Study On Tumor Targeting With Transferrin Modified CPT-PAMAM-PEG Conjugates

Cancer remains one of the world's most devastating diseases,with more than 10 million new cased very year.However,mortality has decreased in the past two years, owing to better understanding of tumor biology and improved diagnostic devices and treatments.Current cancer treatments include surgical intervention,radiation and chemotherapeutic drugs,which often also kill healthy cells and cause toxicity to the patient.It would therefore be desirable to develop chemotherapeutics that can either passively or actively target cancerous cells.Passive targeting exploits the characteristic features of tumor biology that allow nanocarriers to accumulate in the tumor by the enhanced permeability and retention(EPR) effect.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.Although passive targeting approaches form the basis of clinical therapy,they suffer from several limitation because certain tumor do not exhibit the EPR effect and some drugs cannot diffuse efficiently for multiple-drug resistance.One way to overcome these limitations is to programme the nanocarrers so they actively bind to specific cells.This binding cay be achieved by attaching targeting agents such as ligands molecules that bind go specific receptors on the cell surface.In this study,by means of pharmaceutics,macromolecular chemistry, pharmacology and molecular biology,CPT loaded transferrin modified PEG-PAMAM dendrimers were first prepared for effective drug delivery to solid tomor by the combination of passive and active targeting.succinic anhydride as linker,We first synthezised Camptothecin-hemisuccinate. The remain carboxyl group was activated by N-Hydroxysuccinimide.The primary amino groups on the surface of PAMAM were specifically reacted with the NHS groups of the bifunctional PEG derivative.Three different PEG proportion conjugates PP₁₆,PP₃₂ and PP₄₈ were obtained and the average number of PEG chains attached PAMAM were 10.5,20.2 and 28.8 calculated through ¹H NMR spectra.The average diameters of three conjugates were 7.12nm,7.34nm and 8.56nm,respectively and the Zeta potential were 4.84mV,4.43mV and 1.90mV.Camptothecin-SA-NHS ester were reacted with the remains amino groups on the surface of PAMAM,named CPP₁₆, CPP₃₂ and CPP₄₈.The diameter and Zeta potential were also measured by Zetasizer Nano.The release profile of CPT from conjugates showed a slower and sustained fashion(75%of CPT was released within 120 h).Tf was thiolated using Traut's reagent and the extent of thiolation was determined by Ellmann's reagent.The MAL groups of PEG were specifically reacted with the thiol groups of thiolated Tf.The average number of Tf per conjugate was about 1.0 using Braford method.The diameter of CPP₁₆T,CPP₃₂T and CPP₄₈T were lager about 1-fold and the Zeta potential became negative.Tf modified CPT-conjugates displayed the stronger tumor cell cytotoxicity compared with CPT solution and no Tf modified CPT-conjugates in KB,K562 and S180 cells.The mean fluorescence intensity were determined during flow mytometric analysis of the cellular uptake of conjugates against KB cell,K562 cells and S180 cells.The uptake of Tf modified CPT-conjugates were 3.38～3.46 fold in KB cells and 2.52～2.86 fold in K562 cells compared with no Tf modified CPT-conjugates.The uptake of conjugates by KB cells was concentration dependent,which could be inhibited by excess free Tf in the medium,strongly indicating that the uptake mechanism being an energy-driven,transferrin receptor mediated endocytosis process.The endocytosis mechanism of Tf modified conjugates were evaluated.The results of cellular inhibition showed that CPP₄₈ and CPP₄₈T could be taked up by KB cells via clathrin -dependent endocytosis.CPP₄₈T was major mediated via transferrin receptor and Lysosome apparatus were involvement in the cellular distribution.KB cells were incubated with rhodamine labeled conjugates in the presence of lysotracker green,a marker for secondary endosomes and lysosomes,colocalization of rhodamine labeled conjugates in late endosome and lysosome were observed,indicating that cellular transport of conjugate depends on the lysosome process.To measure the apoptosis effect of CPT and conjugates quantificationally, Hochst33342 and PI was used to double stain KB cells.When cells were treated with CPT or conjugates with 1Nm for 12h,the early apoptosis of CPT,CPP₄₈ and CPP₄₈T were 0.76%,2.41%and 4.63%,4.75%,16.02%and 30.91%for 24h,respectively, which was consistent with the above results and indicated that CPP₄₈T induce more apoptosis due to more uptake of CPT,and produce higher cytotoxicity than CPP₄₈ and CPT.The pharmacokinetic study in rats showed that The more PEG chains on surface of the conjugates,the longer t_1/2 of the CPT were observed.However,the half life of conjugates became shorter than that of Tf modified conjugates.The tissue distribution was investigated on S180 tumor bearing mice.CPP₄₈T showed the highest tumor concentration(8.4-fold and 2.7-fold that of CPT solution and CPP₄₈,respectively),largest AUC in tumor(28.7-fold and 3.6-fold that of CPT solution and CPP₄₈,respectively).Good tumor targeting effect of CPP48T was showed by the parameters.Relative targeting efficiency(r_e) was 28.7CPP₄₈T showed the most powerful anti-tumor activity of with the inhibition rate of 65.7%against S180 tumor in mice(1.81-and 1.27-fold that of CPT solution and, respectively),which was attributed to the double effect from passive and active targeting.No significant side effects were observed by weight changes of the mice and the histological examination of the small intestine.Immunohistochemistry of the tumor tissues showed that CPP₄₈T had highest antitumor effects(inhibiting the tumor cells proliferation and inducing the tumor cells apoptosis).