Tumor targeted delivery of doxorubicin in malignant peripheral nerve sheath tumors.

Peripheral nerve sheath tumors are benign tumors that have the potential to transform into malignant peripheral nerve sheath tumors (MPNSTs). Interleukin-13 receptor alpha 2 (IL13Rα2) is a cancer associated receptor expressed in glioblastoma and other invasive cancers. We analyzed IL13Rα2 expression in several MPNST cell lines including the STS26T cell line, as well as in several peripheral nerve sheath tumors to utilize the IL13Rα2 receptor as a target for therapy. In our studies, we demonstrated the selective expression of IL13Rα2 in several peripheral nerve sheath tumors by immunohistochemistry (IHC) and immunoblots. We established a sciatic nerve MPNST mouse model in NIH III nude mice using a luciferase transfected STS26T MPNST cell line. Similarly, analysis of the mouse sciatic nerves after tumor induction revealed significant expression of IL13Rα2 by IHC when compared to a normal sciatic nerve. IL13 conjugated liposomal doxorubicin was formulated and shown to bind and internalized in the MPNST cell culture model demonstrating cytotoxic effect. Our subsequent in vivo investigation in the STS26T MPNST sciatic nerve tumor model indicated that IL13 conjugated liposomal doxorubicin (IL13LIPDXR) was more effective in inhibiting tumor progression compared to unconjugated liposomal doxorubicin (LIPDXR). This further supports that IL13 receptor targeted nanoliposomes is a potential approach for treating MPNSTs.

Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles.

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. STATEMENT OF SIGNIFICANCE: Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease.

HFE genotype affects exosome phenotype in cancer.

Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells.

Application of fullerenes in nanomedicine: an update.

Fullerenes are carbon spheres presently being pursued globally for a wide range of applications in nanomedicine. These molecules have unique electronic properties that make them attractive candidates for diagnostic, therapeutic and theranostic applications. Herein, the latest research is discussed on developing fullerene-based therapeutics as antioxidants for inflammatory diseases, their potential as antiviral/bacterial agents, utility as a drug delivery device and the promise of endohedral fullerenes as new MRI contrast agents. The recent discovery that certain fullerene derivatives can stabilize immune effector cells to prevent or inhibit the release of proinflammatory mediators makes them potential candidates for several diseases such as asthma, arthritis and multiple sclerosis. Gadolinium-containing endohedral fullerenes are being pursued as diagnostic MRI contrast agents for several diseases. Finally, a new class of fullerene-based theranostics has been developed, which combine therapeutic and diagnostic capabilities to specifically detect and kill cancer cells.

Heavy chain ferritin siRNA delivered by cationic liposomes increases sensitivity of cancer cells to chemotherapeutic agents.

Approximately half of all gliomas are resistant to chemotherapy, and new therapeutic strategies are urgently needed to treat this cancer. We hypothesized that disrupting iron homeostasis in glioma cells could block tumor growth, based on an acute requirement for high levels of iron to meet energy requirements associated with their rapid growth. Ferritin is best known as an intracellular iron storage protein, but it also localizes to tumor cell nuclei where it seems to protect DNA from oxidative damage and to promote transcription. In this study, we hypothesize that silencing the H-ferritin (heavy chain ferritin) gene could increase tumor sensitivity to chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to several human cancer cell lines by using cationic liposomes (C-liposome). H-ferritin siRNA decreased protein expression by 80% within 48 hours, and this decrease was associated with more than 50% decrease in the LD(50) for DNA-alkylating agent carmustine (BCNU), which is commonly used to treat glioma in clinic. In a subcutaneous mouse model of human glioma, intratumoral injections of liposomes containing H-ferritin siRNA reduced the effective dose of BCNU needed for tumor suppression by more than 50%. A plasmid supercoil relaxation assay showed that H-ferritin specifically and directly protected DNA from BCNU treatment. H-ferritin siRNA additionally seemed to increase apoptosis in glioma cells in vitro upon H-ferritin knockdown. Overall, our results illustrate how silencing H-ferritin can effectively sensitize tumors to chemotherapy and also show the ability of C-liposomes to serve as a novel in vivo delivery tool for siRNAs.

Protein- and DNA-based active immunotherapy targeting interleukin-13 receptor alpha2.

High-grade astrocytoma (HGA) is an invariably fatal malignancy with a mean survival of 14 months despite surgery, radiation, and chemotherapy. We have found that a restricted receptor for interleukin-13 (IL-13), IL-13 receptor alpha 2 (IL13Ralpha2), is abundantly overexpressed in the vast majority of HGAs but is not appreciably expressed in normal tissue, with the exception of the testes. Therefore, IL-13Ralpha2 is a very attractive target for anti-HGA immunotherapy. In order to test protein and genetic vaccines that target IL13Ralpha2, we developed a G26-IL13Ralpha2-expressing syngeneic immunocompetent murine glioma model. Using this glioma model, mice were immunized with recombinant extracellular IL13Ralpha2 protein (IL13Ralpha2ex) or a DNA expression vector containing the gene for IL13Ralpha2 and were subsequently challenged with IL13Ralpha2( + ) G26 tumors. Mice immunized with either recombinant or genetic IL13Ralpha2, but not mock-immunized controls, demonstrated complete protection against IL13Ralpha2( + ) glioma growth and mortality. Of interest, only the recombinant-protein-based vaccines generated detectable anti-IL13Ralpha2 antibodies. These studies demonstrate the in vivo efficiency of protein- and DNA-based immunotherapy strategies that target IL13Ralpha2 that may play a clinical role to eradicate the residual microscopic HGA cells that inevitably cause disease recurrence and mortality.

Ferritin: a novel mechanism for delivery of iron to the brain and other organs.

Traditionally, transferrin has been considered the primary mechanism for cellular iron delivery, despite suggestive evidence for additional iron delivery mechanisms. In this study we examined ferritin, considered an iron storage protein, as a possible delivery protein. Ferritin consists of H- and L-subunits, and we demonstrated iron uptake by ferritin into multiple organs and that the uptake of iron is greater when the iron is delivered via H-ferritin compared with L-ferritin. The delivery of iron via H-ferritin but not L-ferritin was significantly decreased in mice with compromised iron storage compared with control, indicating that a feedback mechanism exists for H-ferritin iron delivery. To further evaluate the mechanism of ferritin iron delivery into the brain, we used a cell culture model of the blood-brain barrier to demonstrate that ferritin is transported across endothelial cells. There are receptors that prefer H-ferritin on the endothelial cells in culture and on rat brain microvasculature. These studies identify H-ferritin as an iron transport protein and suggest the presence of an H-ferritin receptor for mediating iron delivery. The relative amount of iron that could be delivered via H-ferritin could make this protein a predominant player in cellular iron delivery.

Interleukin-13 receptor-targeted nanovesicles are a potential therapy for glioblastoma multiforme.

The difficulties associated with treatment of malignant brain tumors are well documented. For example, local infiltration of high-grade astrocytomas prevents the complete resection of all malignant cells. It is, therefore, critical to develop delivery systems for chemotherapeutic agents that ablate individual cancer cells without causing diffuse damage to surrounding brain tissue. Here, we describe sterically stable human interleukin-13 (IL-13)-conjugated liposomes, which efficiently bind to the brain cancer cells that overexpress the IL-13 receptor alpha2 protein. The conjugated liposomes bind to glioblastoma multiforme tissue specimens but not to normal cortex. Conjugating the liposomes with human IL-13 allows for specific binding to glioma cells and uptake of the liposomes via endocytosis. Delivering doxorubicin to glioma cells by IL-13-conjugated liposomes results in enhanced cytotoxicity and increased accumulation and retention of drug in the glioma cells compared with delivery of free drug. The therapeutic potential and targeting efficacy of the IL-13-conjugated liposomes carrying doxorubicin was tested in vivo using a s.c. glioma tumor mouse model. Animals receiving i.p. injections of IL-13-conjugated liposomes carrying doxorubicin for 7 weeks had a mean tumor volume of 37 mm3 compared with a mean volume of 192 mm3 in animals injected with nontargeted liposomes. These results strongly suggest that IL-13-conjugated liposomes carrying cytotoxic agents are a feasible approach for creating a nanovesicle drug delivery system for brain tumor therapy.

Interleukin 13 mutants of enhanced avidity toward the glioma-associated receptor, IL13Ralpha2.

Interleukin 13 (IL13) binds a receptor that is highly overexpressed in malignant gliomas, IL13Ralpha2. IL13 protein is composed of four helices: alpha-helix A, B, C, and D, and we found a new "hot spot" in alpha-helix D that is crucial for the binding of IL13 to IL13Ralpha2. Lys-105 plus Lys-106 and Arg-109 represent this hot spot. In the current study, we have made substitutions at these three positions in IL13. We examined both neutralization of an IL13-based cytotoxin's glioma cell killing and direct receptor binding of the new IL13 mutants. We observed that Lys-105 and Arg-109 are critical for IL13 binding to IL13Ralpha2, indeed. However, new mutants of important properties were identified with regard to tumor targeting. IL13.K105R mutant, in which lysine was substituted by arginine, neutralized the killing of IL13Ralpha2-positive cells by IL13-based cytotoxin more efficiently than wild-type IL13. However, IL13.K105L or IL13.K105A was deprived of any such activity. Furthermore, IL13.K105R and IL13.R109K competed 77- and 27-fold better, respectively, with the binding of [(125)I]IL13 to the IL13Ralpha2 binding sites when compared with wild-type IL13. Thus, we have uncovered the first forms of IL13 of higher avidity toward IL13Ralpha2. These mutants should prove useful in the further design of anticancer diagnostics/therapeutics.

Molecular targeting with recombinant cytotoxins of interleukin-13 receptor alpha2-expressing glioma.

A restricted receptor for interleukin 13 (IL-13R alpha2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13R alpha2, we established an IL-13R alpha2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13R alpha2. G-26-IL-13R alpha2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13R alpha2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13R alpha2 in vivo. These characteristics of the G-26-IL-13R alpha2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative of Pseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13R alpha2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxin in vivo, G-26-IL-13R alpha2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13R alpha2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13R alpha2-expressing tumors in a more clinically relevant in vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

Alanine-scanning mutagenesis of alpha-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine.

We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors. We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13. Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction. Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified. The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays. We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor. Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation. Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins. We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.