Construction Of A Nucleolin-targeted Ultrasound Nanoprobe And Its Diagnostic And Therapeutic Efficacy In Triple-negative Breast Cancer

BackgroundFor refractory triple-negative breast cancer(TNBC),there is an urgent need for new and effective ways to solve the challenges of early accurate diagnosis,targeted synergistic treatment,and visualization of efficacy evaluation.Finding effective new targets is the key to breaking through the bottleneck of clinical diagnosis and treatment for TNBC.The latest research has shown that nucleolin(NCL)overexpressed on the surface of tumor cell membranes and tumor neovascular endothelial cell membranes is expected to become a new target for accurate early diagnosis and effective treatment of TNBC.The nucleic acid aptamer AS1411 has attracted extensive attention because of its ability to bind with NCL specifically and to inhibit tumor growth.NCL is an important intermediate link in the process of tumorigenesis and development.The series of diagnosis and treatment strategies for NCL is expected to achieve precise diagnosis and effective treatment of malignant tumors.Compared with other NCL ligands,such as peptides,pseudo-peptides,and immunoagents,nucleic acid aptamers have no immunogenicity.AS1411 can specifically bind to and interact with NCL overexpressed on the surface of tumor cell membranes and tumor neovascular endothelial cell membranes with high affinity.Aptamer AS1411 can inhibit the function of NCL through targeted binding,thus blocking tumor progression involving NCL.At the same time,the interaction between aptamer AS1411 and NCL can stimulate and activate the endocytosis of cells,promote the uptake of therapeutic drugs,and inhibit extravasation and clearance,thereby achieving targeted drug delivery and synergistic treatment.In addition,precise molecular diagnosis based on the NCL-aptamer AS1411 interaction and targeted drug delivery systems are also important research directions.Although some progress has been made in research on NCL and its ligands,there are still some problems;for example,the mechanism of action of NCL is unclear,aptamers have low bioavailability in vivo,and poor effects of aptamers.Therefore,further in-depth research and system optimization are needed before the aptamer AS1411 can be applied in the clinical diagnosis and treatment of TNBC.Ultrasound molecular imaging(USMI)technology can play an important role in the diagnosis and treatment of TNBC.Ultrasound molecular probes are the core of USMI;they can not only achieve specific USMI but also serve as carriers in drug delivery systems.Targeted drug release can also be controlled by ultrasound-targeted microbubble destruction(UTMD)to effectively reduce a drug's toxicity and side effects;that is,it plays therapeutic and diagnostic roles simultaneously and realizes the integration of diagnosis and treatment.Based on this,USMI technology can be used as a key technical way to solve the abovementioned problems using NCL,as a potential high-efficiency target,and its nucleic acid aptamer AS1411 to construct an integrated ultrasound molecular probe for malignant tumor diagnosis and treatment.Through the in-depth study of its USMI characteristics and synergistic treatment mechanism,we can achieve a breakthrough in the clinical diagnosis and treatment of TNBC.Based on previous research,this study aims to design and construct a targeted ultrasound nanoprobe,an aptamer AS1411-functionalized NCL-targeted lipid nanobubble,for TNBC diagnosis and treatment.The specific contrast-enhanced USMI capabilities of the constructed nanobubbles were tested.Then,the therapeutic efficacy of the nanobubbles as anti-tumor drug doxorubicin delivery carriers was evaluated.The purpose of this experiment was to provide a new method for early and accurate diagnosis,effective treatment,and visualization of efficacy evaluation of TNBC.Objectives1.Using NCL over-expressed on the surface of tumor cell membranes and tumor neovascular endothelial cell membranes as a target,construct aptamer AS1411-functionalized NCL-targeted lipid nanobubbles(AS1411-NBs).Systematically evaluate the dual-targeted,contrast-enhanced ultrasound imaging capabilities of AS1411-NBs on tumor tissues and tumor neovascularization.To explore a new approach to early accurate diagnosis and visualization of efficacy evaluation of refractory TNBC.2.Based on previous studies,we focused on the application value of AS1411-NBs in drug delivery systems.NCL-targeted doxorubicin-loaded lipid nanobubbles(AS1411-DOX-NBs)were constructed.Their anti-tumor efficacy and cardioprotective effects were objectively evaluated.The aim was to provide a safe and efficient theranostic ultrasound nanoprobe.Methods1.The construction of a targeted ultrasound nanoprobe and its contrast-enhanced USMI in TNBC.(1)First,the lipid molecule DSPE-PEG2000-COOH and the nucleic acid aptamer AS1411 were covalently linked by an amide condensation reaction to synthesize the lipid–nucleic acid targeted molecule DSPE-PEG2000-AS1411.Combined with other lipid molecules,AS1411-NBs were constructed by membrane hydration,mechanical shaking,and centrifugal floating methods.Denaturing urea polyacrylamide gel electrophoresis and laser scanning confocal microscopy were used to identify whether the targeted molecules and targeted lipid nanobubbles were successfully constructed.We observed and measured the morphologies,particle sizes,and zeta potentials of AS1411-NBs using a light microscope,a transmission electron microscope,and a Malvern nanoparticle size and potential analyzer.(2)In vitro,the 2% agarose model was used to study the contrast-enhanced ultrasound imaging characteristics of AS1411-NBs under physiological conditions at 37°C.The sound intensity changes at different AS1411-NB concentrations in contrast-enhanced ultrasound mode were measured,and the relationship between sound intensity and nanobubble concentration was determined.The difference in in vitro contrast-enhanced ultrasound imaging intensity between AS1411-NBs and nontargeted blank lipid nanobubbles(NBs)at the same concentration was analyzed and compared.(3)The expression of NCL in the cell lines was detected by immunofluorescence assay.Then,the in vitro targeted binding of AS1411-NBs to human TNBC cells(MDA-MB-231 and MDA-MB-468)and human renal proximal tubule epithelial cells(HK-2)were observed by laser scanning confocal microscopy and flow cytometry to determine the targeted binding ability of AS1411-NBs to the membranes NCL-positive cells.(4)We determined the in vivo specific contrast-enhanced ultrasound imaging capability of AS1411-NBs in the nude mouse TNBC xenograft model.The difference between AS1411-NBs and NBs in contrast-enhanced ultrasound imaging of TNBC was compared and analyzed,and the molecular basis of AS1411-NB-specific contrast-enhanced ultrasound imaging was confirmed by histopathological examination.2.Study of the therapeutic efficacy of the targeted drug-loaded ultrasound nanoprobe in treating TNBC.(1)Using AS1411-NBs as carriers of the anti-tumor drug doxorubicin(DOX),NCL-targeted doxorubicin-loaded lipid nanobubbles(AS1411-DOX-NBs)were constructed to identify whether AS1411-DOX-NBs were successfully constructed and to test their ability to carry DOX.(2)The cytotoxicity of AS1411-DOX-NBs on human TNBC cells(MDA-MB-231)was tested in vitro.We also identified whether AS1411-DOX-NBs can effectively increase the uptake of DOX through active targeting and exert a stronger killing effect.(3)In a nude mouse TNBC xenograft model,the effects of AS1411-DOX-NBs and free DOX on the growth curve of tumor-bearing nude mice and tumors were compared and analyzed to determine the anti-tumor efficacy of AS1411-DOX-NBs in vivo.(4)Through echocardiography and histopathological examination,the changes in cardiac function and structure of tumor-bearing nude mice in different treatment groups were compared and analyzed to further evaluate the cardioprotective effect of AS1411-DOX-NBs.Results1.The construction of a targeted ultrasound nanoprobe and its contrast-enhanced USMI in TNBC.(1)Successful synthesis of the lipid–nucleic acid targeted molecule DSPE-PEG2000-AS1411 was confirmed by denaturing urea polyacrylamide gel electrophoresis.AS1411-NBs based on DSPE-PEG2000-AS1411 were also successfully constructed.Under the light microscope,spherical AS1411-NBs of uniform particle size,uniform distribution,and hollowness were observed.The average particle size of the AS1411-NBs was 533.5±19.91 nm,the polydispersity index was 0.095±0.046,and the zeta potential was-3.41±0.17 m V.There was no significant difference in average particle size between AS1411-NBs and NBs(n=3,P>0.05).(2)In the agarose model,the in vitro contrast-enhanced ultrasound imaging results showed that the imaging intensities of both AS1411-NBs and NBs were linearly related to the lipid nanobubble concentrations(n=3,0.8<r<1.0,P<0.05),and there was no significant difference in imaging intensity between the AS1411-NBs and NBs at the same concentration(n=3,P>0.05).(3)Cellular immunofluorescence confirmed that NCL was highly expressed in the cell membranes,cytoplasms,and nucleus of MDA-MB-231 and MDA-MB-468 cells but only in the nuclei of HK-2 cells.Competitive inhibition experiments showed that AS1411-NBs can specifically target and bind to TNBC cells through the interaction of aptamer AS1411 with NCL,and flow cytometry affinity measurements confirmed this result,providing the necessary experimental basis for specific contrast-enhanced ultrasound imaging research with AS1411-NBs in vivo.(4)During initial contrast-enhanced ultrasound imaging in nude mouse xenograft models,there was no significant difference in the peak intensities or the times to peak intensity between the AS1411-NBs and NBs(n=3,P>0.05).However,as time progressed,the area under the time–intensity curve became significantly different(n=3,P<0.001);the duration of AS1411-NBs contrast-enhanced ultrasound imaging was significantly longer than that of the NBs.The histopathological test results confirmed that AS1411-NBs can achieve dual-targeted contrast-enhanced ultrasound imaging of TNBC tissue and neovascularization,effectively prolonging the duration of continuous enhancement imaging.2.Study of the therapeutic efficacy of the targeted drug-loaded ultrasound nanoprobe in treating TNBC.(1)Laser scanning confocal microscopy observation and ultraviolet–visible absorption spectrum detection results confirmed that AS1411-NBs effectively carried DOX,and AS1411-DOX-NBs were successfully constructed.Moreover,the drug-loading and entrapment efficiency of AS1411-DOX-NBs were significantly higher than those of nontargeted doxorubicin-loaded lipid nanobubbles(DOX-NBs)(n=3,P<0.05).The reason for the higher drug-loading and entrapment efficiency of AS1411-DOX-NBs might be that DOX was embedded in both the lipid layer and the quadruplex structure formed by the nucleic acid aptamer AS1411,which significantly improved the ability of AS1411-DOX-NBs to carry and encapsulate DOX.(2)In vitro cytotoxicity testing showed that low-dose nucleic acid aptamers AS1411 and NBs have no obvious cytotoxicity.When carrying DOX,they showed strong cytotoxicity,and AS1411-DOX-NBs showed stronger cytotoxicity than free DOX and DOX-NBs(n=3,P<0.05).Drug uptake experiments further confirmed that AS1411-DOX-NBs could increase the uptake of drugs through active targeting and have a stronger cell-killing effect.(3)In vivo anti-tumor experiments showed that AS1411-DOX-NBs had the same anti-tumor activity as free DOX,which effectively inhibits tumor growth and promotes tumor cell apoptosis and necrosis.At the same time,mice weight and health were better maintained in the AS1411-DOX-NBs treatment group,indicating that the AS1411-DOX-NBs treatment group experienced lower toxicity and fewer side effects than the free DOX treatment group.(4)Echocardiography and histopathological tests confirmed that AS1411-DOX-NBs effectively reduced cardiac injury caused by DOX,although AS1411-DOX-NBs and free DOX did not have significantly different anti-tumor activities in vivo.Conclusions1.In this study,we successfully designed and constructed aptamer AS1411-functionalized NCL-targeted lipid nanobubbles(AS1411-NBs),an ultrasound nanoprobe targeting NCL.AS1411-NBs can specifically recognize and interact with NCL overexpressed on the surface of tumor cell membranes and tumor neovascular endothelial cell membranes to achieve dual-targeted contrast-enhanced USMI of tumor tissue and tumor neovascularization and significantly prolong the duration of continuous contrast-enhanced ultrasound imaging.2.AS1411-NBs can not only be used for specific contrast-enhanced ultrasound imaging but can also efficiently carry and target the delivery of the anti-tumor drug DOX.The constructed AS1411-DOX-NBs can mediate the targeted enrichment of DOX at the tumor site and reduce its exposure to,and influence on,normal tissues.AS1411-DOX-NBs can effectively inhibit tumor growth and significantly reduce the cardiotoxicity of DOX.It is a safe and efficient theranostic ultrasound nanoprobe for the early accurate diagnosis and effective treatment of TNBC.