Study on preparation and slow release properties of Magnesium-Al hydrotalcite with alanine intercalation

. The precursors of Mg-Al hydrotalcite (Mg/Al-LDHs-NO 3 ) were prepared by low saturation coprecipitation method (constant pH method). In addition, alsine-intercalated magnesium-aluminum hydrotalcite (LDHs-Ala) was synthesized by low saturation coprecipitation (constant pH) and nucleation/crystallization isolation methods when the pH value was higher than the isoelectric point of alanine. The release properties of alanine intercalated hydrotalc in simulated gastric fluid (hydrochloric acid solution with pH=1.5) and intestinal fluid (phosphate solution with pH=7.4) were measured by spectrophotometer in vitro. The results showed that LDHs-Ala had good slow-release properties in simulated intestinal fluid and gastric fluid in vitro. Then sodium alginate was used to coated alanine intercalated hydrotalc for slow release in simulated intestinal fluid and simulated gastric fluid. It can be concluded that the coated intercalated hydrotalc has a low slow release rate in gastric fluid, but has a good slow release performance in simulated intestinal fluid. It can be concluded that the coated intercalated hydrotalc is expected to be a intestinal targeted release agent, which can be applied in biomedicine.


Introduction
Hydrotalcite is a kind of anionic intercalated composite with layered structure. Anionic intercalation composites have a certain amount of positive charge on the laminates, so some anions can enter the laminates and act on the laminates with a certain weak chemical force, such as van der Waals force and hydrogen bond, so as to form anionic intercalation composites. There are natural hydrotalcite in nature, but also can be artificial synthesis in the laboratory, and the operation is simple, synthesis methods are more kinds, including coprecipitation method, roasting reduction method, ion exchange method, etc. The magnesium ions on the hydrotalcite laminates can be replaced by other metal ions in the same crystal. The interlayer anions are exchangable, so the hydrotalcite compounds with different functions can be synthesized, which has a good application prospect in the field of functional polymers. In the past, organic matter was used as the skeleton material in the drug slow release system, but there may be shortcomings such as poor mechanical strength, poor thermal stability and secondary pollution of organic solvent. In view of the excellent properties of hydrotalcite, people gradually use hydrotalcite as a drug carrier to form supramolecular structure system. The drug intercalated hydrotalcite obtained by intercalation assembly can improve the thermal stability, acid-base resistance and drug slow release performance of hydrotalcite, and the intercalation assembled hydrotalcite compounds have good biocompatibility and will not produce rejection reaction in human body, so it has been widely concerned by researchers. This topic through the in-depth study of hydrotalcite, according to the special properties of hydrotalcite, amino acids and hydrotalcite intercalation assembly under certain circumstances, expect to prepare a new type of amino acid drug sustained-release agent, so as to study the slow-release performance of amino acid molecules in human body and achieve the targeted release of amino acids. This type of drug release system can be targeted to certain organs to achieve specific efficacy and avoid harming other organs.

Proposal of research questions
Amino acid drugs have some limitations, such as poor acid and alkaline resistance and poor absorption. In view of the advantages of drug intercalated hydrotalcite in the field of drug slow release, I used hydrotalcite as a carrier and inserted alanine into hydrotalcite to study the slow release performance of alanine intercalated hydrotalcite.

Mg/Al-LDHs-NO3 hydrotalcite was prepared by constant pH method
Weighing 20.51g(0.08mol) magnesium nitrate and 15.01g(0.04mol) aluminum nitrate in A certain amount of boiling deionized water is solution A; About 9.6g(0.24mol) of sodium hydroxide was weighed and dissolved in boiled deionized water, denoted as solution B. Solution B and solution A were dripped out at a certain rate, and then added to a large beek to gradually produce milky white precipitation. The pH value of the solution was always adjusted to be between 9 and 10. After dripping, the solution was reflow stirred for 4h at 70℃, the resulting slurry was crystallized for 5h, pumped out and washed with deionized water until neutral. Dry in oven at 65℃ for 24h. Grind and store in a drying oven.

Preparation of alanine intercalated Mg-Al hydrotalcite by constant pH method
Weighing 20.51g(0.08mol) magnesium nitrate and 15.01g(0.04mol) aluminum nitrate in A certain amount of boiling deionized water is solution A; Weighing alanine (Ala) 3.56g(0.04mol) dissolved in a certain amount of boiling deionized water is the solution. Under intense agitation and N2 protection, solution A was added to solution B at a constant rate, keeping the pH equal to 9~10 during the drip process. At pH value, a certain concentration of sodium hydroxide solution was used to adjust the drip, and then the slurry was reflow and stirred at 70℃ for 4h. The resulting slurry was liquid crystalline for 5h, then filtered and washed to neutral with deionized water. Dry in oven at 65℃ for 24h. Grind and store in a drying oven. Denoted as sample LDHs-Ala- [1].

Preparation of alanine intercalated Mg-Al hydrotalcite by nucleation crystallization isolation method
Weighing 20.51g(0.08mol) magnesium nitrate and 15.01g(0.04mol) aluminum nitrate in A certain amount of boiling deionized water is solution A; Weighing alanine (Ala) 3.56g(0.04mol) dissolved in a certain amount of boiling deionized water is the solution. Under intense agitation and N2 protection, solution A was added to solution B at a certain rate, and the pH was always maintained between 9 and 10 during the drip process. The pH was adjusted with sodium hydroxide solution. After drip adding, it was put into a multi-frequency chemical reactor for 40min. Finally, the solution was inverted into a three-necked flask for reflux stirring for 4h. Finally, it was crystallized and filtered, washed to pH less than 8, and dried in a 65℃ oven for 24h. Grind and store in a drying oven. It is denoted as LDHs-Ala- [2].

Generation of sodium alginate coated hydrotalcite
Solution A was obtained by dissolving 4g sodium alginate in a certain amount of deionized water and adding 0.4g or 0.27g alanine intercalated hydrotalcite LDHs-Ala- [2]. 4.4g CaCl 2 ꞏ2H 2 O was dissolved in 100mL deionized water to obtain solution B. After agitation, the solution A was slowly dropped into the solution B. After titration, the solution was filtered, washed with water, and dried at 65℃ in the oven for 24h. After drying, use a mortar to grind the powder as LDHs-Ala(musculo-1:10) or LDHs-Ala(musculo-1:15).

Determination of slow-release properties
The retarded release property of alanine can be determined by ninhydrin chromogenic method. This is the use of ninhydrin and alanine can produce purple or blue complex under a certain pH condition, using 722 spectrophotometer to determine the absorbance of alanine.

Preparation of ninhydrin color developing solution
Mix 100mL anhydrous ethanol with a small amount of water, add 0.5g ninhydrin, add 1.5mL pyridine, and a small amount of ascorbic acid, shake well to form a color solution.

Drawing of alanine standard curve
1g alanine was accurately weighed and diluted with deionized water into a constant volume 1000mL volumetric bottle to prepare 1g/L alanine buffer solution.
A certain volume of alanine mother liquor was absorbed respectively. The pH of 6 phosphate buffer solution was diluted into 25mL colorimetric tube, and alanine standard solution of different concentrations was prepared as 0mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L, 120mg/L, and 1mL color developing agent was added. Cover the stopper of the colorimetric tube and heat in a constant temperature water bath for 20min. The absorbance was measured on a 722 spectrophotometer at 570nm wavelength, and the standard working curve of alanine was drawn.

Sustained release experiment of alanine intercalation Mg-Al hydrotalcite
(1) Determination of the total amount of alanine inserted into hydrotalcite Take 1g of alanine intercalation magnesium aluminum hydrotalc and stir it with 0.3mol/L hydrochloric acid continuously to dissolve it, and dilute it with distilled water into a 100mL volumetric bottle, then accurately measure 2mL of the solution and add it into a 25mL colorimetric tube. Follow the same procedure as the standard curve for color development and measurement of absorbance.
(2) Measurement of slow release properties First, 100mL of simulated gastric fluid and simulated intestinal fluid were prepared, and then 0.5g each of alanine intercalated magnesia al hydrotalc LDHs-Ala- [1], LDHs-Ala- [2] and LDHs-Ala(coating) prepared by constant pH method and nucleation/crystallization isolation method were added, and stirred with magnetic stirring instrument. The temperature of the slow-release system was kept at (37±1)℃. Every once in a while, accurately measure 2mL of the solution, add it to the colorimetric tube, carry out color rendering in the same steps as the standard curve, and measure the absorbance. At the same time, 2mL hydrochloric acid solution with pH=1.5 or phosphate buffer solution with pH=7.4 were added into the simulated sustained release system in vitro. Finally, the sustained release rate at different time can be obtained by calculation. Reference experiment: Weighing 0.110g Mg/Al-LDHs-NO 3 and 0.100g Ala(NO 3 -:Ala=1:1), the two samples were ground evenly in a mortar. The cumulative slow release of alanine (Ala) in hydrochloric acid solution with pH=1.5 and phosphate solution with pH=7.4 was determined by the above method.

Standard curve of alanine
According to the experimental part, the standard curve of alanine (Ala) was drawn, and the equation of the curve was as follows: Y=-0.00393+0.00602X, R2=0.99966.

Determination of the release rate in simulated human gastric juices
According to the experimental part, the propyl acid intercalation magnesium-aluminum hydrotalcite LDHs-Ala- [1], LDHs-Ala- [2] and the physical mixture of magnesium-aluminum nitrate hydrotalcite precursors and alanine prepared by constant pH method and nucleation isolation method were released in hydrochloric acid solution with pH=1.5 for 3h, and the release curve was obtained. As can be seen from Figure 4-1, in the simulated human gastric fluid with pH=1.5, the sample release rate is particularly fast in the first 5 to 10min, which may be because hydrotalcite is layered dihydroxyl hydroxide, which is unstable under acidic conditions. The lamellar may be damaged and partially dissolved and released alanine, and then the release rate gradually slows down as time goes by. In the sustained release experiment of LDHs-Ala- [2], there are 5, 10, 30, 80, 180 In min, the release rates were 50.45%, 61.38%, 72.03%, 85.02% and 92.88%, respectively. The slow release process was a slow release, which may be caused by the exchange of chloride ions in solution with alanine between LDHs-Ala- [2] layers, and chloride ions entered the LDHs layers. However, when alanine enters the solution, the ion exchange rate is fast at the beginning stage, and then decreases gradually after reaching the maximum. Hydrotalc was destroyed in the middle plate of hydrochloric acid solution and the slow release rate of alanine intercalation of hydrotalc was increased. By comparing the two methods for the synthesis of alanine intercalation hydrotalcite, it can be concluded that the release amount of LDHs-Ala- [2] is more than that of LDHs-Ala- [1] at 180 min, indicating that the nucleation crystallization isolation method is easier to insert alanine into the hydrotalcite laminate. It can be seen from Figure 1 that the release process of physical mixture of magnesium aluminum nitrate hydrotalcite precursor and alanine in simulated human gastric fluid shows that the release rate of amino acid increases and decreases rapidly in 5-20min, which may be due to the poor acid resistance of alanine and its partial decomposition in hydrochloric acid solution. It can be concluded that the stability of alanine can be improved when alanine is inserted into hydrotalcite, and the release performance is relatively good.

Determination of release rate in simulated human intestinal fluid
LDHs-Ala- [1], LDHs-Ala- [2] and the physical mixture of precursors and alanine of propyl acid intercalated magnesium and aluminum hydrotalcite prepared by constant pH method and nucleation isolation method, as well as magnesium and aluminum nitrate hydrotalcite, were released in phosphate solution at pH=7.4 for 3 hours, and the release curve was obtained.
As can be seen from Figure 2, in the simulated human intestinal fluid with pH=7.4, the sample is basically released completely in about 30min, and the release rate finally persists at about 98.8%. The release of alanine intercalation hydrotalc synthesized by two different methods in the simulated human intestinal fluid does not occur suddenly from beginning to end, but it is a slow and continuous process. In the sustained release experiment of LDHs-Ala- [2], the release rates at 5, 10, 30, 80 and 180min were 34.94%, 48.55%, 60.40%, 77.03% and 84.99%, respectively. The release process in intestinal fluid is actually the result of the ion exchange between LDHs-Ala and phosphate in intestinal fluid, that is, phosphate ions in the sustained-release solution enter the interlaminates of hydrotalc, and alanine anions between the interlaminates of hydrotalc enter the sustained-release solution. This process is a slow and continuous process. Compared with physical mixtures, alanine intercalated hydrotalcite has better slow release properties. Similar to the simulated human gastric fluid, the slow release performance of the simulated human intestinal fluid is also that the alanine release of the alanine intercalated hydrotalcite prepared by nucleation crystallization isolation method is more and longer, which once again proves that the intercalated hydrotalcite prepared by nucleation crystallization isolation method has better performance.

Determination of the release rate of coated samples in simulated human gastric fluid intestinal fluid
The propyl acid intercalated magnesium aluminum hydrotalc LDHs-Ala- [1] and LDHs-Ala- [2] prepared by constant pH method and nucleation isolation method were coated with sodium alginate at a mass ratio of 1:10 and 1:15, respectively, and then released into the simulated intestinal fluid with pH=7.4 and the simulated gastric fluid with pH=1.5 for 2h to obtain the release curve. The samples coated with sodium alginate, LDHs-Ala (musculo-1:10) or LDHs-Ala (musculo-1:15), were put into simulated human gastric fluid and intestinal fluid respectively for the determination of slow-release properties, and released for two hours. As can be seen from Figure 3, alanine release in simulated human gastric juices is rarely less than 3%. In the simulated human intestinal fluid, alanine can release LDHs-Ala (musculo-1:15) slowly, and the release rates at 5, 10, 30, 80 and 180 min are 29.12%, 37.89%, 51.96%, 70.00% and 73.49%, respectively. The release rates of LDHs-Ala (coating-1:10) at 5, 10, 30, 80 and 180min were 40.74%, 54.21%, 62.63%, 72.8% and 74.38%, respectively. It can be concluded that the release performance of coated amino acid intercalated hydrotalc in human simulated intestinal fluid is better, and it can achieve sustained slow release in a long time, and the acid resistance is significantly improved. Therefore, the coated alanine intercalated hydrotalc is expected to be a targeted release agent of intestinal fluid, which can be widely used in biomedicine.

Conclusion
In this paper, the precursors of nitrate hydrotalcite were prepared by low saturation coprecipitation method (constant pH method), and alanine intercalated magnesium aluminum hydrotalcite was prepared by low saturation coprecipitation method (constant pH method) and nucleation/crystallization isolation method respectively. Then, the slow-release properties of alanine solution with pH=1.5). The results were as follows: Compared with the physical mixture of hydrotalcite and alanine, the hydrotalcite assembled by alanine intercalation can effectively improve the acid resistance of alanine, and has good slow-release performance in the simulated human intestinal and gastric fluid systems. LDHs-Ala coated with sodium alginate is basically not released in simulated human gastric fluid, but has good slow-release performance in simulated human intestinal fluid, that is, it can be released slowly and persistently for a long period of time. Therefore, amino acid intercalation hydrotalc is expected to become a targeted intestinal release agent and be applied in the field of biomedicine.