Uptake Of Album Nanoparticles By Cells

Background:Nanocarrier drug delivery systems is to play the effect, not only concentrate in the rake site, but also ensure drug into the role of intracellular targets. Avoiding the effects of opsonin and lipoproteins, and phagocytosis by macrophages are the important condition for drug targeting, but if the nanocarrier drug delivery systems can not reach the target site within cells in a reasonable manner, and effectively release drugs, can not play a good effect.Targeted nanoparticles reach the target tissue/target organ and release the drug in the extracellular, put free drugs or carried drugs directly into the cell, release the drug in the intracellular targets in a timely manner. The key of former passing way is that the drug release characteristics of the delivery system. Extracellular drug release behavior determine the ultimate efficacy. The processes of deliverying into cells in the latter drug delivery systems involved with endocytosis, intracellular transit, intracellular drug release, the cell efflux and so complex disposal processes.It is concern and challenging parts in pharmacy areas.Most of cells take up exogenous nanoparticles mainly in an endocytosis method, and endocytosis is divided into clathrin-mediated endocytosis, caveolae-mediated endocytosis, pinocytosis and non-caveolae dependent endocytosis. In recent years, caveolae-mediated endocytosis of concern-mediated endocytosis is that deliverying carrier to caveosome with the easing of pH conditions and biological conditions,and then transported to the endoplasmic reticulum, Golgi apparatus and cytoplasm avoiding the classic endosome/lysosomal transported system.Among them, the clathrin-mediated endocytosis the most common. Nanoparticles in the cell into the endosome, further fusion with lysosomes. It is bad to non-lysosomal targeting drugs. Types of particles, surface charge, physicochemical properties and other factors will influence and even change the endocytic pathway of cells. Changing the intracellular distribution in the nano-delivery systems and transport process in the lysosome, thus affec the drug concentration in intracellular targets. Meanwhile, the endocytosis efficiency, cell elimination,intracellular drug release were also influenced by the carrier.Through experimental content and pre-experimental results, we prepared calcein HSA-NPs using calcein as a model drug for hydrophilic drugs with small molecule weight, investigated the impact of nanoparticles wrapped towards cellular uptake and internalization pathways.Purposes:To prepare albumin nanoparticles, and further study the mechanism of endocytosis.Methods:1.Preparation and CharacterizationWe prepared calcein HSA-NPs with desolvation and emulsion cohesion, and determine the optimal formulation and process conditions. Desolvation:first put albumin into aqueous solutions containing the drug to dissolve, then add the organic solvent so that the albumin dehydration condensation shrink into pellets, and finally solidify into the drug-loaded nanoparticles with formaldehyde or glutaraldehyde as a crosslinking agent. Emulsion condensation method:①Emulsion. Drug and albumin dissolved or dispersed in water as the aqueous phase, add which into stired the oil phase so called w/o emulsion.②The curing. Heat curing or chemical crosslinking can be used to make albumin cured after the crosslinking reaction,③The separation. Put the formated nanoparticles in the ether to soluble oil, centrifugal separation.For the colloid solution of nanoparticle was an unstable system,the particles in solution would concentrate together and a part of drugs would seep out after a long time store.So free drying technology was necessary to apply to elevate its stability. Using the scannig electron microscope, the shape and surface of the nanoparticles were observed. The particle size distribution was measures by particle size analyzer. Drug release from Calcein HSA nanoparticles was evaluated by means of a dynamic dialysis technique. The stability test:the freeze drying Calcein HSA nanoparticles were stored at4"C, then the permanent stability was investigated by determination of its appearance、redispersibility and envelopment rate at different times. Classic MTT was measured to assay cytotoxicity of albumin nanoparticles, doxorubicin hydrochloride for injection as a positive control group. Determination of cell viability was investigated by multifunctional microplate reader after1mg mL-1drug solution with the cells for48h.2. Nanoparticles endocytosis mechanismMultifunctional microplate reader, λem=509nm, the laser scanning λex470nm was used to detect the fluorescence intensity of the different time points when cell lysates, the study of cellular uptake and intracellular distribution through confocal microscopy and fluorescence microscopy. For the investigation of the cellular uptake mechanism, cells inoculated at1×106cells/well on6well plates until completely adherent growth after24h, the medium was discarded and replaced by endocytosis inhibitor sodium azide (NaN3),2-deoxyglucose (2DG), chlorpromazine (CPZ) and filipin (Filipin), cells cytochalasin D, sucrose (sucrose) medium, added lmg·mL-1contained calcein nanoparticles after cells incubated30min, cultured for1h. Then washed with PBS, cells were collected after trypsin digestion, washed three times in PBS, the average fluorescence intensity was determined with flow cytometry, ultra-thin sections of cellular uptake was observed by transmission electron microscopy. Finally, the effects of calcein of HSA-NPs cell on F-actin protein were observed using fluorescence microscopy of.Results:1.Preparation and CharacterizationWe Prepared calcein albumin nanoparticles using Desolvation, the best preparation prescription and process was:the concentration of albumin solution1%calcein concentration1mg·mL-1, by adding pH=9buffer, stirring speed of700rpm, the curing time of12h. Centrifugal speed of15000r·min-1,30min, the lyophilized nanoparticles looked better, even color, easy to store. The water shake for a moment that is uniformly dispersed, photon correlation spectrometer measured an average diameter of204.8nm, the poly-dispersion is0.164, and the zeta potential-29.6mV. Most of the particle distribution within the100-300nm range. Nanoparticles was observed for the spherical, smooth surface by TEM. The in vitro release results showed the calcein albumin nanoparticles had a good sustained release,96h released less than80%, while calcein solution released completely about10h. That can guarantee that drugs are not or small quantities of nanoparticles in the releasis at the subsequent cell experiments, to avoid the uptake of free drug that interferes with the experimental results.Stability results showed that albumin nanoparticles had a good appearance.re-dispersion and particle size after one year at4℃refrigerator,, so that the preparation was stable at low temperatures. Cytotoxicity results showed that compared with blank control group, the albumin nanoparticles and calcein albumin nanoparticles had no effect on cell viability, cytotoxicity of doxorubicin solution was more significant. Nanoparticles on the growth of HepG2, L02. HUVEC and A549cells were not affected. 2. Nanoparticles endocytosis mechanismThe semi-quantitative experiments about the cellular uptake showed that the cellular uptake of nanoparticles fluorescence intensity increased following the prolongation of incubation time, cellular uptake of calcein of HSA-NPs were most at4h. According to qualitative experiments,we found that the green fluorescent signal in the cells little.at0.5h,1.5h incubated time, with the prolongation of incubation time, the nanoparticles were more and more closely to the nucleus, nanoparticles clustered around the nucleus at4h, nanoparticles distribution around the nucleus at10h. Cellular eliminated experiments, the nanoparticles had been around the nucleus within4hours.In this study, low temperature and energy metabolism inhibitor2DG and NaN3calcein were to study cell aggregation. The results show that both cases, the calcein uptake were significantly lower than the control group (P<0.05). That calcein HSA-NPs were uptaked by endocytosis.2DG/NaN3, CDs. of CPZ and Sucrose can significantly inhibit intake of calcein HSA-NPs by the L02, the inhibition rates were68.59%,69.65%,71.73%,67.81%. the intake of Filipin by cells had no significant effect.The impact of nanoparticles on cellular F-actin protein, the photos of cell monolayer with albumin nanoparticles incubated2h,4h showed that, compared with the control group, the fluorescence intensity significantly decreased, the fluorescence of the cytoplasm had been unable to identify, and mostly the F-actin protein depolymerizated to form a loose circular ring structure. The structure of F-actin was changed.Conclusions:We prepared calcein HSA-NPs using calcein as a model drug for hydrophilic drugs with small molecule weight by Desolvation, nanoparticles are smaller particle size, round shape, more uniform size. Endocytosis of albumin nanoparticles present time, concentration, temperature dependence, are the active transport process, which can significantly improved the capacity of calcein with the poor transmembrane. The endocytosis method of Calcein HSA-NPs were mainly by clathrin-mediated endocytic pathway and pinocytosis, after internalization mainly concentrated in the lysosomes. The effects of albumin nanoparticles on the cell F-actin protein were that nanoparticles maked cell F-actin protein mostly depolymerization to form a loose circular ring structure, affecting the tight junctions between cells.