CDCP1-targeted nanoparticles encapsulating phase-shift perfluorohexan for molecular US imaging in vitro

Abstract

BACKGROUND:

Molecular targeted contrast-enhanced ultrasound (CEUS) imaging is a potential imaging strategy to improve the diagnostic accuracy of conventional ultrasound (US) imaging. US contrast agents are usually micrometer-sized and non-target gas bubbles while nano-sized and targeted agents containing phase-shift materials absorb more attractions for their size and the liquid core and excellent molecular imaging effect.

METHODS:

PLGA_12k-mPEG_2k-NH₂, DSPE-mPEG_2k and perfluorohexan (PFH) were used to construct a new targeted ultrasound contrast agent with CUB domain-containing protein 1 (CDCP1) receptor for the detection and diagnosis of prostate cancer. The potential of tumor-targeted nanoparticles (CDCP1-targeted perfluorohexan-loaded phase-transitional nanoparticles, anti-CDCP1 NPs) as contrast agents for ultrasound (US) imaging was assessed in vitro. Moreover, studies on the cytotoxicity and the targeting ability of anti-CDCP1 NPs assisted by US were carried out.

RESULTS:

The results showed that anti-CDCP1 NPs had low cytotoxicity, and with the increasing of polymer concentration in anti-CDCP1 NPs, the CEUS imaging of agent gradually enhanced, and enhanced imaging associated with the length of observing time. Furthermore, it was testified that anti-CDCP1 assisted the agent to target cells expressing CDCP1, which demonstrated the active targeting of anti-CDCP1 NPs in vitro.

CONCLUSION:

All in all, the feasibility of using targeted anti-CDCP1 NPs to enhance ultrasound imaging has been demonstrated in vitro, which laid a solid foundation for molecular US imaging in vivo, and anti-CDCP1 NPs might have a great clinical application prospect.

Keywords

Targeted nanoparticle contrast agent prostate cancer

1 Introduction

Ultrasound (US) imaging technology is widely used in clinical diagnosis and cancer treatment monitoring due to its non-invasive, radiation-free and low-cost characteristics [1]. With the appearance and development of ultrasonic molecular imaging, the specificity and sensitivity of ultrasonic imaging for the diagnosis of various diseases have been greatly improved [2, 3]. The basic technique of ultrasonic molecular imaging is contrast agent [4]. Ultrasonic nano-bubble is an acoustic contrast agent with particle size less than 700 nm [5], which can break through the limitation of traditional micron-scale ultrasonic contrast agent pure blood pool imaging, and can be used for external vascular imaging [6, 7]. And through carrying on the nanometer vesicles in view of the specific ligand lesions, especially for tumor cell specificity ligand, able to take advantage of the tumor endothelial cell gap is larger, the lack of basement membrane and lymphatic loop undesirable penetration and the enhanced effect of retention gap into the organization [8 –10] to further combine with tumor cells produce specificity of ultrasound enhanced signal, from the molecular level of tumor diagnosis [11]. Liquid fluorocarbons undergo liquid-gas phase transition in response to external stimuli [12 –14]. The droplet vaporization (ADV) effect can occur in low-intensity focused ultrasound (LIFU) to form ultrasonic nanobubbles, which can achieve the targeted transport positioning and imaging of contrast agents [15 –17]. Nowadays, many investigations about the theranostic platform of nanobubbles have been focused on prostate cancer, which is one of the most common cancers in men. Recent estimates suggest that over a million men are diagnosed with the disease annually [18, 19]. Prostate cancer pathogenesis involves both heritable and environmental factors. The molecular events involved in the development or progression of prostate cancer are still unclear. The application of nanobubbles is hampered for lacking specific target in prostate cancer. Thankfully, a recent research proposed that CUB domain-containing protein 1 (CDCP1) is overexpressed in metastatic prostate cancer, which enables it to be the targeted cell surface glycoproteins for prostate cancer specific analysis [20, 21]. Anti-CDCP1 immunoliposomes (anti–CDCP1 ILs) loaded with chemotherapy suppress prostate cancer growth when administered in combination with enzalutamide(Abdullah Alajati et al. 2020) [22]. The CDCP1 protein is a type I transmembrane glycoprotein containing three CUB domains and multiple glycosylation sites. On the cell surface, CDCP1 can be presented in two forms, the full-length 135 kDa form and the truncated 70 kDa form [23 –25]. CDCP1 is expressed by stem cells or progenitor cells in hematopoietic, mesenchymal, and neural tissues [26, 27] and has been associated with colon [28], breast [29], prostate, and kidney cancers [30]. Therefore, the enhanced expression of CDCP1 in multiple cancer types make it a potential imaging and therapeutic target.

In this study, we developed nanoparticles loaded with liquid fluorocarbon perfluorohexan (PFH) (anti-CDCP1 NPs) for ultrasound imaging [31]. Non-target nanoparticles (NPs) were synthesized by PLGA_12k-mPEG_2k-NH₂, DSPE-mPEG₂₀₀₀ and PFH, and then NPs were further combined with anti-CDCP1 antibodies to form anti-CDCP1 NPs. The characterization and ultrasound imaging of anti-CDCP1 NPs was assessed, and the cytotoxicity and targeting of anti-CDCP1 NPs were also measured.

2 Methods

2.1 Materials

Poly(lactide-co-glycolide_12k) (polyethylene glycol _2k) Amine (PLGA_12k-PEG_2k-NH₂, M_W:14000) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-mPEG_2k) was purchased from Pengshuo Biological Technology, and anti-CDCP1 was from Boster Biological Technology. Chloroform was purchased from Aladdin Industrial Corporation. N-(3-dimethylaminopropyl)-N^′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and perfluorohexane (PFH) were obtained from Sigma-Aldrich (St. Louis, MO). Ham’s F-12K (Kaighn’s) Medium, fetal bovine serum (FBS), penicillin-Streptomycin and phosphate buffered saline (PBS) were acquired from Gibco Life Technologies, Inc. (Carlsbad, CA, USA).

2.2 Preparation of anti-CDCP1 NPs

NPs were produced by an emulsion/solvent evaporation approach [32, 33]. Briefly, 18 mg PLGA_12k-PEG_2k-NH₂ and 12 mg DSPE-mPEG_2k were dissolved in 0.6 mL chloroform and 15μL PFH was added in the solution to form lipophilic phase. Then, the primary emulsion was transferred into 4 mL external aqueous phase of 0.1% Poly (vinyl alcohol) (PVA). The mixture was sonicated on ice (5 minutes, 550 W). At last, the chloroform was removed by evaporation overnight.

The N-hydroxysulfosuccinimide sodium salt (NHS) and N-(3-(dimethylamino)propyl-N^′-ethyl-carbodiimide hydrochloride (EDC) were used to activate the carboxyl groups of Anti-CDCP1 antibodies (80μL, 200μg mL^–¹) at room temperature [34, 35]. Two hours later, NPs were added in the above solution. After the solution stirred for 4 h, anti-CDCP1 NPs which were formed by anti-CDCP1 antibodies connecting to NPs were acquired by centrifugation and washing.

2.3 Characterization of anti-CDCP1 NPs

The size distribution, zeta potential, and polydispersity index (PDI) of anti-CDCP1 NPs were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The morphology of anti-CDCP1 NPs was assessed with transmission electron microscopy (TEM, JEOL 1200EX).

2.4 Ultrasound imaging in vitro

In vitro US imaging of anti-CDCP1 NPs using an Acuson S2000 Ultrasound System unit with a 9 L4 transducer (Siemens Healthcare, Shanghai, China). In order to study the effect of the concentration on in vitro ultrasound imaging, anti-CDCP1 NPs was respectively diluted to 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 4.0 mg/mL and 8.0 mg/mL, with degassed saline as negative control, and then inject the 1 ml samples into rubber gloves. Part of the rubber gloves was embedded in the coupling agent, and the ultrasonic probe was placed on the coupling agent to start ultrasound and adjust ultrasonic parameters in time. The ultrasonic images were recorded. The normal saline group was used as the negative control, and the experiment was repeated for 3 times. The US imaging was performed using the transducer in both contrast-enhanced ultrasound (CEUS) mode (mechanical index, MI = 0.065) and conventional B-mode at the same time.

And then exploring the duration of in vitro ultrasound imaging, anti-CDCP1 NPs was diluted to 1.5 mg/mL with normal saline after degassing, and the other conditions were the same as above. Part of the rubber gloves was embedded in the coupling agent and the ultrasonic probe was placed in the coupling agent. The ultrasonic parameters were adjusted and the ultrasonic signal continued to be weak until it was completely absent. Ultrasound images were recorded continuously. The normal saline group was used as the negative control, and the experiment was repeated for 3 times.

2.5 Cell culture

Prostate adenocarcinoma PC-3 and prostate carcinoma brain metastasis DU145 cell lines were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China). The cells were cultured in Ham’s F-12K (Kaighn’s) Medium supplemented with 10% FBS (v/v) and 5% penicillin-Streptomycin (v/v) at 37°C with 5% CO₂ in a humidified incubator.

2.6 Cytotoxicity of anti-CDCP1 NPs

PC-3 and DU-145 were inoculated with cell density of 5×10³ cells/well in 96-well cell culture plates and incubated overnight. After medium was discarded, anti-CDCP1 NPs of different polymer concentrations diluted with 100μL medium were added and incubated for 24 h, 48 h and 72 h. Culture medium was discarded and 0.5 mg /mL MTT/DMEM without serum was added to 100μL in each well. After 4 h, the 96-well plate was gently inverted onto the absorbent paper to prevent the generated formazan from falling off as much as possible, and 150μL DMSO was added. Shaker 60 r/min, 15 min, ensured uniform purple solution in the hole. Optical density (OD) value at 490 nm wavelength was determined by microplate.

2.7 Western blot

Western blot was used to quantitatively determine the expression of protein CDCP1 in different cells. Firstly, PC-3 and DU145 were seeded into 6-well plates for 24 h and then washed with PBS and lysed in RIPA buffer to harvest proteins. After the protein concentration was determined by a BCA protein assay kit, equal protein from each sample was mixed with a Laemmli sample buffer, loaded, and separated by sodium dodecyl dodecyl-sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on a 8% (v/v) resolving gel. The protein was transferred to the PVDF membrane (bio-rad Laboratories). The imprinting was first incubated at room temperature for 1 hour in a TBS blocking buffer containing 2% milk, and then incubated overnight at 4°C with a primary antibody. The imprints were then washed and incubated in TBST with the appropriate secondary antibody. The blots were obtained by an enhanced chemiluminescence detection system.

2.8 In vitro cell targeting

The intracellular uptake to anti-CDCP1 NPs was observed by confocal laser scanning microscope (CLSM). Cy5-labelling NPs (Cy5 NPs) and Cy5–labelling. Anti-CDCP1 NPs (Cy5 anti-CDCP1 NPs) were prepared. PC-3 and DU145 were inoculated with cell density of 1×10⁴ cells/well on 24-well cell culture plate. After the cells attached to the slides, the culture medium was discarded, and Cy5 NPs and Cy5 anti-CDCP1 NPs (Cy5 20 ug/mL) diluted in medium were added to the wells for further incubation for 2 h, 4 h or 8 h. Next the medium was discarded, and the cells were washed with PBS for 2–3 times. Then 4% paraformaldehyde was added and incubated at 37°C for 10 min to fix cells. After the removal of paraformaldehyde, the cells were washed with PBS for 2–3 times [36, 37]. After incubated with 5 g/mL Hoechst 33342 for 10 min at 37°C, the cell slides were taken out and sectioning, and observed with a CLSM.

2.9 Flow cytometry

Flow cytometry was used to determine the binding rate of antibodies to cells. FITC labelling anti-CDCP1 NPs (FITC anti-CDCP1-NPs) were prepared. PC-3 and DU145 were seeded into 6-well plates at a density of 5×10⁵ cells/well for 24 h. The culture medium was removed, and FITC anti-CDCP1 NPs were added to each well and were incubated for 2 h, 4 h and 8 h at 37°C. After incubation, all reagents were removed, and cells were washed with PBS and collected by trypsin. PBS was added to resuspend the cell pellet. Flow cytometry was performed with a BD FACSCanto II flow cytometer (BD Biosciences). Analysis of flow cytometry data was performed by FlowJo.

2.10 Statistical analysis

All data were presented as mean±standard deviation. Statistical analyses were performed using Student’s t-test. *P<0.05, **P<0.01 were considered statistically significant.

3 Results

3.1 Characterization of anti-CDCP1 NPs

We obtained the anti-CDCP1 NPs via ultrasonic emulsification method as shown in Scheme 1. The mean size of anti-CDCP1 NPs was 172±5.76 nm, as determined by dynamic light scattering, and PDI was 0.19 showing the uniform size distribution (Fig. 2A). The surface charge of the anti-CDCP1 NPs in aqueous solution was –0.041 mV (Fig. 2B).

Scheme. 1

Preparation of anti-CDCP1 NPs. (1–1) PLGA_12k-PEG_2k-NH₂ and DSPE-mPEG_2k were dissolved in chloroform and PFH was added in the solution to form lipophilic phase; (1–2) The primary emulsion was transferred into external aqueous phase of 0.1% (PVA), and the mixture was sonicated on ice (5 minutes, 550 W); (1–3) The NHS and EDC were used to activate the carboxyl groups of anti-CDCP1 antibodies at room temperature. (1–4) The encapsulated PFH undergo phase transition when applied for ultrasound imaging.

Fig. 2

Characterization of anti-CDCP1 NPs. (A) Size distribution and the transmission electron microscopy (TEM) image of anti-CDCP1 NPs; (B) Zeta potential of anti-CDCP1 NPs.

3.2 Ultrasound imaging in vitro

Under two-dimensional ultrasound mode, the imaging of anti-CDCP1 NPs presented high echo and uniform spot, and obvious enhancement under contrast mode. Then, the relationship between ultrasound imaging effect and polymer concentration in anti-CDCP1 NPs was observed (Fig. 3A). The statistical results of gray value showed that when the concentration of anti-CDCP1 NPs was 0.5 1.0 2.0 4.0 and 8.0 mg/mL, the average gray value under the contrast mode was 53.90±0.33, 65.53±1.26, 83.19±2.28, 106.18±4.98 and 127.56±4.62, respectively. Then with time going, the average gray value in the 10-minute contrast mode was 117.76±5.17, the average gray value in the 20-minute contrast mode was 48.37±5.16, and the average gray value in the 30-minute contrast mode was 37.29±3.78.

Fig. 3

US imaging in vitro. (A) (1–6) US imaging of saline (a) and different concentrations of anti-CDCP1 NPs (b-f separately represent 0.5, 1.0, 2.0, 4.0, 8.0 mg/mL). (B) (1–6) US imaging of saline (g) and 2.0 mg/mL anti-CDCP1 NPs after different US exposure time (h-l separately represent US exposing for 1, 5, 10, 20 min and 30 min). Image of each group had B-mode (left) and CEUS (right). (C, D) Grayscale values of the anti-CDCP1 NPs changed along with the changes in concentration and US exposure time. The data are presented as the mean±SD of three independent experiments.

3.3 Cytotoxicity assay

MTT assay was performed to assess the cytotoxicity of anti-CDCP1 NPs. DU145 and PC-3 were treated with different polymer concentrations of anti-CDCP1 NPs and no significant cytotoxicity was observed (Fig. 4).

Fig. 4

Cell viability of DU145 and PC-3 with the treatment of various concentrations of anti-CDCP1 NPs for 24 h, 48 h and 72 h.

3.4 Targeting ability of anti-CDCP1 NPs in vitro

As shown in the Fig. 5, we can clearly see that CDCP1 protein is highly expressed in PC-3 cells and low in DU145 cells. Then, NPs and anti-CDCP1 NPs were respectively co-incubated with DU145 and PC-3 for 2 h, 4 h and 8 h, and fluorescence intensity in cells was observed under a CLSM (Fig. 6A, 6B).The results showed that after 2 h, the fluorescence intensity of the anti-CDCP1 NPs group in PC-3 cells expressing CDCP1 was obviously higher than that of the NPs group, which proved the antibody anti-CDCP1 promoted the uptake to anti-CDCP1 NPs of PC-3, while the red fluorescence intensity of all groups in DU145 cells lack of CDCP1 was weak, which manifested the targeting of anti-CDCP1 from the opposite angle. After 4 h and 8 h, the fluorescence intensity of all groups in both types of cells increased, and the fluorescence intensity of the anti-CDCP1 NPs group in PC-3 was significantly higher than that of NPs group, while there was no significant difference between the NPs group and the anti-CDCP1 NPs group in DU145 cells.

Fig. 5

(A,B) in vitro confocal laser scanning microscopy (CLSM) images of free cy5, cy5 NPs and cy5 anti-CDCP1 NPs (20μg/mL cy5) uptake in PC-3 and DU145 cells. Scale bar 20μm; (C,D) Flow cytometric analysis of PC-3 and DU145 cells treated with anti-CDCP1 NPs (20μg/mL FITC) for 8 h, Scale bar 20μm; (E) Western Blot analyses of CDCP1 expression in PC-3 and DU145 cells.

And the flow cytometry’s results were consistent with the uptake results measured by laser confocal (Fig. 6C, 6D). After 8 h, compared with the free cy5 and cy5 NPs groups, the fluorescence signal of anti-CDCP1 NPs group was stronger, indicating that the uptake of nanoparticles was significantly increased. Meanwhile, the fluorescence intensity of DU145 cells in free cy5 and cy5 NPs groups was not significantly different, and was close to that of PC-3 cells in cy5 NPs group, while that of PC-3 cells in anti-CDCP1 NPs group was significantly higher than that of cy5 NPs group.

4 Discussion

NPs were produced by an emulsion/solvent evaporation approach. Anti-CDCP1 NPs were composed of a perfluorohexane (PFH) liquid core, a hybrid lipid-polymer shell with PLGA_12K-PEG_2K-NH₂ and DSPE-PEG_2K, and an active targeting ligand, the Anti-CDCP1 peptide. The zeta potential made it likely for anti-CDCP1 NPs to circulate longer in blood containing negative charge proteins. And the transmission electron microscopy (TEM) images of the nanoparticles showed a uniform size distribution.

Ultrasound imaging results in vitro (Fig. 3) showed that, the CDCP1-targeted nano-phase-changed ultrasound contrast agent had excellent contrast-enhanced ultrasound (CEUS) imaging when compared with the imaging of saline. As the polymer concentration of anti-CDCP1 NPs increased, the echo intensity of the sample images gradually increased, which meant the contrast effect of anti-CDCP1 NPs gradually increased in contrast mode. And when time goes by, CEUS imaging of anti-CDCP1 NPs gradually enhanced and reached peak at 10 min and then became weaker (Figure 3B). All these results showed that CDCD1-targeted nano-phase-changed ultrasound contrast agent had good CEUS imaging effect and long lasting imaging time in vitro.

MTT experiments indicated that anti-CDCP1 nanoparticles have no significant toxicity to cells, which suggesting a better biocompatibility and laying the foundation for subsequent applications of anti-CDCP1 nanoparticles.

We measured the in vitro internalization of anti-CDCP1 NPs in DU145 and PC-3 cell lines which expression of CDCP1 level are different. These phenomena suggested that the anti-CDCP1 on the surface of anti-CDCP1 NPs could recognize and bind CDCP1 highly expressing on the surface of PC-3 cells, thus promoting the uptake to anti-CDCP1 NPs of PC-3 cells through receptor-mediated endocytosis, so it would be possible for anti-CDCP1 NPs to actively target tumor site expressing CDCP1 for targeted molecular US imaging. And the flow cytometry’s results showed the similar trends. The results mentioned above demonstrated the successful construction of anti-CDCP1 nanoparticles for specific human prostate cancer theranostic.

5 Conclusions

In accordance with the requirements of ultrasound for tumor molecular imaging, we successfully prepared a targeted nano-ultrasound contrast agent with good biocompatibility, and investigated its tumor targeting and imaging ability in vitro. The PFH-contained nanoparticles were prepared by ultrasonic emulsification method and connected with CDCP1 to enhance their targeting. The results of US imaging showed anti-CDCP1 NPs had excellent and lasting contrast-enhanced ultrasound imaging effect, and further investigation of targeting of anti-CDCP1 NPs in vitro manifested that anti-CDCP1 NPs owned good tumor targeting. Considering the good and lasting CEUS imaging of anti-CDCP1 NPs and their targeting, further studies will focus on the biocompatibility of anti-CDCP1 NPs in vivo and their potential for ultrasound imaging in tumor-bearing animal models.

Footnotes

Acknowledgment

This research was supported by the National Natural Science Foundation of China (No.81671708, No. 81773272, No.81771839 and No. 81972886), the State Key Laboratory of Oncogenes and Related Genes (No. ZZ2012RCPY).

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