Dynamic chromatin accessibility tuning by the long noncoding RNA ELDR accelerates chondrocyte senescence and osteoarthritis.

Epigenetic reprogramming plays a critical role in chondrocyte senescence during osteoarthritis (OA) pathology, but the underlying molecular mechanisms remain to be elucidated. Here, using large-scale individual datasets and genetically engineered (Col2a1-CreERT2;Eldrflox/flox and Col2a1-CreERT2;ROSA26-LSL-Eldr+/+ knockin) mouse models, we show that a novel transcript of long noncoding RNA ELDR is essential for the development of chondrocyte senescence. ELDR is highly expressed in chondrocytes and cartilage tissues of OA. Mechanistically, exon 4 of ELDR physically mediates a complex consisting of hnRNPL and KAT6A to regulate histone modifications of the promoter region of IHH, thereby activating hedgehog signaling and promoting chondrocyte senescence. Therapeutically, GapmeR-mediated silencing of ELDR in the OA model substantially attenuates chondrocyte senescence and cartilage degradation. Clinically, ELDR knockdown in cartilage explants from OA-affected individuals decreased the expression of senescence markers and catabolic mediators. Taken together, these findings uncover an lncRNA-dependent epigenetic driver in chondrocyte senescence, highlighting that ELDR could be a promising therapeutic avenue for OA.

In situ production and secretion of proteins endow therapeutic benefit against psoriasiform dermatitis and melanoma.

Genetic medicines have the potential to treat various diseases; however, certain ailments including inflammatory diseases and cancer would benefit from control over extracellular localization of therapeutic proteins. A critical gap therefore remains the need to develop and incorporate methodologies that allow for posttranslational control over expression dynamics, localization, and stability of nucleic acid-generated protein therapeutics. To address this, we explored how the body's endogenous machinery controls protein localization through signal peptides (SPs), including how these motifs could be incorporated modularly into therapeutics. SPs serve as a virtual zip code for mRNA transcripts that direct the cell where to send completed proteins within the cell and the body. Utilizing this signaling biology, we incorporated secretory SP sequences upstream of mRNA transcripts coding for reporter, natural, and therapeutic proteins to induce secretion of the proteins into systemic circulation. SP sequences generated secretion of various engineered proteins into the bloodstream following intravenous, intramuscular, and subcutaneous SP mRNA delivery by lipid, polymer, and ionizable phospholipid delivery carriers. SP-engineered etanercept/TNF-α inhibitor proteins demonstrated therapeutic efficacy in an imiquimod-induced psoriasis model by reducing hyperkeratosis and inflammation. An SP-engineered anti-PD-L1 construct mediated mRNA encoded proteins with longer serum half-lives that reduced tumor burden and extended survival in MC38 and B16F10 cancer models. The modular nature of SP platform should enable intracellular and extracellular localization control of various functional proteins for diverse therapeutic applications.

Resveratrol-induced SIRT1 activation inhibits glycolysis-fueled angiogenesis under rheumatoid arthritis conditions independent of HIF-1α.

OBJECTIVE: This study investigated the impacts of SIRT1 activation on rheumatoid arthritis (RA)-related angiogenesis. METHODS: HUVECs were cultured by different human serum. Intracellular metabolites were quantified by UPLC-MS. Next, HUVECs and rat vascular epithelial cells under different inflammatory conditions were treated by a SIRT1 agonist resveratrol (RSV). Cytokines and biochemical indicators were detected by corresponding kits. Protein and mRNA expression levels were assessed by immunoblotting and PCR methods, respectively. Angiogenesis capabilities were evaluated by migration, wound-healing and tube-formation experiments. To down-regulate certain signals, gene-specific siRNA were applied. RESULTS: Metabolomics study revealed the accelerated glycolysis in RA serum-treated HUVECs. It led to ATP accumulation, but did not affect GTP levels. RSV inhibited pro-angiogenesis cytokines production and glycolysis in both the cells, and impaired the angiogenesis potentials. These effects were mimicked by an energy metabolism interrupter bikini in lipopolysaccharide (LPS)-primed HUVECs, largely independent of HIF-1α. Both RSV and bikinin can inhibit the activation of the GTP-dependent pathway Rho/ROCK and reduce VEGF production. Abrogation of RhoA signaling reinforced HIF-1α silencing-brought changes in LPS-stimulated HUVECs, and overshadowed the anti-angiogenesis potentials of RSV. CONCLUSION: Glycolysis provides additional energy to sustain Rho/ROCK activation in RA subjects, which promotes VEGF-driven angiogenesis and can be inhibited by SIRT1 activation.

Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing.

Endosomal escape remains a fundamental barrier hindering the advancement of nucleic acid therapeutics. Taking inspiration from natural phospholipids that comprise biological membranes, we report the combinatorial synthesis of multi-tailed ionizable phospholipids (iPhos) capable of delivering messenger RNA or mRNA/single-guide RNA for gene editing in vivo. Optimized iPhos lipids are composed of one pH-switchable zwitterion and three hydrophobic tails, which adopt a cone shape in endosomal acidic environments to facilitate membrane hexagonal transformation and subsequent cargo release from endosomes. Structure-activity relationships reveal that iPhos chemical structure can control in vivo efficacy and organ selectivity. iPhos lipids synergistically function with various helper lipids to formulate multi-component lipid nanoparticles (called iPLNPs) for selective organ targeting. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs (respectively) following intravenous administration. This rational design of functional phospholipids demonstrates substantial value for gene editing research and therapeutic applications.

Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and Enhance the Nerve Blockade from Tetrodotoxin.

The efficacy of tetrodotoxin (TTX), a very potent local anesthetic, is limited by its poor penetration through barriers to axonal surfaces. To address this issue, we encapsulated TTX in hollow silica nanoparticles (TTX-HSN) and injected them at the sciatic nerve in rats. TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of the block, and decreased the toxicity compared to free TTX. In animals injected with fluorescently labeled HSN, the imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve and that the penetrating ability of silica nanoparticles was highly size-dependent. These results demonstrated that HSN could deliver TTX into the nerve, enhancing efficacy while improving safety.

Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance.

Drug resistance and toxicity constitute challenging hurdles for cancer therapy. The application of nanotechnology for anticancer drug delivery is expected to address these issues and bring new hope for cancer treatment. In this context, we established an original nanomicellar drug delivery system based on an amphiphilic dendrimer (AmDM), which could generate supramolecular micelles to effectively encapsulate the anticancer drug doxorubicin (DOX) with high drug-loading capacity (>40%), thanks to the unique dendritic structure creating large void space for drug accommodation. The resulting AmDM/DOX nanomicelles were able to enhance drug potency and combat doxorubicin resistance in breast cancer models by significantly enhancing cellular uptake while considerably decreasing efflux of the drug. In addition, the AmDM/DOX nanoparticles abolished significantly the toxicity related to the free drug. Collectively, our studies demonstrate that the drug delivery system based on nanomicelles formed with the self-assembling amphiphilic dendrimer constitutes a promising and effective drug carrier in cancer therapy.

Zinc Oxide Nanoparticles as Adjuvant To Facilitate Doxorubicin Intracellular Accumulation and Visualize pH-Responsive Release for Overcoming Drug Resistance.

Multidrug resistance (MDR) of cancer is a challenge to effective chemotherapeutic interventions. The stimulus-responsive drug delivery system (DDS) based on nanotechnology provides a promising approach to overcome MDR. Through the development of a doxorubicin delivery system based on zinc oxide nanomaterials, we have demonstrated that MDR in breast cancer cell line can be significantly circumvented by a combination of efficient cellular uptake and a pH-triggered rapid drug release due to degradation of nanocarriers in acidic environment. Doxorubicin and zinc oxide nanoparticles, compared with free doxorubicin, effectively enhanced the intracellular drug concentration by simultaneously increasing cell uptake and decreasing cell efflux in MDR cancer cells. The acidic environment-triggered release of drug can be tracked real-time by the doxorubicin fluorescence recovery from its quenched state. Therefore, with the combination of therapeutic potential and the capacity to track release of drug in cancer cells, our system holds great potential in nanomedicine by serving dual roles of overcoming drug resistance and tracking intracellular drug release from the DDS.

Multifunctional metal rattle-type nanocarriers for MRI-guided photothermal cancer therapy.

In the past decade, numerous species of nanomaterials have been developed for biomedical application, especially cancer therapy. Realizing visualized therapy is highly promising now because of the potential of accurate, localized treatment. In this work, we first synthesized metal nanorattles (MNRs), which utilized porous gold shells to carry multiple MR imaging contrast agents, superparamagnetic iron oxide nanoparticles (SPIONs), inside. A fragile wormpore-like silica layer was manipulated to encapsulate 8 nm oleylamine SPIONs and mediate the in situ growth of porous gold shell, and it was finally etched by alkaline solution to obtain the rattle-type nanostructure. As shown in the results, this nanostructure with unique morphology could absorb near-infrared light, convert to heat to kill cells, and inhibit tumor growth. As a carrier for multiple SPIONs, it also revealed good function for T2-weighted MR imaging in tumor site. Moreover, the rest of the inner space of the gold shell could also introduce potential ability as nanocarriers for other cargos such as chemotherapeutic drugs, which is still under investigation. This metal rattle-type nanocarrier may pave the way for novel platforms for cancer therapy in the future.

Functionalized nanoscale micelles improve drug delivery for cancer therapy in vitro and in vivo.

Poor penetration of therapeutic drugs into tumors is a major challenge in anticancer therapy, especially in solid tumors, leading to reduced therapeutic efficacy in vivo. In the study, we used a new tumor-penetrating peptide, CRGDK, to conjugate onto the surface of doxorubicin encapsulated nanoscale micelles. The CRGDK peptide triggered specific binding to neuropilin-1, leading to enhanced cellular uptake and cytotoxicity in vitro and highly accumulation and penetration in the tumors in vivo.

Simplified Lipid Nanoparticles for Tissue- And Cell-Targeted mRNA Delivery Facilitate Precision Tumor Therapy in a Lung Metastasis Mouse Model.

mRNA-based applications have achieved remarkable success in the development of next-generation vaccines and the treatment of diverse liver diseases. Overcoming the challenge of delivering mRNA to extrahepatic tissues, especially specific cells within tissues, is crucial for precision therapy. In this study, a platform is developed for selective mRNA delivery to desired cells within tissues by combining lipid nanoparticle (LNP)-based targeted delivery with mRNA sequence-controlled expression. Through systematic optimization, a three-component LNP platform is developed, enabling targeted mRNA delivery to the lung, liver, and spleen. The incorporation of unique microRNA target sites into the mRNA scaffold further enhances control over protein translation in specific cells within the target tissue. This combined strategy, named SELECT (Simplified LNP with Engineered mRNA for Cell-type Targeting), demonstrates its efficacy in distinguishing mRNA expression between tumor and normal cells based on intracellular microRNA abundance. SELECT encapsulating mRNA encoding a tumor-specific cytotoxic protein, human ELANE, exhibits selective mRNA delivery to tumor lesions and significant inhibition of tumor growth in a mouse model of melanoma lung metastasis. Overall, SELECT has great potential as a new precision tumor treatment approach and also offers promising prospects for other mRNA therapies targeting specific cell types.

Spleen-Targeted mRNA Vaccine Doped with Manganese Adjuvant for Robust Anticancer Immunity In Vivo.

The successful application of mRNA vaccines in preventing and treating infectious diseases highlights their potential as therapeutic vaccines for cancer treatment. However, unlike infectious diseases, effective antitumor therapy, particularly for solid tumors, necessitates the activation of more powerful cellular and humoral immunity to achieve clinical efficacy. Here, we report a spleen-targeted mRNA vaccine (Mn@mRNA-LNP) designed to deliver tumor antigen-encoding mRNA and manganese adjuvant (Mn2+) simultaneously to dendritic cells (DCs) in the spleen. This delivery system promotes DC maturation and surface antigen presentation and stimulates the production of cytotoxic T cells. Additionally, Mn2+ codelivered in the system serves as a safe and effective immune adjuvant, activating the stimulator of interferon genes (STING) signaling pathway and promoting the secretion of type I interferon, further enhancing the antigen-specific T cell responses. Mn@mRNA-LNP effectively inhibits tumor progression in established melanoma and colon tumor models as well as in a model of tumor recurrence after resection. Notably, the combination of Mn@mRNA-LNP with immune checkpoint inhibitors further enhances complete tumor suppression and prolonged the overall survival in mice. Overall, this "All-in-One" mRNA vaccine significantly boosts antitumor immunity responses by improving spleen targeting and immune activation, providing an attractive strategy for the future clinical translation of therapeutic mRNA vaccines.

Surface engineering of lipid nanoparticles: targeted nucleic acid delivery and beyond.

Harnessing surface engineering strategies to functionalize nucleic acid-lipid nanoparticles (LNPs) for improved performance has been a hot research topic since the approval of the first siRNA drug, patisiran, and two mRNA-based COVID-19 vaccines, BNT162b2 and mRNA-1273. Currently, efforts have been mainly made to construct targeted LNPs for organ- or cell-type-specific delivery of nucleic acid drugs by conjugation with various types of ligands. In this review, we describe the surface engineering strategies for nucleic acid-LNPs, considering ligand types, conjugation chemistries, and incorporation methods. We then outline the general purification and characterization techniques that are frequently used following the engineering step and emphasize the specific techniques for certain types of ligands. Next, we comprehensively summarize the currently accessible organs and cell types, as well as the other applications of the engineered LNPs. Finally, we provide considerations for formulating targeted LNPs and discuss the challenges of successfully translating the "proof of concept" from the laboratory into the clinic. We believe that addressing these challenges could accelerate the development of surface-engineered LNPs for targeted nucleic acid delivery and beyond.

Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy.

Messenger RNA (mRNA) has received great attention in the prevention and treatment of various diseases due to the success of coronavirus disease 2019 (COVID-19) mRNA vaccines (Comirnaty and Spikevax). To meet the therapeutic purpose, it is required that mRNA must enter the target cells and express sufficient proteins. Therefore, the development of effective delivery systems is necessary and crucial. Lipid nanoparticle (LNP) represents a remarkable vehicle that has indeed accelerated mRNA applications in humans, as several mRNA-based therapies have already been approved or are in clinical trials. In this review, the focus is on mRNA-LNP-mediated anticancer therapy. It summarizes the main development strategies of mRNA-LNP formulations, discusses representative therapeutic approaches in cancer, and points out current challenges and possible future directions of this research field. It is hoped that these delivered messages can help further improve the application of mRNA-LNP technology in cancer therapy.

Physicochemical pre- and post-treatment of coking wastewater combined for energy recovery and reduced environmental risk.

In this study, physicochemical pre- and post-treatment of highly polluting coking wastewater (CWW) for the removal of refractory compounds and recovery of high-energy substances/components was investigated. An economic optimization model targeting the development of a cost-effective and sustainable treatment technology was proposed. At the post-treatment stage, powdered activated carbon (PAC) was used to separate the refractory and toxic pollutants from the bio-treated CWW, with the adsorption capacity ranging from 50 to 120 mg chemical oxygen demand (COD) g-1 PAC. Then, the spent PAC, together with a coagulant, was reused in the pre-treatment of highly concentrated raw CWW, which lifted the adsorption capacity to 800-1200 mg COD g-1 PAC. Results showed that the adsorbent's high selectivity towards macromolecular and complicated pollutants could remove 25-65 % of COD in both CWW flows. Analysis of pollutants' molecular weight distribution and GC-MS indicated a good affinity between PAC and high-energy pollutants (phenolic compounds and alkanes), which could transfer 144,555 kJ m-3 of energy from CWW to the adsorption-coagulation sludge. The economic optimization model suggested that the cost of the adsorbent was compensated by the net benefits of energy recovery and that profit was achieved when the PAC price was less than 5562 CNY t-1. The proposed two-stage PAC/coagulant approach offers a way to sustainable water quality and sludge management, plus energy recycling, in CWW treatment. It may also be applied to the treatment of other industrial wastewaters.

A combined process model for wastewater treatment based on hydraulic retention time and toxicity inhibition.

Hydraulic retention time (HRT), as an important parameter in the wastewater treatment process, has a great impact on water quality and energy consumption. With the rapid advances in computer technology and deepened understanding of in microbial metabolism, a series of activated sludge models (ASMs) have been developed and applied in wastewater treatment. However, ASMs simulation based on the nexus of HRT, water treatment process, water quality and energy consumption has yet to be verified. In this study, HRT was creatively linked to water treatment process variation. And a novel combined process model (CPM) was developed based on the operational data and treatment performance data from 4 full-scale coking wastewater treatment processes. In the CPM, an array of biological treatment processes were represented by setting the HRT in respective treatment units of the anaerobic-oxic-hydrolytic & denitrification-oxic (A/O/H/O) process. The relationships between HRT, effluent quality and energy consumption were systematically analyzed. Results showed that: (i) for A/O/H/O process, the HRT of first oxic (O1) reactor has a key effect on the effluent water quality and energy consumption, while the impact of the anaerobic (A) reactor HRT was limited; (ii) the O/H/O process has a clear advantage in treating coking wastewater due to the carbon removal and detoxification function of O1 reactor; (iii) the lowest energy consumption (with the total system HRT below 210 h) to meet the biological effluent quality requirements (COD = 200 mg/L, TN = 50 mg/L) is 4.429 kWh/m3. Since the CPM could effectively work out the optimal process configuration and break the boundaries between HRT and process variation, it has enormous potential to be extended to the design of other wastewater treatment processes.

Fate and distribution of phosphorus in coking wastewater treatment: From sludge to its derived biochar.

Due to the phosphorus (P) deficiency in coking wastewater, sufficient P needs to be provided in the treatment process to maintain biotic activity. However, most of the dosed P sources are transferred to the sludge phase out of the chemical equilibrium. After an in-depth investigation of P morphology changes in coking wastewater treatment, it is found that above 71.6 % P applied to the full-scale O/H/H/O (oxic-hydrolytic & denitrification-hydrolytic & denitrification-oxic) process for coking wastewater treatment is ended up in the sludge phase of the aerobic reactors in the forms of non-apatite inorganic phosphorus (NAIP). Theoretical simulations suggest that the P forms precipitates such as FePO4·2H2O, AlPO4·2H2O, MnHPO4 at pH < 7, and Ca5(PO4)3OH at pH > 7. Microbial utilization of P in coking wastewater treatment is swayed by precipitation, pH and sludge retention time (SRT). By pyrolysis treatment of the waste sludge at 700 °C, phosphoric substances in coking sludge are enriched and converted into Ca5(PO4)3OH, Ca5(PO4)3Cl, Ca3(PO4)2, etc. with apatite phosphorus (AP) accounting for 65.7 % of total phosphorus. Moreover, the heavy metals in biochar were below the national standard limits for discharge. This study shows that hazardous waste (coking sludge) can be transformed into bioavailable products (P-rich biochar) through comprehensive management of the fate of P. Combined with the O/H/H/O process, the mechanisms of phosphorus consumption in coking wastewater treatment are revealed for the first time, which will facilitate a reduced consumption of phosphorus and provide a demonstration for other phosphorus-deficient industrial wastewater treatment.

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models.

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery.

A new methodology termed selective organ targeting (SORT) was recently developed that enables controllable delivery of nucleic acids to target tissues. SORT lipid nanoparticles (LNPs) involve the inclusion of SORT molecules that accurately tune delivery to the liver, lungs and spleen of mice after intravenous administration. Nanoparticles can be engineered to target specific cells and organs in the body by passive, active and endogenous targeting mechanisms that require distinct design criteria. SORT LNPs are modular and can be prepared using scalable, synthetic chemistry and established engineering formulation methods. This protocol provides detailed procedures, including the synthesis of a representative ionizable cationic lipid, preparation of multiple classes of SORT LNPs by pipette, vortex and microfluidic mixing methods, physical characterization, and in vitro/in vivo mRNA delivery evaluation. Depending on the scale of the experiments, the synthesis of the ionizable lipid requires 4-6 d; LNPs can be formulated within several hours; LNP characterization can be completed in 2-4 h; and in vitro/in vivo evaluation studies require 1-14 d, depending on the design and application. Our strategy offers a versatile and practical method for rationally designing nanoparticles that accurately target specific organs. The SORT LNPs generated as described in this protocol can therefore be applied to multiple classes of LNP systems for therapeutic nucleic acid delivery and facilitate the development of protein replacement and genetic medicines in target tissues. This protocol does not require specific expertise, is modular to various lipids within defined physicochemical classes, and should be accomplishable by researchers from various backgrounds.

m6A epitranscriptomic regulation of tissue homeostasis during primate aging.

How N6-methyladenosine (m6A), the most abundant mRNA modification, contributes to primate tissue homeostasis and physiological aging remains elusive. Here, we characterize the m6A epitranscriptome across the liver, heart and skeletal muscle in young and old nonhuman primates. Our data reveal a positive correlation between m6A modifications and gene expression homeostasis across tissues as well as tissue-type-specific aging-associated m6A dynamics. Among these tissues, skeletal muscle is the most susceptible to m6A loss in aging and shows a reduction in the m6A methyltransferase METTL3. We further show that METTL3 deficiency in human pluripotent stem cell-derived myotubes leads to senescence and apoptosis, and identify NPNT as a key element downstream of METTL3 involved in myotube homeostasis, whose expression and m6A levels are both decreased in senescent myotubes. Our study provides a resource for elucidating m6A-mediated mechanisms of tissue aging and reveals a METTL3-m6A-NPNT axis counteracting aging-associated skeletal muscle degeneration.

Circulating succinate-modifying metabolites accurately classify and reflect the status of fumarate hydratase-deficient renal cell carcinoma.

Germline or somatic loss-of-function mutations of fumarate hydratase (FH) predispose patients to an aggressive form of renal cell carcinoma (RCC). Since other than tumor resection there is no effective therapy for metastatic FH-deficient RCC, an accurate method for early diagnosis is needed. Although MRI or CT scans are offered, they cannot differentiate FH-deficient tumors from other RCCs. Therefore, finding noninvasive plasma biomarkers suitable for rapid diagnosis, screening, and surveillance would improve clinical outcomes. Taking advantage of the robust metabolic rewiring that occurs in FH-deficient cells, we performed plasma metabolomics analysis and identified 2 tumor-derived metabolites, succinyl-adenosine and succinic-cysteine, as excellent plasma biomarkers for early diagnosis. These 2 molecules reliably reflected the FH mutation status and tumor mass. We further identified the enzymatic cooperativity by which these biomarkers are produced within the tumor microenvironment. Longitudinal monitoring of patients demonstrated that these circulating biomarkers can be used for reporting on treatment efficacy and identifying recurrent or metastatic tumors.