Dual-mode Imaging-guided Targeted Nano-drug Delivery System For Diagnosis And Treatment Of Melanoma

Melanoma is one of the malignant tumors with the highest incidence in skin cancer.Not only has high invasiveness and strong proliferative ability,but also it is prone to lymphatic and blood metastasis and poor prognosis.The incidence has been increasing in recent years.Conventional treatments include radiotherapy,chemotherapy,surgical resection,and immunotherapy,but the therapeutic effect is still hardly ideal.Briefly,the prolongation of survival is not very significant,which has become a huge threat to human health today.Therefore,early diagnosis and treatments are particularly important for melanoma patients.This project aims to establish a two-mode imaging-guided collaborative therapy targeting melanoma nano-delivery system,c RGDyk-2-ME@ICG-TSLs.It uses c RGDyk peptide as the target,thermosensitive liposomes(TSLs)as a carrier,containing the chemotherapeutic drug 2-methoxyestradiol(2-ME)and the photothermal therapeutic agent indocyanine green(ICG),using the photothermal conversion effect of indocyanine green(ICG)to achieve the localized release of chemotherapeutic drugs also produces local thermal effects,increasing the accumulation and uptake of drugs at the tumor site;combining multiple treatment methods such as chemotherapy and hyperthermia to enhance the therapeutic effect of drugs and reducing toxic and side effects.Meanwhile combine fluorescence imaging with photoacoustic imaging,realize the integration of precise diagnosis and treatment under the guidance of dual-mode imaging.The research content of this topic is mainly divided into the following four parts:Firstly: Preparation and characterization of c RGDyk-2-ME@ICG-TSLsThe thin film dispersion method was used to prepare a heat-sensitive lipid nano-delivery system with surface-modified c RGDyk peptides and 2-ME and ICG(c RGDyk-2-ME@ICG-TSLs).The UV-Vis spectrophotometer,transmission electron microscope,and laser nano particle size analyzer proved that the drug delivery system was successfully synthesized.The UV spectrum showed that c RGDyk-2-ME@ICG-TSLs had(2-ME)288 nm and(ICG)780 nm characteristic absorption peak indicates that the drug delivery system co-loaded the chemotherapy drug 2-ME and the photothermal therapeutic agent ICG.The laser particle size analyzer showed that the average particle size of c RGDyk-2-ME@ICG-TSLs was128.4 ± 0.6 nm and the potential was-20.8 ± 1.2 m V.The TEM results showed that the nanoparticles were round in shape and uniform in size.The encapsulation rate and drug loading of 2-ME and ICG were 85.43 ± 0.83%,90.84 ± 0.32%;4.05 ± 0.12%,2.09 ± 0.16%.The in vitro heating experiment showed that the temperature of c RGDyk-2-ME@ICG-TSLs preparation rapidly rose to above 45°C within 3min under the action of 808 nm laser emitter,and the photothermal conversion efficiency was high.These results of in vitro drug release showed that the cumulative release amount of the drug in the incubation environment at 42°C was about twice that at37°C.The drug release rate was significantly accelerated and the blood concentration was increased at the target site.Secondly: Investigation the anti-tumor activity of c RGDyk-2-ME@ICG–TSLs in vitroThe in vitro antitumor activity of c RGDyk-2-ME@ICG-TSLs was investigated using murine melanoma cells B16-F10 as model cells.The MTT method was used to investigate the inhibitory effect of each experimental group on B16-F10 cells.The uptake of B16-F10 to each experimental group was investigated by fluorescence microscopy and flow cytometry.The intracellular activity of each experimental group was analyzed by flow cytometry of oxygen levels,apoptosis,and cell cycle effects.The results showed that the MTT assay showed that the preparation group c RGDyk-2-ME@ICG-TSLs had the strongest tumor inhibition effect under laser conditions compared with other experimental groups;the uptake experiment showed that the preparation group c RGDyk-2-ME@ICG-TSLs under the action of c RGDyk peptide,the uptake effect of B16-F10 was the most obvious,which could effectively promote the entry of lipid nanocarriers into tumor cells;apoptosis experiments showed that the preparation group c RGDyk-2-ME@ICG-TSLs could induce more apoptosis under laser conditions and significantly inhibited cell proliferation;cell cycle results showed preparation c RGDyk-2-ME@ICG-TSLs mainly affects the G2/M phase of the growth cycle to block tumor cell growth;the concentration of reactive oxygen species in the preparation group c RGDyk-2-ME@ICG-TSLs increased significantly under near-infrared light,which will kill tumor cells effectively.Thirdly: NIR-FIR/PAI dual-mode imaging in c RGDyk-2-ME@ICG-TSLsBalb/C tumor-bearing mice as model animals,using a small animal in vivo imager to record the fluorescence imaging effect of c RGDyk-2-ME@ICG-TSLs in mice.At the same time,it was performed imaging of isolated organs with the greatest fluorescence enrichment.The results show that the drug delivery system c RGDyk-2-ME@ICG-TSLs can reach the maximum enrichment at the tumor site within 24 hours.At the same time,the photoacoustic imaging effect of c RGDyk-2-ME@ICG-TSLs was observed by a photoacoustic imaging device in the above animal model.The results showed that c RGDyk-2-ME@ICG-TSLs had a significant time dependence at the tumor site.It reached the maximum enrichment at24 hours with obvious photoacoustic imaging effect,which was consistent with the fluorescence imaging results.Fourthly: Investigation the pharmacokinetics and pharmacodynamics of c RGDyk-2-ME@ICG-TSLs in vivoSD rats were used as model animals to study the pharmacokinetics of the preparation group c RGDyk-2-ME@ICG-TSLs.The results showed that compared with the 2-ME raw material group,c RGDyk-2-ME@ICG-TSLs had a slower plasma clearance rate of the drug in vivo,a significantly longer half-life and average residence time,and significant bioavailability.It indicates that the c RGDyk-2-ME@ICG-TSLs increases the accumulation of drugs at the target site,increases the effective blood concentration,and exerts a more durable antitumor effect.C57 tumor-bearing mice were selected as model animals,and intravenous injection was used to study the antitumor activity of c RGDyk-2-ME@ICG-TSLs in vivo under the action of laser.The results showed that from the tumor volume,tumor weight and other indicators,c RGDyk-2-ME@ICG-TSLs had obvious tumor suppressive effect under the action of laser.The pathological results showed that the laser method did not have obvious toxic and side effects on its organs while enhancing the tumor suppressing effect.It shows that c RGDyk-2-ME@ICG-TSLs has good antitumor activity in vivo and little toxic,which could reduce toxicity and increasing efficiency.