| We used ?-chitin as raw material,reacted with 3-chlorine-2-hydroxypropyl trimethyl ammonium chloride(CTA)at C-6 OH of ?-chitin,afforded O-quaternized chitin.O-quaternized chitosan which degree of deacetylation(DD)was 100% was prepared from O-quaternized chitin via homogeneous deacetylation.With the amino reaction with decanal to form Schiff base and then reduced by sodium borohydride,the hydrophobic decane was grafted at C-2 amino group of O-quaternized chitosan to obtained amphiphilic N-alkylated-O-quaternized chitosan.The structure of intermediates and final N-alkylated-O-quaternized chitosan was characterized by FT-IR,1H NMR,elemental analysis and Mohr titration methods.Thermal performance of N-alkylated-O-quaternized chitosan was characterizatied by TGA.And the preliminary study on carrying Drug/DNA was done.The effects of reaction conditions on substitution degree of quaternary ammonium salt(DSQ)of O-quaternized chitin were studied.The Comparison between conventional heating and microwave heating on the preparation of O-quaternized chitosan was investigated.The effect of substitution degree of alkyl(DSA)of N-alkylated-O-quaternized chitosan on the encapsulating efficiency and release ratio of drug,and gene transfection efficiency were studied.The effect of the molar ratio of ?-chitin to CTA on DSQ of O-quaternized chitin was increased first and then decreased.When the molar ratio was 1:7,the DSQ was the largest(104%).The DSQ increased with the increase of reaction time,and then reached equilibrium.Comparing among Li OH,Na OH and KOH,it was found that KOH could increase the DSQ.But after using KOH,the reaction system was more viscous and the DSQ was not easy to control.So the Na OH was chose as alkalizing reagent.After three time's deacetylation,the O-quaternized chitosan with DD=100% was obtained.And the each reaction was done at 50? for 1 h with 34 wt.% of Na OH solution.The amount of decanal had the greatest effect on DSA of N-alkylated-O-quaternized chitosan,the reaction temperature was the second and the reaction time was the third factor.The effect of amount of decanal on DSA was divided into three stages: a dramatic increase,a smooth growth and equilibrium.The microwave heating could save the reaction time and simplify the reaction operation,and the DSA reached to the extent of 90.5% at 70 °C for 30 min.N-alkylated-O-quaternized chitosan was suitable for heat sterilization because its thermal decomposition temperature was higher than sterilization temperature.The encapsulating efficiency of acetaminophen and vitamin B12 in N-alkylated-O-quaternized chitosan increased as the drug feed increased at first and then reached to equilibrium.When the molar ratio of drug to particles was 3:5,the encapsulating efficiency reached to a limit.With the increase of DSA,the drug loading ratio and the encapsulating efficiency of acetaminophen and vitamin B12 increased.When the DSA of carrier was 67%,the drug loading ratio and the encapsulating efficiency of acetaminophen were 51% and 83%,respectively and the drug loading ratio and the encapsulating efficiency of vitamin B12 were 47% and 80%,respectively.The blank particle size was between 200 nm and 350 nm,the particle size of vitamin B12-loading carrier increased to the extent of 300 nm~700 nm,it showed that the most hydrophobic drug molecules were coated inside the vehicle and just a few gathered outside.The release of acetaminophen and vitamin B12 basically had three stages.The first stage was the burst release and the drug was released very rapidly.The release ratio of acetaminophen and vitamin B12 in phosphate buffer reached 53% and 67% when DSA was 21%.When DSA was 63%,it reached 35% and 43%,respectively during the first 2 h.The second stage was the slow release,and the third stage was the balance stage.both drugs were released completely in 10 h.The Logistic model fitted very well to the experimental results for PCTM-loading carrier with DSA of 63%,where their correlation coefficients(R2)were all recorded as 0.99.In addition to that,the release shapes for logistic and weibull model resembled an S-type release as expected.The Cytotoxicity of N-alkylated-O-quaternized chitosan(DSQ=12%,55%)was lower than PEI's,and the p EGFP transfected efficiency of N-alkylated-O-quaternized chitosan with a DSA of 21% and different DSQ was higher than that of PEI and chitosan.Quaternization of chitosan increased its electrostatic interaction with DNA and cell membrane.However,when the DSQ was higher than 36%,the strong positive charge would limit the desorption of DNA and lower the transfection effect.N-alkylated-O-quaternized chitosan had high DNA transfection efficiency at N/P=8/1,and low at N/P=20/1.At N/P=8/1,the fluorescence intensity was 1.0×10^7 RLU/mg protein for PEI,1×10^11RLU/mg Protein for Lipofectamine 2000,and 1.8×10^8 RLU/mg protein for N-alkylated-O-quaternary quaternized chitosan with a DSQ of 36%.All in all,N-alkylated-O-quaternary quaternized chitosan is expectable to be a novel genetic vector. |
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