Cell-penetrating Peptides Modified Nano-delivery System For Brain And Tumor Drug Delivery

In recent years, some short peptides are found to present cell penetrating function, and designated as cell-penetrating peptides (CPPs). CPPs usually contain multiple positively charged arginine and lysine, and are of strong positive charge. Protein transduction region of9-16amino acids in the CPPs structure play a decisive role in the process of penetrating, and this process does not depend on the transporter protein or receptor-mediated endocytosis. A large number of studies have shown that CPPs (such as Tat, Penetratin, Transportan, VP22, etc.) have strong carrying potential to effectively translocate segments of protein, polypeptides, nucleic acid into the cells of many mammal animals with many methods. So, as a new drug delivery system, CPPs have very good applicable prospect in such research fields as brain targeting, tumor targeting, gene delivery system, transdermal drug delivery and other therapeutic and diagnostic areas. But it still exists a key issue in the application, which is the lack of cell specificity. Some evidence showed that CPPs can be combined with any molecule, non-targeted into the cell, causing drug accumulation in normal tissues or non-targeted organization, producing toxic side effects. To solve this problem, the study constructed three cell-penetrating peptide-modified nano-drug delivery systems by optimizing the route of administration, preferred CPPs with suitable charge and trigger/activatable strategy for penetrating peptides for the brain targeting and tumor targeting respectively, so as to improve targeted treatment of the delivery systems.In the first part, low-molecular-weight protamine (LMWP) was used to modify the PLA nanoparticle (NP). By the intranasal administration, the delivery system reduced the uptake in non-targeted organizations, and further increased the nose-to-brain efficiency. PEG-PLA nanoparticle (NP) was prepared using an emulsion/solvent evaporation technique. LMWP was functionalized to the surface of NP via a maleimide-mediated covalent binding procedure. Important parameters such as particle size distribution, zeta potential and surface content were determined. The obtained LMWP-NP showed a particle size of100nm and zeta potential of2.42mV. Using16HBE14o-cells as the cell model, LMWP-NP was found to exhibit significantly enhanced cellular accumulation than that of unmodified NP via both lipid raft-mediated endocytosis and direct translocation processes without causing observable cytotoxic effects. Following intranasal administration of coumarin-6-loaded LMWP-NP, the AUC0-8h of the fluorescent probe detected in the rat cerebrum, cerebellum, olfactory tract and olfactory bulb was found to be2.03,2.55,2.68and2.82folds, respectively, compared to that of coumarin-6carried by NP. Brain distribution analysis suggested LMWP-NP after intranasal administration could be delivered to the central nervous system along both the olfactory and trigeminal nerves pathways. The findings clearly indicated that the LMWP-functionalized nanoparticle appears as an effective and safe carrier for nose-to-brain drug delivery and can be used for non-invasive treatment of central nervous system diseases.The optimal CPP with relatively low content of basic amino acids——Penetratin was here functionalized to PEG-PLA nanoparticle (NP) in the second part to achieve desirable brain targeting efficiency and pharmacokinetic profiles. The obtained Penetratin-NP showed a particle size of100nm and zeta potential of-4.42mV, and characterized by transmission electron microscopy and surface elemental analysis. In MDCK-MDR cell model, Penetratin-NP significantly enhanced cellular accumulation via both lipid raft-mediated endocytosis and direct translocation processes with the involvement of Golgi apparatus, lysosome and microtubules. The pharmacokinetic results showed that the AUC of Penetratin-NP was1.89times of NP in the brain,0.81-fold of NP in the blood, and the brain targeting index was2.332. Compared with LMWP (CPP with high arginine content)-modified nanoparticle, Penetratin-NP exhibited a significantly enhanced brain uptake and reduced accumulation in the non-target tissues. The results obtained both in vitro and in vivo demonstrated that Penetratin-NP might represent a promising brain-targeting delivery system. These results also provide an important basis for further study of optimizing CPPs, regulating the degree of modification of the nanoparticle surface charge to get a better brain targeting drug delivery efficiency.The third part is the construction of the tumor targeted nanoparticle system, using activatable LMWP (ALMWP) as the targeting ligand. An ALMWP consists of three domains:a polycationic cell-penetrating peptide (LMWP), a cleavable linker (PLGLAG), which could cleaved by matrix metalloproteinase highly expressed by the tumor cell surface, and a polyanionic inhibitory domain (E10). Before the cleavage, the overall charge of ALMWP remains neutral, that greatly reduces the possibility for the monocyte-macrophage system to identify the modified nanoparticle, keeping a long cycle characteristic of the nanoparticle. And when ALMWP-NP has arriving the tumor site through the EPR effect, the linker sequence PLGLAG could be efficiently cleaved by tumor-specific expressed protease, leading to the anion part dissociated out, and cationic part exposed to play strong penetrating ability and tumor tissue infiltration capacity, resulting in nanoparticle uptake by tumor cells. The obtained ALMWP-NP in the third part was generally spherical with a uniform distribution, and showed a particle size of120nm and zeta potential of-26.4mV. The cellular uptake results showed that ALMWP-NP has a significantly higher cellular uptake than the NP, and has a higher cellular uptake than LMWP-NP in the conditions of high concentrations. The cellular interaction mechanism of ALMWP-NP and LMWP-NP could be shared between two general pathways:energy-independent direct translocation and active endocytosis, which is a process of time-, temperature-and concentration-dependant. Endocytosis inhibition experiments showed that both ALMWP-NP and LMWP-NP presenting macropinocytosis-mediated and cytoskeleton-dependent endocytosis. The pharmacokinetic results in HT-1080tumor-bearing mice showed that the ALMWP-NP significantly increased the accumulation of PTX in the tumor site, with the concentration of1.64(P<0.05),1.29,2.03(P<0.05),1.32and1.47(P<0.05) times of LMWP-NP, and2.54,2.08,2.03,2.33and2.15times of NP (P<0.05) at each time point, respectively. The bio-distribution results showed that the LMWP-NP tend to accumulate in the liver and spleen, while ALMWP-NP had no significant difference with NP in the accumulation of non-target tissues, and with a significant increase in tumor drug concentration. The pharmacodynamic experiments demonstrated PTX-ALMWP-NP has better tumor tissue permeation and toxic effects to tumor cells. Administration of tumor-bearing mice though tail vein injection at5mg/kg dose, tumor growth volume of saline group> Taxol group> PTX-NP group> PTX-LMWP-NP group> PTX-ALMWP-NP group; removed the tumor and measured after20days, the tumor weight of five groups in order to0.826,0.574,0.471,0.323and0.225g; the inhibition rate of treatment group were30.53%,43.02%,60.95%and72.72%for Taxol, PTX-NP, PTX-LMWP-NP and PTX-ALMWP-NP group, respectively, suggesting ALMWP-NP can be used as a safe and effective tumor-targeting drug delivery system.