Use of bitter melon exosome and oral medicament prepared therefrom

Abstract

Use of a bitter melon exosome and an oral medicament prepared therefrom. The bitter melon exosome obtained from the bitter melon juice can be used for preparing a pharmaceutical formulation embedding a protein, a polypeptide, a small molecule peptide, a nucleic acid, or a small molecule compound. The compositions of the oral medicament of the present invention comprise the bitter melon exosome and an active pharmaceutical ingredient encapsulated in the bitter melon exosome. The bitter melon exosome is suitable for preparing a drug that is easily damaged by the gastrointestinal tract and is mostly administered by means of an injection route into an oral agent, and the oral agent can produce a similar therapeutic effect to that of the injection administration route by oral administration, and is convenient to use. The extraction method for the bitter melon exosome is simple and has a low cost. Moreover, the bitter melon exosome has no immunogenicity, has relatively high biocompatibility and biosafety, and has relatively strong tolerance to gastric acid and digestive tract proteolytic enzymes, and intestinal barrier permeability.

Description

Application of bitter gourd exosome and oral preparation prepared by the same TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of bitter gourd exosome and oral preparation prepared by the same. BACKGROUND

[0002] Exosomes (EVs) are lipid bilayer particles secreted by cells, including microvesicles, apoptotic bodies and exosomes. One important role of EVs is to transport biomolecules to specific distal locations of the parent cell, and its biological mechanisms and functions in mediating intercellular communication have been gradually revealed.

[0003] EVs have a lipid bilayer structure and can load hydrophilic and hydrophobic substances. In addition, EVs do not contain toxic factors and have no potential safety risks, and have good biocompatibility and biosafety.

[0004] Plant exosomes (PDEVs) can be obtained from a variety of plant cells, and the sources are wide. Compared with animal-derived exosomes, the steps of extracting and purifying PDEVs are simple, and high yield can be easily obtained. In addition, the membrane composition of PDEVs is similar to that of human cells, so it has high biocompatibility and biosafety. At the same time, through surface modification and genetic engineering technology, the composition of surface receptors and ligands of PDEVs can be changed, so as to realize higher targeting and drug delivery efficiency. Therefore, PDEVs have advantages in extraction, purification, yield, biocompatibility and biosafety as drug preparations. Future research will focus on the preparation method of PDEVs, drug delivery mechanism and application potential in the treatment of various diseases.

[0005] Bitter gourd exosomes (BMEVs) contain a variety of active ingredients, including polypeptides, polysaccharides and flavonoids. These components have various pharmacological effects such as anti-tumor, anti-inflammatory, antioxidant and immunomodulatory effects. Studies have shown that exosomes derived from bitter gourd can promote the proliferation of H9C2 cells after radiation, reduce cell apoptosis, inhibit the generation of mitochondrial ROS in H9C2 cells after radiation, restore normal mitochondrial membrane potential and thus alleviate radiation-induced mitochondrial damage; and can also play a certain protective role in radiation-induced cardiac damage and reduce cardiac fibrosis.

[0006] Proteins, nucleic acids and small molecule drugs are destroyed by gastric acid and intestinal alkaline environment, and various enzymes including proteolytic enzymes in the digestive tract when used for drug administration, especially gastrointestinal administration, or are difficult to be absorbed by the intestine; injection administration is inconvenient and costly. Therefore, it is necessary to improve the prior art to solve the above problems. SUMMARY

[0007] The present application aims to provide an application of bitter gourd exosome.

[0008] The application also provides the above-mentioned balsam pear exosome embedded pharmaceutical preparation.

[0009] The technical solution of the application is:

[0010] The balsam pear exosome is used for embedding protein, polypeptide, small molecule peptide, nucleic acid or small molecule compound medicament; the balsam pear exosome is derived from balsam pear and has an average particle size of 140-180 nm.

[0011] Preferably, the balsam pear exosome has an average particle size of 150-160 nm.

[0012] Preferably, the medicament is an oral medicament for gastrointestinal administration.

[0013] Preferably, the balsam pear exosome can be used for preparing an oral insulin preparation.

[0014] An oral medicament, which comprises balsam pear exosome and a pharmaceutical active ingredient wrapped in the balsam pear exosome, the pharmaceutical active ingredient being protein, polypeptide, small molecule peptide, nucleic acid or small molecule compound.

[0015] Preferably, the pharmaceutical active ingredient is insulin.

[0016] Preferably, the insulin drug loading rate is 15%-20%, more preferably 17%-18%.

[0017] The preparation method of the balsam pear exosome is:

[0018] (1) balsam pear is squeezed to obtain balsam pear juice;

[0019] (2) the precipitate in the balsam pear juice is removed by centrifugation, and the supernatant is filtered;

[0020] (3) the supernatant of step (2) is filtered by a 0.1-0.5 μm filter membrane, the filtrate is subjected to ultrahigh speed centrifugation, and the precipitate is the balsam pear exosome.

[0021] Preferably, it further comprises step (4): the precipitate of step (3) is dissolved in buffer or water, and the precipitate is obtained by ultrahigh speed centrifugation again to obtain high-purity balsam pear exosome.

[0022] Preferably, in step (1), fresh balsam pear is used as raw material; the balsam pear is crushed and squeezed, and the juice is collected by passing through 150-800 mesh filter cloth or filter screen; preferably, the balsam pear is crushed and squeezed, and the juice is collected by passing through 200-600 mesh filter cloth or filter screen; in one preferred mode of the application, the balsam pear is crushed and squeezed, and the juice is collected by passing through 400 mesh filter screen or filter cloth.

[0023] In step (2), the precipitates such as plant fibers and cell debris are removed by multiple low-speed and high-speed centrifugation, and the centrifugation conditions are as follows: 200-500 g, 1-3 times, 5-15 min each time; 800 g-1200 g, 1-3 times, 5-15 min each time; 10000-15000 g, 1-3 times, 20-40 min each time. Preferably, 200-400 g, 2-3 times, 8-12 min each time; 800 g-1200 g, 2-3 times, 8-12 min each time; 10000-12000 g, 2-3 times, 25-35 min each time. In a preferred mode of the present application, the differential centrifugation process is as follows: 300 g, 10 min x 2; 1000 g, 10 min x 2; 10000 g, 30 min x 2.

[0024] Preferably, in step (3), the supernatant is filtered with a 0.2-0.5 μm filter membrane. In a preferred mode of the present application, the supernatant is filtered with a 0.22 μm filter membrane.

[0025] In step (3) or step (4), the ultrahigh-speed centrifugation conditions are as follows: 100000-200000 g for 0.5-3 hours, preferably 120000-200000 g for 1-2 hours. In a preferred mode of the present application, the ultrahigh-speed centrifugation conditions are as follows: 150000 g for 1.5 hours.

[0026] The buffer in step (4) has a pH of 7-7.4, preferably a phosphate buffer; more preferably a phosphate buffer with a pH of 7.4.

[0027] The preparation method of the oral agent comprises the following steps:

[0028] (i) mixing the Momordica charantia exosome with the pharmaceutically active ingredient and incubating for 20-60 min;

[0029] (ii) after ultrasonic dispersion, continuing to incubate for 30-120 min to remove the unencapsulated pharmaceutically active ingredient.

[0030] Preferably, in step (i), the incubation is carried out at room temperature.

[0031] Preferably, in step (i), the mass ratio of the Momordica charantia exosome to the pharmaceutically active ingredient is 5-20:1; more preferably 8-12:1.

[0032] Preferably, in step (ii), the incubation is carried out at 35-38°C, more preferably at 37°C.

[0033] Preferably, in step (ii), the unencapsulated pharmaceutically active ingredient is removed by 80-120 kDa ultrafiltration. In a preferred mode of the present application, the unencapsulated pharmaceutically active ingredient is removed by 100 kDa ultrafiltration.

[0034] The beneficial effects of the present application are:

[0035] 1. After using bitter gourd exosomes to embed drugs (such as insulin), oral administration can produce similar therapeutic effects as injection administration. Bitter gourd exosomes have strong resistance to gastric acid and digestive tract proteolytic enzymes and intestinal barrier permeability, and are suitable for making oral preparations of drugs that are easily destroyed by the gastrointestinal tract and are mostly administered by injection. Oral administration can produce similar therapeutic effects as injection administration, and is convenient to use.

[0036] 2. The exosomes used are derived from bitter gourd, which is simple to extract and low in cost, and can be produced on a large scale. The exosomes and the drugs prepared therefrom are non-immunogenic, and also have high biocompatibility and biosafety.

[0037] The present application uses bitter gourd exosomes to encapsulate proteins, nucleic acids, small molecule compounds, etc., especially drugs that are easily destroyed by the digestive tract and are mostly administered by injection, to make oral preparations, which have practical significance and importance. Using bitter gourd exosomes to encapsulate active ingredients of drugs to make oral preparations can protect the active molecules therein from the harsh environment of the gastrointestinal tract and efficiently cross the intestinal epithelial barrier. Moreover, bitter gourd exosomes have good targeting property, biocompatibility, and the presence of multiple active ingredients makes them have potential application value in the treatment of diabetes, tumors, inflammation, and immunotherapy, etc. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a flowchart of the extraction of bitter gourd exosomes and the embedding of drugs in Example 1.

[0039] Figure 2 is the surface biomarker WB (A), transmission electron microscope (TEM) (B, C) and NTA detection atlas of bitter gourd exosomes obtained in Example 1 and the surface biomarker WB (A) after embedding insulin, and the surface potential analysis (D, E) of bitter gourd exosome morphology and particle size distribution.

[0040] Figure 3 is the in vitro biological safety CCK-8 experiment of bitter gourd exosomes and the embedding of insulin in Example.

[0041] Figure 4 is the release of bitter gourd exosomes embedding insulin in simulated gastrointestinal fluid in Example.

[0042] Figure 5 is the pathological section of the biological safety of bitter gourd exosomes and the embedding of insulin in Example.

[0043] Figure 6 is the intestinal villus absorption of bitter gourd exosomes and the embedding of insulin in Example.

[0044] Figure 7 is the in vivo hypoglycemic effect of bitter gourd exosomes (BMEV), bitter gourd exosomes and anionic liposome 18:1 PA-CHOL embedding insulin on type I diabetic mice.

[0045] Figure 8 is the result of an in vitro inhibition experiment of a-glucosidase by balsam pear exosome (BMEV) in an embodiment. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below with reference to the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the described embodiments of the present application, all other solutions obtained by a person of ordinary skill in the art without any inventive effort fall within the scope of protection of the present application.

[0047] Embodiment 1: Separation, purification of balsam pear exosome and preparation of medicament (taking insulin as an example).

[0048] According to the method shown in Figure 1, the steps are as follows:

[0049] (1) Wash fresh balsam pear and remove seeds, squeeze to obtain balsam pear juice, and pass through a 400-mesh filter screen;

[0050] (2) Centrifuge to remove plant fibers, cell debris, etc., and the centrifugal conditions are as follows: 300g, 10minx2; 1000g, 10minx2; 10000g, 30minx2;

[0051] (3) Take the supernatant and pass through a 0.22μm filter membrane; take the filtrate and centrifuge at 150000g for 90min by a HITACHI CP70-ME ultracentrifuge to obtain balsam pear exosome precipitate (crude product);

[0052] (4) Dissolve the above precipitate in pH7.4 PBS, resuspend after PBS, and then centrifuge at 150000g for 90min by ultracentrifuge to obtain purified balsam pear exosome.

[0053] Mix the balsam pear exosome with insulin at a mass ratio of 10:1, incubate at room temperature for 30min; 40% amplitude, ice bath ultrasonication for 2min, 3s ON / 3s OFF, cool for 2minx3; incubate at 37℃ for 1h after ultrasonication to restore the lipid bilayer structure of the exosome; remove the unencapsulated free insulin by ultrafiltration centrifugation (100kDa molecular weight).

[0054] Encapsulation efficiency (ER, %) = (Wt-Wf) / Wt x 100%

[0055] Drug loading rate (LR, %) = (Wt-Wf) / WT x 100%

[0056] Wherein: Wt is the total mass of insulin; Wf is the mass of free insulin; WT is the mass of BMEVs@INS.

[0057] According to the above formula, the concentration of INS in BMEVs@INS was determined using HPLC. Under the condition of BMEVs:INS at 10:1 (mass ratio), the calculated encapsulation efficiency of INS was 68.66%, and the drug loading rate was 17.20%.

[0058] Example 2 Detection of Momordica charantia exosomes and after embedding insulin

[0059] The Momordica charantia exosomes obtained in Example 1 and the Momordica charantia exosomes after embedding insulin were subjected to surface biomarker WB detection, transmission electron microscopy (TEM) and NTA detection, and the atlas is shown in Figure 2.

[0060] First, the Momordica charantia exosomes and the insulin drug suspension samples after embedding insulin were separated by PAGE and transferred to a solid support. Then, the proteins or polypeptides on the solid support were used as antigens to react with the corresponding antibodies. Then, the second antibody labeled with enzyme or isotope was reacted, and the specific protein components expressed by the target gene separated by electrophoresis were detected by substrate color development or autoradiography. Figure 2(A) shows that the Momordica charantia exosomes (BMEVs) have the typical CD63, CD81 and TSG101 biomarkers of EVs, and these markers still exist even after embedding insulin (BMEVs@INS).

[0061] The prepared Momordica charantia exosomes and insulin drug suspension samples were sucked and dropped on the copper mesh with a supporting membrane using a pipette. According to the sample concentration in the suspension, the excess liquid was absorbed from the edge of the liquid bead using filter paper immediately or after placing for a few minutes, and then the staining solution was dropped, with a staining time of 3-5 minutes. Subsequently, the staining solution was absorbed using filter paper to dry it, and transmission electron microscopy was used for observation. According to the TEM results (Figure 2B, C), the Momordica charantia exosomes (BMEVs) and the Momordica charantia exosomes embedding insulin (BMEVs@INS) showed a typical tea tray-like structure. After embedding insulin, the structure of the vesicles did not change significantly.

[0062] Suspension samples (100 μL) of balsam pear exosomes and insulin drug-embedded balsam pear exosomes were filtered, diluted 1 / 400 in PBS, and injected into the analysis chamber of a NanoSight NS300 instrument equipped with a 520 nm laser and a sCMOS camera. Sample analysis was performed at a camera level of 10 and a gain of 250, with a detection threshold of 10 pixels. The settings for blur, minimum track length, and minimum expected size were set to "automatic". Videos were recorded at a temperature of 29°C, at a speed of 30 frames / s, for three replicates of 60 seconds each. All post-acquisition settings were kept constant between samples. Data were processed and analyzed using NTA software v3.0. According to the NTA results (Figures 2D, E), the average size of balsam pear exosomes was about 156.5 nm, and the concentration was about 1.9E+10 particles per milliliter; while the average size of insulin drug-embedded balsam pear exosomes was about 159.2 nm, and the concentration was about 1.9E+10 particles per milliliter. This indicates that there is no significant change in size and particle concentration after embedding insulin drugs in balsam pear exosomes.

[0063] Example 3 In vitro biological safety of balsam pear exosomes and insulin-embedded balsam pear exosomes CCK-8 test

[0064] A 96-well plate was seeded with 100 μL of human macrophage cell suspension (5000 cells / well), and the plate was pre-cultured in an incubator for 24 h; 10 μL of PBS, balsam pear exosome solution, and insulin drug-embedded balsam pear exosome solution were added to the plate; the plate was incubated in an incubator for 6 h; 10 μL of CCK-8 solution was added to each well of the plate; the plate was incubated in an incubator for 2 h; and the absorbance at 450 nm was measured using a microplate reader. After the CCK-8 test, the results are shown in Figure 3. Balsam pear exosomes and insulin-embedded balsam pear exosomes showed no significant drug toxicity and exhibited good biological safety.

[0065] Example 4 Release of insulin-embedded balsam pear exosomes in simulated gastrointestinal fluid

[0066] The release of insulin in gastric juice / intestinal juice after the exosome of Momordica charantia L. encapsulates insulin was investigated by dialysis bag method. Briefly, 1 mL of the solution was placed in a dialysis bag with a molecular weight of 20000 Da, immersed in 50 mL of simulated gastric juice / intestinal juice, and stirred at 37°C and 100 rpm. 20 μL of the solution in the dialysis bag was taken at 0, 10, 30, 60, 120, 180, 240 min, and the remaining insulin content in the system was detected by an insulin kit. The experimental results are shown in Figure 4. In simulated gastric juice, after 240 minutes, the leakage amount of insulin was less than 20% of the total amount; in simulated intestinal juice, after 240 minutes, the leakage amount of insulin was less than 30% of the total amount. This shows that the exosome of Momordica charantia L. can effectively protect insulin from the harsh environment of gastric acid and digestive tract proteolytic enzymes.

[0067] Example 5 Pathological section of biological safety of Momordica charantia L. exosome and its encapsulated insulin

[0068] The I-type diabetic mice were used as a model, the physiological saline group was a blank control group, the experimental group was oral Momordica charantia L. exosome BMEVs and encapsulated insulin drug Momordica charantia L. exosome BMEVs@INS. After the administration was completed, the mouse heart (Heart), liver (Liver), spleen (Spleng), lung (Lung), kidney (Kidney) were taken and immersed in 4% paraformaldehyde, and after embedding section, HE staining was performed and photographs were taken under a upright microscope. The results showed that after the administration was completed, the HE staining results of the mouse tissues of different preparations were shown in Figure 5, and no obvious tissue damage was observed in the important organs in vivo. The above phenomenon shows that BMEVs and BMEVs@INS have good tissue compatibility and high safety.

[0069] Example 6 Intestinal villus absorption of Momordica charantia L. exosome and its encapsulated insulin drug

[0070] The I-type diabetic mice were fasted overnight without water, and the DiO-labeled Momordica charantia L. exosome and the encapsulated insulin drug Momordica charantia L. exosome were injected into the ligated jejunum section for incubation. After 30 min, the incubated intestinal section was taken and fixed in 4% paraformaldehyde for more than 4 h, and then dehydrated with pre-cooled 30% sucrose solution overnight. The frozen section machine was used for sectioning, and the section thickness was 10 μm. After DAPI staining, the section was sealed with 90% glycerol, and the intestinal villus absorption of Momordica charantia L. exosome and the encapsulated insulin drug Momordica charantia L. exosome was observed under a laser confocal microscope. The results are shown in Figure 6, which shows that Momordica charantia L. exosome and the encapsulated insulin drug Momordica charantia L. exosome exhibit good ability to overcome the intestinal epithelial cell barrier in vivo.

[0071] Example 7 In vivo hypoglycemic effect of Momordica charantia exosome and its insulin-embedded counterpart

[0072] Type I diabetic mice were used as a model and randomly divided into 4 groups, 5 mice in each group. The mice were fasted overnight but not deprived of water before the experiment. Insulin s.c. was used as a positive control group, with a dosage of 1 IU / kg. Saline was used as a blank control group. The experimental groups were BMEVs (28 mg / kg) and BMEVs@INS (insulin, 18, 50 IU / kg), respectively. The formulation of 18:1 PA-CHOL@INS was obtained by incubating insulin with anionic artificial liposome 18:1 PA-CHOL using the same method of ultrasonic dispersion, and was used as a control (insulin, 50 IU / kg).

[0073] The blood glucose levels were measured and recorded at 0, 2, 3, 6, 8, and 10 h after administration using a blood glucose meter.

[0074] The experimental results are shown in FIG. 7 (abscissa). The insulin-embedded Momordica charantia exosome drug group and the positive control group showed significant differences in reducing blood glucose compared with the saline control group, the 18:1 PA-CHOL group, and the Momordica charantia exosome group, which indicates that the insulin-embedded Momordica charantia exosome drug has good hypoglycemic effect.

[0075] Alpha-glucosidase is a group of enzymes active in the small intestine, responsible for breaking down complex carbohydrates (such as starch) into monosaccharides (such as glucose) for absorption into the blood by the small intestine. By inhibiting the activity of this enzyme, the rate of carbohydrate breakdown can be slowed down, thereby slowing down the rate of glucose absorption. This effect helps to prevent a sharp rise in blood glucose levels after meals, making blood glucose levels more stable. Momordica charantia exosomes exhibit a hypoglycemic effect in in vitro experiments, as shown in FIG. 8. The in vitro inhibition of alpha-glucosidase by BMEVs showed a clear dose-effect relationship, indicating that BMEVs have a significant inhibitory effect on alpha-glucosidase. However, from the in vivo experiment in mice, the hypoglycemic effect of oral Momordica charantia exosomes was not significant.

[0076] Take test tube E, add 67mmol / L pH6.8 potassium phosphate buffer 1.0mL, 3mmol / L glutathione solution 0.2mL, 0.04mg / mL alpha-glucosidase 0.3mL; add bitter gourd exosome 0.3mL (concentration is 0.228, 0.435, 0.870, 1.305, 1.740, 3.470mg / ml respectively, wherein 3.470mg / ml is the maximum concentration), mix well, 37 degrees Celsius water bath for 10mins, then add 0.1mg / mL PNPG solution 0.5mL, react for 20mins, then add 0.1mol / L sodium carbonate solution 8mL to stop the reaction, measure the absorbance at wavelength 400nm, record as E1.

[0077] Take test tube F, do not add PNPG, the rest of the steps are consistent with test tube E, measure the absorbance at wavelength 400nm, record as E2.

[0078] Take test tube G, do not add bitter gourd exosome solution, the rest of the steps are consistent with test tube E, measure the absorbance at wavelength 400nm, record as E3.

[0079] Take test tube H, do not add PNPG and bitter gourd exosome solution, the rest of the steps are consistent with test tube E, used for zero adjustment of the above E1, E2, E3 absorbance detection, record as Ezero.

[0080] Alpha-glucosidase inhibition rate: enzyme activity inhibition rate (%) = [E3-(E1-E2)] / E3x100(%)

[0081] As shown in Figure 7, when the insulin (INS) dosage is 50IU / kg, the hypoglycemic effect of BMEVs embedded insulin on type I diabetic mice is better than that of anionic liposome PA-CHOL embedded insulin. This shows that BMEVs have characteristics that artificial liposomes do not have, which can effectively protect the embedded INS, and exhibit good intestinal absorption capacity, and the hypoglycemic capacity is similar to that of subcutaneous injection of insulin.

[0082] It should be noted that the above is only a preferred embodiment of the present application, and is not intended to limit the present application; any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Application of Momordica charantia exosome in preparation of a medicament for embedding protein, polypeptide, small molecule peptide, nucleic acid or small molecule compound; the Momordica charantia exosome is derived from Momordica charantia and has an average particle size of 140-180 nm.

2. Use according to claim 1, characterized in that, The medicament is an oral medicament.

3. Use according to claim 1, characterized in that, The Momordica charantia exosome is derived from Momordica charantia and has an average particle size of 150-160 nm.

4. An oral medicament, characterized by, The composition comprises Momordica charantia exosome and a pharmaceutically active ingredient embedded in the Momordica charantia exosome.

5. The oral medicament according to claim 4, characterized in that, The pharmaceutically active ingredient is protein, polypeptide, small molecule peptide, nucleic acid or small molecule compound.

6. The oral medicament according to claim 4, characterized in that, The pharmaceutically active ingredient is insulin.

7. The use according to any one of claims 1 to 3 or the oral medicament according to any one of claims 4 to 6, characterized in that, The preparation method of Momordica charantia exosome comprises the following steps: (1) Momordica charantia is squeezed without seeds, and Momordica charantia juice is obtained; (2) the precipitate in the Momordica charantia juice is removed by centrifugation, and the supernatant is filtered; (3) the supernatant of step (2) is filtered by a 0.1-0.5 μm filter membrane, the filtrate is subjected to ultrahigh speed centrifugation, and the precipitate is Momordica charantia exosome; The centrifugation conditions of step (2) are as follows: 200-500 g, 1-3 times, 5-15 min each time; 800 g-1200 g, 1-3 times, 5-15 min each time; 10000-15000 g, 1-3 times, 20-40 min each time; The ultrahigh speed centrifugation conditions of step (3) are as follows: 100000-200000 g centrifugation for 0.5-3 hours.

8. A process for the preparation of the oral medicament according to any one of claims 4 to 6, characterized in that, It comprises the following steps: (i) mixing Momordica charantia exosome with a pharmaceutically active ingredient and incubating for 20-60 min; (ii) after ultrasonic dispersion, continue to incubate for 30-120 min, and remove the unencapsulated pharmaceutically active ingredient.

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