MPEG-PLGA / RCA nano preparation and application thereof in preparation of T2DM treatment medicine

Abstract

The invention provides an mPEG-PLGA / RCA nano preparation and application thereof in preparation of a medicine for treating T2MD, and relates to the technical field of nano biology.According to the mPEG-PLGA / RCA nano preparation, coptis total alkaloids serve as a tested medicine, mPEG-PLGA serves as a carrier, the carrier is entrapped with the coptis total alkaloids through an emulsion solvent diffusion method, and the mPEG-PLGA / RCA nano preparation is obtained. The mPEG-PLGA / RCA nano-preparation can be applied to preparation of drugs for treating T2MD, the drugs can keep a relatively complete structure in gastrointestinal tracts, slow release of the drugs is realized, side effects caused by direct contact of the drugs with the gastrointestinal tracts are avoided, and compared with direct taking of coptis total alkaloids, the mPEG-PLGA / RCA nano-preparation can prolong the blood circulation time of RCA, improve the blood concentration of RCA and reduce the toxic and side effects of T2MD. The in-vivo bioavailability of the medicine is improved.

Description

Technical Field

[0001] The present application relates to the field of nanobiotechnology, and in particular to mPEG-PLGA / RCA nanoformulations and their application in the preparation of drugs for treating T2MD. Background Art

[0002] With the rapid development of social economy, people's unhealthy lifestyles and eating habits have led to a sharp increase in the incidence of diabetes worldwide. The vast majority of cases are type 2 diabetes mellitus (T2DM). T2DM is a metabolic disease characterized by hyperglycemia caused by insulin resistance and β-cell dysfunction, and is associated with inflammation caused by overnutrition and other environmental factors. It is often accompanied by diffuse complications (damage to multiple organs and sites such as eyes, kidneys, nerves, feet, and cardiovascular system). The occurrence of complications increases the risk of premature death in patients. Existing T2DM treatment drugs such as biguanides, α-glucosidase inhibitors and thiazolidinediones can cause adverse reactions such as flatulence, heart failure and urinary tract infection if used for a long time.

[0003] Traditional Chinese medicine has been used in China for more than a thousand years. There have been many reports on the research of various traditional Chinese medicines and their extracts on diabetes. Coptis chinensis, as a commonly used traditional Chinese medicine for the treatment of diabetes, has the effects of clearing away heat and dampness, purging fire and detoxifying. The efficacy of Coptis chinensis is mainly attributed to its alkaloid components, especially isoquinoline alkaloids, which have the potential to treat T2DM and inhibit the occurrence of T2DM complications. However, factors such as low solubility, poor absorption, and short half-life of these ingredients result in low bioavailability, and they are unable to exert their maximum clinical efficacy. The active ingredients in traditional dosage forms such as granules, capsules, decoctions, and oral liquids have poor water solubility, and there are problems such as large treatment doses, low drug concentrations in the body, unstable release of active ingredients and difficulty in absorption, unstable drug metabolism, and unclear pharmacodynamics. In addition, these poorly soluble active ingredients are not easily effectively absorbed by the gastrointestinal tract when taken orally, resulting in low bioavailability. Therefore, how to use modern formulation technology to improve the absorption defects of active components of Coptis chinensis and improve the bioavailability and targeted distribution of alkaloid components is the key to optimizing the overall synergistic effect of multiple components of Coptis chinensis and improving the effect of treating T2DM. Summary of the invention

[0004] In view of this, the present application provides mPEG-PLGA / RCA nanoformulations and their use in the preparation of drugs for the treatment of T2MD, which solves the problem that in the prior art, Coptis chinensis is a commonly used traditional Chinese medicine for the treatment of diabetes, but its low solubility, poor absorption, short half-life and other factors lead to its low bioavailability and inability to exert its maximum clinical efficacy. The effective ingredients in traditional dosage forms such as granules, capsules, decoctions, and oral liquids have poor water solubility, and there are problems such as large treatment doses, low drug concentrations in the body, unstable release of effective ingredients and difficulty in absorption, unstable drug metabolism, etc., and these poorly soluble active ingredients are not easily effectively absorbed by the gastrointestinal tract when taken orally, resulting in a technical problem of low bioavailability.

[0005] As the first aspect of the present application, the present application provides an mPEG-PLGA / RCA nanoformulation, wherein the mPEG-PLGA / RCA nanoformulation uses total alkaloids of Coptis chinensis as a test drug and mPEG-PLGA as a carrier, and adopts an emulsified solvent diffusion method to encapsulate the total alkaloids of Coptis chinensis in the carrier to obtain the mPEG-PLGA / RCA nanoformulation, wherein the particle size of the mPEG-PLGA / RCA nanoformulation is 145-759nm.

[0006] As a second aspect of the present application, the present application provides a method for preparing an mPEG-PLGA / RCA nanoformulation, which comprises: preparing an organic phase: adding total alkaloids of Coptis chinensis and mPEG-PLGA to an organic solvent, and dissolving them by ultrasonication to form an organic phase; preparing an aqueous phase: dissolving an emulsifier in deionized water to form an aqueous phase; preparing mPEG-PLGA / RCA nanoparticles: dripping the organic phase into the aqueous phase drop by drop, stirring with a homogenizer to form a coarse emulsion, subjecting the coarse emulsion to high-pressure granulation with a homogenizer to obtain a fine emulsion with uniform particle size, pre-cooling and solidifying the fine emulsion to form a nanoparticle suspension, removing the organic solvent from the nanoparticle suspension, and freeze-drying to obtain the mPEG-PLGA / RCA nanoparticles described above.

[0007] In one embodiment, the molecular weight of the mPEG-PLGA is 5000-10000 Da, and the mass ratio of the total alkaloids of Coptis chinensis to mPEG-PLGA is 1:(1-4).

[0008] In one embodiment, the organic solvent is a mixed solution of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is 2:1.

[0009] In one embodiment, the emulsifier is one of SDC and PVA, and the concentration of the emulsifier is 0.1%-1%.

[0010] In one embodiment, the volume ratio of the organic phase to the aqueous phase is 1:(2-10)

[0011] In one embodiment, the homogenization speed is 12000-30000 rpm, and the homogenization time is 30 min-120 min.

[0012] In one embodiment, the homogenization pressure is 5000-15000 psi, and the number of homogenization cycles is 5-15 times.

[0013] In one embodiment, the miniemulsion is dropped into pre-cooled 4°C deionized water to solidify to form a nanoparticle suspension, and then the organic solvent in the nanoparticle suspension is removed by rotary evaporation. The pre-freezing and freeze-drying are performed for 40-50 hours. Preferably, the pre-freezing and freeze-drying time is 48 hours.

[0014] As a third aspect of the present application, the present application provides the use of the mPEG-PLGA / RCA nanoformulation described above in the preparation of a drug for treating type 2 diabetes.

[0015] In one embodiment, the mPEG-PLGA / RCA nanoformulation can be used to prepare an oral drug for treating type 2 diabetes.

[0016] The present application provides an mPEG-PLGA / RCA nanoformulation and its application in the preparation of a drug for treating T2MD, and relates to the field of nanobiotechnology. The mPEG-PLGA / RCA nanoformulation uses total alkaloids of coptis chinensis as a test drug and mPEG-PLGA as a carrier. The total alkaloids of coptis chinensis are encapsulated on the carrier by an emulsified solvent diffusion method to obtain the mPEG-PLGA / RCA nanoformulation, wherein the particle size of the mPEG-PLGA / RCA nanoformulation is 145-759nm. The mPEG-PLGA / RCA nanoformulation can be used to prepare a drug for treating T2MD. The total alkaloids of coptis chinensis are encapsulated in mPEG-PLGA, and a relatively complete structure can be maintained in the gastrointestinal tract, thereby achieving sustained release of the drug and avoiding side effects caused by direct contact of the drug with the gastrointestinal tract. Compared with directly taking the total alkaloids of coptis chinensis, the mPEG-PLGA / RCA nanoformulation can prolong the blood circulation time of RCA, increase the blood drug concentration of RCA, and improve the bioavailability of the drug in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a flow chart of the preparation of mPEG-PLGA / RCA nanoparticles provided in one embodiment of the present application;

[0018] Figure 2 Shown are the TEM images, particle size distribution diagrams, and Zeta potential distribution diagrams of mPEG-PLGA / RCA nanoparticles provided in one embodiment of the present application;

[0019] Figure 3Shown is a cumulative drug release curve of RCA and mPEG-PLGA / RCA nanoparticles provided in Experimental Example 1 of the present application in different media;

[0020] Figure 4 The figure shows the stability comparison of RCA and mPEG-PLGA / RCA nanoparticles in different media provided in Experimental Example 2 of the present application;

[0021] Figure 5 Shown is the tissue distribution diagram of different alkaloid components provided in Experimental Example 3 of this application;

[0022] Figure 6 Shown is a curve chart of average blood concentration of different alkaloids versus time provided in Experimental Example 4 of the present application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. In the description of the present invention, it should be noted that the term "or" is usually used in the sense of including "and / or", unless the content clearly indicates otherwise.

[0024] Example 1

[0025] Figure 1 FIG. 1 is a flow chart of the preparation of mPEG-PLGA / RCA nanoparticles provided in an embodiment of the present application. Figure 1 As shown, the preparation of mPEG-PLGA / RCA nanoparticles includes:

[0026] Preparation of organic phase: 5 mg of drug RCA total alkaloids (RCA) and 20 mg of carrier mPEG-PLGA (molecular weight 15000Da) were accurately weighed into a reaction bottle, 6 mL of methanol and 3 mL of dichloromethane were added, and the organic phase was obtained by ultrasonic dissolution;

[0027] Preparation of aqueous phase: 150 mg of emulsifier sodium cholate (SDC) was weighed and dissolved in 30 mL of deionized water to form an aqueous phase;

[0028] Preparation of mPEG-PLGA / RCA nanoparticles: Under high-speed homogenization stirring conditions at 30,000 rpm, an electronic peristaltic pump was used to drop the organic phase dropwise into the aqueous phase containing the emulsifier. The volume ratio of the organic phase to the aqueous phase was 1:2, and the homogenization was stirred at 30,000 rpm for 30 minutes to form a coarse emulsion; the coarse emulsion was circulated 10 times by a ZEN3600Nano microfluidizer at a pressure of 15,000 psi to obtain a fine emulsion with uniform particle size; the fine emulsion was dropped into pre-cooled 4°C deionized water that accounted for 10 times the volume of the fine emulsion to solidify and form a nanoparticle suspension; the nanoparticle suspension was placed in a rotary evaporator to remove the organic solvent in the nanoparticle suspension, and the unencapsulated drug was removed by ultrafiltration, and then freeze-dried for 48 hours to obtain mPEG-PLGA / RCA nanoparticles.

[0029] 5 mL of deionized water was added to 1 mg of the prepared freeze-dried mPEG-PLGA / RCA nanoparticles to dissolve them to obtain an mPEG-PLGA / RCA nanoparticle aqueous solution, and the mPEG-PLGA / RCA nanoparticle aqueous solution was dropped onto a transmission electron microscope carrier (ultra-thin carbon film), and then dried under an infrared lamp, and then placed under a transmission electron microscope (TEM) to observe its microscopic morphology.

[0030] 1 mg of the prepared freeze-dried mPEG-PLGA / RCA nanoparticles was dispersed in ultrapure water to obtain an mPEG-PLGA / RCA nanoparticle aqueous solution. 1 ml of the mPEG-PLGA / RCA nanoparticle aqueous solution was drawn into a sample pool with a pipette, and the particle size and Zeta potential of the mPEG-PLGA / RCA nanoparticles were measured using a laser particle size analyzer (Malvern Zetasizer Nano ZS).

[0031] Figure 2 The TEM image, particle size distribution diagram and Zeta potential distribution diagram of mPEG-PLGA / RCA provided in one embodiment of the present application are shown. Figure 2 It can be seen from the TEM image that the mPEG-PLGA / RCA nanoparticles are spherical, round and full in appearance, without adhesion. The prepared mPEG-PLGA / RCA nanoparticles are 145 nm and have a Zeta potential of -10.57±0.31 mV.

[0032] Weigh 5 mg of freeze-dried mPEG-PLGA / RCA into a centrifuge tube, add 5 mL of deionized water and ultrasonically dissolve it, then place it in an ultrafiltration tube (10 kDa, Millipore) and centrifuge it at 3000 rpm for 5 min. Take the lower layer of liquid and filter it through a 0.22 μm filter membrane. Use HPLC to analyze the content of coptis alkaloids and six alkaloids (magnolia alkaloids, jatrorrhizine, epiberberine, coptis alkaloids, palmatine, and berberine). HPLC detection conditions: Agilent Extend-C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A is acetonitrile, mobile phase B is 0.05 mol·L- 1 Potassium dihydrogen phosphate solution (45:55) (add 0.4 g sodium dodecyl sulfate to every 100 mL of solution, and adjust the pH to 4.0 with phosphoric acid), flow rate 1.0 mL min -1 , the detection wavelength was 276 nm, the column temperature was 30 °C, and the injection volume was 5 μL.

[0033] The drug loading and encapsulation efficiency were calculated according to formulas (1) and (2).

[0034] Drug loading = (WT-WF) / WNP×100% (1)

[0035] Encapsulation efficiency = (WT-WF) / WT×100% (2)

[0036] Wherein WT is the total amount of Coptis chinensis alkaloids input, WF is the total amount of the six free alkaloids not encapsulated, and WNP is the weight of the mPEG-PLGA / RCA nanoparticles input.

[0037] Example 2

[0038] The implementation method of Example 2 is the same as that of Example 1, except that:

[0039] The ratio of drug RCA and carrier mPEG-PLGA added in Example 2 is 1:2.

[0040] Example 3

[0041] The implementation method of Example 3 is the same as that of Example 1, except that:

[0042] The drug RCA in Example 3 and the carrier mPEG-PLGA (molecular weight 15000 Da) were added in a ratio of 1:1.

[0043] Example 4

[0044] The implementation method of Example 4 is the same as that of Example 2, except that:

[0045] The volume ratio of the organic phase to the aqueous phase in Example 4 is 1:5.

[0046] Example 5

[0047] The implementation method of Example 5 is the same as that of Example 2, except that:

[0048] The volume ratio of the organic phase to the aqueous phase in Example 5 is 1:10.

[0049] Example 6

[0050] The implementation method of Example 6 is the same as that of Example 4, except that:

[0051] The molecular weight of the polymer of Example 6 is 5000 Da.

[0052] Example 7

[0053] The implementation method of Example 7 is the same as that of Example 4, except that:

[0054] The molecular weight of the polymer of Example 7 is 30,000 Da.

[0055] Table 1 Comparison of drug loading and particle size of Examples 1-7

[0056]

[0057] As shown in Example 1, Example 2, and Example 3 in Table 1, when the drug-to-carrier mass ratio increases from 1 / 4 to 1 / 2, the average particle size does not increase significantly, and the drug loading increases from 5.79% to 8.42%. However, when the drug-to-carrier mass ratio reaches 1 / 1, the average particle size increases to 231.60nm, and the drug loading decreases accordingly. This is because when the solution concentration is too high, the number of drug particles increases, which increases the probability of collision and agglomeration between drug particles, so a large number of larger particle agglomerates are generated, which also easily causes drug leakage from the nanoparticles.

[0058] The addition ratio of the organic phase to the aqueous phase in Example 2 is 1:2, the addition ratio of the organic phase to the aqueous phase in Example 4 is 1:5, and the addition ratio of the organic phase to the aqueous phase in Example 5 is 1:10. Compared with Example 2, Example 4 and Example 5, when the ratio of the organic phase to the aqueous phase is 1:5, the minimum particle size is 145.57nm, and the drug loading is relatively high at 13.3%. This is because in the miniemulsion, the size of the water core limits the growth of the particles and determines the size of the nanoparticles. The particle size of the nanoparticles can be controlled by adjusting the amount of water.

[0059] In Example 4, compared with Example 6 and Example 7, when the molecular weight of the carrier is too high, the viscosity of the dissolved organic phase is large and the fluidity is poor, the particle size of the prepared nanoparticles is larger, the distribution becomes wider, and the drug loading capacity is low. Therefore, when the molecular weight of the carrier mPEG-PLGA is 15K, the drug loading capacity of the prepared nanoparticles is 13.3%, the particle size is 145.57nm, and the effect is optimal.

[0060] Example 8

[0061] The implementation method of Example 8 is the same as that of Example 4, except that:

[0062] The emulsifier in Example 8 is SDC (sodium cholate), and the content of SDC in the water phase is 0.1%.

[0063] Example 9

[0064] The implementation method of Example 9 is the same as that of Example 4, except that:

[0065] The emulsifier in Example 9 is SDC (sodium cholate), and the content of SDC in the water phase is 1%.

[0066] Example 10

[0067] The implementation method of Example 10 is the same as that of Example 4, except that:

[0068] The emulsifier in Example 10 is PVA (polyvinyl alcohol), and the content of PVA in the water phase is 0.1%.

[0069] Embodiment 11

[0070] The implementation method of Example 11 is the same as that of Example 4, except that:

[0071] The emulsifier of Example 11 is PVA (polyvinyl alcohol), and the content of PVA in the water phase is 0.5%.

[0072] Example 12

[0073] The implementation method of Example 12 is the same as that of Example 4, except that:

[0074] The emulsifier in Example 12 is PVA (polyvinyl alcohol), and the content of PVA in the water phase is 1%.

[0075] Comparative Example 1

[0076] The implementation method of Comparative Example 1 is the same as that of Example 4, except that:

[0077] In Comparative Example 1, no emulsifier was added.

[0078] Table 2 Comparison of the results of Examples 8-12 and Comparative Examples

[0079]

[0080] The effects of different emulsifiers and their dosage on mPEG-PLGA / RCA nanoparticles are shown in Table 2. Nanoparticles were prepared by emulsification solvent diffusion method. Nanoparticles can also be prepared without adding emulsifiers during the emulsification process. Although the obtained nanoparticles have a larger particle size and a wider distribution, their drug loading is low. Therefore, considering the particle size, drug loading and dispersion coefficient, when the dosage of SDC is 0.5%, the particle size is 196.36nm, and the drug loading is as high as 13.3%, and its particle size and drug loading are optimal.

[0081] Example 13

[0082] The implementation method of Example 13 is the same as that of Example 4, except that:

[0083] The homogenization time of the organic phase and the aqueous phase in Example 13 was 30000 rpm and the homogenization time was 60 min.

[0084] Embodiment 14

[0085] The implementation method of Example 14 is the same as that of Example 4, except that:

[0086] The homogenization time of the organic phase and the aqueous phase in Example 14 was 30000 rpm and the homogenization time was 120 min.

[0087] Embodiment 15

[0088] The implementation method of Example 15 is the same as that of Example 4, except that:

[0089] The homogenization time of the organic phase and the aqueous phase in Example 15 was 12000 rpm and the homogenization time was 30 min.

[0090] Example 16

[0091] The implementation method of Example 16 is the same as that of Example 4, except that:

[0092] The homogenization time of the organic phase and the aqueous phase in Example 16 was 21000 rpm and the homogenization time was 30 min.

[0093] Table 3 Comparison of results of Examples 13-16

[0094]

[0095] The effects of homogenization time and speed on mPEG-PLGA / RCA nanoparticles are shown in Table 3. As the high-speed homogenization time increases from 30 min to 120 min, the average particle size of the nanoparticles increases from 146.30 nm to 395.63 nm, and the drug loading decreases accordingly. Longer homogenization time increases the energy contained in the crude emulsion, breaking the stability of the emulsion, thereby causing the nanoparticles to agglomerate and become larger in size. Therefore, longer homogenization time does not help emulsification. As the homogenization speed increases from 12000 rpm to 30000 rpm, the average particle size of the nanoparticles decreases from 164.23 nm to 146.30 nm, and the drug loading increases accordingly. A larger homogenization speed produces strong shear force and impact force, which makes the particle size of the nanoparticles smaller and the distribution narrower. Therefore, the optimal time for high-speed homogenization is 30 min and the speed of high-speed homogenization is 30000 rpm.

[0096] Embodiment 17

[0097] The implementation method of Example 17 is the same as that of Example 4, except that:

[0098] The crude emulsion of Example 17 was circulated 10 times by a ZEN3600Nano microfluidizer at a pressure of 5000 psi to obtain a fine emulsion with uniform particle size.

[0099] Embodiment 18

[0100] The implementation method of Example 18 is the same as that of Example 4, except that:

[0101] The crude emulsion of Example 18 was circulated 10 times by a ZEN3600Nano microfluidizer at a pressure of 10,000 psi to obtain a fine emulsion with uniform particle size.

[0102] Embodiment 19

[0103] The implementation method of Example 19 is the same as that of Example 4, except that:

[0104] The crude emulsion of Example 19 was circulated 5 times by a ZEN3600Nano microfluidizer at a pressure of 15000 psi to obtain a fine emulsion with uniform particle size.

[0105] Embodiment 20

[0106] The implementation method of Example 20 is the same as that of Example 4, except that:

[0107] The crude emulsion of Example 20 was circulated 15 times by a ZEN3600Nano microfluidizer at a pressure of 15,000 psi to obtain a fine emulsion with uniform particle size.

[0108] Table 4 Comparison of results of Examples 17-20

[0109]

[0110] The effects of homogenization pressure and homogenization times on mPEG-PLGA / RCA nanoparticles are shown in Table 4. The high-pressure homogenizer transfers and transports materials with a high-pressure reciprocating pump, which produces strong shear, impact and cavitation effects under high pressure, making the materials ultra-fine. Generally, as the homogenization pressure increases and the number of cycles increases, the particle size and distribution of the preparation decrease, and tend to be stable after reaching a certain pressure and number of cycles. When the pressure and number of cycles continue to increase, the particle size may increase and the distribution becomes wider. The homogenization pressure and the number of cycles significantly affect the particle size and distribution of the nanoparticles. Therefore, the selection of 15000psi intensity for 10 cycles can obtain nanoparticles with suitable particle size, narrow distribution and relatively high drug loading.

[0111] Experimental Example 1

[0112] The in vitro drug release behavior of free RCA and mPEG-PLGA / RCA nanoparticles was evaluated by dialysis. 1 mg of total alkaloids of Coptis chinensis and 6.25 mg of mPEG-PLGA / RCA nanoparticles were dispersed in release media PBS, SGF, and SIF containing 1% Tween 80, respectively. SGF containing 1% Tween 80, SIF containing 1% Tween 80, and pH 7.4 PBS containing 1% Tween 80 were used as release media to obtain a concentration of 1 mg·mL -1 The free RCA (in terms of RCA) and the concentration of 1 mg mL -1 RCA loaded on mPEG-PLGA / RCA nanoparticles. 1.0 mL of free RCA solution and RCA solution loaded on mPEG-PLGA / RCA nanoparticles were respectively aspirated and sealed in dialysis bags, and then the dialysis bags were placed in 50 mL of PBS, SGF, and SIF release media, and oscillated in a constant temperature water bath oscillator at 37°C and 100 rpm. 1.0 mL of release medium was removed at 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after placement, and then supplemented with an equal volume of release medium to maintain the total volume of the release medium unchanged. The free RCA solution and the RCA solution loaded on the mPEG-PLGA / RCA nanoparticles were filtered through a 0.22 μm microporous membrane after 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h of storage, and the RCA concentration Cd was determined by HPLC. The correction concentration Cc was calculated according to formula (3). The cumulative release rate was calculated according to formula (4). In formula (4), Wt is the total content of the six alkaloids (magnolia alkaloids, jatrophorrhizine, epiberberine, coptisine, palmatine, and berberine) in the mPEG-PLGA / RCA nanoparticles.

[0113]

[0114] Figure 3 Shown is the cumulative drug release curve of RCA and mPEG-PLGA / RCA in different media provided in Experimental Example 1 of this application; Figure 3 As shown in the figure, the cumulative drug release rate refers to the percentage of the superposition of drug amounts at different time points to the total drug amount. Compared with the RCA control solution, the mPEG-PLGA / RCA nanoparticles were released more slowly in SGF, SIF and pH 7.4 PBS. Therefore, the mPEG-PLGA / RCA nanoparticles can maintain a relatively complete structure in the gastrointestinal tract, achieve sustained release of the drug, and avoid side effects caused by direct contact of the drug with the gastrointestinal tract.

[0115] Experimental Example 2

[0116] In order to evaluate the stability of mPEG-PLGA / RCA nanoparticles in different media, mPEG-PLGA / RCA solution was diluted 5 times with pH 7.4 PBS, SGF and SIF, with 6 replicates in each group, and placed in a 37°C constant temperature incubator for 0, 2, 4, 6, 8, 10, 12, and 24 h. The average hydrodynamic diameter and PDI of mPEG-PLGA / RCA nanoparticles were determined by DLS laser scattering particle size analyzer. Figure 4 The figure shows the stability comparison of RCA and mPEG-PLGA / RCA in different media provided in Experimental Example 2 of the present application; Figure 4 As shown in the figure, the hydrodynamic diameter and PDI of mPEG-PLGA / RCA nanoparticles remained unchanged within 24 hours, which means that there was no obvious particle aggregation between the nanoparticles. The drug was encapsulated in the nanoparticles, which, on the one hand, avoided direct contact with the gastrointestinal tract to cause side effects; on the other hand, it could avoid the exclusion effect of P-glycoprotein and improve the bioavailability of the drug. Therefore, mPEG-PLGA / RCA nanoparticles had good stability in SGF, SIF and pH 7.4 PBS.

[0117] Experimental Example 3

[0118] To explore the metabolic rules and tissue distribution characteristics of RCA and mPEG-PLGA / RCA in T2DM C57BL / 6J mice, mice were given RCA and mPEG-PLGA / RCA by oral gavage for 4 weeks. Then, the biodistribution status of the six alkaloids in T2DM C57BL / 6J mice was quantitatively determined by LC-MS / MS (Waters Xevo TQ-S ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer). The six alkaloids were quantitatively analyzed in the heart, liver, spleen, lung, kidney, brain, jejunum, ileum, colon and serum of mice. C57BL / 6J mice have a natural deletion of exons 7-11 of the nicotinamide nucleotide transhydrogenase gene and cannot form the protein of the enzyme, resulting in changes in the steady-state regulation of glucose metabolism, manifested as reduced insulin secretion and mild to moderate hyperglycemia. Therefore, C57BL / 6J mice are often used to model type II diabetes. Six alkaloids in the heart, liver, spleen, lung, kidney, brain, jejunum, ileum, colon and serum of C57BL / 6J mice were quantitatively analyzed. The tissue distribution patterns of the six alkaloids in the RCA-administered group and the mPEG-PLGA / RCA nanoparticle-administered group of C57BL / 6J mice were summarized and compared, in order to provide a reference for the formulation development and clinical application of mPEG-PLGA / RCA.

[0119] Table 5 is the tissue distribution table of different alkaloid components. Figure 5 Shown is the tissue distribution diagram of different alkaloid components provided in Experimental Example 3 of this application; Figure 5 A: Heart; B: Liver; C: Spleen; D: Lung; E: Kidney; F: Brain; G: Jejunum; H: Ileum; I: Colon; J: Serum; Magnoflorine is magnoflorine; Jatrorrhizine is jatrorrhizine; Epiberberine is epiberberine; Coptisine is coptisine; Palmatine is palmatine; Berberine is berberine; Figure 5As shown in Table 5, each alkaloid is widely distributed in tissues. A comprehensive comparison of the alkaloid content of each organ shows that the six alkaloids are mainly distributed in the liver, jejunum, ileum and colon, among which the concentration of RCA (the sum of the six alkaloids) in colon tissue is significantly higher than that in other organs, and the concentration of the six alkaloids in serum is also relatively high. The drug concentrations in the serum and various tissues of the mice in the mPEG-PLGA / RCA group were significantly higher than those in the RCA group, which may be due to the improvement of drug absorption in the intestine caused by the assistance of mPEG-PLGA. Compared with the RCA group, the mPEG-PLGA / RCA group had *P<0.05, **P<0.01, and the difference was considered statistically significant.

[0120] Table 5 Tissue distribution of different alkaloid components

[0121]

[0122] Experimental Example 4

[0123] Twelve SD normal rats were randomly divided into two groups: RCA administration group (RCA) and mPEG-PLGA / RCA nanoparticle administration group, with 6 rats in each group. All mice were intragastrically administered with a high dose once. The intragastrically administered dose of the RCA group was 750 mg kg -1 The mPEG-PLGA / RCA nanoparticle group was intragastrically administered according to the equivalent dosage of RCA converted by 16.04% drug loading. Serum samples were collected from the retro-orbital venous plexus at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after intragastrically administered, and the collected serum samples were placed in EP tubes containing sodium heparin, centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatant serum was taken and stored in a -80°C refrigerator for testing.

[0124] The serum was quantitatively measured by LC-MS / MS to measure the contents of six alkaloids in mice at different time points after intragastric administration. Table 6 shows the contents of alkaloid components at different time points after intragastric administration. Figure 6 The figure shows the curves of the average blood concentration of different alkaloids after intragastric administration and time provided in Experimental Example 4 of the present application. Figure 6 Among them, Magnoflorine is magnoflorine; Jatrorrhizine is jatrorrhizine; Epiberberine is epiberberine; Coptisine is coptisine; Palmatine is palmatine; Berberine is berberine; Figure 6As shown in the figure, the six alkaloids in the RCA group can reach the maximum blood concentration 0.5h after oral administration, and the six alkaloids in the mPEG-PLGA / RCA group can reach the maximum blood concentration 1h after oral administration, which indicates that the six alkaloid components are rapidly absorbed by the gastrointestinal tract after oral administration, and mPEG-PLGA significantly delays the peak time of the six alkaloid components. At the same time, multiple blood concentration peaks were observed in the pharmacokinetics of each alkaloid, which is due to the distribution, reabsorption and enterohepatic circulation of the drug. During the period of 8-12h of administration, the blood concentration of the mPEG-PLGA / RCA group was higher than that of the RCA group. RCA nanoparticles prolonged the blood circulation time of RCA, and PEG-PLGA / RCA nanoparticles increased the blood concentration of RCA and improved the bioavailability of the drug in rats.

[0125] Table 6 Contents of alkaloid components after intragastric administration at different times

[0126]

[0127]

[0128] The data in Table 6 were used to estimate the half-life (T1 / 2), maximum plasma concentration (Cmax), time to peak plasma concentration (Tmax), and area under the concentration-time curve (AUC0-t) using DAS2.0 (Chinese Association of Mathematical Pharmacology, Shanghai, China).

[0129] Table 7 Pharmacokinetic parameters of 6 alkaloids after intragastric administration at different times

[0130]

[0131]

[0132] Table 7 shows the pharmacokinetic parameters of alkaloids after intragastric administration at different times. As shown in Table 7, the C values ​​of magnolia alkaloids, jatrorrhizine, epiberberine, coptisine, palmatine, and berberine in the RCA group were max The values ​​were 68.97±12.15, 202.37±14.47, 96.56±13.59, 132.20±16.36, 102.58±14.30 and 356.17±25.46 ng·mL -1 , while the Cmax of magnolialine, jatrorrhizine, epiberberine, coptisine, palmatine and berberine in the mPEG-PLGA / RCA group were 154.17±27.89, 298.17±22.60, 186.56±23.10, 197.00±24.54, 181.43±13.77, 553.43±41.29 ng·mL -1 , C of mPEG-PLGA / RCA groupmax The T values ​​of each alkaloid in the mPEG-PLGA / RCA group were 2.24, 1.47, 1.93, 1.49, 1.76 and 1.55 times higher than those in the RCA group. max It is about twice that of the RCA group, and the T1 / 2 of each alkaloid in the mPEG-PLGA / RCA group is higher than that in the RCA group. This shows that RCA can indeed change the interaction between the drug and the gastrointestinal tract under the encapsulation of mPEG-PLGA, prolong the retention time of each alkaloid in the body, and increase the oral absorption and utilization of RCA.

[0133] In summary, after the dosage form of RCA was changed, the maximum blood concentration of the six alkaloids in rats increased and the half-life was prolonged, indicating that mPEG-PLGA can increase the residence time of RCA in the body and prolong the in vivo action time of each alkaloid. The pharmacokinetic parameters obtained in this study are of great significance for the clinical application of RCA and its nanoparticles.

[0134] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. mPEG-PLGA / RCA nanoformulation, characterized in that: The mPEG-PLGA / RCA nanoformulation uses total alkaloids of coptis chinensis as a test drug and mPEG-PLGA as a carrier. The total alkaloids of coptis chinensis are encapsulated in the carrier by an emulsified solvent diffusion method to obtain the mPEG-PLGA / RCA nanoformulation, wherein the particle size of the mPEG-PLGA / RCA nanoformulation is 145-759 nm.

2. The preparation method of mPEG-PLGA / RCA nanoformulation is characterized in that: include: Preparation of organic phase: adding total alkaloids of Coptis chinensis and mPEG-PLGA into an organic solvent and dissolving by ultrasonication to form an organic phase; Preparation of aqueous phase: dissolving the emulsifier in deionized water to form an aqueous phase; Preparation of mPEG-PLGA / RCA nanoparticles: the organic phase is dripped into the aqueous phase drop by drop, and a coarse emulsion is formed by stirring with a homogenizer. The coarse emulsion is granulated with a homogenizer under high pressure to obtain a fine emulsion with uniform particle size. The fine emulsion is pre-cooled and solidified to form a nanoparticle suspension. The organic solvent in the nanoparticle suspension is removed and freeze-dried to obtain the mPEG-PLGA / RCA nanoparticles according to claim 1.

3. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The molecular weight of the mPEG-PLGA is 5000-10000Da, and the mass ratio of the total alkaloids of coptis chinensis to the mPEG-PLGA is 1:(1-4).

4. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The organic solvent is a mixed solution of methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is 2:

1.

5. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The emulsifier is one of SDC and PVA, and the concentration of the emulsifier is 0.1%-1%.

6. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The volume ratio of the organic phase to the aqueous phase is 1:(2-10).

7. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The homogenization speed is 12000-30000 rpm, and the homogenization time is 30 min-120 min.

8. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The homogenization pressure is 5000-15000 psi, and the number of homogenization cycles is 5-15 times.

9. The method for preparing the mPEG-PLGA / RCA nanoformulation according to claim 2, characterized in that: The miniemulsion is dropped into pre-cooled 4°C deionized water to solidify and form a nanoparticle suspension, and then the organic solvent in the nanoparticle suspension is removed by rotary evaporation. After pre-freezing, the suspension is freeze-dried for 40-50 hours. Preferably, the freeze-drying time after pre-freezing is 48 hours.

10. Use of the mPEG-PLGA / RCA nanoformulation according to any one of claims 2 to 9 in the preparation of a drug for treating type 2 diabetes.

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