The Mechanism Of Action Of Photoresponsive Liposome Synergistic Anti-microtubule Drugs

Anti-microtubule therapy is one of the hotspots in the field of cancer prevention and treatment.Paclitaxel(PTX)is a common microtubule inhibitor by interfering with microtubule assembly,inhibiting mitosis and producing antitumor effects.However,the clinical application of PTX alone will still result in low cell selectivity and severe side effects.The emergence of bio-nanotechnology can provide a new solution to the clinical problems PTX facing.The use of liposomes as a carrier to carry anti-cancer drugs or genes into the target cells,efficient treatment of the tumor at the same time,can significantly reduce the side effects of chemotherapy drugs.The development of PTX delivery systems,including the use of liposomes,polymer nanoparticles,micelles and cyclodextrin complexes,has attracted the attention of biopharmaceutical experts.The nano drug delivery system(DDS)has the potential to utilize targeted passive tumor enrichment properties and to control drug release at specific sites to improve targeted drug delivery.A more attractive approach is to use light response DDS,which can control the specific release of the drug in time and space in response to a range of light waves.This can be achieved by intracellular optical genetics or by the external loading of photosensitive compounds such as porphyrins.This paper explores a liposome that efficiently produces reactive oxygen species in the case of light radiation,carrying a new photosensitizer,porphyrin sodium(DVDMS)and anti-cancer drug PTX,which is stimulated by photoreaction to release PTX at a specific time and space.And from the mitotic effect of cell mitotic effect,cell energy metabolism and other aspects implied light control liposome synergistic effect of PTX pathway.In this study,we investigated how photochemical catalysis enhances mitochondrial-dependent apoptosis by modulating the phosphorylated Mcl-1 protein expression with increasing the efficacy of PTX,and by measuring mitochondrial respiration(OCR)and glycolysis(extracellular acid release,ECAR)rate.After laser treatment,we analysised co-loaded liposomes'occurrence of energy metabolism changes.Research conclusions can overcome the clinical application of paclitaxel in the side effects and targeted treatment strategies to provide relevant thinking.In this paper,we mainly study from the design and synthesis of co-loaded liposomes,photo-controlled release,in vitro cytotoxicity,in vivo efficacy evaluation and so on.From the following aspects:Methods and ResultsPart 1 The preparation and characterization of liposomesThe standard preparation of PEGylated liposomes was studied as a drug loading platform for photosensitizer DVDMS and microtubule inhibitor PTX.DVDMS was highly water soluble and encapsulated in the hydrophilic core of liposomes.PTX was loaded into hydrophobic lipid bilayer.PTX liposome(PL),DVDMS liposome(DL),DVDMS with PTX loaded liposomes(PDL)were prepared by membrane hydration method.TEM and AFM were used to observe the effects of paclitaxel liposomes.The particle size,potential and dispersion coefficient of different liposomes were determined by the Mark-Nano-ZS90 dynamic light scattering particle size analyzer.Determination of singlet oxygen yield in liposome solution by fluorescence spectrophotometry.The results showed that the preparation procedure of liposomes by nitrogen-blowing thin film was simple and reliable,and the results were stable and reliable.The morphology,particle size,PDI and Zeta potential of PL,DL and PDL preparations were studied.It was found that the three liposomes were homogeneous and the particle size was 100 nm?115 nm.With a small amount of negative charge,is consistent with the liposomes as drug delivery in the conditions of osmotic retention conditions.PL,DL,PDL were effectively overcome the paclitaxel clinical application of poor water solubility,solubility low,toxic side effects and other issues.Part 2 Light control drug releaseThe establishment methods of PTX and DVDMS encapsulation efficiency and light control drug release of drugs from liposomes in vitro.Papers has used UV spectrophotometry and fluorescence spectrophotometry to establish the PTX and DVDMS content determination method,and the linear range was investigated.The results show that this method conforms to the requirements of the analysis.This article also adopt Sephadex G-50 column chromatography to separate the liposomes and free drugs.Liposomes were damaged in DMSO,the usage were determined before and after the separation of the amount of drug in liposome,calculate the encapsulation efficiency of the two drugs.Drug release studies:the same amount of PL,DL,PDL were divided into two groups(light group and the control group),and the light intensity was 30.25 mW/cm2,the light intensity was 4 J/cm2.The control group is not with irradiation,other steps as the above.With different treatment groups would seal dialysis bag in 50 ml centrifuge tubes and took the centrifuge tube cover tight at 37 ? thermostat oscillator oscillating at a speed of 100 prm.In different time,enzyme standard instrument analyzer was used to test every time PTX/DVDMS content in the selected samples;Computational loaded PTX/DVDMS samples with different time points of the accumulative release percentage draw the accumulative release rate-time curve.Results showed:(1)the PTX of encapsulation efficiency was 2.13 ± 3.24%in PL,the DVMS of encapsulation efficiency was 73.28 ±4.25%in DL,while in PDL,PTX and DVDMS envelopment rate close to 52%,the corresponding mass ratio of 1:1.(2)In the 0-12 h,light treatment the PTX in PL went into a state of slow release,and DL,PDL of PTX or DVDMS performed all of a sudden release state.At 12 h drugs release accumulation reached 75.8%,while the control group with the accumulation of drug release was only 12.3%in a slow release state.Part 3 In vitro-PDL anticancer effect and related mechanism researchWe chose human breast cancer cell line MCF-7 as the model.It was mainly determined cell cytotoxicity by MTT method,Viacount,AnnexinV/PI to test PL,DL,PDL with laser.Flow cytometry instrument combining rhodamine 123(Rh123),DCFH-DA tested different processing modes,to detect the change of mitochondrial membrane potential and the generation of reactive oxygen species in the cells.Rh123 instead of PTX,a new liposome of synthetic load with DVDMS,used flow cytometry to detect the take up of loaded liposomes behavior and confocal laser to observe it in the intracellular distribution of PTX/DVDMS in co-liposome and the change of the ?-tubulin.The changes of energy metabolism in mitochondrial respiration and glycolysis energy in different treatment modes were measured by Seahorse energy metabolism analyze.In further,using western blot detection to see MCF-7 cell apoptosis and microtubule associated protein Cleaved caspase 3,Cleaved PARP,Mcl-1,the Bcl-xL expression changes in PL-PDT,DL-PDT,PDL-PDT groups.Results show that:(1)PDL in combination with light had strong killing effect.Comparative analysis confirmed that PDL plus laser treatment significantly reduced(IC50)of PTX and DVDMS,and IC 50 were 94.7 times higher than PTX in PL and 25.8 times of DVDMS in DL group.(2)After laser,the DL,PDL(30ng/ml,50ng/ml)the level of mitochondrial membrane potential all had varying degrees of decline.And in PDL(50 ng/ml),the level of mitochondrial membrane potential decreased more significantly,which prompted the mitochondrial membrane potential changes as sensitive detection indicator may be result from light chemotherapy leading to cell damage.(3)The PL group generated small amount of reactive oxygen species(ROS)and there was no significant difference compared with control group.In DL(50 ng/ml DVDMS)group,40.56±2.31%of the cells present DCF green fluorescence positive,compared with the control group(2.31 ± 1.54%),and had the significant difference(p<0.01).In the PDL(50 ng/ml DVDMS)group,in turn,70.54±4.25%of the cells positive for DCF green fluorescent.(4)The Seahorse tested MCF-7 cells' OCAR and ECAR reflecting mitochondrial oxidative phosphorylation and glycolysis.(5)The presence of light-free laser confocal observation showed that light treatment changed the distribution of liposome-loaded drugs in the cells,which might be related to the release of lysosomes in light-controlled release and liposomes.(6)PDL combined with light treatment seriously damaged the microtubule network structure of MCF-7 cells,enhancing the level of apoptosis-related proteins casepase-3 and PARP,and inhibited the expression of survivin Mcl-1.The DVDMS carried by PDL occurred under light the photodynamic responsing to reslut in the phosphorylation of Mcl-1 Ser 64 locus to promote Mcl-1 ubiquitination degradation,and enhanced PTX-induced cell cycle arrest and apoptosis.Part 4 In vivo studies-the anti-tumor effect of PDLMCF-7 tumor-burdened was established to evaluate following research.living imager determineed tumor's amount of drug largest concentration.Respectively from the tumor volume,weight in mice,main viscera observation,chip morphology observation and immunohistochemical aspects discusses the PL,DL,PDL on MCF-7 transplantation tumor growth inhibition effect.The experimental results showed that:(1)After injection 12h,drugs with maximum amount in tumor.And the various main viscera of fluorescence was the strongest within the tumor.(2)The PDL synergistic with light significantly inhibited tumor growth in tumor-bearing mice.(3)TUNEL detection results showed that cell apoptosis occurs to inhibit tumor growth.(4)Different treatment had no obvious effect on body weight in mice and main organs,which prompted the processing pattern is relatively safe and reliable.ConclusionIn summary,this study designed a new method of photosensitizing liposome-loaded anti-microtubule drugs for the treatment of tumors.The use of new photosensitizer DVDMS responsing light to destruct liposomes,to release PTX at specific sites,while DVDMS caused by the Photodynamic effect can be synergistic gain PTX effect.In vitro,experiments showed that DVDMS-photodynamic energy on the one hand could promote the anti-apoptotic protein Mcl-1 degradation.The inhibition of PTX lead to mitotic blockage might have a slip effect,thereby enhancing the PTX apoptosis induced effect.On the other hand,it is to prevent possible energy conversion to maintain the phenomenon of cell survival by inhibiting cell glycolysis with PTX function.This photochemical therapy can simultaneously inhibit glycolysis and mitochondrial respiration,reduing intracellular ATP production.The potential molecular mechanism still need further study.In vivo experiments showed that light therapy resulted in the local release of PTX in the tumor,which greatly enhanced the effect of PTX treatment and reduced the side effects of the system when the co-loaded liposome reached a relatively high concentration in the tumor.