Construction And In Vivo / In Vitro Evaluation Of Active Components Of Traditional Chinese Medicine Nanocrystal-loaded Liposome Drug Delivery System

In China,many active ingredients of traditional Chinese medicine have significant efficacy and are widely used in clinic,such as andrographolide,paclitaxel,puerarin and so on,but there is a problem of poor water solubility.The poor water solubility of drugs will lead to low bioavailability and insufficient efficacy in vivo,and make intravenous administration challenging.In the past few decades,researchers have launched a variety of strategies to develop intravenous injections of poor water-soluble active ingredients of traditional Chinese medicine,such as nanocrystals,polymer micelles and liposomes.Nanocrystal technology shows great potential in many delivery systems because of its high drug loading rate(up to 100%),easy magnification and organic solvent-free preparation process(Top-down method).Many studies have shown that the preparation of water-insoluble drugs into nanocrystals can improve the in vivo circulation time and bioavailability of drugs.However,other studies have shown that intravenous administration of nanocrystals may not change or improve the pharmacokinetic properties of the drug.As a drug delivery system,liposomes help to improve the behavior of drugs in vivo and prolong their scope of action.When the drug is loaded in the liposome,its pharmacokinetic behavior will be complicated by the mixture of two independent pharmacokinetic processes(Drug and Carrier)after intravenous injection.at the same time,the physical and chemical properties,dose and encapsulated drugs of liposomes will affect their pharmacokinetic behavior.Although compared with traditional intravenous preparations,nanocrystals and liposomes have made great progress in the development and application of intravenous injections of waterinsoluble active components of traditional Chinese medicine,but their pharmacokinetic behavior in vivo is not clear.At the same time,some studies have found that different nanocrystals or liposomes may have different cell behaviors in vitro.In this regard,we selected three drugs with poor water solubility,namely Andrographolide(ADR),Docetaxel(DTX)and Puerarin(PUE),to investigate the pharmacokinetics and in vitro cell behavior of nanocrystalline and liposome drug delivery systems.At the same time,We imagine that a new type of drug delivery system nanocrystal-loaded liposome will be obtained by loading nanocrystals into the liposome of lipids,which will change the pharmacokinetic process after the drug is released from the liposome carrier in the blood circulation..And to explore whether the drug delivery system of nanocrystalloaded liposome will change the original cellular uptake behavior of nanocrystals or liposomes,thus affecting its cellular efficacy in vitro.It can provide reference and basis for the development and application of intravenous injection of new active components of traditional Chinese medicine.In this paper,a method for the determination of andrographolide and docetaxel in liposomes in vitro was established and its methodology was investigated.The results showed that the specificity,linearity,precision and recovery of the two methods were good,and could be used for the determination of andrographolide and docetaxel in liposomes in vitro.to provide a basis for follow-up prescription optimization and quality evaluation.Secondly,the liposome was prepared by thin film hydration method,and the preparation method of liposome was determined by morphology and particle size.The final preparation process was determined as follows: 100 m L eggplant-shaped flask,the ratio of soybean phospholipid to cholesterol at 4:1,anhydrous ethanol as organic solvent,40 ℃ and 100-110 mpa,100 rpm,30 min.Finally,the preparation technology of nanocrystal-loaded liposome was investigated by single factor.The prescription of ADRNC was ADR 25 mg,homogenization pressure was 1000 bar.The prescription of DTXNC was DTX 20 mg,PVP K30 20 mg,homogenization pressure was 1200 bar.The dosage of soybean phospholipid and cholesterol were 100 mg and25 mg,respectively.The physical and chemical properties of drug nanocrystals,drug liposomes and drug nanocrystal-loaded liposome prepared by the optimum process were investigated,including morphology,particle size,entrapment efficiency,drug loading,crystal form,stability and in vitro release.The appearance of the prepared nanocrystal-loaded liposome is milky solution with uniform particle size.The average particle size of Andrographolide nanocrystal-loaded liposome(ADRNC-L)was 221.8 ±2.1 nm,the entrapment efficiency and drug loading were 21.82 ±3.27% and 0.84 ±0.13%,respectively.The average particle size of Docetaxel nanocrystal-loaded liposome(DTXNC-L)was 190.6 ±4.5 nm,the entrapment efficiency and drug loading were 41.02±1.67% and 1.23 ±0.05%,respectively.The average particle size,entrapment efficiency and drug loading of Puerarin nanocrystal-loaded liposome(PUENC-L)were 187.7 ±2.9nm,62.43 ±2.13% and 1.62 ±0.18%,respectively.Both ADR and DTX in nanocrystalloaded liposome exist in amorphous form.Both ADRNC-L and DTXNC-L have good stability.The in vitro release results showed that ADR-L and ADRNC-L had a certain sustained release effect compared with ADR injection in PBS buffer(PH=7.4),and the release behaviors of ADR-L and ADRNC-L were similar.DTX-L and DTXNC-L could promote the release of DTX in PBS buffer(PH=7.4),and it was found that the release rate of liposome loaded DTXNC was slower than that of DTX-L.The pharmacokinetic behaviors of nanocrystals,liposomes and nanocrystalline liposomes in vivo were studied by pharmacokinetic experiments in rats.The results show that the pharmacokinetic behavior of drug nanocrystals will be different due to the differences of drug properties and physicochemical properties of nanocrystals.The particle size of ADR increased due to its poor stability after being prepared into ADRNC and its aggregation after entering the blood circulation.Because of the large saturated solubility of the drug itself and the small particle size after preparation of PUENC,PUE will be dissolved in the blood circulation immediately due to the rapid dilution and stirring of the blood circulation,which can not improve the pharmacokinetic behavior of ADR and PUE in vivo.Used to deliver DTX,DTXNC can maintain better particle size and shape after entering the blood circulation,and it has faster release and better bioavailability in vivo.The main reason for the delivery of ADR,DTX and PUE drugs by the liposome drug delivery system may be that the drug is released from the liposome immediately after the liposome drug delivery system enters the blood circulation,thus causing the pharmacokinetic behavior to be similar to that of drug injection.At the same time,due to the effect of opsins,the unmodified liposome drug delivery system is easily absorbed and cleared by macrophages in MPS from the blood circulation after intravenous injection,resulting in inability to improve the in vivo pharmacokinetic behavior of ADR,DTX and PUE drugs.For nanocrystalloaded liposome,the pharmacokinetic behaviors of ADR,DTX and PUE are similar to those of liposomes,indicating that the pharmacokinetic behavior of drug-loaded nanocrystalline liposomes is similar to that of drug liposomes,which is easy to be ingested and cleared by MPS,and can not further change or improve the pharmacokinetic behavior of liposomes.Finally,the cellular uptake behavior of DTXNC,DTX-L)and DTXNC-L in different tumor cells and the anti-tumor efficacy in vitro were studied by studying the mechanisms of cytotoxicity,cell uptake and cell uptake.The results of cytotoxicity study showed that when liposome was used as drug delivery system,it could significantly improve the anti-tumor effect of DTX on four kinds of tumor cells in vitro,and the anti-tumor effect of DTXNC-L was stronger than that of DTX-L and DTXNC in vitro.The cellular uptake experiments showed that in four kinds of tumor cells,the cell uptake increased with the increase of DTX concentration,and there was no saturation phenomenon at the experimental concentration,indicating that DTX uptake may not need carriers,mainly through concentration-dependent passive diffusion into cells.Free DTX is a small hydrophobic molecule with high cell membrane permeability,which enters the cell through passive diffusion,while DTXNC,DTX-L and DTXNCL are mainly ingested by endocytosis,resulting in faster cellular uptake of DTX within2 hours.The results of the study on the mechanism of cell uptake showed that in A549 cells,the uptake of DTX was mainly through passive diffusion,and the uptake of DTXNC,DTX-L and DTXNC-L was energy-dependent.DTXNC entered the cell mainly through grid protein-mediated pathway,DTX-L entered the cell mainly through caveolin-and non-caveolin-mediated pathway,and DTXNC-L entered the cell mainly through caveolin-mediated pathway.