Preparation method and application of acetylated glucomannan microsphere carrier

Abstract

The present invention discloses a preparation method and application of an acetylated glucomannan microsphere carrier, and relates to the technical field of biomaterials. A preparation method of an acetylated glucomannan microsphere carrier comprises the following steps: A1, mixing chloroform containing acetylated glucomannan with phosphate buffer in a volume ratio of 8 to 12:1, performing a first ultrasonic emulsification to obtain a first emulsion; A2, mixing the first emulsion with a PVA aqueous solution, performing a second ultrasonic emulsification to obtain a second emulsion; A3, removing the organic solvent in the second emulsion to obtain the acetylated glucomannan microsphere carrier. The method has a simple process, and the prepared acetylated glucomannan microsphere carrier has a high drug loading rate and encapsulation rate, and is highly safe, will not cause a significant inflammatory reaction, and will not aggravate the progression of inflammatory diseases.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a preparation method of an acetylated glucomannan microsphere carrier and application thereof. Background Art

[0002] Macrophages are components of innate immunity. They can engulf pathogens or cell fragments and secrete a variety of nutritional factors, immune transmitters, etc. to play a vital role in maintaining tissue homeostasis and immunity. However, they are also involved in the occurrence of various pathological processes, including inflammatory diseases, autoimmune diseases, cancer and other diseases. In short, macrophages are divided into two subtypes, including "classically activated macrophages (M1)" and "selectively activated macrophages (M2)", which exist in the inflammatory environment of various diseases. Among them, M1 macrophages are mainly associated with rheumatoid arthritis (RA), type 2 diabetes (T2D), and atherosclerotic disease (AS). For example, in the context of atherosclerotic disease, long-term inflammatory response caused by macrophage-driven inflammation resolution defects is the main pathogenic factor; in type 2 diabetes, M1 produces proinflammatory factors that aggravate the damage of pancreatic β cells; and in the tumor microenvironment, M2 macrophages inhibit tumor immunity and promote angiogenesis, which contributes to tumor progression. Since the function of macrophages is widely related to the response to various disease states, targeting macrophages is an ideal target for the treatment of various diseases.

[0003] In the related technologies, the use of virus particles with specific targeting ability to macrophages as drug carriers has the risk of off-target immunogenicity and toxicity, low safety, and is difficult to be widely used; the use of fucose and cyclodextrin modified polypeptide carriers as drug carriers has a complex synthesis route, high cost, and low yield; the use of multiple polymers (PLGA or PEG) and mannose for composite surface modification to obtain macrophage-targeted carriers has a complex production process, high cost, and includes non-natural polymers, which are not conducive to metabolism and degradation in the body. In addition, macrophages are prone to secrete inflammatory factors after ingesting foreign matter, leading to increased inflammation levels, thereby aggravating the disease, and the above schemes do not evaluate the pro-inflammatory effect of drug carriers on macrophages.

[0004] Therefore, it is necessary to develop an efficient delivery platform that can target macrophages, has high safety, and does not cause significant inflammatory responses. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for preparing an acetylated glucomannan microsphere carrier, which has a simple process, and the prepared acetylated glucomannan microsphere carrier has a high drug loading rate and encapsulation rate, and is highly safe, does not cause significant inflammatory reactions, and does not aggravate the progression of inflammatory diseases.

[0006] The present invention also provides the acetylated glucomannan microsphere carrier prepared by the above preparation method.

[0007] The present invention also provides the use of the preparation method or the acetylated glucomannan microsphere carrier prepared by the preparation method in the preparation of macrophage targeting drugs.

[0008] A method for preparing an acetylated glucomannan microsphere carrier according to the first aspect of the present invention comprises the following steps:

[0009] A1, mixing chloroform containing acetylated glucomannan with phosphate buffer in a volume ratio of 8 to 12:1, performing a first ultrasonic emulsification to obtain a first emulsion;

[0010] A2, mixing the first emulsion with the PVA aqueous solution, and performing a second ultrasonic emulsification to obtain a second emulsion;

[0011] A3. Removing the organic solvent from the second emulsion to obtain the acetylated glucomannan microsphere carrier.

[0012] The preparation method according to the embodiment of the present invention has at least the following beneficial effects:

[0013] The preparation method of the embodiment is simple, easy to operate, and low in cost. The target product can be obtained through simple modification and double emulsion W1 / O / W2 solvent evaporation method to achieve mass production.

[0014] Compared with the emulsion evaporation method, the double emulsion W1 / O / W2 solvent evaporation method of the embodiment can prepare drug carriers with more uniform size and higher encapsulation rate. The inflammation induced by nanomaterials in the body is often related to the protein corona adsorbed by the nanomaterials. The preparation method of the embodiment uses the medicinal polymer material PVA as an emulsifying stabilizer, which can keep the double emulsion stable on the one hand and prevent excessive adsorption of other proteins on the other hand, thereby maintaining a good particle size and further reducing pro-inflammatory properties.

[0015] According to some embodiments of the present invention, the ultrasonic power of the first ultrasonic emulsification is >100W.

[0016] According to some embodiments of the present invention, the ultrasonic power of the first ultrasonic emulsification is 130 W to 200 W. For example, it can be 130, 140, 150, 160, 170, 180, 190 or 200 W. Thus, the surface morphology of the microsphere carrier may be destroyed due to excessive ultrasonic power.

[0017] According to some embodiments of the present invention, the ultrasound time of the first phacoemulsification is 0.5 to 2 minutes, for example, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9 or 2 minutes.

[0018] According to some embodiments of the present invention, the ultrasonic temperature of the first ultrasonic emulsification is 2-8°C. For example, it can be 2, 4, 6 or 8°C.

[0019] According to some embodiments of the present invention, the ultrasonic power of the second ultrasonic emulsification is >100W.

[0020] According to some embodiments of the present invention, the ultrasonic power of the second ultrasonic emulsification is 130 W to 200 W. For example, it can be 130, 140, 150, 160, 170, 180, 190 or 200 W. It is understandable that the ultrasonic power of the first ultrasonic emulsification and the second ultrasonic emulsification can be kept consistent.

[0021] According to some embodiments of the present invention, the ultrasound time of the second ultrasound emulsification is 0.5 to 2 minutes, for example, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9 or 2 minutes.

[0022] According to some embodiments of the present invention, the ultrasonic temperature of the second ultrasonic emulsification is 2-8°C. For example, it can be 2, 4, 6 or 8°C.

[0023] According to some embodiments of the present invention, the volume ratio of the first emulsion to the PVA aqueous solution is 0.5 to 1:1. For example, it can be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1.

[0024] According to some embodiments of the present invention, in the PVA aqueous solution, the PVA concentration is 0.1 wt% to 1 wt%, for example, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt% or 1 wt%.

[0025] According to some embodiments of the present invention, the method for preparing the acetylated glucomannan comprises the following steps:

[0026] B1, mixing glucomannan with a solvent for reaction, and separating the solid and the liquid to obtain a solid phase;

[0027] The solvent includes pyridine and anhydride;

[0028] B2. After purifying the solid phase, the acetylated glucomannan is obtained.

[0029] According to some embodiments of the present invention, the solid-liquid ratio of the glucomannan to the solvent is 1 g:80-120 mL.

[0030] According to some embodiments of the present invention, in the solvent, the volume ratio of the pyridine to the acid anhydride is 1:0.8-1.2.

[0031] According to some embodiments of the present invention, the glucomannan is further subjected to pretreatment, wherein the pretreatment comprises swelling treatment and drying, and the drying is preferably freeze-drying.

[0032] According to some embodiments of the present invention, the swelling treatment comprises mixing glucomannan with water, wherein the solid-liquid ratio of the glucomannan to the water is 1 g: 150 mL to 250 mL.

[0033] According to some embodiments of the present invention, the purification treatment comprises at least one of an ethanol precipitation treatment and a dialysis treatment.

[0034] According to some embodiments of the present invention, the reaction temperature of step B1 is 40°C to 60°C. For example, it can be 40, 44, 48, 52, 56 or 60°C.

[0035] According to some embodiments of the present invention, the reaction time of step B1 is 60 h to 84 h, for example, 60, 64, 68, 72, 76, 80 or 84 h.

[0036] According to some embodiments of the present invention, the phosphate buffer comprises a drug, and the drug is preferably a macrophage-related disease targeting drug.

[0037] According to some embodiments of the present invention, the drug includes at least one of a polypeptide protein biomacromolecule drug and a small molecule drug. The small molecule drug includes a water-soluble small molecule drug and a poorly water-soluble small molecule drug.

[0038] According to some embodiments of the present invention, the polypeptide protein biomacromolecule drugs include but are not limited to insulin, insulin analogs, interferon, ovalbumin (OVA), recombinant human growth hormone, glucagon-like peptide, glucagon-like peptide analog or immunoglobulin.

[0039] According to some embodiments of the present invention, the small molecule drugs include but are not limited to bupivacaine, lidocaine, mepivacaine, procaine, azathioprine, cyclosporine, D-penicillamine, sodium chloroaurate, hydroxychloroquine, leflunomide, methotrexate, minocycline, sulfasalazine, vildagliptin, saxagliptin, linagliptin or alogliptin.

[0040] According to the second aspect of the present invention, the acetylated glucomannan microsphere carrier is prepared by the preparation method described in the first aspect of the present invention.

[0041] The acetylated glucomannan microsphere carrier according to the embodiment of the present invention has at least the following beneficial effects:

[0042] Glucomannan is derived from natural plants, has a wide range of sources, a clear structure, and is inexpensive. It has the advantages of low immunogenicity, good biocompatibility, low toxicity, and biodegradability. It is more easily internalized and degraded by cells than other polymers. The polysaccharide microsphere carrier prepared from it does not have the problem of limited application due to potential risks. Glucomannan is a polysaccharide composed of D-mannose and D-glucose units. The presence of mannose units in its structure is conducive to increasing the interaction between its polymer and the mannose receptors on the surface of macrophages.

[0043] The acetylated glucomannan microsphere carrier of the embodiment does not cause obvious pro-inflammatory response, and has a certain anti-inflammatory effect at high doses, and is a mild and safe drug carrier; and can deliver drugs to macrophages, which can provide a new approach for the treatment of targeted macrophage drug delivery.

[0044] According to some embodiments of the present invention, the particle size of the acetylated glucomannan microsphere carrier is less than 300 nm, for example, it may be 50, 100, 150, 200, 250 or 300 nm, thereby reducing the pro-inflammatory effect on macrophages.

[0045] According to some embodiments of the present invention, the particle size of the acetylated glucomannan microsphere carrier is 100 nm to 300 nm.

[0046] Use of the above preparation method according to the third aspect of the present invention or the acetylated glucomannan microsphere carrier prepared by the above preparation method in the preparation of macrophage targeting drugs.

[0047] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 1H NMR spectra (A) and (B) of GM and acGM according to an embodiment of the present invention;

[0049] Figure 2 is a chromatogram of the acetylated glucomannan prepared in Example 1 of the present invention;

[0050] Figure 3 is a chromatogram of the acetylated glucomannan prepared in Comparative Example 1 of the present invention;

[0051] Figure 4The particle size distribution diagram (A), transmission electron microscopy diagram (B) and stability test result (C) of the acetylated glucomannan microsphere carrier prepared in Example 1 of the present invention are shown;

[0052] Figure 5 1 is a particle size distribution diagram of the acetylated glucomannan microsphere carrier of Comparative Example 2 (A) and Comparative Example 3 (B) of the present invention;

[0053] Figure 6 This is the cytotoxicity test result of the acetylated glucomannan microsphere carrier prepared in Example 1 of the present invention;

[0054] Figure 7 It is the pro-inflammatory effect of the acetylated glucomannan microsphere carrier prepared in Example 1 of the present invention on normal RAW264.7 cells and inflammatory RAW264.7 cells; wherein A and B are the NO level detection results of the normal RAW264.7 cell group and the inflammatory RAW264.7 cell group, respectively; C and D are the IL-1β expression level detection results of the normal RAW264.7 cell group and the inflammatory RAW264.7 cell group, respectively; E and F are the IL-6 expression level detection results of the normal RAW264.7 cell group and the inflammatory RAW264.7 cell group, respectively; wherein, * indicates a significant difference compared with the LPS+IFN-γ group, P<0.05; ** indicates a significant difference compared with the LPS+IFN-γ group, P<0.01; *** indicates a significant difference compared with the LPS+IFN-γ group, P<0.001; ns indicates no significant difference compared with the LPS+IFN-γ group;

[0055] Figure 8 The acetylated glucomannan microsphere carriers of comparative examples 2 and 3 of the present invention have pro-inflammatory effects on normal RAW 264.7 cells; * indicates a significant difference compared with the Control group, P<0.05; ** indicates a significant difference compared with the Control group, P<0.01; *** indicates a significant difference compared with the Control group, P<0.001, and ns indicates no significant difference compared with the Control group;

[0056] Fig. 9 These are the test results of the encapsulation efficiency (EE) and drug loading rate (DL) of acGMMs@OVA prepared in Examples 2 to 6 of the present invention;

[0057] Fig.10 5 is the distribution detection result of acGMMs@OVA in RAW 264.7 cells and WRL 68 cells of Example 5 of the present invention; wherein A is the blank control group, B, C, and D are the bright field, fluorescence, and merged images of the acGMMs@OVA group, respectively, and E is the flow cytometry detection result;

[0058] Fig.11 This is the test result of the acGMMs@OVA in Example 5 of the present invention promoting the uptake of RAW 264.7 cells; wherein A is the result of fluorescence microscopy detection, B and C are the test results of the uptake of OVA and acGMMs@OVA by normal macrophages and inflammatory macrophages, respectively. DETAILED DESCRIPTION

[0059] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0060] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0061] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0062] In the description of the present invention, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or apparatuses.

[0063] The RAW 264.7 cells and WRL 68 cells in the examples of the present invention were provided by ATCC.

[0064] Unless otherwise specified, "about" in the present invention means that the allowable error is within ±2%.

[0065] Unless otherwise specified, all cell-related cultures and biological experiments in the present invention were performed in a cell culture incubator at 37° C. and 5% CO 2 ; the culture medium was DMEM cell culture medium containing 10% FBS and 1% P / S.

[0066] Example 1

[0067] This embodiment provides a method for preparing acetylated glucomannan microsphere carrier (acGMMs), and the preparation method is as follows:

[0068] S1. Preparation of acetylated glucomannan (acGM):

[0069] Weigh a quantitative amount of 1g of glucomannan (GM) and fully swell it in 200mL of water, freeze-dry it and set it aside; after stirring 100mL of a mixture of pyridine and anhydride (1:1, V / V) at 50°C for half an hour, slowly pour it into a round-bottom flask containing freeze-dried GM, stir and react at 50°C for 72h, filter to obtain a residue; mix the residue with 100mL of a mixture of pyridine and anhydride (1:1, V / V), stir and react at 50°C for 72h, filter to obtain a residue; precipitate the residue with ethanol overnight, filter to obtain a residue; resuspend the residue with ethanol, centrifuge it again, repeat 5 times in total to obtain a residue; dialyze the residue at room temperature for 2 days, use ultrapure water as the dialysis medium, and freeze-dry to obtain the final reaction product acGM.

[0070] S2. Preparation of acetylated glucomannan microsphere carrier:

[0071] Weigh 5 mg of acGM and dissolve it in 800 μL of chloroform, then add 80 μL of phosphate buffer solution PBS (pH 7.4), mix well, emulsify on ice with an ultrasonic probe for 1 min (ultrasonic power 150 W), and stir to obtain a water-in-oil (W1 / O) emulsion; then add 1 mL of 0.5 wt% polyvinyl alcohol (PVA) aqueous solution, continue ultrasonic emulsification on ice for 1 min (ultrasonic power 150 W), open magnetic stirring for 12 h to volatilize the organic solvent, and obtain a W1 / O / W2 emulsified product. After resuspending the W1 / O / W2 emulsified product with deionized water, centrifuge at 10000 rpm and 10 min, repeat 5 times. The acetylated glucomannan microsphere carrier is obtained.

[0072] Example 2

[0073] Example 2 provides a method for preparing an acetylated glucomannan microsphere carrier (acGMMs@OVA) loaded with OVA. The preparation method is basically the same as that of Example 1, and the only difference compared with Example 1 is that the phosphate buffer solution PBS (pH 7.4) in step S2 is replaced by a phosphate buffer solution PBS (pH 7.4) containing 0.2 mg OVA.

[0074] Example 3

[0075] Example 3 provides a method for preparing an acetylated glucomannan microsphere carrier loaded with OVA. The preparation method is basically the same as Example 1, and the only difference compared with Example 1 is that the phosphate buffer solution PBS (pH 7.4) in step S2 is replaced by a phosphate buffer solution PBS (pH 7.4) containing 0.4 mg OVA.

[0076] Example 4

[0077] Example 4 provides a method for preparing an acetylated glucomannan microsphere carrier loaded with OVA. The preparation method is basically the same as that of Example 1, and the only difference compared with Example 1 is that the phosphate buffer solution PBS (pH 7.4) in step S2 is replaced by a phosphate buffer solution PBS (pH 7.4) containing 0.6 mg OVA.

[0078] Example 5

[0079] Example 5 provides a method for preparing an acetylated glucomannan microsphere carrier loaded with OVA. The preparation method is basically the same as Example 1, and the only difference compared with Example 1 is that the phosphate buffer solution PBS (pH 7.4) in step S2 is replaced by a phosphate buffer solution PBS (pH 7.4) containing 0.8 mg OVA.

[0080] Example 6

[0081] Example 6 provides a method for preparing an acetylated glucomannan microsphere carrier loaded with OVA. The preparation method is basically the same as Example 1, and the only difference compared with Example 1 is that the phosphate buffer solution PBS (pH 7.4) in step S2 is replaced by a phosphate buffer solution PBS (pH 7.4) containing 1 mg OVA.

[0082] Comparative Example 1

[0083] Comparative Example 1 provides a method for preparing acetylated glucomannan, which is basically the same as Example 1, and the only difference compared with Example 1 is that the mixture of pyridine and acid anhydride (1:1, V / V) in step S1 is replaced by trifluoroacetic anhydride (TFAA).

[0084] The polysaccharide product obtained by the reaction of pyridine and acid anhydride has fewer broken bonds, a larger molecular weight, a more uniform distribution, and a better uniformity of the microspheres made.

[0085] Comparative Example 2

[0086] Comparative Example 2 provides a method for preparing an acetylated glucomannan microsphere carrier, which is basically the same as Example 1, and the only difference from Example 1 is that the ultrasonic power of the two ultrasonic emulsifications in step S2 is 100W.

[0087] Comparative Example 3

[0088] Comparative Example 3 provides a method for preparing an acetylated glucomannan microsphere carrier, which is basically the same as Comparative Example 2, except that the volume of the phosphate buffer solution PBS (pH 7.4) is 40 L.

[0089] Test Example 1

[0090] 1. By analyzing the changes in the functional groups of glucomannan before and after acetylation, determine whether acetylated glucomannan is obtained. The test method is as follows:

[0091] GM and acGM prepared in Example 1 were mixed with potassium bromide at a mass ratio of 1 / 200, ground into powder and pressed into tablets. The tablets were detected by infrared spectrometer (FI-IR) with a scanning range of 4000-450 cm -1 The characteristic peak of carbonyl (-C=O) was observed at 1735 cm -1 10 mg of GM or acGM were fully dissolved in deuterated chloroform (600 μL) and the changes in 1HNMR at δ=2.1 ppm (-COCH3) were observed.

[0092] Test results such as Figure 1 shown.

[0093] acGM at 1747cm -1 A strong absorption peak appeared near the carbonyl stretching vibration peak at 1372 cm -1 There is an obvious absorption peak near the methyl group, which is at 1237cm -1 A strong absorption peak appears near, which is the absorption peak of saturated ester group. Figure 1 As shown in A. This indicates that there are a large number of acetyl groups (-COCH3) on the acGM molecular chain. The 1H NMR graph also shows that acGM has a peak in the range of δ = 2.1ppm (-COCH3), further proving that the acetylation is successful. Figure 1 As shown in B.

[0094] 2. The molecular weight distribution of the acetylated glucomannan prepared in Example 1 and Comparative Example 1 was determined by high performance gel permeation chromatography (HPGPC). The determination method is as follows:

[0095] The sample to be tested was prepared into a 10 mg / mL solution, centrifuged at 12000 rpm for 10 min, the supernatant was filtered with a 0.22 μm microporous filter membrane, and transferred to a 1.8 ml injection vial for detection. The detection conditions were: chromatographic column: chromatographic column BRT805 gel column; mobile phase: N, N-dimethylacetamide; flow rate: 1 mL / min, column temperature: 40 ° C; injection volume: 50 μL; detector: evaporative light detector A2D.

[0096] Test results such as Figures 2-3 And as shown in Table 1.

[0097] Table 1

[0098]

[0099] The molecular weight distribution of acetylated glucomannan prepared by TFAA is relatively wide, and the distribution is relatively concentrated at a weight average molecular weight of 38871 (accounting for only 29.21%); while the acetylated glucomannan prepared by pyridine and acid anhydride has fewer broken bonds, a larger molecular weight, a more uniform distribution, and better uniformity of the microspheres made. Nearly half (43.96%) of the weight average molecular weight is distributed at 1444154, which is more conducive to the uniformity of the particle size when used in the subsequent preparation of acetylated glucomannan microsphere carriers.

[0100] Test Example 2

[0101] 1. Detect the particle size distribution and morphology of the acetylated glucomannan microsphere carrier prepared in Example 1. The detection method is as follows:

[0102] After ultrasonically resuspending the acetylated glucomannan microsphere carrier with 2 mL PBS, 100 μL was dropped on a 400-mesh copper grid and air-dried, and the morphology and particle size distribution were observed under a transmission electron microscope. The hydrated particle size was measured by dynamic light scattering and a particle size analyzer, and the measurement was continued every other day for 7 days.

[0103] Test results such as Figure 4 shown.

[0104] The average hydrated particle size of the acetylated glucomannan microsphere carrier prepared in Example 1 was 241.3 nm, and the PDI was 0.1638. Within one week, the hydrated particle size was stable at about 200 nm to 300 nm, the PDI was about 0.2, the morphology was normal, spherical, and the stability was good. This shows that the acetylated glucomannan microspheres were successfully prepared by the double emulsion method.

[0105] 2. Detect the particle size distribution of the acetylated glucomannan microsphere carriers prepared in Comparative Examples 2 and 3.

[0106] Particle size distribution diagram Figure 5 shown.

[0107] The average hydrated particle size of the acetylated glucomannan microsphere carrier prepared in Comparative Example 2 was 388.0 nm, and the PDI was 0.1657. The average hydrated particle size of the acetylated glucomannan microsphere carrier prepared in Comparative Example 3 was 845.0 nm, and the PDI was 0.306.

[0108] Based on the particle size parameters of the acetylated glucomannan microsphere carrier prepared in Example 1, it is shown that the greater the ultrasonic power, the more conducive it is to preparing acetylated glucomannan microsphere carriers with small particle size and uniform particle size distribution; the volume ratio of the oil phase to the water phase will affect the particle size of the acetylated glucomannan microsphere carrier, and the greater the volume ratio of the oil phase to the water phase, the larger the particle size of the prepared acetylated glucomannan microsphere carrier.

[0109] 3. Detect the cytotoxicity of the acetylated glucomannan microsphere carrier prepared in Example 1. The detection method is as follows:

[0110] RAW 264.7 cells were cultured at 8 × 10 3 Cells / well were seeded in a 96-well plate and cultured for 24 hours. The culture medium was discarded, and DMEM medium containing 20, 40, and 80 μg / mL acGMMs was added to the wells, incubated for 24 hours, and cell viability was analyzed by CCK-8 assay. Specifically: 10 μL CCK-8 solution was added to each well, and after incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader. Cells treated with PBS without acGMMs were used as the control group (Control).

[0111] Test results such as Figure 6 shown.

[0112] Compared with the control group, 20-80 μg / mL acGMMs treatment did not affect cell viability, and the cell viability was above 90%, indicating that acGMMs had no obvious cytotoxicity, good cell compatibility and safety.

[0113] 4. Detect the pro-inflammatory effect of the acetylated glucomannan microsphere carrier prepared in Example 1 in macrophage RAW 264.7. The detection method is as follows:

[0114] Convert RAW 264.7 to 8×10 4The cells were inoculated on a 6-well plate at a density of 100 cells / well and treated after 24 hours of culture. The treatment method for the normal RAW264.7 cell group was as follows: the culture medium was discarded and the cells were incubated with DMEM medium containing 20, 40, and 80 μg / mL of acGMMs for 24 hours; the treatment method for the inflammatory RAW264.7 cell group was as follows: the culture medium was discarded and the cells were pretreated with DMEM medium containing 20, 40, and 80 μg / mL of acGMMs for 2 hours, and then DMEM medium containing LPS (final concentration of 100 ng / mL) and IFN-γ (final concentration of 10 ng / mL) was added for further incubation for 24 hours. After the cells were treated with DMEM medium without acGMMs for 2 hours, DMEM medium containing LPS (final concentration of 100 ng / mL) and IFN-γ (final concentration of 10 ng / mL) was added for further incubation for 24 hours, which was used as the LPS+IFN-γ group. RAW264.7 cells without any treatment were used as the blank control group. After the incubation, the cell supernatant was collected and the NO level in the cell supernatant was tested using a NO kit (Biyuntian). After washing the cells three times with pre-cooled PBS, total RNA was extracted from the cells using Trizol reagent, and the RNA concentration was quantified using an ultra-micro UV spectrophotometer. 1 μg of RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase, and the expression levels of the target genes IL-1β and IL-6 were detected using a standard SYBRGreen qPCR system on a CFX96TM real-time system.

[0115] Test results such as Figure 7 shown.

[0116] After normal RAW264.7 cells were treated with different doses of acGMMs, the NO expression and the mRNA expression levels of IL-1β and IL-6 did not increase significantly. After pretreatment with 80μg / mL acGMMs, the increase in the mRNA expression levels of IL-1β and IL-6 caused by LPS and IFN-γ stimulation in RAW264.7 cells could be significantly inhibited; after pretreatment with 40μg / mL acGMMs, the increase in the mRNA expression level of IL-1β caused by LPS and IFN-γ stimulation in RAW264.7 cells could be significantly inhibited. This indicates that acGMMs do not cause significant pro-inflammatory responses in macrophages.

[0117] 5. Detect the pro-inflammatory effect of the acetylated glucomannan microsphere carriers in Comparative Examples 2 and 3 in normal RAW 264.7 cells. The detection method is the same as that of the acetylated glucomannan microsphere carriers in Example 1, and the effective concentration of the acetylated glucomannan microsphere carriers is 80 μg / mL.

[0118] Test results such as Figure 8 shown.

[0119] The acetylated glucomannan microsphere carriers of comparative example 2 significantly increased the IL-6 level, and the acetylated glucomannan microsphere carriers of comparative example 3 significantly increased the IL-1β and IL-6 levels. This indicates that the acetylated glucomannan microsphere carriers with a particle size of more than 300 nm will have a certain pro-inflammatory effect on cells.

[0120] Test Example 3

[0121] 1. Detect the encapsulation efficiency (EE) and drug loading rate (DL) of acGMMs@OVA prepared in Examples 2 to 6. The detection method is as follows:

[0122] In step S2, the W1 / O / W2 emulsified product was resuspended with deionized water and centrifuged at 10,000 rpm for 10 min. The supernatant was collected and the protein content in the supernatant was measured using a BCA kit to calculate the encapsulation efficiency and drug loading rate.

[0123] Encapsulated OVA amount = total OVA amount - supernatant protein content;

[0124] Encapsulation efficiency = encapsulated OVA amount / total OVA amount;

[0125] Drug loading rate = amount of encapsulated OVA / mass of acGMMs@OVA.

[0126] Test results such as Fig. 9 shown.

[0127] As the concentration of OVA increases, the encapsulation rate reaches a saturation point. Beyond the saturation point, the amount of encapsulated OVA reaches a certain amount and then stops rising. When the OVA concentration is 0.8 mg, the maximum encapsulation rate is 79.4±1.4%, and the drug loading rate reaches 12.4±0.2%. This indicates that the acetylated glucomannan microsphere carrier can be loaded with OVA.

[0128] 2. Detect the distribution of acGMMs@OVA prepared in Example 5 in RAW 264.7 cells and WRL 68 cells. The detection method is as follows:

[0129] RAW 264.7 cells and WRL 68 cells were cultured at 1.5×10 5 The cells / well were mixed and inoculated on a 24-well plate and cultured for 24 hours to allow them to adhere to the wall. Then the culture medium was removed and DMEM medium containing 80 μg / mL acGMMs@OVA was added. After incubation for another 6 hours, it was recorded as the acGMMs@OVA treatment group. The blank control group was treated with DMEM medium without acGMMs@OVA. The tests shown in (1) and (2) were then performed.

[0130] (1) Fluorescence microscopy: After the culture is completed, the culture medium is removed and the cells are washed three times with PBS. The cells are fixed with paraformaldehyde and fixed in an ice bath for 10 min. The paraformaldehyde is removed and the cells are washed three times with PBS. PBS containing 0.5% BSA is added and fluorescence images are taken under an inverted fluorescence microscope. The fluorescence images are analyzed using image J software.

[0131] (2) Flow cytometry: After the culture was completed, the culture medium was removed and the cells were washed three times with PBS. CD 86-PC7 antibody was added and incubated on ice for 30 min. PBS was added and centrifuged to wash away the antibody. PBS containing 0.5% BSA was added to resuspend the cells and the expression of OVA-FITC and CD 86-PC7 was detected by flow cytometry. Data were analyzed using Flow jo software.

[0132] Test results such as Fig.10 shown.

[0133] Many cells in the acGMMs@OVA treatment group took up acGMMs@OVA, and most of them were macrophages RAW264.7. Macrophages were labeled with CD 86 antibody, and 87% of the cells that took up acGMMs@OVA expressed CD86, that is, 87% of the cells that took up acGMMs@OVA were macrophages. This shows that the microsphere carrier has the ability to selectively target macrophages due to its special mannose unit structure.

[0134] 3. Detect the uptake promoting effect of acGMMs@OVA prepared in Example 5 on RAW 264.7 cells. The detection method is as follows:

[0135] Convert RAW 264.7 to 2.5×10 5 Cells / well were seeded on a 24-well plate and cultured for 24 hours to adhere to the plate, which was used as the normal macrophage group. 100 ng / mL of LPS and 10 ng / mL of IFN-γ were added to the normal macrophage group to treat it, which was used as the inflammatory macrophage group. The normal macrophage group and the inflammatory macrophage group were treated with DMEM medium containing 80 μg / mL acGMMs@OVA, respectively, and incubated for 6 hours, which was used as the acGMMs@OVA group. The cells were treated with DMEM medium containing 10 μg / mL OVA, which was used as the OVA group. The cells were treated with DMEM medium, which was used as the blank control group (control). The tests shown in (1) and (2) were then performed.

[0136] (1) Fluorescence microscopy: After the incubation period, the culture medium was removed and the cells were washed three times with PBS. The cells were fixed with paraformaldehyde and fixed in an ice bath for 10 min. The paraformaldehyde was removed and the cells were washed three times with PBS. 300 μL of DAPI dye was added to each well to stain the cell nucleus. The cells were placed in a 37°C incubator for 10 min, then taken out and washed three times with PBS. PBS containing 0.5% BSA was added, and fluorescence images were taken under an inverted fluorescence microscope. The fluorescence images were analyzed using image J software.

[0137] (2) Flow cytometry: After the culture was completed, the culture medium was removed, the cells were washed three times with PBS, the cells were collected by centrifugation, PBS containing 0.5% BSA was added, and the expression of OVA-FITC was detected by flow cytometry. The data were analyzed using Flowjo software.

[0138] Test results such as Fig.11 shown.

[0139] Compared with free OVA, both normal macrophages and inflammatory macrophages significantly took up acGMMs@OVA. From the right. This indicates that acGMMs microsphere carriers can significantly promote the uptake of the loaded drug by macrophages.

[0140] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for preparing acetylated glucomannan microsphere carrier, characterized in that: The following steps are involved: S1. Preparation of acetylated glucomannan: Weigh a quantitative amount of 1 g of glucomannan and fully swell it in 200 mL of water, freeze-dry it and set it aside; after stirring a mixture obtained by mixing 100 mL of pyridine and anhydride in a volume ratio of 1:1 at 50°C for half an hour, slowly pour it into a round-bottom flask containing the freeze-dried glucomannan, stir it at 50°C for 72 hours, filter it to obtain a residue; mix the residue with a mixture obtained by mixing 100 mL of pyridine and anhydride in a volume ratio of 1:1, stir it at 50°C for 72 hours, filter it to obtain a residue; precipitate the residue with ethanol overnight, filter it to obtain a residue; resuspend the residue with ethanol, centrifuge it again, repeat it 5 times in total, and obtain a residue; dialyze the residue at room temperature for 2 days, use ultrapure water as the dialysis medium, and freeze-dry it to obtain the final reaction product, acetylated glucomannan; S2. Preparation of acetylated glucomannan microsphere carrier: 5 mg of the acetylated glucomannan was weighed and dissolved in 800 μL of chloroform, and then 80 μL of phosphate buffer solution PBS with a pH of 7.4 was added and mixed evenly. The mixture was emulsified on ice with an ultrasonic probe for 1 min at an ultrasonic power of 150 W, and an oil-in-water emulsion was obtained after stirring. Subsequently, 1 mL of 0.5 wt % polyvinyl alcohol aqueous solution was added, and ultrasonic emulsification was continued on ice for 1 min at an ultrasonic power of 150 W. The mixture was magnetically stirred in an open air chamber for 12 h to evaporate the organic solvent, and a W1 / O / W2 emulsified product was obtained. The W1 / O / W2 emulsified product was resuspended in deionized water, and then centrifuged at 10,000 rpm for 10 min, and the mixture was repeated 5 times to obtain an acetylated glucomannan microsphere carrier.

2. An acetylated glucomannan microsphere carrier prepared by the preparation method according to claim 1.

3. Use of the acetylated glucomannan microsphere carrier prepared by the preparation method according to claim 1 in the preparation of macrophage targeting drugs.

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