Biotin-streptavidin-guided two-step pretargeting approach using PLGA for molecular ultrasound imaging and chemotherapy for ovarian cancer

Abstract

Background: Ovarian cancer seriously threatens the lives and health of women, and early diagnosis and treatment are still challenging. Pre-targeting is a promising strategy to improve the treatment efficacy of ovarian cancer and the results of ultrasound imaging.

Purpose: To explore the effects of a pre-targeting strategy using streptavidin (SA) and paclitaxel (PTX)-loaded phase-shifting poly lactic-co-glycolic acid (PLGA) nanoparticles with perfluoro-n-pentane (PTX-PLGA-SA/PFPs) on the treatment and ultrasound imaging of ovarian cancer.

Methods: PTX-PLGA/PFPs were prepared with a single emulsion (O/W) solvent evaporation method and SA was attached using carbodiimide. The encapsulation efficiency of PTX and the release characteristics were assessed with high performance liquid chromatography. The phase-change characteristics of the PTX-PLGA-SA/PFPs were investigated. The anti-carcinoembryonic antigen (CEA) antibody (Ab) was covalently attached to PTX-PLGA/PFPs via carbodiimide to create PTX-PLGA-Ab/PFPs. The targeting efficiency of the nanoparticles and the viability of ovarian cancer SKOV3 cells were evaluated in each group using a microscope, flow cytometry, and cell counting kit 8 assays.

Results: THE PTX-PLGA-SA/PFPs were spheres with a size of 383.0 ± 75.59 nm. The encapsulation efficiency and loading capability of the nanoparticles for PTX were 71.56 ± 6.51% and 6.57 ± 0.61%, respectively. PTX was burst-released up to 70% in 2-3 d. When irradiated at 7.5 W for 3 min, the PTX-PLGA-SA/PFPs visibly enhanced the ultrasonography images (P < 0.05). At temperatures of 45°C and 60°C the nanoparticles phase-shifted into micro-bubbles and the sizes increased. The binding efficiencies of SA and Ab to the PTX-PLGA/PFPs were 97.16 ± 1.20% and 92.74 ± 5.75%, respectively. Pre-targeting resulted in a high binding efficacy and killing effect on SKOV3 cells (P < 0.05).

Conclusions: The two-step pre-targeting process can significantly enhance the targeting ability of PTX-loaded PLGA nanoparticles for ovarian cancer cells and substantially improve the therapeutic efficacy. This technique provides a new method for ultrasonic imaging and precise chemotherapy for ovarian cancer.

Keywords: Liquid-gas Phase-shift; Molecular imaging; PLGA; Paclitaxel; Pre-targeting technology.

Figure 1. Light microscope and electron microscope images of PTX-PLGA-SA/PFPs.

(A) Light microscope image. (B) Inverted fluorescence microscope image of PTX-PLGA-SA/PFPs labeled with DiI showing as red fluorescence. (C) SEM image. (D) TEM image. (E) Size distributions of PTXPLGA/PFPs, PLGA/PFPs, PTX-PLGA-Ab/PFPs, and PTX-PLGA-SA/PFPs. (F) The chromatography results for PTX encapsulated in nanoparticles in HPLC. (G) The release curve for PTX from PTXPLGA/PFPs in vitro. (H) Confocal laser scanning microscope images of PE labeled SA, Dil labeled PTX-PLGA-Ab/PFPs, Nanoparticles without fluorescence, and FITC labeled IgG.

Figure 2. Flow cytometry analysis of the connection efficiency of both SA and Ab bound to PTX-PLGA/PFPs.

(A) Control; (B) SA group; (C) Control; (D) Ab group.

Figure 3. Phase shifting of PTX-PLGA-SA/PFPs.

(A) After exposure to LIFU at different power levels, the B-mode and contrast ultrasonic images of nanoparticles at 3.3, 4.4, 5.5, 6.5, 7.5, and 8.5 W are shown. After irradiation with LIFU at different power levels, the average echo intensities of the nanoparticles are shown. (B) B-mode; (C) CEUS mode. An asterisk (*) indicates there is a significant difference compared with the 0.1 mg/ml group (P < 0.05); a number sign (#) indicates there is no significant difference between the two groups (P > 0.05). An ampersand (&) indicates there is a significant difference compared with the rest of the groups (P < 0.05). The average ultrasonic echo intensity (dB) of the nanoparticles after phase transformation induced by LIFU at different concentrations. (D) B-mode; (E) CEUS. “*” indicates there is a significant difference compared with the 0.1 mg/ml group (P < 0.05); “#” indicates there is no significant difference between the two groups (P > 0.05). (F) Light microscopic images of nanoparticles before and after phase transformation caused by heating.

Figure 4. The schematic diagram of the two-step pre-targeting technology.

(A) The schematic diagram of the two-step pre-targeting technology. (B) Confocal laser scanning microscope images of human ovarian cancer SKOV3 cells showing the results of incubation with FITC-labeled secondary antibodies. Green, FITC. Blue, DAPI.

Figure 5. The survival rate of SKOV3 cells after adding nanoparticles and targeting efficacy of nanoparticles.

(A) Effects of different concentrations of PTX-PLGA/PFPs on the survival rate of SKOV3 cells determined via CCK-8 assays. (B) The average cell fluorescence intensities detected with flow cytometry. An asterisk (*) indicates there is a significant difference compared with the other groups (P < 0.05); an ampersand (&) indicates there is a significant difference compared with these two groups (P < 0.05). Laser confocal microscope images of SKOV3 cells incubated with (C) Drug-loaded pretargeting, (D) Drug-loaded and directly targeting, (E) Drug-loaded and non-targeting, and (F) Antibody blocking.

Figure 6. Cancer cell survival rates determined with CCK-8 assays.

An asterisk (*) indicates there is a significant difference compared with the rest of the groups (P < 0.05).