| Cell penetrating peptides(CPPs)bear net positive charge,they could interact with the negatively charged cell membrane thus assisting nano preparations to enter the cells in faster speed.However,since CPPs are lack of cell specificity,they tend to accumulate in healthy organs causing potential adverse effect which is the main reason that restricts their use.In this manuscript,we constructed a nano drug delivery system called PAR-NLC which consists three motifs:nanostructured lipid carrier(NLC)as the drug cargo;PEG2000-AYELR which is AEYLR attached on the distal end of PEG2000,and AEYLR is an EGFR targeting small peptide which is designed by our research group;and poly-arginine(RRRRRRRR,R8)as a cell penetrating peptide.The structure of PAR-NLC is that PEG2000-AEYLR and R8 are modified on the surface of NLC and the aim of constructing PAR-NLC is to elaborate the synergistic effect of AEYLR and R8,and targetingly deliver antitumor drugs to tumor tissue in a faster speed.At the beginning of our research,to select a suitable R8 density,we systematically investigated the influence of R8 density on various properties of NLC.We built six different R8 density modified NLC naming nR-NLC in which "n" represents the percentage of R8 and the value varies in 0,2,4,6,8 and 10 which means 0%,2%,4%,6%,8%and 10%of R8 modified NLC.Six nR-NLC were all prepared by hot-melt emulsification method after which their size,zeta potential,entrapment efficiency,in vitro drug release profile and morphous were all studied.And results showed that the size and zeta potential of nR-NLC both elevated as the "n"value increase;drug entrapment efficiency and drug loading increased as "n" ascended to 6 and then decreased as "n" continued to add.Besides,we studied the in vitro drug release behavior of PTX loaded nR-NLC,and employed dissolution similarity factor f2 using 0R-NLC as the reference preparation to assess whether R8 density would affect the drug release of NLC.Results have revealed that the f2 value of the other nR-NLC compaired with 0R-NLC are all between 85 to 100,indicating excellent dissolution similarity and also that different R8 modification density rarely affect the drug release of NLC.The transmission electron microscope photograph told us that 0R-NLC and 10R-NLC were both spherical or near spherical solid particle.After the assessment of the pharmaceutical characteristics of nR-NLC,we prepared Cou 6 loaded nR-NLC and studied their cellular uptake using A549 as the model cell line.Clearage method and flow cytometry method were used to quantitatively determine the cellular uptake of nR-NLC.And the results showed that as the "n" increased,the cellular uptake of nR-NLC raised,and when the "n" reached 4,the intracellular amount of nR-NLC improved astonishingly.For example,after 4 h incubation with the cells,the intracellular amount of 4R-NLC was around 5 times of 0R-NLC while the amount of 2R-NLC was only 1.72 times of 0R-NLC.The outcome of confocal laser scanning microscope was identical with cleavage method and flow cytometry method.When the "n" reached 4,the cellular uptake amout increased significantly,and we also noticed that except 0R-NLC,all the other five groups showed obvious Cou 6 signal in the nuclei indicating the cell nuclear location which we believe was aroused by the existence of R8.Next,we assayed the cytotoxicity of blank nR-NLC and PTX loaded nR-NLC.And the results showed that exept blank 10R-NLC,all the other five blank preparations had no inhibition effect to A549 cells in the tested concentration which proved good safety;after the loading of PTX,the cell viability decreased as "n" upgrading,especially when "n" reached 4,the relative cell viability showed Cliff fall,which exerted the antitumor effect of the model drug PTX.We have also studied the tissue distribution of Dir loaded nR-NLC in S180 tumor bearing mice.And the outcomes have revealed that the tissue distribution of nR-NLC with "n"less than 8 were almost identical showing that with less than 8%R8 modification,the in vivo distribution of the cargo would not be influenced;however,when "n" equaled 8,the preparation showed obvious signal in almost all the tested tissues,especially in the heart which indicated potential accurate toxicity;when the "n" value was 10,about 10 min after the administration of 10R-NLC,the mice showed severe uncomfortable behavior like less activity and standing hair which proved our conjecture.According to the above results,we selected 4%R8 modification to build PAR-NLC,the reasons were that 4%R8 modification could improve the cellular uptake significantly while maintain the properties of the cargo NLC.And the amount of AEYLR was according to previous research and the amout of 10%of the total lipid weight.Besides,for the purpose of elaborating the targeting ability of AEYLR first and then the cell penetrating ability of R8,we attached AEYLR to the distal end of PEG2000,hoping to elaborate the right order of the two peptides using the length difference.After the preparation of PAR-NLC using melt-emulsification method,we learnt the pharmaceutical characteristics which showed that PAR-NLC was solid spherical particle with size around 50 nm,zeta potential at+14 mV and sustaind drug release profile.We tested the cellular uptake of PAR-NLC using EGFR positive cells NCI-H1299、S180 and EGFR negative cell K562 as model cell line,employed PA-NLC and PR-NLC as the comparison groups,PA-NLC was NLC modified with PEG2000-AEYLR,and PR-NLC was NLC modified with PEG2000 and R8.The results have showed that in NCI-H1299 cells and S180 cells,PAR-NLC gave the best cellular uptake with a lot more intracellular amount than PA-NLC and PR-NLC;while in EGFR negative cell K562,the cellular uptake of PAR-NLC was similar with PR-NLC and more than PA-NLC suggesting that AEYLR gave no contribution in K562 cells;the outcomes above suggested that PAR-NLC could exert synergistic effect of R8 and AEYLR.Using EGFR positive cell S180 to construct the tumor bearing mice model to test the in vivo tissue distribution of PA-NLC、PR-NLC and PAR-NLC.The results indicated that PAR-NLC could accumulate in tumor area with more amount in a very short time,and accumulated less in the liver and spleen compared with PA-NLC and PR-NLC,although PAR-NLC showed signal in other tissues but the strength was not strong and there was rarely any signal in the heart which implied that PAR-NLC was a relatively safe and effective antitumor drug delivery system.Next,we prepared PTX loaded PAR-NLC,and evaluated its in vivo antitumor efficacy using Taxol(?) as the comparison group.The results showed that after the loading into PAR-NLC,PTX exerted more efficient antitumor effect,more importantly,the potential adverse effect was alleviated which proved that PAR-NLC was indeed a safe and effective antitumor drug delivery system. |
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