Preparation of chitosan/hydroxypropyl methylcellulose thermosensitive hydrogel

Abstract: Thermosensitive hydrogels were prepared using chitosan (CS) and hydroxypropylmethylcellulose (HPMC) as raw materials, and the effects of CS content, HPMC content, HPMC viscosity and glycerol addition on CS/HPMC were analyzed. Effect of low critical solution temperature. The optimal conditions are that the CS content is 1% (m/v), the viscosity of HPMC is 6 mPa·S, the content of HPMC is 7% (m/v), and the content of glycerol is 32% (m/v). The lower critical solution temperature is 32°C, and the viscosity of the system is 1407 mPa·S. Infrared spectroscopy test shows that there is no chemical interaction between cs, HPMC and glycerin, and rheological test shows that the system can gel at 36°C. MTT test results showed that CS/HPMC/Gyl had no toxic and side effects and had good biocompatibility. The research results show that the system is a good injectable thermosensitive hydrogel.

Key words:chitosan; hydroxypropyl methylcellulose; thermosensitive hydrogel

Thermosensitive hydrogels are used in drug delivery systems and tissue engineering, and have the characteristics of injectable, moldable, controllable release and degradable. Thermosensitive hydrogels based on natural polysaccharide polymers, especially chitosan (CS), have attracted extensive attention due to their simple preparation process, low cost, good biocompatibility, biodegradability and antibacterial properties.

Chitosan itself is not temperature-sensitive, and needs to be mixed with substances that produce temperature-sensitive properties to form a temperature-sensitive hydrogel. Among them, sodium glycerophosphate (GP) is the most studied, but the hydrogel structure is relatively loose. Can not well meet the application requirements of drug sustained release and tissue scaffolding, because the polymer solution concentration is low (<2%), thus the strength of the gel skeleton formed is low, the water content is high and easy to run off. Therefore, increasing the concentration of polysaccharide polymers and reducing water loss is an effective solution. However, chitosan is difficult to increase the concentration because of the dissolution problem. Another polysaccharide derivative, hydroxypropyl methylcellulose (HPMC), not only has reversible thermal gel properties, but also has good water solubility, pH stability, water retention, adhesion and formability, and has been widely used Used in tablets, drops and ointments. Since the lower critical solution temperature (LCST) of HPMC is generally above 60 °C, its research is mainly applied to solid materials, and there are few reports on temperature-sensitive hydrogels in the body temperature range.

In this paper, a new polysaccharide thermosensitive hydrogel was prepared by blending CS, HPMC and glycerol. Among them, HT-E series low-viscosity HPMC was selected to increase the concentration of polysaccharide polymer to more than 8%; adding a certain amount of glycerol not only lowered the LCST of the system to below the body temperature (37°C), but also further reduced the water content of the system. The content and loss are beneficial to the stability of the gel network. Moreover, the system has good fluidity and is an injectable temperature-sensitive hydrogel. In this paper, the preparation and influencing factors of thermosensitive hydrogels were analyzed.

1. Experimental part

1.1 Raw materials

Chitosan: relative molecular mass 100,000-300,000, degree of deacetylation 85%, Chengdu Kelong Chemical Reagent Factory; hydroxypropyl methylcellulose: methoxyl content 28%-30%, hydroxypropyl content 7 ．0%～12%, viscosity values are 6 mPa·S, 15 mPa·S, 30 mPa·S, 50 mPa·S, KIMA CHEMICAL CO.,LTD; glacial acetic acid, NaOH, glycerin: analytically pure, Chuandong, Chongqing Produced by Chemical Co., Ltd.; MTT kit: Jiangsu Biyuntian Biotechnology Research Institute.

1.2 Preparation of thermosensitive hydrogel

Heat the 0.2 mol/L dilute acetic acid solution to 80°C, then add the homogeneously mixed CS powder and HPMC powder into the dilute acetic acid solution, stir magnetically until the solution is transparent, and let it stand at room temperature. Use 5 mol/L NaOH solution to adjust the pH value to about 6.8, then add a certain amount of glycerin to the solution dropwise, and stir slowly until uniform.

1.3 Determination of LCST

The gel temperature of the solution was determined by the tube inversion method. Add the prepared solution into a glass test tube, then place it in a constant temperature water bath, take out the test tube after 10 minutes, turn it upside down, and observe whether the liquid in the test tube flows. If it does not flow, this temperature is the minimum gel temperature of the sample; if it flows, continue to increase the temperature by 1°C each time, and then observe whether the liquid flows after 10 minutes, and so on until the LCST of the sample is found.

1.4 Infrared spectrum test

Dry the sample at 100°C, grind it into a fine powder, mix it with potassium bromide and press it into tablets, and use a Speclmm GX infrared spectrometer (PE company in the United States) to test its infrared spectrum. The wave number is 4000-500 cm-1, and the resolution is 4 cm-1.

1.5 Rheological test

DHR-1 rheometer (TA Instruments, USA) was used to test the rheological properties of the CS/HPMC crucible system. The test system is a 40mm flat panel with a spacing of 1 mm. Take an appropriate amount of the solution and put it between the plates, set the heating program at 20-80°C, and measure the relationship between the modulus and the temperature. Then, the frequency was changed while the scanning temperature was 37° C., and the gelation time of the solution was measured.

1.6 MTT experiment

MTT experiments were performed using L929 cells. Add 200 μL each of human experimental samples and negative control samples (10% bovine serum albumin DMEM medium) to a 96-well cell culture plate, and operate 5 wells in parallel in each group, and place the culture plate at 37°C in an atmosphere of 5% CO2 Under these conditions, they were cultured in the incubator for 1 d, 3 d, and 5 d respectively. According to the instructions of the MTT kit, the absorbance value (O.D) at a wavelength of 570 nm was measured with a Multiskan MK3 microplate reader (Thermo Company, USA).

2. Results and Discussion

CS and HPMC can dissolve well and maintain fluidity within a certain concentration range, indicating that there is no chemical cross-linking between macromolecules. With the increase of their concentration, there are more and more overlaps and entanglements between macromolecules, and more and more intermolecular and intramolecular forces, the viscosity of CS/HPMC solution also increases sharply, and the fluidity becomes worse and worse. , even at normal temperature (25°C), it gels quickly, which no longer meets the requirements of thermosensitive hydrogels.

2.1 The effect of CS content on the lower critical solution temperature

The four varieties of HPMC are represented by "HPMC" plus the corresponding viscosity value (the same below), and the concentration is 6%. With the increase of CS content, the LCST value of CS/HPMC solution showed a linear decreasing trend. When the Cs content increased from 0.5% to 2.5%, the LCST of the system basically dropped only 5℃, and all of them were higher than 50℃. However, increasing the CS content is not only difficult to dissolve, but also the viscosity of the system is very high, which is not suitable. For the CS/HPMC system of HPMC with different viscosities, the LCST corresponding to HPMC50 is the highest, followed by HPMC6, HPMCl5 and HPMC30 are the lowest and basically consistent, and the LCST difference between adjacent layers is not large, basically 1 °C.

Regardless of the viscosity of HPMC, the increase of CS content will lead to the decrease of the lower critical solution temperature of CS/HPMC system. The increase of CS concentration increases the overlapping and entanglement of macromolecules in the system, and macromolecules are more likely to form multi-chain aggregates due to molecular thermal motion. In addition, CS has many hydrophilic groups, which can deprive water molecules from HPMC. The increase of CS content promotes the dehydration of HPMC, and the exposure of hydrophobic groups of HPMC molecules increases. As the temperature rises, the thermal movement of water molecules intensifies, resulting in the breaking of the hydrogen bonds between HPMC and water molecules, and the increase of hydrophobic bonds between HPMC macromolecules, and finally the gel network is formed at a lower temperature. But it can also be seen that the role of CS is very limited. The reason is that a large number of CS macromolecules are interspersed between HPMC molecules, and its large steric hindrance increases the difficulty of HPMC molecular movement and reduces the binding points between HPMC molecules. Played a certain counterproductive.

2.2 The effect of HPMC content on the lower critical solution temperature

The content of HPMC directly affects the strength of the interaction between molecules and the size of the structure. It is the hydrophilic and hydrophobic interactions between a large number of HPMC molecules that lead to its temperature sensitivity, so a higher content of HPMC is required. Experiments have found that several HPMCs are difficult to form gels when the content is lower than 4%. With the increase of HPMC content, the LCST of the system decreased gradually. The initial LCST of HPMC50 is high, but with the increase of its content, its LCST declines the fastest. When the content is 8%, the LCST drops to 54℃. The LCST of HPMC6 and HPMCl5 cannot be gelled when the content is 4%, and the content is increased. At a high concentration of 12%, their LCSTs all decreased to 51°C. In contrast, the LCST of HPMC30 is always at the lowest with the increase of the content, but its decline is not large. The lowest level of HPMC50 is 54°C when the concentration is 6%, and the lowest LCST value is 50°C when the concentration is 12%.

The content of HPMC has a great influence on the lower critical solution temperature of the system, and it should also be viewed in combination with its viscosity. For HPMC, the higher the viscosity value, the greater the relative molecular mass. When the mass is constant, the number of moles of HPMC with a large relative molecular mass will be small. When the concentration is low, there are more intramolecular effects in HPMC, so higher energy is needed to release the intramolecular effects and transform them into intermolecular effects, so the initial LCST of HPMC50 is the highest. However, when the concentration increases, the interaction between HPMC molecules is significantly enhanced. The larger the relative molecular mass of HPMC, the more network nodes, the larger the size, and the stronger the gelling ability. Therefore, the LCST of HPMC50 has the fastest downward trend. However, due to the large relative molecular mass of HPMC50, increasing the content of HPMC50 over 8% will make the system very viscous and difficult to flow. The relative molecular weight of HPMC6 and HPMC15 is relatively low, the number of molecules is relatively large, and there are many interactions between molecules, so it is easy to form a large number of small-sized aggregates. However, HPMC30 is in the middle, and has the comprehensive advantages of relative molecular mass and molar number, and correspondingly obtains the lowest LCST.

2.3 The effect of glycerol on the lower critical solution temperature

Studies have shown that many salts, surfactants, small molecule alcohols, etc. can reduce the lower critical solution temperature of HPMC. The effect of glycerol on the lower critical solution temperature of CS/HPMC system was studied by using glycerol as an additive. When the glycerol content is lower than 10%, the LCST of the system is still above 50°C. With the increase of glycerol content, the LCST of the system began to decrease significantly. When the glycerol content reaches 30%, the LCST of the CS/HPMC system drops below 37°C. Continue to increase the glycerol content to 33%, the state of the CS/HPMC solution is still stable, and the LCST of the system can drop to 32°C. If the glycerin content is further increased, the system becomes unstable, turbid and difficult to form a gel.

Glycerin is very hydrophilic. When its content is low, the small molecules of glycerin disperse and interact with free water molecules, without destroying the hydration layer of HPMC, but interspersed around the macromolecules of HPMC, and even play a certain role of lubrication and protection, so the gelation temperature No lowering. When the glycerol content increased to a certain level, glycerol began to compete for water molecules on HPMC, resulting in dehydration, which reduced the dehydration energy of HPMC, so the LCST of the system began to decrease significantly. In addition, the polyhydroxy groups of glycerol will form a large number of hydrogen bonds with CS macromolecules and free water molecules, promote the formation and stability of molecular network structures, and accelerate the gelation of the system.

2.4 Orthogonal experiment

Based on the above experiments, the concentration of CS was selected as 1%, and the HPMC viscosity (A), concentration (B) and glycerol addition (C) were used as variables, and a three-factor four-level L16 (43) orthogonal experiment was designed. The order of primary and secondary factors is HPMC content > glycerol content > HPMC. Viscosity, the optimal combination is A3B4C4, that is, the viscosity of HPMC is 30 mPa·S, the content of HPMC is 7%, and the content of glycerin is 32%. Since there is no such combination in the orthogonal experiment, the verification experiment is carried out with this combination, the experiment number is V, and the LCST of the system is 32°C, which is the same as the result of the experiment number 4. Comparing the factors, it can be seen that the only difference between the two experimental conditions is the viscosity of HPMC, which also confirms that the influence of the viscosity of HPMC among the three factors is not significant.

In addition, it can also be seen that the lower critical solution temperatures of the six experiments numbered 3, 4, 7, 8, 10, and 12 are all lower than 37°C. From the requirements of injectability, the viscosity of the solution should be low, so the viscosity of the 6 groups of experimental samples and the samples of group V were tested. The highest viscosity of group V is 7.67Pa. S, exceeding 5 Pa·S, is not conducive to injection. The viscosity of experiment 4 with the same LCST is only 1.41 Pa·S. Considering comprehensively, experiment No. 4 was selected as the optimal scheme, that is, the viscosity of HPMC was 6 mPa·S, the content of HPMC was 7%, and the content of glycerol was 32%. At the same time, the pH value of the system was tested with accurate pH test paper to be about 6.8, which is within the acceptable range of the human body, and the system is stable without precipitation, which meets the requirements of biomedical materials.

2.5 Infrared spectral analysis

Theoretically speaking, HPMC and CS can form intermolecular hydrogen bonds to form a compatible system. The kinetic process of phase separation in this system is quite slow, so a blend with more stable structure and properties can be obtained. Glycerol is a small hydrophilic molecule, and it is also easy to form hydrogen bonds with the CS/HPMC system.

2.6 Gelation temperature characterization

When the sample is at room temperature and below, it is clear and transparent, has good fluidity, and can be stored for a long time; at 32°C, it will gel, and at human body temperature (37°C) and above, it can be stored in a short period of time (by visual inspection). About 15 rain) completely gelled.

2.7 Cytotoxicity analysis

The MTT test is based on the absorbance value of the test sample to characterize the toxic effect of the sample on the cells. According to the different RGR values, the evaluation criteria can be divided into 0-5 grades. A higher rating indicates a more toxic material.

3. Conclusion

The blending of CS and HPMC can make thermosensitive hydrogel. Chitosan macromolecule is the skeleton material for forming hydrogel, which has certain influence on gelation temperature and gelation time. HPMC has the greatest impact on the lower critical solution temperature of hydrogel. HPMC with lower viscosity cannot gel when the content in the system is low (<4%), and at a higher concentration (5% to 10%) , the LCsT of the system showed a slight downward trend, but it was still above 50℃. The addition of glycerol can significantly reduce the lower critical solution temperature of CS/HPMC to below the body temperature (37°C). The system has good fluidity and stability at room temperature (25°C), can gel in a short period of time at human body temperature, has good biocompatibility, and is suitable for injectable thermosensitive in situ Development of gel products.