Synergistic anti-tumor effect of dual drug co-assembled nanoparticles based on ursolic acid and sorafenib.

Both ursolic acid (UA) and sorafenib (Sora) have been generally utilized in cancer treatment, and the combination of the two has also shown a good anti-tumor effect. However, single-agent therapy for Hepatocellular carcinoma (HCC) has the disadvantages of multi-drug resistance, poor water solubility and low bioavailability, and the application of traditional nanocarrier materials is limited due to their low drug loading and low carrier-related toxicity. Therefore, we prepared US NPs with different proportions of UA and Sora by solvent exchange method for achieving synergistic HCC therapy. US NPs had suitable particle size, good dispersibility and storage stability, which synergistically inhibited the proliferation of HepG2 cells, SMMC7721 cells and H22 cells. In addition, we also proved that US NPs were able to suppress the migration of HepG2 cells and SMMC7721 cells and reduce the adhesion ability and colony formation ability of these cells. According to the results, US NPs could degrade the membrane potential of mitochondrial, participate in cell apoptosis, and synergistically induce autophagy. Collectively, the carrier-free US NPs provide new strategies for HCC treatment and new ideas for the development of novel nano-drug delivery systems containing UA and Sora.

Dual-drug controllable co-assembly nanosystem for targeted and synergistic treatment of hepatocellular carcinoma.

The effectiveness of chemotherapeutic agents for hepatocellular carcinoma (HCC) is unsatisfactory because of tumor heterogeneity, multidrug resistance, and poor target accumulation. Therefore, multimodality-treatment with accurate drug delivery has become increasingly popular. Herein, a cell penetrating peptide-aptamer dual modified-nanocomposite (USILA NPs) was successfully constructed by coating a cell penetrating peptide and aptamer onto the surface of sorafenib (Sora), ursolic acid (UA) and indocyanine green (ICG) condensed nanodrug (USI NPs) via one-pot assembly for targeted and synergistic HCC treatment. USILA NPs showed higher cellular uptake and cytotoxicity in HepG2 and H22 cells, with a high expression of epithelial cell adhesion molecule (EpCAM). Furthermore, these NPs caused more significant mitochondrial membrane potential reduction and cell apoptosis. These NPs could selectively accumulate at the tumor site of H22 tumor-bearing mice and were detected with the help of ICG fluorescence; moreover, they retarded tumor growth better than monotherapy. Thus, USILA NPs can realize the targeted delivery of dual drugs and the integration of diagnosis and treatment. Moreover, the effects were more significant after co-administration of iRGD peptide, a tumor-penetrating peptide with better penetration promoting ability or programmed cell death ligand 1 (PD-L1) antibody for the reversal of the immunosuppressive state in the tumor microenvironment. The tumor inhibition rates of USILA NPs + iRGD peptide or USILA NPs + PD-L1 antibody with good therapeutic safety were 72.38 % and 67.91 % compared with control, respectively. Overall, this composite nanosystem could act as a promising targeted tool and provide an effective intervention strategy for enhanced HCC synergistic treatment.

CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.

CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.

Differential development and electrophysiological activity in cultured cortical neurons from the mouse and cynomolgus monkey.

In vitro cultures of primary cortical neurons are widely used to investigate neuronal function. However, it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons, and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions. Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods, our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice. We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons, and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey. Although cultured monkey cortical neurons developed slowly in vitro, they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin (HTT), the Huntington's disease protein. A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites. Therefore, compared with the primary cortical neurons of mice, cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity, such as electrophysiological activity. Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease, and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration. All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China (IACUC Approval No. 20200512-04) on May 12, 2020. All monkey experiments were approved by the IACUC protocol (IACUC Approval No. LDACU 20190820-01) on August 23, 2019 for animal management and use.

[Interaction among peroxisome proliferators-activated receptor alpha, cytochrome P450 oxysterol 7α-hydroxylase and estrogen receptor and its association with intrahepatic cholestasis in pregnant rats].

OBJECTIVE: To investigate the relationship between interaction of peroxisome proliferators-activated receptor alpha (PPARα), cytochrome P450 oxysterol 7α-hydroxylase (CYP7B1) and estrogen receptor (ER) and intrahepatic cholestasis in pregnant rats. METHODS: Eighty clean SD pregnant rats were selected and divided into four groups randomly with 20 in each. Since the 13th day of pregnancy, rats in the control group was injected subcutaneously with refined vegetable oil 2.0 ml×kg(-1)×d(-1), those in the low-dose, moderate-dose and high-dose groups received 17-α-ethynylestradiol (EE) 1.0 mg×kg(-1)×d(-1), 1.25 mg×kg(-1)×d(-1) and 1.5 mg×kg(-1)×d(-1), respectively. All rats were sacrificed at the 21(st) day of pregnancy and maternal hepatic tissues were collected. The serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bile acid (TBA) and bilirubin (BIL) were determined by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of PPARα, CYP7B1, ERα and ERß in maternal rat livers were examined by real-time PCR. RESULTS: (1) Biochemical indicators: the serum levels of ALT, AST, TBA and BIL were significantly lower in the control group than in the rest 3 groups, respectively [control group: (41.1 ± 2.8) U/L, (44.4 ± 3.6) U/L, (26.4 ± 5.6) µmol/L and (2.8 ± 0.2) U/L; low-dose group: (48.2 ± 3.4) U/L, (47.9 ± 3.7) U/L, (36.4 ± 4.2) µmol/L and (4.2 ± 0.2) U/L; moderate-dose group: (70.4 ± 5.3) U/L, (68.4 ± 5.6) U/L, (64.3 ± 3.8) µmol/L and (6.2 ± 1.2) U/L; high-dose group: (72.4 ± 7.6) U/L, (70.2 ± 3.8) U/L, (72.4 ± 7.8) µmol/L and (8.2 ± 2.2) U/L, P < 0.05], and those in the moderate or high-dose groups were higher than in the low-dose group (P < 0.05). (2) mRNA expression of ERα and ERß: the mRNA expression of ERα in pregnant rat livers increased in a dose-dependent manner, which were all significantly higher than that in the control group, respectively (low-dose group: 0.76 ± 0.02); moderate-dose group: (0.99 ± 0.04; high-dose group: 1.21 ± 0.01; control group: 0.65 ± 0.01, P < 0.05), but no difference was found among the 4 groups in the mRNA expression of ERß (P > 0.05). (3) mRNA expression of CYP7B1 and PPARα: the mRNA expression of CYP7B1 in pregnant rat livers increased from the low-dose group to the high-dose group, and were all higher than that of the control group (low-dose group: 0.93 ± 0.01; moderate-dose group: 0.99 ± 0.06; high-dose group: 1.22 ± 0.04; control group: 0.75 ± 0.02, P < 0.05). However, the mRNA expression of PPARα decreased from the low-dose group to the high-dose group, and were all lower than that of the control group (low-dose group: 0.83 ± 0.05; moderate-dose group: 0.71 ± 0.02; high-dose group: 0.64 ± 0.03; control group: 1.35 ± 0.05; P < 0.05). CONCLUSIONS: The down regulated mRNA expression of PPARα, caused by higher dose of estrogen, may increase the expression of CYP7B1 due to the ineffectiveness of the inhibition of PPARα on CYP7B1, which may further stimulate the ERα activity and then induce intrahepatic cholestasis. Abnormal expression of PPARα, CYP7B1 and ER may play a role in the pathogenesis of estrogen-induced intrahepatic cholestasis.

[Expression of FXR mRNA, PPAR alpha mRNA and bile acid metabolism related genes in intrahepatic cholestasis of pregnant rats].

OBJECTIVE: To study the expressions of FXR, PPARa and Bile acid metabolism related genes in intrahepatic cholestasis of pregnant rats. METHODS: 60 clean SD pregnant rats were selected and divided randomly into three groups. Since the 13th day of pregnancy rats in control group were injected subcutaneously with refined vegetable oil 2.0 mg/kg/d Rats in no-treated group were injected subcutaneously with the 17-a-ethynylestradiol (EE) 1.25 mg/kg/d until the 17th day. Those rat ih treated group were injected subcutaneously with the 17-a-ethynylestradiol (EE) 1.25 mg/kg/d until the 17th day and then were treated with fenofibrate for another four days until the 21th day. All rats were killed at the 21th day and livers were collected for study. The levels of serum TBA were examined by ELISA. The mRNA expressions of PPARa, FXR, CYP7A1, CYP27A1 and CYP8B1 were examined by real-time PCR. (1) RESULTS: The levels of TBA were significantly higher in no-treated group (68.7+/-4.2)mumol/L and treated group (69.5+/-3.8)mumol/L compared with that of control group (26.6+/-2.3)mumol/L at the 17th day (P value is less than 0.05) and no difference found between treated and no-treated groups (P value is more than 0.05). The levels of TBA were higher in no-treated group (69.4+/-3.7)mumol/L and treated group (48.5+/-4.8)mumol/L as compared to control group (27.1+/-3.2)mumol/L at the 21th day (P value is less than 0.05). The lever of TBA was significantly lower in Treated group compared with No-treated group (P value is less than 0.05). (2) The mRNA expressions of CYP7A1, FXR, CYP27A1 and CYP8B1 increased in No-treated group (1.55+/-0.03, 1.75+/-0.02, 2.45+/-0.01, 2.15+/-0.01, respectively) and were all higher as compared to control group (0.75+/-0.02, 1.25+/-0.03, 0.65+/-0.03, 1.50+/-0.02, respectively) (P value is less than 0.05). However, the mRNA expression of PPARa decreased in No-treated group (0.85+/-0.02) compared with control group (1.45+/-0.02) (P value is less than 0.05). The mRNA expressions of CYP27A1, PPARa and CYP8B1 increased in treated group (1.25+/-0.01, 1.65+/-0.05, 1.65+/-0.02, respectively) and were all higher than that of control group (P value is less than 0.05). CONCLUSION: Abnormal expressions of CYP7A1, FXR, CYP27A1, CYP8B1 and PPARa may play a role in pathogenesis of estrogen-induced intrahepatic cholestasis. Activator of PPARa may be used as therapeutical drug for ICP.