Studies On Tumor Targeting Of Stealth Nanoparticles Loading Recombinant Human Tumor Necrosis Factor-alpha

In this study, poly (methoxy-polyethyleneglycol cyanoacrylate-co-n-hexadecyl cyanoacrylate) (PEG-PHDCA) was synthesized and used as drug carriers. The PEG-PHDCA nanoparticles loading recombinant human tumor necrosis factor-α (rHuTNF-α) with different monomethoxy polyethylene glycol (MePEG, Mr=2000, 5000, 10000) or particle size were prepared using double emulsion method. Their physicochemical characteristics, in vitro biological behaviors including complement consumption, serum protein adsorption and macrophage uptake were studied. The pharmacokinetics in rats and biodistribution in S180-bearing mice were examined and the tumor targeting were evaluated and compared. Finally, the influence of particle size and MePEG molecular weight of the stealth nanoparticles on the in vivo tumor targeting were summarized and the in vivo-in vitro correlations between them were discussed.In the first chapter, PEG-PHDCA with different MePEG were synthesized. The polyhexadecyl cyanoacrylate (PHDCA) was also obtained and used as a control. The FTIR, ¹H-NMR and ¹³C-NMR were consistent with their structures. Their molecular weight and distribution were determined by gel permeation chromatography (GPC), and their polydispersity indexes were all very low (less than 1.1).In the second chapter, the content of rHuTNF-a was assayed by UV method and its concentration was 23.5mg/mL. The in vitro biological activity determined by L929 cells toxicity method was 4.94 × 10³IU/mg. ¹²⁵I-rHuTNF-α was prepared with Iodogen method. The labeling yield was above 93% and radiochemical purity above 99%. The recovery of ¹²⁵I-rHuTNF-α was 91.5%.In the third chapter, under the basis of single factor experiment, PEG-PHDCA nanoparticles loading rHuTNF-α with different MePEG or particle size were prepared using double emulsion method. The entrapment efficiencies were optimized by uniform design. The nanoparticles were characterized in terms of particle size, zeta potential, entrapment efficiency, drug loading, fixed aqueous layer thickness (FALT) and MePEG density. The result showed that nanoparticles were spherical, and particle sizes were about 80nm, 170nm and 240nm, respectively. As MePEG molecular weight increased, zeta potential decreased, FALT increased and MePEG density reduced. As particle size decreased, zeta potential reduced, FALT increased andMePEG density increased. Particle size and MePEG molecular weight had dramatic influence on the release of rHuTNF-cc in PBS and rat plasma. The release rate and cumulative release ratio increased with the decrease of particle size or the increase of MePEG molecular weight. The release mechanism of PEG-PHDCA nanoparticles in PBS was diffusion-controlled model, with in diffusion and corrosion controlled pattern in rat plasma.In the forth chapter, the influence of particle size and MePEG molecular weight on the in vitro complement consumption, serum protein adsorption and macrophage uptake were evaluated. As the particle size decreased or MePEG molecular weight increased, the effect of complement activation was weakened, the ability of repulsing serum protein was enhanced and the amount of uptake by macrophages was decreased. PEG5000-PHDCA nanoparticles (80.0nm) had the strongest ability of reducing complement consumption, serum protein absorption and macrophage uptake.In the fifth chapter, the pharmacokinetics in rats, biodistribution in S180-bearing mice and tumor targeting were evaluated. The results showed that the elimination half-life of rHuTNF-a in rats increased with the decrease of particle size. The half-lives of rHuTNF-a delivered by PEG5000- PHDCA nanoparticles (80.0nm, 170.9nm and 242.9nm) were 9.97h, 6.83h and 4.06h, which were 35.6, 28.5 and 22.6 times as that by control PHDCA nanoparticles, 25.6, 17.5 and 10.4 times as that of free rHuTNF-a (0.39h), respectively. The AUC of PEG5000-PHDCA nanoparticles (80.0nm, 170.9nm and 242.9nm) in rat plasma were 14.8,9.0 and 6.58 times as that of PHDCA nanoparticles, 15.8, 8.4 and 4.8 times as that of free rHuTNF-a, respectively.The elimination half-life of rHuTNF-a in rat was prolonged with the increase of MePEG molecular weight. The half-lives of rHuTNF-a delivered by PEG2000-PHDCA, PEG5ooo- PHDCA and PEG10000-PHDCA nanoparticles were 4.11h, 6.83h and 7.37h, respectively, which were 17.1, 28.5 and 30.7 times as that by control PHDCA nanoparticles, 10.5, 17.5 and 18.9 times as that of free rHuTNF-a, respectively. The AUC of PEG2000-PHDCA, PEG5000-PHDCA and PEG10000-PHDCA nanoparticles in rat plasma were 5.7, 9.0 and 12.5 times as that of PHDCA nanoparticles, 5.3, 8.4 and 11.7 times as that of free rHuTNF-a, respectively.With free rHuTNF-a as control, both the relative tumor tissue exposures(re) and the ratios of rHuTNF-a peak concentration (Ce) in tumors for PEG-PHDCA nanoparticles were higher than one, the tumor targeting efficiency increased with the decrease of particle size or increase of MePEG molecular weight. In view of targeting...