Molecule Design And Assembly Of Cisplatin Thermosensitive Magnetioliposomes

Cisplatin, a potent cell cycle nonspecific antineoplastic drug, is widelyused against various solid tumors, such as melanomas, head and neck tumors,endometrial cancer cell, as well as non-small cell lung cancer. Althoughtreatment with this drug is often effective, its therapeutic exploitation islimited by its severe toxicities, including nephrotoxicity, ototoxicity, as well asother side effects such as nausea and vomiting. Therefore, the significantresearches of cisplatin are selecting delivery to tumour cells, reducing drugtoxicity, and improving the therapeutic index.Thermosensitive magnetoliposomes, used as a targeted drug deliverysystem, trap magnetic nanoparticles （MNs） and drug in liposomes. Liposomestrapped with MNs allow the liposomes to be concentrated in a desired area inthe organ of a patient by magnetic force; meanwhile, the drugs could display arapid release under the local hyperthermia. Compared with ordinary liposomes,thermosensitive magnetoliposomes can play a partial destruction effects,reduce the impact of normal tissues, and improve the therapeutic effect.Objective: Assembly of cisplatin magnetic liposomes was designedusing a suitable phospholipid precursor, and a reasonable preparation processwhich could improve the drug encapsulation efficiency and targeting ofliposomes. Furthermore, the interaction of dipalmitoyl phosphatidylcholine（DPPC） with a variety of metal ions and the mechanism of the metalion-assisted cisplatin loading model were investigated, which could provide anew approach to improve drug loading efficiency.Methods:The Fe3O4MNs coating by organic functional group on surface weresynthesized by a one-step modified hydrothermal method, then, weresubjected to XRD, TEM, IR characterizations. Using egg lecithin （EPC） as phospholipid material, the magnetic liposomes were prepared by differentmethods. Using selected film dispersion method, the optimized prescription ofmagnetic liposomes was selected by orthogonal test in which encapsulationefficiency of cisplatin was used as index and graphite furnace atomicabsorption spectrophotometry （AAS） was used to determine cisplatin.Based on EPC phospholipid material, and film dispersion method,magnetic liposomes with different microstructure were prepared by the twodifferent procedures. In procedure I, MNs were combined with phospholipidsduring film formation, and in procedure II, MNs were mixed with drugsduring hydration. The microstructures of liposomes were observed bytransmission electron microscope （TEM） to seek for a reasonable assembly ofmagnetic liposomes. The release of cisplatin from liposomes was evaluated invitro system.DPPC based cisplatin thermosensitive magneticliposomes were preparedusing two different procedures. From the molecular level point of view, theinfluence of the single component phospholipid precursor on physiochemicalproperties of liposomes was investigated. The stability and drug releasecharacter of DPPC-based liposomes in vitro were evaluated, and the magnetictargeting was investigated by animal experiments. The interactions betweenfour kinds of metal ions (Zn²⁺, Cu²⁺, Mn²⁺, Mg²⁺) and DPPC were analyzed bydifferential thermal analysis （DSC）, infrared spectroscopy （IR） and Ramanspectra. Liposomes prepared using metal ions-assisted loading model and themechanism of its higher drug encapsulation efficiency were investigated.Results:The Fe3O4MNs were synthesized by a one-step modified solvothemalmethod at low temperatures and short times in a N2-free environment. Themean particle size of superparamagnetic MNs is8.9nm. The oleicacid-coating MNs could change their surface properties, improve thelipophilic character, and increase their combination ability with phospholipids.Liposomes were synthesized by film dispersion method and theencapsulation was determined using sephadex G-50column as separating instrument. The optimized prescription of magnetic liposomes was:100mg·mL^-1 of phospholipids,1mg·mL^-1 of cisplatin, EPC:CH=7:1（w/w） andEPC: Fe3O4=5:1（w/w）.The microstructures of EPC-based magnetic liposomes synthesized bythe two different procedures were observed by TEM. In procedure I, MNswere embedded in a phospholipid bilayer. In procedure II, MNs werecontained in an interior aqueous compartment. MNs-loaded liposome byprocedure I was superior over procedure II both in cisplatin encapsulationefficiency and MN content; the encapsulation efficiency of cisplain inprocedure I and II liposomes were34.90±3.31%and28.34±4.72%,respectively, while, MN content in procedure I and II liposomes were4.19±1.70and3.05±3.13mg·mL^-1respectively. Encapsulation efficiency ofcisplain in both I and II magnetic liposome were higher than MNs-freeliposome. The release profile of all the three different liposomes in vitro fittedwith a first-order equation which would ensure sustained-release character.When DPPC was used as a heat-sensitive phospholipid material,liposomes showed thermosensitive at the ratio of DPPC/CH lower than7:1.Thermosensitive magnetic liposomes prepared by procedure I also trappedwell-dispersed MNs in the bilayer; the encapsulation efficiency of cisplain andthe content of MN were33.51±3.30%and2.34±0.09mg·mL^-1, respectively.Compared with EPC-based magnetic liposomes, the mean size of liposomevesicles was smaller, and vesicles uniformity and magnetic particles dispersityin the phospholipid layer are better. Furthermore, as-prepared thermosensitivemagnetic liposomes showed fine heat sensitivity proved by DSC and in vitrothermal release experiment, meanwhile, showed the magnetic targeting in vivowhich can delivery cisplatin to the target location proved by animalexperiments.The electrostatic interation between the metal ions and phospholipid acylgroup of DPPC resulted in the change of DPPC conformation and thehydrocarbon chain of lipid molecules arranged closer than before. The TmofDPPC is increased. Followed the increasing atomic number and decreasing radius of metal ions, the charge density of cations increase, and theelectrostatic interaction between metal ions and DPPC strengthen. Theencapsulation efficiency of the metal ion-assisted loading liposomes wassignificantly higher than metal ion-free liposomes, and still maintained goodthermal release.Conclusion: The Fe3O4MNs synthesized by a one-step modifiedhydrothermal method were used to prepare magnetic liposomes. The magneticliposomes were prepared by procedure I in which combined with MNs andphospholipids during film formation. MNs were embedded in a phospholipidbilayer and the encapsulation efficiency of cisplain and the content of MNwere higher than those of conventional liposome. Thermosensitive magneticliposomes prepared by thermosensitive phospholipid material showed the dualeffect both of heat sensitivity and targeting. It would ensure sustained-releasecharacter in vitro and vivo. The investigations on magnetic liposomes in thispaper can provide a strong reference for targeted drug delivery system. Inaddition, the research on the interaction between metal ions and DPPC offereda theoretical foundation for further investigations of metal ion-assisted drugloading liposomes.