Simuitaneous Delivery Of P53and Doxorubicin Using Hydroxyapatite Against Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), a form of very aggressive primary liver cancer, isthe sixth most prevalent neoplasm and the third most frequent cause of cancer-relateddeath. Surgical resection or liver transplantation are potentially curative treatmentoptions, but the majority of patients are not eligible for either resection ortransplantation, due to advanced disease stages. Traditional chemotherapy agents, inparticular doxorubicin (Dox), have been used in the past several decades. Besidessevere side-effects, their clinical benefits remain debatable, since HCC iswell-characterized by a remarkable failure of conventional treatments. However,dosage fine-tuning with improved delivery method or even combining with otherregimens may potentially argue the therapeutic efficacy and attenuate side effects ofchemotherapy agents, such as Dox. Gene therapy, aiming at modifying the geneticprogram of a cell towards a therapeutic effect, has emerged as an alternative approachfor treating cancer. The wild-type p53tumor suppressor gene plays an important rolein controlling cell cycle. Cell DNA damage can trigger activation of p53leading tocell cycle termination and apoptosis. However, p53is the most frequently mutatedgene in human cancer, including HCC, and alterations have been found in virtuallyevery region of the protein. Re-introducing wild-type p53by means of adenoviralvector-based gene delivery approach has reached clinical phase to treat various typesof cancers, including for HCC patients. Besides restoration of a prominent tumorsuppressing pathway, delivered P53protein could also sensitively respond to stresssignals including DNA damage induced by chemotherapeutic drugs as such Dox.Combining p53gene therapy with chemotherapy has shown favorable response incancer patients. The major challenge of combining gene therapy with chemotherapy isfinding an efficient but safe vehicle that can simultaneously deliver both regimens.Recently, inorganic nano-materials have gained more favor. Among those,hydroxyapatite (HAp) possesses several advantages, including superiorbiocompatibility and biodegradability, susceptibility to acid, lacking of toxicity and stable chemical properties. It is able to deliver a broad range of genes or drugs to avariety of biological systems. Nevertheless, the transfection efficiency of HAp isrelatively low. We previously have demonstrated that modifying HAp withpolyethylenimine (PEI) could dramatically enhanced intracellular transfectionefficiency without notable cytotoxicity.Methods：1) A novel HAp nanoparticles were synthesized and characterized by fieldemission scanning electron microscope (FE-SEM), X-ray diffraction measurement(XRD), fourier transform infrared spectroscopy (FTIR), transmission electronmicroscope (TEM), selected area electron diffraction (SAED) and atomic forcemicroscopy (AFM) to evaluate the morphology, size, crystallinity,etc., and developeda stable synthetic technology of HAp.2) The HAp nanoparticles were modified with PEI to form HAp-PEI （HP）complexes. The cytotoxic effects of HAp and HP on hepatocellular carcinoma cellline HuH-7,Hep-3B, SMMC-7721and hepatocyte line L-02cells were determinedwith the CCK-8cell viability assays.3) Compared the transfection efficiency of HP with pEGFP-C1-p53on L-02withHuH-7, Hep-3B, SMMC-7721.4) Evaluated the encapsulation of HP to p53and Dox, and developed thecomplexes HP-p53/Dox. The release of p53and Dox was measured in two normalsaline systems (pH=7.4and6.5).5) Inversed fluorescent microscope was used to compare the intracellular uptakeand investigate the cellular distribution of p53and Dox.6）Evaluated the anticancer efficiency of HP-p53/Dox to HCC in vitro and in vivo.7) Evaluated the anticancer efficiency of HP-p53/Dox to other cancer cells invitro.Results:1) HAp were nanorod particles with length ranged from20to50nm and width ofapproximately20nm, had a low degree of crystallinity and excellent dispersibility inaqueous suspension. The morphology of HP was same to HAp. HAp and HP carrier system itself exhibited no clear cytotoxicity on cells.2) HP with pEGFP-C1-p53had highest transfection efficiency in HuH-7cellsthan Hep-3B and SMMC-7721. HP almost had no transfection in L-02cells.3) HP had excellent ability to encapsulate p53and Dox. The gene and drugslowly released from HP-p53/Dox in vitro, and the amount of p53or Dox releasedfrom HP-p53/Dox at pH6.5was higher than at pH7.4.4) The fluorescent signal of p53and Dox in HuH-7cells were predominatelyoverlaid with the nucleus staining6hrs after transfection. Notably, the primarydistribution of both P53protein and Dox in the nucleus will be important to exerttheir functionality.5) Using CCK-8assays, enhanced cancer cell cytotoxic effects were observed inthe combination group in a dose-dependent manner, compared with p53or Dox alone.Simultaneous delivery of p53and Dox by HP resulted in the best antitumor activitywith high safety profile in mice.6) HAp and HP also had no clear cytotoxicity on other cancer cells, andHP-p53/Dox enhanced the cytotoxic effects.