| Head and neck cancers account for about 30% of systemic malignant tumours.There were about 225000 new cases in China in 2015,causing 77500 cases of death.Head and neck squamous cell carcinoma(HNSCC)is the most common pathological type among various malignancies originating in the head and neck regions.It often occurs in the mucosal epithelium of nose,sinuses,oral cavity,pharynx and throat,with strong concealment and aggressiveness,easy recurrence and metastasis,and poor prognosis with high mortality and disability rates.Combination treatment modality consisted of surgery and adjuvant radiochemotherapy effectively improved the quality of patient's life,however,it did not significantly improve postoperative 5-year survival rate.As a result,the researchers are turning their focus to target treatment strategy to improve treatment outcomes.In recent years,with the successive development of nano-medicine,there are many reports about nano-carrier medicine that can improve the diagnosis and treatment of HNSCC.The advantages of nano-carrier drugs include:(1)nano-drugs by chemical or physical modification,such as by tumor-specific antibodies,peptides,small molecules and oligonucleotides,can achieve targeted drug delivery;(2)nano-carrier can improve drug biologic compatibility,i.e.,preparation of drug insoluble injection formulation,can improve uptake rate by the targeting cells;(3)nao-carrier can reduce the dosage and thus effectively reduce the toxic side effects of chemotherapeutic drugs;(4)nano-carrier can preserve drug activities by avoiding degradation and destruction of certain active composition of drugs.A large number of in vitro or in vivo experiments confirmed that DHA has a killing effect on a variety of tumors,include HNSCC.However,to develop DHA into a real anti-tumor drug,there are several restrictions from physical and chemical properties of DHA.For instances,DHA is insoluble in water but soluble in lipid,common formulations for drug delivery can not achieve effective blood concentrations;its half life in plasma(T1/2)is only 34 min to 90 min.Much effort and more intensive studies must be paid to promote anti-tumor actions of DHA.Presumably,combination of DHA with a nano-carrier is an effective means.Based on the acknowledgement above,magnetic-targeting DHA nanoparticles is designed for the purpose of increasing the DHA blood concentration,prolongating DHA T1/2 and providing experimental basis to develope DHA into a clinical antitumor drug.The present study is divided into three parts: Part ?: Preparation and characterization of magnetic-targeting DHA nano-liposomeObjective: To optimize the synthesis conditions of magnetic-targeting DHA nano-liposome(DHA-Mag-NL),detect the important characteristics of DHA-Mag-NL to meet the requirements of intravenous medication.Methods:1 Fe304 was synthesized through modified chemical precipitation method,and the magnetic response and magnetic stability were observed.2 PEG-DHA was synthesized through esterification reaction and quantification.4 The standard curve equation of DHA was established by HPLC.5 DHA-Mag-NL was synthesized by reverse evaporation method.The recipe was optimized by detecting the encapsulation efficiency.4 Important characterization parameters of DHA-Mag-NL were detected according to the drug delivery system evaluation index.Results:1 Magnetic Fe3O4 nanoparticles exhibited satisfactory magnetic response and super paramagnetism.2 The established DHA solution standard concentration curve regression equation was y = 55.48x-1.571,R2 = 0.999,with good linearity.3 The optimized synthesis conditions calculated through L9(34)orthogonal design was PC: CH(w/w)4:1,PC: DHA(w/w)20: 1,PC: Mag(w/w)10: 1,DHA(w/w): PEG(m/m)1:10.DHA-Mag-NL nanoparticles with high encapsulation efficiency were obtained.The importance of each influencing factor affecting the encapsulation efficiency was described as: PC: CH(factor A)> DHA: PEG(factor D)> PC: Mag(factor C)> PC: DHA(factor B).4 The encapsulation efficiency of DHA-Mag-NL was 82.12 ± 0.91%;the drug loading was 5.87 ± 0.06%;the dosage of DHA was 2.46 ± 0.03mg;particle size was 209.10 ± 4.92 nm,particle size was uniform(PDI = 0.25 ± 0.02);particle charge was-37.13±1.01 mV.5 DHA-Mag-NL nanoparticles were stored at 4 ? for 3 months with good stability.There were no significant differences in the particle size and entrapment efficiency with new synthesized DHA-Mag-NL nanoparticles(P> 0.01).However,when DHA-Mag-NL nanoparticles were stored at-20 ? or at room temperature(25 ?)for 3 months,the two key physical parameters,particle size and entrapment efficiency,changed greatly,both of which were significantly different from the initial synthesis(P <0.01).Conclusions:DHA-Mag-NL drug delivery system was successfully synthesized,with suitable size and adequate encapsulation efficiency.The stability of the synthesized nanoparticles was good at 4 ?,which can be used for subsequent experiments.Part ?: Study on magnetic targeting efficientcy of DHA-Mag-NL nanoparticlesObjective: To observe the targeted distribution of DHA-Mag-NL nanoparticles under magnetic field and verify the targeting effect of DHA-Mag-NL.Methods:1 Hep-2 and Cal-27 cells are treated with DiI-labeled DHA-Mag-NL and DAPI;magnetic targeting of DHA-Mag-NL was observed by fluorescence microscope.2 Mice are injected with DIG-labeled DHA-Mag-NL suspension in the tail vein.The left kidney was exposed to the 4500 Gaussian magnetic field for 1 h,and iron staining in tissue sections was compared between the left and right kidney.Frozen sections were used to observe fluorescence stain.Results:1 In 4500 Gaussian magnetic field,Hep-2 and Cal-27 cells were treated with DiI fluorescence-labeled DHA-Mag-NL suspension for 1 h,the DiI fluorescence intensity and the number of cell-bound magnetic particles(Fe3O4)was significantly higher than those under no magnetic action.2 After the mice were injected with DiI fluorescence-labeled DHA-Mag-NL suspension,significant difference was observed in the fluorescence intensity between left kidney exposed to magnetic field and the right kidney without magnetic action.The tissue sections from kidneys were colored with iron stain,significant difference was noted between the left kidney exposed magnetic field and the right kidney under no magnetic action.The results suggest the strong magnetic targeting capabilitiy of the DHA-Mag-NL naoparticles both in vitro and in vivo.Conclusions:Magnetic targeting-based DHA-Mag-NL nanoparticles have been shown to have in vitro targeting effects.Part ?: Inhibitory effects DHA-Mag-NL nanoparticles on human tongue squamous cell carcinoma Cal-27 cell line and laryngeal carcinoma Hep-2 cell line in vitroObjective: To observe the inhibitory effect of DHA-Mag-NL nanoparticles on Cal-27 and Hep-2 cell lines in vitro.Methods:1 MTT assay was used to detect the proliferation inhibition effects of DHA-Mag-NL on Hep-2 and Cal-27 cells at different concentration and different time intervals.2 The experiment groups were categorized as control,Mag-NL,DHA-Mag-NL and DHA-Mag-NL in the presence of magnetic field.Flow cytometry was used to detect the effects of 40?M DHA on cell cycles in Hep-2 and Cal-27.3 The experiment groups were set up as control,Mag-NL,DHA-Mag-NL and DHA-Mag-NL with exposure to magnetic field.Flow cytometry was used to detect the effects of 60?M DHA on apoptosis in Hep-2 and Cal-27 cell.Results:1 MTT assay showed that DHA-Mag-NL could inhibit the proliferation of Hep-2 and Cal-27 cells at 24 h and 48 h,IC50 values were 28.66?M and 15.67?M,and 41.75?M and 11.86?M,respectively.DHA-Mag-NL could inhibit the proliferation of tumor cells in dose-and time-dependent manners.After 24 h of application,DHA-Mag-NL demonstrated less inhibitory effect on Cal-27 cells then on Hep-2 cells.2 The cell cycle was detected by FCM after 24 hours drug action,with the concentration of DHA is 60?M.Comparing with the control group,the two groups of DHA and DHA-Mag-NL in magnetic field had the definitely effect of G0 / G1 phase arrest of two HNSCC cells(P<0.01).DHA-Mag-NL had the effect of G0 / G1 phase arrest compared with the control group in Hep-2 cells(P<0.01);Cal-27 cells under DHA-Mag-NL intervention,cell cycle distribution had no significant difference with the control group(P>0.05).The results showed that DHA-Mag-NL had a weak inhibitory effect on Cal-27 cells.3 The apoptotic of cells were detected by FCM after 24 hours drug action,with the concentration of DHA is 60?M.Comparing with control group,the groups of DHA DHA-Mag-NL and DHA-Mag-NL in magnetic field had the effect of promoting apoptosis of the two HNSCC cells(P<0.01).The effect of DHA-Mag-NL on cell apoptosis was significantly lower than that of DHA and DHA-Mag-NL targeting intervention(P<0.01).Statistical analysis showed that the apoptotic ratio of DHA-Mag-NL targeting intervention Cal-27 tumor cells was significantly weaker than DHA(P <0.01).Conclusions:DHA-Mag-NL can inhibit the proliferation of Hep-2 and Cal-27 cells,and promote cell apoptosis in dose-and time-dependent manners. |
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