Enhancing the Inhibition of Breast Cancer Growth Through Synergistic Modulation of the Tumor Microenvironment Using Combined Nano-Delivery Systems.

Purpose: Breast cancer is a prevalent malignancy among women worldwide, and malignancy is closely linked to the tumor microenvironment (TME). Here, we prepared mixed nano-sized formulations composed of pH-sensitive liposomes (Ber/Ru486@CLPs) and small-sized nano-micelles (Dox@CLGs). These liposomes and nano-micelles were modified by chondroitin sulfate (CS) to selectively target breast cancer cells. Methods: Ber/Ru486@CLPs and Dox@CLGs were prepared by thin-film dispersion and ethanol injection, respectively. To mimic actual TME, the in vitro "condition medium of fibroblasts + MCF-7" cell model and in vivo "4T1/NIH-3T3" co-implantation mice model were established to evaluate the anti-tumor effect of drugs. Results: The physicochemical properties showed that Dox@CLGs and Ber/Ru486@CLPs were 28 nm and 100 nm in particle size, respectively. In vitro experiments showed that the mixed formulations significantly improved drug uptake and inhibited cell proliferation and migration. The in vivo anti-tumor studies further confirmed the enhanced anti-tumor capabilities of Dox@CLGs + Ber/Ru486@CLPs, including smaller tumor volumes, weak collagen deposition, and low expression levels of α-SMA and CD31 proteins, leading to a superior anti-tumor effect. Conclusion: In brief, this combination therapy based on Dox@CLGs and Ber/Ru486@CLPs could effectively inhibit tumor development, which provides a promising approach for the treatment of breast cancer.

A pH­responsive hyaluronic acid nano­vehicle co­encapsulating doxorubicin and all­trans retinoic acid for the inhibition of hepatic stellate cell­induced tumor growth and metastasis.

All­trans retinoic acid (ATRA) has been implicated in the differentiation of hepatic stellate cells (HSCs). In the present study, the liver­targeting hyaluronic acid micelles (ADHG) were prepared for co­delivery of ATRA and doxorubicin (DOX) to block the HSC­hepatoma interrelation. To simulate the tumor microenvironment, an in vitro dual­cell model and an in vivo co­implantation mouse model were established for anticancer studies. The experimental methods involved the MTT assay, wound­healing assay, cellular uptake, flow cytometry and and in vivo antitumor study. The results revealed that the HSCs in the research models notably promoted tumor proliferation and migration. Furthermore, ADHG were readily internalized by cancer cells and HSCs simultaneously, and widely distributed in cancer regions. The in vivo antitumor studies demonstrated that ADHG could notably decrease HSC activation and extracellular matrix deposition, as well as constrain tumor growth and metastasis. Therefore, ATRA could facilitate DOX­induced anti­proliferation and anti­metastasis effects, and ADHG are a promising nano­sized formulation for the combination therapy of hepatocellular carcinoma.

X-linked BCOR variants identified in Chinese Han patients with congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) frequently manifests as a complex phenotype and approximately one-third of cases may be caused by genetic factors. BCOR, an X-linked gene encoding the corepressor of BCL6, has been demonstrated to be closely involved in human heart development. However, whether BCOR variants represent the genetic etiology underlying CHD needs further investigation. METHODS: We performed whole exome sequencing on CHD nuclear families and identified a candidate gene, BCOR, by robust bioinformatic analysis and medical literature searches. Targeted DNA sequencing of the candidate gene was conducted and then the association between variants and the risk of developing CHD was analyzed. The effects of BCOR mutations on gene expression, localization, protein interaction, and signaling pathways were evaluated in vitro. RESULTS: We identified a BCOR hemizygous missense variant (c.1448C>T, p.Pro483Leu) in a male proband presented with CHD/heterotaxy. Sanger sequencing confirmed that this variant was inherited from his asymptomatic mother. Interestingly, through literature searches, we observed another novel BCOR hemizygous missense variant (c.1619G>A, p.Arg540Gln) in a CHD patient with heterotaxy, supporting the pathogenic evidence of BCOR variants. Functional experiments conducted in vitro revealed that the variant p.Pro483Leu altered the subcellular localization of BCOR protein, disrupted its interaction with BCL6, and significantly promoted cell proliferation, whereas the variant p.Arg540Gln displayed no obvious effects. Nevertheless, transcriptional analysis revealed that down-regulation of BCOR substantially enhanced the activities of mitogen-activated protein and phosphoinositide 3-kinase-AKT signaling pathways, which are closely attributed to heart development. Targeted sequencing of 932 sporadic CHD patients enriched nine variants of BCOR predicted as likely rare and damaging and a septal defect was present in 81.8% (9/11) of them, including the two probands, which was consistent with the possible phenotype caused by BCOR defects. CONCLUSIONS: The findings of the present study indicate that variants in BCOR may predispose individuals to CHD in the Chinese Han population.

Copy number variant analysis for syndromic congenital heart disease in the Chinese population.

BACKGROUND: Syndromic congenital heart disease (CHD) is among the most severe conditions in the pediatric population. Copy number variant (CNV) is an important cause of syndromic CHD, but few studies focused on CNVs related to these patients in China. The present study aimed to identify pathogenic CNVs associated with syndromic CHD in the Chinese population. METHODS: A total of 109 sporadic patients with syndromic CHD were applied chromosomal microarray analysis (CMA). Phenotype spectrum of pathogenic or likely pathogenic CNVs was analyzed. CHD-related genes were prioritized from genes within pathogenic or likely pathogenic CNVs by VarElect, OVA, AMELIE, and ToppGene. RESULTS: Using CMA, we identified 43 candidate CNVs in 37/109 patients. After filtering CNVs present in the general population, 29 pathogenic/likely pathogenic CNVs in 24 patients were identified. The diagnostic yield of CMA for pathogenic/likely pathogenic CNVs was 23.1% (24/104), excluding 5 cases with aneuploidies or gross chromosomal aberrations. The overlapping analysis of CHD-related gene lists from different prioritization tools highlighted 16 CHD candidate genes. CONCLUSION: As the first study focused on CNVs in syndromic CHD from the Chinese population, this study reveals the importance of CMA in exploring the genetic etiology of syndromic CHD and expands our understanding of these complex diseases. The bioinformatic analysis of candidate genes suggests several CHD-related genes for further functional research.

Curcumin and berberine co-loaded liposomes for anti-hepatocellular carcinoma therapy by blocking the cross-talk between hepatic stellate cells and tumor cells.

Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment (TME). In hepatocellular carcinoma (HCC), quiescent hepatic stellate cells (HSCs) could be activated to become CAFs, which play a critical role in tumor progression and drug resistance. Therefore, recent efforts have been focused on combining anti-HSC and pro-apoptotic activities to improve anti-tumor efficacy of drugs. In this study, glycyrrhetinic acid and hyaluronic acid-modified liposomes (GA-HA-Lip) were prepared for co-delivery of curcumin (CUR) and berberine (BBR) for the treatment of HCC. Furthermore, we established the LX-2+BEL-7402 co-cultured cell model and implanted the m-HSCs+H22 cells into a mouse to evaluate the anti-tumor effect of CUR&BBR/GA-HA-Lip both in vitro and in vivo. The results showed that CUR&BBR/GA-HA-Lip could accumulate in tumor tissues and be taken up by HSCs and BEL-7402 cells simultaneously. Compared with free CUR, the combination therapy based on GA-HA-Lip exhibits stronger pro-apoptotic and anti-proliferation effect both in vitro and in vivo. The anti-tumor mechanistic study revealed that CUR&BBR/GA-HA-Lip could inhibit the activation of HSCs and restrain drug resistance of tumor cells. In summary, CUR&BBR/GA-HA-Lip could be a promising nano-sized formulation for anti-tumor therapy.

Identification of mite-specific eosinophils in the colon of patients with ulcerative colitis.

The pathogenesis of ulcerative colitis (UC) is unclear. House dust mite (HDM) is associated with immune inflammation in the body. This study is designed to identify the association between HDM and UC clinical symptoms. UC patients (n = 86) and non-UC control (NC) subjects (n = 64) were recruited. Colon lavage fluids (CLF) were collected from HDM skin prick test positive patients during colonoscopy, and analyzed by immunological approaches. HDM was detected in fecal samples, which was positively correlated with UC clinical symptoms. HDM-specific eosinophils and Th2 cells were detected in CLF, which could be specifically activated by exposing to HDM in the culture. Direct exposure to HDM induced eosinophil activation in the colon of UC patients. UC patients displayed elevated levels of Th2 cytokines in the serum. UC clinical symptom scores were positively correlated with serum levels of Th2 cytokines. HDM was detected in UC patients' stools, which was positively correlated with UC clinical symptoms. Direct exposure to HDM could trigger eosinophilic activation of the colon.

Therapeutic Response of Multifunctional Lipid and Micelle Formulation in Hepatocellular Carcinoma.

Hepatic stellate cells (HSCs), as an important part of the tumor microenvironment (TME), could be activated by tumor cells as cancer-associated fibroblasts (CAFs), thereby promoting the production of extracellular matrix (ECM) and favoring the development of tumors. Therefore, blocking the "CAFs-ECM" axis is a promising pathway to improve antitumor efficacy. Based on this, we developed a multifunctional nanosized delivery system composed of hyaluronic acid-modified pH-sensitive liposomes (CTHLs) and glycyrrheic acid-modified nanomicelles (DGNs), which combines the advantages of targeted delivery, pH-sensitivity, and deep drug penetration. To mimic actual TME, a novel HSCs+BEL-7402 cocultured cell model and a m-HSCs+H22 coimplanted mice model were established. As expected, CTHLs and DGNs could target CAFs and tumor cells, respectively, and promote the drug penetration and retention in tumor regions. Notably, CTHLs+DGNs not only exhibited a superior antitumor effect in three-level tumor-bearing mice but also presented excellent antimetastasis efficiency in lung-metastatic mice. The antitumor mechanism revealed that the lipid&micelle mixed formulations effectively inhibited the activation of CAFs, reduced the deposition of ECM, and reversed the epithelial-mesenchymal transition (EMT) of tumor cells. In brief, the nanosized delivery system composed of CTHLs and DGNs could effectively improve the therapeutic effect of liver cancer by blocking the "CAFs-ECM" axis, which has a good clinical application prospect.

GA&HA-Modified Liposomes for Co-Delivery of Aprepitant and Curcumin to Inhibit Drug-Resistance and Metastasis of Hepatocellular Carcinoma.

Background: Tumor microenvironment (TME) plays a vital role in the development of hepatocellular carcinoma (HCC). Mounting evidence indicates that peripheral nerves could induce a shift from quiescent hepatic stellate cells (HSCs) to cancer-associated fibroblasts (CAFs) by secreting substance P (SP). The anti-tumor strategy by targeting "SP-HSCs-HCC" axis might be an effective therapy to inhibit tumor growth and metastasis. Objective: In this study, we prepared novel liposomes (CUR-APR/HA&GA-LPs) modified with hyaluronic acid (HA) and glycyrrhetinic acid (GA) for co-delivery aprepitant (APR) and curcumin (CUR), in which APR was chosen to inhibit the activation of HSCs by blocking SP/neurokinin-1 receptor (NK-1R), and CUR was used to induce apoptosis of tumor cells. Results: To mimic the TME, we established "SP+HSCs+HCC" co-cultured cell model in vitro. The results showed that CUR-APR/HA&GA-LPs could be taken up by CAFs and HCC simultaneously, and inhibit tumor cell migration. Meanwhile, the "SP+m-HSCs+HCC" co-implanted mice model was established to evaluate the anti-tumor effect in vivo. The results showed that CUR-APR/HA&GA-LPs could inhibit tumor proliferation and metastasis, and reduce extracellular matrix (ECM) deposition and tumor angiogenesis, indicating a superior anti-HCC effect. Conclusion: Overall, the combination therapy based on HA&GA-LPs could be a potential nano-sized formulation for anti-HCC therapy.

PTPN11 Gene Mutations and Its Association with the Risk of Congenital Heart Disease.

Congenital heart disease (CHD) is the most common congenital birth defect, with a prevalence of 8.98‰ of all live births in China. PTPN11 has been known to be closely involved in heart developments. In this research, we carried out whole-exome sequencing in nine CHD families and identified eight rare deleterious missense variants of PTPN11 gene in nine probands by stringently filtering criteria. Sanger sequencing of these probands and their unaffected familiar members revealed that six damaging variants were de novo in seven CHD families. Then, targeted sequencing was used to assess the PTPN11 exon variants in 672 sporadic CHD cases and 399 unrelated controls and identified 7 deleterious missense variants in 8 patients. Fisher's exact test reveals a significant association of PTPN11 variations with CHD (P = 0.0289). We observed the distribution of different subtypes in CHD patients with PTPN11 variants and found atrial septal defect (ASD) is a prominent phenotype (58.8%, 10/17). In vitro functional assays revealed that the predicted PTPN11 variants disturb RAS-mitogen-activated protein kinase signaling activity by influencing the phosphorylation level of pathway proteins and increasing the proliferation and migration abilities of cardiomyocytes to different extents. Our findings demonstrated that PTPN11 variants were associated with increased risk of CHD development and may be served as an important susceptible genetic event for CHD, especially the ASD subphenotype.

Propionic acid regulates immune tolerant properties in B Cells.

Interleukin 10 (IL-10)-producing B cells (B10 cells) are a canonical cell fraction for regulating other activities of immune cells. Posttranscriptional modification of IL-10 in B10 cells is not yet fully understood. Short-chain fatty acids play an important role to regulate the functions of immune cells. This study aims to clarify the role of propionic acid (PA), a short-chain fatty acid, in regulating the expression of IL-10 in B10 cells. Blood samples were collected from patients with food allergy (FA) and healthy subjects. Serum and cellular components were prepared with the samples, and analysed by enzyme-linked immunosorbent assay and flow cytometry, respectively. The results showed that serum PA levels were lower in FA patients. PA concentrations were negatively correlated with serum cytokine Th2 concentrations, specific IgE concentrations in serum and skin prick test results. The peripheral frequency of B10 cells and the production of IL-10 in B cells were also associated with serum PA concentrations. Activation of B cells by CpG induced the production of IL-10 and tristetretrprolin (TTP), in which TTP caused the spontaneous decay of IL-10 mRNA. PA was necessary to stabilize the IL-10 mRNA in B cells by inducing the production of granzyme B, which resulted in the degradation of the IL-10 mRNA. Administration of PA attenuated FA response in mice by maintaining homeostasis of B10 cells. In conclusion, PA is needed to stabilize the expression of IL-10 in B10 cells. PA administration can mitigate experimental FA by maintaining B10 cell functions.

CpG site hypomethylation at ETS1­binding region regulates DLK1 expression in Chinese patients with Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart malformation accounting for ~10% of cases. Although the pathogenesis of TOF is complex and largely unknown, epigenetics plays a huge role, specifically DNA methylation. The protein δ like non­canonical Notch ligand 1 (DLK1) gene encodes a non­canonical ligand of the Notch signaling pathway, which is involved in heart development. However, the epigenetic mechanism of DLK1 in the pathogenesis of TOF is yet to be elucidated. Therefore, the present study aimed to clarify its specific mechanism. In this study, immunohistochemistry was used to detect the protein expression of DLK1 and the methylation status of the DLK1 promoter was measured via bisulfite sequencing PCR. Dual­luciferase reporter assays were performed to examine the influence of transcription factor ETS proto­oncogene 1 (ETS1) on DLK1 gene expression. The electrophoretic mobility shift assay and chromatin immunoprecipitation assay, both in vivo and in vitro, were used to verify the binding of the ETS1 transcription factor to the DLK1 promoter as well as the influence of methylation status of DLK1 promoter on this binding affinity. The expression of DLK1 in the right ventricular outflow tract was significantly lower in patients with Tetralogy of Fallot (TOF) than that in controls (P<0.001). Moreover, the methylation level of CpG site 10 and CpG site 11 in the DLK1_R region was significantly decreased in TOF cases compared with controls (P<0.01). The integral methylation levels of DLK1_R and the methylation status of the CpG site 11 were both positively associated with DLK1 protein expression in TOF cases. ETS1 was found to inhibit DLK1 transcriptional activity by binding to the CpG site 11 and this affinity could be influenced by the methylation level of the DLK1 promoter. These findings demonstrated that the hypomethylation of the DLK1 promoter could increase the binding affinity of ETS1 transcription factor, which in turn inhibited DLK1 gene transcriptional activity and contributed to the development of TOF.

CD38+ B cells affect immunotherapy for allergic rhinitis.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the mainstay in the treatment of allergic diseases, but the therapeutic effects of AIT need to be improved. CD38+ B cells are an immune cell fraction involved in the pathogenesis of allergic diseases as well as in immune regulation. OBJECTIVE: We sought to elucidate the role of antigen-specific CD38+ B cells in AIT. METHODS: An analysis was carried out on AIT results of 48 patients with perennial allergic rhinitis (AR), among which peripheral blood immune cells were analyzed by flow cytometry; serum cytokine levels were determined by ELISA. An AR murine model was developed to test the role of CD38+ B cells in AIT. RESULTS: A fraction of antigen-specific CD38+ B cell was detected in AR patients. CD38+ B-cell frequency was negatively correlated with the therapeutic effects of AIT. A negative correlation was detected between the CD38+ B-cell frequency and regulatory T-cell frequency in AR patients treated with AIT. Exposure to specific antigens induced CD38+ B cells to produce IL-6, that converted Treg cells to TH17 cells. Coadministration of anti-CD38 antibody significantly promoted the therapeutic effects of AIT. CONCLUSIONS: Antigen-specific CD38+ B cells compromise AIT effects by producing IL-6 to convert regulatory T cells to TH17 cells. Inhibition of CD38+ B cells promotes the effects of AIT.

Combined anti-hepatocellular carcinoma therapy inhibit drug-resistance and metastasis via targeting "substance P-hepatic stellate cells-hepatocellular carcinoma" axis.

Peripheral nerves have emerged as the important components in tumor microenvironment (TME), which could activate hepatic stellate cells (HSCs) by secreting substance P (SP), leading to hepatocellular carcinoma (HCC) invasion and metastasis. Herein, we proposed a novel anti-HCC concept of blocking "SP-HSCs-HCC" axis for omnidirectional inhibition of HCC development. To pursue this aim, the novel CAP/GA-sHA-DOX NPs were developed for targeted co-delivery of capsaicin (CAP) and doxorubicin (DOX) using glycyrrhetinic acid (GA) modified sulfated-HA (sHA) as nanocarriers. Among that, CAP could inhibit the activation of HSCs as an inhibitor of SP. Notably, to real mimic "SP-HSCs-HCC" axis for in vitro and in vivo evaluation, both "SP + LX-2+BEL-7402" co-cultured cell model and "SP + m-HSC + H22" co-implantation mice model were attempted for the first time. Furthermore, in vivo anti-HCC effects were performed in three different tumor-bearing models: subcutaneous implantation of H22 or "SP + m-HSC + H22", intravenous injection of H22 for lung metastasis, and orthotopic implantation of H22 for primary HCC. Our results showed that CAP/GA-sHA-DOX NPs could be efficiently taken up by tumor cells and activated HSCs (aHSCs) simultaneously, and effectively inhibit tumor drug-resistance and migration by blocking SP-induced HSCs activation. In addition, CAP/GA-sHA-DOX NPs exhibited low ECM deposition, less tumor angiogenesis, and superior in vivo anti-HCC effects. The anti-HCC mechanisms revealed that CAP/GA-sHA-DOX NPs could down-regulate the expression level of Vimentin and P-gp, reverse epithelial-mesenchymal transition (EMT) of tumor cells. In brief, the nano-sized combination therapy based on GA-sHA-DOX polymers could effectively inhibit drug-resistance and metastasis of HCC by blocking "SP-HSCs-HCC" axis, which provides a promising approach for cancer therapy.

Co-Delivery of Curcumin and Capsaicin by Dual-Targeting Liposomes for Inhibition of aHSC-Induced Drug Resistance and Metastasis.

Recent research studies have shown that the low survival rate of liver cancer is due to drug resistance and metastasis. In the tumor microenvironment (TME), activated hepatic stellate cells (aHSCs) have been proven to favor the development of liver cancer. Hence, the combination therapy dual-targeting aHSCs and tumor cells might be an effective strategy for treatment of liver cancer. In this study, the novel multifunctional liposomes (CAPS-CUR/GA&Gal-Lip) were prepared for co-delivery of curcumin (CUR) and capsaicin (CAPS), in which glycyrrhetinic acid (GA) and galactose (Gal) were chosen as targeting ligands to modify the liposomes (Lip) for dual-targeting liver cancer. To mimic TME, a novel HSCs+HepG2 (human hepatoma cell line) cocultured model was established for the antitumor effect in vitro. The results showed that, compared to HepG2 cells alone, the cocultured model promoted drug resistance and migration by upregulating the expression of P-glycoprotein (P-gp) and Vimentin, which were effectively inhibited by CAPS-CUR/GA&Gal-Lip. The efficacy of the in vivo antitumor was evaluated by three mice models: subcutaneous H22 (mouse hepatoma cell line) tumor-bearing mice, H22+m-HSC (mouse hepatic stellate cell) tumor-bearing mice, and orthotopic H22 cells-bearing mice. The results showed that CAPS-CUR/GA&Gal-Lip exhibited lesser extracellular matrix (ECM) deposition, lesser tumor angiogenesis, and superior antitumor effect compared with the no- and/or Gal-modified Lip, which was attributed to the simultaneous blocking of the activation of HSCs and inhibition of the metastasis of tumor cells. The dual-targeting method using Lip is thus a potential strategy for liver cancer treatment.

A Non-coding HES1 Variant Predisposes Children to Congenital Heart Disease in Chinese Population.

Background: As a key component in the NOTCH signaling pathway, HES1 plays an important role in vertebrate heart development. Variants in the HES1 coding sequence are known to be associated with congenital heart disease (CHD). However, little is known about HES1 non-coding sequence variants and their association with the risk of developing CHD. Method and Results: We initially analyzed the non-coding sequence of the HES1 gene in 12 unrelated CHD families by direct sequencing and identified a previously unreported promoter region variant (NM_005524.4: c.-1279-1278 insAC, rs148941464) in the HES1 gene in four CHD families. The homozygous variant in patients was inherited from carrier parents with normal phenotypes, indicating a likely recessive genetic model. Given that the HES1 gene is predicted to be likely to exhibit haploinsufficiency (%HI: 11.44), we hypothesized that the HES1 homozygous variant is a genetic risk factor underlying CHD. We then carried out sequencing of this HES1 variant in 629 sporadic non-syndromic CHD cases and 696 healthy controls and performed association analysis. Interestingly, we observed a significant association of the homozygous HES1 promoter variant with CHD (18.92% of cases vs. 9.91% of controls; OR: 2.291, 95% CI: 1.637-3.207, p = 9.72 × 10-7). No significant association with CHD was observed for the HES1 promoter heterozygous variant (p > 0.05). However, association analysis tests of the HES1 homozygous variant with each subtype of CHD revealed that this homozygous variant was strongly associated with transposition of the great arteries (TGA) (OR: 3.726, 95% CI: 1.745-7.956, p = 0.0003). Moreover, the prevalence of HES1 homozygous variants in CHD patients with TGA (27.66%) was significantly higher than that in patients with other CHD subtypes or controls. Similar results were observed in a replication group of TGA (n = 64). Functional studies demonstrated that the homozygous variant in the HES1 promoter can disrupt its ability to bind RXRA, an inhibitory transcription factor, which results in abnormally high expression of the HES1 gene, indicating that this variant harbors gain-of-function effects. Conclusions: Our findings reveal that the non-coding homozygous variant in the HES1 promoter has a gain-of-function effect and is associated with an increased risk of CHD development, especially the severe TGA subtype.

Galactose Modified Liposomes for Effective Co-Delivery of Doxorubicin and Combretastatin A4.

BACKGROUND: Tumor angiogenesis plays a crucial role in tumor development, and recent efforts have been focused on combining proapoptotic and antiangiogenic activities to enhance antitumor therapy. METHODS: In this study, galactose-modified liposomes (Gal-LPs) were prepared for co-delivery of doxorubicin (DOX) and combretastatin A4 phosphate (CA4P). The co-cultured system composed of BEL-7402 and human umbilical vein endothelial cells (HUVEC) cells was established to effectively evaluate in vitro anti-tumor activity through cell viability and cell migration assay. Furthermore, both in vivo bio-distribution and anti-hepatoma effect of DOX&CA4P/Gal-LPs were investigated on H22 tumor cell-bearing mice. RESULTS: The results showed that DOX&CA4P/Gal-LPs were spherical with a mean particle size of 143 nm, and could readily be taken up by BEL-7402 cells. Compared with a mixture of free DOX and CA4P, the DOX&CA4P/Gal-LPs were more effective in inhibiting cell migration and exhibited stronger cytotoxicity against BEL-7402 cells alone or a co-cultured system. The in vitro studies showed that the co-cultured system was a more effective model to evaluate the anti-tumor activity of combination therapy. Moreover, DOX&CA4P/Gal-LPs exhibited a greater anti-hepatoma effect than other drug formulations, indicating that Gal-LPs could promote drug accumulation in the tumor region and improve the anti-tumor activity. CONCLUSION: Gal-LPs co-loaded with chemotherapeutic and antiangiogenic drugs are a promising strategy for anti-hepatoma therapy.

Characteristics of electrophysiological changes in the process of astrocytes pyroptosis after hyperoxia exposure. / 高氧损伤性星形胶质细胞焦亡过程的电生理变化特征.

OBJECTIVES: To observe the electrophysiological changes of astrocytes in the process of hyperoxia induced apoptosis and analyze the relationship between electrophysiological characteristics and morphological changes. METHODS: Astrocytes were exposed to 90% hyperoxia for 12-72 h. The electrophysiological characteristics of astrocytes in each group were detected by patch clamp technique, and the morphological characteristics of astrocytes were observed at the same time. Then the same batch of astrocytes were collected, and the expression levels of caspase-1, caspase-3, gasdermin D (GSDMD) and gasdermin E (GSDME) were detected by Western blotting. RESULTS: From 12 h to 72 h after hyperoxia exposure, the inward current was significantly lower than that of the control group (P<0.05), while the outward current was significantly decreased at 12 h and increased at 48 h (P<0.05). There was no significant difference between 24 h or 72 h after hyperoxia exposure and the control group (P>0.05). At each time point, the morphology of cells changed correspondingly. Western blotting showed that the expression of caspase-1 was increased significantly at 24 h and decreased significantly at 72 h after hyperoxia exposure (P<0.05); the expression of GSDMD was increased at 12 h and decreased gradually from 24 h to 72 h after hyperoxia exposure (P<0.05); the expression of caspase-3 did not change significantly at 12 h and 24 h after hyperoxia exposure (P>0.05), but began to decrease at 48 h (P<0.05); GSDME increased gradually at 24 h after hyperoxia exposure (P<0.05). CONCLUSIONS: Under hyperoxia exposure, the ion channels of astrocytes are damaged, which can maintain the dysfunction of ion homeostasis, activate GSDME, induce the damaged cells to break away from the apoptotic pathway, and mediate the pyroptosis.

Liver-Targeting and pH-Sensitive Sulfated Hyaluronic Acid Mixed Micelles for Hepatoma Therapy.

BACKGROUND: The tumor-targeting ability and pH-sensitive properties of intelligent drug delivery systems are crucial for effective drug delivery and anti-tumor therapy. METHODS: In this study, sHA-DOX/HA-GA mixed micelles were designed with the following properties: sulfated hyaluronic acid (sHA) was synthesized to block cell migration by inhibiting HAase; sHA-DOX conjugates were synthesized via pH-sensitive hydrazone bond to realize DOX-sensitive release. The introduction of HA-GA conjugate could improve active-targeting ability and cellular uptake. RESULTS: The results showed that the mixed micelles possessed a nearly spherical shape, nanoscale particle size (217.70±0.89 nm), narrow size distribution (PDI=0.07±0.04), negative zeta potential (-31.87±0.61 mV) and pH-dependent DOX release. In addition, the sHA-DOX/HA-GA micelles exhibited concentration-dependent cytotoxicities against liver carcinoma cells (HepG2) and HeLa cells, and were shown to be effectively taken up by HepG2 cells by confocal microscopy analysis. Furthermore, the in vivo anti-tumor study showed that mixed micelles had a superior anti-tumor effect compared to that of free DOX. Further evidence obtained from the hematoxylin-eosin staining and immunohistochemistry analysis also demonstrated that sHA-DOX/HA-GA exhibited stronger tumor inhibition and lower systemic toxicity than free DOX. CONCLUSION: The sHA-DOX/HA-GA mixed micelles could be a potential drug delivery system for anti-hepatoma therapy.

Liver-targeted liposomes for codelivery of curcumin and combretastatin A4 phosphate: preparation, characterization, and antitumor effects.

BACKGROUND: Recent efforts have been focused on combining two or more therapeutic approaches with different mechanisms to enhance antitumor therapy. Moreover, nanosize drug-delivery systems for codelivering two drugs with proapoptotic and antiangiogenic activities have exhibited great potential in efficient treatment of cancers. METHODS: Glycyrrhetinic acid (GA)-modified liposomes (GA LPs) for liver-targeted codelivery of curcumin (Cur) and combretastatin A4 phosphate (CA4P) were prepared and characterized. In vitro cellular uptake, cytotoxicity, cell migration, in vivo biodistribution, antitumor activity, and histopathological studies were performed. RESULTS: Compared with unmodified LPs (Cur-CA4P LPs), Cur-CA4P/GA LPs were taken up effectively by human hepatocellular carcinoma cells (BEL-7402) and showed higher cytotoxicity than free drugs. In vivo real-time near-infrared fluorescence-imaging results indicated that GA-targeted LPs increased accumulation in the tumor region. Moreover, Cur-CA4P/GA LPs showed stronger inhibition of tumor proliferation than Cur, Cur + CA4P, and Cur-CA4P LPs in vivo antitumor studies, which was also verified by H&E staining. CONCLUSION: GA-modified LPs can serve as a promising nanocarrier for liver-targeted co-delivery of antitumor drugs against hepatocellular carcinoma.

Liver-Targeted Combination Therapy Basing on Glycyrrhizic Acid-Modified DSPE-PEG-PEI Nanoparticles for Co-delivery of Doxorubicin and Bcl-2 siRNA.

Combination therapy based on nano-sized drug delivery system has been developed as a promising strategy by combining two or more anti-tumor mechanisms. Here, we prepared liver-targeted nanoparticles (GH-DPP) composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-polyetherimide (DSPE-PEG-PEI) with Glycyrrhetinic acid-modified hyaluronic acid (GA-HA) for co-delivery of doxorubicin (DOX) and Bcl-2 siRNA. Particles size, zeta potential and morphology were determined for the drug-loaded GH-DPP nanoparticles (siRNA/DOX/GH-DPP). Cellular uptake and in vitro cytotoxicity were analyzed against HepG2 cells. In vivo bio-distribution and anti-tumor therapeutic effects of siRNA/DOX/GH-DPP were evaluated in H22-bearing mice. The results showed that siRNA/DOX/GH-DPP nanoparticles were nearly spherical and showed dose-dependent cytotoxicity against HepG2 cells. Compared to Glycyrrhetinic acid-free co-delivery system (siRNA/DOX/DPP) and GH-DPP nanoparticles for delivery of DOX or Bcl-2 siRNA alone, siRNA/DOX/GH-DPP nanoparticles could induce more cellular apoptosis, and showed higher anti-tumor effect. Herein GH-DPP nanoparticles could simultaneously deliver both chemotherapy drugs and siRNA into the tumor region, exhibiting great potential in anti-tumor therapy.