Study On The Construction And The Intracellular Delivery Process Of Multifunctional Self-Assembly Nanocarriers

Due to the complexity of tumorigenesis and the difficulty of cancer therapy, combined delivery of drug and gene has emerged as an exciting method of treating cancer which possesses a synergistic effect of increasing drug efficacy or enhancing gene transfection efficiency, thereby increasing the efficiency of cancer treatment and prolong the survival time of cancer patients. However, one of the most critical challenges for highly efficient gene/drug co-delivery is the design and development of co-delivery systems. For an effective delivery system, there are various in vivo or intracellular barriers must to be crossed in order to selectively deliver therapeutic drugs to their target site and exert their anti-tumor effect. Therefore, effective drug delivery system must have multifunction. For example, a shielding component could prolong the circulation time of drug delivery system in the blood; targeting moieties on the surface of a nano-system loading gene/drug could increase the specific binding between nanoparticles and targeted cells; some other functional components could increase the biocompatibility of the nanoparticles or enhance the ability of nanoparticles to escape from endosomes, etc. Owing to the difficulty of synthesizing one delivery carrier that meets all the requirements mentioned above, construction of a highly efficient vector with multiple functionality via procedurally assemble has drawn increasing attentions. Firstly, various functional units were synthesized. Using the supramolecular assembly technologies, functional units were then procedurally assembled into a nano-system with suitable size, controllable structure and good biocompatibility, which could across series barriers like the blood vessel wall, cell membrane, endosome membrane and the nuclear membrane and so on.In this study, we have developed multifunctional self-assembly nanocarriers by the procedural assembly integrated with passive and active tumor targeting, cell membrane translocation, pH-triggered drug release and co-delivery strategies simultaneously. Poly (ethyleneimine)(PEI)-polyethylene glycol (PEG) copolymer was synthesized with coupling TAT to the distal end of PEG to obtain PEI-PEG-TAT (PPT) with membrane penetration activity. The functional amino group of PEI in PPT was used to chemically conjugate doxorubicin (DOX) via pH-sensitive hydrazone linkage to obtain pH sensitive pro-drug, DOX-PPT (DPPT). The pH sensitive DPPT could effectively control the release of the drug in the acidic environment, and improve the selectivity and the accumulation of the drug in the tumor tissue. Meanwhile, the cationic PEI backbone of DPPT could condense DNA and fabricate DOX and DNA co-loaded self-assembly nanocarriers (DDN). To achieve active targeting, NGR (asparagines-glycine-arginine) peptide was selected as target ligand. Then, pH sensitive targeted shell functioned with NGR was conferred by electrostatic adsorption of sulfamerazine (SA)-PEG-NGR (SPN) on the surface of DDN to obtain targeted DOX and DNA co-loaded self-assembly nanocarriers (TDDN). NGR could specifically target to the over-expressed aminopeptidase N (APN/CD13) on tumor neovascular endothelial cells and tumor cells. Thus, the multifunctional self-assembly nanocarrier was expected to achieve better therapeutic effects by virtue of multifunction such as long circulation time, active targeting, pH sensitivity, endosomal escape, efficient penetration of the cell membrane and synergistic effect of gene therapy with chemotherapy.The elucidation of the intracellular mechanisms of carriers is very important. The design of novel drug delivery systems must base on the fully understanding of their cellular processes. In this study, based on the construction of carriers, we further studied the interaction between the carriers and targeted cells and their intracellular delivery process, especially focused on the caveolae-mediated endocytosis. The co-localization of CD13with caveolin-1(CAV-1) on the HUVEC membrane were investigated to verify that NGR could target to the caveolae-mediated endocytosis and efficiently co-deliver drug and gene to targeted cells. To evaluate the target capacity of TDDN to tumor cells after passing through vascular endothelial cell monolayer, a model of the blood vessel wall was developed in vitro using HUVEC cells, and a co-culture non-contact model of HUVEC and tumor cells was established to mimic the microenvironment of tumor. The results of the experiments indicated that TDDN could be uptaked by tumor cells after going through HUVEC monolayer.Above all, this study would make a contribution to the gene and drug co-delivery research field not only by constructing such a novel self-assembly multifunctional nano-carrier, but also by carrying out the basic researches on the exploration of the intracellular and intercellular mechanisms of this novel nanocarriers. The main methods and results were as follows:1The synthesis and characterization of novel functional materialsTo construct mltifunctinal drug/gene codelivery nanocarriers, first of all, the functional units should be prepared. Doxorubicin (DOX) was selected as model drug and PEI-PEG-TAT (PPT) was synthesized firstly. The structure of resulting PPT was verified by1H NMR and the integration ratio of PEI, PEG and TAT indicated that the mole ratio between PEI, PEG and TAT approximately to be1:13:2. A heterobifunctional crosslinker, C6-SANH was used to conjugate the C-13ketone group of DOX to the amino group of PPT by pH sensitive hydrazone bond to obtain DOX-PPT (DPPT). The1H NMR spectrum indicated that DOX were successfully conjugated to PPT. The amount of DOX loaded in DPPT determined by UV-Vis spectrophotometry was8.01±1.22%(weight ratio of DOX to polymer). The results of in vitro drug release of DOX from DPPT indicated that the release of DOX from DPPT exhibited a pH-dependent characteristics and was considerably lower at pH7.4(48h,32.8±3.25%) than pH5.0(48h,70.7±5.78%)(P<0.01). The results of acid-base titration indicated that DPPT exhibited excellent buffering capacity (21.5%) but a little lower than PEI25K (25.8%). suggesting DPPT possessed the endosome buffering capability. Cytotoxicity of free DOX and the DPPT conjugates was evaluated by MTT assay. At the test concentrations, PPT presented much lower cell cytotoxicity than PEI in MCF-7cells, HepG2cells and HUVEC (P<0.01). DPPT exhibited higher cytotoxicity than PPT in MCF-7, HepG2cells (P<0.05), which indicated that conjugating DOX to PPT did not affect the efficacy of DOX. While as, DPPT was less cytotoxic than DOX in MCF-7and HepG2cells. The phenomenon could be attributed to the gradual DOX release from DPPT. The cellular uptake of DPPT and DOX in HUVEC, HepG2and MCF-7cells under different pH was investigated using flow cytometry and fluorescence microscope. Compared with pH7.4, it was much easier for DPPT to release drug at pH5and the fluorescence intensity in cells was much stronger, which indicated that the pH sensitivity of DPPT. In addition, SPN was also synthesized and the chemical structure was confirmed by1H NMR, MTT results indicated that no obvious cytotoxicity of SA, SP and SPN was observed against any of the three cell lines tested, respectively. Above all, the synthesized materials possessed the required function, which were ready for the further construction of multi-functional self-assembly nanocarriers.2Construction of multifunctional self-assembly nanocarriers and the drug/gene codelivery in vitroIn this part, multifunctional self-assembly nanocarriers were constructed by the self-assembly method. First, DPPT was condensed with DNA via electrostatic force leading to the formation of DDN. Then, SPN was further assembled onto the surface of DDN to obtain TDDN. The results of agarose gel retardation assay indicated that when the weight ratio of DPPT to DNA up to25:16, all of DNA was effectively condensed by DPPT. And SPN could not affect the stability of DDN at the test concentration. The particle size, zeta potential, in vitro cytotoxicity and transfection of DDN and TDDN were measured at various weight ratios of DPPT to DNA or SPN to DNA. The DDN prepared at a weight ratio of DPPT/DNA12.5was chosen for the further coating of SPN. In addition, SPN at a weight ratio of SPN/DNA6.25was chosen to fabricate TDDN. Finally, the optimum composition of TDDN was DPPT/DNA/SPN=50:4:25. Both DDN and TDDN in optimum composition were shown analogous spherical shape and good stability which could protect DNA from the degradation of nuclease and plasma. The average particle sizes of DDN and TDDN were108.4±4.2nm and199.8±9.2nm, respectively. The zeta potentials were+10.08±0.58mV and+4.390±0.83mV, respectively. At the transfection dose, the transfection efficiency of DDN and TDDN in CD13positive MCF-7cells was38.34±2.3%and28.54±3.1%, respectively. TDDN showed about1.3fold more efficiency than DDN (P<0.05). In CD13negative HepG2cells, the transfection efficiency of DDN and TDDN was22.89±4.5%and19.49±5.6%, respectively. The cellular delivery of TDDN to MCF-7cells was about1.7fold compared with those of HepG2cells (P<0.05). These results indicated that NGR modified TDDN indeed enhanced the transfection efficiency in the CD13positive MCF-7cells. More importantly, DOX and pEGFP plasmid DNA were successfully co-delivered into the MCF-7and HepG2cells by TDDN, which indicated that TDDN hold the potential for drug/gene co-delivery.3Study on the cell uptake and endocytosis mechanism of multifunctional self-assembly nanocarriersThe reasonable construction of drug delivery systems was based on the thorough understanding of their endocytosis pathway and the drug release mechanism. In this part, CD13positive HUVEC and MCF-7cells, and CD13negative HepG2cells were chosen as cell models for the following studies. The results of cellular uptake indicated that both DDN and TDDN could be internalized in HUVEC, MCF-7and HepG2cells efficiently. A significant increase in DOX accumulation of TDDN in CD13positive HUVEC and MCF-7cells compared to that in HepG2cells was observed (P<0.05). suggesting the target capability of TDDN to CD13. However, for DDN. there was no significant difference of red fluorescence between HUVEC, MCF-7and HepG2cells (P>0.05). further implied the non-specific cellular penetration of DDN. To further evaluate the cellular uptake and intracellular drug release behaviors of DDN and TDDN in HUVEC, the cell nuclei were stained with Hoechst33342(blue). The red fluorescence of DOX accumulated in cytoplasm treated with TDDN or DDN suggested the efficient release of DOX inside cells. The results of cellular uptake kinetics studies at4℃and37℃demonstrated that the entering of TDDN into cell was time and energy depended process. To test the specificity of NGR-mediated uptake, competitive inhibition was performed with excess free NGR (1mg/mL). It was shown that the cellular uptake of TDDN in the presence of excess free NGR in HUVEC cells was suppressed significantly (P<0.01), pointing to the NGR mediated active targeting mechanism was involved in the enhanced uptake of TDDN into CD13positive cells. In the further experiments with endocytosis inhibitors, it was identified that the internalization of TDDN into HUVEC was a combine process of clathrin-mediated and caveolae-mediated endocytosis, and the latter was the main process. While, TDDN entering HepG2was clathrin-mediated endocytosis combined with macropinocytosis and the former was the main process. These results indicated that both the type of nanoparticles and cell affected the pathway of internalization. NGR modification was the key factor which affected the mechanism and the pathway of TDDN internalization.4NGR mediated the multifunctional self-assemble nanocarriers entering the cell through caveolae mediated endocytosisCaveolae-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 CD13co-localized with CAV-1in most cells. We therefore hypothesized that NGR might be able to mediate the carriers into CD13positive cells via the caveolae-mediated endocytosis. In the present study, we selected HUVEC as the test cell to verify the hypothesis using flow cytometry and confocal laser scanning technology. Both CD13and CAV-1expression on HUVEC at the same time was identified by flow cytometric analysis. Punctate distribution of CD13on HUVEC was observed under fluorescence microscope. It was also shown that CD13was concentrated on sites where cell membrane project into filaments. CD13was also observed in the filaments themselves. In the co-localization experiment, anti-CD13antibody and anti-CAV-1antibody were used to label CD13and CAV-1on HUVEC, respectively. When HUVEC were incubated with an anti-CD13antibody and fixed without warming, red fluorescence was observed evenly on the cell surfaces. In the same cells, labeling for CAV-1was seen in green. There was no co-localization happened between CD13and CAV-1. When cells bound with the antibodies were incubated for10min at37℃, the labeling of CD13and CAV-1on HUVEC surface showed in uniform punctate distribution and no co-localization between CD13and CAV-1was observed. When incubation time was30min at37℃, the labeling of CD13and CAV-1on HUVEC surface showed in spots and small extent of co-localization between CD13and CAV-1was observed. With the extending of incubation time at37℃, co-localization of CD13with CAV-1became more frequent after60min of incubation at37℃. When the incubation time extended to2h and3h, CD13and CAV-1gathered into clusters and co-localized extensively. When evaluated the influence of TDDN on the co-localization of CD13and CAV-1, it was found that incubation with TDDN would accelerate the speed and enhance the degree of the co-localization of CD13and CAV-1. These results demonstrated that TDDN could bind to CD13and entry into HUVEC through CD13mediated endocytosis. Moreover, TDDN could bind to CAV-1and entry into HUVEC through the caveolae-endocytosis. Thus, we could infer that TDDN could cause the cluster of CD13. subsequently inspire the co-localized of CD13and CAV-1, and finally internalized via caveolae-mediated endosytosis. The result of inhibition study of CD13enzyme activity indicated that the internalization of TDDN to HUVEC was not dependent on the enzyme activity of CD13. To further investigate the important effect of CAV-1on the intracellular entry of TDDN, MPCD 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 TDDN. Furthermore, co-localization between CD13and CAV-1was significantly decreased in cells that were treated by M(3CD suggesting the important effect of caveolae on the internalization of TDDN.5The in vivo target delivery process study of multifunctional self-assembly nanocarriers on an intro model of tumor microenvironmentThe internalization process of TDDN involved both clathrin mediated endosytosis and caveolae mediated entosytosis. Based on this, in this part, we further studied in vivo target delivery process study of multifunctional self-assembly nanocarriers on an intro model of tumor microenvironment, so as to provide a theoretical basis for the efficient design of gene/drug co-delivery systems. HUVEC monolayer was constructed as the model of blood vessel wall, and a co-culture non-contact model of HUVEC and tumor cells was established to mimic the microenvironment of tumor. The results of intracellular process demonstrated that TDDN had experienced a pH decreased process in HUVEC suggesting the involvement of clathrin mediated endocytosis, which was consistent with the previous results. Then, in order to imitate the actual pH of tumor microenvironment, tumor MCF-7cells were cultured in slightly acidic media of pH6.5. Only the red fluorescence of DOX existed in the MCF-7cells cultured under pH6.5, suggesting the SPN shell has been already dissociated before TDDN entering into MCF-7cells. Therefore. NGR could actively target TDDN to accumulate at tumor microenvironment. Parts of them could extravasate and localize in the tumor tissue interstitial space by the enhanced permeation and retention (EPR) effect, subsequently, the outer shell deshielded at the acid microenvironment and TAT was exposed to facilitate the tumor cellular uptake. Besides, parts of TDDN could bind to CD13on tumor vascular endothelial cells via NGR and subsequently internalized into HUVEC. Among of the internalized TDDN in HUVEC, some could be transport to the tumor stroma by the unique caveolae-mediated transcytosis of endothelial cells, then actively targeted to CD13over-expressed tumor cells and exerted drug efficacy to them. While as, another part of TDDN could also underwent the clathrin-mediated encytosis. Once internalized in target cells, DOX and DNA was released at the acidic pH due to hydrolysis of hydrazone linkage and escape from endosomes with the help of the "proton sponge" effect of PEI, allowing cytoplasmic and nuclear accumulation of released DOX and DNA, resulting in DOX toxicity and gene expression in target cells.Thus, the multifunctional self-assembly nanocarriers co-loading with drug and gene could act on both tumor cells and tumor vasculature endothelial cells through passive and active drug targeting. Besides, they could exert the cell-penetrating capability and pH sensitive drug release. All in all, the multifunctional self-assembly nanocarrier achieved the true sense of the multi-functional and provided a new concept, new tool and new strategy to tumor therapy.