Clathrin Modified Lipid Nanoparticle-salinomycin Nanoscale Drug Delivery System

Cancer is a major disease threatening human health.It is almost impossible to cure cancer with current clinical treatment and even more difficult to deal with highly malignant advanced cancer or multiple metastases.Most anti-cancer drugs are highly cytotoxic.Once distributed systemically through circulation,the cancer inhibition effects of these drugs accompanied with massive casualty of normal cells,which leads to serious intolerable systemic toxicity,even accelerate the death of patient.With the development of nanotechnology and oncology,the phenomenon of nanoparticle selectively distributed and retained in tumor tissue has attracted great attention.Further studies illustrated that the nature of this phenomenon is the interaction between nanoparticle and nanoscale structure of tumor tissue and the phenomenon was termed enhanced permeability and retention(EPR)effect.On the theoretical basis of EPR effect,the concept of nanoscale drug delivery system(NDDS)was proposed.NDDSs achieved better treatment effects and reduced toxicity through selectively deliver drugs into tumor tissues with reduced systemic distribution.However,the two decades of researches revealed that the application results of NDDS were far from ideal state that predicted by EPR effect,the full potential of EPR effect has not been achieved.The drug delivery of NDDS is in complicated body fluid environment,during which,the premature drug release(PDR)is unavoidable under varies of internal and external pressures.For the purpose of reducing PDR,the NDDS must have steady structure and slow drug release profile.On the contrary,NDDS have to release the loaded drug at fast rate to form effective free drug concentration to exert anti-cancer effect in tumor cells.The drug release dilemma is the major challenge faced by NDDS.Objective:For the purpose of reducing PDR while ensuring fast intracellular drug release and fully realize the potential of EPR effect to eliminate cancer stem cell(CSC)with a novel nanoscale drug delivery platform,we used clathrin protein with natural activity as modification material of NDDS.Clathrin coat was formed on the surface of lipid nanoparticle(LN)loaded with salinomycin(LN-SAL)by taking advantage of the self-assembly property of clathirin to construct a novel NDDS,clathrin modified LN-SAL(CMLN-SAL).The research contains the following experiments:(1)Preparation and characterization of CMLN-SAL.The film-sonication method was used to prepare LN-SAL.Intact clathrin coated vesicle(CCV)was isolated from rat liver cells by ultracentrifugation and free clathrin was extracted from CCV.CMLN-SAL was incubated with clathrin in a simulated intercellular environment to polymerize clathrin on the particle surface.The purification method was established to study the drug loading property of LN-SAL and CMLN-SAL and analyze the main content of CMLN-SAL.The hydrodynamic diameter(HD)and Zate potential of LN-SAL and CMLN-SAL were measured by dynamic light scattering(DLS).(2)The morphology and rigidity of CMLN-SAL.LN-SAL and CMLN-SAL nanoparticles were observed under transmission electron microscopy(TEM)and atomic force microscopy(AFM)to measure particle diameter and height.The rigidity of particle was calculated by comparing particle diameters and heights measured by different methods.(3)Drug release kinetics of CMLN-SAL.Drug release kinetics of LN-SAL and CMLN-SAL were studied under varies of conditions including still plasma,sonication,intracellular environment and external pressure.(4)Intracellular uptake of CMLN-SAL.Propidium iodide(PI)was used as fluorescent probe to prepare fluorescence labeled LN-PI and CMLN-PI.The intracellular uptake efficiency of LN-PI and CMLN-PI was studied by flow cytometry and confocal microscope.The further investigation of intracellular uptake mechanism was performed using chlorpromazine,nystatin,amiloride hydrochloride,monensin and colchicine as cellular uptake inhibitor.(5)Anti-tumor effects of CMLN-SAL.CSCs in HepG2 and Huh-7 cell lines were marked by fluorescent antibodies and the inhibition effects of LN-SAL and CMLN-SAL on CSC subpopulations were examined by flow cytometry.The study of inhibitory effects of LN-SAL and CMLN-SAL on cultured HepG2 cells was performed.LN-SAL and CMLN-SAL induced cancer cell apoptosis was detected by flow cytometry.The results of experiments are as following:(1)The methodology of quantitative analysis of SAL was established.The linear equation of SAL is A=0.02457 C+0.01156(R~2=0.99802).The methodology of purification of LN-SAL and CMLN-SAL through mini Sephadex G25column-centrifugation was established.The elution curve indicated that in the first 3times of elution,only 2.2%free SAL and 4.6%free protein was eluted,while LN-SAL and CMLN-SAL was completely collected.This method was fully capable to separate nanoparticles from their constructing materials in solution.The clathirin nature of the protein coat on CMLN-SAL was confirmed by SDS-PAGE and western-blot.Based on the purification of LN-SAL and CMLN-SAL,the analysis of their drug loading profile and chemical content was performed.The encapsulation efficiency(EE)and drug loading content(DLC)of LN-SAL was 97.96±1.67%and 12.81±0.19%respectively.The EE and DLC of CMLN-SAL was 93.50±1.93%and 10.14±0.22%respectively and the CMLN-SAL particle also contained 17.58±0.3%protein and 72.29±0.08%lipids.The hydrodynamic diameter of LN-SAL and CMLN-SAL measured by DLS was198.83±1.07 nm and 310.13±2.99 nm respectively and Zeta potential was-33.53±1.52 mv and-36.63±0.23 mv respectively.(2)The success of forming clathrin coat on the surface of CMLN-SAL was further confirmed by TEM observation morphologically.The TEM diameter(TEMD)of LN-SAL and CMLN-SAL was 123±16 nm and 312±33 nm respectively.Under AFM,the diameter(AFMD)of LN-SAL and CMLN-SAL was 31±3 nm and 231±54 nm respectively and the height(AFMH)of LN-SAL and CMLN-SAL was 12±2 nm and31±3 nm respectively.The particle rigidity was calculated by comparing particle diameters and heights measured by different methods.The results indicated the rigidity of CMLN-SAL was significantly higher than that of LN-SAL.(3)The results of drug release kinetics experiments showed that under the circumstances of still plasma,sonication and external pressure,the drug release of CMLN-SAL was significantly slower than that of LN-SAL.In the intracellular environment,the clathrin shell of CMLN-SAL was hydrolyzed by HSC70 and the drug release rate of CMLN-SAL was significantly accelerated to the level of LN-SAL.(4)The intracellular uptake of LN-PI and CMLN-PI was analyzed qualitatively and quantitatively.The results revealed that during the same period,the CMLN-PI treated cells showed significantly stronger fluorescent intensity.Mechanism study of cellular uptake found that chlorpromazine was more effective to inhibit CMLN-SAL uptake than LN-SAL uptake.In chlorpromazine treated cells,the uptake of CMLN-SAL and LN-SAL was reduced by 38.1%and 20.9%respectively.The inhibitory effect of colchicine was stronger on cellular uptake of LN-SAL.The presence of colchicine reduced uptake of LN-SAL and CMLN-SAL by 27.9%and 12%respectively.The results indicated that the intracellular uptake of CMLN-SAL was more clathrin-mediated endocytosis dependent and the uptake of LN-SAL was more macropinocytosis dependent.(5)Among three SAL formulations(LN-SAL,CMLN-SAL and free SAL),CMLN-SAL was the most effective inhibitor against CSC subpopulation.In the experiment of inducing tumor cell apoptosis,the early apoptosis,late apoptosis and cell death in CMLN-SAL treated cells were significantly higher than that of LN-SAL and free SAL.CMLN-SAL showed strong anti-tumor abilities.Conclusion:Our research used clathrin as modification material to construct a novel NDDS CMLN-SAL.With the higher rigidity arise from its clathrin shell,the CMLN-SAL was capable of resisting multiple internal and external pressures and reducing the drug loss in transportation.In cytoplasm,the clathrin shell of CMLN-SAL was hydrolyzed by HSC70 and the loaded drug was rapidly released.Additionally,CMLN-SAL had higher intracellular uptake efficiency,which allowed the NDDS to deliver more drugs into tumor cells to form effective drug concentration rapidly in cytoplasm and cause more damage to tumor cells.In conclusion,CMLN-SAL is an efficient NDDS,which resolved the drug release dilemma and possess significant value in research and clinical application.Our works established the foundation for the further research and clinical application of CMLN-SAL.