Fabrication Of Nano Layered Double Hydroxide Co-delivery System And Its Synergistic Mechanism Study Of Anti-resistance To 5-fluorouracil In Hepatocellular Carcinoma

Hepatocellular carcinoma is the fifth most commonly occured malignant tumor and remains the third leading cause of cancer-related death worldwide.It is widely accepted that surgical treatment is the optimal choice for the early staged HCC patients.However,the concealed characteristics and advanced-stage diagnosis of HCC led to the loss of surgical indications.In addition,clinical data shows that the patients with early surgical resection also suferred a high rccueerence rates.Therefore,systemic pharmacotherapy and intervention are essential for HCC patients.Most of HCC patients suffered different degrees of liver fibrosis,leading to a weakened hepatic alexipharmic functions.Direct expose of chemotherapeutic agents,such as 5-fluorouracil causes acute or chronic hepatic damage.What's more,with the extensive use of traditional chemotherapeutic drugs,multidrug resistance?MDR?has become a huge obstacle for chemotherapy of cancer patients.Literatures showed that MDR is related to many mechanisms,such as glycoprotein on cell membrane,upregulation of anti-apoptotic gene,autophagy and so on.Addressing the problem of drug resistance is key to successful chemotherapy for cancer patients.Common methods include increasing drug doses to achieve an effective therapeutic concentration,but it brings higher risk of side effects.In recent years,combined chemotherapy brings new hope for the HCC patients,which can synergistically kill cancer cells by combining effects of different drugs.However,there exists many uncertain factors of combining different chemotherapeutic drugs,which may bring greater side effects.Curcumin is a kind of traditional drugs derived from food,which has many advantages,such as low cost,non-toxic,etc.Studies have indicated that curcumin has multiple pharmacological activities in anti-inflammation,anti-oxidation and anti-tumor.It was reported that the combination of chemotherapy drugs with curcumin could effectively reverse MDR in tumors.However,curcumin has some shortcomings,such as poor water solubility,fast in vivo clearance.It is worth exploring to give full play of the powerful curcumin.In recent years,the fast development of nanotechnology provides a new direction for constructing novel drug formulations.Nano technology is advantageous in many aspects:The pH and thermal sensitivities of nanocarriers help achieve controlled drug release,thus enhancing bioavailability and reducing adverse effects caused by direct drug exposure to normal tissues;The thermal-responsiveness and modification of different ligands of nanocarriers slow release of the drug can effectively realize the active and passive target:ing;Codelivery of drugs via nanocarriers is not merely a combination of two drugs,but an integration of the sequential and synergistic effects of chemotherapy drugs.Layered double hydroxide?LDH?is a kind of anionic clay with a double layered structure.Due to positive charge and abimdant hydroxyl in the outer layer,LDH is able to incorporate drugs via forming hydrogen bonds.The negative charge between the layers can be applied for anionic exchange,which allows incorporation of anionic drugs and small molecular nucleic acids.LDH thus attracts extensive attention due to non-toxicity,high drug-loading and pH-sensitivity.Based on previous experiments and literature reports,we fabricated a codelivery system that loaded Fu and Cur between the layers of LDH?LDH-Fu-Cur?.Bel 7420/Fu resistant hepatocellular cancer cell line were selected to investgate the effects of the codelivery system.We first examine the efFcects of LDH-Fu-Cur orn normal liver cells L02 and Bel 7402/Fu cell.We firstly detected the proliferation inhibiton effects of LDH-Fu-Cur on L02?normal hepatic cells?and Bel 7420/Fu.Then apoptosis effects of LDH-Fu-Cur were examined by flow cytometry and the underlying mechanisms were further confirmed by western blot.The endocytosis and autophagy induced by LDH-Fu-Cur were visualized by confocal microscopy.We detected the effect of autophagy in Bel 7420/Fu by autophagic agents.Finally,the anti-tumor effects of LDH-Fu-Cur was evaluated through the in vivo xenograft model.Results showed that we have successfully prepared the LDH co-delivery system with high drug-loading capacity and biosafety.The Fu loading efficiency measured by UV spectrophotometer in LDH-Fu and LDH-Fu-Cur was 20.3%±0.4%and 19.7%±0.2%respectively,with 15.7%±0.6%Cur loaded in LDH-Fu-Cur.It was as expected and met the requirements for further experiment.Our results also suggested that LDH-Fu-Cur showed an obvious inhibitory effects against Bel 7402/Fu tumor cells in a dose-dependent way while posing few influence against normal liver LO₂ cells.Flow cytometry and western blot results confirmed that compared with Cur,LDH-Fu-Cur facilitated cell apoptosis dramatically.Further experiments revealed that nano drugs down regulated the level of Bcl-2 which may explain to the apoptosis effect of drug resistance cells.Interestingly,autophagy,which is a "double-edged sword",also involved in the inhibitory effects of LDH-Fu-Cur.We confirmed that LDH-Fu-Cur could initiate autophagy,which contributed collaboratively to promote cell death.Eventually,we built Bel 7402/Fu xenograft model to assess the anti-tumor effects of LDH-Fu-Cur on fluorouracil resistance hepatocellular carcinoma.Compared with PBS control group,free Fu group and free Cur group,LDH-Fu-Cur could significantly inhibit the growth of Bel 7402/Fu cells in nude mice.It indicated that LDH-Fu-Cur was effective in suppressing fluorouracil resistance hepatoma cancer cells.In conclusion,we synthesized nano layered double hydroxide-based codelivery system which owns a desired structure and biosafety.LDH-Fu-Cur shows good effects on inhibition of fluorouracil-resistance hepatocellular carcinoma in vitro and in vivo,meanwhile promoting autophagy.The new formulation provides a new approach for designing anti-MDR drugs in HCC.