| The development of formulation for water insoluble active compounds is a huge challenge.Nowadays,approximately 70?80%of the discovered drug active ingredient(API)suffered the defects of poor water solubility.Therefore,it is highly desired to improve the bioavailability of the poor water soluble drug active compounds.In recent decades,the nanocrystal technology has emerged as a new pharmaceutical development technology,and has been applied in pharmaceutical industry.The drug nanocrystal suspension is submicron colloidal dispersion containing pure drug particles stabilised by a small amount of excipients,which can significantly improve the solubility and dissolution rate of poor water soluble drugs.Theoratically,the drug nanocrystals could achieve 100%drug loading capacity.In addition,comparing with other nanocarrier-based drug delivery systems,nanocrystals exhibited superior drug delivery ability,improved stability and decreased side-effects.Moreover,nanocrystals can be administered in a variety of administration ways,and thus can provide more options for their clinical applications.Up to now,17 types of drug nanocrystal products has been introduced into the market,and more than 20 types of products are undergoing clinical trials.The nanocrystal technology is considered as the most successful pharmaceutical nanotechnology in the past twenty years.At present,the researches about nanocrystals mainly focused on the preparation and characterization.However,the preparation and optimization of nanocrystal formulations were mostly based on the operational feasibility,but were not closely correlated to the in vivo performance of drug nanocrystals.Without the study of the absorption mechanism of nanocrystals,it is difficult to support and guide the development of reasonable nanocrystal prescriptions.Nanocrystals are usually provided as oral formulations,but the absorption mechanism of gastrointestinal tract is still rarely reported.It is generally believed that the increased intestinal absorption and subsequent increased bioavailability can be mostly attributed to increased nanocrystal dissolution rate.What's more,some researchers tend to believe that the mechanism of in vivo uptake of nanocrystals is similar to those of other nanocarrier-based delivery systems.However,there is no compelling in vivo evidence to directly support these proposed mechanisms.Consequently,it is necessary to systematically investigate the absorption mechanism of drug nanocrystals in gastrointestinal tract.An overall understanding of the transmembrane characteristics and oral absorption mechanism of drug nanocrystals will play an important role to guide the rational development and optimization of nanocrystal oral formulations.In this paper,a systematic study on the oral absorption mechanism of nanocrystals was carried out.Firstly,the process of transmembrane of nanocrystals was analyzed at the cell level.The mechanism of cell surface adsorption,apical membrane uptake,organelle localization and basolateral membrane excretion were elucidated.Based on this,the transport mechanism and characteristics of nanocrystals were investigated in different intestinal segments at organ level.Finally,the correlation between in vivo and in vitro results was verified at the whole animal level.Furthermore,the biological effects caused by the decreased nanocrystal size were also verified.The transmembrane characteristics and oral absorption mechanism of nanocrystals were systematically investigated by a gradually progressive study strategy.The main experiments and results of this project are summarized as follows:1.Three types of drug nanocrystals with different sizes were prepared by utilizing the precipitation method or wet-milling method.Spherical Coumarin-6 nanocrystals with positive zeta potential were prepared by the precipitation method,and a fluorescence method was established for quantitative analysis of Coumarin-6.Rectangular Aprepitant nanocrystals with negative zeta potential were prepared by a wet milling method,and an LC-MS/MS method was established for the quantitative analysis of Aprepitant.Conoidal Quercetin nanocrystals with negative zeta potential were prepared by a wet milling method.Generally,for coumarin-6,Aprepitant and Quercetin,nanocrystals with size of around 200 nm,500 nm,3 ?m were all synthesized.For all the nanocrystal samples,the size distribution was narrow,the quantitative analysis method exhibited good linear performance,and lower detection limit could satisfy the requirement of all the experiments.2.The interaction between the nanocrystals and the surface of MDCK cells was studied.The experiment results showed that the drug nanocrystals can be rapidly and efficiently adsorbed onto cell surfaces and can initiate subsequent interactions with cells.The cell adsorption effect of drug nanocrystals increased with the decreasing of the nanocrystal sizes,and was greatly influenced by the adhesion agent and surface charge of the nanocrystals.As to the aprepitant nanocrystals,due to the presence of adhesion agent HPC-SL,the cell adsorption of the three groups of aprepitant nanocrystals with different sizes did not exhibited obvious differences.On the other hand,even if the adhesion agent was present,the adsorption efficiency of the aprepitant nanocrystals remained low due to the negative surface charge of the nanocrystals.3.The cellular uptake processe of drug nanocrystals were observed.Nanocrystals can be endocytosed into the cells in the form of nanocrystal particles,and these process included multiple protein-mediated pathways.The endocytosis efficiency of nanocrystals increased with the decrease of their sizes and also depended on the concentrations and incubation times of nanocrystals.The size of nanocrystals not only affected the speed and extent of cellular uptake,but also influenced the protein-mediated pathway of cell endocytosis.The micro-sized drug crystals were mostly uptaken by cells through macrocytosis.While the nano-sized drug crystals were uptaken by various endocytic pathways,including clathrin-mediated,caveolin-meidated,macrocytosis and actin-involved cell endocytosis process.These diverse endocytic pathways may be the reason of the high cellular uptake efficiency of nanocrystals.4.The intracellular colocalization of the organelle and nanocrystals was observed.The drug nanocrystals were mainly distributed in lysosomes after endocytosis,while their colocalization with Golgi and endoplasmic reticulum was not obvious.The drug nanocrystals were mostly metabolized by lysosomes in cells,and thus,the efficiency of transmembrane was limited.5.The process of nanocrystals piercing the cell bottom membrane was studied.Nanocrystals were transported in the form of whole nanocrystal particles,and the bypass pathway was not obvious.The nanocrystal formulation could improve the permeability of the poor water soluble drug,and the permeability increased with the decreasing of the nanocrystal size,involving passive diffusion of drugs and multiple protein-mediated endocytosis pathways of nanocrystals.However,the overall transmembrane efficiency of nanocrystals was still low.This may be related to limited re-modification of the endoplasmic reticulum and Golgi in cells.6.The intestinal transport characteristics of nanocrystals were studied by in vitro intestinal valgus model and in vivo intestinal perfusion model.The 200 nm and 500 nm aprepitant nanocrystals both showed largest absorption in the duodenum.However,the 3 ?m aprepitant nanocrystals showed largest absorption in jejunum.Nanocrystals can improve the speed and extent of the intestinal adsorption of the poor water soluble drug,and smaller nanocrystal size lead to better performance.Besides,the absorption of nanocrystals can be saturated at high concentrations,which also indicated that the absorption of nanocrystals in the intestinal tract included passive diffusion and protein-mediated endocytosis pathways.7.The pharmacokinetic characteristics of nanocrystals were investigated in rat model.Nanocrystals can increase the oral absorption and bioavailability of poorly soluble drugs.Aprepitant nanocrystals significantly increased the AUC and Cmax,and reduced the Tmax of aprepitant.Nanocrystals improved the rate and extent of absorption of poorly soluble drugs,and smaller nanocrystal size lead to better performance.8.Nanocrystals can improve the biological effects of poorly soluble drugs.Quercetin nanocrystals can inhibit the proliferation,adhesion,migration and invasion of tumor cells in a concentration-and time-dependent manner,and their antitumor mechanism may be related to inhibition of STAT3 protein expression.The nanocrystal formulation can significantly enhance the biological effects of quercetin at the molecular and cellular levels,and the smaller the particle size was the more obvious the effect was,which may be due to the increase of solubility and transmembrane transport capacity of quercetin..In conclusion,nanocrystals can improve the oral absorption and bioavailability of insoluble drugs,which is the result of the increase of solubility and permeability of insoluble drugs.The mechanism is related to the passive diffusion of drugs and multiple protein-mediated endocytosis pathways in gastrointestinal tract.For BCS IV drugs with poor permeability,nanocrystals can not only improve their solubility,but also improve the permeability of these drugs.However,the efficiency of improvement needs to be further improved.In view of this,the design can be optimized from specific aspects of transmembrane transport of nanocrystals.During the adhesion process between nanocrystals and cell surface,the passive adsorption,charge modification,addition of adhesive and the ligand-receptor interaction targeting cell membrane specific receptors can enhance the cell adhesion effects of nanocrystals,and then induce the cell endocytosis of nanocrystals.In the transmembrane transport of nanocrystals,for the cellular bypass pathway,ingredients like chitosan and polyacrylate can open the tight junction and increase the permeability of cells.For the piercing membrane pathway,on one hand,ligand modification can be carried out on the surface of nanocrystals to increase the efficiency of cell endocytosis,and on the other hand,materials with lysosome escape function can be used.For example,polyethyleneimine and dendritic polymers are introduced into the nanocrystal structure to reduce the metabolism of nanocrystals in lysosome,which improve the transmembrane transport efficiency of nanocrystals,and then enhance the oral absorption and bioavailability of insoluble drugs.In this study,the mechanism of increasing transmembrane transport and oral absorption of insoluble drugs by nanocrystals were discussed,and an important idea was provided for the rational design and optimization of nanocrystals.At the same time,it also laid a certain theoretical foundation for the application of nanocrystals in the field of biomedicine.The novel points and new findings of this project are listed as follows:1.In this project,the transmembrane transport characteristics and oral absorption mechanism of nanocrystals were systematically studied at macroscopical level,including cell level,organ level and animal level.It is revealed that nanocrystals can realize transmembrane transport in the whole form,and it is verified in vitro and in vivo that the improvement of transmembrane transport and oral absorption of insoluble drugs is the result of the increase of solubility and endocytosis.These esults provided important direct evidence for the mechanism of the transmembrane transport of nanocrystals.2.The characteristics of nanocrystals in cell surface adsorption,cell apical membrane uptake,organelle localization and transmembrane transport pathway were preliminarily expounded.According to the influencing factors of these parts,the design of nanocrystals can be optimized.It provides reasonable guidance for the development and application of nanocrystals3.The effects of submicron particle sizes on the transmembrane transport and oral absorption of nanocrystals were clarified,which pointed out the way for the selection and optimization of nanocrystal particle sizes. |
PDF Full Text Request