A non-phospholipid lipid membrane modified yeast microcapsule carrier, its preparation method and application

Abstract

This invention discloses a non-phospholipid lipid membrane-modified yeast microcapsule carrier, its preparation method, and its application, belonging to the field of drug carrier technology. The invention obtains yeast microcapsules through ethanol dehydration, adsorbs and loads drugs to obtain drug-loaded yeast microcapsules, and then modifies these drug-loaded yeast microcapsules with a cationic lipid membrane to obtain yeast microcapsules modified with a non-phospholipid cationic lipid membrane coating. The non-phospholipid lipid membrane-modified yeast microcapsule carrier prepared by this invention possesses the acid stability, pH responsiveness, and ion responsiveness of cationic lipid membranes. It successfully solves the problems of poor stability, excessive drug loss in gastric juice, and low bioavailability of drugs such as berberine hydrochloride due to their ineffective retention in the intestine, thus improving the oral bioavailability of drugs such as berberine and promoting drug absorption in the intestinal environment. This provides a new approach for the research and development and use of drugs with low bioavailability in oral formulations.

Description

Technical Field

[0001] This invention belongs to the field of drug carriers, specifically relating to a method for preparing a nonphospholipid-modified yeast microcapsule as an oral sustained-release drug. This method achieves drug protection and loading in an acidic gastric environment and responsive sustained release of the drug in an alkaline intestinal environment, thereby effectively enhancing the absorption rate and bioavailability of the drug in the intestine. Background Technology

[0002] Oral drug delivery, as a common method of drug delivery, offers advantages such as safety, convenience, and low cost. It also provides greater flexibility and better patient compliance, reducing the physical pain and psychological fear associated with injections. Therefore, it is the preferred route of administration and treatment for chronic diseases requiring long-term medication management. Oral drug formulations typically incorporate carriers to maintain drug concentrations within an appropriate range, preventing excessively high concentrations that exceed tolerable levels. This plays a crucial role in improving treatment efficacy and reducing side effects. Therefore, good absorption and high bioavailability are essential for the therapeutic effect of oral drugs, and the development and utilization of novel oral drug carriers are receiving increasing attention.

[0003] Cell-based drug delivery systems, based on cells or cell derivatives, are used for targeted drug delivery. They possess superior active targeting capabilities and good biocompatibility, attracting significant attention in the field of drug delivery. Whether animal cells, plant cells, or microorganisms, as cellular structures, they all possess natural structural morphologies. This simplifies drug delivery by eliminating many cumbersome steps in carrier synthesis, leading researchers to increasingly focus on the applications of cell-based drugs.

[0004] Yeast cells, as an emerging biological delivery vehicle, possess immense potential in encapsulating hydrophobic and hydrophilic compounds and bioactive molecules, protecting against external environmental stresses, controlling release, and exhibiting biocompatibility and biodegradability. Furthermore, most microorganisms struggle to survive and maintain intact cell morphology in the extremely acidic environment of gastric juice with its low pH. However, yeast cells persist in the digestive tract under these conditions, with *Saccharomyces cerevisiae* strains exhibiting particularly strong tolerance to these highly acidic conditions. This provides a prerequisite for *Saccharomyces cerevisiae* to serve as an oral drug delivery vehicle.

[0005] However, existing oral drug delivery systems for yeast cells still suffer from a series of problems, such as low mucosal adhesion, low drug load, and short release cycles for some drugs. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide a non-phospholipid lipid membrane modified yeast microcapsule carrier, its preparation method and application, to solve the problems of poor stability of drugs such as berberine hydrochloride, excessive loss of drug volume in gastric juice, and low utilization due to ineffective retention in the intestine, while endowing the carrier with ion response and pH response properties, thereby improving the oral utilization of drugs such as berberine.

[0007] The objective of this invention is achieved through the following means:

[0008] This invention provides a non-phospholipid lipid membrane modified yeast microcapsule carrier. The yeast microcapsule carrier is obtained by ethanol dehydration to obtain Saccharomyces cerevisiae microcapsules (YCMs), which are loaded with drugs of different properties to obtain drug-loaded yeast microcapsules. The drug-loaded yeast microcapsules are then modified with a cationic lipid membrane to obtain non-phospholipid cationic lipid membrane coated yeast microcapsules (SYCMs).

[0009] Based on the above technical solution, the cationic lipid membrane further comprises cholesterol and octadecylamine.

[0010] Another aspect of the present invention provides a method for preparing the above-mentioned non-phospholipid lipid membrane modified yeast microcapsule carrier, which mainly includes the following steps:

[0011] (1) Inoculate the brewer's yeast in YPD medium at a ratio of 1:1000 and culture it to resuscitate and activate it. Then, transfer it to fresh YPD medium at a ratio of 1:100 for expansion culture. When the logarithmic growth phase is reached, collect the cells by centrifugation. Wash with excess ethanol aqueous solution, then wash with excess deionized water, and finally remove the water. Then freeze-dry the obtained precipitate to obtain the dried substance, grind it into powder, and obtain yeast microcapsules.

[0012] (2) Prepare a drug solution containing the drug to be loaded, add the yeast microcapsules obtained in step (1) for adsorption and drug loading, centrifuge to collect the precipitate after completion, freeze dry to obtain drug-loaded yeast microcapsules;

[0013] (3) Cholesterol and octadecylamine were dissolved in a mixture of benzene and methanol in a certain proportion, freeze-dried and resuspended in the solution. After 1 to 10 freeze-thaw cycles, a cationic lipid membrane was obtained by self-assembly.

[0014] (4) The cationic lipid membrane obtained in step (3) is mixed with an aqueous solution containing the drug-loaded yeast microcapsules from step (2) at a certain mass ratio, stirred evenly and then centrifuged. The resulting precipitate is freeze-dried to obtain the yeast microcapsule carrier.

[0015] Based on the above technical solution, further, the conditions for resuscitation and activation in step (1) are 25℃~30℃, 120rpm~350rpm, and activation time is 12~18h. The most preferred conditions for resuscitation and activation are 30℃, 200rpm, and 18h.

[0016] Based on the above technical solution, further, the expanded culture conditions in step (1) are 25℃~30℃, 120rpm~350rpm, and the culture time is 18~24h. The most preferred expanded culture conditions are 30℃, 200rpm, and 22h.

[0017] Based on the above technical solution, further, the conditions for centrifuging and collecting bacterial cells in step (1) are 3500-10000 rpm and 1-20 min; the most preferred conditions are 8000 rpm and 10 min.

[0018] Based on the above technical solution, further, the volume percentage concentration of the ethanol aqueous solution in step (1) is 1% to 40%, and it is washed 1 to 5 times. The most preferred condition is an ethanol aqueous solution with a volume percentage concentration of 10% and washing 2 times.

[0019] Based on the above technical solution, further, in step (1), after washing with deionized water, the bacterial solution is centrifuged at 2000-10000g for 1-10min and washed 1-3 times. The most preferred conditions are 2500g, centrifuged for 5min, and washed once.

[0020] Based on the above technical solution, the freeze-drying time in step (1) is further 24h to 96h.

[0021] Based on the above technical solution, further, the drugs mentioned in step (2) include drugs classified as Class II, III, and IV by the Biopharmaceutics Classification System (BCS), drugs with the properties of BCS Class IV drugs, and biological macromolecular drugs. BCS Class II drugs are those with low solubility and high permeability, and their bioavailability is limited by their dissolution rate, such as cyclosporine, griseofulvin, lignins, flavonoids, and terpenoids. BCS Class III drugs are those with high solubility and low permeability, such as atenolol, cimetidine, metformin, phenolic acids, and phthalides, and the bioavailability of these drugs is limited by the permeability of the gastrointestinal membrane. BCS Class IV drugs are those with low solubility and low permeability, and the active ingredient has a low dissolution rate from the formulation and a low transmembrane transport rate, such as isoflavone derivatives such as puerarin, alkaloids such as berberine, and paclitaxel, and have the properties of BCS Class II drugs. Drugs classified as Class IV include saponins such as notoginsenoside R1 and glycyrrhizic acid, and biopharmaceuticals including proteins, enzymes, and nucleic acids; the mass ratio of the drug to be loaded to yeast microcapsules is 1:100 to 1:1. The conditions described are mixing and stirring for 15 minutes to 48 hours.

[0022] Based on the above technical solution, further, the conditions for adsorption and drug loading in step (2) are mixing and stirring for 15 min to 48 h.

[0023] Based on the above technical solution, further, the centrifugation conditions in step (2) are 2000-10000g centrifugation for 1-10 min, and the most preferred conditions are 8000rpm for 10 min.

[0024] Based on the above technical solution, further, the molar ratio of cholesterol to octadecylamine in step (3) is 1:5 to 5:1, and the volume ratio of benzene to methanol is 1:9 to 9:1.

[0025] Based on the above technical solution, the solution in step (3) further includes deionized water and MES-Tris buffer, with a preferred buffer concentration of 30-500 mM and a buffer pH range of 4-11.

[0026] Based on the above technical solution, further, the freeze-thaw cycle in step (3) is specifically an alternating cycle under two conditions: liquid nitrogen freezing and water bath, with vortex oscillation during each cycle; the preferred liquid nitrogen freezing time is at least 0.5 minutes, more preferably at least 10 minutes; the water bath temperature range is 25-90℃; the preferred water bath time is at least 5 minutes; the preferred number of alternating cycles is at least 3 times, and the most preferred reaction conditions are liquid nitrogen freezing for 1 minute, 70℃ water bath for 10 minutes, and alternating cycles for 5 times; the preferred vortex oscillation time is not less than 10 seconds, and the more preferred time is 25-35 seconds.

[0027] Based on the above technical solution, in step (4), the mass ratio of lipid membrane to drug-loaded yeast microcapsules is (1-5):(5-1), and the more preferred mass ratio is 1:1.

[0028] Based on the above technical solution, in step (4), the stirring time after mixing the lipid membrane and the drug-loaded yeast microcapsules is 5 min to 48 h, and the stirring time is more preferably 15 min.

[0029] Based on the above technical solution, further, the centrifugation conditions in step (4) are 2000-10000g centrifugation for 1-10 min, and the most preferred conditions are 8000rpm for 10 min.

[0030] The present invention also provides the application of the above-mentioned non-phospholipid lipid membrane modified yeast microcapsule carrier as an oral formulation.

[0031] The advantages of this invention over the prior art are as follows:

[0032] The non-phospholipid lipid membrane-modified yeast microcapsule carrier prepared by this invention possesses the acid stability, pH responsiveness, and ion responsiveness of cationic lipid membranes. It successfully solves the problems of poor stability, excessive drug loss in gastric juice, and low bioavailability of drugs such as berberine hydrochloride due to their inability to effectively remain in the intestine. This improves the oral bioavailability of drugs such as berberine and promotes drug absorption in the intestinal environment, providing a new approach for the research and development and use of drugs with low bioavailability in the field of oral formulations. Attached Figure Description

[0033] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0034] Figure 1 The images show SEM images of yeast microcapsules before and after loading with berberine hydrochloride in Example 1, where (a) is a yeast microcapsule and (b) is a drug-loaded yeast microcapsule.

[0035] Figure 2 The images show the FTIR spectra of berberine hydrochloride-yeast microcapsules, berberine hydrochloride, and yeast microcapsules in Example 1, where (a) is the total reflectance spectrum and (b)-(c) are the transmission spectra.

[0036] Figure 3 Thermogravimetric analysis of berberine hydrochloride-yeast microcapsules, berberine hydrochloride and yeast microcapsules in Example 1, wherein (a) is the percentage of weight loss and (b) is the weight loss rate;

[0037] Figure 4The images show the in vitro release curves of berberine hydrochloride-yeast microcapsules and berberine hydrochloride in Example 1, where (a) is the in vitro release curve in water and (b) is the in vitro release curve of berberine hydrochloride-yeast microcapsules at different pH values.

[0038] Figure 5 To test the stability of the lipid membrane components in Example 2 under different environments, (a) simulated gastric juice (pH 1.2); (b) simulated intestinal juice (pH 7.4); and (c) Tris-NaCl solution (pH 7.4);

[0039] Figure 6 FTIR analysis of yeast microcapsules, lipid membrane, and yeast microcapsules after lipid membrane modification in Example 2, wherein (a) is 4000-500 cm⁻¹ -1 Range; (b) 2000-500cm -1 scope;

[0040] Figure 7 The images shown are SEM images of the drug-loaded yeast microcapsules modified with lipid membranes in Example 2. (a)-(b) are drug-loaded yeast microcapsules modified with lipid membranes; (c) is drug-loaded yeast microcapsules modified with lipid membranes after release from simulated gastric juice (pH 1.2); and (d) is drug-loaded yeast microcapsules modified with lipid membranes after release from simulated intestinal juice (pH 7.4).

[0041] Figure 8 The images show the release curves of the drug-loaded yeast microcapsules with and without lipid membrane modification in Example 2. (a) is the release curve in simulated gastric juice (pH 1.2); (b) is the release curve of the drug-loaded yeast microcapsules with lipid membrane modification in simulated intestinal juice (pH 7.4).

[0042] Figure 9 The toxicity of the lipid membrane-modified yeast microcapsules to J774A.1 cells in Example 2 is shown in (a) for 24 hours and (b) for 48 hours. Detailed Implementation

[0043] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from chemical companies.

[0044] Example 1: Preparation of Berberine Hydrochloride-Yeast Cell Microcapsules (BRH-YCMs)

[0045] Saccharomyces cerevisiae (ATCC9763) culture was diluted 1:1000 in YPD medium (100 μL of culture solution per 100 mL of medium) and activated at 30°C and 200 rpm for 16 h. The culture was then transferred to fresh YPD medium (1:100, i.e., 10 mL of activated culture solution per 1 L of fresh medium) and cultured at 30°C and 200 rpm for 22 h. The culture was then collected by centrifugation at 8000 rpm for 10 min. The collected cells were washed twice with excess 10% (v / v) ethanol solution and once with excess deionized water. After each wash, the culture was centrifuged at 2500 g for 5 min, and then centrifuged again to remove excess water. The precipitate obtained after centrifugation was then freeze-dried and ground into a white powder. Using a precise analytical balance, berberine hydrochloride technical grade was weighed and dissolved in hot water at 80°C. The solution was then placed on a magnetic stirrer and heated and stirred at medium speed until the berberine hydrochloride was completely dissolved, preparing a drug solution with a concentration of 5 mg / mL. The magnetic stirrer temperature was adjusted to 50°C, and 1 g of lyophilized yeast microcapsules were added for adsorption and drug loading (carrier to drug mass ratio = 5:1). After stirring at a constant speed for 16 h, loading was completed. The solution was collected by centrifugation at 8000 rpm for 10 min, and the precipitate was placed in a lyophilizer for freeze-drying and storage for later use. By detecting the unloaded drug in the supernatant at 228 nm using a UV spectrophotometer, the drug loading was calculated to be 8.71 ± 0.39%.

[0046] The lyophilized unloaded and drug-loaded yeast microcapsules were observed by scanning electron microscopy, and the results are as follows: Figure 1 As shown. Yeast usually has a relatively rounded ellipsoidal structure. After freeze-drying, the surface is slightly wrinkled. After drug loading, textures begin to appear on the surface of the yeast microcapsules, which means that the drug is adsorbed due to swelling during the drug loading process.

[0047] To further confirm that berberine hydrochloride was absorbed into the carrier by the yeast microcapsules, rather than adsorbed onto their surface, FTIR analysis was performed on the yeast microcapsules before and after berberine hydrochloride loading. The results are as follows: Figure 2 As shown; Figure 2 (a) shows the FTIR total reflectance spectrum of berberine hydrochloride-yeast microcapsules. The drug-loaded and unloaded yeast microcapsules showed similar FTIR total reflectance spectra, indicating that berberine hydrochloride did not remain on the carrier surface. Figure 2 (b) shows the FTIR transmission spectra of both instruments. Analysis revealed that the transmission spectral density (TIR) ​​values ​​at 3353.258, 2841.16, 1635.366, 1507.121, 1362.484, 1304.649, 925.197, and 899.163 cm⁻¹ were significantly higher. -1The characteristic infrared peak of berberine hydrochloride was observed at [insert value here]. Compared to unloaded yeast microcapsules, its peak was 1635.366 cm⁻¹. -1 The absorption peaks were significantly enhanced. Overall, the total reflectance and transmission spectra of FTIR indicate that berberine hydrochloride was loaded into the yeast microcapsule carrier.

[0048] Based on thermogravimetric analysis results Figure 3 As can be seen, all samples exhibited some weight loss between 30-100℃. This is because, between 30-70℃, the weight loss was primarily attributed to the evaporation of free water molecules; however, between 70-110℃, bound water molecules began to be lost. Berberine hydrochloride showed a second wave of weight loss starting at 180℃, and the significant thermal loss of berberine hydrochloride-yeast microcapsules occurred at 290℃. This means that below 290℃, the weight loss rate of berberine hydrochloride was much greater than that of berberine hydrochloride-yeast microcapsules. This indicates that the carrier can reduce the thermal loss of the drug within a certain temperature range. Considering that the weight loss percentage and rate were similar for unloaded and loaded yeast microcapsules, it can be concluded that within a certain range, yeast microcapsules can provide some protection for the drug and improve the thermal stability of berberine hydrochloride.

[0049] The main reason why berberine hydrochloride has very poor oral bioavailability (less than 1%) is that it is only easily soluble in hot water, does not dissolve well at room temperature, and has poor permeability. Figure 4 (a) The release of berberine hydrochloride and berberine hydrochloride-yeast cell microcapsules in water. The results show that, under conditions conducive to sufficient drug dissolution, encapsulation of berberine hydrochloride by YCMs improves the bioavailability of the drug. In the aqueous phase (deionized water), berberine hydrochloride (4.0 mg) was rapidly released within 2 h, with a release rate exceeding 60% during this period. After 2 h, the drug release gradually increased, reaching 80% of the maximum value (i.e., 3.2 mg) around 8 h, and maintained until 42 h. In contrast, berberine hydrochloride was released more slowly from the carrier, with only 5% of the initial drug amount (4.85 mg) released within 2 h. The release gradually increased over time, reaching 71.03 ± 1.40% after 48 h, corresponding to a release of 3.5 mg, slightly higher than the total drug release without a carrier. Overall, these results demonstrate that yeast microcapsules do not limit drug release and can achieve sustained drug release. Figure 4The results in (b) indicate that the amount of drug released by BRH-YCMs in simulated intestinal fluid (pH 7.4) was significantly higher than that in simulated gastric fluid (pH 1.2). Since the drug is administered orally, its residence time in gastric fluid is 3–4 hours. During this period, 13.20 ± 4.23% of berberine hydrochloride is released from the carrier, meaning that most of the drug is preserved and can be absorbed in the intestine. In simulated intestinal fluid, the drug release reached 92.82 ± 2.29% within 48 hours. Therefore, YCM encapsulation enhances the stability of berberine hydrochloride in gastric fluid. While the original drug dissolves by more than 50% within 4 hours in an acidic environment, the release from the drug-loaded yeast capsules under these conditions is only 13.20 ± 4.23%, a reduction of approximately 37%, thus ensuring the effective amount of drug released in intestinal fluid and improving drug utilization.

[0050] Example 2: Preparation of lipid membrane-modified berberine hydrochloride yeast microcapsules (BRH-SYCMs)

[0051] Cholesterol and octadecylamine were dissolved in a mixed solution of benzene and methanol in an equimolar ratio (V(benzene):V(methanol) = 9:1). The solution was dissolved by sonication, lyophilized, and a certain amount of sample was weighed and resuspended in deionized water to make the lipid film concentration 10 mg / mL. The solution was then rapidly frozen with liquid nitrogen and then bathed in a water bath at 70°C for 10 min. The liquid nitrogen freezing and water bath process was repeated 5 times to obtain the desired lipid film. The lipid film was then added to a yeast microcapsule solution loaded with berberine hydrochloride at a mass ratio of 1:1 to the yeast microcapsules. The mixture was rapidly stirred for 15 min, centrifuged at 8000 rpm for 10 min, and the precipitate was lyophilized for later use.

[0052] Cationic lipid membranes exhibit some pH responsiveness and can remain stable under acidic conditions. However, they rapidly degrade when the pH exceeds 10. The oral environment does not reach pH 10, and in the intestinal fluid environment, the ionic environment is primarily phosphate-rich, with phosphate ions having a significant destructive impact on lipid membrane stability. Figure 5 The prepared cationic lipid membrane exhibits good stability in the acidic environment of simulated gastric juice and is relatively stable and uniformly dispersed in Tris-NaCl solution (pH 7.4). However, in the simulated intestinal juice system (pH 7.4), the lipid membrane stability is rapidly disrupted, with unstable floating matter appearing on the surface. This demonstrates that the phosphate content in the simulated intestinal juice system has a significant impact on the stability of the cationic lipid membrane. This phenomenon is beneficial for improving the oral bioavailability of the lipid-modified yeast microcapsules. It is expected to maintain stability in an acidic environment, reducing drug release, while the disruption of the lipid membrane coating in simulated intestinal juice promotes drug release and improves the effective utilization of the drug in the intestinal juice.

[0053] FTIR analysis was performed on the yeast microcapsules modified with cationic lipid membranes. Figure 6 ), at 4000-500cm -1 Overall, the lipid membrane can be observed at 2917.336 and 2850.802 cm. -1 The CH stretching vibration peak at 2000-500 cm⁻¹ indicates that lipid membrane modification is present on the surface of yeast microcapsules, while the peak at 2000-500 cm⁻¹... -1 Within this range, the infrared spectrum showed more variations, with the modified yeast microcapsules exhibiting changes at 1465.177, 1023.068, and 719.812 cm⁻¹, respectively. -1 New characteristic peaks appeared at all locations, corresponding to the CC stretching vibrations in the cationic lipid membrane (1465.177 cm⁻¹). -1 CN stretching vibration (1023.068cm) -1 ) and in-plane bending vibrations of olefin CH (719.812 cm) -1 ).

[0054] Figure 7 The images show SEM images of yeast microcapsules modified with a lipid membrane before drug release and after release in simulated gastric and intestinal fluids. It can be seen that lipid membrane modification leaves a coating mark on the surface of the yeast microcapsules; the morphology of the yeast microcapsules themselves is still faintly visible, but the coating modification effect is more obvious. After release in simulated gastric fluid, due to the stability of the lipid membrane in an acidic environment, the coating remains intact on the surface of the yeast microcapsules. After release in simulated intestinal fluid, due to the ionic responsiveness of the coating to phosphate, the coating rapidly disintegrates. The SEM images after release show the appearance of the ellipsoidal morphology of the yeast microcapsules themselves, and the lipid membrane coating disappears due to instability, further demonstrating the ionic responsiveness of the lipid membrane.

[0055] Yeast microcapsules with added lipid membrane modification exhibit significantly better protective effects against drugs in the acidic environment of the stomach, such as... Figure 8 (a) The final drug release rate of the lipid-membrane-modified drug-loaded yeast microcapsules within 48 hours was 31.50±0.66%, while the drug release rate of the unmodified yeast microcapsules within 48 hours was 78.05±6.89%. In comparison, the modified yeast microcapsules reduced drug loss in the gastric acid environment by 40-50% within 48 hours. In the first 4 hours, the drug release rate of the modified drug-loaded yeast microcapsules was only 8.82±0.33%, minimizing drug waste and ensuring an effective drug concentration in the intestine. Under simulated gastric acid conditions, the drug release of the lipid-membrane-modified yeast microcapsules was controlled at approximately 30% within 48 hours. In simulated intestinal fluid, the drug release rate was 55.21±1.51% at 24 hours and 65.27±3.96% at 48 hours (see Table 1). The lipid-membrane-modified drug-loaded yeast microcapsules exhibited a long-lasting sustained-release effect in simulated intestinal fluid, with a superior sustained-release effect.

[0056] Table 1. Release results of lipid membrane-modified drug-loaded yeast microcapsules in simulated intestinal fluid.

[0057]

[0058] Example 3: Cytotoxicity of lipid membrane-modified yeast microcapsules

[0059] Cytotoxicity assays were performed using the MTT assay. J774A.1 cells were cultured until they reached 80%-90% confluence. After trypsin digestion, the cells were pipetted into suspension. The cell suspension was then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended to prepare a single-cell suspension. The concentration was adjusted to 5 × 10⁻⁶ cells / mL. 4 Cells / mL. The prepared cell suspension was slowly and gently mixed, and 100 μL of the suspension was added to each well. The cells were incubated overnight. Once a suitable cell density was reached, pre-sterilized yeast microcapsules and lipid-modified yeast microcapsules (dissolved in PBS buffer) were added to achieve carrier concentrations of 1, 2, 3, 5, 8, and 10 μg / mL. Cell viability was measured by MTT assay after 24 or 48 hours of incubation. After co-culture, the supernatant was slowly aspirated, and 90 μL of fresh culture medium was added, followed by 10–20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT). Incubation was continued for 4 hours. The culture was terminated, the supernatant was discarded, and 110 μL of Formazan dissolving solution was added to each well. The cells were shaken at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm, and cell viability was calculated.

[0060] from Figure 9 It can be seen that the yeast microcapsules have low cytotoxicity before and after lipid membrane modification. Within 24h and 48h, the cell survival rate is good, ensuring a certain degree of oral safety. Moreover, the changes in low cytotoxicity between the unmodified and modified yeast microcapsules within 48h are not significant.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A non-phospholipid lipid-modified yeast microvesicular carrier, characterized in that, The yeast microcapsule carrier is obtained by ethanol dehydration method, and the drug-loaded yeast microcapsule is obtained after loading drugs with different properties, and the non-phospholipid cationic lipid film-coated yeast microcapsule is obtained by modifying the drug-loaded yeast microcapsule with a cationic lipid film. The cationic lipid film comprises cholesterol and octadecylamine. The preparation method of the yeast microcapsule carrier comprises the following steps: (1) inoculate Saccharomyces cerevisiae in YPD culture medium for culture, perform recovery activation, then transfer into fresh YPD culture medium for expansion culture, collect the bacterial bodies by centrifugation, wash with excessive ethanol aqueous solution, then wash with excessive deionized water, finally remove the water, then freeze-dry the obtained precipitate, grind into powder, and obtain the yeast microcapsule; (2) prepare a drug solution containing a drug to be loaded, add the yeast microcapsule obtained in step (1) for adsorption and drug loading, collect the precipitate by centrifugation, freeze-dry, and obtain the drug-loaded yeast microcapsule; (3) dissolve the cholesterol and octadecylamine in a mixture of benzene and methanol according to a certain proportion, resuspend after freeze-drying, and self-assemble the cationic lipid film after 1-10 freeze-thaw cycles; (4) mix the cationic lipid film obtained in step (3) with the aqueous solution containing the drug-loaded yeast microcapsule of step (2) according to a certain mass ratio, stir uniformly, then centrifuge, and freeze-dry the obtained precipitate to obtain the yeast microcapsule carrier.

2. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, The drug of step (2) is selected from the biological pharmaceutical classification system (BCS) II, III, IV, drugs with BCS IV drug properties, and biological macromolecular drugs, BCS II is selected from cyclosporine, griseofulvin, lignin, flavonoids and terpenoids; BCS III is selected from atenolol, cimetidine, metformin, phenolic acid, phthalide compound; BCS IV is selected from alkaloids; drugs with BCS IV drug properties are selected from notoginsenoside R1, glycyrrhizic acid, and biological macromolecular drugs are selected from proteins, enzymes, and nucleic acids; the mass ratio of the drug to be loaded to the yeast microcapsule is 1:100-1:1; the adsorption and drug loading condition is mixing and stirring, and the time is 15 min-48 h.

3. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, The molar ratio of cholesterol to octadecylamine in step (3) is 1:5-5:1, and the volume ratio of benzene to methanol is 1:9-9:1; the solution comprises deionized water and MES-Tris buffer.

4. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, The freeze-thaw cycle in step (3) is specifically alternating cycles under liquid nitrogen freezing and water bath conditions; the liquid nitrogen freezing time is at least 0.5 minutes; the water bath temperature is 25-90℃, and the water bath time is at least 5 min; the alternating cycle number is at least 3 times.

5. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, The recovery activation condition in step (1) is 25-30℃, 120-350 rpm, and the activation time is 12-18 h; the expansion culture condition is 25-30℃, 120-350 rpm, and the culture time is 18-24 h.

6. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, The centrifugal collection conditions of the bacteria in step (1) are 3500-10000 rpm for 1-20 min; the volume percentage concentration of the ethanol aqueous solution is 1%-40%, and the washing is performed for 1-5 times; after washing with deionized water, the bacteria solution is centrifuged at 2000-10000 g for 1-10 min, and the washing is performed for 1-3 times; and the freeze-drying time is 24 h-96 h.

7. The non-phospholipid liposome-modified yeast microvesicular carrier of claim 1, wherein, In step (4), the mass ratio of the lipid film to the drug-loaded yeast microcapsule is (1-5):(5-1); the stirring time of the mixture of the lipid film and the drug-loaded yeast microcapsule is 5 min-48 h; and the centrifugal conditions are 2000-10000 g for 1-10 min.

8. Use of the non-phospholipid lipid film modified yeast microcapsule carrier in claim 1 or 2 in the preparation of an oral preparation.

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