Studies On Tumor Targeting Of Tranferrin Modified Stealth Nanoparticles Encapsulating PEGylated Hydroxycamptothecin Conjugates

Cancer has become one of the most terrible nightmares for human being.It is the second leading cause of morbidity and mortality in the world and the incidences are expected to continue on an upward trend for at least the near future.Nevertheless, over the past few decades significant advances have been made in fundamental cancer biology,allowing for remarkable progresses in diagnosis and therapy of cancer. However,the majority of therapeutic agents fail to be successfully delivered to the target site,causing adverse damage resulting from systemic administration.Moreover, systemic drug delivery hinges largely on physicochemical properties of the drug.The development of tumor targeting nanoscale drug delivery systems has opened the doors for the diagnosis and therapy of cancer.Polymeric drug delivery system has been one of the hot research fields not only because of its tremendous versatility in polymer matrices allowing for tailoring of the system properties to meet the specific intended need,but also its ease of surface modification,high encapsulation efficiency of the drug,drug protection,control over the release of drugs,and feasibility of scale-up and manufacturing.In this study,by means of pharmaceutics,macromolecular chemistry, pharmacology and molecular biology,HCPT loaded transferrin modified PEG-niosomes(Tf-PEG-NS) were first prepared for effective drug delivery to solid tomor by the combination of passive and active targeting.For the purpose of most efficient tumor targeting and antitumor effects enhancement,a novel drug delivery system was developed in the further studies.PEG was covalently linked to the 10-hydroxyl group of HCPT to produce PEG-HCPT conjugates.The conjugates were encapsulated into transferrin modified PEG-nanoparticles(Tf-PEG-NP) so as to exploit the possiblility of combination of the functions of passive and active targeting by Tf-PEG-NP,as well as sustained drug release in tumor from PEGylated drug for most efficient tumor targeting and antitumor effects enhancement.In the first chapter,poly(amino-poly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate)(NH₂PEG-PHDCA) was synthesized usingα-t-Butyloxycarbonyl- amino-ω-hydroxy poly(ethylene glycol)(t-Boc-NH-PEG-OH) as native materials. Poly(methoxy-poly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate (MePEG- PHDCA) and polyhexadecyl cyanoacrylate(PHDCA) were also synthesized as control materials.¹H-NMR,¹³C-NMR and FT-IR spectra of the copolymers were consistent with their structures.The molecular weight and distribution of copolymers were determined by gel permeation chromatography (GPC),and whose polydispersity indexes were relative low(less than 1.4).In the second chapter,HCPT loaded PEG-niosomes(PEG-NS) were prepared by thin-film hydration and ultrasound method;the periodate-oxidated Tf was coupled to terminal amino group of PEG to produce the active targeting vesicles(Tf-PEG-NS) under optimized condition with a molar ratio of oxidized transferrins:PEG at 1:2 for 12 h incubation.The niosomes were spherical in shape with average diameters of (115.4±9.1) nm.The zeta potential was(3.76±0.03) mV,the encapsulation rate and drug loading coefficient were(93.00±0.38)%and(2.74±0.01)%,respectively.The PEG density on the surface ofniosomes was 0.45 PEG/nm² and there were average 59 Tf molecules coupled to each noisome.The release profile of HCPT from Tf-PEG-NS showed a slower and sustained fashion(90%of HCPT was released within 60 h).Tf-PEG-NS displayed the strongest tumor cell cytotoxicity compared with HCPT injection,non-NS and PEG-NS.The uptake of Tf-PEG-NS by KB cells was time and concentration dependent,which could be inhibited by both low temperature and excess free Tf in the medium,strongly indicating that the uptake mechanism being an energy-driven,transferrin receptor mediated endocytosis process.In the study of intracellular drug distribution,it was found that the relative uptakes for all noisome groups into nuclei were more than those into cytoplasm,especially for Tf-PEG-NS, which was 79-,5.9-,1.7-fold than that of injection,non-NS and PEG-NS in nuclei at 12 h,respectively.While HCPT injection tends to accumulate in the cytoplasm and maintained in low degree.The pharmacokinetic study in rats showed that Tf-PEG-NS and PEG-NS could extend the half life(t_1/2β) of HCPT from 0.91 h to 10.39 and 11.32 h,respectively.The areas under the HCPT concentration-time curve(AUC) were 7.49 and 8.23-fold that of HCPT injection,respectively,while there was no significant difference between Tf-PEG-NS and PEG-NS.The tissue distribution and antitumor acticity were investigated on S180 tumor bearing mice.Tf-PEG-NS showed the highest tumor concentration(4.00-,2.83-and 1.51-fold that of HCPT injection,non-NS and PEG-NS, respectively),largest AUC in tumor(16.97-,3.32-and 1.45-fold that of HCPT injection,non-NS and PEG-NS,respectively) and the most powerful anti-tumor activity with the inhibition rate of 71%against S180 tumor in mice(16.97-,3.32-and 1.45-fold that of HCPT injection,n-NS and PEG-NS,respectively),which was attributed to the double effect from passive and active targeting.In the third chapter,PEG-HCPT conjugate was synthesized by selective acylation at the 10-OH moiety of HCPT with MePEG₂₀₀₀-COOH.The product structure was confirmed using TLC,¹H-NMR,¹³C-NMR as well as FT-IR,and product purity more than 98%using HPLC analysis.The absolute molecular mass of the conjugate was 2500.56Da measured by MALDI-TOF/MS.It could be concluded that one PEG polymer molecule carried exactly one HCPT molecule from the difference between MALDI-TOF spectra of MePEG-COOH and PEG-HCPT.As expected,PEGylation substantially increased the solubility of HCPT.The conjugate appeared to be more stable in acidic media(pH 4.0).For long time incubation(72h), the PEG-HCPT conjugate prepared in this study demonstrated cytotoxicity similar to native drug on KB cells.In the forth chapter,PEG-HCPT-loaded transferrin modified PEG-nanoparticles were prepared using the double emulsion/solvent evaporation method(Tf-PEG-NP) with the mean size of(109.80±5.26) nm.The differences of particle size,drug loading coefficient and average Tf number per particle between PEG-HCPT/Tf-PEG-NP and HCPT/Tf-PEG-NS were not significant.The zeta potential of PEG-HCPT/ Tf-PEG-NP was significantly lower compared with HCPT/Tf-PEG-NS.The PEG density on the surface of PEG-HCPT/Tf-PEG-NP was significantly greater than Tf-PEG-NS(HCPT),which may further influence their in vivo behavior.The release of HCPT from PEG-HCPT/Tf-PEG-NP was much slower than HCPT/Tf-PEG-NS with less than 60%of HCPT released within 72 h.The in vitro cytotoxity studies on KB cells showed that PEG-HCPT/Tf-PEG-NP was less active than HCPT/Tf-PEG-NS for 24h incubation.But for 48h incubation, the differences of cytotoxicity between the two systems were not significant.The uptake profiles of PEG-HCPT/Tf-PEG-NP in cytoplasm and nuclear were gradually increased.Its uptake index in nuclear was 1.61-,1.38- and 3.72-fold that of HCPT/Tf-PEG-NS,PEG-HCPT/PEG-NP and PEG-HCPT/non-NP for 48h incubation, respectively.On the contrary,the uptake of HCPT/Tf-PEG-NS,both in cytoplasm and nuclear,reached peaks at 12h,then dropped.The endocytosis mechanism of Tf modified niosomes and nanoparticles were further evaluated.The results of cellular inhibition showed that Tf-PEG-NS and Tf-PEG-NP could be taked up by KB cells via both clathrin- and caveolae-dependent endocytosis.Lysosome and Golgi apparatus were involvement in the cellular distribution.PEG-HCPT/Tf-PEG-NP exhibited extraordinary performance in in vivo pharmacokinetics,biodistribution and antitumor effect studies.The circulation half life was significantly prolonged(t_1/2β was up to 20.92h) due to the greater PEG density on the surface of PEG-HCPT/Tf-PEG-NP,which facilitate the carrier to accumulate in tumor tissue owing to EPR effect.The C_max and AUC of PEG-HCPT/Tf-PEG-NP in tumor were 1.66-fold and 2.12-fold that of PEG-HCPT/PEG-NP,respectively,with no significant differences of pharmacokinetic parameters between them in plasma. This phenomenon indicated transferrin played an important role in the drug delivery to tumor site.PEG-HCPT/Tf-PEG-NP showed the most selective to tumor with the highest value of t_e.In addition,the drug level in tumor of PEG-HCPT/Tf-PEG-NP group remained highest compared with other systems during the elimination phase. The AUC of Tf-PEG-NP loading PEG-HCPT was 1.45-fold that of the counterpart loading HCPT.The inhibition rate of Tf-PEG-NP was 93.43%,which was 1.23-, 1.25-,1.53-,1.85- and 2.79-fold of HCPT/Tf-PEG-NS,PEG-HCPT/ PEG-NP, PEG-HCPT/non-NP,PEG-HCPT solution and HCPT injection,indicating that PEG-HCPT/Tf-PEG-NP possessed the most powerful anti-tumor activity.The advantages of PEG-HCPT/Tf-PEG-NP included 1)prolonging drug residence time in circulation and increasing EPR effect by the stealthing action of PEG-PHDCA nanoparticles,2)active targeting function of transferrin by transferrin receptor-mediated endocytosis and 3)sustained releasing drug in tumor by PEGylation of the drug.