CTLA-4 silencing could promote anti-tumor effects in hepatocellular.

Preclinical and clinical research showed that immune checkpoint blockade provides beneficial effects for many patients with liver cancer. This study aimed to assess the effect of CTLA-4-specific siRNA on the proliferation, cell cycle, migration, and apoptosis of HePG2 cells. Transfection of siRNA was performed by electroporation. The viability of cells was determined through MTT assay. Flow cytometry was performed to investigate the cell cycle and apoptosis rate, and the wound-healing assay was used to determine HepG2 cells migration. The expression levels of CTLA-4, c-Myc, Ki-67, BCL-2, BAX, caspase-9 (CAS9), and MMP-2,9,13 were measured by qRT-PCR. Transfection of specific CTLA-4-siRNA significantly inhibited the expression of the CTLA-4 gene. Also, our results revealed that CTLA-4 silencing diminished the proliferation and migration as well as induced the apoptosis of HePG2 cells. CTLA-4-siRNA transfection induced the cell cycle arrest in G2 phase. Moreover, CTLA-4-siRNA transfection reduced the expression levels of c-Myc, Ki-67, BCL-2, MMP-2,9,13, and elevated the expression levels of BAX and caspase-9. Our results suggest that silencing CTLA-4 through specific siRNA may be a promising strategy for future therapeutic interventions for treating liver cancer.

Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

RNA-guided RNA silencing by an Asgard archaeal Argonaute.

Argonaute proteins are the central effectors of RNA-guided RNA silencing pathways in eukaryotes, playing crucial roles in gene repression and defense against viruses and transposons. Eukaryotic Argonautes are subdivided into two clades: AGOs generally facilitate miRNA- or siRNA-mediated silencing, while PIWIs generally facilitate piRNA-mediated silencing. It is currently unclear when and how Argonaute-based RNA silencing mechanisms arose and diverged during the emergence and early evolution of eukaryotes. Here, we show that in Asgard archaea, the closest prokaryotic relatives of eukaryotes, an evolutionary expansion of Argonaute proteins took place. In particular, a deep-branching PIWI protein (HrAgo1) encoded by the genome of the Lokiarchaeon 'Candidatus Harpocratesius repetitus' shares a common origin with eukaryotic PIWI proteins. Contrasting known prokaryotic Argonautes that use single-stranded DNA as guides and/or targets, HrAgo1 mediates RNA-guided RNA cleavage, and facilitates gene silencing when expressed in human cells and supplied with miRNA precursors. A cryo-EM structure of HrAgo1, combined with quantitative single-molecule experiments, reveals that the protein displays structural features and target-binding modes that are a mix of those of eukaryotic AGO and PIWI proteins. Thus, this deep-branching archaeal PIWI may have retained an ancestral molecular architecture that preceded the functional and mechanistic divergence of eukaryotic AGOs and PIWIs.

Targeted delivery of CEBPA-saRNA for the treatment of pancreatic ductal adenocarcinoma by transferrin receptor aptamer decorated tetrahedral framework nucleic acid.

Pancreatic cancer, predominantly pancreatic ductal adenocarcinoma (PDAC), remains a highly lethal malignancy with limited therapeutic options and a dismal prognosis. By targeting the underlying molecular abnormalities responsible for PDAC development and progression, gene therapy offers a promising strategy to overcome the challenges posed by conventional radiotherapy and chemotherapy. This study sought to explore the therapeutic potential of small activating RNAs (saRNAs) specifically targeting the CCAAT/enhancer-binding protein alpha (CEBPA) gene in PDAC. To overcome the challenges associated with saRNA delivery, tetrahedral framework nucleic acids (tFNAs) were rationally engineered as nanocarriers. These tFNAs were further functionalized with a truncated transferrin receptor aptamer (tTR14) to enhance targeting specificity for PDAC cells. The constructed tFNA-based saRNA formulation demonstrated exceptional stability, efficient saRNA release ability, substantial cellular uptake, biocompatibility, and nontoxicity. In vitro experiments revealed successful intracellular delivery of CEBPA-saRNA utilizing tTR14-decorated tFNA nanocarriers, resulting in significant activation of tumor suppressor genes, namely, CEBPA and its downstream effector P21, leading to notable inhibition of PDAC cell proliferation. Moreover, in a mouse model of PDAC, the tTR14-decorated tFNA-mediated delivery of CEBPA-saRNA effectively upregulated the expression of the CEBPA and P21 genes, consequently suppressing tumor growth. These compelling findings highlight the potential utility of saRNA delivered via a designed tFNA nanocarrier to induce the activation of tumor suppressor genes as an innovative therapeutic approach for PDAC.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge.

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

Role of Piezo2 in Schwann Cell Volume Regulation and Its Impact on Neurotrophic Release Regulation.

BACKGROUND/AIMS: Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aß-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aß and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. CONCLUSION: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.

RNAi-Mediated Silencing in the Insect Cell-Baculovirus Expression System.

RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.

New challenges in hepatocellular carcinoma: A role for PIWI-interacting RNAs?

Hepatocellular carcinoma (HCC) is the most common and deadliest subtype of liver cancer worldwide and, therefore, poses an enormous threat to global health. Understanding the molecular mechanisms underlying the development and progression of HCC is central to improving our clinical approaches. PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind to PIWI family proteins to regulate gene expression at transcriptional and post-transcriptional levels. A growing body of work shows that the dysregulation of piRNAs plays a crucial role in the progression of various human cancers. In this editorial, we report on the current knowledge of HCC-associated piRNAs and their potential clinical utility. Based on the editorial by Papadopoulos and Trifylli, on the role and clinical evaluation of exosomal circular RNAs in HCC, we highlight this other emerging class of non-coding RNAs.

[Regulation of Retrotransposons in Drosophila melanogaster Somatic Tissues].

Regulation of retrotransposon activity in somatic tissues is a complex mechanism that has still not been studied in detail. It is strongly believed that siRNA interference is main mechanism of retrotransposon activity regulation outside the gonads, but recently was demonstrated that piRNA interference participates in retrotransposon repression during somatic tissue development. In this work, using RT-PCR, we demonstrated that during ontogenesis piRNA interference determinates retrotransposon expression level on imago stage and retrotransposons demonstrate tissue-specific expression. The major factor of retrotransposon tissue-specific expression is presence of transcription factor binding sites in their regulatory regions.

piRNA associates with immune diseases.

PIWI-interacting RNA (piRNA) is the most abundant small non-coding RNA in animal cells, typically 26-31 nucleotides in length and it binds with PIWI proteins, a subfamily of Argonaute proteins. Initially discovered in germ cells, piRNA is well known for its role in silencing transposons and maintaining genome integrity. However, piRNA is also present in somatic cells as well as in extracellular vesicles and exosomes. While piRNA has been extensively studied in various diseases, particular cancer, its function in immune diseases remains unclear. In this review, we summarize current research on piRNA in immune diseases. We first introduce the basic characteristics, biogenesis and functions of piRNA. Then, we review the association of piRNA with different types of immune diseases, including autoimmune diseases, immunodeficiency diseases, infectious diseases, and other immune-related diseases. piRNA is considered a promising biomarker for diseases, highlighting the need for further research into its potential mechanisms in disease pathogenesis.

[Pharmacological and clinical profile of inclisiran sodium, a long-acting LDL cholesterol lowering siRNA, LEQVIO for s.c. injection syringe 300†|mg].

Inclisiran sodium (Brand name: LEQVIO® for s.c. injection syringe 300 |mg, hereinafter referred to as inclisiran), a small interfering ribonucleic acid (siRNA) product that targets the mRNA that encodes the proprotein convertase subtilisin/kexin type 9 (PCSK9) protein was approved on September 25, 2023 for the indication of "Familial hypercholesterolemia, hypercholesterolemia" in Japan. Inclisiran is conjugated on the sense strand with triantennary N-acetylgalactosamine to facilitate uptake by hepatocytes. In vitro and in vivo pharmacology studies demonstrated the lowering effects of PCSK9 and LDL-C in hepatocytes and cynomolgus monkeys. It was considered unlikely to cause clinically significant risks due to toxicities arising from complementary binding to non-target RNA sequences (hybridization-dependent off-target effects). Clinical trials conducted globally including Japan in patients with familial hypercholesterolemia and hypercholesterolemia who did not reach the LDL-C target showed that inclisiran sodium 300 |mg dosed at Day 1, Day 90 and then every 6 months demonstrated significant LDL-C reduction and the efficacy sustained long. The majority of patients achieved the guideline recommended LDL-C targets. Inclisiran sodium 300 |mg was well tolerated and there were no specific safety concerns. Therefore, inclisiran is expected to be a new therapeutic option for the patients with familial hypercholesterolemia and hypercholesterolemia.

Advancements and Future Prospects in Molecular Targeted and siRNA Therapies for Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is an oncological myeloproliferative disorder that accounts for 15 to 20% of all adult leukemia cases. The molecular basis of this disease lies in the formation of a chimeric oncogene BCR-ABL1. The protein product of this gene, p210 BCR-ABL1, exhibits abnormally high constitutive tyrosine kinase activity. Over recent decades, several targeted tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1 have been developed and introduced into clinical practice. These inhibitors suppress BCR-ABL1 activity through various mechanisms. Furthermore, the advent of RNA interference technology has enabled the highly specific inhibition of BCR-ABL1 transcript expression using small interfering RNA (siRNA). This experimental evidence opens avenues for the development of a novel therapeutic strategy for CML, termed siRNA therapy. The review delves into molecular genetic mechanisms underlying the pathogenesis of CML, challenges in CML therapy, potential molecular targets for drug development, and the latest results from the application of siRNAs in in vitro and in vivo CML models.

PIWI-Interacting RNAs: A Pivotal Regulator in Neurological Development and Disease.

PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs (sncRNAs) with 24-32 nucleotides (nt), were initially identified in the reproductive system. Unlike microRNAs (miRNAs) or small interfering RNAs (siRNAs), piRNAs normally guide P-element-induced wimpy testis protein (PIWI) families to slice extensively complementary transposon transcripts without the seed pairing. Numerous studies have shown that piRNAs are abundantly expressed in the brain, and many of them are aberrantly regulated in central neural system (CNS) disorders. However, the role of piRNAs in the related developmental and pathological processes is unclear. The elucidation of piRNAs/PIWI would greatly improve the understanding of CNS development and ultimately lead to novel strategies to treat neural diseases. In this review, we summarized the relevant structure, properties, and databases of piRNAs and their functional roles in neural development and degenerative disorders. We hope that future studies of these piRNAs will facilitate the development of RNA-based therapeutics for CNS disorders.

Potential molecular mechanisms and clinical implications of piRNAs in preeclampsia: a review.

Preeclampsia is a multisystem progressive condition and is one of the most serious complications of pregnancy. Owing to its unclear pathogenesis, there are no precise and effective therapeutic targets for preeclampsia, and the only available treatment strategy is to terminate the pregnancy and eliminate the clinical symptoms. In recent years, non-coding RNAs have become a hotspot in preeclampsia research and have shown promise as effective biomarkers for the early diagnosis of preeclampsia over conventional biochemical markers. PIWI-interacting RNAs, novel small non-coding RNA that interact with PIWI proteins, are involved in the pathogenesis of various diseases at the transcriptional or post-transcriptional level. However, the mechanisms underlying the role of PIWI-interacting RNAs in the pathogenesis of preeclampsia remain unclear. In this review, we discuss the findings of existing studies on PIWI-interacting RNA biogenesis, functions, and their possible roles in preeclampsia, providing novel insights into the potential application of PIWI-interacting RNAs in the early diagnosis and clinical treatment of preeclampsia.

The role of cyclin-dependent kinase inhibitor 3 in the proliferation and migration of renal cell carcinoma.

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, is over expressed in renal cell carcinoma (RCC). However, the cell biology functions of RCC are not well understood. The present study aimed to verify the ability of CDKN3 to promote the proliferation and migration of RCC through in vitro experiments. Subsequently, the clinical prognostic effects were analyzed using The Cancer Genome Atlas (TCGA; https://www.cancer.gov/) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The chelators, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an analogue of the anti-tumor agent, were screened through bioinformatics analysis. The expression of CDKN3 is positively correlated with the IC50 of Dp44mT. In two RCC cell lines, 786-0 and Caki-1, we conducted small interfering RNA (siRNA) knockdown of CDKN3 and overexpression of CDKN3 by transfection plasmid. Subsequently, we administered Dp44mT to examine the resulting alterations in cell proliferation, migration, and apoptosis, thereby elucidating the role of CDKN3 and Dp44mT in these processes. The results of the experiment revealed a positive association between CDKN3 expression and the proliferation of RCC cell lines. Down-regulating CDKN3 significantly increased the apoptosis rate and inhibited cell migration in 786-0 and Caki-1 cells. Furthermore, bioinformatics analysis revealed a high expression of CDKN3 in RCC and a negative association between CDKN3 expression and survival. Gene set enrichment analysis (GSEA) revealed a significant association between high CDKN3 expression and the cell cycle pathway. Furthermore, we identified Dp44mT as a drug highly correlated with CDKN3 through the database. Subsequent addition of Dp44mT resulted in similar findings to those observed upon CDKN3 knockdown. Our findings have important implications for the diagnosis and treatment of CDKN3 in RCC. Additionally, Dp44mT is likely to be a promising candidate for future clinical applications.

Polyethyleneimine-mediated assembly of DNA nanotubes for KRAS siRNA delivery in lung adenocarcinoma therapy.

Self-assembled DNA nanostructures hold great promise in biosensing, drug delivery and nanomedicine. Nevertheless, challenges like instability and inefficiency in cellular uptake of DNA nanostructures under physiological conditions limit their practical use. To tackle these obstacles, this study proposes a novel approach that integrates the cationic polymer polyethyleneimine (PEI) with DNA self-assembly. The hypothesis is that the positively charged linear PEI can facilitate the self-assembly of DNA nanostructures, safeguard them against harsh conditions and impart them with the cellular penetration characteristic of PEI. As a demonstration, a DNA nanotube (PNT) was successfully synthesized through PEI mediation, and it exhibited significantly enhanced stability and cellular uptake efficiency compared to conventional Mg2+-assembled DNA nanotubes. The internalization mechanism was further found to be both clathrin-mediated and caveolin-mediated endocytosis, influenced by both PEI and DNA. To showcase the applicability of this hybrid nanostructure for biomedical settings, the KRAS siRNA-loaded PNT was efficiently delivered into lung adenocarcinoma cells, leading to excellent anticancer effects in vitro. These findings suggest that the PEI-mediated DNA assembly could become a valuable tool for future biomedical applications.

VmRDR2 of Valsa mali mediates the generation of VmR2-siR1 that suppresses apple resistance by RNA interference.

Cross-kingdom RNA interference (RNAi) is a crucial mechanism in host-pathogen interactions, with RNA-dependent RNA polymerase (RdRP) playing a vital role in signal amplification during RNAi. However, the role of pathogenic fungal RdRP in siRNAs generation and the regulation of plant-pathogen interactions remains elusive. Using deep sequencing, molecular, genetic, and biochemical approaches, this study revealed that VmRDR2 of Valsa mali regulates VmR2-siR1 to suppress the disease resistance-related gene MdLRP14 in apple. Both VmRDR1 and VmRDR2 are essential for the pathogenicity of V. mali in apple, with VmRDR2 mediating the generation of endogenous siRNAs, including an infection-related siRNA, VmR2-siR1. This siRNA specifically degrades the apple intracellular LRR-RI protein gene MdLRP14 in a sequence-specific manner, and overexpression of MdLRP14 enhances apple resistance against V. mali, which can be suppressed by VmR2-siR1. Conversely, MdLRP14 knockdown reduces resistance. In summary, this study demonstrates that VmRDR2 contributes to the generation of VmR2-siR1, which silences the host's intracellular LRR protein gene, thereby inhibiting host resistance. These findings offer novel insights into the fungi-mediated pathogenicity mechanism through RNAi.

Novel Lipid Nanocomplex Co-Carrying Bcl2 siRNA and Quantum Dots for EGF Receptor-Targeted Anti-Cancer Theranosis.

Many different types of nanoparticles have been suggested for tumor-targeted theranosis. However, most systems were prepared through a series of complicated processes and could not even overcome the blood-immune barriers. For the accurate diagnosis and effective treatment of cancers, herein we suggested the lipid micellar structure capturing quantum dot (QD) for cancer theranosis. The QD/lipid micelles (QDMs) were prepared using a simple self-assembly procedure and then conjugated with anti-epidermal growth factor receptor (EGFR) antibodies for tumor targeting. As a therapeutic agent, Bcl2 siRNA-cholesterol conjugates were loaded on the surface of QDMs. The EGFR-directed QDMs containing Bcl2 siRNA, so-called immuno-QDM/siBcl2 (iQDM/siBcl2), exhibited the more effective delivery of QDs and siBcl2 to target human colorectal cancer cells in cultures as well as in mouse xenografts. The effective in vivo targeting of iQDM/siBcl2 resulted in a more enhanced therapeutic efficacy of siBcl2 to the target cancer in mice. Based on the results, anti-EGFR QDM capturing therapeutic siRNA could be suggested as an alternative modality for tumor-targeted theranosis.

Photosensitive and dual-targeted chromium nanoparticle delivering small interfering RNA YTHDF1 for molecular-targeted immunotherapy in liver cancer.

Tumor-associated macrophages (TAMs) are a promising target for cancer immunotherapy, but delivering therapeutic agents to TAMs within the tumor microenvironment (TME) is challenging. In this study, a photosensitive, dual-targeting nanoparticle system (M.RGD@Cr-CTS-siYTHDF1 NPs) was developed. The structure includes a shell of DSPE-modified RGD peptides targeting integrin receptors on tumor cells and carboxymethyl mannose targeting CD206 receptors on macrophages, with a core of chitosan adsorbing m6A reading protein YTHDF1 siRNA and chromium nanoparticles (Cr NPs). The approach is specifically designed to target TAM and cancer cells, utilizing the photothermal effect of Cr NPs to disrupt the TME and deliver siYTHDF1 to TAM. In experiments with tumor-bearing mice, M.RGD@Cr-CTS-siYTHDF1 NPs, when exposed to laser irradiation, effectively killed tumor cells, disrupted the TME, delivered siYTHDF1 to TAMs, silenced the YTHDF1 gene, and shifted the STAT3-STAT1 equilibrium by reducing STAT3 and enhancing STAT1 expression. This reprogramming of TAMs towards an anti-tumor phenotype led to a pro-immunogenic TME state. The strategy also suppressed immunosuppressive IL-10 production, increased expression of immunostimulatory factors (IL-12 and IFN-γ), boosted CD8 + T cell infiltration and M1-type TAMs, and reduced Tregs and M2-type TAMs within the TME. In conclusion, the dual-targeting M.RGD@Cr-CTS-siYTHDF1 NPs, integrating dual-targeting capabilities with photothermal therapy (PTT) and RNA interference, offer a promising approach for molecular targeted cancer immunotherapy with potential for clinical application.

Cationic Polythiophene as Gene Carrier and Sonosensitizer for Sonodynamic Synergic Gene Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal and highly malignant tumors. Sonodynamic therapy (SDT) is a new cancer treatment method. One of its unique advantages lies in the treatment of deep tumors due to its excellent tissue penetration ability caused by ultrasound (US). However, most sonosensitizers suffer from weak sonodynamic activity and poor tumor-targeting ability. In addition, small interfering RNA (siRNA) is a promising anticancer drug, and the efficacy of siRNA-based gene therapy largely depends on the cell impermeability of the gene carrier. Here, we designed and synthesized a cationic polythiophene derivative (PT2) that can be used as a siRNA carrier for gene therapy. Moreover, PT2 could generate singlet oxygen (1O2) and hydroxyl radicals (O2•-) under US irradiation, which suggests that PT2 could be used for SDT. Our study discovered that NUDT1 promoted HCC proliferation and inhibited intracellular ROS production. Therefore, si-NUDT1 was designed and synthesized. NUDT1 silencing can inhibit the proliferation of tumor cells and increase the production of intracellular ROS to further improve the efficacy of SDT. Then, si-NUDT1 assembled with PT2 and DSPE-PEG-FA to prepare a novel tumor-targeting nanodrug (PT2-siRNA@PEG-FA) for synergic SDT and gene therapy of HCC.