Intracellular Transport Of Nanocarrier Drug Delivery System

The intracellular transport and cellular kinetics in taregeted cells is vital to nanocarrier drug delivery systems. However, systematic studys concerned with intracellular kintecis is limited. In this work, some representative nanocarriers loaded with hydrophobic drugs or hydrophilic drugs were prepared to investigate their transport into tumor cells and the following intracellular trafficking processes based on the study of uptake kinetics, uptake mechanism, nanocarrier intracellular transportation, cellular elimination kinetics and anti-tumor activity in vitro. We compared drug cellular delivery mediated with different nanocarriers in a similar experimental condition, and the effects of pharmaceutical factors on drug transport in vitro and in vivo were also studied.We chose the fluorescent probe coumarin 6 as a hydrophobic modle drug to study the cellular delivery meditaed with three types of widely used nanocarriers. We prepared liposomes composed of SPC and cholesterol, PLGA nanoparticles and lipid emulsions with comparable particle size, zeta potential and loading efficiency. The results of CLSM indicated that there was no significant difference between cellular drug distributions delivered by these nanocarriers and drug solution. Coumarin 6 was located extensively in cytoplasm but isolated by nuclear membrane. The heterogeneous intracellular distribution suggested the different drug affinity with various intracellular components. The presence of serum in culture medium would decrease the cell uptake. Adrop of 20～40% was observed in nanoparticle, liposome and solution group, while lipid emulsions were hardly affected by serum.Cellular uptake kinetic experiments showed that solution group had the highest coumarin 6 intracellular level which was 1.2,1.6 and 4 times of liposomes, nanoparticle and emulsion group. Solution, liposomes and emulsions delivered the drug into cells fast. Intracellular accumulative coumarin 6 increased with time and reached a plateau within 1.5 h except for nanoparticle group which kept growing up still 4 h. In the elimination process, bright perinuclear region disappeared and fluorescence dispersed extensively in cytoplasm after elimination for an hour. Elimination kinetic results indicated that emulsion group had signifacant difference between other groups. We established a kinetic model which divided into fast and slow elimination phases to mathematically describe the cellular elimination process for the first. Solution and all nanocarriers shared a similar fast elimination rate constant. The slow elimination constants were almost the same in solution, liposome and nanoparticle group. However, emulsions had a maximum MRT which was 2-4 times of other groups. The elimination kinetic parameters suggested the emulsions had the strongest intracellular retention ability.Endocytosis inhibition showed coumarin 6 in liposomes and nanoparticles entered into cells via diffusion or direct contact while a part of intracellular coumarin 6 was uptaken through clathrin-mediated endocytosis within emulsions which was attributed to the slow elimination of emulsions.Then the effects of lipid emulsion formulation factors which including oil type, co-surfactants and zeta potential on coumarin 6 uptake were investigated. The results showed oil phase and surface modification with Tween had no significant effect on cell uptake. Intracellular drug level was enhanced by Span series among which Span 20 with HLB 8.6 was the best enhancer. Cationic emulsions could significantly increase the cell uptake and accelerate elimination. Its uptake and elimination rate constant were 4 times and 1.5 times of conventional emulsions, respectively. Endocytosi inhibition test demostrated cationic emulsions upatake enhancement was achieved mainly by cell membrane contact. Living aninal imagine results showed cationic emulsions could speed up coumarin 6 distribution and elimination in vivo.We prepared liposomes using calcein as a model drug for hydrophilic drugs with small molecule weight. The effects of encapsulation into liposomes on the endocytosis of calcein were studied. The encapsulation significantly enhanced the uptake of calcein. Macropinocytosis and clathrin-mediated endocytosis were the main routes by which calcein liposomes entered into cells. After internalization, calcein almost accumulated in lysosomes.Cisplatin (CDDP) is a widely used anti-tumor agent which enters into cells mainly via diffusion and active transport. It makes sense to study nanocarrier drug delivery systems loaded with calcein to some extent. We prepared CDDP-liposomes composed of HSPC and cholesterol and gelatin-CDDP nanoparticles (GPs-Pt) with comparable particle size, zeta potential and loading efficiency. Assaying method of CDDP in nanocarriers by HPLC and method of intracellular Pt by ICP-MS were established.In the uptake experiment, we used trypsinization to discriminate intracellular and membrane associated drug.The results of CDDP-nanocarriers cellular kinetic showed that uptake of liposomes and nanoparticles was lower than CDDP solution in A549 cells, and the intracellular Pt concentration of nanoparticle group was higher than liposomes. Cellular membrane had the strongest adsorption with GPs-Pt. Then the elimination kinetics was analyzed by the compartmental model. The results showed a similar elimination rate constant of liposome and solution which was significantly smaller than GPs-Pt. The MRT of nanoparticles was about 1/3 of the other two groups. The intracellular retention ability of liposomes was much greater than GPs-Pt which was prone to be efflux out. It might be attributed to the quick release of liposomes but slow release of GPs-Pt intracellularly. The order of cellular AUC was CDDP solution> liposomes> GPs-Pt. The following MTT assay was in accordance with the results of AUC, antitumor activity of CDDP-liposomes was much higher than GPs-Pt and its IC50 was lower than CDDP solution. These results indicated that nanocarriers are not always valid to water soluble drugs which have endocytosis mechanisms instinctively.The results of uptake mechanism demonstrated that CDDP-liposomes were internalized by clathrin-mediated endocytosis and macropinocytosis and Pt uptake amount through above two pathways was almost equivalent. GPs-Pt was uptaken mainly by clathrin-mediated endocytosis and the rest part was attributed to macropinocytosis.It was necessary to study the effect of liposomes surface modification on cell uptake and intracellular trafficking considering the little enhancement of liposomes to cell uptake. We tried to introduce gene transfection agent polyethyleneimine (PEI) into anti-tumor drug delivery system. The synthesized amphipathic compound cationic polymer (PEI-Cholesterol) was modified onto surface of CDDP loaded neutral liposomes. We compared the drug transport intracellularly mediated with cationic liposomes, neutral liposomes and CDDP solution and their anti-tumor activity in vitro and in vivo was also compared.The cationic liposomes significantly increased the cellular uptake and membrane adsorption; total cellular and intracellular Pt concentrations were 9 times and 6.5 times of those delivered by neutral liposomes. Compared with CDDP solution, total cellular and intracellular Pt levels also increased up to 5 times and 3 times in cationic liposomes. At the same time, PEI-Chol modified cationic liposomes changed the endocytosis pathway of CDDP-liposomes. Neutral liposomes entered into A549 cells via clathrin-mediated endocytosis and macropinocytosis while cationic ones were internalized by caveolae-mediated endocytosis and macropinocytosis. CLSM further confirmed that cationic liposomes could deliver drugs into intracellular compartments besides lysosomes which were closely related to degradation in endosomal/lysosomal system. MTT test demostrated that cationic CDDP-liposomes could significantly enhance the anti-tumor activity compared with neutral liposomes and CDDP solution. The results of anti-tumor activity in tumor-bearing mice were in highly accordance with the MTT results. Compared with neutral liposomes in vivo, cationic liposomes enhanced the anti-tumor activity which was comparable with CDDP solution. Meanwhile, cationic liposomes could increase the administration safety which efficiently decreased the toxicity caused by CDDP solution.In conclusion, the effects of nanocarriers on the drug cellular transport are related to the instinct of drugs. As to the hydrophobic drugs with high partition coefficiency which could diffuse into cells easily like coumarin 6 or hydrophilic drugs with instinct endocytosis mechanisms like cisplatin, nanocarriers are not always efficient for the drug delivery. After the internalization of nanocarriers, intracellular drug release was important to the pharmacological effect. High uptake doesn't always mean the high anti-tumor activity. The types and components of nanocarriers will influence endocytosis pathways and their intracellular transport and eventually change the cellular kinetics. This study accomplished a part work of nanocarrier transport and their intracellular kinetics. It can provide some information on rational choices and design of nanocarrier drug delivery systems.