c(RGDfK)

Active targeting strategies are currently being extensively investigated in order to enhance the selectivity of photodynamic therapy. The aim of the present research was to evaluate whether the external decoration of nanopolymeric carriers with targeting peptides could add more value to a photosensitizer formulation and increase antitumor therapeutic efficacy and selectivity. To this end, we assessed PLGA-PLA-PEG nanoparticles (NPs) covalently attached to a hydrophilic photosensitizer 5-[4-azidophenyl]-10,15,20-tri-(N-methyl-4-pyridinium)porphyrinato zinc (II) trichloride (ZnTriMPyP) and also to c(RGDfK) peptides, in order to target αvβ3integrin-expressing cells. In vitro phototoxicity investigations showed that the ZnTriMPyP-PLGA-PLA-PEG-c(RGDfK) nanosystem is effective at submicromolar concentrations, is devoid of dark toxicity, successfully targets αvβ3integrin-expressing cells and is 10-fold more potent than related nanosystems where the PS is occluded instead of covalently bound.

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell-cell and cell-matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αvβ3integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of the αvβ3integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The αvβ3antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10).Most of the cyclic RGD peptides are composed of five amino acids. Haubner et al. (11) reported that various cyclic RGD peptides exhibit selective inhibition of binding to αvβ3(50% inhibition concentration (IC50), 7-40 nM) but not to αvβ5(IC50, 600-4,000 nM) or αIIbβ3(IC50, 700-5,000 nM) integrins. Various radiolabeled cyclic RGD peptides and peptidominetics have been found to have high accumulation in tumors in mice (12, 13). Out of these developments [18F]Galacto-c(RGDfK) has been evaluated in a number of clinical studies for imaging of αvβ3in cancer patients (14-19). Li et al. (20) used 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) as a bifunctional chelator for labeling Glu-[cyclo(RGDyK)]2(RGD2) to form (68Ga-NOTA-RGD2) for positron emission tomography (PET) imaging of αvβ3receptors in nude mice bearing human glioblastoma U87MG tumors.

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3).Several families of MMPs are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastases (4-7). MMP expression is highly regulated in normal cells, such as trophoblasts, osteoclasts, neutrophils, and macrophages. Elevated levels of MMPs have been found in tumors associated with a poor prognosis for cancer patients (8). There are four members of endogenous tissue inhibitors of metalloproteinases (TIMPs), which regulate the activity of MMPs leading to inhibition of tumor growth and metastasis (9, 10). TIMP-2 (TIMP2) is a bifunctional inhibitor of angiogenesis by inhibition of proteinase activity of MMPs and endothelial cell proliferationviabinding to α3β1(the N-terminal domain) and by MMP-independent anti-angiogenic activity (the C-terminal domain) (11, 12). Kang et al. (13) fused the N-terminal domain of TIMP2 to the C-terminus of human serum albumin (HSA) to form HSA/TIMP2 fusion protein (HSA-TIMP2), which is readily secreted by the transfected yeastSaccharomyces cerevisiae. HSA-TIMP2 retains its anti-angiogenic activity at the C-terminal domain with little MMP inhibitory activity at the N-terminal domain. Lee et al. (14) have evaluated Cy5.5-HSA/TIMP2 (Cy5.5-HSA-TIMP2) forin vivonear-infrared (NIR) fluorescence imaging of rat prostate MLL tumors in nude mice showing maximum tumor accumulation at 2 d after injection.Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell-cell and cell-matrix interactions (15). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The αvβ3integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (16-21). Expression of the αvβ3integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. The peptide sequence Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. The αvβ3antagonists are being studied as anti-tumor and anti-angiogenic agents (18, 22, 23). Various radiolabeled RGD peptides (antagonists) have been introduced for imaging of tumors and tumor angiogenesis (24). Choi et al. (25) conjugated multiple c(RGDfK) peptides to HSA-TIMP2 to enhance the binding capacity of the protein (RGD-HSA-TIMP2) to tumors and their vasculatures.123I-RGD-HSA-TIMP2 and123I-HSA-TIMP2 have been studied as potential single-photon emission computed tomography (SPECT) probes for imaging αvβ3receptors in nude mice bearing human glioblastoma U87MG tumors.