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

Materials

PLGA-COOH (molecular weight 12,000) was obtained from Daigang Biological Engineering Co., LTD (Jinan, Shandong, China). PTX was purchased from Haoxuan Biological Co., LTD (Xi’an, Shanxi, China). Polyvinyl alcohol (PVA), 1-ethyl-3-(3-dimethylamino) propyl carbodiimide, hydrochloride (EDC), N-hydroxysuccinimide (NHS), Dimethyl sulfoxide (DMSO), 2-(N-morpholino) ethanesulfonic acid (MES) buffer, and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG were purchased from Sigma (St. Louis, MO, USA). SA, phycoerythrin (PE)-labeled SA (PE-SA), anti-CEA antibody biotinylated anti-CEA antibody (Bio-Ab), the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI), and the fluorescent dye DiI were obtained from Boorson Biotechnology Co., LTD (Beijing, China). Phosphate-buffered saline (PBS), 4% paraformaldehyde, and cell counting kit 8 (CCK-8) kits were purchased from Baodu Biological Engineering Co., LTD (Wuhan, Hubei, China). PFP was obtained from Strem Chemicals (USA). Dichloromethane (CH2CL2) was purchased from Beibei Chemical Reagent Company (Chongqing, Sichuan, China). Sodium azide was obtained from Huanyu Biotechnology Co., LTD (Beijing, China). Fetal bovine serum (FBS), RPMI-1640 medium, and 0.25% Trypsin were purchased from Hyclone Biochemical Products Co., LTD (Wuhan, Hubei, China).

Preparation of PTX-loaded PLGA nanoparticles and pure PLGA nanoparticles

PTX-loaded phase-change PLGA nanoparticles (PTX-PLGA/PFPs) were prepared via emulsification (O/W). Briefly, 50 mg and 5 mg of PTX were dissolved in two mL of dichloromethane and placed in an ice bath. Afterward, PFP and 5% PVA aqueous solution was added and the mixture was sonicated at 100 W for 6 min, followed by the addition of 20 mL of 2% (v/v) isopropanol aqueous solution. The reaction was stirred with a magnetic stirrer for 2–3 h to volatilize the dichloromethane, centrifugated and washed with water several times to obtain PTX-PLGA/PFPs, then stored at 4 °C. Pure nanoparticles without PTX (PLGA/PFPs) were prepared using the same protocol.

Preparation of PTX-PLGA-SA/PFPs and PTX-PLGA-Ab/PFPs

The prepared PTX-PLGA/PFP nanoparticles were suspended in MES buffer (0.1 mol/L, pH 5.5). EDC and NHS were added and the mixture was incubated in a shaker for 45 min. The reaction was centrifuged and washed three times with water to remove EDC and NHS followed by resuspension in MES buffer (0.1 mol/L, pH 8.0). PTX-PLGA/PFP and SA were mixed at a mass ratio of 1:1. Ab was added to PTX-PLGA/PFP (PTX-PLGA/PFP: Ab = 20:1) and the mixture was stirred for 2 h at 4 °C, followed by washing with water several times to obtain PTX-PLGA-SA/PFPs and PTX-PLGA-Ab/PFPs. For fluorescence observation, PE-SA was used to prepare PTX-PLGA-SA-PE/PFPs in the same manner in the dark. PTX-PLGA-Ab/PFPs stained with DiI were incubated with FITC-labeled IgG for 1 h in a shaker at 4 °C and washed with water. The binding of SA and Ab to the PTX-PLGA/PFPs was confirmed using a confocal laser scanning microscope (CLSM) and flow cytometer.

Characteristic of nanoparticles

The morphology of PTX-PLGA-SA/PFPs was observed with a transmission electron microscope (TEM, Hitachi, Tokyo, Japan). The particle size and zeta potential of the PTX-PLGA/PFPs, PLGA/PFPs, PTX-PLGA-Ab/PFPs, and PTX-PLGA-SA/PFPs were determined with a Malvern particle size analyzer (Brookhaven Instruments Co., Holtsville, USA). High-performance liquid chromatography (HPLC, Waters E2695, Germany) was used to detect the encapsulation efficiency and drug loading of PTX in the PTX-PLGA/PFPs. The liquid-phase conditions were as follows. The mobile phase was acetonitrile water at a ratio of 45:55, the detection wavelength was 227 nm, and the detector was the UV2489 C18 Xbridge (150 × 4.6 mm, 3.5 µm). The volume of the organic phase was two mL, the temperature was 4 °C, and the stirring speed was 800 rpm. The power capacity of the sonicator was 100 W in pulse mode, which was applied for 5 s every 5 s, with a total sonicating time of 6 min. PTX was dissolved in methanol to prepare a standard solution at concentrations of 12.5, 25, 50, 75, and 100 µg/mL and used to create a standard curve. The PTX-PLGA/PFPs were demulsified using methyl chloride and the PTX content was determined using HPLC. PTX encapsulation rate (%) = (Detection amount/total dose) ×100%. Drug loading capacity (%) = (Detection amount/total mass of nanoparticles) ×100%.

In vitro release characteristics of PTX-loaded nanoparticles

Two mL of PTX-PLGA/PFPs nanoparticles (resuspended in PBS) was added to a dialysis bag and placed in 150 mL of medium containing 30% ethanol, 0.01% TWEEN-80, and 0.02% azide sodium. Subsequently, the mixture was placed on a shaker at 37 °C and shaken at 150 rpm. Then, one mL of the solution was removed at 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 h, respectively, and one mL of fresh solution was added. The PTX content was determined using HPLC.

Low-intensity focused ultrasound (LIFU)-induced PTX-PLGA-SA/PFPs phase change for ultrasound imaging in vitro and thermally induced phase change

PTX-PLGA-SA/PFPs nanoparticles were placed in the wells of a gel model and irradiated with LIFU at different power levels (3.3, 4.4, 5.5, 6.5, 7.5, 8.5 W) for 3 min before being observed in normal mode (B-mode) and contrast-enhanced ultrasound (CEUS). The average echo intensities of the ultrasound images before and after the phase change were compared using the DFY image analysis system. After determining the optimal power, LIFU irradiation was performed at 0.1, 0.5, 1.0, 2.0, and 5.0 mg/mL of the nanoparticle concentration (based on the quality of PLGA). In addition, the heating plate method was used to observe the thermally induced phase transition process for the nanoparticles under a microscope.

Cell culture

Human ovarian cancer SKOV3 cells were cultured with RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin with 5% CO₂ at 37 °C. The cells were cultured in a constant temperature incubator (the incubation processes described later were performed under this condition). The cells were digested with 0.25% trypsin and centrifuged at 1,000 rpm for 5 min.

Cellular immunofluorescence experiment

SKOV3 cells in the logarithmic growth phase were cultured in a disposable 15 mm glass-bottom dish, incubated with Ab for 2 h, and washed with PBS, followed by adding FITC-labeled IgG (secondary antibody) for 1 h. The cells were fixed with 4% paraformaldehyde for 15–20 min, washed with PBS three to five times, incubated with DAPI for 5–8 min, washed with PBS three to five times, and observed under a CLSM.

Cytotoxicity of PTX-PLGA/PFPs

SKOV3 cells were seeded in a 96-well plate at a concentration of 10⁴ cells/well and incubated with PTX-PLGA/PFPs at concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/mL (calculated based on PLGA). Cells without any treatment were used as the negative controls and each group had five replicates. After 24 h or 48 h, the cells were washed with PBS three to five times, and the cell survival rate was assessed with the CCK-8 assay according to the instructions for the kit. Cell survival rate (v%) = (optical density (OD) of experimental group −  OD of zero adjustment group)/(OD of negative group −  OD of zero adjustment group) * 100%.

In vitro targeting experiment

Cells were cultured in a disposable dish and divided into a drug-loaded pre-targeting group, drug-loaded direct-targeting group, drug-loaded non-targeting group, and antibody-blocking group. In the pre-targeting group, the cells were first incubated with 200 µL of Bio-Ab at a concentration of 100 µg/mL for 2 h, washed with PBS three to five times, and then incubated with one mL of DiI-labeled PTX-PLGA-SA/PFPs. In the drug-loaded direct-targeting group, nanoparticles and the same amount of DiI-PTX-PLGA-Ab/PFPs were added. In the drug-loaded non-targeting group, DiI-PTX-PLGA/PFPs were added. The procedure for the antibody blocking group was the same as that for the drug-loaded pre-targeting group, but Bio-Ab was replaced with the same amount of Ab. All cells were further incubated for 45 min, washed with PBS several times, fixed with 4% paraformaldehyde for 20 min, washed with PBS three to five times, stained with DAPI, washed with PBS, and observed under a CLSM. In addition, the cells were cultured in a six-well plate with approximately 10⁶ cells per well. The blank control group consisted of cells without any treatment. After adding the corresponding nanoparticles or reagents to each group, the cells were cultured for 45 min and washed with PBS. The cells were digested with 0.25% trypsin, centrifuged at 800 rpm, resuspended in one mL of PBS, and visualized with flow cytometry.

In vitro toxicity to SKOV3 cells

SKOV3 cells were seeded in 96-well plates at a concentration of 10⁴ cells/well and divided into a drug-loaded pre-targeting group, drug-loaded direct-targeting group, drug-loaded non-targeted group, antibody-blocking group, nanoparticle only group, free drug group, and blank control group. Each group had five replicates. In the drug-loaded pre-targeting group, the cells were first incubated with 25 uL of Bio-Ab at a concentration of 100 ug/mL. After incubating for 2 h, the cells were washed with PBS three to five times and incubated with 100 uL PTX-PLGA-SA/PFPs. The procedure for the antibody blocking group was the same as that for the drug-loaded pre-targeting group, but Bio-Ab was replaced with the same amount of Ab. In the drug-loaded direct targeting group, PTX-PLGA-Ab/PFPs were added. In the drug-loaded non-targeting group, PTX-PLGA/PFPs were added. In the nanoparticle only group, the same amount of PLGA/PFPs was added to each well. In the free drug group, an equal amount of fresh 1640 medium containing PTX (10 ug/mL) was added to each well. In the blank control group, 100 uL of fresh 1640 medium was added to each well. After adding the corresponding nanoparticles or reagents, the cells were cultured for 45 min, washed with PBS three to five times, and 100 uL of fresh RMPI 1640 medium was added. After 24 h or 48 h of incubation, the CCK-8 assay was used to determine the cell viability.

Statistical analysis

All statistical analysis was performed using SPSS 19.0 statistical analysis software. The independent sample t test was used for comparison between two groups and single factor analysis of variance was used to compare the means of multiple groups. The least significant difference (LSD) test was used for pairwise comparisons between multiple groups and P < 0.05 was considered as statistically significant.

Result

Preparation and characterization of PLGA

The prepared PTX-PLGA-SA/PFPs were spheres with a uniform size (Figs. 1A–1D). The diameters of the PTX-PLGA/PFPs, PLGA/PFPs, PTX-PLGA-Ab/PFPs, and PTX-PLGA-SA/PFPs were 333.0 ±  52.27, 364.9 ±  106.9, 397.7 ±  99.96, and 383.0 ±  75.59 nm, respectively (Fig. 1E). The zeta potentials of the PTX-PLGA/PFPs, PLGA/PFPs, PTX-PLGA-Ab/PFPs, and PTX-PLGA-SA/PFPs were −15.70 ±  3.07, −32.7 ±  6.81, −0.93 ±  3.43, and −5.66 ±  3.46 mV, respectively. The zeta potentials of the PTX-PLGA/PFPs without SA or Ab and PLGA/PFPs without drugs were significantly higher than those for the other nanoparticles (P < 0.05).

PTX load and release

The PTX encapsulation rate and loading capacity were 71.56 ±  6.51% and 6.57 ±  0.61%, respectively (Fig. 1F). PTX was released relatively quickly in the first 48 h and the drug release rate exceeded 70% after nearly 50 h. After this period, the drug release rate became significantly slower, showing obvious burst-release characteristics with a high drug release rate (Fig. 1G).

Connection between SA and Ab and PTX-PLGA/PFPs

Orange fluorescence was observed when the PE-SA attached to the fluorescent PTX-PLGA/PFPs, indicating that SA successfully attached to the nanoparticles. The fluorescence was yellow when FITC-labeled IgG attached to the DiI-labeled PTX-PLGA/PFPs, demonstrating the Ab successfully attached to the PTX-PLGA/PFPs (Fig. 1H). The mean connection rate for SA to PTX-PLGA/PFPs measured with flow cytometry was 97.16 ±  1.20%, and that of Ab was 92.74 ±  5.75% (Figs. 2A–2D).

In vitro LIFU-induced PTX-PLGA/PFPs phase transition for ultrasound imaging

After 3 min of LIFU with ultrasound irradiation with a power of 3.3, 4.4, 5.5, 6.5, 7.5, and 8.5 W, each group exhibited significantly higher echo intensity in B-mode, and the echo intensity was highest at 6.5 W and 7.5 W (P < 0.05). However, the difference between the echo intensity at 6.5 W and 7.5 W was not statistically significant (P > 0.05). In CE-mode, the echo intensity increased with the increase of ultrasound power, reaching the highest value at 7.5 W (P < 0.01) but decreased at 8.5 W (Figs. 3A–3C). The echo intensity for different concentrations of nanoparticles (0.1, 0.5, 1.0, 2.0, 5.0 mg/mL) irradiated with ultrasound at 7.5 W for 3 min was investigated. The results showed that the phase change caused by LIFU had a certain relationship with the concentration. When the concentration exceeded 1.0 mg/mL, more obvious images could be obtained in B-mode, and when the concentration exceeded 2.0 mg/mL, the resolution of the CE-mode was significantly enhanced (Figs. 3D, 3E). There was no significant difference in the echo intensity between 2.0 and 5.0 mg/mL in the B-mode and CE-mode (P > 0.05).

Heat-induced PTX-PLGA-SA/PFPs phase transition

The particle size of PTX-PLGA-SA/PFPs before heating was at the nanometer level and the distribution was uniform. When the temperature of the heating plate reached 45 °C, some of the nanoparticles began to undergo a phase change and gradually increased in size from nanometers to micrometers. When the temperature of the heating plate reached 60 °C, most of the nanoparticles underwent a phase change, with different particle sizes ranging from a few microns to tens of microns. Some of the microbubbles ruptured after increasing in size to a certain extent (Fig. 3F).

Cellular immunofluorescence and viability of cells incubated with PTX-PLGA/PFPs

The products from the two-step biotin-streptavidin pre-targeting technology were combined with PTX-loaded phase-change PLGA multifunctional nanoparticles to treat ovarian cancer (Fig. 4A). Green fluorescence was observed, indicating that the FITC-labeled IgG adhered to the cell membrane and the SKOV3 cells expressed a large amount of CEA antigens (Fig. 4B). CCK-8 assay results showed that the PTX-PLGA/PFPs killed the SKOV3 cells in a concentration-dependent manner. As the concentration of the nanoparticles increased, the survival rate of the SKOV3 cells at 24 h and 48 h gradually decreased (Fig. 5A).

Cell targeting and killing effect on tumor cells for nanoparticles in vitro

Stronger red fluorescent could be observed with CLSM, demonstrating that more DiI-labeled nanoparticles adhered to the SKOV3 cells compared with the drug-loaded direct-targeting group and the other groups (Fig. 5B). Flow cytometry results showed that the fluorescence intensity of the drug-loaded pre-targeting group was significantly stronger than that for the other groups, and the difference was statistically significant (P < 0.05) (Figs. 5C–5F). The fluorescence intensity of the drug-loaded direct-targeting group was higher than that for the drug-loaded non-targeting group (P < 0.05).

The results of the CCK-8 assay showed that when incubated for 24 h, the cell survival rate for the free drug group was the lowest (P < 0.05) and the difference was statistically significant. The cell survival rate for the drug-loaded pre-targeting group was close to 40%, which was lower than that of the drug-loaded direct-targeting group and the other groups except for the free drug group. The difference was statistically significant (P < 0.05). At 48 h, the cell survival rate for the drug-loaded pre-targeting group decreased to about 5%, which was lower than that for the other groups except the free drug group, and the difference was statistically significant (P < 0.05). At both 24 h and 48 h, the cell survival rate for the free drug group was the lowest (P < 0.05) (Fig. 6).