Study On The Preparation And Biomedical Applications Of Environment-responsive And Physically Crosslinked Hydrogel Materials

Hydrogel is an aqueous three-dimensional network composed of hydrophilic or amphipathic polymer,which is usually crosslinked by chemical bonds or physical interactions between polymers.Physically crosslinked hydrogels usually have the advantages of dynamicity,reversibility,shear-thinning property and self-healing property,thus have received extensive attention and applications.Currently physically crosslinked hydrogels mainly contain the following cross-linking mechanisms:hydrophobic interaction,ionic interaction,polymer crystallization,formation of stereocomplexes,coordination bond formation,hydrogen bonding interaction,antigenantibody interactions,π-π stacking,host-guest interaction,etc.Hydrogels prepared through hydrophobic interaction of amphiphilic polymer materials or ionic interaction of polyelectrolytes,are always sensitive to environmental conditions such as temperature,ionic solution and pH.And they can respond to changes of physiological environment.In addition,it’s relatively easy to achieve the manipulation of temperature,ionic strength and pH.Therefore,taking advantage of the hydrophobic packing and ionic interaction of polymers,we designed three types of environment-responsive and physically crosslinked hydrogels or nanogels,respectively,and investigated the effect of polymer composition on the properties and functions of the gels:1.A hydrogel composed of polyethylene glycol-poly(y-(2-(2-ethoxyethoxy)ethyl)L-glutamate(PEG-P(EEO2LG)),a type of temperature-sensitive and rapidly reversible block copolymer,was prepared by forming a physical aggregation network of amphiphilic block copolymers.The application and performance of the hydrogel as a material designed for three-dimensional cell culture scaffold and the temperaturecontrolled rapid cell collection were studied.By using PEG-NH2,NH2-PEG-NH2 and 4arm PEG-NH2 as initiators to initiate polymerization of γ-(2-(2-ethoxyethoxy)ethyl)L-glutamate-N-carboxyanhydride(EEO2LG NCA),diblock,triblock and four-arm star shaped hydrogels were synthesized respectively.By adjusting the block lengths of PEG and P(EEO2LG),it was found that both of the block length of P(EEO2LG)and the PEG/P(EEO2LG)block ratio affected the hydrophilc-hydrophobic balance of the hydrogel,thereby determining the performance of the hydrogel.And the polymers with PEG/P(EEO2LG)block ratio of 1:1.5～1:2 showed better gelation behavior,including lower critical gelation temperature,wider gelation range,higher storage modulus and higher stability.The prepared polymer solution can undergo a sol-gel transition within 5～60 seconds at elevated temperatures,while it can undergo a gel-to-sol transition within 10～70 seconds when the temperature was reduced to 0℃.The gelation of the hydrogel attributed to the formation of an amphiphilic physical aggregation network which is caused by the decrease of the hydrophilicity of the PEG segments and the enhanced hydrophobic interaction of the polypeptide segments under the condition of elevated temperature.This temperature-sensitive reversible hydrogel exhibits good biocompatibility and faster biodegradability.In vitro cell culture experiments proved that the reversible hydrogel can be used as a 3D cell culture scaffold,and cells can be collected by cooling and centrifugation,which avoids the effect of trypsin on cells.2.An ionically crosslinked nanogel composed of arginine grafted chitosan(CSArg)and tris(2-carboxyethyl)phosphine(TCEP)were prepared by forming a reversible crosslinking network through ionic interactions between cationic polymers and polyvalent and anionic small molecules.The morphology and solubility of nanogels vary with pH changes,and the nanogels can interact with anions and cations in aqueous solutions.The application and performance of CS-Arg/TCEP(CAT)nanogels in mucolytic thearpy,antibacterial property and treatment of animal model of asthma were studied.The ionically crosslinked network of the nanogel can show gradual and reversible dissociation under physiological conditions,and release components of CSArg and TCEP.The positively charged CS-Arg has the character of antibacterial and mucoadhesive properties,and can interact with mucins electrostatically to displace calcium ions to thin the mucus.TCEP is a negatively charged small-molecular reducing agent,which has better reducing ability and stability than dithiositol(DTT)and glutathione(GSH),thus can break the disulfide bonds effectively in mucins.The antibacterial experiments proved that,at a concentration of 5 mg/mL,CAT nanogels could inhibit the growth of Escherichia coli and Staphylococcus aureus without obvious cytotoxicity within 24 hours.By establishing an OVA-induced asthma mouse model and nebulizing the nanogel aqueous solution,the airway mucus accumulation was effectively alleviated and airway inflammation was relieved.3.The cationic antibacterial polymer ε-poly(L-lysine)(ε-PLL)was further introduced into the CS-Arg/TCEP ionically crosslinked network to prepare the ionically crosslinked ε-PLL/CS-Arg/TCEP(ECAT)nanogels with enhanced antibacterial activity.And their in vitro antibacterial properties at lower concentrations were investigated.The nanogels can dissociate gradually under physiological conditions and release the active ingredients.By co-culturing ECAT nanogels with Gram-positive bacteria Staphylococcus aureus or Gram-negative bacteria Escherichia coli,the minimum inhibitory concentration can be as low as 0.02～0.5 mg/mL.And the nanogel with a concentration of 0.05 mg/mL can show good antibacterial effect against the untypeable Haemophilus influenzae,a common pathogen in the airway of asthmatic patients.In cell culture experiments,0.5 mg/mL ECAT-1 nanogels did not show significant cytotoxicity after 48 hours of co-incubation with cells.In addition,the ECAT nanogels exhibited certain mucolytic ability.