| The HCC was the main disease with a high incidence in worldwide, is one of severalmalignancies with the highest fatality rate, currently. About the treatment of liver cancer,the radical resection is the most effective means for the treatment of liver cancer.Unfortunately, just about20%patients have a good physiological basis for this surgery.About80%patients has no a chance to received radical resection due to rapidly spread ofcancer or severe liver dysfunction, the mortality rate is very high. In addition, the statisticsdata of authority department showed that the5-year survival rate of liver cancer just5%, oseek more efficient and safe treatment for liver cancer in imminent. The pathologicalfeatures of HCC in areas mainly as incidence of occult, the rapid rate of deterioration,metastasis and recurrence rate, etc., brought many difficulties for the clinical treatment ofliver cancer. In recent years, although hepatic arterial embolization radiotherapy caneffectively reduce the recurrence rate of patients with liver cancer and5-year mortality, butthe treat means still unable to meet the demand for the clinical treatment of liver cancerdue to the high economic costs and targeting is not ideal.The nanotechnology was a new cutting-edge technology form1980s, had been givena more significant development in many subject areas, the study of nano-materials withintelligent features is also deeply in current. Nano pharmacy as a branch ofnano-technology, has been got a rapidly development in pharmacy, mainly involvingnanocarriers drugs, and the research about the active ingredients research with nanoscale,etc, and provided an possibility for the targeted treatment for some diseases. TheNanocarriers drugs include nano-liposomes, nano-polymeric micelles, nanoparticles, etc, the nano-drug not only can to achieve sustained and targeted release of BCSI drugs, andcould effectively improve the water-solubility of BCSII drugs.Liver as the vital organs formetabolism, a substantial weaken of the liver function would endanger the lives of patients,the damage to normal liver cells during chemotherapy for liver cancer was an importantfactor we should consider during the period of treatment. Based on above, the targetedtherapy is critical for the patients with liver cancer. The targeted therapy could increase theconcentration of anti-hepatoma drugs at the tumor site, and reduce the adverse reaction,could minimize the toxic of the chemotherapy drug for the normal liver cells of adjacentorgans. The results of clinical studies in abroad showed that there are some specificreceptors in the surface of liver, such as galactose receptor, mannose receptor and theasialoglycoprotein receptors, the receptor provide a natural target for the nano-durg.Chitosan, seaweed polysaccharides, galactose and other polymer materials has beenapplied widely in the preparation of targeted nano-dug, due to them good biocompatibilityand biodegradable, etc, and could specific bind with the receptor in the surface of liver, toreceive a targeted effect. In addition, the human liver is blood-rich that could absorb thedrug quickly, an optimum particle size is benefit to absorbing by the liverreticuloendothelial system (RES) and mononuclear phagocytic system (MPS) for the drugwhich could not bind to the receptor, and then receive a targeted treatment.In the research field of targeting agents, the galactose and chitosan always been usedas a polymer material, and could meet the request of specific receptor bind, all of thenano-system which be modified by polymer could receive a target effect in liver. Thus, inthis study, the The myristic acid (Myristic acid, MA) as the hydrophobic component andgrafted to the hydrophilic O-carboxymethyl chitosan (O-Carboxymethyl Chitosan, CC),then prepared two kinds of amphiphilic chitosan derivative nanoparticles, byabove-mentioned polymerization self-assemble in water. The nano-system was furthermodified with galactose residues containing lactose acid and mannose residues containingseaweed polysaccharide, respectively. Finally obtained lactose tetradecanoyloxycarboxymethyl chitosan(LMCC) and seaweed polysaccharides tetradecanoyloxy carboxymethyl chitosan(AMCC). The chemical composition, crystal form, and thermalstability of AMCC and LMCC were detected by infrared spectroscopy (FTIR), nuclearmagnetic resonance (1HNMR), X-ray diffraction, differential thermal analysis (DTA) andother analytical methods. The release function and targeting were studied by usingdoxorubicin as a model drug, to make a deeply and comprehensive evaluation for the twonao-system.Chapter I The preparation and characterization of the amphiphilicchitosan contains carbohydrate ligands with liver targetingChitosan as carrier material, and was condensation reaction with the carboxyl ofmyristic acid which was activated by chloride method, to prepared thetetradecanoyloxy-carboxymethyl chitosan (MCC) with nano-scale. For got a bettertargeting with the chitosan nanoparticles, the chitosan nanoparticles was modified bygalactose residues containing lactose acid and mannose residues containing seaweedpolysaccharide, prepared lactose tetradecanoyloxy carboxymethyl chitosan(LMCC) andseaweed polysaccharides tetradecanoyloxy carboxymethyl chitosan(AMCC).Then thenano-system received some qualitative analysis. The results of FTIR and1H-NMR showedthat, all of the nano-polymers were target polymer. The calculated results about thestructure of LMCC showed that the DS of myristic acid was63, the DS of lactose residueswas44; while the DS of myristic acid was54and the DS of polysaccharide residues was43in AMCC. The results of X-ray showed that LMCC and AMCC was turned toamorphous structure form the crystal structure of carboxymethyl chitosan. It suggested thatthe water solubility of the two nano-partices would increased further more, and could meetthe demand of targeting treatment. The results of TGA showed that the hermodynamicproperties of LMCC and AMCC had a great different form original chitosan, thehermodynamic and the structure also changed. Chapter II The study about the pharmacy performan andcharacterization of the amphiphilic chitosan contains carbohydrateligands with liver targetingIn this chapter, the drug loading and release properties of LMCC and AMCC werecharacterized by doxorubicin as a model drug. The hydrophobic carboxymethyl chitosanmolecule chains and the carboxyl groups were the key factors for the amphiphilic ofLMCC and AMCC, the hydrogen bonds among or in the chitosan molecules weredestroyed, the rigidity of molecules structure also changed, so the polymer could be formhydrophobic self-assembled nanoparticles. Then prepared the LMCC and AMCC, usedtransmission electron microscopy (TEM) to investigate the morphology of nanoparticles,used the dynamic laser light scattering (DLLS) to investigate particle size and Zetapotential of LMCC and AMCC. Prepared LMCC and AMCC nanoparticles which loadedAMD, then investigate the rate of drug loading and release behavior in vitro. Thenanoparticles with loading drug and blank nanoparticles were prepared by dialysis, theresults of TEM showed the morphology of AMCC and LMCC was rounded, and theaverage particle size was20-30nm. The average particle size after hydration of LMCC andAMCC was67.5±11.1nm，80.4±13.1nm. The Zeta potential of LMCC and AMCC was-17.5±3.7mV，-18.7±5.4mV respectively in pure water. After loaded ADM, the particlesize increased to86.8±10.3nm and102.5±18.9nm respectively, the Zeta potential did notchange. The rate of loaded of LMCC and AMCC was81.6%±1.2%，70.3%±0.6%, theproportion of loaded drug was3.8%±0.3%and3.4%±0.2%, it suggested LMCC andAMCC could bundle AMD well, and could improved the water-solubility of ADM. Thenanoparticles which loaded ADM showed a sensitive for pH, the release rate of ADM inthe medium at pH5.5was much higher than in the medium at pH7.4. The characteristicsabout the drug release of LMCC and AMCC was slow release followed by burst release,the release rate of drug at0-4h was the fast, the accumulated rate could up to50%to-60%,the release speed slow down during4~24h, and the accumulated rate could up to70%~96%. Under the same conditions, the release speed of LMCC was faster than AMCC slightly.Chapter III The study about the anti-tumor effect、targeting andsafety of amphiphilic nanoparticles loaded ADMThe LMCC and AMCC were marked by rhodamine B isothiocyanate, preparedRITC-LMCC and RITC-AMCC, investigated the uptake rate of above nano-particles byHU-h human hepatoma cell. The mice with H22xenografts were as a study model forassessed the effect of anti-tumor and the tissue distribution of LMCC and AMCC. Thesafety of LMCC and AMCC was evaluated by acute toxicity test and hemolysis test.The hepatoma cells and HT22hippocampal neurons cell as subjects, the results oftransfected cell tests showed that, the fluorescence intensity of hepatoma cells was strongerthan HT22hippocampal neurons cell at1h,2h and4h. The results suggested that LMCCand AMCC had a good selectivity for hepatoma cells, could receive a targeting. The resultsof transfected cell tests with different doses((concentration was2mg/ml, dose was75μl,150μl,300μl)) nanoparticle loaded ADM showed that the amount of nanoparticlesabsorbed by hepatoma cells was increased with the concentration of nanoparticles, itsuggested the amount of nanoparticles absorbed by hepatoma cells wasconcentration-dependent.The results of a study about the absorb time of nano-particles showd that, the amountof nano-praticles be absorbed by hepatoma cells was very low at1h while higher at2h, andthe amount was most higher at4h. It suggested the amount of nanoparticles absorbed byhepatoma cells was also time-dependent, a sustained release pharmaceutical preparationswas more conducive to the target organ.The mice with H22xenograft as the objects for the study about the anti-tumor effectof LMCC and AMCC loaded ADM. The results showed the rate of anti-tumor of LMCCgroup was62.7%±5.4%while the ADM group was51.2%±7.7%, the effect of anti-tumorwas very good. The anti-tumor rate of AMCC group was42.5%±14.7%, had no differentwith ADM group(P>0.05). The results found the anti-tumor effect of ADM could beenhanced by LMCC,but the AMCC was not ideal. The results of H22tumor tissue distribution in mice showed: compared todoxorubicin hydrochloride solution, the LMCC nanoparticles by modified by lactose–modified have a good targeting for liver cell and tumor cell, the AMCC nano-particle hasa good targeting for liver cells but has a very weak targeting for H22hepatoma cells. Theconcentration of ADM in lung and spleen was also higher than the same organs indoxorubicin hydrochloride group. Both LMCC and AMCC nanoparticle could reduce thetoxicity on cardiac and renalThe hemolysis rate was less than5%at the highest concentration (10mg/ml), no ratdied after treated with LMCC and AMCC at a high doses was2000mg·kg-1. It foundLMCC and AMCC has a good safety for treated subjects. |
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