| BackgroundMalignant tumors are currently one of the most common diseases in human beings,which seriously threaten human life and health.The cancer incidence and number of cancer deaths are increasing every year.Early detection has an important impact on the therapeutic effect of malignant tumors.With the continuous development of imaging technology,imaging examination methods play an irreplaceable role in the diagnosis of malignant tumors,which can determine the location and size of malignant tumors in vivo.Ultrasound examination is one of the commonly used imaging methods in the clinical diagnosis of malignant tumors,which has the advantages of real-time dynamic imaging,safety,non-radioactivity and easy operation.However,conventional ultrasound examination can only detect the changes in anatomical structure and hemodynamics of malignant tumors,which cannot find the changes in molecular expression in malignant tumors at an early stage.Therefore,conventional ultrasound examination cannot really realize the early diagnosis of malignant tumors.Contrast-enhanced ultrasound technology is the third revolution in the field of ultrasound.After intravenous injection of ultrasound contrast agents(UCAs),the contrast of ultrasound imaging between malignant tumors and surrounding normal tissues is significantly enhanced,and the accuracy of tumor diagnosis is improved.However,UCAs currently used can only achieve intravascular ultrasound imaging of malignant tumors,and cannot achieve extravascular ultrasound imaging of malignant tumors.Because of the enhanced permeability and retention(EPR)effect of malignant tumors,nanoscale UCAs can accumulate in the extravascular space through tumor blood vessels,and achieve extravascular ultrasound imaging of malignant tumors.Nanobubble is a new type of nanoscale UCA.Specific ligands against extravascular target molecules of malignant tumors can be loaded on the surface of nanobubbles,which can further enh ance the aggregation and binding ability of nanobubbles in the extravascular space of tumor tissues,specifically enhance ultrasound imaging of malignant tumors,effectively monitor the changes in molecular expression in the extravascular space of tumor tissues in vivo,and thus improve the accuracy of early diagnosis and prognosis evaluation of malignant tumors.Carbonic anhydrase IX(CAIX)is highly expressed on the cell membranes of various tumors(such as lung cancer,kidney cancer,cervical cancer,breast cancer,colon cancer,liver cancer,head and neck tumors,and so on),which is an important protein molecule in the development of malignant tumors.Related studies have found that molecular probes for CAIX can achieve molecular imaging of malignant tumors,and inhibition of CAIX activity can achieve targeted therapy of malignant tumors.In our previous studies,anti-CAIX monoclonal antibody and nanobody were loaded on the surface of nanobubbles to construct targeted nanobubbles,which could be used for ultrasound molecular imaging(USMI)of kidney cancer.Compared with blank nanobubbles,targeted nanobubbles could specifically bind to 786-O cells in vitro and had higher enhanced ultrasound imaging intensity in transplanted tumor model.However,our previous research was limited to USMI of kidney cancer and only focused on the enhanced ultrasound imaging effect of targeted nanobubbles in transplanted tumor tissues.The characteristics and morphological basis of USMI with targeted nanobubbles in various tumor tissues have not been studied.Polypeptides are amino acid sequences composed of α-amino acids linked by peptide bonds.Because of their small molecular weight,simple chemical structure,wide distribution and low immunogenicity,polypeptides have been widely used in molecular diagnosis and drug treatment.The polypeptide PGLR-P1 is a specific ligand for CAIX proteoglycan region.Therefore,targeted nanobubbles constructed by coupling PGLR-P1 to lipid nanobubbles can achieve USMI in malignant tumors from various organs and monitor the expression of CAIX in tumor tissues,which is beneficial to improve the accuracy of early diagnosis of various malignant tumors.Relevant studies have shown that targeted UCAs carrying polypeptide can cause an immune response after repeated injection,so their clinical application is limited.Aptamers consist of RNA or single-stranded DNA sequences that can specifically bind to cells and biological macromolecules.Aptamers have the advantages of diverse structure,wide range of application,easy modification and no immunogenicity,which have played an important role in molecular recognition,experimental diagnosis and disease treatment.Therefore,the construction of aptamer-mediated targeted UCAs with small particle size,high safety and strong specificity,and the effect and morphological basis of USMI in vivo still need to be further explored.Based on the above research progress,we carried out experimental studies on USMI of targeted nanobubbles for CAIX in malignant tumors from two parts.Firstly,targeted nanobubbles carrying CAIX polypeptide were constructed,and their binding ability to CAIX-positive tumor cells in vitro and their enhanced ultrasound imaging effect in CAIX-positive transplanted tumor tissues were investigated.Secondly,in view of the shortcomings of targeted nanobubbles carrying CAIX polypeptide,aptamer-mediated targeted nanobubbles were constructed by coupling CAIX aptamer on the surface of nanobubbles,and their ability to enhance ultrasound imaging in transplanted tumor tissues and the morphological basis of USMI were investigated.Objectives1.The ability of targeted nanobubbles carrying CAIX polypeptide to specifically bind to CAIX-positive tumor cells in vitro and to enhance ultrasound imaging of CAIX-positive transplanted tumors in vivo were studied,and the reason for their distribution differences in transplanted tumor tissues was explored.Therefore,this study provides a new type of targeted UCAs with small size and good enhanced ultrasound imaging effect for USMI of various malignant tumors,and also offers the research methods for targeted UCAs to achieve USMI of tumor parenchymal cells from various organs.2.In view of the shortcomings of targeted nanobubbles carrying CAIX polypeptide,the specific binding ability of targeted lipid nanobubbles coupled with CAIX aptamer to tumor cells and the effect in contrast-enhanced ultrasound imaging were evaluated.The morphological basis of enhanced ultrasound imaging with targeted nanobubbles in vivo was demonstrated.Therefore,this study not only offers a molecular probe with high safety and specificity for USMI of malignant tumors,but also lays the research foundation for targeted nanobubbles in the diagnosis of malignant tumors.Methods1.Enhanced ultrasound imaging of malignant tumors with targeted nanobubbles carrying CAIX polypeptide(1)The polypeptide PGLR-P1 was synthesized by chemical solid-phase synthesis method.Lipid nanobubbles were prepared by mechanical oscillation method,and CAIX polypeptide was coupled to the surface of lipid nanobubbles by biotin-avidin method.Fluorescence labeling was used to observe the distribution of CAIX polypeptide on the surface of targeted nanobubbles,and the particle size,potential,morphology,distribution,cytotoxicity and stability of targeted nanobubbles were evaluated.(2)Optical microscopy and flow cytometry were used to measure the ability of targeted nanobubbles to specifically bind to tumor cells in vitro.The enhanced ultrasound imaging effects and decay rates of targeted and blank nanobubbles in vitro were examined.The change in enhanced ultrasound imaging intensity of targeted nanobubbles after ultrasound irradiation with high mechanical index was also analyzed.(3)786-O,HeLa and BxPC-3 transplanted tumor models in nude mice were established.The enhanced ultrasound imaging effects of targeted and blank nanobubbles in transplanted tumor tissues were examined,and their enhanced ultrasound imaging effects in liver and kidney tissues were analyzed.(4)Immunofluorescence technique was used to observe the distribution of targeted nanobubbles in the intravascular and extravascular space of transplanted tumor tis sues and muscle tissues.Immunohistochemical staining was used to detect the expression of CAIX in transplanted tumor tissues,and the reason for the distribution differences of targeted nanobubbles in transplanted tumor tissues was investigated.2.CAIX aptamer-functionalized targeted nanobubbles for USMI of malignant tumors(1)A single-stranded DNA strand against CAIX protein was screened by the systematic evolution of ligands by exponential enrichment in vitro,and the sequence of CAIX aptamer was analyzed by high-throughput sequencing.The specific binding ability of CAIX aptamer at the cellular level in vitro was evaluated by immunofluorescence technique and flow cytometry.(2)On the basis of lipid nanobubbles,targeted nanobubbles coupled with CAIX aptamer were prepared by a maleimide-thiol coupling reaction.The ability of targeted nanobubbles to couple CAIX aptamer was determined by fluorescence labeling,and the basic characteristics and cytotoxicity of targeted nanobubbles were evaluated.The enhanced ultrasound imaging effects of targeted and non-targeted nanobubbles in vitro were compared.(3)The ability of targeted and non-targeted nanobubbles to bind to tumor cells was observed under confocal laser scanning microscopy.The contrast-enhanced ultrasound imaging effects of targeted and non-targeted nanobubbles were compared in tumor-bearing nude mice.Fluorescence imaging of live small animals was used to further evaluate the aggregation ability of targeted nanobubbles in transplanted tumor tissues.(4)The distribution of targeted nanobubbles in the intravascular and extravascular space of transplanted tumor tissues,and the ability to couple CAIX aptamer and to bind to tumor cells in transplanted tumor tissues were studied on frozen sections.The histological characteristics of transplanted tumor tissues were observed by H&E staining.Western Blot(WB)was used to measure the expression of CAIX in transplanted tumor tissues,and the morphological basis for targeted nanobubbles to achieve USMI in malignant tumors was demonstrated.Results1.Enhanced ultrasound imaging of malignant tumors with targeted nanobubbles carrying CAIX polypeptide(1)The polypeptide PGLR-P1 was successfully synthesized.CAIX polypeptide was evenly loaded on the surface of targeted nanobubbles.The particle size of targeted nanobubbles was 503.7±78.5 nm,which had the advantages of uniform distribution,regular morphology,high stability and low cytotoxicity.(2)Targeted nanobubbles could specifically bind to 786-O and HeLa cells,rather than BxPC-3 cells,and blank nanobubbles could not bind to the three types of tumor cells in vitro.The enhanced ultrasound imaging effects and decay rates were not significantly different between targeted and blank nanobubbles in vitro(P>0.05),and the enhanced ultrasound imaging intensity of targeted nanobubbles significantly decreased after ultrasound irradiation with high mechanical index(P<0.05).(3)There was no significant difference in peak time,peak intensity and duration time between targeted and blank nanobubbles in liver and kidney tissues of nude mice(P>0.05).The enhanced ultrasound imaging effect of targeted nanobubbles was significantly different from that of blank nanobubbles in 786-O and HeLa transplanted tumor tissues,which had higher peak intensity,longer duration time and larger area under the curve(AUC)(P<0.05).However,the enhanced ultrasound imaging effects of targeted and blank nanobubbles in BxPC-3 transplanted tumor tissues were not significantly different(P>0.05).(4)After intravenous injection,targeted nanobubbles could be distributed in the intravascular and extravascular space of transplanted tumor tissues,and the number of targeted nanobubbles in 786-O and HeLa transplanted tumor tissues was significantly more than that in BxPC-3 transplanted tumor tissues.However,targeted nanobubbles only could be distributed in blood vessels of muscle tissues.Immunohistochemical staining showed that CAIX was expressed in 786-O and HeLa transplanted tumor tissues,but not in BxPC-3 transplanted tumor tissues.Therefore,the differences in CAIX expression led to the distribution differences of targeted nanobubbles in transplanted tumor tissues.2.CAIX aptamer-functionalized targeted nanobubbles for USMI of malignant tumors(1)A single-stranded DNA strand against CAIX protein was obtained.Immunofluorescence technique and flow cytometry found that CAIX aptamer had good binding ability to 786-O and HeLa cells,but had no binding ability to BxPC-3 cells,and nonsense aptamer of the same sequence length had no binding ability to the three types of tumor cells.(2)The surface of targeted nanobubbles was loaded with CAIX aptamer uniformly,which had small particle size,uniform distribution,similar size and high safety.There was no significant difference in the enhanced ultrasound imaging effects between targeted and non-targeted nanobubbles in vitro(P>0.05).(3)Targeted nanobubbles could specifically aggregate around 786-O and HeLa cells,but not around BxPC-3 cells,and non-targeted nanobubbles could not aggregate around the three types of tumor cells in vitro.Compared with non-targeted nanobubbles,enhanced ultrasound imaging of targeted nanobubbles in 786-O and HeLa transplanted tumor tissues had higher peak intensity and larger AUC(P<0.05),while there was no significant difference in BxPC-3 transplanted tumor tissues(P>0.05).Fluorescence imaging of live small animals showed that targeted nanobubbles could specifically aggregate in 786-O and HeLa transplanted tumor tissues.(4)Confocal laser scanning microscopy found that targeted nanobubbles distributed in the extravascular space of transplanted tumor tissues could still load CAIX aptamer,and the number of targeted nanobubbles in 786-O and HeLa transplanted tumor tissues was significantly more than that of non-targeted nanobubbles,while the number of targeted and non-targeted nanobubbles was not significantly different in BxPC-3 transplanted tumor tissues.WB showed that CAIX was highly expressed in 786-O and HeLa transplanted tumor tissues,but not in BxPC-3 transplanted tumor tissues.Therefore,targeted nanobubbles could specifically enhance ultrasound imaging of transplanted tumors under the action of CAIX aptamer.Conclusions1.Targeted nanobubbles carrying CAIX polypeptide could specifically accumulate around CAIX-positive tumor cells in vitro and enhance ultrasound imaging of CAIX-positive transplanted tumors in vivo,which provides a novel targeted UCA for USMI of tumor parenchymal cells from various organs.2.Targeted nanobubbles coupled with CAIX aptamer had the ability to specifically bind to various tumor cells in vitro,and had a good imaging effect in USMI of tumor parenchymal cells,which offers the experimental methods and research basis for aptamer-mediated targeted UCAs to enhance ultrasound imaging of malignant tumors.3.After intravenous injection,targeted nanobubbles can diffuse into the extravascular space of transplanted tumor tissues through the EPR effect of malignant tumors,actively bind to tumor cells express target molecule under the action of specific ligands on their surface,significantly enhance ultrasound imaging of malignant tumors,and thus achieve USMI of tumor parenchymal cells. |
