NMR Studies On Organic Drug Molecular Complexes And Gene Delivery System

The interactions between cyclodextrin and organic drug molecules,degradation pathway of polymers including a cross-linked polyethyleneimine(c-PEI)andγ-polyglutamic acid(γ-PGA)suitable as gene carders and the interaction and morphology of polyethylenimine/DNA complexes were studied by PFG NMR measurement combined with Monte Carlo simulation,¹H-¹H ROESY and solid-state high-resolution ¹³3C NMR spectroscopy.The main results include:1.The interactions between cyclodextrin and several drug molecules were studied by NMR diffusion measurement.The diffusion coefficients of the ligands in solution,which reflects the affinity of association between cycodextrin and them, were found to decrease as the concentration of cyclodextrin increased.Together with 2D-ROESY experiments,it was demonstrated that informing inclusion complex,the hydrophobic interaction is predominant among various possible interactions between cyclodextrin and the ligands.Based on the above experimental results,the potential of application of CD/drug molecule system in drug release was discussed.2.The degradation pathway of a cross-linked polyethyleneimine(c-PEI) suitable as a gene carrier was studied at different pH values by real time ¹H NMR and diffusion-weighted ¹H NMR spectroscopy.The c-PEI was synthesized by cross-linking PEI segments with 2,4-pentanediol diacrylate(PDDA).The experimental results show that under basic,neutral and acidic conditions,the degradation of c-PEI occurs through hydrolysis of the ester moieties of the PDDA linkers.The degradation half-life of the polymer is 22,48 and more than 720 h at pH 10.2,7.4 and 4.6,respectively,showing that the apparent molecular weight of c-PEI in the degradation process was monitored.Furthermore,a Monte Carlo simulation was applied to simulate the relation between the average molecular weight and the number of chain ends during the degradation process of a model system of c-PEI.By comparing the NMR results with those obtained from simulation,the mechanism of degradation under pH conditions and Monte Carlo simulation is a useful strategy for characterizing the degradation process of degradable polymers.3.The degradation pathway ofγ-polyglutamic acid(γ-PGA)suitable as a polymeric carrier was studied at acidic condition by real time ¹H NMR and PFG NMR techniques.By using a modified version of diffusion-weighted ¹H NMR experiment,the variation of the weight-averaged molecular weight(MW)ofγ-PGA during the degradation process was monitored.Meanwhile,the PFG NMR response curve ofγ-PGA reveals a non-exponential behavior which is excellently fitted by a stretch exponential function.By performing an inverse Laplace transformation of the stretch exponential function a distribution curve of the diffusivity is obtained which is enables the molecular weight distribution to be extracted.The results show that the width of the molecular weight distribution increasing as the average molecular weight decreases during the degradation process.The decrease in average molecular weight with degradation time,as determined by PFG NMR,agrees with the corresponding results obtained by Monte Carlo simulations,suggesting that the degradation is a random process.The above method can be used as a general method of determining molecular weight distribution of polymers.4.Solid-state ¹H wide-line NMR and ³¹p magic angle spinning(MAS)NMR were applied to a series of PEI/DNA complex samples with different PEI molecular weights and nitrogen/phosphate(N/P)molar ratios,in order to disclose the influence of various structural factors on the interactions and morphology.The results of ¹H NMR spectra showed that the linewidth of the PEI signal was greatly broadened with increasing the N/P ratio.Meanwhile,in the ³¹p M_AS experiment,the signal was found to shift upfield with the increase of the N/P ratio.Both ¹H and ³¹P NMR experiments demonstrated that the interaction between two constituents and subsequently the morphology of the complexes vary with the N/P ratio.By carrying out ¹H spin-diffusion experiments,the domain sizes of DNA in the complexes were determined.The domain size of DNA is found to decrease greatly when the N/P ratio changes from 0.5 to 3 and changes only slightly with further increase of the N/P ratio.After making a thorough analysis and discussion on drug control release and gene delivery systems with general NMR techniques,we presents a method of 1D PFG NMR technique to realize quick observation on the Mw change of polymer during its degradation process.Moreover,according to the inverse Laplace transformation theory and based on a function of diffusivity distribution of polymer in solution,2D PFG NMR technique is developed to measure the Mw distribution of polymer.Combined with Monte Carlo simulation,the degradation mechanism can be figured out.The results of the experiments indicate that the PFG NMR methods can better investigate the interactions,morphology and dynamics information of systems including drug control release and gene delivery systems in our work and is accordingly more convenient,nondestructive and applicable compared with other methods,i.e.GPC.