Preparation Of Staged Tumor Targeting PAMAM-lipid Composite Nanosize Drug Delivery System And Studies On Its Mechanism Of Action

The combination of "tumor-cell targeting mechanism" and "tumor-angiogenesis'targeting mechanism" is the tumor targeting mechanism for tumor lesions with high specificity,and thus it has received extensive attention of academic circles in recent years.However,in terms of the combination of the two mechanisms,the existing mechanism of"Dual tumor targeting mechanism" possesses the defects of "being difficult to realize high endocytosis efficiency in tumor cells and long-circulating effect simultaneously(PEG dilemma)" and "the declined tumor-cell endocytosis efficiency caused by the saturation of receptors on tumor-cell surface".To address this issue,a staged tumor targeting PAMAM-Iipid composite nanosize drug delivery system(c[RGDyk]-PEG2000-PSL[PAMAM G5.0-AC80-FITC5-FA5/DOX]," cRGD-PEG-PSL[ND/DOX]" for short)based on functional PAMAM dendrimers packaged by c[RGDyk]modified pH sensitive long-circulating liposome,was constructed to to overcome the defects of the abovementioned "Dual tumor targeting mechanism" and increase the tumor targeting efficiency of drug delivery system efectively by means of its "Staged tumor targeting mechanism"(Targeting tumor tissues firstly,and then targeting tumor cells).This paper mainly includes seven parts:(1)Synthesis and characterization of PAMAM G5.0 dedrimers;(2)Synthesis and characterization of chemically modified functional PAMAM G5.0 dedrimers(PAMAM G5.0-AC80-FITC5-FA5,"ND"for short)and intigrin ???3 guiding phospholipid(DSPE-PEG2000-c[RGDyk]);(3)construction and characterization of the staged tumor targeting PAMAM-lipid composite nanosize drug delivery system;(4)in vitro study on the "staged tumor targeting mechanism" of the staged tumor targeting PAMAM-lipid composite nanosize drug delivery system;(5)in vivo pharmacokinetics and biodistribution of the doxorubicin-load:ing staged tumor targeting PAMAM-lipid composite nanosize drug delivery system;(6)in vitro and in vivo pharmacodynamics of the doxorubicin-loading staged tumor targeting PAMAM-lipid composite nanosize drug delivery system;(7)in vitro cytotoxicity of blank staged tumor targeting PAMAM-lipid composite nanosize drug delivery system.PAMAM dendrimers were synthesized by divergent method,and dialysis method was employed to a whole generation of PAMAM synthesis product for purification.Fourier transform infrared spectrometry(FT-IR),nuclear magnetic resonance hydrogen spectrum(1H-NMR)were employed·to characterize the structure of the product;electrospray time-of-flight mass spectrometry(ESI-TOF-MS),matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),and gel permeation chromatography(GPC)were employed to characterize the molecular weight and polydispersion of the product;potentiometric titration were employed to determine the number of amino groups("-NH2")on the product surface.Chemically modified functional PAMAM G5.0 dedrimers(PAMAM G5.0-AC80-FITC5-FA5,"ND" for short)and intigrin ???3 guiding phospholipid(DSPE-PEG2000-c[RGDyk])were synthesized,respectively.The obtained ND pruduct was characterized by GPC,1-NMR,particles chemiluminescence immunoassay(CLIA),fluorescence spectrophotometry(SPF),ultraviolet-visible spectrophotometry(UV-Vis),Zeta potential measurement,and transmission electron microscopy(TEM)technology.T hin layer chromatography(TLC)was employed to track the synthetic process of DSPE-PEG2000-c[RGDyk]and MALDI-TOF-MS was employed to characterize the molecular weight of the product.Organic solvent volatilization method were employed to prepare the doxorubicin-loading functional PAMAM G5.0 dedrimers(ND/DOX),and thin-film dispersion method was employed to prepare the doxorubicin-loading PAMAM-lipid composite nanosize drug delivery system on the basis of ND/DOX.The encapsulation rate(ER%)and drug loading rate(DL%)were used to determine the best prescription for the preparation of cRGD-PEG-PSL[ND/DOX]:mole ratio of ND and DOX(1:2),lipid concentration(29.4?mol/mL),mole ratio of lipid and ND(410:1).At the same time,the leading-in of 5%(mole ratio)of c[RGDyk]-PEG2000-DSPE was proved to have no significant influence on the drug loading effect of cRGD-PEG-PSL[ND/DOX].Dynamic light scattering method(DLS),modulated differential scanning calorimetry(DSC),transmission electron microscopy(TEM)and Zeta potential method were employed for the structure characterization of cRGD-PEG-PSL[ND/DOX].The results showed:the newly prepared PAMAM-lipid composite nanosize drug delivery system was a vesicle structure,and its size was 106.1±18.4nm,Zeta potential was 6.00 mV,and it could maintain basically stable within 48h;DOX was amorphous in the preparation,which would be conducive to the.releasing of doxorubicin.Fluorescence spectrophotometry(SPF)was employed to investigate the in vitro release characteristics of cRGD-PEG-PSL[ND/DOX],the results proved its pH sensitivity and stability against plasma.In the nearly neutral environment(pH=7.4)simulating the internal enviroment of the human body,the releasing speed of this drug delivery system was very low,and it was stable against plasma.However,this drug delivery system could release ND/DOX as a whole very quickly in the weak acid environment(pH=6.5)simulating the interstitial fluid in tumor,which could provide premise conditions to the tumor cell targeting performance under the guidance of folic acid group.Flow cytometry and fluorescence microscopy were empolyed for a qualitative investigation on the blank staged tumor targeting PAMAM-lipid composite nanosize drug delivery system(cRGD-PEG-PSL[ND]).The results showed:cRGD-PEG-PSL[ND]and ND showed relatively high KB-cell uptake efficiency;The uptake of ND was specific in KB cells,while cRGD-PEG-PSL[ND]had similar uptake efficiency to KB cells and HUVEC cells.Fluorescence spectrophotometry(SPF)was employed for a quantitative investigation on the endocytosis of cRGD-PEG-PSL[ND].The results showed:the endocytosis of ND to KB cells was an active process,the clathrin mediated endocytosis pathway played a major role,and the folate ligands on ND surface played a significant role in promoting it endocytosis in KB cells;the endocytosis of cRGD-PEG-PSL[ND]to KB cells was also an active process,the giant pinocytosis and clathrin mediated endocytosis were the most important,and the c[RGDyk]ligands on the surface of cRGD-PEG-PSL[ND]played a significant role in promoting it endocytosis in KB cells.In addition,cRGD-PEG-PSL[ND]was pH sensitive,ND could release in the weak acid environment(pH=6.5)simulating the interstitial fluid in tumor,and ND could further improve the tumor endocytosis efficiency of cRGD-PEG-PSL[ND]by means of the KB-cell targeting drug delivery under the guidance of folic acid ligands on its surface,which could not be hindered by the saturation of integrin ???3(c[RGDyk]receptor).Laser confocal microscopy was empolyed to investigate the staged tumor targeting mechanism of cRGD-PEG-PSL[ND/DOX].The results showed:in the nearly neutral environment(pH=7.4)simulating the internal enviroment of the human body,ND/DOX and cRGD-PEG-PSL[ND/DOX]had similar staged tumor targeting performance,which was superior to other contrast agents.However,cRGD-PEG-PSL[ND/DOX]should have obviously higher in vivo tumor targeting efficiency then ND/DOX without long-circulating effect.At the same time,in the weak acid environment(pH=6.5)simulating the interstitial fluid in tumor,cRGD-PEG-PSL[ND/DOX]could release ND/DOX and realize the KB-cell targeting drug delivery under the guidance of folic acid ligands on the surface of the latter,which could not be influenced by c[RGDyk]ligands.Therefore,this drug delivery system could effectively overcome the defects of the existing mechanism of "Dual tumor targeting mechanism",such as "PEG dilemma" and "the saturation of receptors on tumor-cell surface".In addition,the monitoring results of the TEER changes of HUVEC single cell layer showd:The superiority of cRGD-PEG-PSL[ND/DOX]on staged tumor targeting efficiency was closely related to the increasing the permeability of the HUVEC cell monolayer achieved by the "anti-angiogenesis effect" of this drug delivery system,and this performance would also be conducive to the tumor targeting distribution of the drug delivery system during in vivo applications.The results of the in vivo pharmacokinetic study on cRGD-PEG-PSL[ND/DOX]showed that the drug delivery system could significantly increase the half-life of doxorubicin and blood drug concentration,and thus has gaven play to good sustained release effect and long-circulating effect.In vivo optical imaging of tumor-bearing mice was exploited to study the time-dependent biodistribution of different drug delivery systems,and the results showd that cRGD-PEG-PSL[ND/DIR]could not only manifest much better tumor targeting effect then its contrast agents(MPEG-PSL[ND/DER]and ND/DIR),but also reduce the distribution of drug delivery system to the vital organs of(such as liver,spleen,lung,kidney).Results of the in vitro pharmacodynamics research on cRGD-PEG-PSL[ND/DOX]showed:in the nearly neutral environment(pH=7.4)simulating the internal enviroment of the human body,the system can obviously enhance the cytotoxicity of doxorubicin;while in the weak acid environment(pH=6.5)simulating the interstitial fluid in tumor,cRGD-PEG-PSL[ND/DOX]could release ND/DOX and enhance the cytotoxicity of doxorubicin under the guidance of folic acid ligands on the surface of the latter.The S180 tumor-bearing mice modle was established,and tumor size,tumor weight and body weight was chosen as the indexes to investigate the in vivo tumor inhibition rate and the overall toxicity of cRGD-PEG-PSL[ND/DOX].The results showed that this drug dilivery system could significantly increase the in vivo tumor inhibition rate of doxorubicin,and also could significantly reduce the overall toxicity.Results of the in vitro cytotoxicity research on the blank staged tumor targeting PAMAM-lipid composite nanosize drug delivery system(cRGD-PEG-PSL[ND])showed that in the experimental concentration range,thecytotoxity of cRGD-PEG-PSL[ND]on HUVEC cells and KB cells were very low and could be ignored.In addition,the results of the hemolytic research on cRGD-PEG-PSL[ND]showed that the drug delivery system conformed to the requirements to the hemolysis degree of intravenous fluid preparations,and was suitable to be employed as Intravascular injection.In conclusion,it has been demonstrated that the doxorubicin-loading staged tumor targeting PAMAM-lipid composite nanosize drug delivery system(cRGD-PEG-PSL[ND/DOX])could effectively overcome the defects of the existing mechanism of "Dual tumor targeting mechanism"("PEG dilemma" and "the declined tumor-cell endocytosis efficiency caused by the saturation of receptors on tumor-cell surface"),depending on its long-circulating effect,tumor-angiogenesis targeting effect,tumor-cell targeting effect,and pH sensitive drug releasing effect in tumor tissue,and thus it could realize the staged tumor targeting delivery effectively in a way of "targeting tumor tissues firstly,and then targeting tumor cells".