self-Assembly of Ionisable Lipids from COVID-19 Vaccines and In Situ Nucleic Acid Complexation

Ionisable amino-lipid is a key component in lipid nanoparticles (LNPs), which plays a crucial role in the encapsulation of RNA molecules, allowing efficient cellular uptake and then releasing RNA from acidic endosomes. Herein, we present direct evidence for the remarkable structural transitions, with decreasing membrane curvature, including from inverse micellar, to inverse hexagonal, to two distinct inverse bicontinuous cubic, and finally to a lamellar phase for the two mainstream COVID-19 vaccine ionisable ALC-0315 and SM-102 lipids, occurring upon gradual acidification as encountered in endosomes. The millisecond kinetic growth of the inverse cubic and hexagonal structures and the evolution of the ordered structural formation upon ionisable lipid-RNA/DNA complexation are quantitatively revealed by in situ synchrotron radiation time-resolved small angle X-ray scattering coupled with rapid flow mixing. We found that the final self-assembled structural identity, and the formation kinetics, were controlled by the ionisable lipid molecular structure, acidic bulk environment, lipid compositions, and nucleic acid molecular structure/size. The implicated link between the inverse membrane curvature of LNP and LNP endosomal escape helps future optimisation of ionisable lipids and LNP engineering for RNA and gene delivery.     

Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP‐based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability‐indicating making it suitable for both process and formulation development.     

Mechanism Study on Nanoparticle Negative Surface Charge Modification by Ascorbyl Palmitate and Its Improvement of Tumor Targeting Ability

Surface charge polarity and density influence the immune clearance and cellular uptake of intravenously administered lipid nanoparticles (LNPs), thus determining the efficiency of their delivery to the target. Here, we modified the surface charge with ascorbyl palmitate (AsP) used as a negatively charged lipid. AsP-PC-LNPs were prepared by dispersion and ultrasonication of AsP and phosphatidylcholine (PC) composite films at various ratios. AsP inserted into the PC film with its polar head outward. The pK_a for AsP was 4.34, and its ion form conferred the LNPs with negative surface charge. Zeta potentials were correlated with the amount and distribution of AsP on the LNPs surface. DSC, Raman and FTIR spectra, and molecular dynamics simulations disclosed that AsP distributed homogeneously in PC at 1–8% (w/w), and there were strong hydrogen bonds between the polar heads of AsP and PC (PO²⁻), which favored LNPs’ stability. But at AsP:PC > 8% (w/w), the excessive AsP changed the interaction modes between AsP and PC. The AsP–PC composite films became inhomogeneous, and their phase transition behaviors and Raman and FTIR spectra were altered. Our results clarified the mechanism of surface charge modification by AsP and provided a rational use of AsP as a charged lipid to modify LNP surface properties in targeted drug delivery systems. Furthermore, AsP–PC composites were used as phospholipid-based biological membranes to prepare paclitaxel-loaded LNPs, which had stable surface negative charge, better tumor targeting and tumor inhibitory effects.     

脂质纳米颗粒的制备及在基因治疗中的应用

基因治疗已经成为人类治疗疾病的一种重要手段.然而,为了将基因药物用于临床,需要更加复杂的递送系统.脂质纳米颗粒(LNPs)系统是目前领先的非病毒递送系统,在治疗诊断学方面取得了许多令人鼓舞的进展,其具有实现基因药物临床治疗应用的潜力.由于LNPs纳米尺寸的优势及类脂化合物的生物相容性和生物降解性,LNPs能够克服阻碍基...     

LDL-mimetic lipid nanoparticles prepared by surface KAT ligation for in vivo MRI of atherosclerosis

Low-density lipoprotein (LDL)-mimetic lipid nanoparticles (LNPs), decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachment of apolipoprotein-mimetic peptide (P), Gd(iii)-chelate (Gd), and sulforhodamine B (R) moieties on the LNP surface. The functionalized LNPs were prepared using the amide-forming potassium acyltrifluoroborate (KAT) ligation reaction. The KAT groups on the surface of LNPs were allowed to react with the corresponding hydroxylamine (HA) derivatives of P and Gd to provide bi-functionalized LNPs (PGd-LNP). The reaction proceeded with excellent yields, as observed by ICP-MS (for B and Gd amounts) and MALDI-TOF-MS data, and did not alter the morphology of the LNPs (mean diameter: ca. 50 nm), as shown by DLS and cryoTEM analyses. With the help of the efficient KAT ligation, a high payload of Gd(iii)-chelate on the PGd-LNP surface (ca. 2800 Gd atoms per LNP) was successfully achieved and provided a high r₁ relaxivity (r₁ = 22.0 s⁻¹ mM⁻¹ at 1.4 T/60 MHz and 25 °C; r₁ = 8.2 s⁻¹ mM⁻¹ at 9.4 T/400 MHz and 37 °C). This bi-functionalized PGd-LNP was administered to three atherosclerotic apoE^−/− mice to reveal the clear enhancement of atherosclerotic plaques in the brachiocephalic artery (BA) by MRI, in good agreement with the high accumulation of Gd in the aortic arch as shown by ICP-MS. The parallel in vivo MRI and ex vivo studies of whole mouse cryo-imaging were performed using triply functionalized LNPs with P, Gd, and R (PGdR-LNP). The clear presence of atherosclerotic plaques in BA was observed by ex vivo bright field cryo-imaging, and they were also observed by high emission fluorescent imaging. These directly corresponded to the enhanced tissue in the in vivo MRI of the identical mouse.

LDL-mimetic lipid nanoparticles, decorated with MRI contrast agents and fluorescent dyes, were prepared by the covalent attachments of an apoB100-mimetic peptide, Gd(iii)-chelate, and rhodamine to enhance atherosclerosis in the in vivo imaging.     

Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation

The discovery of potent new materials for in vivo delivery of nucleic acids depends upon successful formulation of the active molecules into a dosage form suitable for the physiological environment. Because of the inefficiencies of current formulation methods, materials are usually first evaluated for in vitro delivery efficacy as simple ionic complexes with the nucleic acids (lipoplexes). The predictive value of such assays, however, has never been systematically studied. Here, for the first time, by developing a microfluidic method that allowed the rapid preparation of high-quality siRNA-containing lipid nanoparticles (LNPs) for a large number of materials, we have shown that gene silencing assays employing lipoplexes result in a high rate of false negatives (~90%) that can largely be avoided through formulation. Seven novel materials with in vivo gene silencing potencies of >90% at a dose of 1.0 mg/kg in mice were discovered. This method will facilitate the discovery of next-generation reagents for LNP-mediated nucleic acid delivery.     

Time‐Controllable Lipophilic‐Drug Release System Designed by Loading Lipid Nanoparticles into Polysaccharide Hydrogels

A hybrid hydrogel composed of solid lipid nanoparticles (LNPs) entrapped within chemically cross‐linked carboxymethylcellulose (CMC) is developed to achieve localized and sustained release of lipophilic drugs. The analysis of LNP stability as well as the hydrogel swelling and mechanical properties confirm the successful incorporation of particles up to a concentration of 50% w/w_CMC. The initial LNP release rate can be prolonged by increasing the particle diameter from 50 to 120 nm, while the amount of long‐term release can be adjusted by tailoring the particle surface charge or the cross‐linking density of the polymer. After 30 d, 58% of 50 nm diameter negatively charged LNPs escape from the matrix while only 17% of positively charged nanoparticles are released from materials with intermediate cross‐linking density. A mathematical diffusion model based on Fick's second law is efficient to predict the diffusion of the particles from the hydrogels.     

Visible Light Conjugation with Triazolinediones as a Route to Degradable Poly(ethylene glycol)–Lipids for mRNA Lipid Nanoparticle Formulation

Polyethylene glycol (PEG) is considered as the gold standard for colloidal stabilization of nanomedicines, yet PEG is non-degradable and lacks functionality on the backbone. Herein, we introduce concomitantly PEG backbone functionality and degradability via a one-step modification with 1,2,4-triazoline-3,5-diones (TAD) under green light. The TAD-PEG conjugates are degradable in aqueous medium under physiological conditions, with the rate of hydrolysis depending on pH and temperature. Subsequently, a PEG-lipid is modified with TAD-derivatives and successfully used for messenger RNA (mRNA) lipid nanoparticle (LNP) delivery, thereby improving mRNA transfection efficiency on multiple cell cultures in vitro. In vivo, in mice, mRNA LNP formulation exhibited a similar tissue distribution as common LNPs, with a slight decrease in transfection efficiency. Our findings pave the road towards the design of degradable, backbone-functionalized PEG for applications in nanomedicine and beyond.     

Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG-lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed-phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines.     

Lignin nanoparticles as a novel carrier for efficacious delivery of toll like receptor 7/8 agonist: Physicochemical and in-vitro evaluation

Imidazoquinolinone (IMDQ) derivatives are known TLR7/8 agonists and are approved by the FDA for antiviral and skin cancer treatment. Their use as innate immune system activating molecules has been limited by poor pharmacokinetic properties and toxicities associated with systemic administration. In the present study, an IMDQ derivative, 1-(3-(aminomethyl) benzyl)-2-butyl-1H-imidazo[4,5-c] quinolin-4-amine (meta-aminomethyl BBIQ, 4), was encapsulated in biopolymer lignin nanoparticles (LNPs) to develop slow-release delivery system and to enhance its immune activating properties. A co-precipitation method was used to synthesize LNPs of alkali lignin. Characterization studies demonstrated the formation of spherical shaped nanoparticles of ∼150 nm size. The encapsulation efficiency and loading capacity for meta-aminomethyl BBIQ in LNPs was found to be 99% and 70%, respectively. In-vitro release studies showed 78% release over 24 h at pH 7.4 followed by sustained release. Kinetic modelling studies showed the release profile followed Weibull order kinetics with β value ≤ 0.75 corresponding to Fickian diffusion. Moreover, blank as well as meta-aminomethyl BBIQ-loaded LNPs were non-hemolytic and did not show significant cytotoxicity in RAW 264.7 and MDA-MB-23 cells at all tested concentrations. This study confirms the potential to use LNPs as a drug delivery system for immune modulators or vaccine adjuvants.     

Semi-elastic core-shell nanoparticles enhanced the oral bioavailability of peptide drugs

The rigidity of nanoparticles was newly reported to influence their oral delivery. Semi-elastic nanoparticles can enhance the penetration in mucus and uptake by epithelial cells. However, it is still challenging and unclear that the semi-elastic core-shell nanoparticles can enhance the oral bioavailability of peptide drugs. This study was for the first time to validate the semi-elastic core-shell poly(lactic-co-glycolic acid) (PLGA)-lipid nanoparticles (LNPs) as the carrier of the oral peptide drug. The antihypertensive peptide Val-Leu-Pro-Val-Pro (VP5) loaded LNPs (VP5-LNPs) were prepared by a modified thin-film ultrasonic dispersion method. Uptake experiment was performed in Caco-2 and HT-29 cells and monitored by high content screening (HCS) and flow cytometric (FCM). Pharmacokinetics of VP5-LNPs was carried out in Sprague-Dawley (SD) rats and analyzed by DAS 2.0. The optimal VP5-LNPs had an average particle size of 247.3 ± 3.8 nm, zeta potential of −6.57 ± 0.45 mV and excellent entrapment efficiency (EE) of 89.88% ± 1.23%. Transmission electron microscope (TEM) and Differential scanning calorimeter (DSC) further confirmed the core-shell structure. VP5-LNPs could increase the cellular uptake in vitro and have a 2.55-fold increase in AUC0-72 h, indicating a great promotion of the oral bioavailability. The semi-elastic LNPs remarkably improved the oral availability of peptide and could be a promising oral peptide delivery system for peptide drugs in the future.     

Established Liposome-Coated IMB16-4 Polymeric Nanoparticles (LNPs) for Increasing Cellular Uptake and Anti-Fibrotic Effects In Vitro

As a global health problem, liver fibrosis still does not have approved treatment. It was proved that N-(3,4,5-trichlorophenyl)-2(3-nitrobenzenesulfonamide) benzamide (IMB16-4) has anti-hepatic fibrosis activity. However, IMB16-4 displays poor water solubility and poor bioavailability. We are devoted to developing biodegraded liposome-coated polymeric nanoparticles (LNPs) as IMB16-4 delivery systems for improving aqueous solubility, cellular uptake, and anti-fibrotic effects. The physical states of IMB16-4−LNPs were analyzed using a transmission electron microscope (TEM), high-performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and differential scanning calorimeter (DSC). The results show that IMB16-4−LNPs increased the drug loading compared to liposomes and enhanced cellular uptake behavior compared with IMB16-4−NPs. In addition, IMB16-4−LNPs could repress the expression of hepatic fibrogenesis-associated proteins, indicating that IMB16-4−LNPs exhibited evident anti-fibrotic effects.     

Dissecting Functional Biological Interactions Using Modular RNA Nanoparticles

Nucleic acid nanoparticles (NANPs) are an exciting and innovative technology in the context of both basic and biomedical research. Made of DNA, RNA, or their chemical analogs, NANPs are programmed for carrying out specific functions within human cells. NANPs are at the forefront of preventing, detecting, and treating disease. Their nucleic acid composition lends them biocompatibility that provides their cargo with enhanced opportunity for coordinated delivery. Of course, the NANP system of targeting specific cells and tissues is not without its disadvantages. Accumulation of NANPs outside of the target tissue and the potential for off-target effects of NANP-mediated cargo delivery present challenges to research and medical professionals and these challenges must be effectively addressed to provide safe treatment to patients. Importantly, development of NANPs with regulated biological activities and immunorecognition becomes a promising route for developing versatile nucleic acid therapeutics. In a basic research context, NANPs can assist investigators in fine-tuning the structure-function relationship of final formulations and in this review, we explore the practical applications of NANPs in laboratory and clinical settings and discuss how we can use established nucleic acid research techniques to design effective NANPs.     

Current Status and Trends in Nucleic Acids for Cancer Therapy: A Focus on Polysaccharide-Based Nanomedicines

The efficacious delivery of therapeutic nucleic acids to cancer still remains an open issue. Through the years, several strategies are developed for the encapsulation of genetic molecules exploiting different materials, such as viral vectors, lipid nanoparticles (LNPs), and polymeric nanoparticles (NPs). Indeed, the rapid approval by regulatory authorities and the wide use of LNPs complexing the mRNA coding for the spark protein for COVID-19 vaccination paved the way for the initiation of several clinical trials exploiting lipid nanoparticles for cancer therapy. Nevertheless, polymers still represent a valuable alternative to lipid-based formulations, due to the low cost and the chemical flexibility that allows for the conjugation of targeting ligands. This review will analyze the status of the ongoing clinical trials for cancer therapy, including vaccination and immunotherapy approaches, exploiting polymeric materials. Among those nanosized carriers, sugar-based backbones are an interesting category. A cyclodextrin-based carrier (CALAA-01) is the first polymeric material to enter a clinical trial complexed with siRNA for cancer therapy, and chitosan is one of the most characterized non-viral vectors able to complex genetic material. Finally, the recent advances in the use of sugar-based polymers (oligo- and polysaccharides) for the complexation of nucleic acids in advanced preclinical stage will be discussed.     

Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next‐generation delivery materials for RNA therapeutics.     

Cyclodextrin-Containing Polymers for Gene Delivery

Cyclodextrin-containing polymers are now being explored as vehicles for delivering nucleic acids into cells. The structures of the cyclodextrin-containing polycations affect the nucleic acid delivery efficiencies and their toxicities. Of interest is the fact that the cyclodextrin-containing polymers reveal lower toxicities than polymers that lack the cyclodextrins. The cyclodextrins endow the nucleic acid delivery vehicles with the ability to be modified by compounds that form inclusion complexes with the cyclodextrins, and these modifications can be performed without disruption of the polymer-nucleic acid interactions. Thus, cyclodextrin-containing polymers provide unique properties for gene delivery.     

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.     

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

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