Experimental Study Of SERS Imaging Based On AuNPs Material For Laryngeal Cancer Diagnosis And Treatment

【Objective】Laryngeal carcinoma(LC)is one of the common malignant tumours of the head and neck,with squamous cell carcinoma being the most common,and it ranks second in the incidence of head and neck cancer.Currently,most clinical treatments are based on a combination of surgical resection with adjuvant radiotherapy,but the survival rate at advanced stages is still less than 50%.Therefore,there is an urgent need to develop more sensitive and efficient treatment tools in order to improve the survival rate of laryngeal cancer patients.In this study,a surfaceenhanced Raman Scattering(SERS)probe based on gold nanoparticles(Au NPs)for targeted delivery of si RNA was developed,which utilises a p H-responsive i-motif DNA structure to release si RNA for targeting abnormal gene silencing for laryngeal cancer and surface-enhanced Raman Scattering signal imaging.【Methods】1.Use RNA interference technology to design and prepare si RNA that can silence matrix metalloproteinase 2(MMP2),and use q PCR to verify the silencing effect of si RNA on MMP2 gene in laryngeal cancer cells.2.Three pairs of imotif DNA with different p H responses were designed and prepared,and the response of i-motif DNA to different p H gradients was verified by polyesterose gel electrophoresis.3.Gold nanoparticles with a particle size of 50 nm were synthesized by seed growth method and characterized by TEM/DLS for size,particle size,and uniformity.4.Confocal Raman microscopy was used to detect the Raman signals of Au NPs connected to i-motif DNA/linker DNA under different p H responses and screen them.5.The probe was synthesized through chemical synthesis methods,and the purified SERS probe was subjected to surface enhanced Raman scattering signals with different p H responses in vitro.The solution was scanned on a UV visible spectrophotometer to obtain UV absorption spectra,and characterized by projection electron microscopy(TEM).6.Exploring the proliferation of laryngeal cancer cells by SERS probe delivery of si RNA and unbound carrier delivery of si RNA through CCK-8 cell experiments.7.Analyze the effect of SERS probe delivery of si RNA on MMP2 protein expression through cell surface protein specific expression detection experiments.8.To understand the influence of SERS probe delivering si RNA and non binding vector delivering si RNA on the migration and invasion of laryngeal cancer cell migration through scratch test.9.Detecting the imaging ability of p H responsive SERS probes in laryngeal cancer cells through surface enhanced Raman scattering signals.【Results】1.Through q PCR experiments,it was verified that three primer si RNAs can be used to silence MMP2 abnormal genes in laryngeal cancer cells,among which si RNA-1 has the best silencing effect on MMP2 genes in laryngeal cancer cells.2.The results of polyesterose gel electrophoresis showed that the response of three imotif DNA to different p H gradients was feasible 3.Through particle size(DLS),projection electron microscopy(TEM),and UV absorption spectroscopy,it was confirmed that our synthesized gold nanoparticles have a particle size of about 50 nm,good uniformity and stability.4.The p H responsive SERS probe based on i-motif DNA was used to detect its surface enhanced Raman scattering signal under different p H conditions,among which Au NPs i-motif DNA3 had the best surface enhanced Raman scattering signal effect.5.The probe was synthesized through chemical synthesis methods,and the purified SERS probe was subjected to surface enhanced Raman scattering signals with different p H responses in vitro.The solution was scanned on a UV visible spectrophotometer to obtain UV absorption spectra,and characterized by projection electron microscopy(TEM).6.The si RNA delivered by SERS probes and the si RNA delivered by unbound carriers have inhibitory effects on the proliferation of laryngeal cancer cells.7.The si RNA delivered by SERS probes and the si RNA delivered by unbound carriers have knockdown effects on MMP2 abnormal genes in laryngeal cancer cells.8.The cell scratch test verified that the si RNA delivered by SERS probe and the si RNA delivered by non binding carrier had inhibitory effects on the migration and invasion of laryngeal cancer cell migration.9.The SERS probe was co incubated with laryngeal cancer cells for 0,1,3,5,and 8hours,and after 3 hours,the Raman signal could achieve good detection results.【Conclusion】In this study,a p H responsive SERS probe based on Au NPs targeted delivery of si RNA was successfully designed and synthesized.The SERS probe utilizes a p H responsive i-motif DNA structure to release si RNA,which has dual functions of targeted gene silencing therapy for laryngeal cancer and surface enhanced Raman scattering signal imaging.It can be used for the diagnosis and treatment of laryngeal cancer research.