Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide

Abstract

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.

Figure 1

Chemical structures of HWGF peptide, DOTA-derivatized peptide (M₁) and dual-conjugate (M₂).

Figure 2

HPLC traces for M₁, IRDye 800CW and M₂. Traces were acquired at 280 nm or with a fluorescence detector.

Figure 3

MMP-9 enzyme binding assays. Shown are the fluorescent scans acquired on the LICOR infrared imaging system. In all cases, 10 ng of purified MMP-9 was used and binding was resolved by SDS gel electrophoresis. The binding experiments shown are as follows: MMP-9 was incubated with increasing amounts of M₂ (A) and processed M₂ (B) at concentrations of 0, 0.1, 0.2, 0.4, 1, 2, 4 and 10 μM (lanes 1–8). For the blocking study (C), MMP-9 was incubated with M₁ atconcentrations of 0.2, 0.5, 1, 2, 20, 200, 500 and 1000 μM (lanes 1–8). M₂ (2 μM) was then added and incubated for 1 h. Bands resolved at 62 kDa correspond to M₂ binding with MMP-9.

Figure 4

HPLC chromatograms for ⁶⁸Ga-M₂: UV at 280 nm (A), fluorescent (B), and radiometric (C).

Figure 5

Stability studies for ⁶⁸Ga-M₂ in PBS, DTPA challenge and serum.

Figure 6

In vitro characterization of BMP-2 expression from adenovirus transduced cells.

Figure 7

Multimodality imaging of mice with fracture putty implants. Human fibroblast cells transduced with AdBMP2 were injected into the right hind limb of NOD/SCID mice. Mice were injected with ⁶⁸Ga-M₂ and imaged at 18 h. NIR images (A,C) were acquired on day 4 post-implantation and follow-up CTs (B,D) were taken on day 11. Solid arrows indicate site of new bone formation and agent accumulation. Dashed arrows designate control (empty cassette) injection sites in the contralateral limb.