Studies On Targeting Modifications Of Chitosan-stearic Acid Conjugate Micelles

Polymer micelles are colloidal carrier system and have nanoscaled size(between 10～100 nm).The core of micelles can solubilize hydrophobic drugs by physically entrapped and the shell can retain the stabilization in aqueous envirment.Polymer micelles have many advantages in the drug targeting delivery system.Due to the leakiness of the tumor blood vessels(relative to the healthy blood vessels in most healthy organs),colloidal particles preferentially accumulate in the tumor through enhanced permeability and retention effect.After chemical binding("grafted") with ligand or antibody(folic acid et al.),polymer micelles can target to particular tissues, cells(especially tumor cells).Researchs in recent years show that polymer micelles can selectively target to particular cellular organelles,such as cytochondriome and Golgi's apparatus et al.This special characterisct of polymer micelle makes it a powerful carrier in gene delivery system.Chitosan(CSO) is a cationic polymer comprising of glucosamine with excellent biocompatibility,low toxicity and biodegradability.A number of researches revealed that chitosan and its derivate can protect biomacromolecule drugs.The chitosan carring polypeptide,protein and nucleic acid as perioral preparation has been widely researched,and getting more and more attention.Recently chitosan is being used to condense plasmid DNA in the research of gene therapy.Chitosan with low molecular weight does not cause accumulation in vivo.Thus investigating chitosan with low molecular weight as drug carrier is very useful.Amino groups of chitosan and carboxyl group of stearic acid was reacted using 1-ethyl-3-(3-dimethylaminopropl) carbodiimide(EDC) as coupling agent,to obtain stearic acid gafted chitosan(CSO-SA).The CSO-SA could form micells by self-aggregation.In order to reduce the uptake of CSO-SA micelles by macrophage and prolong the circulation time in plasma,polyethylene glycol(PEG) was grafted onto CSO-SA.For the active targeting purpose,galactose was also coupled to CSO-SA to increase the uptake of micelle by hepatoma cells.To increase the accumulation of drug in target site by reducing the initial release rate of drug from micelles,adenotriphos was added into micelles.Referring to the electrostatic interaction between DNA and cationic carriers,electrostatic interaction between the negative charge of adenotriphos and the positive charge of CSO-SA will make the micelles with more compact,followed by the decrease in drug release late.Therefore more drugs is brough to the interesting tissue.In order to find out the optima condition of PEGylation,PEGylated CSO-SA (PEG-CSO-SA) with different mole ratio(PEG to CSO-SA) was synthesized.The graft of PEG to CSO-SA was confirmed using H¹-NMR.The critical micelle concentration(CMC) of PEG-CSO-SA was determind using pyrene as fluorescent probe.Size and zeta potencial were determind using Zetasizer 3000HS.The number of hydrophobic micro-domains per CSO-SA or PEG-CSO-SA molecule was estimated by steady-state fluorescence quenching method,using 1-Dodecylpyridinium chloride(DPC) as quencher.Quantification of cellular uptake was conducted in RAW264.7cells to find out whether PEGylation will decrease the uptake of micelles by macrophage.To study the difference in uptake of CSO-SA and PEG-CSO-SA micelles by tumor tissue and normal tissue,quantifications of cellular uptake was conducted in HepG2 and mouse immortalization liver cells BRL-3A cells were also conducted.Using mitomycin C(MMC) as a model drug,in vitro anti-tumor activities of the micelles loading drug were investigated to find out whether PEGylation will affect the cytotoxicity of MMC-loaded micelles.Compared with CSO-SA,the H¹-NMR spectrum of the PEG-CSO-SA showed a sharp signals atδ=3.5-3.65ppm,which was the chemical shifts of the proton of -CH₂CH₂O- for PEG,indicating that PEG was coupled to CSO-SA.After PEGylation of CSO-SA,the critical micelle concentration(CMC) had no significant change,remaining at the same level with CSO-SA.The CMC values of PEG-CSO-SA varied from 11.5μg/mL to 12.5μg/mL,and that of CSO-SA was 13.8μg/mL, implying PEGylation has no effect on micelle forming of CSO-SA.To determine the graft ratio of SA and PEG,the degrees of amino-substitution (DS) of CSO-SA and PEG-CSO-SA were measured using TNBS method.The DS of CSO-SA was about 8.8±0.2%,and the DS of PEG-CSO-SA increased with increasing the charge ratio of PEG,varying from 11.8%to 19.8%.The results also confirmed the graft reaction of SA to CSO and the PEGylation of CSO-SA.To investigate spatial structure of the micelles after the PEGylation,the aggregation number of SA groups per hydrophobic micro-domain(n_SA) and the number of hydrophobic micro-domain per copolymer chain(n_domain) was estimated by the steady-state fluorescence quenching method using DPC as a fluorescence quencher for quenching of pyrene fluorescence.For CSO-SA,n_SA was 4.5±0.3,n_domain was 3.2±0.2.For PEG-CSO-SA,n_SA was 5.4～7.8,n_domain was 2.7～1.8.The results indicated that the introduction of hydrophilic PEG chain into the CSO-SA reduced the number of micro-domain in micelle.However,even using the higher PEG modification ratio,the PEG-CSO-SA had special spatial structure with multi hydrophobic micro-domains.It was found the cellular uptake percentage of CSO-SA micelles in normal liver cells and tumor cells were almost same,and the PEGylation of CSO-SA did not affect the cellular uptake of these micelles.However,the cellular uptake of PEG-CSO-SA micelles in macrophage reduced significantly.About 58.4±0.63%CSO-SA micelles was uptaken by RAW264.7 in 24 h.Only 17.7±0.94%PEG-CSO-SA micelles was internalized after the CSO-SA was modified with five molar times PEG.The results indicated that PEG modification can significantly reduce the uptake of the CSO-SA micelles by macrophage,while the uptaken amount of CSO-SA and PEG-CSO-SA by HepG2 and BRL-3A remained almost the same. Using MTT method,the IC₅₀ values of CSO-SA and PEG-CSO-SA were firstly determined as about 400μg/mL,indicating the present micelles can be used as a safe drug carrier.PEG modification of CSO-SA did not increase the cytotoxicities of the micelles.The IC₅₀ value of free MMC was about 1.97±0.2μg/mL.The IC₅₀ value was decreased to 0.13±0.02μg/mL after 32.3±2.3%drug was loaded into CSO-SA micelles.This means the same in vitro anti-tumor activity can be reached when CSO-SA micelles were used as drug transport carrier,and the drug dosage was reduced about 14 times.The IC₅₀ value did not changed obviously after the PEGylation of CSO-SA,which might due to the same cellular uptake and drug entrapment efficiency.Asialoglycoprotein receptor(ASGP-R) is a hepatic binding protein(HBP) that can specificly recognize glycoprotein which has galactose residue at the end of the molecule,followed by binding to the residue.It has been reported that there is plenty of ASGP-R on the surface of HepG2 cells and few ASGP-R is found on the surface of A549 cells.In this study galactose was coupled to CSO-SA,and the uptake of galactose grafted CSO-SA micelles by HepG2 and A549 cells was then visualized using fluorescence inverted microscope.The results were compared with that of CSO-SA micelles.It was showed that the internalization amount of galactose grafted CSO-SA micelles was more than that of CSO-SA micelles.This phenomenon was not seen in A549 cells.Furthermore,the effects of adenotriphos amount on the size,zeta potencial and in vitro release of hydroxycamptothecine-loaded micelles were investigated.As the adenotriphos amount increasing,the size and zeta potencial of hydroxycamptothecine-loaded micelles decreased.The release rate of hydroxycamptothecine from the adenotriphos modified micelles was obviously slower than that of free hydroxycamptothecine and unmodified micelles,when the mass ratio of adenotriphos to hydroxycamptothecine was 0.4:1(m/m).