| Injectable hydrogels were used extensively in the field of biomedicine,such as drug controlled release, tissue engineering and biosensor. For the reasons that they can be cross-linked in situ quickly and their properties are highly analogous to softer tissues of body. In spite of the significant advantages and excellent potential in biology applications mentioned above,the poor dynamic stability of hydrogels limits their practical application seriously. Under the constant low shear force of body hydrogels can be destroyed and then diluted by tissue fluids. At present, exploring approaches to improve the mechanical property of injectable hydrogel is imperative. In this paper, injectable, de-crosslinkable and thermosensitive poly(N-isopropylacrylamide) (PNIPAM) hydrogels were prepared via hydrazide-aldehyde crosslinking reaction, which can carried out rapidly at room temperature. And organic-based aldehyde-functionalized dextrin oxidized oligomers played as macromolecular crosslinkers. In order to improve the mechanical property of hydrogels, organic-based starch nanoparticles (SNPs) were chosen for their unique characteristics of no toxicity, biodegradability, and cytocompatibility. The main contents are as follows:1. Preparation and characterization of PNIPAM precursors poly(NIPAM-co-AA)-hdz. The copolymers of poly(NIPAM-co-AA) were synthesized through free radical copolymerization using N-isopropylacrylamide(NIPAM) as main monomers and acrylic acid (AA) as functional monomers. Then, poly(NIPAM-co-AA)-hdz was prepared by dehydration condensation reaction between -COOH in poly(NIPAM-co-AA)and - NHNH2 in adipic acid dihydrazide (ADH) in the presence of coupling agent. The structure of copolymers, molecular weight and polydispersity index(PDI) were characterized by Fourier Transform Infrared Spectroscopy (FTIR)and Gel Permeation Chromatography (GPC). The content of acrylic acid groups was characterized by conductometric titration. The results showed that copolymers of poly(NIPAM-co-AA) owned a molecular weight of Mn ?19100 g-mol-1, Mw = 38300 g-mol-1 (PDI = 2.00), which were below the renal cutoff. Then content of hydrazine was calculated by residual acrylic acid groups through conductometric titration. The results indicated that the consumption of acrylic acid groups was 98.9%, giving rise to 39.81×10-4 mol·g-1 of hydrazine groups content of functionalized polymers.2. Preparation and characterization of macrocrosslinker dialdehyde dextrin. A series of aldehyde-functionalized dextrin with different -CHO groups content were prepared by using natural, non-toxic and biodegradable dextrin as raw material and sodium periodate (NaI04) as oxidant agent. Effects of reaction temperature, reaction time and amount of oxidant agent on oxidation degree were investigated in detail. Then structure of dextrin with or without oxidation was tested by FTIR. The results indicated that the oxidation degree was 80.86% and polymers contained 40.30×10-4 mol·g-1 of aldehyde groups when the mass ratio of dextrin to NaI04 was 15:8. In addition, the oxidation degree of aldehyde dextrin can be controlled by controlling the ratio of NaI04 to ortho-hydroxyl groups of dextrin.3. Preparation and characterization of starch nanoparticles (SNPs). SNPs with different particle size and particle size distribution were obtained taking natural polysaccharide starch as raw material, boric acid (B(OH)3) as crosslinking agent and Span 80 as surfactant, through inverse microemulsion process. Effects of ratio and amount of toluene-chloroform mixtures, amount of surfactant and crosslinking agent, reaction temperature and time on the particle size and their distribution of SNPs were investigated. The morphology of SNPs was shown by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) exhibited the dispersibility of nanoparticles in water. Meanwhile, the particle size and particle size distribution were tested through Zeta potential and laser particle size tester precisely. According to the results it indicated that the more the amount of surfactant and the less the amount of cross-linking agent were, the smaller the particle size of SNPs was. Moreover, when amount of surfactant was 2% of mixed solvent and amount of B(OH)3 was 0.05% of starch mass, SNPs with average particle size of 56-100 nm and polydispersity of 0.229 were obtained.4. Preparation and characterization of hydrogels. The PNIPAM composite hydrogels were prepared via aldehyde-hydrazine crosslinking reaction at 37?using SNPs as reinforcing nanoparticles and aldehyde dextrin as macrocrosslinker. Effects of reactive groups content, concentration of PNIPAM precursor solution and macrocrosslinker solution on the gelation time of hydrogel were investigated. The microcosmic morphology of hydrogel after CO2 critical point drying was characterized by SEM. As indicated in the results that uniform and small pores were observed obviously in the three dimension structure and sphere particles dispersed homogeneous throughout the 3D structure of hydrogels. Additionally, the higher the concentration of the solution of precursor was, the shorter the gelation time of hydrogel was. The curing gelation time of hydrogel exhibited a trend of decline followed by a rise with the rose in temperature.5. Performance testing of hydrogels. In this experiment, the properties of dynamic modulus, thermosensitivity, decrosslinking, drug release and cytotoxicity of reinforced composite hydrogels or unreinforced hydrogels were tested in detail.(1) Property of dynamic modulus. A parallel-plate rheometer was used to measure the dynamic modulus of noncomposite and SNPs composite hydrogels. Influences of concentration of precursors solution on storage modulus of hydrogel and amount of SNPs on storage modulus of composite hydrogel were investigated. It can be seen from the results that the dynamic modulus of unreinforced hydrogels kept increasing when the concentration of precursor solutions was from 4.5 wt % to 11.0 wt %. Especially when the concentration was 9.0 wt % hydrogel maintained faster gelation rate than other samples. In addition, when the concentration of aldehyde dextrin solution was from 0.5 wt % to 4.0 wt %, the storage modulus of composite hydrogels exhibited a trend of ascent followed by a descent with the increase of SNPs in dosage. Moreover, the dynamic modulus of SNPs composite hydrogel raised to the maximum value of 399.2 KPa when the concentration of nanoparticles was 1.5 wt %. Comparing the modulus of hydrogels reinforced or unreinforced with SNPs it can be seen that the maximum storage modulus of SNPs composite hydrogel was 399.2 KPa and a dramatic improvement(343.4%) in storage modulus was indicated when the concentration of precursor was 9.0 wt %. All in all, a remarkable improvement in dynamic modulus was observed through the addition of SNPs.(2) Property of thermosensitivity. Thermosensitivity behavior of wet hydrogel was measured in 10 mmol·L-1 phosphate buffered saline (PBS)solution at alternating cyclic temperature of 25 °C (below LCST) and 37 °C(above LCST). The results showed that water absorption ability of SNPs composite hydrogel decreased with the addition of SNPs. Although the decrease in water absorption, both noncomposite and SNPs composite PNIPAM hydrogels showed perfect thermosensitive performance at alternating cyclic temperature of 25 °C and 37 °C. Moreover, all the PNIPAM hydrogels showed the state of contraction at 37 °C.(3) Property of decrosslinking. Decrosslinking performance and mechanism of hydrogels were assessed via incubating hydrogels in 5 mL of 1.0 mol·L-1, 0.5 mol-L-1, and 0.1 mol·L-1 HCl standard titration solutions respectively at 37 °C. Effects of H+ concentration and the addition of SNPs on decrosslinking performance of hydrogels were investigated. Based on the results it revealed that hydrogels exhibited perfect decrosslinking properties in hydrochloric acid solution. With the increase of H+ concentration, the decrosslinking rate of hydrogel was accelerated obviously. Moreover,according to testing results that mixing SNPs to the 3D network of hydrogel had little influence on decrosslinking performance of hydrogels.(4) Property of drug release. Antitumor drug doxorubicin hydrochloride was selected as model drug and drug loaded hydrogels were incubated in 5 mL of 10 m mol·L-1 PBS at 37 °C to measure their performance of drug release.Influences of drug concentration on drug release properties of drug loaded hydrogels were investigated in detail. It is clear from the results that drugs could be released by hydrogels continuously, and with the increase of drug concentration, drug release rate of hydrogels slowed down gradually. In general, the sustained drug release time of hydrogel was more than 25 days and the peaking amount of sustained released drug was more than 95%.(5) Property of cytotoxicity. The cytotoxicity of precursor of poly(NIPAM-co-AA)-hdz, macrocrosslinker of aldehyde dextrin, reinforcing material of SNPs and 9.0 wt % hydrogels were tested via a CCK-8 assay using L929 fibroblast cells as testing cells. Effects of polymer concentration, SNPs concentration and the method of cell seeding on proliferation of L929 cells were investigated minutely. The cytotoxicity of hydrogels was investigated via leaching method by immersing bulk hydrogel in DMEM media for 24 h with the concentration of 0.2 mg·mL-1. The results showed that poly(NIPAM-co-AA)-hdz polymer had no significant effect on cell proliferation with the increase of polymer concentration. And the cytotoxicity test results of SNPs were analogous to poly(NIPAM-co-AA)-hdz precursors.Almost synchronously grown between experimental groups and control groups of SNPs were observed obviously. But aldehyde dextrin solution indicated obvious cytotoxicity when the concentration was greater than 800?g·mL-1. Meanwhile, as can be seen from the cytotoxicity results of hydrogels after cells seeding, the number of cells increased significantly with the growth of incubation time. Comparing cell counts between experiment and control groups, the relative cells viability was 0.73 ±0.15 on the fifth day and 0.78 ±0.18 on the seventh day. |
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