Experimental Study Of Ultrasound Nanobubble Coupled With Anti-PSMA Nanobody In The Targeting Diagnosis Of Prostate Cancer

BackgroundProstate cancer has become one of the serious threats to the health of older men, andits detection rate also increased with the popularity of prostate-specific antigen (PSA)screening and continuously changing living habits of countrymen in our country. Earlydiagnosis of prostate cancer is important for patients’ prognosis. However, there are nothigh sensitivity and strong specificity for early diagnosis of prostate cancer in the currentimaging techniques. Molecular imaging, as an effective supplement to traditional imaging,can occur in functional changes before the discovery of the anatomical structure of lesions.Combined with convenience, quickness, real time, safety and no radiation of ultrasoundtechnology, contrast-enhanced ultrasound (CEUS) can improve the early diagnosis ofprostate cancer. Nanobubble, as a kind of ultrasound contrast agent (UCA), has a smallparticle size, good stability and strong ability to penetrate. And it can achieve substantialimaging of tumors via the enhanced permeability and retention effect (EPR), which meansthat the basement membrane is incomplete, tumorigenesis vascular endothelial gap iswidened and peripheral lymphatic drainage is poor. Additionally, prostate-specificmembrane antigen (PSMA) is the more sensitive marker than PSA. It is located in the cellmembrane and thus makes it an important target widely used in many studies of molecularimaging. Our group found that nanobubbles carrying anti-PSMA monoclonal antibody(mAb) could be used in the targeted imaging of prostate cancer, but we also found thatthese targeted nanobubble had the larger size, which may affect the performance ofnanobubbles’ penetration. So we want to seek a new substance targeted to PSMA.Nanobody, first found in the camel, is the variable region of heavy chain antibody in theabsence of CH1region and the light chain, and it has the characteristics of strong stability, asmall molecular weight and low immunogenicity and so on. Therefore, we expect that the nanobubble coupled with anti-PSMA nanobody can be used in molecular imaging ofprostate cancer through the EPR effect of tumor, and thus our study will provide alternativemeans for the diagnosis, monitoring treatment and prognosis evaluation of prostate cancer.ObjectiveThe anti-PSMA nanobody obtained from non-immune phage display library isbiotinylated and then is connected with biotinylated nanobubble through streptavidin-biotinmethod (SABC) to construct the targeted nanobubble. Through data analysis andmorphological observation, nanobubbles could enter into the tussue spaces. On this basis,the ability of nanobubbles to target prostate cancer cell and the imaging effect ofnanobubbles in xenografts of the prostate cancer could be explored.Methods1. The extracellular region of PSMA was reconstructed using the RT-PCR method inthe eukaryotic cells. Thereafter, four rounds of solid-phase panning yielded the phagespecific to the PSMA extracellular region in a non-immune nanobody phage display library.Both cell enzyme-linked immunosorbent assay (ELISA) and immunofluorescence methodwere used in vetifying the specificity of positive phage clone in the cell level. The regioncoding nanobody from the positive phage was intergrated in the prokaryote. And then thespecific nanobody was expressed and biotinylated. ELISA was used in comparing theaffinity with the biotinylated mAb.2. The nanobubbles were prepared and their physical properties were assayed. Theimaging effects (including the arrival time, the time to peak, the peak intensity, and theduration of contrast imaging) of the nanobubbles and microbubbles (SonoVue) on gastriccancer xenografts in nude mice and their in vitro imaging effects in the agarose gel modelwere analyzed via the L12-5transducer on the iu22system. After the exclusion of theimpact of the nanobubbles in the blood vessels through saline flush, the in vivo and in vitrodistributions of the nanobubbles in the tissue of the gastric cancer xenografts in nude micewere investigated using ultrasound burst and frozen sectioning. Finally, transmissionelectron microscopy was applied to verify the presence of nanobubbles in the tissue spacesof the transplanted tumor.3. Western blotting was used to detect PSMA expression in3cell lines (LNCaP, C4-2and MKN45). Nanobubbles were generated using the SABC method and validated via immunofluorescence evaluations. Nanobubble targeting of cells was investigated viamicroscopy and the results were expressed as the “nanobubble number/cell” and “adhesionrate”; the latter represented the percentage of cells with no less than4nanobubbles. Finally,ultrasound imaging was performed to evaluate nanobubble localization in3animalxenografts (LNCaP, C4-2and MKN45) before statistically analyzing the ultrasonographyindicators (arrival time, peak time, peak intensity and enhanced duration).Results1. The recombinant DNA sequence coding with extracellular domain of PSMA wascorrect in sequence and the western blot method proved the protein expression to besuccessful. The phage clones with binding activity has got effective enrichment by fourbiopannings, and the positive ratio was increased from8.3%to64.6%. The result of cellELISA showed that the absorption of LNCaP cells with the OD450value (0.62±0.04) wassignificantly higher than that of MKN45cells with the OD450value (0.15±0.01)(P <0.01),and immunoflurescence also showed that the phage clone from the above could bindspecifically to the PSMA-positive LNCaP cells. Using whole-cell ELISA, the nanobodyproduced by prokaryotes could bind specifically to the LNCaP cells after biotinylation,which was coincided with the biotinylated mAb in our prvious study.2. The particle size of the prepared nanobubbles was435.2±60.53nm, and theenhanced imaging effect of nanobubbles in vitro at the different concentrations wassignificantly stronger than that of SonoVue. Four indicators including the arrival time, thetime to peak, the peak intensity, and the duration of imaging obtained with the nanobubbleswere significantly higher than those obtained with the SonoVue microbubbles (every Pvalue less than0.01). After saline flush, the statistical analysis of the ultrasound signalintensities showed that the nanobubbles in80%(4/5) xenografted tumors could be detectedand the result obtained with the frozen sectioning technique revealed that the nanobubbleswere able to enter the tissue space. Furthermore, transmission electron microscopy alsoconfirmed that the nanobubbles could pass through the nascent vascular endothelial gap.3. The western blot results revealed that LNCaP cells had the highest PSMA levels,followed by C4-2cells while MKN45cells did not produce PSMA. The nanobubblesconstructed via the SABC method, had an average diameter with (487.60±33.55) nm,which was less than the average diameter with (644.30±55.85) nm of nanobubbles coupled with the mAb. And these nanobubbles could carry nanobody, as demonstrated byimmunofluorescence. A cell-binding experiment revealed that the adhesion rates of LNCaPcells and C4-2cells were more than90%. In contrast, no binding was detected on MKN45cells. In vivo ultrasound imaging of nude mouse xenografts revealed that in LNCaP andC4-2xenografts, targeted nanobubbles exhibited significantly different peak values andenhanced durations (P<0.01) from those of blank nanobubbles. A comparison of MKN45and C4-2xenografts revealed significant differences in all4indicators (P<0.05).Additionally, a comparison of LNCaP and C4-2xenografts showed that the arrival time andpeak value differed significantly (P<0.01).Conclusion1. The PSMA extracellular domain was successfully prepared, and its specificnanobody was obtaind from a non-immune phage display library. And the nanobody couldbind specifically to the LNCaP cells after biotinylation compared with the biotinylatedmAb.2. The nanobubbles exhibited a superior imaging effect compared with the SonoVue microbubbles in vitro and in vivo. In addition, the nanobubbles were able to pass throughthe tumor neovascular endothelial gap. These results provide a morphological basis for theextravascular ultrasound imaging of tumors and an experimental basis for the application oflipid nanobubbles in extravascular tumor-targeted ultrasound diagnostics and ultrasoundtherapy.3. The targeted anti-PSMA nanobody-loaded nanobubbles constructed in this studybound specifically to prostate cancer cells in vitro and produced distinctive images inprostate cancer xenografts with the characteristics of higher peak intensity and longerduration of imaging, which was accorded with nanobubbles coupled with anti-PSMA mAb.And this study provides the method for nanobody-coupled nanoscale ultrasound contrastagent in the ultrasound molecular imaging of tumors.