Optimized lipid nanoparticles (LNPs) for organ-selective nucleic acids delivery in vivo.

Nucleic acid therapeutics offer tremendous promise for addressing a wide range of common public health conditions. However, the in vivo nucleic acids delivery faces significant biological challenges. Lipid nanoparticles (LNPs) possess several advantages, such as simple preparation, high stability, efficient cellular uptake, endosome escape capabilities, etc., making them suitable for delivery vectors. However, the extensive hepatic accumulation of LNPs poses a challenge for successful development of LNPs-based nucleic acid therapeutics for extrahepatic diseases. To overcome this hurdle, researchers have been focusing on modifying the surface properties of LNPs to achieve precise delivery. The review aims to provide current insights into strategies for LNPs-based organ-selective nucleic acid delivery. In addition, it delves into the general design principles, targeting mechanisms, and clinical development of organ-selective LNPs. In conclusion, this review provides a comprehensive overview to provide guidance and valuable insights for further research and development of organ-selective nucleic acid delivery systems.

Spatiotemporal-Controlled NIR-II Immune Agonist Sensitizes Cancer Immunotherapy.

The integration of nanomedicine and immunotherapy has presented a promising opportunity for the treatment of cancer and diverse diseases. However, achieving spatiotemporal controllable immunotherapy with excellent efficacy and safety performances remains a significant challenge. This study develops a biodegradable near-infrared II (NIR-II) photothermal response polymer nanoparticle (PTEQ) system. This platform exhibits intrinsic immunostimulatory properties while concurrently delivering siRNA for Programmed Death-Ligand 1 (siPD-L1), leveraging enhanced immune responses and immune checkpoint blockade for safe and effective cancer therapy. In the CT26 tumor-bearing mouse model, PTEQ, as an immune stimulant, significantly boosts the infiltration of CD4+ and CD8+ T cells within the tumor microenvironment (TME). The PTEQ/siPD-L1+laser group not only initiates NIR-II photothermal therapy but also promotes the activation and infiltration of T cells, M1 macrophage polarization, and maturation of dendritic cells in the TME, resulting in the complete elimination of tumors in 7/10 cases, achieving a 100% survival rate. In another in vivo vaccine experiment, all tumors on the right side are completely eliminated in the PTEQ/siPD-L1+laser group, reaching a 100% tumor eradication rate. These findings underscore the potential of this strategy to overcome the current immunotherapeutic limitations and achieve immune therapy normalization.

Ultra-efficient delivery of CRISPR/Cas9 using ionic liquid conjugated polymers for genome editing-based tumor therapy.

Emerging CRISPR-Cas9 systems can rebuild DNA sequences in the genome in a spatiotemporal manner, offering a magic tool for biological research, drug discovery, and gene therapy. However, low delivery efficiency remains a major roadblock hampering the wide application of CRISPR-Cas9 gene editing talent. Herein, ionic liquid-conjugated polymers (IL-CPs) are explored as efficient platforms for CRISPR-Cas9 plasmid delivery and in vivo genome editing-based tumor therapy. Via molecular screening of IL-CPs, IL-CPs integrated with fluorination monomers (PBF) can encapsulate plasmids into hybrid nanoparticles and achieve over 90% delivery efficiency in various cells regardless of serum interference. In vitro and in vivo experiments demonstrate that PBF can mediate Cas9/PLK1 plasmids for intracellular delivery and therapeutic genome editing in tumor, achieving efficient tumor suppression. This work provides a new tool for safe and efficient CRISPR-Cas9 delivery and therapeutic genome editing, thus opening a new avenue for the development of ionic liquid polymeric vectors for genome editing and therapy.

pH-Responsive polymer boosts cytosolic siRNA release for retinal neovascularization therapy.

Small interfering RNA (siRNA) has a promising future in the treatment of ocular diseases due to its high efficiency, specificity, and low toxicity in inhibiting the expression of target genes and proteins. However, due to the unique anatomical structure of the eye and various barriers, delivering nucleic acids to the retina remains a significant challenge. In this study, we rationally design PACD, an A-B-C type non-viral vector copolymer composed of a hydrophilic PEG block (A), a siRNA binding block (B) and a pH-responsive block (C). PACDs can self-assemble into nanosized polymeric micelles that compact siRNAs into polyplexes through simple mixing. By evaluating its pH-responsive activity, gene silencing efficiency in retinal cells, intraocular distribution, and anti-angiogenesis therapy in a mouse model of hypoxia-induced angiogenesis, we demonstrate the efficiency and safety of PACD in delivering siRNA in the retina. We are surprised to discover that, the PACD/siRNA polyplexes exhibit remarkable intracellular endosomal escape efficiency, excellent gene silencing, and inhibit retinal angiogenesis. Our study provides design guidance for developing efficient nonviral ocular nucleic acid delivery systems.

Aptamers targeting SARS-CoV-2 nucleocapsid protein exhibit potential anti pan-coronavirus activity.

Emerging and recurrent infectious diseases caused by human coronaviruses (HCoVs) continue to pose a significant threat to global public health security. In light of this ongoing threat, the development of a broad-spectrum drug to combat HCoVs is an urgently priority. Herein, we report a series of anti-pan-coronavirus ssDNA aptamers screened using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). These aptamers have nanomolar affinity with the nucleocapsid protein (NP) of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and also show excellent binding efficiency to the N proteins of both SARS, MERS, HCoV-OC43 and -NL63 with affinity KD values of 1.31 to 135.36 nM. Such aptamer-based therapeutics exhibited potent antiviral activity against both the authentic SARS-CoV-2 prototype strain and the Omicron variant (BA.5) with EC50 values at 2.00 nM and 41.08 nM, respectively. The protein docking analysis also evidenced that these aptamers exhibit strong affinities for N proteins of pan-coronavirus and other HCoVs (-229E and -HKU1). In conclusion, we have identified six aptamers with a high pan-coronavirus antiviral activity, which could potentially serve as an effective strategy for preventing infections by unknown coronaviruses and addressing the ongoing global health threat.

Dietary cobalt oxide nanoparticles alleviate aging through activation of mitochondrial UPR in Caenorhabditis elegans.

Mitochondrial unfolded protein response (UPRmt), which is a mitochondrial proteostasis pathway, orchestrates an adaptive reprogramming for metabolism homeostasis and organismal longevity. Similar to other defense systems, compromised UPRmt is a feature of several age-related diseases. Here we report that dimercapto succinic acid (DMSA)-modified cobalt oxide nanoparticles (Co3O4 NPs), which have received wide-spread attention in biomedical fields, is a promising UPRmt activator and, more importantly, provides a gate for extending healthy lifespan. Methods: UPRmt activation by Co3O4 NPs was tested in transgenetic Caenorhabditis elegans (C. elegans) specifically expressing UPRmt reporter Phsp-6::GFP, and the underlying mechanism was further validated by mitochondrial morphology, mtDNA/nDNA, metabolism-related genes' expression, mitonuclear protein imbalance, oyxgen assumption and ATP level in C. elegans. Then therapeutic response aganist senescence was monitored by lifespan analysis, lipofusin contents, MDA contents, Fe accumulation, pharyngeal locomotion performance as well as athletic ability (head thrashes and body bends) at different developmental stages of C. elegans. RNAi towards ubl-5 or atfs-1 in UPRmt pathway was applied to clarify the role of UPRmt in Co3O4 NPs -mediated anti-aging effects. Finally, the effect of Co3O4 NPs on mitochondrial homeostasis and D-galactose-induced cell viability decline in mammalian cells were studied. Results: Co3O4 NPs was revealed as a bona fide activator of the UPRmt signaling pathway, through fine-tuning mitochondrial dynamics and inducing a stoichiometric imbalance between OXPHOS subunits encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) at early life stage of C. elegans. Phenotypically, Co3O4 NPs treatment protect C. elegans from external stresses. More importantly, dietary low level of Co3O4 NPs effectively extend lifespan and alleviate aging-related physiological and functional decline of worms, demonstrating its potential roles in delaying aging. While the protective effect exerted by Co3O4 NPs was compromised in line with atfs-1 or ubl-5 RNAi treatment. Further studies verified the conservation of Co3O4 NPs in activating UPRmt and exerting protective effects in mammalian cells. Conclusions: The results reveal beneficial effects of Co3O4 NPs on mitochondrial metabolic control, thus presenting their potential efficacy in anti-aging care.

Photoactivatable Silencing Extracellular Vesicle (PASEV) Sensitizes Cancer Immunotherapy.

Immunotherapy has delivered impressive outcomes in combating tumor malignancies. However, insufficient immune infiltration and poor immunogenicity within the tumor microenvironment (TME) greatly compromise patient response rates. Here, a photoactivatable silencing extracellular vesicle (PASEV) is developed for sensitized cancer immunotherapy. p21-Activated kinase 4 (PAK4) is a newly identified tumor-cell-intrinsic "guard" associated with immune exclusion. Small interfering RNA against PAK4 (siPAK4) is designed and assembled with a photoactivatable reactive-oxygen-species (ROS)-sensitive polymer to form the nanocomplex core, which is further camouflaged by extracellular vesicles from M1 macrophages. The PASEV not only serves as a vehicle for packaging, tumor accumulation, and ROS-responsive release of siPAK4 for potent PAK4 silencing, but also primes the TME through immunogenic phototherapy, thereby simultaneously boosting intratumoral infiltration and immune activation. The combined immunotherapy elicits robust anticancer immunity, thus showing great promise for fighting cancers. This work opens a new avenue to simultaneously boost intratumoral infiltration and immune activation for sensitized cancer immunotherapy.

Conscription of Immune Cells by Light-Activatable Silencing NK-Derived Exosome (LASNEO) for Synergetic Tumor Eradication.

Exosomes derived from natural killer (NK) cells (NEO) constitute promising antineoplastic nano-biologics because of their versatile functions in immune regulation. However, a significant augment of their immunomodulatory capability is an essential need to achieve clinically meaningful treatment outcomes. Light-activatable silencing NK-derived exosomes (LASNEO) are orchestrated by engineering the NEO with hydrophilic small interfering RNA (siRNA) and hydrophobic photosensitizer Ce6. Profiling of genes involved in apoptosis pathway with Western blot and RNA-seq in cells receiving NEO treatment reveals that NEO elicits effective NK cell-like cytotoxicity toward tumor cells. Meanwhile, reactive oxygen species (ROS) generation upon laser irradiation not only triggers substantial photodynamic therapy effect but also boosts M1 tumor-associated macrophages polarization and DC maturation in the tumor microenvironment (TME). In addition, ROS also accelerates the cellular entry and endosomal escape of siRNA in TME. Finally, siRNAs targeting PLK1 or PD-L1 induce robust gene silencing in cancer cells, and downregulation of PD-L1 restores the immunological surveillance of T cells in TME. Therefore, the proposed LASNEO exhibit excellent antitumor effects by conscripting multiple types of immune cells. Considering that its manufacture is quite simple and controllable, LASNEO show compelling potential for clinical translational application.

Label-Free Fluorescent Nanofilm Sensor Based on Surface Plasmon Coupled Emission: In Situ Monitoring the Growth of Metal-Organic Frameworks.

We have proposed a universal label-free fluorescent nanofilm sensor based on surface plasmon coupled emission (SPCE). A metal-dye-dielectric (MDD) structure was fabricated to mediate the label-free monitoring based on SPCE. The nonfluorescent dielectric film smartly borrowed the fluorescence signal from the bottom dye layer and led to a new SPCE response through the adjacent metal film. The fluorescence emission angle and polarization strongly depended on the thickness of the nonfluorescent dielectric film on the MDD structure. As a demonstration, the growth of a two-dimensional zeolitic imidazolate framework film (ZIF-L) was in situ monitored in the liquid phase by MDD-SPCE for the first time. The label-free fluorescent sensors are facilely prepared by a spin coating technique, with the potential to be widely spread for in situ studies, especially toward nanomaterial growth processes.

mRNA vaccines for COVID-19 and diverse diseases.

Since its outbreak in late 2019, the novel coronavirus disease 2019 (COVID-19) has spread to every continent on the planet. The global pandemic has affected human health and socioeconomic status around the world. At first, the global response to the pandemic was to isolate afflicted individuals to prevent the virus from spreading, while vaccine development was ongoing. The genome sequence was first presented in early January 2020, and the phase I clinical trial of the vaccine started in March 2020 in the United States using novel lipid-based nanoparticle (LNP), encapsulated with mRNA termed as mRNA-1273. Till now, various mRNA-based vaccines are in development, while one mRNA-based vaccine got market approval from US-FDA for the prevention of COVID-19. Previously, mRNA-based vaccines were thought to be difficult to develop, but the current development is a significant accomplishment. However, widespread production and global availability of mRNA-based vaccinations to combat the COVID-19 pandemic remains a major challenge, especially when the mutations continually occur on the virus (e.g., the recent outbreaks of Omicron variant). This review elaborately discusses the COVID-19 pandemic, the biology of SARS-CoV-2 and the progress of mRNA-based vaccines. Moreover, the review also highlighted a detailed description of mRNA delivery technologies and the application potential in controlling other life-threatening diseases. Therefore, it provides a comprehensive view and multidisciplinary insights into mRNA therapy for broader audiences.

Biosafety materials: Ushering in a new era of infectious disease diagnosis and treatment with the CRISPR/Cas system.

Despite multiple virus outbreaks over the past decade, including the devastating coronavirus disease 2019 (COVID-19) pandemic, the lack of accurate and timely diagnosis and treatment technologies has wreaked havoc on global biosecurity. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has the potential to address these critical needs for tackling infectious diseases to detect viral nucleic acids and inhibit viral replication. This review summarizes how the CRISPR/Cas system is being utilized for the treatment and diagnosis of infectious diseases with the help of biosafety materials and highlights the design principle and in vivo and in vitro efficacy of advanced biosafety materials used to deal with virus attacks.

Thermostable ionizable lipid-like nanoparticle (iLAND) for RNAi treatment of hyperlipidemia.

Small interfering RNA (siRNA) therapeutic is considered to be a promising modality for the treatment of hyperlipidemia. Establishment of a thermostable clinically applicable delivery system remains a most challenging issue for siRNA drug development. Here, a series of ionizable lipid-like materials were rationally designed; 4 panels of lipid formulations were fabricated and evaluated on the basis of four representative structures. The lead lipid (A1-D1-5) was stable at 40°C, and the optimized formulation (iLAND) showed dose and time dual-dependent gene silencing pattern with median effective dose of 0.18 mg/kg. In addition, potent and durable reduction of serum cholesterol and triglyceride were achieved by administering siRNAs targeting angiopoietin-like 3 or apolipoprotein C3 (APOC3) in high-fat diet-fed mice, db/db mice, and human APOC3 transgenic mice, respectively, accompanied by displaying ideal safety profiles. Therefore, siRNA@iLAND prepared with thermostable A1-D1-5 demonstrates substantial value for siRNA delivery, hyperlipidemia therapy, and prevention of subsequent metabolic diseases.

Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy.

CRISPR/Cas9-based gene editing has emerged as a powerful biotechnological tool, that relies on Cas9 protein and single guided RNA (sgRNA) to edit target DNA. However, the lack of safe and efficient delivery carrier is one of the crucial factors restricting its clinical transformation. Here, we report an ionizable lipid nanoparticle (iLP181, pKa = 6.43) based on iLY1809 lipid enabling robust gene editing in vitro and in vivo. The iLP181 effectively encapsulate psgPLK1, the best-performing plasmid expressing for both Cas9 protein and sgRNA targeting Polo-like kinase 1 (PLK1). The iLP181/psgPLK1 nanoformulation showed uniformity in size, regular nanostructure and nearly neutral zeta potential at pH 7.4. The nanoformulation effectively triggered editing of PLK1 gene with more than 30% efficiency in HepG2-Luc cells. iLP181/psgPLK1 significantly accumulated in the tumor for more than 5 days after a single intravenous injection. In addition, it also achieved excellent tumor growth suppression compared to other nucleic acid modalities such as siRNA, without inducing adverse effects to the main organs including the liver and kidneys. This study not only provides a clinically-applicable lipid nanocarrier for delivering CRISPR/Cas system (even other bioactive molecules), but also constitutes a potential cancer treatment regimen base on DNA editing of oncogenes.

Progress of Photodynamic and RNAi Combination Therapy in Cancer Treatment.

Photodynamic therapy (PDT) is a noninvasive and effective local treatment for cancers that produces selective damage to target tissues and cells. However, PDT alone is unlikely to completely inhibit tumor metastasis and/or local tumor recurrence. RNA interference (RNAi) is a phenomenon of gene silencing mediated by exogenous or endogenous double-stranded RNA (dsRNA). RNAi has entered a golden period of development, with the approval of four treatments employing RNAi. PDT in combination with RNAi therapy to inhibit related targets has been a research hotspot, with better clinical outcomes than monotherapy. In this review, the progress of PDT and small interfering RNA (siRNA) targeting different genes is discussed, while the achievements of the combined immunotherapy are reviewed.

Advances of mRNA vaccines for COVID-19: A new prophylactic revolution begins.

Image, graphical abstract.

Core Role of Hydrophobic Core of Polymeric Nanomicelle in Endosomal Escape of siRNA.

Efficient endosomal escape is the most essential but challenging issue for siRNA drug development. Herein, a series of quaternary ammonium-based amphiphilic triblock polymers harnessing an elaborately tailored pH-sensitive hydrophobic core were synthesized and screened. Upon incubating in an endosomal pH environment (pH 6.5-6.8), mPEG45-P(DPA50-co-DMAEMA56)-PT53 (PDDT, the optimized polymer) nanomicelles (PDDT-Ms) and PDDT-Ms/siRNA polyplexes rapidly disassembled, leading to promoted cytosolic release of internalized siRNA and enhanced silencing activity evident from comprehensive analysis of the colocalization and gene silencing using a lysosomotropic agent (chloroquine) and an endosomal trafficking inhibitor (bafilomycin A1). In addition, PDDT-Ms/siPLK1 dramatically repressed tumor growth in both HepG2-xenograft and highly malignant patient-derived xenograft models. PDDT-Ms-armed siPD-L1 efficiently blocked the interaction of PD-L1 and PD-1 and restored immunological surveillance in CT-26-xenograft murine model. PDDT-Ms/siRNA exhibited ideal safety profiles in these assays. This study provides guidelines for rational design and optimization of block polymers for efficient endosomal escape of internalized siRNA and cancer therapy.

Membrane-destabilizing ionizable lipid empowered imaging-guided siRNA delivery and cancer treatment.

One of the imperative medical requirements for cancer treatment is how to establish an imaging-guided nanocarrier that combines therapeutic and imaging agents into one system. siRNA therapeutics have shown promising prospects in controlling life-threatening diseases. However, it is still challenging to develop siRNA formulations with excellent cellular entry capability, efficient endosomal escape, and simultaneous visualization. Herein, we fabricated multifunctional ionizable lipid nanoparticles (iLNPs) for targeted delivery of siRNA and MRI contrast agent. The iLNPs comprises DSPC, cholesterol, PEGylated lipid, contrast agent DTPA-BSA (Gd), and ionizable lipid termed iBL0104. siRNA-loaded iLNPs (iLNPs/siRNA) could be decorated with a tumor targeting cyclic peptide (c(GRGDSPKC)) (termed GARP), or without targeting modification (termed GAP). Data revealed that GARP/siRNA iLNPs exhibited significantly higher cellular entry efficiency than GAP/siRNA iLNPs. GARP/siRNA iLNPs rapidly and effectively escaped from endosome and lysosome after internalization. Compared with GAP/siPLK1, GARP/siPLK1 exhibited better tumor inhibition efficacy in both cell-line derived xenograft and liver cancer patient derived xenograft murine models. In addition, GARP formulation displayed ideal MRI effect in tumor-bearing mice, and was well tolerated by testing animals. Therefore, this study provides an excellent example for achieving imaging-guided and tumor-targeted siRNA delivery and cancer treatment, highlighting its promising potential for translational medicine application.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle.

mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.