Lymph-Node-Targeted Cholesterolized TLR7 Agonist Liposomes Provoke a Safe and Durable Antitumor Response.

Toll-like receptor (TLR) agonists as the potent stimulants of an innate immune system hold promises for applications in anticancer immunotherapy. However, most of them are limited in the clinical translation due to the uncontrolled systemic inflammatory response. In the current study, 1V209, a small molecule TLR7 agonist, was conjugated with cholesterol (1V209-Cho) and prepared into liposomes (1V209-Cho-Lip). 1V209-Cho-Lip exerted minimal toxic effects and enhanced the transportation ability in lymph nodes (LNs) compared with 1V209. 1V209-Cho-Lip treatment inhibited tumor progression in CT26 colorectal cancer, 4T1 breast cancer, and Pan02 pancreatic ductal cancer models through inducing effective DC activation and eliciting CD8+ T cell responses. Furthermore, 1V209-Cho-Lip induced tumor-specific memory immunity to inhibit cancer recurrence and metastasis. These results indicate that cholesterol conjugation with 1V209 is an effective approach to target lymph nodes and to reduce the adverse effects. This work provides a rational basis for the distribution optimization of TLR agonists for potential clinical use.

Inhibition of ROCK ameliorates pulmonary fibrosis by suppressing M2 macrophage polarisation through phosphorylation of STAT3.

BACKGROUND: Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti-PF therapeutic method has promising effect in its treatment. Rho-associated coiled-coil kinases (ROCK) inhibition significantly ameliorates bleomycin-induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation-induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages. METHODS: Bleomycin-induced PF was induced by intratracheal instillation and radiation-induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real-time reverse transcription-polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow-derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265. RESULTS: Therapeutic administration of ROCK inhibitor ameliorates bleomycin-induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti-fibrotic effect in macrophages-depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin-induced PF compared with positive drugs. In radiation-induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co-cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin-4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p-signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors. CONCLUSION: Inhibiting ROCK could significantly attenuate bleomycin- and radiation-induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.

Tripterin liposome relieves severe acute respiratory syndrome as a potent COVID-19 treatment.

For coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 15-30% of patients are likely to develop COVID-19-related acute respiratory distress syndrome (ARDS). There are still few effective and well-understood therapies available. Novel variants and short-lasting immunity are posing challenges to vaccine efficacy, so finding antiviral and antiinflammatory treatments remains crucial. Here, tripterin (TP), a traditional Chinese medicine, was encapsulated into liposome (TP lipo) to investigate its antiviral and antiinflammatory effects in severe COVID-19. By using two severe COVID-19 models in human ACE2-transgenic (hACE2) mice, an analysis of TP lipo's effects on pulmonary immune responses was conducted. Pulmonary pathological alterations and viral burden were reduced by TP lipo treatment. TP lipo inhibits SARS-CoV-2 replication and hyperinflammation in infected cells and mice, two crucial events in severe COVID-19 pathophysiology, it is a promising drug candidate to treat SARS-CoV-2-induced ARDS.

Biodegradable self-assembled PEG-PCL-PEG micelles for hydrophobic honokiol delivery: I. Preparation and characterization.

This study aims to develop self-assembled poly(ethylene glycol)-poly(epsilon-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) micelles to encapsulate hydrophobic honokiol (HK) in order to overcome its poor water solubility and to meet the requirement of intravenous administration. Honokiol loaded micelles (HK-micelles) were prepared by self-assembly of PECE copolymer in aqueous solution, triggered by its amphiphilic characteristic assisted by ultrasonication without any organic solvents, surfactants and vigorous stirring. The particle size of the prepared HK-micelles measured by Malvern laser particle size analyzer were 58 nm, which is small enough to be a candidate for an intravenous drug delivery system. Furthermore, the HK-micelles could be lyophilized into powder without any adjuvant, and the re-dissolved HK-micelles are stable and homogeneous with particle size about 61 nm. Furthermore, the in vitro release profile showed a significant difference between the rapid release of free HK and the much slower and sustained release of HK-micelles. Moreover, the cytotoxicity results of blank micelles and HK-micelles showed that the PECE micelle was a safe carrier and the encapsulated HK retained its potent antitumor effect. In short, the HK-micelles were successfully prepared by an improved method and might be promising carriers for intravenous delivery of HK in cancer chemotherapy, being effective, stable, safe (organic solvent and surfactant free), and easy to produce and scale up.

Co-delivery honokiol and doxorubicin in MPEG-PLA nanoparticles.

The combination chemotherapy is an important protocol in cancer therapy. Honokiol shows synergistic anticancer effect with doxorubicin. In this paper, honokiol and doxorubicin co-loaded MPEG-PLA nanoparticles were prepared. The particle size, morphology, in vitro release profile, cytotoxicity and cell proliferation study were studied in detail. The results indicated that honokiol and doxorubicin could be efficiently loaded into MPEG-PLA nanoparticles simultaneously, and could be released out in an extended period in vitro. Meanwhile, honokiol and doxorubicin in MPEG-PLA nanoparticle could efficiently suppress cancer cell proliferation in vitro. The described honokiol and doxorubicin co-loaded MPEG-PLA nanoparticle might be a novel anticancer agent.

Jumonji domain-containing protein 6 protein and its role in cancer.

The jumonji domain-containing protein 6 (JMJD6) is a Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses lysine hydroxylation and arginine demethylation of histone and non-histone peptides. Recently, the intrinsic tyrosine kinase activity of JMJD6 has also been reported. The JMJD6 has been implicated in embryonic development, cellular proliferation and migration, self-tolerance induction in the thymus, and adipocyte differentiation. Not surprisingly, abnormal expression of JMJD6 may contribute to the development of many diseases, such as neuropathic pain, foot-and-mouth disease, gestational diabetes mellitus, hepatitis C and various types of cancer. In the present review, we summarized the structure and functions of JMJD6, with particular emphasis on the role of JMJD6 in cancer progression.

Targeting folate receptor ß positive tumor-associated macrophages in lung cancer with a folate-modified liposomal complex.

Tumor-associated macrophages (TAMs) facilitate cancer progression by promoting tumor invasion, angiogenesis, metastasis, inflammatory responses, and immunosuppression. Folate receptor ß (FRß) is overexpressed in TAMs. However, the clinical significance of FRß-positive macrophages in lung cancer remains poorly understood. In this study, we verified that FRß overexpression in lung cancer TAMs was associated with poor prognosis. We utilized a folate-modified lipoplex comprising a folate-modified liposome (F-PLP) delivering a BIM-S plasmid to target both lung cancer cells and FRß-positive macrophages in the tumor microenvironment. Transfection of LL/2 cells and MH-S cells with F-PLP/pBIM induced cell apoptosis. Injection of F-PLP/pBIM into LL/2 and A549 lung cancer models significantly depleted FRß-positive macrophages and reduced tumor growth. Treatment of tumor-bearing mice with F-PLP/pBIM significantly inhibited tumor growth in vivo by inducing tumor cell and macrophage apoptosis, reducing tumor proliferation, and inhibiting tumor angiogenesis. In addition, a preliminary safety evaluation demonstrated a good safety profile of F-PLP/pBIM as a gene therapy administered intravenously. This work describes a novel application of lipoplexes in lung cancer targeted therapy that influences the tumor microenvironment by targeting TAMs.

Redox/pH dual-stimuli responsive camptothecin prodrug nanogels for "on-demand" drug delivery.

At present, chemotherapy remains to be one of the most important therapeutic approaches for malignant tumors. The tumor microenvironment(TME)-responsive intelligent drug delivery systems are still the hot research topics in delivering chemotherapeutic drugs. Camptothecin (CPT) possesses very strong antitumor activities, but its clinical application is hindered by its poor water-solubility and serious toxic side effects. Herein, a new intelligent and TME-responsive P(CPT-MAA) prodrug nanogel was developed for delivering CPT and reducing its side effects. P(CPT-MAA) prodrug nanogels were prepared with methacrylic acid (MAA), CPT monomer (CPTM) and N,N'-methylenebisacrylamide (Bis) via distillation-precipitation polymerization, in which CPT was covalently conjugated into the nanogels via redox-responsive disulfide linker. The as-prepared nanogels were spherical shapes with uniform size and narrow size distribution. With the help of redox-responsive property of disulfide linker and pH-responsive property of PMAA, the release of CPT from prodrug nanogels was redox/pH-dual dependent and could be accelerated by the increased concentration of GSH and the decreased pH value, which were favorable to realize the "on-demand" drug release in tumor cell and tumor tissue microenvironment. Furthermore, P(CPT-MAA) prodrug nanogels exhibited superior antitumor activity both in vitro and in vivo without observed side effects. Hence, the prepared P(CPT-MAA) prodrug nanogels may be a promise delivery system for chemotherapeutic agents.

An Endogenous Vaccine Based on Fluorophores and Multivalent Immunoadjuvants Regulates Tumor Micro-Environment for Synergistic Photothermal and Immunotherapy.

Recently, near-infrared (NIR) light-based photothermal therapy (PTT) has been widely applied in cancer treatment. However, in most cases, the tissue penetration depth of NIR light is not sufficient and thus photothermal therapy is unable to completely eradicate deep, seated tumors inevitably leading to recurrence of the tumor. Due to this significant limitation of NIR, improved therapeutic strategies are urgently needed. Methods: We developed an endogenous vaccine based on a novel nanoparticle platform for combinatorial photothermal ablation and immunotherapy. The design was based on fluorophore-loaded liposomes (IR-7-lipo) coated with a multivalent immunoadjuvant (HA-CpG). In vitro PTT potency was assessed in cells by LIVE/DEAD and Annexin V-FITC/PI assays. The effect on bone marrow-derived dendritic cells (BMDC) maturation and antigen presentation was evaluated by flow cytometry (FCM) with specific antibodies. After treatment, the immune cell populations in tumor micro-environment and the cytokines in the serum were detected by FCM and Elisa assay, respectively. Finally, the therapeutic outcome was investigated in an animal model. Results: Upon irradiation with 808 nm laser, IR-7-lipo induced tumor cell necrosis and released tumor-associated antigens, while the multivalent immunoadjuvant improved the expression of co-stimulatory molecules on BMDC and promoted antigen presentation. The combination therapy of PTT and immunotherapy regulated the tumor micro-environment, decreased immunosuppression, and potentiated host antitumor immunity. Most significantly, due to an enhanced antitumor immune response, combined photothermal immunotherapy was effective in eradicating tumors in mice and inhibiting tumor metastasis. Conclusion: This endogenous vaccination strategy based on synergistic photothermal and immunotherapy may provide a potentially effective approach for treatment of cancers, especially those difficult to be surgically removed.

Hyaluronan Reduces Cationic Liposome-Induced Toxicity and Enhances the Antitumor Effect of Targeted Gene Delivery in Mice.

Cationic nanocarriers are reported to induce cell necrosis, especially in the lungs upon systemic administration. The release of damage-associated molecular patterns, such as mitochondrial DNA from the injured cell may result in the inflammatory toxicity of the nanocarrier, which has largely limited its clinical application. Partial blocking of the surface charge of cationic nanocarriers might improve their safety. As hyaluronan (HA) is an anionic polysaccharide that is widely used for specific binding to CD44 to improve the cellular uptake efficiency in tumor-targeting therapy, in this study, we modified cationic liposomes (LP) with the negatively charged HA at a mass ratio of 10% to prepare targeted HA-modified cationic liposomes (HALP). Cationic liposomes modified with hyaluronan showed significantly less cytotoxicity due to the blockage of their surface charge than the unmodified liposomes. In addition, HA modification helped to reduce cell necrosis in lung tissue and reduced the amount of mitochondria subsequently released, which alleviated pulmonary inflammation in mice. HA-modified liposomes also improved the survival of mice injected with a fatal dose of HALP compared with mice injected with cationic LP. In addition, both serological biochemical analysis and histological examination proved that a liposome modified with HA is a safer carrier for systemic administration than an unmodified liposome. Furthermore, HALP/survivin exhibited an enhanced antitumor effect by inhibiting tumor growth and promoting tumor cell apoptosis compared with the unmodified LP group. In conclusion, compared to the properties of cationic liposomes, liposomes modified with 10% HA (HALP) might be gene vectors with lower toxicity and higher tumor targeting efficiency.

Negative regulation of cationic nanoparticle-induced inflammatory toxicity through the increased production of prostaglandin E2 via mitochondrial DNA-activated Ly6C+ monocytes.

Rationale: Cationic nanocarriers present with well-known toxicities, including inflammatory toxicity, which limit their clinical application. How the cationic nanocarrier-induced inflammatory response is negatively regulated is unknown. Herein, we found that following a sublethal dose of cationic nanocarriers, the induced inflammatory response is characterized by early neutrophil infiltration and spontaneous resolution within 1 week. Methods: C57BL/6 mice were intravenously injected with a dosage of 1-100 mg/kg cationic DOTAP liposomes as well as other cationic materials. Cell necrosis was detected by flow cytometry. Release of mitochondrial DNA was quantified by qPCR via Taqman probes. Signal proteins were detected by Western blotting. PGE2 production in the supernatant was quantitated using an enzyme immunoassay (EIA). The infiltrated inflammatory cells were observed in WT mice, Ccr2-/- mice, Sting-/- mice and Tlr9-/- mice. Results: The early stage (24-48 h) inflammatory neutrophil infiltration was followed by an increasing percentage of monocytes; and, compared with WT mice, Ccr2-/- mice presented with more severe pulmonary inflammation. A previously uncharacterized population of regulatory monocytes expressing both inflammatory and immunosuppressive cytokines was identified in this model. The alteration in monocyte phenotype was directly induced by mtDNA release from cationic nanocarrier-induced necrotic cells via a STING- or TLR9-dependent pathway. Neutrophil activation was specifically inhibited by PGE2 from Ly6C+ inflammatory monocytes, and intravenous injections of dual-phenotype monocytes beneficially modified the immune response; this inhibitory effect was abolished after treatment with indomethacin. Moreover, we provide clear evidence that mitochondrial DNA activated Ly6C+ monocytes and increased PGE2 production through TLR9- or STING-mediated MAPK-NF-κB-COX2 pathways. Conclusion: Our findings suggest that Ly6C+ monocytes and mtDNA-induced Ly6C+ monocyte PGE2 production may be part of a feedback mechanism that contributes to the resolution of cationic nanocarrier-induced inflammatory toxicity and may have important implications for understanding nanoparticle biocompatibility and designing better, safer drug delivery systems.

Cholesterol-modified Hydroxychloroquine-loaded Nanocarriers in Bleomycin-induced Pulmonary Fibrosis.

An increasing number of reports have suggested the use of hydroxychloroquine (HCQ) as an adjunct anti-cancer treatment to enhance the chemotherapeutic response, as well as for the treatment of several fibrotic skin diseases and cystic fibrosis. In this study, we synthesized a cholesterol-modified HCQ (Chol-HCQ) and hypothesized that a systemic delivery system with Chol-HCQ nanocarriers could be effective for the treatment of bleomycin-induced pulmonary fibrosis. Chol-HCQ significantly inhibits the proliferation of rat lung fibroblasts, regulates inflammation and ameliorates bleomycin-induced pulmonary fibrosis in rats. It regulates the expression of pro-inflammatory cytokines, such as TNF-α; reduces the infiltration of inflammatory neutrophils; and inhibits the phosphorylation of NF-κB. Chol-HCQ also reduces the expression of connective tissue growth factor (CTGF) and phosphorylation of extracellular regulated protein kinase (p-ERK) in rats with bleomycin-induced pulmonary fibrosis. Chol-HCQ nanocarriers reduce early pulmonary inflammation and inhibit the CTGF/ERK signalling pathway in bleomycin-induced pulmonary fibrosis. These results demonstrate that Chol-HCQ liposomes suppress pulmonary inflammation and reduce pulmonary fibrosis induced by bleomycin. The systemic administration safety of Chol-HCQ liposomes was confirmed after intravenous administration for 28 days in rats. The present study provides evidence that Chol-HCQ liposomes may be a potential therapeutic agent for inflammation associated with pulmonary fibrosis.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.

Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our results indicate that treatment of collagen membranes with appropriate concentrations of EGCG has an anti-inflammatory effect and shows promise for GBR applications.

A folate receptor-targeted lipoplex delivering interleukin-15 gene for colon cancer immunotherapy.

Interleukin-15 has been implicated as a promising cytokine for cancer immunotherapy, while folate receptor α (FRα) has been shown to be a potentially useful target for colon cancer therapy. Herein, we developed F-PLP/pIL15, a FRα-targeted lipoplex loading recombinant interleukin-15 plasmid (pIL15) and studied its antitumor effects in vivo using a CT26 colon cancer mouse model. Compared with control (normal saline) treatment, F-PLP/pIL15 significantly suppressed tumor growth in regard to tumor weight (P < 0.001) and reduced tumor nodule formation (P < 0.001). Moreover, when compared to other lipoplex-treated mice, F-PLP/pIL15-treated mice showed higher levels of IL15 secreted in the serum (P < 0.001) and ascites (P < 0.01). These results suggested that the targeted delivery of IL15 gene might be associated with its in vivo antitumor effects, which include inducing tumor cell apoptosis, inhibiting tumor proliferation and promoting the activation of immune cells such as T cells and natural killer cells. Furthermore, hematoxylin and eosin staining of vital organs following F-PLP/pIL15 treatment showed no detectable toxicity, thus indicating that intraperitoneal administration may be a viable route of delivery. Overall, these results suggest that F-PLP/pIL15 may serve as a potential targeting preparation for colon cancer therapy.

Ovarian cancer treatment with a tumor-targeting and gene expression-controllable lipoplex.

Overexpression of folate receptor alpha (FRα) and high telomerase activity are considered to be the characteristics of ovarian cancers. In this study, we developed FRα-targeted lipoplexes loaded with an hTERT promoter-regulated plasmid that encodes a matrix protein (MP) of the vesicular stomatitis virus, F-LP/pMP(2.5), for application in ovarian cancer treatment. We first characterized the pharmaceutical properties of F-LP/pMP(2.5). The efficient expression of the MP-driven hTERT promoter in SKOV-3 cells was determined after an in-vitro transfection assay, which was significantly increased compared with a non-modified LP/pMP(2.5) group. F-LP/pMP(2.5) treatment significantly inhibited the growth of tumors and extended the survival of mice in a SKOV-3 tumor model compared with other groups. Such an anti-tumor effect was due to the increased expression of MP in tumor tissue, which led to the induction of tumor cell apoptosis, inhibition of tumor cell proliferation and suppression of tumor angiogenesis. Furthermore, a preliminary safety evaluation demonstrated a good safety profile of F-LP/pMP(2.5) as a gene therapy agent. Therefore, FRα-targeted lipoplexes with therapeutic gene expression regulated by an hTERT promoter might be a promising gene therapy agent and a potential translational candidate for the clinical treatment of ovarian cancer.

Combined Delivery and Anti-Cancer Activity of Paclitaxel and Curcumin Using Polymeric Micelles.

Paclitaxel (PTX) is efficacious in treating various solid tumors. However, the severe adverse effects of its present formulation (Cremophor EL and ethanol) and the development of drug resistance by the activation of nuclear factor-κB (NF-κB) reduce the anti-tumor activities of PTX. Curcumin (Cur) demonstrates anti-tumor activity by means of antiangiogenesis and induction of apoptosis as well as suppression of the activity of NF-κB. Therefore, to improve its antitumor activity and eliminate the toxicity of the commercial formulation of PTX, we prepared biodegradable monomethoxy poly(ethyleneglycol)-poly(ε-caprolactone) (MPEG-PCL) micelles to co-deliver PTX and Cur using a solid dispersion method. The mixed PTX and Cur polymeric micelles (PTX-Cur-M) produced were monomorphous micelles of 38 nm in diameter that released PTX and Cur for an extended period of time and induced cell apoptosis in vitro. In addition, the PTX-Cur-M exhibited anti-angiogenic activity in vitro and in vivo. Furthermore, the therapeutic efficacy of PTX-Cur-M in a mouse model of colon cancer was evaluated. PTX-Cur-M micelles produced significantly more inhibition of tumor growth than Cur micelles (Cur-M) and PTX micelles (PTX-M) alone at the same dose (P < 0.05 and P < 0.05, respectively). Immunohistochemical and immunofluorescent analyses demonstrated that PTX-Cur-M enhanced tumor cell apoptosis and inhibited angiogenesis to a greater extent than control treatment. Our data suggested that PTX-Cur-M may have potential clinical applications in cancer therapy.

Folate-Modified Lipoplexes Delivering the Interleukin-12 Gene for Targeting Colon Cancer Immunogene Therapy.

The incidence and mortality rate of colorectal cancer increase every year, making it a serious threat to human health. Targeted immunogene therapy is a novel method of treating this type of cancer. Colon cancer overexpresses folate receptor α (FRα) and folate-modified liposomes for colon cancer immunogene therapy may suppress tumor growth effectively. In this study, F-PLP/pIL12, an FRα-targeted lipoplex loading plasmid interleukin-12 (pIL12) was prepared and its physicochemical properties were characterized. Then the antitumor effect of F-PLP/pIL12 was studied in an in vivo model of CT-26 colon cancer. F-PLP/pIL12 was associated with about 56.6% tumor growth inhibition compared with the saline control. The production of malignant ascites was significantly less pronounced than in controls, and there were fewer tumor nodules and less overall tumor mass (P < 0.01). There was more IL12 expression and IFN-γ secretion in F-PLP/pIL12-treated tumor tissues, but there was less FRα expression. The antitumor mechanisms involved inducing tumor cell apoptosis, reducing microvessel density, and stimulating TNF-α secretion. In addition, there were fewer M2 macrophages in the tumor microenvironment of tissues stimulated with F-PLP/pIL12, which also activated the natural killer cells. H&E staining of vital organs suggested that F-PLP/pIL12 is safe for use in intraperitoneally administered cancer therapy. It was here concluded that F-PLP/plL12 may be a suitable targeting formulation for colon cancer immunogene therapy.

Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response.

Nanocarriers with positive surface charges are known for their toxicity which has limited their clinical applications. The mechanism underlying their toxicity, such as the induction of inflammatory response, remains largely unknown. In the present study we found that injection of cationic nanocarriers, including cationic liposomes, PEI, and chitosan, led to the rapid appearance of necrotic cells. Cell necrosis induced by cationic nanocarriers is dependent on their positive surface charges, but does not require RIP1 and Mlkl. Instead, intracellular Na(+) overload was found to accompany the cell death. Depletion of Na(+) in culture medium or pretreatment of cells with the Na(+)/K(+)-ATPase cation-binding site inhibitor ouabain, protected cells from cell necrosis. Moreover, treatment with cationic nanocarriers inhibited Na(+)/K(+)-ATPase activity both in vitro and in vivo. The computational simulation showed that cationic carriers could interact with cation-binding site of Na(+)/K(+)-ATPase. Mice pretreated with a small dose of ouabain showed improved survival after injection of a lethal dose of cationic nanocarriers. Further analyses suggest that cell necrosis induced by cationic nanocarriers and the resulting leakage of mitochondrial DNA could trigger severe inflammation in vivo, which is mediated by a pathway involving TLR9 and MyD88 signaling. Taken together, our results reveal a novel mechanism whereby cationic nanocarriers induce acute cell necrosis through the interaction with Na(+)/K(+)-ATPase, with the subsequent exposure of mitochondrial damage-associated molecular patterns as a key event that mediates the inflammatory responses. Our study has important implications for evaluating the biocompatibility of nanocarriers and designing better and safer ones for drug delivery.