A new prototype of nanoconjugate, Polycefin, was synthesized for targeted delivery of antisense oligonucleotides and monoclonal antibodies to brain tumors. The macromolecular carrier contains: 1. biodegradable, nonimmunogenic, nontoxic β-poly(L-malic acid) of microbial origin; 2. Morpholino antisense oligonucleotides targeting laminin α4 and β1 chains of laminin-8, which is specifically overexpressed in glial brain tumors; 3. monoclonal anti-transferrin receptor antibody for specific tissue targeting; 4. oligonucleotide releasing disulfide units; 5. L-valine containing, pHsensitive membrane disrupting unit(s), 6. protective poly(ethylene glycol); 7. a fluorescent dye (optional). Highly purified modules were conjugated directly with N-hydroxysuccinimidyl esteractivated β-poly-(L-malic acid) at pendant carboxyl groups or at thiol containing spacers via thioether and disulfide bonds. Products were chemically validated by physical, chemical, and functional tests. In vitro experiments using two human glioma cell lines U87MG and T98G demonstrated that Polycefin was delivered into the tumor cells by a receptor-mediated endocytosis mechanism and was able to inhibit the synthesis of laminin-8 α4 and β1 chains at the same time.Inhibition of laminin-8 expression was in agreement with the designed endosomal membrane disruption and drug releasing activity. In vivo imaging showed the accumulation of intravenously injected Polycefin in brain tumor tissue via the antibody-targeted transferrin receptor-mediated endosomal pathway in addition to a less efficient mechanism known for high molecular mass biopolymers as enhanced permeability and retention effect. Polycefin was nontoxic to normal and tumor astrocytes in a wide range of concentrations, accumulated in brain tumor, and could be used for specific targeting of several biomarkers simultaneously.

A new prototype of polymer-derived drug delivery system, the nanoconjugate Polycefin, was tested for its ability to accumulate in tumors based on enhanced permeability and retention (EPR) effect and receptor mediated endocytosis. Polycefin was synthesized for targeted delivery of Morpholino antisense oligonucleotides into certain tumors. It consists of units that are covalently conjugated with poly(β-L-malic acid) (M w 50,000, M w /M n 1.3) highly purified from cultures of myxomycete Physarum polycephalum. The units are active in endosomal uptake, disruption of endosomal membranes, oligonucleotide release in the cytoplasm, and protection against enzymatic degradation in the vascular system. The polymer is biodegradable, non-immunogenic and non-toxic. Polycefin was also coupled with AlexaFluor 680 C2-maleimide dye for in vivo detection.Nude mice received subcutaneous injections of MDA-MB 468 human breast cancer cells into the left posterior mid-dorsum or intracranial injections of human glioma cell line U87MG. Polycefin at concentration of 2.5 mg/kg was injected via the tail vein. In vivo fluorescence tumor imaging was performed at different time points, 0-180 min up to 24 h after the drug injection. The custom-made macro-illumination imaging MISTI system was used to examine the in vivo drug accumulation in animals bearing human breast and brain tumors. In breast tumors the fluorescence signal in large blood vessels and in the tumor increased rapidly until 60 min and remained in the tumor at a level 6 times higher than in non-tumor tissue (180 min) (p < 0.003). In brain tumors drug accumulated selectively in 24 h without any detectable signal in non-tumor areas. The results of live imaging were corroborated histologically by fluorescence microscopic examination of various organs. In addition to tumors, only kidney and liver showed some fluorescent signal.

Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow-derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow-derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, β-galactosidase-labeled bone marrow-derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow-derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow-derived neural progenitor/stem cells with a blocking anti-CXCR4 antibody, suggesting a CXCR4-dependent tracking mechanism. Glioma tracking bone marrow-derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow-derived neural progenitor/ stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors.

No abstract

<div>Abstract<p>Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow–derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow–derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, β-galactosidase-labeled bone marrow–derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow–derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow–derived neural progenitor/stem cells with a blocking anti–CXCR4 antibody, suggesting a CXCR4–dependent tracking mechanism. Glioma tracking bone marrow–derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow–derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors. [Mol Cancer Ther 2009;8(9):2746–53]</p></div>

<div>Abstract<p>Malignant gliomas manifest frequent tumor recurrence after surgical resection and/or other treatment because of their nature of invasiveness and dissemination. The recognized brain tumor-tracking property of neural progenitor/stem cells opened the possibility of targeting malignant brain tumors using neural progenitor/stem cells. We and others have previously shown that fetal neural progenitor/stem cells can be used to deliver therapeutic molecules to brain tumors. Our recent work has further shown that gene delivery by bone marrow–derived neural progenitor/stem cells achieves therapeutic effects in a glioma model. In this study, we isolate and characterize bone marrow–derived neural progenitor/stem cells, which also express the chemokine receptor chemokine CXC receptor 4 (CXCR4). We show that CXCR4 is required for their chemotaxis and extracellular matrix invasion against a gradient of glioma soluble factors. Furthermore, β-galactosidase-labeled bone marrow–derived neural progenitor/stem cells implanted in the contralateral side of the brain were shown to track gliomas as early as day 1 and increased through days 3 and 7. Intracranial glioma tracking by bone marrow–derived neural progenitor/stem cells is significantly inhibited by preincubation of bone marrow–derived neural progenitor/stem cells with a blocking anti–CXCR4 antibody, suggesting a CXCR4–dependent tracking mechanism. Glioma tracking bone marrow–derived neural progenitor/stem cells were found to express progenitor/stem cell markers, as well as CXCR4. Although bromodeoxyuridine incorporation assays and proliferating antigen staining indicated that tumor tracking bone marrow–derived neural progenitor/stem cells were mostly nonproliferating, these cells survive in the local tumor environment with little apoptosis. Elucidating the molecular mechanism of brain tumor tracking by adult source stem cells may provide basis for the development of future targeted therapy for malignant brain tumors. [Mol Cancer Ther 2009;8(9):2746–53]</p></div>

Supplementary Data from Chemokine CXC receptor 4–mediated glioma tumor tracking by bone marrow–derived neural progenitor/stem cells

Supplementary Data from Chemokine CXC receptor 4–mediated glioma tumor tracking by bone marrow–derived neural progenitor/stem cells