Amphiphilic Molecular Umbrellas: Synthesis, Characterization And Cell Penetrability

Cell membranes serving as selective natural barriers permit the passage of some necessary molecules and ions while inhibit the entray of many biologically-active molecules that have therapeutic potential,especially those that are highly hydrophilic or charged,e.g.,antisense oligonucleotides,DNA,protein,and certain peptides.To design a new drug vector for promoting the agents across cellular membrane and releasing at the target issues is a persistent challenge.A novel molecular umbrella vector with amphiphilic faces may transport many biotherapeutic molecules across the cell membrane and solve the low efficiency and security problems accounted in the application of some drug carriers potentially.A molecular umbrella carring 5-carboxyfluorescein was synthesized in this paper,the ability of molecular umbrella to transport 5-carboxyfluorescence across cell membrane and its cytotoxicity were also studied.The translocation mechanism of molecular umbrella was discussed in detail.The results were as follows:1.Synthsis and characterization of molecular umbrella(1) The N-hydroxysuccinimide ester of cholic acid was synthesized from cholic acid and N-hydroxysuccinimide with dicyclohexylcarbodiimide in anhydrous organic solution.Reaction of this ester with spermidine was carried out in anhydrous DMF to yield N₁,N₃-dicholeamidospermidine.(2) 5-carboxyfluorescein was preactived with 3-hydroxy-1,2,3-benzotriazin-4(3H)-one(Dhbt) and dicyclohexylcarbodiimide in anhydrous DMF to gain the Dhbt ester of 5-carboxyfluorescein.The ester was further coupled to the N₁,N₃-dicholeamidospermidine in the present of triethylamine to form the 5-carboxyfluorescein molecular umbrella(5-cf-mu).(3) The structure and molecular weitht of N-hydroxysuccinimide ester of cholic acid,N₁,N₃-dicholeamidospermidine and 5-cf-mu were confirmed by IR,NMR, ESI-MS;their melt points were also recorded by a microscopic melting point apparatus;and the purity of 5-cf-mu was analyized by HPLC.The characterization results were consistent with their molecular structures and molecular weitht and the purity of 5-cf-mu was above 95%.2.The cell penetrability of molecular umbrella (1) The cytotoxicity of 5-cf-mu toward L929 cells after 24 and 48 h treatment with 5-cf-mu was tested by MTT assays.The concentrations of 5-cf-mu were 25,50, 100 and 200μM respectively.5-cf-mu exhibited no toxicity to L929 cells at concentrations from 25 to 200μM following 24 h incubation.Although the concentration of 5-cf-mu from 50 to 200μM exhibited some inhibition to cells after 48 h incubation,high cell viability was achieved and 5-cf-mu showed nontoxic to L929 cells at 25μM at all even after 48 h.The cytotoxicity of 5-cf-mu toward Hela cells after 24 h treatment with 5-cf-mu was also tested.No toxicity of 5-cf-mu was detected to Hela cells when the concentration of 5-cf-mu was below 100μM.The MTT assay results showed that the molecular umbrella was a biocompatible vector.(2) The intracellular distribution of 5-cf-mu in Hela cells and L929 cells was analyzed by fluorescent microscopy and confocal microscopy.The punctuated fluorescence of 5-cf-mu was detected mostly in cytoplasm and around the nucleus,suggesting that 5-cf-mu had entered the interior of Hela cells and L929 cells.Results showed that molecular umbrella was an appropriate vector for intracellular delivery.(3) The internalization of 5-cf-mu was analyzed by FACS at different concentrations,temperatures and incubation times.The cellular ATP inhibitors were also introduced to evaluate the uptake mechanism of 5-cf-mu.The uptake of 5-cf-mu is a concentration-,temperature-,time-dependent and energy-independent process.3.Uptake mechanism of molecular umbrella(1) A mathematical model for the membrane translocation of molecular umbrella was proposed.According to the results of flow cytometry,the mean fluorescence intensity(MFI) inside cells has linear relationship with the square root of the incubation time(t^1/2) and initial concentration(C_o^1/2),and the correlation coefficients were 0.985 and 0.994,respectively.(2) The process of 5-cf-mu across the cell membrane fitted to the proposed model, indicating that 5-cf-mu transported across the cell membrane by the solution-diffusion mechanism.