Tuning The Cell Internalization Ability Of Micelle Through Variations Of The Tat Density On The Surface

As one of the most representative cell-penetrating peptides(CPP),Tat(HIV-1 trans-activator of transcription)peptide has been widely used in the surface modification of nanocarriers in recent years.Due to a large number of positively charged guanidine groups in the peptide structure,Tat peptide displays an excellent affinity with negatively charged heparan sulfate proteoglycan(HSPG)on the surface of cell membrane which effectively promotes the cellular internalization of the Tat modified nanocarriers.On the other hand,polymeric micelles emerged as ideal carriers for targeted drug delivery because of their good biocompatibility,large drug loading capacity,controllable structure and modifications.Although numerous research groups reported the use of Tat peptide to enhance the internalizations of polymeric micelles,the influence of the Tat density on the cell internalization and the mechanism of Tat-mediatied micelles still remained unclear.In this thesis,we set to use PCL-PEG(poly(?-caprolactone)-block-poly(ethylene glycol))micelles as the carriers to systematically study the effect of Tat peptides on the internalization behavior and possible mechanism of micelles by tuning Tat density,incubation concentration,incubation time and other factors.Firstly,we synthesized PCL-ss-PEG-Tat and PCL-mPEG copolymers,the structures of the products were confirmed by ~1H nuclear magnetic resonance spectroscopy(~1H NMR),fourier transform infrared spectroscopy(FT-IR)and gel permeation chromatography(GPC).And then,we changed the propotion of PCL-ss-PEG-Tat copolymers to prepare mixed micelles with different Tat density.The data of dynamic light scattering(DLS),transmission electron microscope(TEM),ultraviolet-visible spectroscopy(UV-vis),fluorescence spectrophotometer demonstrated the relative uniformity of these mixed micelles in terms of the particle size,morphology,the loading content(LC),the encapsulation efficiency(EE),and critical micelle concentration(CMC).These data provided a required precondition for the following studies.Besides,we investigated the reductive response of mixed micelles under high concentration of glutathione(GSH,10 mM)in vitro,which demonstrated the reduction-response release behavior of PCL-ss-PEG-Tat mixed micelles.And through the investigation of the cell compatibility of blank micelles,the safety of the materials used under the experimental conditions was next proved.Compared to the unmodified group,the Tat-modified drug-loaded micelles displayed 3-fold higher antitumor activity.Finally,we systematically investigated the internalization behavior of Tat-mediated micelles by fluorescence activated cell sorter(FACS)and confocal laser scanning microscope(CLSM).By tuning the Tat density,incubation concentration,incubation time and other factors as well as changing the temperature or adding endocytosis inhibitors,we explored the process and possible mechanism of the internalization of Tat-mediated micelles.Combined with the experimental results,we drew the following conclusions:As the Tat density increases,the Zeta potential of micelles gradually increases,which subsequently enhances the electrostatic interaction between the positively charged groups of Tat-modified micelles and the negatively charged groups of the cell membrane.The Tat-modified micelles firstly accumulated on the surface of the cells.And then the micelles were internalized into the cells through both energy-dependent(such as endocytosis)or energy-independent pathways(such as cell membrane penetration).The rate of cellular uptake also increased with the increase of Tat density.