Aminoglycoside Derivatives Smart Hydrogel As A Drug Carrier

Due to plentiful crosslinking network and favorable expansibility,hydrogels have been studied extensively for application as drug delivery carrier.In the present study,a novel microgel was designed by the incorporation of temperature sensititive N-isopropylacrylamide(NIPAM) and pH sensitive acrylic acid(AAc) to copolymerize with glucosamine derivative,acrylamido-2-deoxy-glucose(AADG). The novel microgel has favorable biocompatibility,tissue-targeting and stimuli-responsibility to pH and temperature.Moreover,a novel in situ cross-linked hydrogel was prepared under physiological conditions,based on self-assembly of natural amino polysaccharide derivatives with homo-bifunctional PEG derivatives using Michael type addition,and the natural amino polysaccharide hydrogel also has some temperature sensitivity.The in vitro macromolecular drug delivery experiment suggested the favorable intelligent drug release abilities of these two gels,and they have potential to be novel intelligent drug delivery system.Ⅰ.Preparation of gels1.Preparation of glucosamine derivative microgelAcrylic acid was attached to glucosamine via an amide bond between the carboxylic group and a free primary group.The microgels of the obtained functional glucosamine-AADG with NIPAM and AAc as the comonomer were prepared by the method of a free radical precipitation polymerization.2.Preparation of natural amino polysaccharide hydrogelFour homo-bifunctional PEG derivatives were synthesized:acrylate,bromoacetate, iodoacetate and maleimide functional PEG..And they were examined as cross-linking agents for thiol-modified derivatives of chitosan.The hydrogels were prepared by Michael type addition between the bifunctional PEG derivatives and thiol-modified chitosan.Ⅱ.Characterization of gels1.Size and morphology of microgel The morphology of the resulting microgel was investigated by SEM.It is evident that microgels are well dispersed as individual particles with spherical shapes.It can be seen that the size of the microgels is around 50～60 nm in diameter in the solid state,and the microgels exhibit a narrow size distribution with an average diameter of around 100 nm in the wet state.2.Pore size and morphology of hydrogelThe SEM images of freeze-dried hydrogels exhibited a highly macroporous spongelike structure and the average mesh size is about 10μm.After being dipped in PBS for 24 h and 7 d,the chains in the network degraded and the pores of the hydrogel increased3.Thermal stability of hydrogelAll the hydrogels showed a two-stage thermal decomposition,the first-stage decomposition temperature was similar to that of chitosan,suggesting the introduction of PEG via Michael type addition did not decrease thermal stability of chitosan.And the weight loss results in the second-stage thermal decomposition were similar to the contents of PEG in hydrogels and indicate that the second-stage decomposition was caused by PEG.The second-stage degradation temperature increased when decreasing the content of PEG,which may be due to the inhibition of crystal growth of PEG caused by the stiff chitosan chain.4.RheologyThe resulting of oscillatory stress sweep and frequency sweep indicated the higher mechanical strength and elasticity of hydrogel can be achieved by increasing the cross-linking density.5.Swelling and degradationAll the hydrogels swelled rapidly and reached equilibrium within 0.5 h at 37℃, but within 4 h at 25℃.After reaching swelling equilibrium,all the hydrogels were rapidly degraded in the first 24 h,and then the degradation became slowly and hydrogels did not completely dissolve within six weeks.Ⅲ.Intelligent drug release and biocompatibility of gels1.Temperature sensitivityThe results of dynamic light scattering measurement show that the size of the microgels significantly increased with an increase in the temperature.And drug release profile represents the temperature sensitivity,under the same pH value,at 41℃,about 88%of drug was released within the initial 2 h;at 37℃,only about 79% of drug was released after 2 d.The temperature sensitivity of hydrogel is also remarkable,the equilibrium swelling ratio of hydrogel at 25℃decreased to 25%of that at 37℃.For drug release profile,at 37℃,drug was completely released within 3 d;at 25℃,only about 60%of drug was released after 3 d.2.pH sensitivityWhen pH＜5.0,the diameters of microgels were nearly invariable regardless of the change of pH,and when pH≥5.0,the diameters became larger when the pH value was increased.And drug release profile represents the pH sensitivity,under the same temperature,at pH 6.9,about 88%of drug was released within the initial 2 h;at pH 7.4,about 79%drug was released within the initial 3 h.3.Tissue-targetingThe drug release from microgel was two-stage under the tumor-surrounding environment,the initial burst release and subsequent slow release;and for the drug release under physiological conditions,the diffusing process was sustained slow and mild.Therefore,the release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions.The gelation time of hydrogel was determined by the thiols contents and concentration of CS-NAC,the terminal group type,molecular weight and the amount of cross-linking agents,and the reaction temperature.The controllability of hydrogel gelation time make macro form gel have tissue-targeting ability.4.BiocompatibilityThe cell viability study showed that the use of glucosamine may improve the biocompatibility of the microgels.The cell culture results illustrate that cells can migrate into the hydrogel networks and remain viable and maintain their 3D morphology during the 3 days culture.The secondary structures of insulin and BSA as determined by CD spectra and SDS-PAGE were preserved in all gels. Ⅵ.Mechanism of intelligent drug release1.ShrinkageThe drug release from microgel has an initial burst,which was correlated to the NIPAM segment in the microgels.As the temperature increased up to a certain point, the water contained in the microgels was expelled due to the disruption of hydrogen bonding between water and the hydrophilic amide groups,then the hydrophobic isopropyl groups began to associate.And following the structural change and consequent enhanced surface hydrophobicity,the microgels shrunk and aggregated, the insulin was squeezed out.2.SwellingFitted by semi-empirical mathematical model,mechanism of drug release from hydrogel was determined for Fick diffusion,and the release type was belong to swelling dominating drug release by further inference.Therefore,the crosslinking density determined the release rate of different hydrogels.3.DegradationDegradation of hydrogel did not have significant effect on drug release,which may be due to the huge difference between hydrodynamic diameter of BSA(d_h=7.2 nm) and hydrogel pore size(10μm).