Folic Acid Modified Paclitaxel Long Circulating Half-solid Lipid Nanoparticles

The number and activity of folate receptors on the membrane surface of tumor cells are significantly higher than that of normal cells.Hence,folate can be used together with drug delivery system to achieve the targeting delivery of drug to the tumor tissues by the specific interaction between folate and receptors.In this study, paclitaxel(PTX) is used as a model drug,and PTX semi-solid lipid nanoparticles (SSLN) modified with folate and PEG are prepared.This kind of SSLN with certain long-circulating and tumor-targeting functions is a promising tumor-targeting drug delivery system.1 Preparation and characterization of monomethoxypolyethylene glycol-phosphatidylethanolamine(MPEG-PE)SC-MPEG with activity degree of 96.2%is prepared using TEA as the catalyst. The activated PEG is allowed to react with PE,followed by addition of anhydrous ethyl ether to precipitate MPEG-PE.The coarse MPEG-PEG is purified using silica gel chromatography with a yield about 54.9%.Result of TLC shows that the polarity of MPEG-PE is between SC-MPEG and PE.The melting range of the self-prepared MPEG-PE is 51.7-64.6℃,which is similar to that of the standard substance of MPEG-PE(55.8-63.6℃).The electrophoretic mobility ratio of MPEG-PE in SDS-PAGE gel is lower than that of MPEG.The FTIR spectrum of SC-MPEG displays a peak at 1715cm^-1 which is specific for the inner lactam.MPEG-PE has a FTIR peak of ester bond at 1738cm^-1 instead of the peak at 1715cm^-1,which shows that the amino group of PE has displaced the succinimide group in SC-MPEG.The ¹H-NMR spectrum of MPEG-PE exhibits a chemical shift at 5.34ppm that is induced by the hydrogen of amide linkage in MPEG-PE.As stated above,MPEG-PE has been prepared.2 Preparation and characterization of the folate-PEG-PE compositeFolate-PEG-PE is synthesized with PEG-bis-NH₂ and PE through two-stepped reactions.The reaction mixture is dealt with anhydrous ethyl ether to precipitate folate-PEG-PE,and the coarse product is further purified by Sephadex G-25 gel filtration chromatography.It is showed that folate-PEG-NH₂,folate-PEG-folate and folate-PEG-PE are well separated from each other.TLC of the fraction of folate-PEG-PE displays a single spot with R_f of 0.85.There is a strong peak at 1720cm^-1 in the FTIR spectrum of folate-PEG-PE,which is specific for amide linkage, while all the reactants have no such peak.The ¹H-NMR spectrum of folate-PEG-PE exhibits chemical shifts at 4.21ppm,2.48ppm and 3.64ppm that can be ascribed toα-CH₂,β,γ-CH₂ in Glutamate moiety of folate and the repeated units of PEG, respectively.In summary,folate-PEG-PE has been prepared using the above method with a yield of 7.5%.3 Preparation and characterization of PTX loaded SSLNThe formula and technology for preparation of PTX-SSLN by the procedure of "emulsification at a high temperature and solidification at a low temperature"are established in this study.Five kinds of SSLN including blank-SSLN,Span60-SSLN, Myrj59-SSLN,PEG-PE-SSLN and folate-PEG-SSLN are prepared.Span60-SSLN appears to be polygon under TEM,and the other kinds of SSLN are in round shape with good dispersion.Besides,the PEG "shells"can be observed on the surface of Myrj59-SSLN,PEG-PE-SSLN and Folate-PEG-SSLN.The entrapment efficiencies of these SSLNs are all over 95%.The mean particle sizes of folate-PEG-SSLN, PEG-PE-SSLN,Span60-SSLN and Myrj59-SSLN are 207.2nm,213.3nm,275.5nm and 236.7nm,respectively.The particle sizes of folate-PEG-SSLN and PEG-PE-SSLN are smaller than that of Span60-SSLN and Myrj59-SSLN under the same preparing conditions,which can be attributed to the better emulsification effect of MPEG-PE.All the five SSLNs are negatively charged.The Zeta potentials of folate-PEG-SSLN,PEG-PE-SSLN,Span60-SSLN and Myrj59-SSLN are -34.92mV, -37.54mV,-29.21mV and -28.16mV,respectively.The absolute value of Zeta potentials of folate-PEG-SSLN and PEG-PE-SSLN are higher than that of Span60-SSLN and Myrj59-SSLN,which indicate that the former two SSLN may hold more stability.In addition,the viscosities of all the five SSLNs are low.4 In vitro drug releasing and cell phagocytosis experiments of PTX loaded SSLNAlmost all the drug is released from Span60-SSLN in 6d.The drugs released from PEG-PE-SSLN and Myrj59-SSLN in 12d are both over 85%and 78%, respectively,and the drug released from folate-PEG-SSLN in 15d exceeds 54%.The drug released from Span60-SSLN is much faster than that from PEG-PE-SSLN, Myrj59-SSLN and folate-PEG-SSLN,which attached to PEG chains.The stereo hindrance effect of PEG chains prevents collisions between nanoparticles and therefore decreases drug releaseing by diffusing through the solid lipid matrix.The drugs quickly release from Span60-SSLN,PEG-PE-SSLN,Myrj59-SSLN and Folate-PEG-SSLN during 24h,which are 43%,29.6%,23.5%and 10.7%, respectively.After that,the drug release becomes slower.The MPM uptake rates of Span60-SSLN,PEG-PE-SSLN and Myrj59-SSLN in 30min are 2.31%,0.66%and 0.07%,respectively.After adding blood plasma,the intake rates become 3.2%,1.62%and 0.14%,respectively.This result demonstrates that opsonization by plasma proteins can promote the phagocytosis of nanoparticles by macrophage,while PEG can interfere with the uptake process.5 Pharmacokinefics study of PTX loaded folate-PEG-SSLN in ratsThe analytical RP-HPLC method is established by using Diazepam as the internal standard to determine the concentration of PTX in rats' plasma.The folate-PEG-SSLN,PEG-PE-SSLN and Span60-SSLN are intravenously administered through tail vein of rats.24h after administration,the PTX concentration in plasma is below the limit of determination for the group of Span60-SSLN,while for the groups of PEG-PE-SSLN and folate-PEG-SSLN,the PTX concentration in plasma are 375ng·mL^-1 and 258.7ng·mL^-1,respectively.The mean plasma concentration of PTX-time curves show that the decreasing rate of plasma concentration of PTX is slower in PEG-PE-SSLN and folate-PEG-SSLN groups with PEG than in Span60-SSLN group without PEG.The increase of the AUC of folate-PEG-SSLN and PEG-PE-SSLN and the elongation of MRT and t_1/2β indicate that folate-PEG-SSLN and PEG-PE-SSLN possess long-circulating effect in some degree,which maybe do favor to target tumor sites.6 The tissue distribution and tumor inhibition rate of PTX loaded folate-PEG-SSLN in S-180 bearing miceThe drug distribution rates of the groups of folate-PEG-SSLN and PEG-PE-SSLN in tumor are higher than that of the group of PTX solution at different time intervals from 0.25h to 12h,which suggests that the two SSLN have certain tumor targetability.Comparing with the PTX solution,the tumor AUC_0-12 of folate-PEG-SSLN group increases by 25%.And the lung AUC₉0-12) of folate-PEG-SSLN and PEG-PE-SSLN groups decreases by 86.5%and 90.9%, respectively.The liver AUC_0-12 decreases by 36.1%and 70.6%,respectively,which indicate that the phagocytosis of folate-PEG-SSLN and PEG-PE-SSLN by liver macrophage decreases in some degree.The heart AUC_0-12 decrease by 94%and 78.9%,respectively,which probably indicate that the toxicities of folate-PEG-SSLN and PEG-PE-SSLN to heart are lower.The plasma AUC_0-12 of folate-PEG-SSLN and PEG-PE-SSLN respectively are 2.47 and 2.33 times of that of PTX solution,which demonstrate that they have certain long-circulating characteristics and can be targeted to tumor tissues much more.The relative uptake rates of folate-PEG-SSLN and PEG-PE-SSLN by tumor tissues are 1.25 and 0.85,respectively,which demonstrate that folate-PE-SSLN has certain tumor targetability.The target efficiencies of folate-PEG-SSLN and PEG-PE-SSLN are less than 1,which shows their poor tissue selectivity.The relative tumor target efficiencies of folate-PEG-SSLN as well as PEG-PE-SSLN to PTX solution are 5.77 and 5.26,respectively,which displays that the two SSLN have better tumor targetability than PTX solution,and the tumor targetability of folate-PEG-SSLN is slightly better than that of PEG-PE-SSLN.The tumor inhibition rates of folate-PEG-SSLN and PTX solution in the same dose are 44.38%and 37.38%,respectively.The histomorphological study shows that folate-PEG-SSLN administered in the dose of 6mg·kg^-1 can evidently inhibit the growth of tumor cells.7 Preparation and the preliminary stability study of lyophilized powder of PTX loaded folate-PEG-SSLNThe trehalose is selected as the freeze-drying protectant.The folate-PEG-SSLN and PEG-PE-SSLN can keep in good morphology and dispersion using 5%trehalose solution as protectant.There is slight increase in particle sizes as well as slight decrease in Zeta potentials of the SSLN.Results of DSC show that PTX presents as amorphous state in the SSLN.Results of ¹H-NMR also demonstrate that some structural changes of the lipids and PTX take place in the SSLN,which displays new thermodynamic properties.The preliminary stability study demonstrates that the SSLN should be stored at 4℃.After storing at 4℃for 3 months,the morphology, particle size and encapsulation efficiency of the SSLN change a little,and the dispersion of the SSLN retains good.