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.

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.

Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors.

Bioimaging plays an important role in cancer diagnosis and treatment. However, imaging sensitivity and specificity still constitute key challenges. Nanotechnology-based imaging is particularly promising for overcoming these limitations because nanosized imaging agents can specifically home in on tumors via the "enhanced permeation and retention" (EPR) effect, thus resulting in enhanced imaging sensitivity and specificity. Here, we report an original nanosystem for positron emission tomography (PET) imaging based on an amphiphilic dendrimer, which bears multiple PET reporting units at the terminals. This dendrimer is able to self-assemble into small and uniform nanomicelles, which accumulate in tumors for effective PET imaging. Benefiting from the combined dendrimeric multivalence and EPR-mediated passive tumor targeting, this nanosystem demonstrates superior imaging sensitivity and specificity, with up to 14-fold increased PET signal ratios compared with the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). Most importantly, this dendrimer system can detect imaging-refractory low-glucose-uptake tumors that are otherwise undetectable using [18F]FDG. In addition, it is endowed with an excellent safety profile and favorable pharmacokinetics for PET imaging. Consequently, this dendrimer nanosystem constitutes an effective and promising approach for cancer imaging. Our study also demonstrates that nanotechnology based on self-assembling dendrimers provides a fresh perspective for biomedical imaging and cancer diagnosis.

Clinical advances of siRNA therapeutics.

Small interfering RNA (siRNA) enables efficient target gene silencing by employing a RNA interference (RNAi) mechanism, which can compromise gene expression and regulate gene activity by cleaving mRNA or repressing its translation. Twenty years after the discovery of RNAi in 1998, ONPATTRO™ (patisiran) (Alnylam Pharmaceuticals, Inc.), a lipid formulated siRNA modality, was approved for the first time by United States Food and Drug Administration and the European Commission in 2018. With this milestone achievement, siRNA therapeutics will soar in the coming years. Here, we review the discovery and the mechanisms of RNAi, briefly describe the delivery technologies of siRNA, and summarize recent clinical advances of siRNA therapeutics.

A Dual Targeting Dendrimer-Mediated siRNA Delivery System for Effective Gene Silencing in Cancer Therapy.

Small interfering RNA (siRNA) is emerging as a novel therapeutic for treating various diseases, provided a safe and efficient delivery is available. In particular, specific delivery to target cells is critical for achieving high therapeutic efficacy while reducing toxicity. Amphiphilic dendrimers are emerging as novel promising carriers for siRNA delivery by virtue of the combined multivalent cooperativity of dendrimers with the self-assembling property of lipid vectors. Here, we report a ballistic approach for targeted siRNA delivery to cancer cells using an amphiphilic dendrimer equipped with a dual targeting peptide bearing an RGDK warhead. According to the molecular design, the amphiphilic dendrimer was expected to deliver siRNA effectively, while the aim of the targeting peptide was to home in on tumors via interaction of its warhead with integrin and the neuropilin-1 receptor on cancer cells. Coating the positively charged siRNA/dendrimer delivery complex with the negatively charged segment of the targeting peptide via electrostatic interactions led to small and stable nanoparticles which were able to protect siRNA from degradation while maintaining the accessibility of RGDK for targeting cancer cells and preserving the ability of the siRNA to escape from endosomes. The targeted system had enhanced siRNA delivery, stronger gene silencing, and more potent anticancer activity compared to nontargeted or covalent dendrimer-based systems. In addition, neither acute toxicity nor induced inflammation was observed. Consequently, this delivery system constitutes a promising nonviral vector for targeted delivery and can be further developed to provide RNAi-based personalized medicine against cancer. Our study also gives new perspectives on the use of nanotechnology based on self-assembling dendrimers in various biomedical applications.

Label-free aptasensor based on ultrathin-linker-mediated hot-spot assembly to induce strong directional fluorescence.

We have demonstrated the proof-of-concept of a label-free biosensor based on emission induced by an extreme hot-spot plasmonic assembly. In this work, an ultrathin linking layer composed of cationic polymers and aptamers was fabricated to mediate the assembly of a silver nanoparticles (AgNPs)-dyes-gold film with a strongly coupled architecture through sensing a target protein. Generation of directional surface plasmon coupled emission (SPCE) was thus stimulated as a means of reporting biorecognition. Both the biomolecules and the nanoparticles were totally free of labeling, thereby ensuring the activity of biomolecules and allowing the use of freshly prepared metallic nanoparticles with large dimensions. This sensor smartly prevents the plasmonic assembly in the absence of targets, thus maintaining no signal through quenching fluorophores loaded onto a gold film. In the presence of targets, the ultrathin layer is activated to link NPs-film junctions. The small gap of the junction (no greater than 2 nm) and the large diameter of the nanoparticles (~100 nm) ensure that ultrastrong coupling is achieved to generate intense SPCE. A >500-fold enhancement of the signal was observed in the biosensing. This strategy provides a simple, reliable, and effective way to apply plasmonic nanostructures in the development of biosensing.

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.

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.

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.

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.

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.

Prism-based surface plasmon coupled emission imaging.

A prism-based surface plasmon coupled emission (SPCE) imaging apparatus with a reverse Kretschmann (RK) configuration was developed and applied to dye-doped polymer films. Highly polarized, directional and enhanced fluorescence images were obtained. The angular distribution of the SPCE images was in accordance with the validated theoretical calculation performed using Fresnel equation. Prism-based SPCE imaging combined with microarray technology appears to be a promising platform for rapid and high-throughput analysis, especially for high-density arrays. We believe that prism-based SPCE imaging has potential applications in biochemical research.

A covalently crosslinked polysaccharide hydrogel for potential applications in drug delivery and tissue engineering.

The development of non-cytotoxic hydrogels that can allow for the controlled release of molecules has important clinical and therapeutic applications. In this paper, we developed a series of in situ hydrogels by combining N,O-carboxymethyl chitosan and oxidized alginate without additional crosslinking agents. The rheological properties of these hydrogels as well as their gelling time, swelling ratio, and in vitro degradation behavior were investigated. We observed that although gelation was rapid at physiological temperature, it was even faster in the presence of higher oxidization degree of alginate. In vitro cytotoxicity study showed that the developed hydrogels were not cytotoxic after 24 h of culturing with NIH-3T3 cells. Additionally, bovine serum albumin was released from the hydrogels initially by diffusion at early stages followed by a degradation-dependent mechanism at later stages. In conclusion, the developed hydrogel might have potential application in the drug delivery system and tissue engineering.

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.

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.

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.

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.

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.