Construction Of Multifunctional Nanoprobes And Their Applications In The Diagnosis And Treatment Of Osteosarcoma

The early diagnosis of cancer and non-toxic treatment with high efficiency are the greatest challenges in cancer diagnosis and treatment.The rapid development of nanotechnology has exhibited prospects for cancer diagnosis and treatment.Nanomaterials possess optical,electrical,magnetic,thermal properties and so on.Furthermore,they can be passively enriched in the tumor sites through enhanced permeability and retention effect（EPR）.Therefore,nanomaterials have shown great potential and advantages in tumor diagnosis and treatment.Osteosarcoma（OS）is the most common primary bone malignant tumor in skeletal diseases,with strong recurrence and metastasis.In this work,a series of multifunctional nanoprobes were constructed for the diagnosis and treatment of osteosarcoma.The content and innovation are summarized as follows:1.Matrix metalloproteinase 2（MMP-2）plays an essential role in tumor invasion and metastasis.Indeed,elevated MMP-2 activity levels are closely associated with invasiveness,angiogenesis,and metastasis of cancer progression.Detection of MMP-2activity by nanoprobe is of great significance in tumor screening,diagnosis,prognosis,monitoring treatment and recurrence.In the present work,a core-satellite magnetic-fluorescent-plasmonic nanoprobe（FMNS@Au）has been constructed through biological self-assembly to generate high density SERS“hot spots”and an efficient fluorescence resonance energy transfer（FRET）system for the sensitive determination of MMP-2 activity in a SERS-fluorescence dual-mode assay.In this nanoprobe,a biotin-labeled peptide containing a specific MMP-2 substrate（PLGVR）was employed as a bridge for the assembly of gold nanoparticles（Au NPs）and avidin functionalized fluorescent-magnetic nanospheres（FMNS）.The rhodamine B modified on FMNS served as a Raman reporter and a donor of FRET,while the Au NPs assembled on FMNS acted as SERS substrates and acceptors of FRET.In the presence of MMP-2,the SERS“hot spot”effect was weakened and the FRET system was disrupted through enzymatic cleavage of PLGVR,resulting in a reduction of SERS signal and the recovery of fluorescence emission.Importantly,this combination of SERS and fluorescence methods in the FMNS@Au nanoprobe extended the detection range of MMP-2 for 1-200 ng m L^-1,with a limit of detection of 0.35 ng m L^-1.Compared with traditional single-mode detection methods,our novel nanoprobe affords semi-quantitative sensing of MMP-2 by fluorescence observation and accurate detection of MMP-2 by dual-mode analysis.In addition,MG-63 cell line was utilized as a model to study the expression level of MMP-2 under different conditions.The effectiveness and practicability of the nanoprobe were also confirmed by determination of MMP-2 activity in human serum samples.The as-designed FMNS@Au nanoprobe provides a new technique for clinical diagnosis of MMP-2 related diseases such as osteosarcoma tumors.2.Caspases,a unique family of cysteine-containing aspartate proteases,play important roles in the initiation and execution of apoptosis.Caspase-9 is a prominent member of caspase family and an upstream activator in the caspase cascade.Real-time monitoring of caspase-9 activity in vivo is of great significance for the prognosis of diseases and the development of anticancer drugs.In the present work,peptide-functionalized upconversion@silica nanoparticle（UCNP@SiO₂@Cy5-pep）-based FRET nanoprobe has been fabricated for the detection of caspase-9 activity in vitro and in vivo.A Cy5 labeled peptide containing specific motif LEHD for caspase-9 cleavage was designed and conjugated with UCNP@SiO₂.The red upconversion luminescence（UCL）emission of UCNP@SiO₂ can be quenched by Cy5 while the green UCL emission of UCNP@SiO₂ remains unchanged.After the cleavage of LEHD by caspase-9,the Cy5 detached from UCNP@SiO₂ surface,resulting in a recovery of red UCL emission.Based on this sensing principle,UCNP@SiO₂@Cy5-pep was used to monitor the changes of caspase-9 activity levels in cisplatin-induced apoptosis cancer cells（osteosarcoma cells and colorectal cancer cells）.Under the same experimental conditions,it is found that the intracellular caspase-9 activity level of cisplatin-treated MG-63 cells is higher than that of cisplatin-treated SW480 cells.Furthermore,UCNP@SiO₂@Cy5-pep can also be used for in vivo visual imaging of caspase-9activity in cisplatin-treated mouse-bearing MG-63 tumors.Thus,the evaluation of therapeutic effect in osteosarcoma tumors under the guidance of imaging can be realized.3.Caspase-3,another important member of the caspase family,is a typical biomarker of irreversible apoptosis and is often used to evaluate the therapeutic effect of cancer.Real-time monitoring and evaluation of caspase-3 activity in the cancer treatment by using the theranostic nanoprobe is the basis for realizing precision medicine.The theranostic nanoprobes can be used to monitor the therapeutic effect and adjust the drug dosages at any time in the treatment process,which is beneficial for achieving the best therapeutic effect.Based on the previous work,a six-in-one theranostic nanoprobe（UCNP@PDA@Cy3-pep@STS）has been fabricated for in situ monitoring the anticancer efficacy of photothermal-chemotherapy and chemotherapy in real time through detection of caspase-3 activity.The upconversion nanoparticle（UCNP）was utilized as an internal reference,which can be used for ratiometric fluorescence detection.The PDA shell was employed as an acceptor of FRET system,photothermal therapy（PTT）agent,loading agent of chemical drug and immobilization platform of Cy3 labeled peptide（Cy3-pep）.The Cy3-pep contains a specific caspase-3substrate（DEVD）and an active tumor targeting motif（PSP）.In the presence of caspase-3,the FRET system was destructed through the enzymatic cleavage of DEVD,resulting in a recovery of Cy3 fluorescence emission.Based on this sensing principle,mouse-bearing MG-63 tumor was still used as a model,and the response of MG-63tumor to PTT and chemotherapy was evaluated non-invasively in real time through monitoring of caspase-3 activity.Moreover,the theranostic nanoprobe has achieved a good therapeutic effect in the synergistic treatment of photothermal and chemotherapy for osteosarcoma,realizing the combined treatment under the guidance of imaging.The experimental results indicate that the six-in-one nanoprobe can be used as an ideal theranostic tool for the diagnosis and treatment of osteosarcoma.