Targeted Delivery of Circular Single-Stranded DNA Encoding IL-12 for the Treatment of Triple-Negative Breast Cancer.

Interleukin-12 (IL-12) is a critical cytokine with notable anticancer properties, including enhancing T-cell-mediated cancer cell killing, and curbing tumor angiogenesis. To date, many approaches are evaluated to achieve in situ overexpression of IL-12, minimizing leakage and the ensuing toxicity. Here, it is focused on circular single-stranded DNA (Css DNA), a type of DNA characterized by its unique structure, which could be expressed in mammals. It is discovered that Css DNA can induce sustained luciferase expression for half a year by intramuscular injection and showed effective antitumor results by intratumoral injection. Motivated by these findings, a folate-modified LNP system is now developed for the delivery of Css DNA expressing IL-12 for the therapy of 4T1 triple-negative breast cancer (TNBC). This delivery system effectively activates anti-cancer immune responses, slows tumor growth, significantly prolongs survival in animal models, and prevents tumor recurrence. After 6 months of long-term observation, the elevated level of IL-12 is still detectable in the lymph nodes and serum of the cured mice. This study highlights the long-term sustained expression capacity of Css DNA and its ability to inhibit recurrence, and the potential of tumor-targeted LNPs for Css DNA-based cancer therapy, providing a new insight into gene overexpression strategy.

Co-delivery of PROTAC and siRNA via novel liposomes for the treatment of malignant tumors.

Targeted elimination of damaged or overexpressed proteins within the tumor serves a pivotal role in regulating cellular function and restraining tumor cell growth. Researchers have been striving to identify safer and more effective methods for protein removal. Here, we propose the synergistic employment of a small molecule degrading agent (PROTAC) and siRNA to attain enhanced protein clearance efficiency and tumor therapeutic effects. Co-delivery liposomes were prepared to facilitate the efficient encapsulation of PROTAC and siRNA. Specifically, the cationic liposome significantly improved the solubility of the insoluble PROTAC (DT2216). The cationic polymer (F-PEI) achieved efficient encapsulation of the nucleic acid drug, thereby promoting endocytosis and enhancing the therapeutic impact of the drug. Both in vivo and in vitro experiments demonstrated remarkable degradation of target proteins and inhibition of tumor cells by the co-delivery system. In conclusion, the co-delivery liposomes furnished a nano-delivery system proficient in effectively encapsulating both hydrophilic and hydrophobic drugs, thereby presenting a novel strategy for targeted combination therapy in treating tumors.

Ligand-free biodegradable poly(beta-amino ester) nanoparticles for targeted systemic delivery of mRNA to the lungs.

Non-viral nanoparticles (NPs) have seen heightened interest as a delivery method for a variety of clinically relevant nucleic acid cargoes in recent years. While much of the focus has been on lipid NPs, non-lipid NPs, including polymeric NPs, have the possibility of improved efficacy, safety, and targeting, especially to non-liver organs following systemic administration. A safe and effective systemic approach for intracellular delivery to the lungs could overcome limitations to intratracheal/intranasal delivery of NPs and improve clinical benefit for a range of diseases including cystic fibrosis. Here, engineered biodegradable poly (beta-amino ester) (PBAE) NPs are shown to facilitate efficient delivery of mRNA to primary human airway epithelial cells from both healthy donors and individuals with cystic fibrosis. Optimized NP formulations made with differentially endcapped PBAEs and systemically administered in vivo lead to high expression of mRNA within the lungs in BALB/c and C57 B/L mice without requiring a complex targeting ligand. High levels of mRNA-based gene editing were achieved in an Ai9 mouse model across bronchial, epithelial, and endothelial cell populations. No toxicity was observed either acutely or over time, including after multiple systemic administrations of the NPs. The non-lipid biodegradable PBAE NPs demonstrate high levels of transfection in both primary human airway epithelial cells and in vivo editing of lung cell types that are targets for numerous life-limiting diseases particularly single gene disorders such as cystic fibrosis and surfactant deficiencies.

Dihydroxyphenylalanine-conjugated high molecular weight polyethylenimine for targeted delivery of Plasmid.

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4-dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5-12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4-dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier.

"All in one" lipid-polymer nanodelivery system for gene therapy of ischemic diseases.

Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.

Nanoparticle-mediated gene delivery of TRAIL to resistant cancer cells: A review.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), also known as APO2L, has emerged as a highly potential anticancer agent because of its capacity to effectively trigger apoptosis in tumor cells by specifically binding to either of its death receptors (DR4 or DR5) while having no adverse effects on normal cells. Nevertheless, its practical use has been hindered by its inefficient pharmacokinetics characteristics, the challenges involved in its administration and delivery to targeted cells, and the resistance exhibited by most cancer cells towards TRAIL. Gene therapy, as a promising approach would be able to potentially circumvent TRAIL-based cancer therapy challenges mainly through localized TRAIL expression and generating a bystander impact. Among different strategies, using nanoparticles in TRAIL gene delivery allows for precise targeting, and overcoming TRAIL resistance by combination therapy. In this review, we go over potential mechanisms by which cancer cells achieve resistance to TRAIL and provide an overview of different carriers for delivering of the TRAIL gene to resistant cancer cells, focusing on different types of nanoparticles utilized in this context. We will also explore the challenges, and investigate future perspectives of this nanomedicine approach for cancer therapy.

Biosurfactant Nanomicelles and Peptide Integration: Novel Approaches to Targeted Gene Delivery in Colon Cancer Treatment.

A notable breakthrough in the treatment of colon cancer involves the utilisation of a cutting-edge drug delivery technology known as biosurfactant-derived nanomicelles. These nanomicelles, composed of natural biosurfactant molecules, possess the distinct capability to enclose pharmaceuticals or genetic material, such as DNA, siRNA, or mRNA, within spherical formations. With a size ranging from 10 to 100 nanometers, these nanomicelles exhibit precision targeting capabilities towards colon cancer cells, hence minimising the occurrence of side effects typically associated with treatment. Upon being specifically targeted, the nanomicelles liberate their cargo into cancer cells, resulting in enhanced therapy efficacy. This novel strategy utilises the specific attributes of the tumour microenvironment to administer precise and focused treatment. These nanomicelles improve the absorption by cells and reduce harm to healthy tissues by imitating important nutrients or utilising compounds that specifically target tumours. Furthermore, the incorporation of stimuli-responsive components allows for regulated medication release in reaction to the acidic environment seen in tumours. The review focuses on examining the use of biosurfactants and natural peptides in nanomicellar carriers as ways to fight against colon cancer. Folate-coated nanomicelles incorporating curcumin facilitate precise gene delivery, while the partnership of biosurfactants, such as surfactin from Bacillus subtilis and natural peptides, enables the transportation of particular cyclopeptides into the tumour network. Peptides, similar to bombesin, direct nanomicelles to specific places, while peptides based on curcumin control the release of medicinal substances. While preclinical investigations demonstrate promise, obstacles remain in formulation and regulatory issues. However, biosurfactant-based nanomicelles, particularly folate-coated carriers loaded with curcumin, show tremendous potential in overcoming biological barriers and delivering medicines efficiently to colon cancer cells.

Carbon quantum dots for the diagnosis and treatment of ophthalmic diseases.

Carbon quantum dots (CQDs), an emerging nanomaterial, are gaining attention in ophthalmological applications due to their distinctive physical, chemical, and biological characteristics. For example, their inherent fluorescent capabilities offer a novel and promising alternative to conventional fluorescent dyes for ocular disease diagnostics. Furthermore, because of the excellent biocompatibility and minimal cytotoxicity, CQDs are well-suited for therapeutic applications. In addition, functionalized CQDs can effectively deliver drugs to the posterior part of the eyeball to inhibit neovascularization. This review details the use of CQDs in the management of ophthalmic diseases, including various retinal diseases, and ocular infections. While still in its initial phases within ophthalmology, the significant potential of CQDs for diagnosing and treating eye conditions is evident.

DOTAP Modified Formulations of Aminoacid Based Cationic Liposomes for Improved Gene Delivery and Cell Viability.

The liposomal systems proved remarkably useful for the delivery of genetic materials but enhancing their efficacy remains a significant challenge. While structural alterations could result in the discovery of more effective transfecting lipids, improving the efficacy of widely used lipid carriers is also crucial in order to compete with viral vectors for gene delivery. Herein, we developed formulations of commercially available lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) with synthetic cationic lipids containing amino acids,  cystine (CTT) or arginine (AT) in the head group. These lipids were used to formulate with different co-lipid compositions and were broadly categorised into two types: amino acid-based liposomes without DOTAP (CTTD and ATD) and those with DOTAP (DtATD and DtCTTD). Optimized lipid-DNA complexes of DOTAP-incorporated formulations (DtATD and DtCTTD) exhibited enhanced efficacy in transfection compared to formulations lacking DOTAP as well as commercial formulations such as DOTAP:DOPE. Notably, DtCTTD displayed superior transfection capabilities in prostate cancer (PC3) and lung cancer (A549) cell lines when compared to the widely used commercial transfection reagent, Lipofectamine. Collectively, the findings from this study suggest that DOTAP-incorporated formulations derived from amino acid-based liposomes, hold promise as effective tools for improving transfection efficacy with reduced toxicity, offering potential advancements in gene delivery applications.

Biodegradable silica nanoparticles for efficient linear DNA gene delivery.

Targeting, safety, scalability, and storage stability of vectors are still challenges in the field of nucleic acid delivery for gene therapy. Silica-based nanoparticles have been widely studied as gene carriers, exhibiting key features such as biocompatibility, simplistic synthesis, and enabling easy surface modifications for targeting. However, the ability of the formulation to incorporate DNA is limited, which restricts the number of DNA molecules that can be incorporated into the particle, thereby reducing gene expression. Here we use polymerase chain reaction (PCR)-generated linear DNA molecules to augment the coding sequences of gene-carrying nanoparticles, thereby maximizing nucleic acid loading and minimizing the size of these nanocarriers. This approach results in a remarkable 16-fold increase in protein expression six days post-transfection in cells transfected with particles carrying the linear DNA compared with particles bearing circular plasmid DNA. The study also showed that the use of linear DNA entrapped in DNA@SiO2 resulted in a much more efficient level of gene expression compared to standard transfection reagents. The system developed in this study features simplicity, scalability, and increased transfection efficiency and gene expression over existing approaches, enabled by improved embedment capabilities for linear DNA, compared to conventional methods such as lipids or polymers, which generally show greater transfection efficiency with plasmid DNA. Therefore, this novel methodology can find applications not only in gene therapy but also in research settings for high-throughput gene expression screenings.

Enhancing RNA-lipid nanoparticle delivery: Organ- and cell-specificity and barcoding strategies.

Recent advancements in RNA therapeutics highlight the critical need for precision gene delivery systems that target specific organs and cells. Lipid nanoparticles (LNPs) have emerged as key vectors in delivering mRNA and siRNA, offering protection against enzymatic degradation, enabling targeted delivery and cellular uptake, and facilitating RNA cargo release into the cytosol. This review discusses the development and optimization of organ- and cell-specific LNPs, focusing on their design, mechanisms of action, and therapeutic applications. We explore innovations such as DNA/RNA barcoding, which facilitates high-throughput screening and precise adjustments in formulations. We address major challenges, including improving endosomal escape, minimizing off-target effects, and enhancing delivery efficiencies. Notable clinical trials and recent FDA approvals illustrate the practical applications and future potential of LNP-based RNA therapies. Our findings suggest that while considerable progress has been made, continued research is essential to resolve existing limitations and bridge the gap between pre-clinical and clinical evaluation of the safety and efficacy of RNA therapeutics. This review highlights the dynamic progress in LNP research. It outlines a roadmap for future advancements in RNA-based precision medicine.

Current knowledge of hybrid nanoplatforms composed of exosomes and organic/inorganic nanoparticles for disease treatment and cell/tissue imaging.

Exosome-nanoparticle hybrid nanoplatforms, can be prepared by combining exosomes with different types of nanoparticles. The main purpose of combining exosomes with nanoparticles is to overcome the limitations of using each of them as drug delivery systems. Using nanoparticles for drug delivery has some limitations, such as high immunogenicity, poor cellular uptake, low biocompatibility, cytotoxicity, low stability, and rapid clearance by immune cells. However, using exosomes as drug delivery systems also has its own drawbacks, such as poor encapsulation efficiency, low production yield, and the inability to load large molecules. These limitations can be addressed by utilizing hybrid nanoplatforms. Additionally, the use of exosomes allows for targeted delivery within the hybrid system. Exosome-inorganic/organic hybrid nanoparticles may be used for both therapy and diagnosis in the future. This may lead to the development of personalized medicine using hybrid nanoparticles. However, there are a few challenges associated with this. Surface modifications, adding functional groups, surface charge adjustments, and preparing nanoparticles with the desired size are crucial to the possibility of preparing exosome-nanoparticle hybrids. Additional challenges for the successful implementation of hybrid platforms in medical treatments and diagnostics include scaling up the manufacturing process and ensuring consistent quality and reproducibility across various batches. This review focuses on various types of exosome-nanoparticle hybrid systems and also discusses the preparation and loading methods for these hybrid nanoplatforms. Furthermore, the potential applications of these hybrid nanocarriers in drug/gene delivery, disease treatment and diagnosis, and cell/tissue imaging are explained.

Chitosan-based smart stimuli-responsive nanoparticles for gene delivery and gene therapy: Recent progresses on cancer therapy.

Recent cancer therapy research has found that chitosan (Ch)-based nanoparticles show great potential for targeted gene delivery. Chitosan, a biocompatible and biodegradable polymer, has exceptional properties, making it an ideal carrier for therapeutic genes. These nanoparticles can respond to specific stimuli like pH, temperature, and enzymes, enabling precise delivery and regulated release of genes. In cancer therapy, these nanoparticles have proven effective in delivering genes to tumor cells, slowing tumor growth. Adjusting the nanoparticle's surface, encapsulating protective agents, and using targeting ligands have also improved gene delivery efficiency. Smart nanoparticles based on chitosan have shown promise in improving outcomes by selectively releasing genes in response to tumor conditions, enhancing targeted delivery, and reducing off-target effects. Additionally, targeting ligands on the nanoparticles' surface increases uptake and effectiveness. Although further investigation is needed to optimize the structure and composition of these nanoparticles and assess their long-term safety, these advancements pave the way for innovative gene-focused cancer therapies.

Advancements of gene therapy in cancer treatment: A comprehensive review.

Cancer is the main contributor for mortality in the world. Conventional therapy that available as the treatment options are chemotherapy, radiotherapy and surgery. However, these treatments are hardly cell-specific most of the time. Nowadays, extensive research and investigations are made to develop cell-specific approaches prior to cancer treatment. Some of them are photodynamic therapy, hyperthermia, immunotherapy, stem cell transplantation and targeted therapy. This review article will be focusing on the development of gene therapy in cancer. The objective of gene therapy is to correct specific mutant genes causing the excessive proliferation of the cell that leads to cancer. There are lots of explorations in the approach to modify the gene. The delivery of this therapy plays a big role in its success. If the inserted gene does not find its way to the target, the therapy is considered a failure. Hence, vectors are needed and the common vectors used are viral, non viral or synthetic, polymer based and lipid based vectors. The advancement of gene therapy in cancer treatment will be focussing on the top three cancer cases in the world which are breast, lung and colon cancer. In breast cancer, the discussed therapy are CRISPR/Cas9, siRNA and gene silencing whereas in colon cancer miRNA and suicide gene therapy and in lung cancer, replacement of tumor suppressor gene, CRISPR/Cas9 and miRNA.

Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier's intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.

Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety.

INTRODUCTION: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.). OBJECTIVES: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro. METHODS: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis. RESULTS: The particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %-40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.

Peptide ligands for the universal purification of exosomes by affinity chromatography.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

Self-Produced O2 CNs-Based Nanocarriers of DNA Hydrophobization Strategy Triggers Photodynamic and Mitochondrial-Derived Ferroptosis for Hepatocellular Carcinoma Combined Treatment.

Hypoxia can aggravate tumor occurrence, development, invasion, and metastasis, and greatly inhibit the photodynamic therapy (PDT) effect. Herein, carbon nitride (CNs)-based DNA and photosensitizer co-delivery systems (BPSCNs) with oxygen-producing functions are developed to address this problem. Selenide glucose (Seglu) is used as the dopant to prepare red/NIR-active CNs (SegluCNs). The tumor-targeting unit Bio-PEG2000 is utilized to construct BPSCNs nanoparticles through esterification reactions. Furthermore, DNA hydrophobization is realized via mixing P53 gene with a positively charged mitochondrial-targeted near-infrared (NIR) emitting photosensitizer (MTTPY), which is encapsulated in non-cationic BPSCNs for synergistic delivery. Ester bonds in BPSCNs@MTTPY-P53 complexes can be disrupted by lipase in the liver to facilitate P53 release, upregulated P53 expression, and promoted HIF-1α degradation in mitochondria. In addition, the oxygen produced by the complexes improved the hypoxic microenvironment of hepatocellular carcinoma (HCC), synergistically downregulated HIF-1α expression in mitochondria, promoted mitochondrial-derived ferroptosis and enhanced the PDT effect of the MTTPY unit. Both in vivo and in vitro experiments indicated that the transfected P53-DNA, produced O2 and ROS by these complexes synergistically led to mitochondrial-derived ferroptosis in hepatoma cells through the HIF-1α/SLC7A11 pathway, and completely avoiding PDT resistance caused by hypoxia, exerting a significant therapeutic role in HCC treatment.

Enhanced delivery of CRISPR/Cas9 system based on biomimetic nanoparticles for hepatitis B virus therapy.

The persistent presence of covalently closed circular DNA (cccDNA) in hepatocyte nuclei poses a significant obstacle to achieving a comprehensive cure for hepatitis B virus (HBV). Current applications of CRISPR/Cas9 for targeting and eliminating cccDNA have been confined to in vitro studies due to challenges in stable cccDNA expression in animal models and the limited non-immunogenicity of delivery systems. This study addresses these limitations by introducing a novel non-viral gene delivery system utilizing Gemini Surfactant (GS). The developed system creates stable and targeted CRISPR/Cas9 nanodrugs with a negatively charged surface through modification with red blood cell membranes (RBCM) or hepatocyte membranes (HCM), resulting in GS-pDNA@Cas9-CMs complexes. These GS-pDNA complexes demonstrated complete formation at a 4:1 w/w ratio. The in vitro transfection efficiency of GS-pDNA-HCM reached 54.61%, showing homotypic targeting and excellent safety. Additionally, the study identified the most effective single-guide RNA (sgRNA) from six sequences delivered by GS-pDNA@Cas9-HCM. Using GS-pDNA@Cas9-HCM, a significant reduction of 96.47% in in vitro HBV cccDNA and a 52.34% reduction in in vivo HBV cccDNA were observed, along with a notable decrease in other HBV-related markers. The investigation of GS complex uptake by AML-12 cells under varied time and temperature conditions revealed clathrin-mediated endocytosis (CME) for GS-pDNA and caveolin-mediated endocytosis (CVME) for GS-pDNA-HCM and GS-pDNA-RBCM. In summary, this research presents biomimetic gene-editing nanovectors based on GS (GS-pDNA@Cas9-CMs) and explores their precise and targeted clearance of cccDNA using CRISPR/Cas9, demonstrating good biocompatibility both in vitro and in vivo. This innovative approach provides a promising therapeutic strategy for advancing the cure of HBV.

TIPE2 gene transfer ameliorates aging-associated osteoarthritis in a progeria mouse model by reducing inflammation and cellular senescence.

Osteoarthritis (OA) pain is often associated with the expression of tumor necrosis factor alpha (TNF-α), suggesting that TNF-α is one of the main contributing factors that cause inflammation, pain, and OA pathology. Thus, inhibition of TNF-α could potentially improve OA symptoms and slow disease progression. Anti-TNF-α treatments with antibodies, however, require multiple treatments and cannot entirely block TNF-α. TNF-α-induced protein 8-like 2 (TIPE2) was found to regulate the immune system's homeostasis and inflammation through different mechanisms from anti-TNF-α therapies. With a single treatment of adeno-associated virus (AAV)-TIPE2 gene delivery in the accelerated aging Zmpste24-/- (Z24-/-) mouse model, we found differences in Safranin O staining intensity within the articular cartilage (AC) region of the knee between TIPE2-treated mice and control mice. The glycosaminoglycan content (orange-red) was degraded in the Z24-/- cartilage while shown to be restored in the TIPE2-treated Z24-/- cartilage. We also observed that chondrocytes in Z24-/- mice exhibited a variety of senescent-associated phenotypes. Treatment with TIPE2 decreased TNF-α-positive cells, ß-galactosidase (ß-gal) activity, and p16 expression seen in Z24-/- mice. Our study demonstrated that AAV-TIPE2 gene delivery effectively blocked TNF-α-induced inflammation and senescence, resulting in the prevention or delay of knee OA in our accelerated aging Z24-/- mouse model.