UNASSIGNED: Heterologous immunization using different vaccine platforms has been demonstrated as an efficient strategy to enhance antigen-specific immune responses. In this study, we performed a head-to-head comparison of both humoral and cellular immune response induced by different prime-boost immunization regimens of mRNA vaccine and adjuvanted protein subunit vaccine against varicella-zoster virus (VZV) in middle-aged mice, aiming to get a better understanding of the influence of vaccination schedule on immune response.
UNASSIGNED: VZV glycoprotein (gE) mRNA was synthesized and encapsulated into SM-102-based lipid nanoparticles (LNPs). VZV-primed middle-aged C57BL/6 mice were then subjected to homologous and heterologous prime-boost immunization strategies using VZV gE mRNA vaccine (RNA-gE) and protein subunit vaccine (PS-gE). The antigen-specific antibodies were evaluated using enzyme-linked immunosorbent assay (ELISA) analysis. Additionally, cell-mediated immunity (CMI) was detected using ELISPOT assay and flow cytometry. Besides, in vivo safety profiles were also evaluated and compared.
UNASSIGNED: The mRNA-loaded lipid nanoparticles had a hydrodynamic diameter of approximately 130 nm and a polydispersity index of 0.156. Total IgG antibody levels exhibited no significant differences among different immunization strategies. However, mice received 2×RNA-gE or RNA-gE>PS-gE showed a lower IgG1/IgG2c ratio than those received 2×PS-gE and PS-gE> RNA-gE. The CMI response induced by 2×RNA-gE or RNA-gE>PS-gE was significantly stronger than that induced by 2×PS-gE and PS-gE> RNA-gE. The safety evaluation indicated that both mRNA vaccine and protein vaccine induced a transient body weight loss in mice. Furthermore, the protein vaccine produced a notable inflammatory response at the injection sites, while the mRNA vaccine showed no observable inflammation.
UNASSIGNED: The heterologous prime-boost strategy has demonstrated that an mRNA-primed immunization regimen can induce a better cell-mediated immune response than a protein subunit-primed regimen in middle-aged mice. These findings provide valuable insights into the design and optimization of VZV vaccines with the potentials to broaden varicella vaccination strategies in the future.

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy.

UNASSIGNED: The anticancer potential of indomethacin and other nonsteroidal anti-inflammatory drugs (NSAIDs) in vitro, in vivo, and in clinical trials is well known and widely reported in the literature, along with their side effects, which are mainly observed in the gastrointestinal tract. Here, we present a strategy for the application of the old drug indomethacin as an anticancer agent by encapsulating it in nanostructured lipid carriers (NLC). We describe the production method of IND-NLC, their physicochemical parameters, and the results of their antiproliferative activity against selected cancer cell lines, which were found to be higher compared to the activity of free indomethacin.
UNASSIGNED: IND-NLC were fabricated using the hot high-pressure homogenization method. The nanocarriers were physicochemically characterized, and their biopharmaceutical behaviour and therapeutic efficacy were evaluated in vitro.
UNASSIGNED: Lipid nanoparticles IND-NLC exhibited a particle size of 168.1 nm, a negative surface charge (-30.1 mV), low polydispersity index (PDI of 0.139), and high encapsulation efficiency (over 99%). IND-NLC were stable for over 60 days and retained integrity during storage at 4 °C and 25 °C. The potential therapeutic benefits of IND-NLC were screened using in vitro cancer models, where nanocarriers with encapsulated drug effectively inhibited the growth of breast cancer cell line MDA-MB-468 at dosage 15.7 μM.
UNASSIGNED: We successfully developed IND-NLC for delivery of indomethacin to cancer cells and confirmed their antitumoral efficacy in in vitro studies. The results suggest that indomethacin encapsulated in lipid nanoparticles possesses high anticancer potential. Moreover, the presented strategy is highly promising and may offer a new alternative for future therapeutic drug innovations.

Millions of people worldwide have hereditary genetic disorders, trauma, infectious diseases, or cancer of the eyes, and many of these eye diseases lead to irreversible blindness, which is a major public health burden. The eye is a relatively small and immune-privileged organ. The use of nucleic acid-based drugs to manipulate malfunctioning genes that target the root of ocular diseases is regarded as a therapeutic approach with great promise. However, there are still some challenges for utilizing nucleic acid therapeutics in vivo because of certain unfavorable characteristics, such as instability, biological carrier-dependent cellular uptake, short pharmacokinetic profiles in vivo (RNA), and on-target and off-target side effects (DNA). The development of lipid nanoparticles (LNPs) as gene vehicles is revolutionary progress that has contributed the clinical application of nucleic acid therapeutics. LNPs have the capability to entrap and transport various genetic materials such as small interfering RNA, mRNA, DNA, and gene editing complexes. This opens up avenues for addressing ocular diseases through the suppression of pathogenic genes, the expression of therapeutic proteins, or the correction of genetic defects. Here, we delve into the cutting-edge LNP technology for ocular gene therapy, encompassing formulation designs, preclinical development, and clinical translation.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 μL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100-200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.

Rationale: NLRP3 inflammasome is critical in the development and progression of many metabolic diseases driven by chronic inflammation, but its effect on the pathology of postmenopausal osteoporosis (PMOP) remains poorly understood. Methods: We here firstly examined the levels of NLRP3 inflammasome in PMOP patients by ELISA. Then we investigated the possible mechanisms underlying the effect of NLRP3 inflammasome on PMOP by RNA sequencing of osteoblasts treated with NLRP3 siRNA and qPCR. Lastly, we accessed the effect of decreased NLRP3 levels on ovariectomized (OVX) rats. To specifically deliver NLRP3 siRNA to osteoblasts, we constructed NLRP3 siRNA wrapping osteoblast-specific aptamer (CH6)-functionalized lipid nanoparticles (termed as CH6-LNPs-siNLRP3). Results: We found that the levels of NLRP3 inflammasome were significantly increased in patients with PMOP, and were negatively correlated with estradiol levels. NLRP3 knock-down influenced signal pathways including immune system process, interferon signal pathway. Notably, of the top ten up-regulated genes in NLRP3-reduced osteoblasts, nine genes (except Mx2) were enriched in immune system process, and five genes were related to interferon signal pathway. The in vitro results showed that CH6-LNPs-siNLRP3 was relatively uniform with a dimeter of 96.64 ± 16.83 nm and zeta potential of 38.37 ± 1.86 mV. CH6-LNPs-siNLRP3 did not show obvious cytotoxicity and selectively delivered siRNA to bone tissue. Moreover, CH6-LNPs-siNLRP3 stimulated osteoblast differentiation by activating ALP and enhancing osteoblast matrix mineralization. When administrated to OVX rats, CH6-LNPs-siNLRP3 promoted bone formation and bone mass, improved bone microarchitecture and mechanical properties by decreasing the levels of NLRP3, IL-1β and IL-18 and increasing the levels of OCN and Runx2. Conclusion: NLRP3 inflammasome may be a new biomarker for PMOP diagnosis and plays a key role in the pathology of PMOP. CH6-LNPs-siNLRP3 has potential application for the treatment of PMOP.