Pretargeting Technology For Ultrasound Molecular Imaging

IntroductionRecently, ultrasound molecular imaging, which involves the use of molecularly targeted contrast agents, has begun to receive an increasing amount of attention. As a noninvasive targeting approach, ultrasound molecular imaging combines the advantages of contrast-enhanced ultrasound (including the lack of ionizing radiation, noninvasiveness, and high spatial and temporal sensitivity) with the ability to characterize neoplastic processes on a molecular level. Ultrasound molecular imaging provides a new approach to the non-invasive diagnosis of tumors. The majority of preclinical research using ultrasound molecular imaging has involved the use of microbubbles as contrast agents. To achieve tumor targeting, ideal tumor-targeted contrast agents should satisfy the following criteria:(i) stability in circulation, (ii) prolonged circulation time to allow for effective accumulation in the targeted tissue, and (iii) a size that allows extravasation through the tumor microvasculature (380-780 nm). However, at present, the size of commonly used targeted ultrasound microbubbles is 2-8 μm. At this size, the targeted microbubbles cannot pass through the tumor microvasculature. The solution to the above problems may lie in developing nanoscale microbubble precursors that can effectively accumulate in tumor tissue and then convert into microbubbles in situ after tumor accumulation. Some scholars have developed perfluoropentane (PFP) nanoparticles that convert into microbubbles under heating to hyperthermic conditions or ultrasound irradiationTargeted contrast agents that target tumor cells have primarily been developed based on the direct targeting method, which is achieved through contrast agents binding antibodies on their surfaces and incubating with the tumor cells. This method is simple and convenient but cannot achieve high-efficiency targeting. Antibodies tend to bind to hydrophobic surfaces through the constant region (Fc) of the molecule, leaving the antigen-binding sites free to interact with the antigen. However, some studies have demonstrated that during the preparation of targeted contrast agents, the orientation of antibodies absorbed on the surfaces of these agents is random, with either the Fc regions or the antigen-binding sites binding to the surface. As a result, the contrast agents may be coated in antibodies but unable to appropriately target to the tumor. Pre-targeting has been widely used in preclinical studies of tumor radioimmunoimaging and radioimmunotherapy. In pre-targeting, a bifunctional mAb that can bind not only to a tumor-associated antigen but also to a second reagent (radiolabeled ligand, toxin, drug, etc.) is first injected. This second reagent is injected at a later time. In the second step of this method, a high-affinity binding system such as the avidin-biotin system is used.Methods1. PLGA/PFPs were prepared via an emulsion evaporation process. The optimization of parameters (such as, the amount of PFP, the intensity of LIFU and the duration of time.) of nanoparticles was measured by LIFU. The streptavidin and the antibodies were covalently bound to the surfaces of the PLGA nanoparticles through a carbodiimide reaction. Two factors that induced the droplet-to-bubble transition in the nanodroplets:heating (thermal factor) and sonication (thermal and/or mechanical factor) were identified. Heat-induced droplet vaporization was visualized by heating nanodroplet samples using a microscope heating plate. An ultrasound-induced droplet-to-bubble transition is referred to as acoustic droplet vaporization (ADV) was visualized by LIFU.2. Confocal laser scanning microscopy, flow cytometry and bicinchoninic acid (BCA) protein assay were used to confirm the presence of streptavidin and antibodies on the nanoparticle surfaces:3. Two different targeting approaches were studied:a direct targeting method using antibody-labeled PLGA/PFPs (PLGA-Ab/PFPs) targeted to SKOV3 ovarian cancer cells and a pre-targeting method using the avidin-biotin system. In the first step of the pre-targeting method, biotinylated anti-CEA antibodies were added, and streptavidin-coated PLGA/PFPs (PLGA-SA/PFPs) were added in the second step.Result1. The three types of nanoparticles exhibited very similar characteristics with respect to particle size and dispersibility. The mean sizes of the PLGA-mAb/PFPs, PLGA-SA/PFPs and PLGA/PFPs were 346.2±74.49 nm, 395.2±60.25 nm and 310.7±91.87 nm, respectively. Under microscope, all types of nanoparticles were round, uniformly dispersed, non-aggregated and some holes on the surface.2. According to the flow cytometry results, the effective binding between the PLGA/PFPs and streptavidin was 95.02±0.62%, and the effective binding with the antibody was 99.69±0.2%, indicating that nearly all of the PLGA/PFPs were coated with streptavidin or antibodies. Under confocal laser scanning microscopy, the PE-labeled streptavidin coated PLGA/PFPs (PLGA-SA-PE/PFPs) exhibited red fluorescence on their surfaces. An FITC-labeled secondary antibody coated PLGA-Ab/PFPs (PLGA-Ab-FITC/PFPs) exhibited yellow fluorescence on their surfaces. The numbers of streptavidin and antibody molecules bound to each PLGA nanoparticle were 4648 and 1780, respectively.3. As a result of the optimization of parameters of nanoparticles, the amount of PFP was 4001, the intensity of LIFU was 8.5W and the duration time was 3min can obviously increase ultrasound imaging. The three types of nanoparticles exhibited very similar characteristics with respect to the heat-induced droplet-to-bubble transition and ultrasound-induced droplet-to-bubble transition. As the temperature increased, new bubbles were formed. After ultrasound treated, the sound intensity of the ultrasound images were increased.4. As a result of cell targeting, compared with the other groups, the pre-targeting group exhibited a statistically significant difference, either in the stationary state or flow state.Conclusion1. The prepared nanoparticles can phase-shift under heat and ultrasound induced. The effective coupling of the nanoparticles with the streptavidin and antibody was demonstrated via confocal laser scanning microscopy, flow cytometry and a BCA protein assay, nearly all of the PLGA/PFPs were coated with streptavidin or antibodies.2. At the present stage of this investigation, the pre-targeting strategy was found to be superior for the delivery of the nanoparticles to the tumor cells. However, further work is required to define which targeting method is superior in an in vivo study.