论文部分内容阅读
Caveolae-mediated pathway has attracted tremendous attention in gene therapy since it has the ability to avoid lysosomal degradation of delivered genes.As the significance of caveolae for gene delivery has emerged, the approach of introducing ligands into the polymer-based carriers is promising for the construction of non-viral gene vectors to target caveolae-mediated endocytosis.NGR peptide showed highly specific recognition to CD13.In addition, it has been reported that aggregated labeling of CD13 co-localized with CAV-1 in most cells, suggesting the critical role of caveolae that provides an environment to assemble essential signaling components in mediating NGR functioned nano-carriers transactivation.We therefore hypothesized that NGR might be able to mediate the NGR functioned multifunctional nanocarriers into CD13 positive cells via the caveolae-mediated endocytosis.In the present study, to verify the hypothesis and exploit the relationships between CD13, NGR and CAV-1, HUVEC was selected as the test cell and the previously formulated DOX loaded targeted PIC micelles (TPIC) in our group were selected as the test nanocarriers.Using the monoclonal antibody specifically recognizing CD13 and CAV-1 respectively, both CD 13 and CAV-1 expressed on HUVEC at the same time was identified by flow cytometric analysis.In the co-localization experiment, when HUVEC were incubated with an anti-CD13 antibody and fixed without warming, there was no co-localization happened between CD13 and CAV-1.When incubation time was 30 min at 37℃, the labeling of CD13 and CAV-1 on HUVEC surface showed in spots and small extent of co-localization between CD 13 and CAV-1 was observed.With the extending of incubation time at 37℃, co-localization of CD13 with CAV-1 became more frequent after 60 min of incubation at 37℃.When the incubation time extended to 2 h and 3 h, CD13 and CAV-1 gathered into clusters and co-localized extensively.That is to say, the antibody to CD 13 bound to the cell surface evenly when incubated on ice, but the bound antibodies became sequestered to caveolin-l-positive patches when the temperature was raised to 37℃.When evaluated the influence of TPIC on the co-localization of CD13 and CAV-1, it was found that incubation with TPIC could cause the cluster of CD 13 and would accelerate the speed and enhance the degree of the co-localization of CD13 and CAV-1.The results of co-localization and co-internalization experiments of CD 13 with TPIC demonstrated that TPIC could bind to CD 13 and entry into HUVEC together with CD13, suggesting that TPIC entering into HUVEC was CD13 mediated endocytosis.Similarly, TPIC could bind to CAV-1 and entry into HUVEC together with CAV-1, suggesting that TPIC entering into HUVEC was caveolae mediated endocytosis.The result of inhibition study of CD 13 enzyme activity indicated that the internalization of TPIC to HUVEC was not dependent on the enzyme activity of CD 13.To further investigate the important effect of CAV-1 on the intracellular entry of TPIC, MβCD was used to deplete cholesterol from cell membrane, thereby selectively destroyed the formation of caveolae.The result demonstrated that cholesterol depletion could inhibit the intracellular entry of TPIC.Furthermore, co-localization between CD13 and CAV-1 was significantly decreased in cells that were treated by MβCD suggesting the important effect of caveolae on the internalization of TPIC.Thus, we could infer that through the interaction between CD13 and NGR, TPIC could change the distribution of CD13 on HUVEC, cause the cluster of CD 13, subsequently inspire the co-localized of CD 13 and CAV-1, and finally internalized via caveolae-mediated endosytosis.