Porous Collagen Sponge Loaded with Large Efficacy-Potentiated Exosome-Mimicking Nanovesicles for Diabetic Skin Wound Healing.

Diabetic skin wounds are difficult to heal quickly due to insufficient angiogenesis and prolonged inflammation, which is an urgent clinical problem. To address this clinical problem, it becomes imperative to develop a dressing that can promote revascularization and reduce inflammation during diabetic skin healing. Herein, a multifunctional collagen dressing (CTM) was constructed by loading large efficacy-potentiated exosome-mimicking nanovesicles (L-Meseomes) into a porous collagen sponge with transglutaminase (TGase). L-Meseomes were constructed in previous research with the function of promoting cell proliferation, migration, and angiogenesis and inhibiting inflammation. CTM has a three-dimensional porous network structure with good biocompatibility, swelling properties, and degradability and could release L-Meseome slowly. In vitro experiments showed that CTM could promote the proliferation of fibroblasts and the polarization of macrophages to the anti-inflammatory phenotype. For in vivo experiments, on the 21st day after surgery, the wound healing rates of the control and CTM were 83.026 ± 4.17% and 93.12 ± 2.16%, respectively; the epidermal maturation and dermal differentiation scores in CTM were approximately four times that of the control group, and the skin epidermal thickness of the CTM group was approximately 20 µm, which was closest to that of normal rats. CTM could significantly improve wound healing in diabetic rats by promoting anti-inflammation, angiogenesis, epidermal recovery, and dermal collagen deposition. In summary, the multifunctional collagen dressing CTM could significantly promote the healing of diabetic skin wounds, which provides a new strategy for diabetic wound healing in the clinic.

Orientational Nanoconjugation with Gold Endows Marked Antimicrobial Potential and Drugability of Ultrashort Dipeptides.

Antibiotic resistance is a global threat. Antimicrobial peptides (AMPs) are highly desirable to treat multidrug-resistant pathogen infection. However, few AMPs are clinically available, due to high cost, instability, and poor selectivity. Here, ultrashort AMPs (2-3 residues with an N-terminal cysteine) are designed and assembled as gold nanoparticles. Au-S conjugation and ultrashort size restrict nonspecific reactions and peptide orientation, thus concentrating positively charged residues on the surface. The nanostructured assemblies enormously enhance antimicrobial abilities by 1000-6000-fold and stability. One representative (Au-Cys-Arg-NH2, Au_CR) shows selective antibacterial activity against Staphylococcus aureus with 10 nM minimal inhibitory concentration. Au_CR has comparable or better in vivo antimicrobial potency than vancomycin and methicillin, with low propensity to induce resistance, little side effects, and high stability (17.5 h plasma half-life). Au_CR acts by inducing collapse of membrane potential and rupture of the bacterial membrane. The report provides insights for developing AMP-metal nanohybrids, particularly tethering nonspecific reactions and AMP orientation on the metal surface.

Combination of irinotecan silicasome nanoparticles with radiation therapy sensitizes immunotherapy by modulating the activation of the cGAS/STING pathway for colorectal cancer.

Our previous clinical trial (Identifier: NCT02605265) revealed that addition of irinotecan (IRIN) to neoadjuvant chemoradiotherapy for rectal cancer could improve the curative effect. However, the adverse effects caused by IRIN limited the wide application of IRIN chemoradiotherapy. This study aimed to explore the mechanism under the synergistic effects of IRIN plus radiation therapy in colorectal cancer (CRC) cells and optimization of IRIN delivery via a silicasome nanocarrier in vivo. Our results revealed that compared with single IRIN or radiation treatment, IRIN combined with radiation therapy remarkably activated the intracellular cGAS/STING pathway, and promoted the expression levels of major histocompatibility complex class I (MHC-I) and programmed death ligand 1 (PD-L1). Further, a silicasome (mesoporous silica nanoparticle coated with lipid bilayer) nanocarrier was utilized to improve the delivery of IRIN with enhanced efficacy and reduced side effects. In the MC38 CRC syngeneic tumor model, IRIN silicasome combined with radiation therapy demonstrated a greater antitumor efficacy than free IRIN plus radiation therapy. Flow cytometry showed the increased number of CD4+ T cells, CD8+ T cells, and dendritic cells (DCs) in tumor in the IRIN silicasome plus radiation group. The immunofluorescence staining further confirmed the activated immune microenvironment with the elevated interferon-γ (IFN-γ) deposition. Besides, the antitumor effect of IRIN silicasome plus radiation therapy was synergistically enhanced by anti-PD-1 immunotherapy. These findings indicated that the combination of IRIN silicasome with radiation therapy could sensitize immunotherapy by manipulating the cGAS/STING pathway serving as a new strategy for CRC treatment.

Lactate oxidase nanocapsules boost T cell immunity and efficacy of cancer immunotherapy.

Cancer immunotherapy has reshaped the landscape of cancer treatment. However, its efficacy is still limited by tumor immunosuppression associated with the excessive production of lactate by cancer cells. Although extensive efforts have been made to reduce lactate concentrations through inhibition of lactate dehydrogenase, such inhibitors disrupt the metabolism of healthy cells, causing severe nonspecific toxicity. We report herein a nanocapsule enzyme therapeutic based on lactate oxidase, which reduces lactate concentrations and releases immunostimulatory hydrogen peroxide, averting tumor immunosuppression and improving the efficacy of immune checkpoint blockade treatment. As demonstrated in a murine melanoma model and a humanized mouse model of triple-negative breast cancer, this enzyme therapeutic affords an effective tool toward more effective cancer immunotherapy.

Inflammation-Stimulated MSC-Derived Small Extracellular Vesicle miR-27b-3p Regulates Macrophages by Targeting CSF-1 to Promote Temporomandibular Joint Condylar Regeneration.

Small extracellular vesicles (sEVs) secreted by mesenchymal stem cells (MSCs) have been extensively studied in recent years. sEV contents change with the secreting cell state. When MSCs are exposed to an inflammatory environment, they release more functional growth factors, exosomes, and chemokines. Herein, MSCs are stimulated to alter sEV cargos and functions to regulate the inflammatory microenvironment and promote tissue regeneration. Sequencing of sEV miRNAs shows that certain RNAs conducive to cell function are upregulated. In this study, in vitro cell function experiments show that both inflammation-stimulated adipose-derived MSC (ADSC)-derived sEV (IAE) and normal ADSC-derived sEV (AE) promote cell proliferation; IAE also significantly improves cell migration. Regarding macrophage polarization regulation, IAE significantly promotes M2 macrophage differentiation. RNA-sequencing analysis indicates that high miR-27b-3p expression levels in IAE may regulate macrophages by targeting macrophage colony-stimulating factor-1 (CSF-1). In vivo, a rabbit temporomandibular joint (TMJ) condylar osteochondral defect model shows that both AE and IAE promote TMJ regeneration, with IAE having the most significant therapeutic effect. Therefore, the authors confirm that exposing MSCs to an inflammatory environment can feasibly enhance sEV functions and that modified sEVs achieve better therapeutic effects.

Multifunctional Lipid Bilayer Nanocarriers for Cancer Immunotherapy in Heterogeneous Tumor Microenvironments, Combining Immunogenic Cell Death Stimuli with Immune Modulatory Drugs.

In addition to the contribution of cancer cells, the solid tumor microenvironment (TME) has a critical role in determining tumor expansion, antitumor immunity, and the response to immunotherapy. Understanding the details of the complex interplay between cancer cells and components of the TME provides an unprecedented opportunity to explore combination therapy for intervening in the immune landscape to improve immunotherapy outcome. One approach is the introduction of multifunctional nanocarriers, capable of delivering drug combinations that provide immunogenic stimuli for improvement of tumor antigen presentation, contemporaneous with the delivery of coformulated drug or synthetic molecules that provide immune danger signals or interfere in immune-escape, immune-suppressive, and T-cell exclusion pathways. This forward-looking review will discuss the use of lipid-bilayer-encapsulated liposomes and mesoporous silica nanoparticles for combination immunotherapy of the heterogeneous immune landscapes in pancreatic ductal adenocarcinoma and triple-negative breast cancer. We describe how the combination of remote drug loading and lipid bilayer encapsulation is used for the synthesis of synergistic drug combinations that induce immunogenic cell death, interfere in the PD-1/PD-L1 axis, inhibit the indoleamine-pyrrole 2,3-dioxygenase (IDO-1) immune metabolic pathway, restore spatial access to activated T-cells to the cancer site, or reduce the impact of immunosuppressive stromal components. We show how an integration of current knowledge and future discovery can be used for a rational approach to nanoenabled cancer immunotherapy.

Reconstruction of Epidural Fat to Prevent Epidural Fibrosis After Laminectomy in Rabbits.

Laminectomy can effectively decompress the spinal cord and expand the vertebral canal. However, the fibrosis that appears may cause adherence and recompression of the spinal cord or/and nerve root, which may cause failed back syndrome (FBS) and make the reexposure process more difficult. Reconstruction of the epidural fat may be an ideal method to achieve satisfactory results. Thirty-six New Zealand rabbits were randomly divided into three groups: control, extracellular matrix (ECM), and ECM+aMSCs groups. Saline, ECM gel, and ECM+aMSC complex were placed, respectively, at the fifth lumbar vertebrate of the rabbits. Epidural fat and fibrosis formation were detected by magnetic resonance imaging (MRI) and histologically at the 4th, 8th, and 12th weeks. Quantitative RT-PCR was used to detect the expression of interleukin 6 (IL-6) and transforming growth factor ß (TGF-ß). MRI and Oil Red O staining revealed epidural fat formation at the 12th week in the ECM+aMSCs group. Hematoxylin and eosin staining showed that the numbers of fibroblasts in the ECM gel and ECM+aMSCs groups were less than the control group at the 4th and 8th weeks (p < 0.05). Masson's trichrome staining showed that the proportion of collagen fibers in the ECM gel and ECM+aMSCs group was lower than the control group (p < 0.05). Quantitative RT-PCR showed the expressions of TGF-ß and IL-6 were lower in the ECM gel and ECM+aMSCs group than those in the control group (p < 0.05) at the 4th week, but higher at the 8th week. We successfully reconstructed the epidural fat with ECM gel and aMSC complex; additionally, IL-6 and TGF-ß cytokines were lower at early stage after laminectomy. Impact statement Our study revealed that epidural fat formed at the 12th week in the extracellular matrix (ECM) plus mesenchymal stem cell (MSC) group after laminectomy in rabbits; additionally, transforming growth factor ß (TGF-ß) (fibrosis) and interleukin 6 (IL-6) (inflammation) expression was reduced. Thus, we believe that our study makes a significant contribution to the literature because we were able to successfully reconstruct the epidural fat with an ECM gel combined with MSCs and reduce local inflammation.

Prodrug nanoparticles rationally integrating stroma modification and chemotherapy to treat metastatic pancreatic cancer.

Accumulating evidence suggests that stromal modifications improve chemotherapeutic outcomes in pancreatic ductal adenocarcinoma (PDAC). However, combination regimens of stroma-modifying agents and small-molecule cytotoxic drugs have achieved only limited improvements in the clinic, probably due to unsatisfactory pharmacokinetic profiles and restricted drug distribution in tumors. Here, we developed self-assembled prodrug nanoparticles integrating a stromal reprogramming inducer, calcipotriol (CAL), and a potent chemotherapeutic agent, 7-Ethyl-10-hydroxycamptothecin (SN38), to treat PDAC. While SN38 is conjugated to the block polymer backbone, CAL is loaded into the inner hydrophobic space during polymer self-assembly into nanoparticles. To achieve an efficient drug co-package, a planar and hydrophobic cholesterol domain was introduced to stabilize the hydrophobic CAL. Notably, the blood circulation time of CAL significantly improved as CAL|SN38 nanoparticle (CAL|SN38 NP). In addition, CAL|SN38 NP treatment significantly decreased the expression of N-cadherin, collagen, and fibronectin in tumors, which play critical roles in PDAC metastasis. Potent inhibition of primary tumor growth and vigorous anti-metastasis effects were observed after systemic administration of CAL|SN38 NP to stroma-rich PDAC orthotopic tumor-bearing mice. These findings provide a promising paradigm for developing tailor-made nanoparticles with potent stroma-modification capability to combat metastatic cancer.

Nanocellulose Length Determines the Differential Cytotoxic Effects and Inflammatory Responses in Macrophages and Hepatocytes.

Nanocellulose including cellulose nanocrystal (CNC) and cellulose nanofiber (CNF) has attracted much attention due to its exceptional mechanical, chemical, and rheological properties. Although considered biocompatible, recent reports have demonstrated nanocellulose can be hazardous, including serving as drug carriers that accumulate in the liver. However, the nanocellulose effects on liver cells, including Kupffer cells (KCs) and hepatocytes are unclear. Here, the toxicity of nanocellulose with different lengths is compared, including the shorter CNCs (CNC-1, CNC-2, and CNC-3) and longer CNF (CNF-1 and CNF-2), to liver cells. While all CNCs triggered significant cytotoxicity in KCs and only CNC-2 induced toxicity to hepatocytes, CNFs failed to induce significant cytotoxicity due to their minimal cellular uptake. The phagocytosis of CNCs by KCs induced mitochondria ROS generation, caspase-3/7 activation, and apoptotic cell death as well as lysosomal damage, cathepsin B release, NLRP3 inflammasome and caspase-1 activation, and IL-1ß production. The cellular uptake of CNC-2 by hepatocytes is through clathrin-mediated endocytosis, and it induced the caspase-3/7-mediated apoptosis. CNC-2 shows the highest levels of uptake and cytotoxicity among CNCs. These results demonstrate the length-dependent mechanisms of toxicity on liver cells in a cell type-dependent fashion, providing information to safely use nanocellulose for biomedical applications.

Lateral size of graphene oxide determines differential cellular uptake and cell death pathways in Kupffer cells, LSECs, and hepatocytes.

As a representative two-dimensional (2D) nanomaterial, graphene oxide (GO) has shown high potential in many applications due to its large surface area, high flexibility, and excellent dispersibility in aqueous solutions. These properties make GO an ideal candidate for bio-imaging, drug delivery, and cancer therapy. When delivered to the body, GO has been shown to accumulate in the liver, the primary accumulation site of systemic delivery or secondary spread from other uptake sites, and induce liver toxicity. However, the contribution of the GO physicochemical properties and individual liver cell types to this toxicity is unclear due to property variations and diverse cell types in the liver. Herein, we compare the effects of GOs with small (GO-S) and large (GO-L) lateral sizes in three major cell types in liver, Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatocytes. While GOs induced cytotoxicity in KCs, they induced significantly less toxicity in LSECs and hepatocytes. For KCs, we found that GOs were phagocytosed that triggered NADPH oxidase mediated plasma membrane lipid peroxidation, which leads to PLC activation, calcium flux, mitochondrial ROS generation, and NLRP3 inflammasome activation. The subsequent caspase-1 activation induced IL-1ß production and GSDMD-mediated pyroptosis. These effects were lateral size-dependent with GO-L showing stronger effects than GO-S. Amongst the liver cell types, decreased cell association and the absence of lipid peroxidation resulted in low cytotoxicity in LSECs and hepatocytes. Using additional GO samples with different lateral sizes, surface functionalities, or thickness, we further confirmed the differential cytotoxic effects in liver cells and the major role of GO lateral size in KUP5 pyroptosis by correlation studies. These findings delineated the GO effects on cellular uptake and cell death pathways in liver cells, and provide valuable information to further evaluate GO effects on the liver for biomedical applications.

Combination Chemo-Immunotherapy for Pancreatic Cancer Using the Immunogenic Effects of an Irinotecan Silicasome Nanocarrier Plus Anti-PD-1.

There is an urgent need to develop new life-prolonging therapy for pancreatic ductal adenocarcinoma (PDAC). It is demonstrated that improved irinotecan delivery by a lipid bilayer coated mesoporous silica nanoparticle, also known as a silicasome, can improve PDAC survival through a chemo-immunotherapy response in an orthotopic Kras-dependent pancreatic cancer model. This discovery is premised on the weak-basic properties of irinotecan, which neutralizes the acidic lysosomal pH in PDAC cells. This effect triggers a linked downstream cascade of events that include autophagy inhibition, endoplasmic reticulum stress, immunogenic cell death (ICD), and programmed death-ligand 1 (PD-L1) expression. ICD is characterized by calreticulin expression and high-mobility group box 1 (HMGB1) release in dying Kras-induced pancreatic cancer (KPC) cells, which is demonstrated in a vaccination experiment to prevent KPC tumor growth on the contralateral site. The improved delivery of irinotecan by the silicasome is accompanied by robust antitumor immunity, which can be synergistically enhanced by anti-PD-1 in the orthotopic model. Immunophenotyping confirms the expression of calreticulin, HMGB1, PD-L1, and an autophagy marker, in addition to perforin and granzyme B deposition. The chemo-immunotherapy response elicited by the silicasome is more robust than free or a liposomal drug, Onivyde. The silicasome plus anti-PD-1 leads to significantly enhanced survival improvement, and is far superior to anti-PD-1 plus either free irinotecan or Onivyde.

Development of Facile and Versatile Platinum Drug Delivering Silicasome Nanocarriers for Efficient Pancreatic Cancer Chemo-Immunotherapy.

In this study a mesoporous silica nanoparticle (MSNP) based platform is developed for high-dose loading of a range of activated platinum (Pt) chemo agents that can be attached to the porous interior through the use of electrostatic and coordination chemistry under weak-basic pH conditions. In addition to the design feature for improving drug delivery, the MSNP can also be encapsulated in a coated lipid bilayer (silicasome), to improve the colloidal stability after intravenous (IV) injection. Improved pharmacokinetics and intratumor delivery of encapsulated activated oxaliplatin (1,2-diamminocyclohexane platinum(II) (DACHPt)) over free drug in an orthotopic Kras-derived pancreatic cancer (PDAC) model is demonstrated. Not only does IV injection of the DACHPt silicasome provide more efficacious cytotoxic tumor cell killing, but can also demonstrate that chemotherapy-induced cell death is accompanied by the features of immunogenic cell death (ICD) as well as a dramatic reduction in bone marrow toxicity. The added ICD features are reflected by calreticulin and high-mobility group box 1 expression, along with increased CD8+ /FoxP3+ T-cell ratios and evidence of perforin and granzyme B release at the tumor site. Subsequent performance of a survival experiment, demonstrates that the DACHPt silicasome generates a significant improvement in survival outcome, which can be extended by delayed administration of the anti-PD-1 antibody.

Immune checkpoint inhibition in syngeneic mouse cancer models by a silicasome nanocarrier delivering a GSK3 inhibitor.

Checkpoint blocking antibodies that interfere in the PD-1/PD-L1 axis provide effective cancer immunotherapy for tumors that are immune inflamed or induced to become "hot". It has also been demonstrated that a small molecule inhibitor of the signaling hub kinase GSK3 can interfere in the PD-1/PD-L1 axis in T-cells by suppressing PD-1 expression. This provides an alternative approach to intervening in the PD-1/PD-L1 axis to provide cancer immunotherapy. In this communication, we demonstrate the remote loading of GSK3 inhibitor AZD1080 into the porous interior of mesoporous silica nanoparticles coated with a lipid bilayer (a.k.a. silicasomes). In a MC38 colon cancer model, intravenous injection (IV) of silicasome-encapsulated AZD1080 significantly improved biodistribution and drug delivery to the tumor site. The improved drug delivery was accompanied by cytotoxic MC38 tumor cell killing by perforin-releasing CD8+ T-cells, exhibiting reduced PD-1 expression. IV injection of encapsulated AZD1080 also resulted in significant tumor shrinkage in other syngeneic mouse tumor models, including another colorectal tumor (CT26), as well as pancreas (KPC) and lung (LLC) cancer models. Not only was the therapeutic efficacy of encapsulated AZD1080 similar or better than anti-PD-1 antibody, but the treatment was devoid of treatment toxicity. These results provide proof-of-principal demonstration of the feasibility of using encapsulated delivery of a GSK3 inhibitor to provide cancer immunotherapy, with the possibility to be used as a monotherapy or in combination with chemotherapy or other immunomodulatory agents.

Liposomal Delivery of Mitoxantrone and a Cholesteryl Indoximod Prodrug Provides Effective Chemo-immunotherapy in Multiple Solid Tumors.

We developed a custom-designed liposome carrier for codelivery of a potent immunogenic cell death (ICD) stimulus plus an inhibitor of the indoleamine 2,3-dioxygenase (IDO-1) pathway to establish a chemo-immunotherapy approach for solid tumors in syngeneic mice. The carrier was constructed by remote import of the anthraquinone chemotherapeutic agent, mitoxantrone (MTO), into the liposomes, which were further endowed with a cholesterol-conjugated indoximod (IND) prodrug in the lipid bilayer. For proof-of-principle testing, we used IV injection of the MTO/IND liposome in a CT26 colon cancer model to demonstrate the generation of a robust immune response, characterized by the appearance of ICD markers (CRT and HMGB-1) as well as evidence of cytotoxic cancer cell death, mediated by perforin and granzyme B. Noteworthy, the cytotoxic effects involved natural killer (NK) cell, which suggests a different type of ICD response. The immunotherapy response was significantly augmented by codelivery of the IND prodrug, which induced additional CRT expression, reduced number of Foxp3+ Treg, and increased perforin release, in addition to extending animal survival beyond the effect of an MTO-only liposome. The outcome reflects the improved pharmacokinetics of MTO delivery to the cancer site by the carrier. In light of the success in the CT26 model, we also assessed the platform efficacy in further breast cancer (EMT6 and 4T1) and renal cancer (RENCA) models, which overexpress IDO-1. Encapsulated MTO delivery was highly effective for inducing chemo-immunotherapy responses, with NK participation, in all tumor models. Moreover, the growth inhibitory effect of MTO was enhanced by IND codelivery in EMT6 and 4T1 tumors. All considered, our data support the use of encapsulated MTO delivery for chemo-immunotherapy, with the possibility to boost the immune response by codelivery of an IDO-1 pathway inhibitor.

Use of ratiometrically designed nanocarrier targeting CDK4/6 and autophagy pathways for effective pancreatic cancer treatment.

Aberrant cell cycle machinery and loss of the CDKN2A tumor suppressor locus make CDK4/6 a potential target in pancreatic ductal adenocarcinoma (PDAC). However, a vast majority of PDAC cases do not harbor a durable response to monotherapy of CDK4/6 inhibitor. Utilizing remote loading to co-encapsulate CDK4/6 inhibitor palbociclib (PAL) and an autophagy inhibitor hydroxychloroquine (HCQ), we demonstrate a ratiometrically designed mesoporous silica nanoformulation with synergistic efficacy in subcutaneous and orthotopic PDAC mouse models. The synergism is attributed to the effective intratumoral buildup of PAL/HCQ, which otherwise exhibit distinctly different circulatory and biodistribution profile. PAL/HCQ co-delivery nanoparticles lead to the most effective shrinkage of PDAC compared to various controls, including free drug mixture. Immunohistochemistry reveals that PAL/HCQ co-delivery nanoparticles trigger anti-apoptotic pathway after repetitive intravenous administrations in mice. When combined with a Bcl inhibitor, the performance of co-delivery nanoparticles is further improved, leading to a long-lasting anti-PDAC effect in vivo.

Safety Considerations of Cancer Nanomedicine-A Key Step toward Translation.

The rate of translational effort of nanomedicine requires strategic planning of nanosafety research in order to enable clinical trials and safe use of nanomedicine in patients. Herein, the experiences that have emerged based on the safety data of classic liposomal formulations in the space of oncology are discussed, along with a description of the new challenges that need to be addressed according to the rapid expansion of nanomedicine platform beyond liposomes. It is valuable to consider the combined use of predictive toxicological assessment supported by deliberate investigation on aspects such as absorption, distribution, metabolism, and excretion (ADME) and toxicokinetic profiles, the risk that may be introduced during nanomanufacture, unique nanomaterials properties, and nonobvious nanosafety endpoints, for example. These efforts will allow the generation of investigational new drug-enabling safety data that can be incorporated into a rational infrastructure for regulatory decision-making. Since the safety assessment relates to nanomaterials, the investigation should cover the important physicochemical properties of the material that may lead to hazards when the nanomedicine product is utilized in humans.

Mechanistic Differences in Cell Death Responses to Metal-Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes.

The mononuclear phagocyte system in the liver is a frequent target for nanoparticles (NPs). A toxicological profiling of metal-based NPs is performed in Kupffer cell (KC) and hepatocyte cell lines. Sixteen NPs are provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences to study the toxicological effects in KUP5 (KC) and Hepa 1-6 cells. Five NPs (Ag, CuO, ZnO, SiO2 , and V2 O5 ) exhibit cytotoxicity in both cell types, while SiO2 and V2 O5 induce IL-1ß production in KC. Ag, CuO, and ZnO induced caspase 3 generated apoptosis in both cell types is accompanied by ion shedding and generation of mitochondrial reactive oxygen species (ROS) in both cell types. However, the cell death response to SiO2 in KC differs by inducing pyroptosis as a result of potassium efflux, caspase 1 activation, NLRP3 inflammasome assembly, IL-1ß release, and cleavage of gasdermin-D. This releases pore-performing peptide fragments responsible for pyroptotic cell swelling. Interestingly, although V2 O5 induces IL-1ß release and delays caspase 1 activation by vanadium ion interference in membrane Na+ /K+ adenosine triphosphate (ATP)ase activity, the major cell death mechanism in KC (and Hepa 1-6) is caspase 3 mediated apoptosis. These findings improve the understanding of the mechanisms of metal-based engineered nanomaterial (ENM) toxicity in liver cells toward comprehensive safety evaluation.

circ_ARF3 regulates the pathogenesis of osteosarcoma by sponging miR-1299 to maintain CDK6 expression.

Increasing evidence suggests that circular RNAs are emerging biomarkers or targets for early cancer diagnosis and treatment. However, the studies of circular RNA in osteosarcoma (OS) are limited. In this study we found that circ_ARF3 were highly expressed in osteosarcoma cell lines and tumor tissues. Knocking down circ_ARF3 greatly ceased OS cell growth, impaired cell colony formation and halted cell cycle transition from G1 to S phase. Bioinformatic analysis suggested that miR-1299 is the target of circ_ARF3. Luciferase assay and biotin labeled circ_ARF3 pull down assay confirmed their interactions in OS cells. The regulatory roles of circ_ARF3 on miR-1299 was also investigated. Further bioinformatic analysis showed that CDK6 is the target of miR-1299. Overexpressing miR-1299 in OS cells decreased CDK6 expression and arrested OS cell growth and cell cycle progression. However, the roles of miR-1299 in regulating CDK6 expression, OS cell growth and cell cycle progression were greatly impaired in the presence of circ_ARF3. In general, our study demonstrated that in the OS, highly expressed circ_ARF3 acts as a sponge of miR-1299 to inhibit miR-1299 mediated CDK6 downregulation which further promoted OS pathogenesis. circ_ARF3 could be a potential target for OS treatment in the future.

Circular RNA 0060428 sponges miR-375 to promote osteosarcoma cell proliferation by upregulating the expression of RPBJ.

Osteosarcoma (OS) is the most common primary bone tumor, and mainly occurs in children and early adults. The overall pathogenesis of osteosarcoma remains unclear. Circular RNAs (circRNAs) have been demonstrated to be one of the regulators in tumors as non-coding RNAs. In present study, we identified an elevated expressed circ-0060428 in osteosarcoma cells. The results of CCK-8 and flow cytometry assay showed that circ-0060428 promoted cell proliferation and reduced cell apoptosis of osteosarcoma cells. Bioinformatics analyses predicted the binding site between miR-375 to RPBJ and circ-0060428, and dual luciferase reporter assay verified this prediction. Furthermore, miRNA inhibitor of miR-375 could recover the influence of cell proliferation and apoptosis by knocking down the expression of circ-0060428. Meanwhile, knockdown of both circ-0060428 and RBPJ resulted in a lower apoptosis rate than circ-0060428 alone. Western blot analyses indicated that circ-0060428 regulated the expression of apoptosis related proteins (Bax, Bcl-2, and cleaved-Caspase-3) by upregulating RPBJ expression in osteosarcoma cells. Altogether, we confirmed the up-regulated circ-0060428 could improve the proliferation and survival of osteosarcoma cells by sponging miR-375 to upregulate RBPJ expression. This finding supported a novel clinically biomarker and treatment target for osteosarcoma therapy.