Peptide-mediated Drug Delivery System For Targeted Glioma Therapy

Glioma is the most common form of tumor in the central nervous system in humans, which accounts for approximately 40% of all primary brain tumors. Glioma usually grows at many important parts in brain, including central sulcus and thalamus, which increase the difficulty of surgery to remove the tumor tissue. Furthermore, the peripheral infiltrating part can not be completely eradicated due to the poor cellular differentiation of glioma. Chemotherapy seems essential in the auxiliary treatment of glioma. Research shows that many chemotherapeutics (e.g. PTX, Doxorubicin) exhibited excellent in vitro anti-glioma efficacy. However, the efficiency of chemotherapy in clinic is far from satisfaction mainly owing to the drug delivery problems:(1) For high-grade glioma, the Blood-Brain Barrier (BBB) has been impaired during the development of glioma. While the new barrier Blood-Brain Tumor Barrier which is formed by tumor neovascular and tumor cells still resist the penetration of chemotherapeutics. (2) Due to the infiltrating growth of glioma cells, there is poor cellular differentiation of glioma cells and normal cells. The chemotherapeutics will kill both glioma cells and normal cells due to the poor selectivity of chemotherapeutics when they get into brain tissue. (3) High-grade glioma is solid tumor, which has high density of cells, high inside pressure, hypoxia and acidic pH. Besides, theis is over 100μm gap between tumor neovascular and tumor tissue. All this physiological feature increase the difficulty of penetration of chemotherapeutics and enhance the possibility of recurrence of glioma after chemotherapy. Therefore, the development of a drug delivery strategy which can mediate efficient glioma targeting together with high cellular internalization is essential and important for glioma treatment.According to the pathological conditions of glioma, we explored several novel methods that might have the potential use in enhancing the therapy for glioma. In the first part, we constructed a CGKRK-decorated nanoparticulate drug delivery system which target both tumor neovascular and tumor cells to overcome the barrier of BBTB. In the second part, we develop a penetration-faciliating peptide tLyp-1 modified nanoparticulate drug delivery system to enhance the permeability of blood vessels and penetration into tumor. In the third part, we explore a new drug delivery syetem which is using F3-modified nanoparticle co-administrated with tLyp-1 peptide strategy to enhance enhance glioma-specific drug accumulation and penetration. In the fourth part, by taking advantage of ECM receptor, we constructed the ATWLPPR peptide and CGKRK peptide dual-decorated nanoparticulate drug delivery system to target the infiltrating section of glioma and decrease the possibility of recurrence.In the first part, a dual-targeting drug delivery system was developed, using CGKRK as the targeting ligand. The receptor of CGKRK peptide is reported to be heparan sulfate-a sulfated polysaccharide which was found on the surface of neovascular endothelial cells and tumor cells. By taking advantage of overexpressed heparan sulfate, the CGKRK-NP can achieve enhanced accumulation at glioma site and improve the therapy efficiency. The obtained CGKRK-functionalized PEG-co-PCL nanoparticles (CGKRK-NP) with a particle size of 117.28 nm and zeta potential of-15.7 mV, exhibited an enhanced accumulation via an energy-dependent, lipid raft/caveolae-mediated endocytosis with the involvement of microtubules in human umbilical vein endothelial cells (HUVEC) and an energy-dependent, lipid raft/caveolae-mediated endocytosis with the participation of Golgi apparatus in human U87MG cells. Using coumarin-6 as the fluorescence probe, in vitro U87MG tumor spheroids assays showed that CGKRK-NP effectively penetrated into the tumor spheroids. Selective accumulation and extensive bio-distribution of CGKRK-NP at tumor site was confirmed by in vivo imaging and tumor section analysis. After drug loading, CGKRK-NP enhanced cytotoxicity and apoptosis induction activity of the loaded PTX on both HUVEC cells and U87MG cells and improved its inhibition effect on the growth of U87MG tumor spheroids. The smallest tumor volume was achieved by those mice bearing subcutaneous U87MG tumor following the treatment of PTX-loaded CGKRK-NP. The findings here indicated that CGKRK peptide-functionalized nanoparticulate DDS could be used as an effective tumor angiogenic blood vessels and tumor cells dual-targeting DDS and might provide a great promising approach for reducing the disadvantages of antiangiogenic therapy alone.The second part is the construction of a penetration-faciliating peptide tLyp-1-modified nanoparticulate drug delivery system. By taking advantage of the excessively upregulated expression of neuropilin (NRP) on the surface of both glioma cells and endothelial cells of angiogenic blood vessels, the ligand of NRP with high affinity of tLyp-1 peptide, was functionalized to the surface of PEG-PLA nanoparticules (tLyp-1-NP) to mediate its tumor homing, vascular extravasation and deep penetration into the glioma parenchyma. The tLyp-1-NP was prepared via a maleimide-thiol coupling reaction with uniformly spherical shape under TEM and particle size of 111.30±15.64 nm. tLyp-1-NP exhibited enhanced cellular uptake in both human umbilical vein endothelial cells and Rat C6 glioma cells, increased cytotoxicity of the loaded PTX, and improved penetration and growth inhibition in avascular C6 glioma spheroids. Selective accumulation and deep penetration of tLyp-1-NP at the glioma site was confirmed by in vivo imaging and glioma distribution analysis. The longest survival was achieved by those mice bearing intracranial C6 glioma treated with PTX-loaded tLyp-1-NP. the medium survival time for mice treated with saline, Taxol(?), NP-PTX and tLyp-1-NP-PTX was 18,23,28,37 days, respectively. tLyp-1-NP-PTX significantly prolonged animal survival time when compared with NP-PTX (p< 0.01), Taxol(?) (p< 0.001) and saline (p< 0.001). The findings here strongly indicate that tLyp-1 peptide-functionalized nanoparticulate DDS could significantly improve the efficacy of paclitaxel glioma therapy.In the third part, we constructed a F3 peptide-modified nanoparticulate drug delivery system co-administrated with tLyp-1 peptide to enhance the accumulation and penetration of drug delivery system. To achieve this goal, F3 peptide which specifically bind to nucleolin, which is highly expressed on the surface of both endothelial cells of glioma blood vessels and glioma cells, is utilized to decorate nanoparticulate drug delivery system and realize dual-targeting, drug controlled release propose. Furthermore, tLyp-1 peptide which presents the motif of (R/K)XX(R/K) and specially bind to neuropilin as a transmembrane protein receptor is co-administrated to improve the penetration of DDS from blood vessels into glioma region. The F3-conjugation via a maleimide-thiol coupling reaction was confirmed by XPS analysis with size of 125 nm and zeta potential of-13.3 mV. F3-NP showed enhanced cellular interaction with C6 cells, improved penetration in 3D multicell tumor spheroids, increased the cytotoxicity of the loaded paclitaxel. Following co-administration with tLyp-1 peptide, the F3-functionalized nanoparticles displayed enhanced accumulation at the tumor site and deep penetration into the glioma parenchyma and achieved the longest survival in mice bearing intracranial C6 glioma. The medium survival was 19 days for those animals treated with saline,24 days with Taxol,27 days with NP,32 days with F3-NP,31 days with NP with tLyp-1, and 42 days with F3-NP and tLyp-1. Additionally, the log-rank analysis showed that F3-NP with co-administration of tLyp-1 significantly prolonged the survival of mice bearing glioma when compared with F3-NP (P< 0.05), NP with tLyp-1 (P< 0.01), NP (P< 0.001), Taxol (P< 0.001), saline (P< 0.001).In the fourth part, we used ATWLPPR peptide and CGKRK peptide to construct a dual-decorated nanoparticulate drug delivery system. By targeting the BBTB and tumor ECM, the dual-decorated nanoparticulate drug delivery system can enhance the accumulation of drug at glioma site and decrease the possibility of recurrence. ATWLPPRCCGKRK (AC) peptide was synthesized by using cycsteine to conjugate the ATWLPPR and CGKRK peptdie. The obtained AC-functionalized PEG-PLA nanoparticles (AC-NP) with a particle size of 123 nm and zeta potential of-11.4 mV, exhibited an enhanced accumulation via an energy-dependent, lipid raft/caveolae-mediated endocytosis with the involvement of lysosome in human umbilical vein endothelial cells (HUVEC) and an energy-dependent, lipid raft/caveolae-mediated endocytosis with the participation of Golgi apparatus, microtubulin and lysosome in human U87MG cells. Using coumarin-6 as the fluorescence probe, in vitro U87MG tumor spheroids assays showed that AC-NP effectively penetrated into the tumor spheroids. Selective accumulation and extensive bio-distribution of AC-NP at tumor site was confirmed by in vivo imaging and tumor section analysis. After drug loading, AC-NP enhanced cytotoxicity and apoptosis induction activity of the loaded PTX on U87MG cells and improved its inhibition effect on the growth of U87MG tumor spheroids. The median survival of mice treated with AC-NP-PTX was significantly longer than those of mice treated with PTX formulations.In this study, we develop a peptide-mediated drug delivery strategy which can mediate efficient glioma targeting together with high cellular internalization. The nanoparticulate drug delivery system can improve the efficiency of glioma treatment.