Size-tailorable Nanoparticles:Synthesis And Their Applications In Nanomedicine

With the development of nanotechnology, nanoparticles as drug delivery system for cancer treatment have received great attention in rencent years. One of the advantages of the nanoparticles is to improve the pharmacokinetics (PK) and tissue distribution compared with traditional drug carriers, thus efficiently enhancing cancer therapy and reducing normal tissue toxicity. Preliminary studies have shown that the pegylated outer layer, stability and size of polymeric nanoparticles are the most important parameters determining their pharmacokinetics and tumor drug accumulation efficiency in cancer drug delivery carriers. However, preparation of such stable and pegylated nanoparticles with well-defined sizes mostly requires multisteps. In this study, we report a facile one-pot synthesis of crosslinked nanoparticles (CNPs) with tunable sizes and polyethylene glycol (PEG) shells via click reactions. By simply adjusting the contents of the macromonomer (PEG monoacrylate) in the reaction of ethylene diacrylate and a corsslinker containing hexa-thiols groups, the sizes of the resulting pegylated crosslinked nanoparticles could be easily tuned from 10 to 90 nm. The resulting nanoparticles were easily dispersible in water, stable and nontoxic. Their cores could encapsulate hydrophobic drugs such as doxorubicin (DOX), and the remaining thiol and acrylate groups could be used for drug conjugation or labeling. Thus, the nanoparticles provide a multifunctional platform for drug delivery.The cancer drug delivery process of nanomedicine can be divided into three stages:the drug-loaded nanocarriers arrive at the tumor by the EPR effect, penetrate the tumor tissue and interact with the cancer cells, and finally enter into the cells, traverse to the targeted subcellular sites and release the carried drug cargo. This process can encounter multistage barrier:the system barrier, the tumor barrier and cancer cell barrier. Therefore, the nanocarriers must overcome these barriers in order to enhance efficacy against tumors and reduce side effects. However, most of these investigations have concerned the intracellular localization of nancarriers mainly in the cytoplasm and rarely in the cell nucleus. Many clinical anticancer drugs, including anthracyclines, camptothecin (CPT) and cisplatin, induce cell death (apoptosis) mainly by oxidative DNA damage and topoisomerase Ⅱ hibition in the nucleus. However, cancer cells have many intracellular drug-resistance mechanisms to limit the access of cytosolic drugs to the nucleus, leading to the drug-resistance and failture of the cancer treatment. Recently, charge-reversal drug carriers based on P-carboxyl acid amidized cationic polymers, such as PEI, have been developed to deliver drugs into cancer cell nuclei. In the second part of this dissertation, a poly (β-aminoester) dendrimer (G4.5-DCA) with charge-reversal ability was synthesized and doxorubicin (DOX) loaded into the dendrimer. Then, the tunable-size nanoparticles were prepared with G4.5-DCA(DOX) and PEG-b-PLL by electrostatic self-assembly. The nanoparticles with PEG outer shell not only could effectively prevent the burst drug release at physiological pH condition, but also minimize recognition and uptake by macrophages of the reticuloendothelial system (RES), prolonging the blood circulation time and enhancing enhanced permeability and retention (EPR) effect. Once in an acidic environment, the amides of the G4.5-DCA hydrolyze to regenerate the amine groups and the self-assemble nanoparticles released the dendrimer carrying cationic charges. The positive dendrimers could escape from the lysosomes and deliver the drug into the nucleus. In vitro MTT assays showed much improved cytotoxicity to cancer cells compared to free DOX.This dissertation can be categorized into six parts as described below:The first Chapter I briefly summed up the classification and development of nanomedicine.The Chapter Ⅱ focused on the synthesis and characterization of the CNPs, and their drug loading or conjugating. We firstly synthesized the size-tunable CNPs (10 to 90 nm) via the thiol-acrylate reaction of a macromonomer mPEG-AC, a diacrylate monomer EGDA, and a crosslinker TLA-6SH. Dynamic light scattering (DLS), atomic force microscopy (AFM),1H-NMR were used to characterize the CNPs. The CNPs could be nanoscale containers encapsulating hydrophobic drugs, as verified that hydrophobic doxorubicin (DOX) could be loaded into the CNPs with high loading contents (about 14%). The thiol groups were activated by the reaction with 2, 2’-dithiodipyridine, and then reacted with a thiolated anticancer drug (SN38-SH) and anchored the drug to the nanoparticle core via a disulfide bond. The acrylate can be easily convertd to amine group by reacting with cysteamine, and the Cy7 NHS ester could easily conjugate to the CNPs. The Cy7 labelled CNPs (CNPs-Cy7) were used to study the in vivo behaviors of CNPs in the following experiments.In Chapter Ⅲ, in vitro drug release, the subcellular distribution and in vitro antitumor activity of the CNPs/DOX were evaluated. Acid-sensitive release experiments show that the CNPs/DOX had a pH-sensitive release behavior. In vitro MTT assays demonstrated that the CNPs had no cytotoxicity and the three sizes of CNPs/DOX could efficiently inhibit the growth rate of the breast cancer cell BCap37. Furthermore, the intracellular distributions of CNPs/DOX were observed by confocal laser scanning microscopy (CLSM). The results showed the CNPs/DOX could quickly enter the cancer cells, release and deliver the DOX efficiently to the nuclei of cells in vitro.In Chapter IV, we evaluated the biodistribution, in vivo antitumor activity and safety of those CNPs/DOX. Firstly, we studied the relationship between the size of CNPs/DOX and the half-life of blood clearance. The blood clearance results demonstrated that CNPs/DOX significantly improved pharmacokinetic characteristics compared with free DOX and the prolonged half-life of blood clearance with the size increased. Besides, we found significant plasma clearance behavior difference between CNPs/DOX (encapsulation) and CNPs-Cy7 (conjugation). Ex vivo fluorescent images showed that the accumulation of CNPs/DOX in tumor enhanced with the size increased, in agreement with blood clearance results. The CNPs/DOX had ideal in vivo anti-tumor effects, HE staining of the heart and tumor tissue results showed that CNPs/DOX significantly reduced the toxic side effect and increased in vivo safety compared with free DOX.In Chapter V, biodegradable polyester dendrimer with butanediamine as the core molecule having uniform structure were prepared by the click reaction. Gel Permeation Chromatography (GPC) and 1-NMR were used to characterize the dendrimer. In the following experiments, we used 3,4,5,6-tetrahydrophthalic anhydride (DCA) and succinicanhydride (SA) to modify the surface of the dendrimer. The amidization of the polyester dendrimers not only can enhance its stability, but also can improve its water solubility. At acid conditions, the β-carboxylic-acid amides of the G4.5-DCA could be hydrolyzed into primary amines and they had very good hemolysis. The following experimental studies demonstrated that the G4.5-DCA and PEG-b-PLL could form stable nanoparticles through the electrostatic self-assembly at physiological pH conditions. Furthermore, the size of the nanoparticles can be adjusted by controlling the chain length of the PLL.In Chapter VI, free DOX was then loaded into the carboxyl polyester dendrimer via hydrophobic interactions. The G4.5-DCA encapsulated of DOX and PEG-b-PLL could form nanoparticles with different size through the electrostatic self-assembly. In the following experiment, in vitro release of drug, the subcellular distribution and in vitro antitumor activity of the nanoparticles were evaluated. In vitro MTT assays demonstrated that the nanoparticles efficiently inhibited the growth rate of the BCap37 and Hela cancer cells. Acid-sensitive drug release experiments show that the nanoparticels have a pH-sensitive release behavior and the out layer of the PEG-b-PLL could effectively prevent the burst drug release at physiological pH condition. The CLSM results indicated the nanoparticles could release and deliver the DOX efficiently to the nuclei of cells in vitro. The blood clearance results demonstrated that the nanoparticles improved pharmacokinetic characteristics compared with free DOX and G4.5-DCA (DOX).