Method for preparing layered double hydroxide@poloxamer 188 nanohybrids and applications thereof

Abstract

The application discloses a preparation method and application of layered double hydroxide@poloxamer 188 nanohybrid, and the method comprises the following steps: dissolving a divalent metal soluble salt and a trivalent metal soluble salt to obtain a mixed salt solution; slowly dropping the mixed salt solution into a NaOH solution, adjusting the pH value to 9-10 after the dropping is completed, continuously stirring at room temperature, removing supernatant by centrifugation, and washing the precipitate with water until neutral; resuspending the precipitate, pouring into a crystallization kettle, freezing and drying after crystallization to obtain layered double hydroxide powder; dissolving the layered double hydroxide powder into formamide to obtain a layered double hydroxide suspension, and carrying out delamination; adding a NaOH solution containing an antitumor drug into the delaminated layered double hydroxide suspension, adding a poloxamer 188 micelle solution, removing the formamide, resuspending with water, and aging at room temperature to obtain a layered double hydroxide@poloxamer 188 nanohybrid solution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a preparation method and application of layered double hydroxide@poloxamer 188 nanohybrid. BACKGROUND

[0002] Layered double hydroxides (LDHs) are typical two-dimensional nanomaterials. Due to their inherent physicochemical properties, including structural tunability, biocompatibility, and pH sensitivity, they have shown great potential in nanodiagnostic and nanotherapeutic platforms, including drug and gene delivery, photothermal and chemical kinetic therapy, and magnetic resonance imaging diagnosis. However, one of the problems that needs to be solved for LDHs as delivery carriers is that bare drug-loaded LDHs are prone to aggregation, have poor stability, and are easily adsorbed by negatively charged macromolecules in physiological environments, which leads to poor delivery behavior and limits their clinical application. Therefore, it is imperative to modify the surface with a coating layer that can increase the circulation time in the body and has good biocompatibility.

[0003] In existing research, some polymer electrolytes such as low molecular weight heparin, bovine serum albumin, high molecular block polymers, chitosan, liposomes, and PEGylated hyaluronic acid can be used as LDH colloidal stabilizers to reduce toxicity caused by aggregation. However, in the preparation process of polymer electrolytes as LDH colloidal stabilizers, improper use of preparation methods results in less drug insertion between layers and more adsorption on the surface, which leads to burst release of drug-loaded LDHs in physiological media and reduces drug utilization.

[0004] Therefore, it is of great significance to solve the problems of stable dispersion and burst release of drug-loaded LDHs through rational structure design and preparation methods. SUMMARY

[0005] To overcome the deficiencies in the prior art, the purpose of the present application is to prepare a layered double hydroxide@poloxamer 188 nanohybrid with good dispersion stability, drug release ability, and cell uptake effect by a delamination-reconstruction method, to improve drug release effect and improve drug utilization.

[0006] A preparation method of a layered double hydroxide@poloxamer 188 nanohybrid, comprising,

[0007] dissolving a divalent metal soluble salt and a trivalent metal soluble salt in CO2-removed deionized water to obtain a mixed salt solution;

[0008] The NaOH solution is magnetically stirred under N2 atmosphere at a speed of 500-600 rpm, and the mixed salt solution is slowly added dropwise into the stirring NaOH solution, after the dropwise addition is completed, the pH value is adjusted to 9-10, and the stirring is continued at room temperature for 0.5-2 h, then centrifuged at a speed of 4000-5000 r / min, and the supernatant is removed, the precipitate is resuspended with water, and the resuspension is repeated for multiple times to wash the precipitate to neutral;

[0009] After the precipitate is resuspended with water, it is poured into a crystallization kettle, and the crystallization kettle is placed in an oven at 90-120℃ for crystallization for 10-14 h, then frozen at -15 to -25℃ for at least 8 h, and then vacuum dried for at least 72 h to obtain a layered double hydroxide powder;

[0010] The layered double hydroxide powder is dissolved in formamide to obtain a layered double hydroxide suspension, and the layered double hydroxide suspension is ultrasonicated for 6-8 h for delamination;

[0011] NaOH solution containing an antitumor drug is added to the delaminated layered double hydroxide suspension, ultrasonicated for 0.5-2 h, then poloxamer 188 micelle solution is added, and ultrasonicated for 0.5-2 h, then nitrogen is filled to seal, and after standing, the precipitate is centrifuged and washed with water to remove formamide, and then resuspended with water, and then aged at room temperature to obtain a layered double hydroxide-poloxamer 188 nanohybrid solution.

[0012] Further, the above preparation method, wherein the divalent metal soluble salt is a combination of one or more of magnesium nitrate, magnesium chloride and manganese nitrate.

[0013] Further, the above preparation method, wherein the trivalent metal soluble salt is a combination of one or more of aluminum nitrate, aluminum chloride, gadolinium nitrate and europium nitrate.

[0014] Further, the above preparation method, wherein the molar ratio of metal ions of the divalent metal soluble salt and the trivalent metal soluble salt is 2:1-3:1.

[0015] Further, the above preparation method, wherein the antitumor drug is an anionic drug.

[0016] Further, the above preparation method, wherein the content of the antitumor drug in the NaOH solution containing the antitumor drug is 2.5-9 mg / mL.

[0017] Further, the above preparation method, wherein the concentration of the layered double hydroxide suspension is 0.1-0.25 g / mL.

[0018] Further, the preparation method described above, wherein the concentration of the poloxamer 188 micelle solution is 0.8-8 mg / mL.

[0019] Further, the preparation method described above, wherein the ratio of the volume of the total water in the poloxamer 188 micelle solution and the NaOH solution dissolving the antitumor drug to the volume of the formamide is 1:1.

[0020] The application also provides an application of the layered double hydroxide@poloxamer 188 nanohybrid, which is applied to drug delivery.

[0021] The application utilizes the electrically neutral polymer micelle poloxamer 188 to improve the stability of the LDHs colloid. The poloxamer 188 is a kind of triblock copolymer copolymerized by polyoxyethylene and polyoxypropylene ether, belongs to the amphiphilic polymer micelle, has good biocompatibility (non-toxic, non-antigenic, non-irritating, non-sensitizing, stable chemical properties, non-hemolysis) and water phase dispersibility, and also has the effect of repairing cell membranes, can promote the absorption of drugs as a pharmaceutical excipient. In addition, it can self-assemble into nanomicelles in water, can effectively reduce the risk of carrier material being removed by the reticuloendothelial system, and improve the in vivo stability of the drug-loaded system.

[0022] The application has the following advantages:

[0023] First, the poloxamer 188 is compounded with the layered double hydroxide, which solves the problem that the layered double hydroxide is easy to aggregate in high ionic strength physiological fluid, so that the drug-loaded nanohybrid product can be stably dispersed in the physiological medium.

[0024] Second, the exfoliation and reconstruction method is adopted to obtain nanoscale hybrid particles with uniform particle size distribution, and the product has good drug release capacity.

[0025] Additional aspects and advantages of the application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is the XRD pattern of the layered double hydroxide in Example 1;

[0027] Figure 2 It is the TEM pattern of the LDHs-MTX@poloxamer 188 nanohybrid in Example 1;

[0028] Figure 3 It is the TEM pattern of the LDHs-5Fu@poloxamer 188 nanohybrid in Example 2;

[0029] Figure 4In-vitro release profile of LDHs-MTX@poloxamer 188 nanohybrid for Example 3;

[0030] Figure 5 In-vitro release profile of LDHs-5Fu@poloxamer 188 nanohybrid for Example 4;

[0031] Figure 6 Cellular uptake profile of LDHs-FITC@poloxamer 188 nanohybrid for Example 5. DETAILED DESCRIPTION

[0032] In order to make the objects, features and advantages of the present application more clear and obvious, the specific embodiments of the present application are described in detail below. In the examples, several embodiments of the present application are given. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0033] Example 1

[0034] The example 1 of the present application provides a preparation method of layered double hydroxide@poloxamer 188 nanohybrid, mainly including two steps: preparing layered double hydroxide; and preparing layered double hydroxide@poloxamer 188 nanohybrid based on the layered double hydroxide.

[0035] Specifically, the preparation of the layered double hydroxide includes the following steps:

[0036] (1) 1.5112 g (5.89 mmol) of magnesium nitrate hexahydrate and 0.7891 g (2.1 mmol) of aluminum nitrate nonahydrate were weighed respectively and dissolved in 20 mL of CO2-free deionized water to obtain a mixed salt solution;

[0037] (2) 0.7 g (0.0175 mol) of NaOH was dissolved in 100 mL of water, and the mixed salt solution was slowly added to the stirring lye under N2 atmosphere at 600 rpm. After the addition was completed, the pH value of the mixed solution was adjusted to 9.5, and the stirring was continued at room temperature for 1 h. Then, the solution was centrifuged at 4500 r / min for 10 min to remove the supernatant, and the precipitate was resuspended with water. The cycle was repeated 4 times to wash the precipitate to near neutral.

[0038] (3) The precipitate was resuspended with water to a constant volume of 75 mL, then poured into a crystallization kettle, and then placed in an oven. The crystallization was carried out at 100℃ for 12 h. Then, the crystallized solution was frozen at -20℃ for more than 8 h, and then placed in a vacuum drying machine with a cold trap temperature of -60℃ for drying for 72 h to obtain a layered double hydroxide powder.

[0039] The crystal form characteristics of the layered double hydroxide of the present embodiment are shown in Figure 1 As shown in the figure, the characteristic diffraction peaks of the (003), (006) and (009) crystal planes are respectively located near 11.4°, 22.9° and 34.5°, and the degrees between the diffraction angles have a good multiple relationship, indicating that the product has a regular layered structure.

[0040] The layered double hydroxide@poloxamer 188 (LDHs-MTX@poloxamer 188) nanohybrid is prepared by the following steps:

[0041] The layered double hydroxide powder 0.4 g is added to 20 mL formamide, and delamination is performed by ultrasonic for 7 h. Then, 10 mL of NaOH solution (0.01 mol / L) dissolving 50 mg of methotrexate (MTX) is added, and ultrasonic is performed for 1 h. Then, 10 mL of poloxamer 188 micelle solution with a concentration of 2 mg / mL is added, and ultrasonic is continued for 1 h. After that, nitrogen is filled for sealing, and the mixture is left overnight. The precipitate is repeatedly centrifuged and washed with water for 6 times to remove formamide. Finally, freeze-drying is performed to obtain the LDHs-MTX@poloxamer 188 nanohybrid.

[0042] The antitumor drug used in the present embodiment is methotrexate, and the obtained layered double hydroxide@poloxamer 188 nanohybrid is a layered double hydroxide@poloxamer 188 loaded methotrexate (LDHs-MTX@poloxamer 188) nanohybrid. The TEM image of the prepared LDHs-MTX@poloxamer 188 nanohybrid is shown in Figure 2 As shown in the figure, the particle size of the product is mostly not more than 100 nm, and the product is uniformly dispersed.

[0043] Example 2

[0044] In the present embodiment, the method for preparing the layered double hydroxide is the same as in the first embodiment. However, in the present embodiment, the antitumor drug is 5-fluorouracil (5Fu), and the obtained layered double hydroxide@poloxamer 188 loaded 5-fluorouracil (LDHs-5Fu@poloxamer 188) nanohybrid is prepared by the following steps:

[0045] The layered double hydroxide powder 0.4 g is added to 20 mL formamide, and delamination is performed by ultrasonic for 7 h. Then, 10 mL of NaOH solution (0.01 mol / L) dissolving 90 mg of 5-fluorouracil is added, and ultrasonic is performed for 1 h. Then, 10 mL of poloxamer 188 micelle solution with a concentration of 2 mg / mL is added, and ultrasonic is continued for 1 h. After that, nitrogen is filled for sealing, and the mixture is left overnight. The precipitate is repeatedly centrifuged and washed with water for 6 times to remove formamide. Finally, freeze-drying is performed to obtain the LDHs-5Fu@poloxamer 188 nanohybrid.

[0046] The TEM image of the prepared LDHs-5Fu@poloxamer 188 nanohybrid is shown in Figure 3 As shown in the figure, the particle size of the product is within 100 nm, and the dispersion is uniform.

[0047] It should be noted that in another embodiment of the present application, the speed of the magnetic stirring in the step of preparing the layered double hydroxide can also be 500 rpm. After slowly adding the mixed salt solution to the stirring NaOH solution, the pH value is adjusted to 10, stirring at room temperature for 2 h, the crystallization temperature is 120℃, the crystallization time is 14 h, and the other conditions are the same as in the first embodiment. The required nanohybrid can also be prepared.

[0048] In still another embodiment of the present application, in the step of preparing the layered double hydroxide@poloxamer 188 nanohybrid based on the layered double hydroxide, the concentration of the poloxamer 188 micelle solution can be 8 mg / mL, and the other conditions are the same as in the first embodiment. The required nanohybrid can also be prepared.

[0049] Example 3

[0050] The in vitro drug release experiment of the LDHs-MTX@poloxamer 188 nanohybrid includes the following steps.

[0051] (1) Prepare sodium acetate buffer solution with pH = 4.6 and potassium dihydrogen phosphate buffer solution with pH = 7.4, respectively, as the simulated medium for in vitro drug release.

[0052] (2) Measure 1.0 mL of the LDHs-MTX@poloxamer 188 nanohybrid before freeze-drying in Example 1, put it into a dialysis bag, seal the two ends with a fine line, and put the dialysis bag into a buffer containing 20 ml, and release in a constant temperature shaker at a speed of 100 r / min and a temperature of 37±0.5℃.

[0053] (3) Every 0.25 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 7 h, 8 h, 10 h, 12 h, 24 h, take out 3 mL of the medium containing the drug and supplement 3 mL of the release medium. After sampling at the fixed point, the medium containing MTX at different time periods is determined by ultraviolet-visible spectrophotometer to determine the ultraviolet absorbance at 265 nm, and the corresponding drug concentration and release percentage are calculated according to the standard curve to draw the cumulative release curve.

[0054] The MTX release behavior of the LDHs-MTX@poloxamer 188 nanohybrid in different pH value simulated media is shown in Figure 4As shown, the drug-loaded system has good sustained-release effect in both pH environments, and in the medium with pH = 7.4 (simulating the neutral environment in vivo), the release of MTX is very slow because the layer structure is stable, and the cumulative release rate in 24h is 40.4%, while in the medium with pH = 4.6 (simulating the tumor lysosome environment), the layer structure collapses gradually because the layer plate with alkaline is gradually dissolved, so that the MTX is completely released in 24h, achieving the purpose of full drug release of the drug-loaded system in the lesion area.

[0055] Example 4

[0056] The in vitro drug release experiment of the LDHs-5Fu@poloxamer 188 nanohybrid includes the following steps.

[0057] (1) Prepare a sodium acetate buffer solution with pH = 4.6 and a potassium dihydrogen phosphate buffer solution with pH = 7.4, respectively, as the simulated medium for in vitro drug release.

[0058] (2) Measure 1.0mL of the LDHs-5Fu@poloxamer 188 nanohybrid before freeze-drying in Example 2, put it into a dialysis bag, seal the two ends with a thin wire, and put the dialysis bag into a buffer solution containing 20ml, and release in a constant temperature oscillator, keeping the speed at 100r / min and the temperature at 37±0.5℃.

[0059] (3) Take out 3mL of the medium containing the drug every 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 7h, 8h, 10h, 12h, 24h and supplement 3mL of the release medium. After sampling at the fixed point, the medium containing 5Fu at different time periods is determined by ultraviolet-visible spectrophotometer to determine the release amount of 5Fu, and the ultraviolet absorbance is measured at 265nm. The corresponding drug concentration and release percentage are calculated according to the standard curve, and the cumulative release curve is drawn.

[0060] The 5Fu release behavior of the LDHs-5Fu@poloxamer 188 nanohybrid in different pH simulated media is as shown in Figure 5 As shown, the drug-loaded system has good sustained-release effect in both pH environments, and in the medium with pH = 7.4 (simulating the neutral environment in vivo), the release of MTX is very slow because the layer structure is stable, and the cumulative release rate in 24h is 40.4%, while in the medium with pH = 4.6 (simulating the tumor lysosome environment), the layer structure collapses gradually because the layer plate with alkaline is gradually dissolved, so that the MTX is completely released in 24h, achieving the purpose of full drug release of the drug-loaded system in the lesion area.

[0061] Example 5

[0062] The cell uptake experiment of the LDHs-FITC@poloxamer 188 nanohybrid includes the following steps.

[0063] (1) 0.4 g layered double hydroxide powder (obtained in Example 1) was added to 20 mL formamide, and delamination was performed by ultrasonic treatment for 7 h, 10 mL of a NaOH solution (0.01 mol / L) in which 9 mg of fluorescein isothiocyanate (FITC) was dissolved was added, and ultrasonic treatment was performed for 1 h, then 10 mL of a poloxamer 188 micelle solution having a concentration of 2 mg / mL was added, and ultrasonic treatment was continued for 1 h, then nitrogen was filled to seal, and the mixture was left to stand overnight, and the precipitate was repeatedly washed with water by centrifugation 6 times to remove the formamide, to obtain a LDHs-FITC@poloxamer 188 nanohybrid suspension.

[0064] (2) Human osteosarcoma cells (MG63 cells) were selected as experimental cells, the suspended MG63 cells were inoculated in a cell culture plate, and were cultured in a 5% CO2 incubator for 24 h, after the cells adhered, 1 mL of free FITC having a FITC content of 20 μg / mL and the LDHs-FITC@poloxamer 188 nanohybrid suspension were added respectively, and were cultured for 24 h, the residual FITC and nanohybrid in the hole were removed by washing with PBS (polybutylene succinate) 3 times, the cells were fixed with 4% paraformaldehyde in the dark for 20 min, then were washed with PBS 3 times, then Hoechst 33342 (5 μg / mL) was added to stain the nucleus in the dark for 20 min, and the cells were washed with PBS 3 times, and then the treated cells were observed under a laser confocal microscope to observe the cell morphology and cell uptake.

[0065] The uptake of free FITC and the LDHs-FITC@poloxamer 188 nanohybrid by MG63 cells is shown in FIG. 2. Figure 6 As can be seen from the comparison, the green fluorescence of the LDHs-FITC@poloxamer 188 group is brighter, which indicates that the FITC of the simulated drug is more easily taken up by the target cells and enters the cells after being combined with the carrier material, and the layered double hydroxide@poloxamer 188 nanocarrier hybrid as a drug carrier material plays a role.

[0066] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0067] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A process for the preparation of layered double hydroxide@poloxamer 188 nanohybrid, characterized by, The application relates to a preparation method of layered double hydroxide@poloxamer 188 nanohybrid solution. The bivalent metal soluble salt and the trivalent metal soluble salt are magnesium nitrate hexahydrate and aluminum nitrate nonahydrate respectively; The mixed salt solution is slowly added into the stirring NaOH solution under the N2 atmosphere, the magnetic stirring is carried out at the rotating speed of 500-600 rpm, the pH value is adjusted to 9-10 after the addition is completed, the stirring is continuously carried out at room temperature for 0.5-2 h, the centrifugal separation is carried out at the speed of 4000-5000 r / min, the supernatant is removed, the precipitate is resuspended with water, and the resuspension is repeated for multiple times to wash the precipitate to neutral. The precipitate is resuspended with water, poured into a crystallization kettle, and then the crystallization kettle is placed in an oven at 90-120 DEG C for crystallization for 10-14 h, frozen at-15 to-25 DEG C for at least 8 h, and then vacuum dried for at least 72 h to obtain layered double hydroxide powder. The layered double hydroxide powder is dissolved in formamide to obtain a layered double hydroxide suspension, and the layered double hydroxide suspension is ultrasonically treated for 6-8 h to carry out delamination. The NaOH solution containing an antitumor drug is added into the delaminated layered double hydroxide suspension, ultrasonically treated for 0.5-2 h, then the poloxamer 188 micelle solution is added, ultrasonically treated for 0.5-2 h, then nitrogen is filled to seal, and after standing, the precipitate is centrifugally washed to remove formamide, resuspended with water, and then aged at room temperature to obtain the layered double hydroxide@poloxamer 188 nanohybrid solution, wherein the concentration of the poloxamer 188 micelle solution is 0.8-8 mg / mL.

2. The production method according to claim 1, wherein The molar ratio of metal ions of the bivalent metal soluble salt to the trivalent metal soluble salt is 2:1-3:

1.

3. The production method according to claim 1, wherein The antitumor drug is an anionic drug.

4. The production method according to claim 1, wherein The content of the antitumor drug in the NaOH solution containing the antitumor drug is 2.5-9 mg / mL.

5. The production method according to claim 1, wherein The concentration of the layered double hydroxide suspension is 0.1-0.25 g / mL.

6. The production method according to claim 1, wherein The ratio of the total volume of water in the poloxamer 188 micelle solution and the NaOH solution containing the antitumor drug to the volume of the formamide is 1:

1.

7. Use of a layered double hydroxide®poloxamer 188 nanohybrid for the preparation of a drug delivery formulation, characterized in that, The layered double hydroxide@poloxamer 188 nanohybrid is obtained by the preparation method in any one of claims 1 to 6.

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