Precise Margin Evaluation In Breast-conserving Surgery Guided By NIR Fluorescence Imaging Targeting α_vβ₃ Receptor

Breast-conserving surgery(BCS)is the most commonly used surgical method for early-stage breast cancer,and positive postoperative margin is the key influencing factor of tumor local recurrence.Therefore,obtaining a tumor-free surgical margin is crucial for improving patient prognosis.However,due to the lack of effective methods for intraoperative precise evaluation of margin status in real-time,the reoperation rate of BCS patients is as high as 20-55%.Notably,near-infrared fluorescence(NIR)imaging is an emerging imaging technology with the advantages of real-time and convenient operation.It can assist surgeons to visualize tumor boundaries in real-time and is expected to guide the precise evaluation of surgical margins.Moreover,the development of fluorescent molecular probes with optimal biosafety that can fulfill the requirements of high-resolution and high-sensitive imaging is crucial to achieving precise intraoperative navigation.Nevertheless,a series of probes currently entering clinical studies have some shortcomings,such as insufficient delivery efficiency and physicochemical stability,low imaging resolution and penetration depth,which need to be further optimized and improved.Herein,we constructed two novel near-infrared(NIR)fluorescent nanoprobes based on the unique structural characteristics and in vivo delivery superiority of nanoparticles,and further preclinical explored its biosafety and effectiveness in the precise margins evaluation.The main contents are as follows:(1)Construction and characterization of targeted NIR fluorescent probe RGDHBc160/ICG and its application in NIR-I fluorescent surgical navigation.Purpose:A novel NIR fluorescent nanoprobe was constructed based on tailored hepatitis B viral-like protein particles(HBc VLP).The tumor-specific targeting and the accuracy and biosafety of visually-guided tumor resection were further evaluated.Methods:The hepatitis B virus-like fusion protein RGD-HBc160 with the characteristics of integrin αvβ3 receptor-targeted was prepared by genetic engineering method followed encapsulation of ICG via disassembly-reassembly to construct a novel NIR fluorescent probe RGD-HBc160/ICG.After fully characterizing the physicochemical properties and safety of the probe,the targeting binding ability to αvβ3 was tested in the MDA-MB-231 breast cancer cell line,subcutaneous transplanted tumor mouse model,and MMTV-PyVT spontaneous mammary tumor transgenic mouse model.Subsequently,the effectiveness of RGD-HBc160/ICG-based fluorescence image-guided surgery was evaluated in an infiltrative tumor-bearing mouse model and MMTV-PyVT transgenic mouse model.Finally,the immunogenicity and safety of the probe were preliminarily evaluated in BALB/c mice.Results:The prepared RGD-HBc160/ICG possessed stable physicochemical properties,and showed specific αvβ3 targeting binding ability in the MDA-MB-231 cell and subcutaneous transplanted tumor mouse model.Moreover,compared with traditional white-light guided surgery,the RGD-HBc160/ICG-based NIR-I fluorescence imaging can effectively identify residual tumor lesions,and improved the postoperative survival of the mice.In addition,the enrichment of the probe in the breast tumor tissues of MMTV-PYVT transgenic mice was significantly higher than that in the normal breast tissue and showed high accuracy in tumor diagnosis(AUC=0.846;95%CI:0.952,1.0).Interestingly,the ingenious tailoring of HBc VLP could not only endow its tumor-targeting ability towards integrin αvβ3 but also significantly reduce the in vivo immune responses.Conclusion:The novel NIR fluorescent probe RGD-HBc160/ICG,with characteristics of stable physicochemical properties and tumor-specific targeting,was fully demonstrated well biosafety and ability to guide precise tumor resection and has potential clinical translation application value.(2)Construction and characterization of targeted NIR-Ⅱ/MR multimodal imaging probe R&HV-Gd@ICG and its application in NIR-Ⅱ fluorescent surgical navigation.Purpose:To further improve the fluorescence imaging resolution and penetration,and provide more comprehensive tumor location information,we further expand the application of ICG in NIR-Ⅱ surgical navigation and combine it with the metal nanoparticles imaging functionalization to construct a novel NIR-Ⅱ/MR multimodal imaging probe.The multimodal imaging characteristics of the probe and visual navigation of breast cancer surgery were further evaluated.Methods:We constructed a Gd-based degradable hollow virus-like nanoparticle via the hard template of virus-like mesoporous SiO2 with loading ICG and modified the nanoparticle surface with c(RGDfK)peptides to develop NIR-Ⅱ/MR multimodal probe R&HV-Gd@ICG.After fully characterizing the physicochemical properties of the probe,the targeted binding properties to αvβ3 receptor were detected in the 4T1 mouse breast cancer cell line,subcutaneously transplanted tumor mice,and MMTV-PYVT transgenic mouse model,and the probe properties of achieving NIR-Ⅱ/MR imaging in vivo were further studied.Subsequently,an intraoperative residual tumor mouse model was constructed to evaluate the accuracy of the NIR-Ⅱ fluorescence image-guided recognition of residual tumors.Results:The R&HV-Gd nanoparticles remarkably improved both photostability and photobleaching of ICG.And the R&HV-Gd@ICG is specifically bound to integrin receptor αvβ3 achieving high signal-to-noise ratio tumor imaging in 4T1 tumor-bearing mice.Additionally,the nanoprobe-based NIR-Ⅱ fluorescence image guidance facilitated complete tumor resection and effectively discriminated between benign and malignant breast tissues in MMTV-PyVT transgenic mice(AUC=0.978;95%CI:0.952,1.0).Moreover,the nanoprobe acts as an MRI contrast agent,which outperforms clinically used gadoteric acid meglumine(GAM),which is expected to achieve intraoperative NIR-Ⅱ/MR multimodal imaging in breast cancer.Noteworthy,the nanoprobes also show pH-responsive degradability both in vivo and in vitro,and exhibited accelerated bodily clearance.Conclusion:R&HV-Gd@ICG multimodal imaging probe with the characteristics of stability,specific targeting,and biodegradable,accurate identification and excision of breast tumor in the mouse model,which was expected to provide a visual surgical navigation platform for breast cancer patients.It also provides a new train of thought and direction for the clinical translation of inorganic nanoparticles.