Study On The Preparation And Targeted Characteristics In Vivo Of The Carboplatin-Fe@C Nanocage-loaded Chitosan Nanoparticles

Due to the incapability of targeted delivery and sustained release, chemotherapeutic agent brings markedly toxicity to normal tissue while it kills the tumor cell, which hinders its curative effect. Magnetic drug nanoparticles are composed of chemotherapeutic agent, superparamagnetic materials and coating polymers, and possesses the long circulating ability. When it is injected into body, it can accumulate at the target region under the magnetic field, and then drug is released smoothly to kill the tumor cell avoiding systematic toxicity. In this study the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles (C-Fe@CN-CN) are prepared by reverse microemulsion method, which consist of distinct Fe@C nanocage absorbed with drug as magnetic core, chitosan as matrix and carboplatin as model drug loaded with dual physical mechanisms. Its characteristics in vitro is observed. The blood drug level and Pharmacokinetics parameter and the distribution performance of C-Fe@CN-CN injection by hepatic artery in rat or rat with transplanted liver tumor are also investigated.Article I : Preparation and characteristics in vitro of the carboplatin-Fe@C nanocage-loaded chitosan nanoparticlesObjectives: To prepare the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles using dual physical drug-loaded mechanisms, optimize the conditions of preparation, observe its characteristics and observe the inhibitive effect on the proliferation of human hepatoma cell line HepG2 in vitro. Methods: The Fe@Cnanocage was made from the Fe@C nanopowder using the diluted HCl. The carboplatin-Fe@C nanocage-loaded chitosan nanoparticles was prepared by the reverse microemulsion method. The prepared nanoparticles consisted of Fe@C nanocage as magnetic core and chitosan as matrix. The carboplatin was loaded into the nanoparticles by dual physical mechanisms including encapsulation of matrix and adsorption of nanocage. The orthogonal experimental design was applied to optimize the preparation procedure. The shape, size, drug content, entrapment efficiency, magnetic responsivity and drug release in vitro were determined. The inhibitive effect on the proliferation of the HepG2 cell was measured by MTT colorimetry, IC50 was calculated. Results: Compared with Fe@C nanopowder, BET of Fe@C nanocage increased from 20.98m²/g to 40.38m²/g, pore volume of Fe@C nanocage increased from 70mm³/g to 110mm³/g, and the content of absorption for carboplatin reached 10.7%. The carboplatin-Fe@C nanocage-loaded chitosan nanoparticles exibited fairly smooth and spherical in shape and good magnetic responsivity. The average size was 207nm±21nm with narrow distribution. The drug content was (11.40±1.31)%. The drug release in vitro lasted 5d, and the cumulative release percent were 51 % 68%, 80% , 87% , 91%, respectively. C-Fe@CN-CN could apparently inhibit the proliferation of HepG2 cell in the dose-dependent and time-dependent manner. The inhibitive effect of C-Fe@CN-CN in 24 hour was lower than that of original carboplatin, and were equal to original carboplatin in 48 hour and 72 hour. Bland nanoparticles had no cytotoxicity on HepG2 cell. Conclusions: The favorable magnetic core and the rational cooperation of multiple physical drug-loaded mechanisms can improve the performance of nanoparticles, and the ideal magnetic drug-loaded nanoparticles can be prepared. C-Fe@CN-CN can effectively inhibit the proliferation of HepG2 cell and blank nanoparticles indicate favourable biocompatibility in vitro.Article II: Studies on Pharmacokinetics and tissue distribution ofC-Fe@CN-CN by hepatic artery injection in rat.Objectives: To investigate the blood drug level and Pharmacokinetics parameter and to detect the distribution performance of C-Fe@CN-CN injection by hepatic artery in rat. Metheods: Eighty normal rats were randomly divided into two groups: Group A, Free carboplatin Group, Group B, C-Fe@CN-CN with magnetic field Group. Abdominal exposure was carried out through a midline abdominal incision, and a cannula was inserted into the hepatic artery and fixed. In Group A, Free carboplatin was injected into hepatic artery, and in Group B, C-Fe@CN-CN was injected into hepatic artery and the left liver lobe was under 5000Gs magnetic field as targeted region for 30min. The doses of carboplatin in both groups were 5mg/kg body weight. At post injection time of 15min, 30min, 1h, 3h, 6h, 12h, 24h and 48h respectively the rats were sacrificed. The drug levels in plasma, targeted liver, non-targeted liver, kidney, spleen, lung and heart were measured by flameless atomic absorption spectrophotometry. The data was analysed by 3P87 computer program. The tissues of liver, kidney, spleen, lung and heart was stained by HE to observe the distribution of C-Fe@CN-CN. C-Fe@CN-CN was also labeled with⁹⁹Tc, and the distribution of radioactivity in different tissue in vivo was detected by ECT. Results: Two-compartment model was adapted to describe the pharmacokinetic characteristics of C-Fe@CN-CN and free carboplatin injected by hepatic artery. The Cmax of plasma in Group B was 7.85 mg/L, which was 73 percent of that in Group A (10.74 mg/L). The plasma concentration at 48h in Group B was 0.304 mg/L, which was 11.3 times higher than that in Group A (0.027 mg/L). The AUC, t_1/2β , and MRT values in Group B were 57.82, 30.20, and 19.04, which was 3, 1.4 and 2.6 times higher than those in Group A (19.03, 21.75,7.44) . The CL in Group B (0.087) was 33 percent of that in Group A (0.26) . The Cmax of targeted liver in Group B (38.47μg/g) was 2.6 times higher than that in non-targeted liver (14.79μg/g) , and 7.7 times higher than that of liver in Group A (4.98μg/g) . The concentration of targeted liver at 48h in Group B (3.11μg/g) was 8.9 times than that of non-targeted liver (0.35μg/g) , and that inGroup A was too low to be detected. The AUC of targeted liver in Group B (421.34) was 5 times higher than that of non-targeted liver (84.68) , and was 27.6 times higher than that in Group A (15.24) .The Cmax of kidney, spleen, lung and heart in Group B (29.94μg/g, 1.54μg/g, 1.76μg/g) were significantly lower that those in Group A (37.78μg/g, 2.14μg/g, 2.34μg/g). The histology examination demonstrated that a lot of C-Fe@CN-CN was accumulated among liver cells and in the hepatic sinusoid in the magnetic field. On the contrast, there was no C-Fe@CN-CN in non-targeted region. The photograph of radioacticity also showed C-Fe@CN-CN was accumulated in the targeted liver. Conclusions: C-Fe@CN-CN shows long-circulation and controlled release in vivo. C-Fe@CN-CN also has remarkable magnetic target, which can be specially accumulated among cells of targeted region under magnetic field, slowly release drug, increase the levels of drug, prolong the active time and decrease the Cmax in other organs.Article III: Study on Pharmacokinetics and tissue distribution of C-Fe@CN-CN by hepatic artery injection in rat with transplanted liver cancerObjectives: To investigate the blood drug level and Pharmacokinetics parameter and to detect the distribution performance of C-Fe@CN-CN injected by hepatic artery in rat with transplanted liver cancer. Metheods: Forty rats with transplanted liver cancer were composed of GroupB. Abdominal exposure was carried out through a midline abdominal incision, and a cannula was inserted into the hepatic artery and fixed. C-Fe@CN-CN was injected into hepatic artery and the liver tumor was under 5000Gs magnetic field as targeted region for 30min. The doses of carboplatin were 5mg/kg body weight. At post injection time of 15min, 30min, 1h, 3h, 6h, 12h, 24h and 48h respectively the rats were sacrificed. The drug levels in plasma, liver tumor, non-targeted liver, kidney, spleen, and lung were measured by flameless atomicabsorption spectrophotometry. The data was analysed by 3P87 computer program. The tissues of tumor, liver, kidney, spleen, lung and heart was stained by HE to observe the distribution of C-Fe@CN-CN. Another forty normal rats were composed of GroupB as control group, the left liver lobe was as magnetic field, and received the same treatment. Results: The histology examination demonstrated that a lot of C-Fe@CN-CN was accumulated among tumor cell and some small arteries were embolismed due to the accumulation of C-Fe@CN-CN in the magnetic field. On the contrast, there was no C-Fe@CN-CN in non-targeted region. The Cmax of tumor in GroupC (65.21μg/g) was 1.7 times higher than that in targeted liver in Group B (38.47μg/g). The concentration of tumor at 48h in Group C (7.27μg/g) was 2.3 times than that of targeted liver in Group B (3.11μg/g). The AUC of tumor in Group C (906) was 2.2 times higher than that of targeted liver in Group B (421.34). Both group displayed the similar Pharmacokinetics performance in vivo. Conclusions: C-Fe@CN-CN with long-circulation and controlled release performance in vivo shows more remarkable magnetic target to tumor, increase the levels of drug, prolong the active time, compared with normal liver tissue under magnetic field.