Multifunctional Theranostic Nanoparticles for Brain Tumors
Authors: Yuhua Wang, Leaf Huang
Journal: Molecular Therapy (2012)

Antiangiogenic approaches have been extensively exploited to provide a rationally designed therapy for the treatment of brain cancer. The brain tumor endothelium, with characteristics of high proliferation, high permeability, and high expression of proangiogenic factors (such as vascular endothelial growth factor, VEGF), is a particularly appealing therapeutic target for this strategy. Many antibody drugs, which primarily target the interaction between VEGF and its receptors, have been investigated in clinical trials but have shown only modest effects. Recent research published by Agemy et al.1Agemy L Friedmann-Morvinski D Kotamraju VR Roth L Sugahara KN Girard OM et al.Targeted nanoparticle enhanced proapoptotic peptide as potential therapy for glioblastoma.Proc Natl Acad Sci USA. 2011; 108: 17450-17455Crossref PubMed Scopus (278) Google Scholar has alternatively harnessed a tumor-homing peptide (CGKRK) to specifically deliver multifunctional theranostic nanoparticles composed of iron oxide nanoworm (NW) and mitochondria-targeted cytotoxic peptide D[KLAKLAK]2 to glioblastoma multiforme (GBM) endothelium. Moreover, with the help of a tumor-penetrating peptide (iRGD), the NWs were capable of infiltrating the tumor tissue after extravasation for tumor cell eradication. This strategy was evaluated in lentivirus-induced, transplantable, and orthotopic brain tumor models. Complete tumor ablation was achieved in the first model and significant prolongation of survival was observed in the latter two models, suggesting promise for eventual clinical application. The CGKRK tumor-homing peptide was previously identified using a combination of in vivo and ex vivo phage display systems.2Hoffman JA Giraudo E Singh M Zhang L Inoue M Porkka K et al.Progressive vascular changes in a transgenic mouse model of squamous cell carcinoma.Cancer Cell. 2003; 4: 383-391Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar The peptide was found to accumulate in the endothelium and parenchyma of tumors and dysplastic foci with high specificity after intravenous administration. Moreover, the peptide was demonstrated to home to the endothelium of different types of subcutaneously grown transplanted tumors. The homing property was especially apparent in a subcutaneous tumor model implanted by Matrigel in combination with VEGF and basic fibroblast growth factor. It is believed that the combination of these two angiogenesis-related growth factors is sufficient to elicit the expression of the binding moieties for this homing peptide, which are strategically exploited as the target in antiangiogenic therapy. Agemy et al. harnessed the tumor-homing property of the CGKRK peptide to deliver a previously identified proapoptotic peptide, D[KLAKLAK]2, as a chimeric peptide.3Ellerby HM Arap W Ellerby LM Kain R Andrusiak R Rio GD et al.Anti-cancer activity of targeted pro-apoptotic peptides.Nat Med. 1999; 5: 1032-1038Crossref PubMed Scopus (822) Google Scholar The peptide was covalently linked to an iron oxide NW with a 5K–polyethylene glycol linker. The D[KLAKLAK]2 is designed in such a way that this a-helix-forming, cationic peptide disrupts the membrane once the cationic amino acids are attached to the head groups of anionic phospholipids in the membrane. However, the peptide preferentially disrupts the mitochondria membranes because of their higher content of anionic phospholipids than that of cytoplasm membranes, which contain mostly zwitterionic phospholipids. Therefore, the peptide gives rise to minimal toxicity outside of the cell. Once internalized, the peptide causes cell death by destabilizing the mitochondria membranes and subsequently inducing mitochondria-dependent apoptosis.3Ellerby HM Arap W Ellerby LM Kain R Andrusiak R Rio GD et al.Anti-cancer activity of targeted pro-apoptotic peptides.Nat Med. 1999; 5: 1032-1038Crossref PubMed Scopus (822) Google Scholar The exploitation of elongated iron oxide NWs, which have been demonstrated to be more effective, in terms of targeting capacity, than spherical nanoparticles, additionally provided a signal for diagnostic imaging with magnetic resonance imaging. Agemy and colleagues assembled this multifunctional theranostic nanoparticle from a collection of state-of-the-art elements. The tumor accumulation of the NWs coated with the chimeric peptide composed of CGKRK and D[KLAKLAK]2 indicated that the function of the targeting peptide was not compromised in the chimeric form or after conjugation to the NWs. The D[KLAKLAK]2 peptide maintained mitochondrial targeting and apoptosis-inducing capacity in the NWs as well. These results suggest that the elegantly designed nanoparticles include all the essential elements of an advanced theranostic reagent without compromising the individual functionalities of the components. Indeed, the half-maximal inhibitory concentration (IC50) of free CGKRKD[KLAKLAK]2 was much higher (>200-fold) than that of the NW-linked peptide, suggesting that coating of the peptide on the NWs enhanced its activity, probably because of the increased local concentration. To evaluate the delivery specificity and therapeutic efficacy of the NWs in vivo, Agemy and colleagues chose a GBM model, the most frequent primary and malignant brain tumor model, by targeting the endothelium—the very supply for tumor survival and growth.4Jain RK di Tomaso E Duda DG Loeffler JS Sorensen AG Batchelor TT Angiogenesis in brain tumours.Nat Rev Neurosci. 2007; 8: 610-622Crossref PubMed Scopus (1050) Google Scholar They studied three mouse GBM models that closely resemble human GBMs in their aggressiveness and diffusion pattern into normal brain tissue. The first model was created by injection of a lentiviral vector carrying the H-Ras V12 oncogene and a short hairpin RNA targeting p53 into the hippocampus of mice. This tumor shows all the features of GBM, including hypervascularity.5Soda Y Marumoto T Friedmann-Morvinski D Soda M Liu F Michiue H et al.Transdifferentiation of glioblastoma cells into vascular endothelial cells.Proc Natl Acad Sci USA. 2011; 108: 4274-4280Crossref PubMed Scopus (398) Google Scholar After systemic administration every other day for 3 weeks, the tumors were eradicated in 9 of 10 mice treated, with no histologically detectable tumor tissue in the brain. Although such a high frequency of repetitive administration might arouse some translational concerns due to the possibility of generation of antibody to the peptide-coated NWs or liver and kidney toxicity due to nonspecific uptake, this result was particularly impressive in view of the fact that these tumor cells have been reported to differentiate into endothelial cells in a VEGF-independent manner in this GBM model.5Soda Y Marumoto T Friedmann-Morvinski D Soda M Liu F Michiue H et al.Transdifferentiation of glioblastoma cells into vascular endothelial cells.Proc Natl Acad Sci USA. 2011; 108: 4274-4280Crossref PubMed Scopus (398) Google Scholar Thus, the tumor was potentially resistant to VEGF-targeted antiangiogenic therapy. The authors also tested a transplantable tumor model—the GBM 005 cell line—that was generated by a lentiviral vector expressing the oncogenes H-Ras and Akt and by knocking down p53. Although the administration of the NWs substantially prolonged survival, the treatment was non­curative in this setting. Despite the complete dysfunction of blood vessels observed from the tumor tissue harvested at the end point, the mice died. The authors speculated that fluid-conducting channels lined by tumor cells could provide blood supply to maintain tumor progression. It is also reported that GBM 005 cells contain tumor-initiating stem cells with extremely high tumor-forming ability. Only 100 cells are sufficient to form a tumor in the brain of nude mice.6Marumoto T Tashiro A Friedmann-Morvinski D Scadeng M Soda Y Gage FH et al.Development of a novel mouse glioma model using lentiviral vectors.Nat Med. 2009; 15: 110-116Crossref PubMed Scopus (242) Google Scholar A similar result was observed in another orthotopic model generated by the injection of U87 human GBM cells, which also contain tumor-initiating cells with the ability of self-renewal and multipotency.7Yu SC Ping YF Yi L Zhou ZH Chen JH Yao XH et al.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.Cancer Lett. 2008; 265: 124-134Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar Taken together with the results in the first model, the findings clearly suggest that a single antiangiogenic therapy is not sufficient to eradicate GBM, particularly if tumor-initiating cells are present. Although the authors attempted to enhance the therapeutic efficacy in the GBM 005 model with the aid of an iRGD peptide, which has been shown to facilitate tumor penetration of the NWs, only prolonged survival was achieved.8Sugahara KN Teesalu T Karmali PP Kotamraju VR Agemy L Greenwald DR et al.Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs.Science. 2010; 328: 1031-1035Crossref PubMed Scopus (848) Google Scholar This result is understandable, since the primary target cells of the NWs were the endothelium with the binding moieties presented as entries to the cells. Without endocytosis, the peptide is nontoxic to the cells. Therefore, the extravascular NWs in the tumor tissue were less effective in killing tumor cells, consistent with in vitro results showing that the IC50 of NWs for GBM cells (T3 and U87) was three times that for human umbilical vein endothelial cells. In summary, Agemy et al. have successfully assembled multiple elements into a single theranostic NW without compromising their individual functions. The NWs could effectively target tumor vasculature and kill the endothelium with a proapoptotic peptide. Although the NWs showed exceptional efficacy in eliminating a lentivirus-induced GBM model, which is potentially refractory to antiangiogenic therapy targeting VEGF-VEGFR, it could not completely inhibit GBM growth induced by orthotopic inoculation of a GBM cell line. Although all three models share common characteristics of GBM, such as hypervasculature, the latter two tumor models contain tumor-initiating cells in the cell culture, suggesting a possible mechanism for resistance to antiangiogenic strategies in treating GBM. There is little doubt that the targeted NWs hold promise for treating brain cancer in an antiangiogenic therapy. However, it is also noteworthy that tumor-initiating cells are the evil roots that need to be taken care of for the eventual cure of GBM.