PEGylated Poly (Trimethylene Carbonate)Nanoparticles Functionalized With Small Moleculor For Differential Gliomas Targeted Drug Delivery

Glioma is the most common form of tumor in the central nervous system in humans. Based on histopathologic assessment, cerebral gliomas are categorized into low-grade (â…  and â…¡ glioma) and high-grade gliomas (â…¢ and â…£ glioma), where low-grade astrocytomas account for20-30%and glioblastoma multiforme for50%of the cases. As gliomas grow and progress to a higher grade, the vascular supply is no longer adequate to support the increasing metabolic demands of the rapidly proliferating tumor cells. Regional hypoxia then ensues, leading to the upregulation of vasoactive endothelial growth factor (VEGF) and the promotion of new blood vessel formation from the existing vasculature, a phenomena known as angiogenesis. The newvessels that are formed often lack the complex structure of the normal brain vasculature and result in endothelial permeability. Tumor growth can also damage the existing vasculature and promote blood brain-barrier breakdown (BBB) which results in microvascular leakage. It could cause edema, which is the consequence of a local disruption of the BBB by impaired capillary endothelial tight junctions. While the blood-brain tumor barrier (BBTB) between the brain tumor and vessel is known to be leaky compared with normal brain, it still offers signicant resistance to chemotherapeutic agents, thus impeding the entry of these drugs into malignant gliomas. According to the pathological conditions of glioma in different grades, the different strategies should be taked to the design of the drug delivery system targeting glioma. When the glioma is still low-grade, the BBB is intact and the drug delivery system should be able to cross the BBB and further target to glioma. However, when it develops to advanced-grade, BBTB are major factors limiting the access of many therapeutic agents to brain tumors, and this barrier will become even more signicant with the development of new drug delivery system.In present work, low molecular MPEG-PTMC was synthesized by ring-opening polymerization. The diblock MPEG-PTMC copolymer was composed of high percentage of PEG segment and a hydrophobic PTMC block which is a biodegradable polyester and has attracted much research interest due to their tunable biodegradability and biocompatibility. PTX, as a model water insoluble drug, was incorporated in MPEG-PTMC nanoparticle delivery system by the emulsion/solvent evaporation technique. This system contains a hydrophobic drug in the core and a long PEG chain in the shell, which endows the nanoparticles with a lower level of the reticuloendothelial system (RES) uptake and hence a prolonged circulation half-life. In order to increase the drug delivery efficiency to advanced-grade glioma and low-grade glioma, the micellar-like nanoparticles were functionalized with two different small targeting moleculor, a cyclic RGD peptide and a hexose, respectively.In the first part, a series of different molecular MPEG-PTMC copolymers was synthesized through ring-opening polymerization. Biodegradable nanoparticles were prepared in the present study from synthesized MPEG-PTMC copolymer by the emulsion/solvent evaporation technique for controlled release of an antineoplastic drug (PTX). The optimized formulation showed the particle size around67nm (Z-Average by Malvern) with ideal drug loading content (DLC%:6.17%) and encapsulation efficiency (EE%:94.3%). In vitro, drug-loaded nanoparticles (NP/PTX) showed sustained release profiles and noticeable anti-tumor efficacy against U87MG glioblastoma cells a little higher or similar to Taxol. However, in contrast to Cremophor EL, no cytotoxicity of the polymers was observed at the concentration of1,000μg/ml. In intracranial xenograft tumor-bearing mice, biodistribution and intracranial tumor accumulation of DiR-labeled NP were evaluated by non-invasive and real-time NIR imaging systems. The accumulation of NP in tumor tissues increased distinctly after12h post i.v. MPEG-PTMC nanoparticles showed long-circulating effects in circulation and the enhanced permeability and retention (EPR) effect in malignant gliomas. Glioma interstitial fluid sample was collected by the microdialysis method, and determined by LC-MS method to evaluate its concentration in target site. Free PTX AUC0-12h of NP/PTX group in glioma interstitial fluid was2.17times compared to Taxol group, with statistical significant difference. NP/PTX was proved to accumulate in malignant gliomas by passive targeting.In the second part, a novel amphiphilic diblock copolymer, a-carboxyl poly(ethylene glycol)-poly(trimethylene carbonate)(HOOC-PEG-PTMC), was synthesized by ring-opening polymerization. The c(RGDyK) ligand, a cyclic RGD peptide that can bind to the integrin proteins predominantly expressed on the surface of tumor cells with high affinity and specificity, was conjugated to the NHS-Activated PEG terminus of the copolymer. The c(RGDyK)-functionalized PEG-PTMC micellar nanoparticles encapsulating PTX (c(RGDyK)-NP/PTX) was fabricated by the emulsion/solvent evaporation technique and was used as the platform for actively targeted PTX delivery to integrin αvβ3-rich malignant gliomas. In vitro physiochemical characterization of that c(RGDyK)-NP/PTX showed satisfactory encapsulation efficiency and loading capacity and size distribution, without statistical significant difference compared to NP/PTX. Coumarin6was used as fluorenscent probe to investigate the in vitro and in vivo glioma targeting ablity of c(RGDyK)-NP. Cellular uptake of c(RGDyK)-NP was found to be higher than that of NP due to the integrin protein-mediated endocytosis effect. And after internalized by U87MG cells, Glu-NP was delivered to endosome through clathrin-mediated endocytosis and caveolae-mediated endocytosis. In vitro cytotoxicity, cell apoptosis and cell cycle arrest studies and immunofluorescence analysis of U87MG cells treated with distinct PTX formulations also revealed that c(RGDyK)-NP/PTX was more potent than those of NP/PTX and Taxol. In vivo multispectral fluorescent imaging indicated that c(RGDyK)-NP had high specificity and efficiency in tumor active targeting in both subcutaneous xenograft nude mice model and intracranial xenograft glioma-bearing mice. In vitro,3D U87MG glioma spheroids were developed to evaluate the solid tumor penetration ability, and c(RGDyK)-NP exhibited the strongest activity in tumor penetration and reduction of U87MG glioma tumor spheroid volume and in the apoptosis of the spheroids. Furthermore, the in vivo antitumoral activity of c(RGDyK)-NP/PTX against both subcutaneous U87MG xenograft and intracranial glioma was assessed. c(RGDyK)-NP/PTX reflected the strongest tumor growth inhibition among the studied paclitaxel formulations. The anti-glioblastoma effect exhibited the median survival time of c(RGDyK)-NP/PTX treated mice (32days) was significantly longer than those of mice treated with Taxol (23days), physiological saline (21days) and NP/PTX (27days). Brain tumor slices stained by hematoxylin and eosin (H&E) were analyzed via optical microscopy. Qualitatively, necrosis area in the c(RGDyK)-NP/PTX-treated tumor is explicitly the largest among all of the tested groups. All of these results demonstrated that c(RGDyK)-decorated PEG-PTMC NP could penetrate BBTB and deliver hydrophobic chemotherapeutic agents to integrin-rich malignant gliomas.In the third part, D-glucosamine (Glu), a small moleculor of hexose, was conjugated to the NHS-Activated PEG terminus of the copolymer. The Glu-modified PEG-PTMC micellar nanoparticles encapsulating PTX (Glu-NP/PTX) was adopted to dually delivery PTX to lower grade gliomas, especially to grade â…¡ glioma. RBITC was used as fluorenscent probe to investigate the in vitro and in vivo glioma targeting ablity of Glu-NP. Both bEnd.3and RG-2cellular uptake of Glu-NP obviously enhanced, and These uptake could be inhibited by glucose and phlorhizin indicating that the internalization of Glu-NP by either bEnd.3cells or RG-2cells was mediated by glucose transporter. After internalized by RG-2cells, Glu-NP was delivered to endosome.Mice brain tissue section revealed that Glu-NP could accumulate in cerebral, lateral cerebral ventricle, hippocampus and third vetricle, but the conventional NP could not, indicating that Glu-NP could be transported across BBB, and accumulated in brain like a small hexose.In vitro cytotoxicity and immunofluorescence analysis of RG-2cells treated with various PTX formulations experiments, Glu-NP/PTX showed the strongest anti-tumor effect. A bEnd.3monolayer and RG-2glioma3D spheroids co-culture model was developed to evaluate the dual actively drug delivery function. The results revealed that Glu-NP exhibited the strongest ability in glioma spheroid penetration after crossing the bEnd.3monolayer. In vivo NIR fluorescent imaging indicated that Glu-NP could actively home to brain tumor in intracranial RG-2xenograft glioma-bearing mice, however neither conventional NP nor free fluorescent probe could accumulate in brain. In vivo antitumor pharmacological effect in intracranial RG-2glioma-bearing mice was evaluated. Glu-NP/PTX could significantly enhance anti-glioma efficacy in RG-2glioma-bearing mice compare with physiological saline, however, both Taxol and NP/PTX could not. In summary, hexose’s conjugation could assist NP to delivery antineoplastic agents across BBB and actively accumulate in lower grade glioma.In the final part, the pharmacokinetic parameters for PTX in plasma were estimated by compartmental method. The c(RGDyK)-NP/PTX, Glu-NP/PTX and NP/PTX showed initial high blood circulating levels compared to Taxol, while paclitaxel formulated in Taxol was quickly removed from the circulating system at6h after administration. On the contrary, c(RGDyK)-NP/PTX, Glu-NP/PTX and NP/PTX exhibited a markedly delayed blood clearance. Compartmental analysis of the plasma concentrations showed a significant change in pharmacokinetic parameters of PTX in nanoparticles compared to that of commercial formulation. It was shown that c(RGDyK)-NP/PTX, Glu-NP/PTX and NP/PTX could extend the elimination half-life (t1/2β) of Taxol from4.19h to11.16h,11.02h and12.19h, respectively (P<0.01). Meanwhile, AUC0â†'t increased by about4.70-fold for c(RGDyK)-NP/PTX,4.48-fold for Glu-NP/PTX and4.73-fold for NP/PTX compared to Taxol. In addition, MRT for the formulations of c(RGDyK)-NP/PTX, Glu-NP/PTX and NP/PTX were5.61-fold, 5.34-fold and5.85-fold higher than Taxol separately (P<0.01). These results suggest that the moderate modification of small moleculor such as RGD peptide and hexose did not evidently influence the in vivo long-circulating property of the polymeric nanoparticles.The biocompatibility and toxicity test was performed by measuring by histopathology, blood cell counts and clinical chemistry parameters (total bilirubin, alanine transferase, aspartate transferase, blood urea nitrogen and creatinine), and the primary study revealed that neither c(RGDyK)-NP nor Glu-NP display any side effect.Taken together, these results illustrated the potential utility of both of the above small moleculor functionalized nanoparticles as long circulating reservoirs for hydrophobic anticancer agents targeted delivery to differential glioma.