The Construction And Preliminary Evaluation Of Nanostructured Lipid Carriers For Gene Delivery That Modified By Oligomeric Positively Charged Amino Acid

Objective:Liposomes have become the most important carrier of drugs, genetic,imaging agents, but there are still problems such as low load rate of biologicalmacromolecules, low cellular uptake efficiency and poor performance of theintracellular release,etc. Therefore, this paper focuses on designing apluripotent efficient biological macromolecular transport carrier.We synthesized DC-Chol that can efficiently improves transfectionefficiency, and a lipid material CHEMS that offers liposomes withpH-sensitive performance. The DSPE-PEG (2000) is connected with the CPPto obtain guidance lipid material DSPE-PEG-CPP,in order to enhance theuptake of liposomes to cells ability. Construction, evaluation of generalnon-compressed drug-loaded liposome, CPP-modified non-compresseddrug-loaded liposome, ordinary liposomes containing compressed matter, CPPmodified liposomes containing compressed matter.Methods:1Cholesterol chloroformate and excess N,N-dimethylethylenediamine aredissolved in the chloroform without alcohol respectively,and reaction in thecondition of0-4℃ice water bath.The reaction progress was monitored byTLC,until cholesterol chloroformate was complete,terminated thereaction(about2h).then organic solvent was evaporated to dryness by rotaryevaporation, recrystallized twice from hot absolute ethanol and dried,andvacuum drying.2Cholesterol with an excess of succinic anhydride was dissolved in20mLtoluene, DMAP as a catalyst, and the reaction was heated to reflux for3hours.The products were dried after organic solvent was evaporated by rotary evaporation. Finally, the crude product after dried was recrystallized twicefrom hot absolute ethanol and dried in vacuum to give the pure gray.3The solution of MAL-PEG-DSPE in chloroform-methanol (3:1, v/v),revolved and evaporated using rotary evaporator, producing an even thin film.The film was hydrated by HBS (pH6.5) solution3.0mL and ultrasonic to formtranslucent system. Then the translucent system was mixed with CPP peptidesolution(5mg CPP was dissolved in HBS (pH6.5)2.0mL), and stirred48h ininlucifugal condition. After an appropriate amount cysteine was added to thesystem, stirring continuously for4h, in nlucifugal condition dialysis2d, andfreeze-dried.4Bank liposomes single factor investigation, by fixing the total molarconcentration of membrane, select the molar ratio of DOPE and CHEMS; theeffect after adding equal proportion of Chol with CHEMS on liposomes;thechoice of hydrated fluid;ultrasonic time of choose to determine the blankliposome preparation methods.5Construction of non-compression drug-loaded liposomes (NL1/CL1) andliposomes containing compressed material (NL2and CL2), and the study ofparticle size, potential and electron microscopy.The method of preparation as follows：Respectively using DSPE-PEG, DSPE-PEG(CPP) to form lipids film,taking off the film by DEPC hydrated fluid, and sonicating for preparation ofM1, M2.The preparation of non-compression drug-loaded liposomes (NL1/CL1)：using SPC/Chol/DC-Chol as the membrane material to form a thin lipid film,taking off the film by DEPC hydrated fluid, which including siRNA, bathsonicating for10-15min, to get the ordinary drug-loaded liposomes,whichincubation with M1and M2respectively for4h,using by after the insertionmethod，and then get NL1/CL1at last.Liposomes containing compressed material (NL2/NL2):usingDOPE/CHEMS/Chol as the membrane material to form a thin lipid film,taking off the film by DEPC hydrated fluid, sonicating for10-15min, to get the blank liposomes. Blank liposomes were incubation with siRNA, adjust pH topH7.4-7.8,at the ice condition.Then using by after-inserting-method and thenget CL2/NL2,at last.6Analysis of MCF-7cells which were treated by N-L/C-L, N-L’/C-L’respectively, the percentage of positive cells and mean fluorescence intensityof intracellular (MFI) as the marke to evaluate cellular uptake efficiency.Result:1Synthesis of DC-Chol：to give a white solid powder1.621g,and the yieldwas60.7%;the melting point:107-108℃;TLC(Chloroform-methanol=65:35(v/v),chromogenic agent was10%H2SO4) showed that product was pure; MS+:the molecular ion peak m/z501.60in the spectrum were identical with thetheoretical value501.08(M+H+).All the results proved correct structure, andthe resulting product is the DC-Chol.2Synthesis CHEMS: to give a pale gray solid powder0.49g,and the yieldwas78%;Melting point:173-176℃;TLC assay (chloroform: methanol (10:1,v/v), to generate new spot Rf=0.10,shown as a pure product;MS+： ion peakin the mass spectrum m/z485.5,was consistent with calculated value485.5(M-H+). The product proved correct structure.3Synthesis of DSPE-PEG(2000)-CPP: white flocculent; the response rateof CPP was84.4%,as measured by the Ellman reagent; MS+: molecular ionpeak m/z4469.17,was consistent with calculated value of the design ofpeptides;confirmed that the product of the title compound as DSPE-PEG-CPP.4By single factor determining preparation method of liposomesThe precise amount of DOPE,CHEMS, Chol5:2:2(mol/mol/mol) lipidmaterial solution, forming lipid thin film, taking off the film by DEPChydrated fluid2mL,sonicating for10-15min,obtained with light blueopalescent translucent system, measuring particle size.5Constructs containing liposomes uncompressed (NL1/CL1) and liposomescontaining compressed material (NL2and CL2), and thus the particle size,potential and electron microscopy characterization.NL1, CL1particle size were226.8nm,262.4nm; latter PdI was small, narrow particle size distribution; potentials were37.0,55.7mV, systemstability; electron microscopy to a particle size of about200nm or so.NL2,CL2diameter respectively289.8nm,293.2nm; the PdI of twoformulations is very small, narrow particle size distribution; potentialwas-29.6mV,-29.2mV, negatively charged surface of the liposome, thestabilizing system; the electron microscope, particle size of about around200nm.6Using flow cytometry analysis cell suspensions that were treatment bypharmaceutical preparations.Compared to the free siRNA and the negative control group, the group ofnon-compressed formulations N-L and C-L, fluorescence intensity ofintracellular X-mean were9.1,10.4, improved the cellular uptake of about25times. Whereby the non-compressed described CPP modified liposomes (C-l),Common uncompressed liposomes (N-L) have improved cellular uptake, CPPuncompressed modified liposomes have higher cell uptake.The group of containing compressed group N-L ’, C-L’,whose percentageof positive cells was6.4%,7.9%, improving the efficiency of cellular uptakeof about10times, indicating that both preparations containing compressedimproved cellular uptake efficiency, and C-L ’having a higher cell uptake.Conclusion:Experimental results show that, the synthetic routes of DC-Chol andCHEMS are reliable, having high reproducibility. The yields are60.3%,78%,respectively, product having high purity.Successfully connect the CPP to DSPE-PEG (2000) in the distal end ofPEG, obtaining guidance lipid material DSPE-PEG-CPP.Successfully prepared CPP modified gene delivery nanostructured lipidcarriers CL1,CL2.Four agents were improved cellular uptake efficiency. cellular uptake ofnormal liposomes and CPP modified liposomes relatively high,but thedifference was not significant. Possible reasons for this result are such as thatthere are many remaining lipid materials in the Synthesis product of DSPE-PEG-CPP.Thus, the reaction conditions should be further optimized toimprove product purity.