Synthesis, characterization, and biological evaluation of integrin alphavbeta3-targeted PAMAM dendrimers

Abstract

Ligand size and valency strongly influence the receptor uptake and clearance of tumor angiogenesis imaging agents. The structures of successful imaging agents exhibit a high degree of variability, encompassing small monovalent arginine-glycine-aspartic acid (RGD)-containing peptides, multivalent RGD-oligomers, and a monoclonal antibody against integrin alpha-v-beta-3 (alpha-v-beta-3). We have pursued a nanoscale approach to imaging of angiogenesis using rationally designed polyamidoamine (PAMAM) dendrimers covalently adorned with RGD-cyclopeptides. An orthogonal oxime-ligation strategy was applied to chemoselectively effect conjugation of the PAMAM dendrimers with RGD-cyclopeptides for targeting alpha vbeta 3. Fluorescent dyes for optical imaging and chelates for gadolinium-based magnetic resonance (MR) imaging were subsequently appended to create robust multimodal macromolecular imaging agents. Fluorescence microscopy revealed selective binding of the resulting RGD peptide-bearing dendrimer with empty chelates to alpha-v-beta-3-expressing cells, but somewhat reduced selectivity was observed following Gd(III) complexation. The expected incomplete saturation of chelates with Gd(III) ions permitted radiometal complexation, and an in vivo tissue distribution of the resulting agent in M21 melanoma tumor-bearing mice showed mostly renal and reticuloendothelial accumulation, with the tumor:blood ratio peaking (3.30+/-0.03) at 2 h postinjection.

Figure 1

Structural representation (half-section) of modified PAMAM dendrimer 7. The PAMAM dendrimer core appears in black, the oxime-ligated α_vβ₃-targeting peptide, c(RGDfK), in orange, the 1B4M chelating agent in green, complexed Gd(III) as yellow spheres, and coordinating H₂O is blue.

Figure 2

General schematic representation of the stepwise modification of PAMAM dendrimers with cyclic-RGD-peptides, conjugation of Alexa Fluor 594 dye, saturation of remaining terminal amines with 1B4M, and chelation of Gd(III).

Figure 3

Fluorescence confocal images of M21 cells incubated for 30 min (peptides, (A–B)) or 150 min (6a, (C)) in the absence (A–B) or presence (C) of LM609-Alexa Fluor 488 (anti-α_vβ₃ mAb) and (A) 1 µM c(RGDfK)-Alexa Fluor 594, (B) 1 µM c(RADfK)-Alexa Fluor 594, or (C) 1 µM 6a. Each image displays Alexa Fluor 594 fluorescence represented in red (top left), the differential interference contrast (DIC) image (bottom left), and the overlay (bottom right). In image (C), Alexa Fluor 488 fluorescence is represented in green (top right), and yellow indicates co-localization.

Figure 4

Fluorescence microscopy overlay images of M21 cells incubated for 120 min in the presence of 50 µg LM609-ALexa Fluor 488 (anti-α_vβ₃ mAb) in (A) 10 µM 6a, (B) 10 µM 6b, (C) 10 µM 7a, and (D) 10 µM 7b. Alexa Fluor 488 conjugate is unmixed in green, Alexa Fluor 594 conjugates appear as red, and yellow indicates co-localization.

Figure 5

Biodistributions of ¹¹¹In-7a (top) and ¹¹¹In-CHX-A’’-c(RGDfK) (bottom) following i.v. injection in athymic mice bearing s.c. M21 melanoma tumors.