Preparation Of Chitosan Nanoparticles Loading¹²⁵IUdR And Application In Therapy Of Rabbit Hepatic Carcinoma

Objective1. To prepare high deacelation degree and low molecular weight Chitosan, andfurther development chitosan nanoparticles and¹²⁵I-UdR-Chitosan nanoparticles. Theoptimum process conditions were established, and the physical and chemical properties,biocompatibility and drug release properties were identified. Further more, thepharmacokinetics and tissue distribution of¹²⁵I-UdR-CS-NP in New Zealand rabbitwere analyzed.2. To validate the passive targeting property of¹²⁵I-UdR-CS-NP to in situ livercancer models, especially the improvement of liver targeting ability through hepaticartery interventional delivering drug.3. To observe the internalization pathways and distribution, transshipment anddegradation of CS-NP in subcellular level.Mehtod1. High deacelation degree and low molecular weight Chitosan was preparedusing intermittent hydrolysis and radiation degradation method, and its infraredspectrum was analyzed.2. Using ion cross-linking method for preparation of CS-NP, and optimizing thepreparation techniques through the orthogonal test.3. The physical properties and characterization of CS-NP were analyzed usingtransmission electron microscope, laser particle size analyzer and zeta potentiometer.4. The biocompatibility of CS-NP was evaluated through the MTT method andflow cytometry instrument.5. Using the ion cross-linking method for the preparation of¹²⁵I-UdR-CS-NP, andthrough the single factor analysis method to determine the optimum process conditions.6. The in vitro drug release properties of¹²⁵I-UdR-CS-NP were analyzed by dynamic dialysis method.7. The pharmacokinetic characteristics and drug organization distribution of¹²⁵I-UdR-CS-NP injected via peripheral vein of the rabbits were analyzed.8. The side effects of¹²⁵I-UdR-CS-NP were analyzed using in vivo experiments.9. Rabbit in situ liver cancer models were prepared using ultrasound-guidedpercutaneous puncture technology.10. The in vitro cell uptake experiments were performed to verify the liver cancertargeting ability, including the comparisons on the uptake volume of¹²⁵I-UdR-CS-NPand time effection, dose effection between the liver cancer cells and normal liver cells.11. The fluorescein isothiocyanate marked chitosan nanoparticles were preparedusing ion cross-linking method.12. As drug carrier, the liver cancer targeting property of CS-NP were validated,through continuous observing the dynamic distribution of FITC-CS–NP in liver cancercells and normal liver cells with laser confocal microscope.13. The process of liver cancer cells and normal liver cells internalizedFITC-CS–NP were dynamically record via the time sequence scanning technology.14.¹²⁵I-UdR-CS-NP were perfused in hepatic artery of rabbits with liver cancer insitu model using Seldinger micro catheter under the CT guided, and the distribution andliver targeting property were observed in deferent period by SPECT.15. The organization distribution of¹²⁵I-UdR-CS-NP been perfused in rabbithepatic artery were analyzed to verify its tumor targeting property.16. When the FITC-CS–NP were uptake under different temperature, the cells’behavior characteristics were observed by Laser confocal microscope.17. The transmembrane process of FITC-CS-NP entering into hepatoma cellsthrough different ways and its distribution characteristics within the cell was observedwith the help of laser scanning confocal microscopy.18. The dynamic distribution, transportation and degradation process ofFITC-CS-NP in subcellular level was observed by adopting multicolor stainingtechnology.Result1. The deacetylation degree of chitosan prepared by intermittent hydrolysis andradiation degradation was93.062±2.384%, with the molecular weight of3kDa. It was dark yellow powder, which can be completely dissolved in water of PH7.0, to presentdark yellow clarified translucent liquid. IR spectra showed that the processed chitosanamide bond was hydrolyzed with more exposure of free amino groups, while the degreeof deacetylation was markedly improved.2. The CS-NP was successfully prepared. The orthogonal experiment showed thatthe influences on CS nano-particle size were arranged in a decreasing manner, the CSmolecular weight, TPP concentration, stirring speed and CS concentration. Optimizedpreparation technological condition had CS concentration of1g/L, stirring speed of600rpm, TPP concentration of2g/L, CS molecular weight of3kDa, average preparedparticle diameter of CS-NP of70.39±5.12nm, PDI of0.16±0.012, and zeta potentialof+32.5±3.3. Its appearance through TEM observation presented a regular sphere withsmooth surface, uniformed size, and good dispersity.3. MTT method and flow cytometry results showed that CS-NP had no celltoxicity on human normal liver cell line HL-7702and QSG-7701; when the CS-NPconcentration was equal or greater than200μg/mL, there was certain toxicity for thehuman hepatoma cell line HepG2and SMMC-7721.4.¹²⁵I-UdR-CS-NP was successfully prepared in the study. The optimizedpreparation condition required reagent quantity of1.2-2.4mg/mL, PH5.5, and theaverage particle diameter of the prepared¹²⁵I-UdR-CS-NP was175.8nm. Compared tothe blank CS-NP, its traits was slightly changed with the increase of the particle size anddecrease of uniformity, loose of structure, and irregular of the surface morphology.5. Dissolution curve of the¹²⁵I-UdR-CS-NP in PH5.3to7.4fit Higuchi equation,with the characteristics of long-acting preparation and distinct sustained releasefunction.6. After peripheral intravenous administration, the pharmacokinetics of¹²⁵I-UdR-DLN was in line with the characteristics of the two-compartment model. Thehalf-life of the first phase and the second phase were1.1±0.69h and1.5±0.16hrespectively, and the area under the curve （AUC0-∞） was11.2±1.4mg/L h. Comparedto¹²⁵I-UdR, the biological distribution of¹²⁵I-UdR-CS-NP tended to be on the liver andspleen, showing obvious delay by renal excretion, and its distribution of marrow andgastrointestinal mucosa was less.7. Intravenous injection of¹²⁵I-UdR-CS-DLN was as high as7.4MBq （6mg） indosage, with no significant abnormality of blood biochemical indicators or distinct bone marrow suppression found. While the bone marrow of the control group of the¹²⁵I-UdRshowed that the¹²⁵I-UdR had an inbibitional effect on the hematopoietic cells ofproliferation phase in2d, which indicated that¹²⁵I-UdR-CS-DLN could significantlyreduce the inhibitory effect on bone marrow hematopoietic cells.8. The in-vitro cells uptake experiment revealed that¹²⁵I-UdR-CS-NP had a cleartargeting on the above two hepatoma cells, the amount of which entering to thehepatoma cells was10times of normal liver cells, presenting time effect and doseeffects. The intracellular drug radioactivity intensity would reach saturation30min afterdosing or when the dose was greater than185kBq/mL.9. The CS-NP （FITC-CS-NP） labeled by isothiocyanate fluorescein wassuccessfully prepared, with an average particle size of158.02±12.30nm, and zetapotential of+28.8±3.1. The observation of its appearance by electron microscopy wasregular sphere with its surface structure being loose.10. In the cellular uptake experiment, FITC-CS-NP can be largely absorbed by theabove two hepatoma cells and unevenly distributed within the cytoplasm; whileFITC-CS-NP entering to the two normal liver cells was significantly reduced, more ofthem were adsorbed on the cell membrane, and dissociated after30min.11. ROI scanning curve demonstrated that the FITC-CS-NP, after contacting withhepatoma cell HepG2, completed processes of cell membrane adsorption,transmembrane internalization and entering cytoplasm. The peak time of cell membraneadsorbing FITC-CS-NP was15min, and that of intracytoplasmic FITC-CS-NPfluorescence was30min. FITC-CS-NP can also be largely absorbed on the surface ofnormal liver cells HL-7702within15min, however, the amount entering the cells wassmaller with clear slowing down of the process.12. Liver VX2tumor model was established successfully in rabbit, all of whichwere left lobe of liver tumor lesions with an average diameter of9.2±1.3mm.13. Micro-catheter using Seldinger technique made super-selection to the lefthepatic artery through the arteria cruralis. After infusion of¹²⁵I-UdR-CS-DLN, SPECTday parting image showed that the¹²⁵I-UdR-CS-DLN could be massively accumulatedin the liver and tumor by virtue of the precise positioning of Seldinger micro-catheterand liver first-pass effect.¹²⁵I-UdR-CS-DLN in the liver tissue could spread over thewhole body to maintain at an average level after2h, while the tumor body stillpresented clear¹²⁵I-UdR-CS-DLNconcentration, and the T/NT ratio in the ROI interest area reached4.49±1.22, which indicated that the¹²⁵I-UdR in-CS-DLN had clearpassive targeting on the in-situ liver tumor of the rabbit living body with sustainedrelease characteristics.14.48h after hepatic artery infusion of¹²⁵I-UdR-CS-DLN, the drug tissuedistribution revealed that the radioactivity intensity in the tumor was over12times morethan that of the normal liver tissue, and about50times more than the muscle tissue,exhibiting longer drug release time, which further proved the passive targeting andsustained release characteristics of¹²⁵I-UdR-CS-DLN in rabbit living body on in-situliver tumor. However, the gastrointestinal mucosa drug was slightly higher or lowerthan¹²⁵I-UdR.15. As indicated by the laser scanning confocal microscopy results, the entering ofFITC-CS-NP to tumor cells was mainly through the temperature-dependent activeuptake mechanisms requiring cellular energy consumption.16. Uptaking of¹²⁵I-UdR-CS-DLN by hepatoma cell involved a variety of ways,mainly by the giant endocytosis pathway for43%, and then the clathrin-classicalpathway for34%. The caveolae-pathway accounted for only12%. The three pinocytosismechanisms were independent with no synergy, but with a certain amount ofcompensation.17. Laser scanning confocal microscopy verified the function of three endocytosismeans for FITC-CS-NP entering to the hepatoma cells by morphological andquantitative means. Moreover, the locations of endocytic vesicles distributed in theinner cell membrane and cytoplasm can also be observed.18. The observation by the multicolor fluorescence staining techniques in dayparting showed that the endocytic FITC-CS-NP by clathrin-classical pathway had fastertransmembrane speed to move towards the nucleus after endocytosis and integrate withthe lysosomes. The secondary lysosomes ultimately released the FITC-CS-NP or debrisaround the nuclear; endocytic FITC-CS-NP by giant endocytosis pathway had slowendocytosis speed, and can be scattered in the cytoplasm after transmembrane for a longtime, which was conducive to the delayed release of drugs for the purpose of sustainedintracellular release.Conclusion1. This paper firstly loads¹²⁵I-UdR into nano-drug carrier to be the targeting sustained release preparation. The chitosan nano-uniform particle prepared by optimizedtechnological condition is with uniformed size, regular morphology, as well as goodbiocompatibility; the release curve fits the Higuchi equation, with the characteristics oflong-acting preparation and distinct sustained release function.2. The paper creatively applies the administration technology of hepatic arteryintervention into the improvement of the passive targeting of nanoparticles. In vitro andin vivo experiments demonstrated that¹²⁵I-UdR-CS-DLN had obvious hepatomapassive targeting. After the administration by hepatic artery intervention,¹²⁵I-UdR-CS-DLN can be largely accumulated in liver tumor, and the T/NT can be upto12within48h.3.¹²⁵I-UdR-CS-DLN enters to the tumor cells mainly through large pinocytosisand clathrin-classical pathway, and the giant pinocytosis body can reside in thecytoplasm for a long time. It was first observed in the study that the FITC-CS-NPentering into the cytoplasm via clathrin-pathway can be centralized and released aroundthe nucleus after its integration and digestion by the lysosomes.All in all, the development of¹²⁵I-UdR-CS-DLN here has an obvious targeting ofHEPATOMA cells, whose intratumoral drug accumulation can be improvedsignificantly through the administration by hepatic artery intervention. It can stay in thehepatoma cells for a long time with some of the particles released around the nucleus.Therefore,¹²⁵I-UdR-CS-DLN is expected to become the new formulation of the internalradiotherapy of tumor subcellular level for the achievement of better therapeutic effect.