Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle

Abstract

Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin alphav beta3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting alphav beta3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin alphav beta3 compound (IA) which has superior binding affinity to alphav beta3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targ alphav beta3 was observed. These ITNPs also were rapidly taken up by cells that express alphav beta3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.

Figure 1

(a) Schematic diagram outlining the formation of the ITNPs by self-assembly of the appropriate lipids. The lipid with targeting agent (10 mole%) was combined with base lipid (90 mole%) in a solution of chloroform and methanol (1:1). The mean diameter of the ITNPs was 104.4 ± 3.3 nm, as determined by dynamic light scattering, and the zeta potential was approximately −43.8 mv. The ITNPs were stable for weeks without any observable changes in their physical and biological properties when formulated for use in vivo. (b) Schematic diagram outlining the formation of the Control-NPs by self-assembly of the appropriate lipids. The mean diameter of the nanoparticle was 101.2 ± 2.4 nm, as determined by dynamic light scattering, and the zeta potential was approximately −32.6 mv.

Figure 2

Transmission electron microscopic image of integrin targeted nanoparticles.

Figure 3

ITNPs binding affinity to the integrin α vβ 3 protein was quantified by the α_vβ₃ plate binding assay, as in method description. The concentration of inhibitor producing 50% inhibition (IC50) of vitronectin binding to α_vβ₃ was calculated based on a curve fitting model using KaleidaGraph 3.5 (Synergy Software, Reading, PA). ITNPs and targeting agent IA have a closed binding affinity to the α_vβ₃ protein.

Figure 4

Cellular uptake of the ITNPs by M21 cells in vitro. The ITNPs were added to the chamber yielding a final concentration of 12 μmol/ml. After 5, 15, and 30 minutes of incubation, the cells were washed, stained with DAPI and observed by fluorescence microscopy.

Figure 5

Angiogenic vessel targeting by the ITNPs. A: SCC-7 Cell nucleus stained by DAPI, B: Functional angiogenic vessels stained by injected FITC-Lectin, C: angiogenic vessels targeting by ITNPs observed by Rhodamine, D: Merge signals of the ITNPs targeted vessels and functional angiogenic vessels labeled by Lectin, E: Merge signals of the ITNPs targeted vessels, functional angiogenic vessels labeled by Lectin, and cell nuclear staining by DAPI. (scale bar = 50 um).

Scheme 1

Schematic illustration for the chemical synthesis of targeting lipid (BisT-PE-EDTA-IA).