Composition, preparation method and application of bionic nanosystem loaded with amphotericin B
Amphotericin B is encapsulated through the PLGA nanoparticle carrier wrapped in vaginal epithelial cell membrane, solving the problems of drug targeting and poor retention time, reducing toxic side effects, and significantly improving the therapeutic effect.
Patent Information
- Application Number
- CN202210250345.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-03-15
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2042-03-15
AI Technical Summary
The existing amphotericin B antifungal drugs have targeting and poor retention time when treating vulvovaginal candidiasis, resulting in the need for large doses of injections and causing toxic side effects.
Using PLGA nanoparticle carrier wrapped in vaginal epithelial cell membrane, amphotericin B is encapsulated on its surface, separated and encapsulated by hypotonic and mechanical breaking methods, combined with nanocoprecipitation method to encapsulate amphotericin B to improve the drug in Candida Targeting and retention time of infection site.
It significantly increased the concentration and duration of amphotericin B in the infection site of Candida, reduced toxic side effects, enhanced the killing effect on Candida, and showed good biocompatibility and low cytotoxicity.
Smart Images
Figure CN114668772B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to biomedicine, and in particular to a composition of a bionic nanosystem loaded with amphotericin B constructed based on vaginal epithelial cell membrane, a preparation method and application thereof. Background Art
[0002] Vulvovaginal candidiasis (VVC) is one of the most common diseases in women of childbearing age, of which 85%-90% are caused by Candida albicans infection. Patients will experience symptoms such as vulvar redness, intense itching, and increased vaginal discharge. According to statistics, 75%-80% of women have had at least one Candida albicans infection in their lifetime, which is a conditional pathogen. In clinical treatment, VVC can be treated with topical or oral antifungal drugs. Amphotericin B (AmB) has been shown to bind to ergosterol on the cell membrane of Candida albicans, increase cell membrane permeability, and cause the outflow of intracellular ions such as K+ and Mg2+, destroying the normal metabolism of the fungus and inhibiting its growth.
[0003] Studies have shown that Candida albicans is relatively sensitive to amphotericin B (AmB), with a minimum inhibitory concentration (MIC) of ≤1μg / mL. However, due to the similarity between ergosterol and cholesterol, AmB can also bind to cholesterol on the surface of normal human cell membranes, causing toxic side effects, such as acute renal damage. In addition, AmB is derived from Streptomyces and can be recognized by Toll-like receptor 2 (TLR2) and transmembrane signaling protein CD14 on the surface of monocytes, causing a series of adverse reactions such as chills, nausea, vomiting, and headaches.
[0004] In recent years, nanotechnology has provided new ideas for improving the therapeutic index of AmB. A large number of literature reports show that nanomaterials can effectively improve the solubility of AmB, prolong its half-life and reduce its immunogenicity, such as nanoliposomes loaded with AmB, polylactic acid-co-glycolic acid (PLGA) nanoparticles, etc. However, most nanoformulations loaded with AmB have failed to effectively improve the targeting and retention time of the drug at the site of Candida infection, and can only achieve a certain therapeutic effect by injecting a large dose of nanomaterials containing AmB, which inevitably causes dose-dependent toxic side effects.
[0005] Therefore, the existing treatment options are actually not sufficient to achieve very good treatment effects, and there are still problems of poor treatment effects or obvious side effects. Summary of the invention
[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a composition, preparation method and application of a bionic nanosystem loaded with amphotericin B, which can overcome the poor targeting and retention time of drugs in the preparations of the prior art and reduce the problem of toxic side effects caused by injecting large doses of drugs for therapeutic effects.
[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing a bionic nanosystem loaded with amphotericin B, comprising the following steps:
[0008] Isolation and purification of vaginal epithelial cell membranes;
[0009] Encapsulation of AmB into PLGA nanoparticles;
[0010] The cell membrane of vaginal epithelial cells was wrapped onto the surface of PLGA nanoparticles containing AmB.
[0011] As a further improvement of the present invention, the separation and purification of cell membranes adopts hypotonic and mechanical disruption methods.
[0012] As a further improvement of the present invention, AmB is encapsulated into PLGA nanoparticles by nano-coprecipitation method.
[0013] As a further improvement of the present invention, the vaginal epithelial cells are cultured before being isolated and purified.
[0014] As a further improvement of the present invention, the cell membrane of the vaginal epithelial cells is wrapped by water bath ultrasound.
[0015] As a further improvement of the present invention, the encapsulation is completed by water bath ultrasound with 120W ultrasound for 5 minutes.
[0016] A composition loaded with amphotericin B biomimetic nanosystem comprises AmB, PLGA nanoparticles and vaginal epithelial cell membranes; wherein the AmB is encapsulated in the PLGA nanoparticles, and the entire encapsulation is wrapped by the vaginal epithelial cell membranes.
[0017] An application of a bionic nanosystem loaded with amphotericin B, wherein the bionic nanosystem is obtained by the above-mentioned preparation method or adopts the above-mentioned composition and is used for treating Candida albicans infection.
[0018] The beneficial effects of the present invention are that the prepared vaginal epithelial cell bionic nanomaterial can bind to Candida through the unique adhesion molecules on the surface of vaginal epithelial cells, thereby improving the targeting and retention time of the bionic nanomaterial at the Candida infection site, increasing the local AmB concentration, prolonging the action time of AmB, reducing the toxic side effects of AmB, and ultimately enhancing the Candida killing effect of AmB. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Characterization of the AmB@PLGA@VEM bionic nanosystem of the present invention (A is a measurement diagram of AmB@PLGA nanoparticles and AmB by ultraviolet spectrometer; B is a surface morphology diagram of the nanosystem; C is a hydrodynamic size and surface potential diagram of the nanosystem);
[0020] Figure 2 The diagram is a diagram showing the targeting and adhesion effects of the AmB@PLGA@VEM biomimetic nanosystem of the present invention on Candida albicans in vitro;
[0021] Figure 3 The diagram is a diagram of the targeting and adhesion effects of the AmB@PLGA@VEM bionic nanosystem of the present invention on the in vivo Candida albicans mouse infection model;
[0022] Figure 4 This is a diagram showing the therapeutic effect of the AmB@PLGA@VEM bionic nanosystem of the present invention on a mouse model infected with Candida albicans;
[0023] Figure 5 The biocompatibility of the AmB@PLGA@VEM bionic nanosystem of the present invention (A is a diagram showing the effect of the AmB@PLGA@VEM bionic nanosystem on hemolysis of red blood cells; B&C are test diagrams showing the cytotoxicity of the AmB@PLGA@VEM bionic nanosystem on vaginal epithelial cells and macrophages). DETAILED DESCRIPTION
[0024] The present invention will be further described below in detail with reference to the embodiments shown in the accompanying drawings.
[0025] Reference Figure 1-5 As shown,
[0026] Example 1
[0027] Extraction of vaginal epithelial cell membrane
[0028] Cultivation of vaginal epithelial cells VK2 / E6E7 (ATCC CRL-2616): VK / E6E7 cells were cultured in a T175 culture flask using a keratinocyte-serum-free medium (GIBCO-BRL 17005-042) containing 0.1 ng / mL human recombinant EGF, 0.05 mg / mL bovine posterior pituitary extract, and 44.1 mg / L calcium chloride until the cell confluence reached more than 80%, and the cells were digested with 0.02% EDTA solution to collect the vaginal epithelial cells. Prepare a buffer containing D-mannitol, sucrose and Tris-HCl, add it to the cells, centrifuge at 800×g for 5 minutes to wash the cells to remove impurities, add protease inhibitors and phosphatase inhibitors to the buffer after the cells are cleaned, resuspend the cells with it, homogenize with a homogenizer, centrifuge at 7600rpm for 30 minutes to remove the nucleus, organelles, etc. in the cells, and finally centrifuge at 29600rpm for 50 minutes to collect the precipitate, which is the vaginal epithelial membrane (VEM). The protein concentration is detected by the BCA protein quantification kit and stored at -80 degrees for subsequent experiments.
[0029] Example 2
[0030] Preparation and characterization of AmB@PLGA@VEM biomimetic nanoparticles
[0031] AmB@PLGA particles were prepared by nano-coprecipitation method: 50:50 viscosity, carboxyl-terminally modified PLGA (0.67 dL / g) was dissolved in acetone to 7.5 mg / mL for standby use. 0.65 mL of 4.6 mg / mL amphotericin B (AmB) in dimethyl sulfoxide (DMSO) solution was mixed with 2 mL of PLGA solution, and then 5 mL of 0.5% (w / v) polyvinyl alcohol (PVA) aqueous solution was quickly added to the mixture. After stirring to evaporate the organic solvent, the mixture was centrifuged at 13300 rpm for 30 min to remove excess AmB and DMSO, and AmB@PLGA nanoparticles were collected. The ultraviolet spectrometer determined that AmB@PLGA nanoparticles had the same characteristic peaks as AmB, indicating that AmB had been encapsulated into PLGA nanoparticles ( Figure 1 -A). Subsequently, AmB@PLGA nanoparticles were mixed with vaginal epithelial membrane (VEM) at a mass ratio of PLGA to VEM membrane protein of 2:1, and VEM was coated on the surface of AmB@PLGA nanoparticles using water bath ultrasound (120W ultrasound for 5 min) to construct AmB@PLGA@VEM biomimetic nanosystem. The prepared AmB@PLGA@VEM was subjected to basic physical characterization, including observation of the surface morphology of the nanosystem using transmission electron microscopy (TEM). Figure 1 -B) and the use of a particle size potential analyzer to determine the hydrodynamic size and surface potential of the nanosystem ( Figure 1 -C). The results showed that AmB@PLGA@VEM exhibited a core-shell structure, with a hydrodynamic size of approximately 101.8 nm and a surface potential of approximately -28.4 mV ( Figure 1 ).
[0032] Example 3
[0033] Targeting and adhesion effects of AmB@PLGA@VEM biomimetic nanosystem on Candida albicans in vitro
[0034] DiD fluorescently labeled AmB@PLGA-DiD@VEM was prepared according to the mass ratio of DiD to PLGA of 2:1000. Similarly, DiD fluorescently labeled AmB@PLGA-DiD@RBCM (RBCM is red blood cell membrane) and AmB@PLGA-DiD@F127 (F127 is lipid membrane) biomimetic nanosystems were prepared. The bacterial load of Candida albicans was adjusted to 3×10 7CFU / mL were incubated with AmB@PLGA-DiD@VEM, AmB@PLGA-DiD@RBCM and AmB@PLGA-DiD@F127 with the same lipid concentration for 30 minutes, and then washed with double distilled water to remove the nanosystems that did not bind to Candida albicans. The targeted adhesion effects of the three biomimetic nanosystems on Candida albicans were observed using scanning electron microscopy (SEM). The results showed that the AmB@PLGA-DiD@VEM nanosystem bound most to Candida albicans, the AmB@PLGA-DiD@RBCM nanosystem bound less, and the AmB@PLGA-DiD@F127 group nanosystem was almost unobservable ( Figure 2 ).
[0035] Example 4
[0036] Targeting and adhesion effects of AmB@PLGA@VEM biomimetic nanosystem on Candida albicans infection model in vivo in mice
[0037] Construction of Candida albicans infection model: The amount of Candida albicans was adjusted to 7×10 6 CFU / mL, and 100 μL was injected into the vagina of BalB / C female mice and subsequent experiments began on the third day. The same lipid dose of AmB@PLGA-DiD@VEM, AmB@PLGA-DiD@RBCM bionic nanosystem and PBS were injected as negative control groups. After 48 hours, the vaginal tissue of female mice was exposed, and the intensity of DiD fluorescence signal in each group was detected using a small animal live imaging device. The results of small animal live imaging showed that the fluorescence signal in the vaginal tissue of female mice in the AmB@PLGA-DiD@VEM nanosystem group was stronger, while the fluorescence signal of the AmB@PLGA-DiD@RBCM nanosystem group was close to that of the negative control group, indicating that the AmB@PLGA-DiD@VEM nanosystem has a targeting and enrichment effect on the vaginal area infected with Candida albicans compared with the AmB@PLGA-DiD@RBCM nanosystem ( Figure 3 ).
[0038] Example 5
[0039] Therapeutic effect of AmB@PLGA@VEM biomimetic nanosystem on Candida albicans infection mouse model
[0040] A vaginal infection model of Candida albicans in BalB / C female mice was constructed. PBS, AmB, AmB@PLGA, and AmB@PLGA@VEM bionic nanosystems (containing equal doses of AmB) were injected into the vagina of model mice, and the bacterial counts of Candida albicans in vaginal fluid and vaginal tissue homogenate were measured 24 hours later. The results showed that the effect of AmB@PLGA@VEM nanosystem in treating Candida albicans-infected mice was significantly better than that of other groups, indicating that AmB@PLGA@VEM nanosystem has a better therapeutic effect on Candida albicans infection ( Figure 4 ).
[0041] Example 6
[0042] Biocompatibility of AmB@PLGA@VEM biomimetic nanosystem
[0043] Effect of AmB@PLGA@VEM bionic nanosystem on hemolysis of erythrocytes. Fresh whole blood of mice was collected, erythrocytes were separated, and diluted to 5% (v / v) with PBS for subsequent experiments. AmB@PLGA and AmB@PLGA@VEM containing different AmB concentrations and the corresponding free AmB were added to the 5% erythrocyte suspension, incubated in a 37°C water bath for 30 minutes, centrifuged at 13300 rpm for 5 minutes, and then the absorbance at 540 nm was detected using an enzyme marker. The results showed that free AmB can cause hemolysis at 1.88 μg / mL, while AmB@PLGA and AmB@PLGA@VEM groups will not cause hemolysis even if the concentration reaches 15 μg / mL, indicating the safety of AmB@PLGA@VEM ( Figure 5 -A).
[0044] Cytotoxicity of AmB@PLGA@VEM biomimetic nanosystem on vaginal epithelial cells and macrophages. VK2 / E6E7 human vaginal epithelial cells and RAW264.7 mouse macrophages were plated in 96-well plates until the confluence reached more than 80%, and then different concentrations of AmB, AmB@PLGA and AmB@PLGA@VEM nanosystems were added (based on the concentration of AmB). The fresh culture medium group served as the negative control group. After 24 hours of culture, the cytotoxicity was detected using the Cell Counting Kit-8 (CCK-8) method. The results showed that when the AmB concentration was higher than 3.75 μg / mL, free AmB produced obvious cytotoxicity to both cells ( Figure 5 -B&C). At the same time, AmB concentrations higher than 3.75 μg / mL in AmB@PLGA also produced certain cytotoxicity to RAW264.7 mouse macrophages ( Figure 5-C). However, even at 15 μg / mL, the AmB@PLGA@VEM nanosystem did not produce any cytotoxicity to both cells, indicating that the AmB@PLGA@VEM nanosystem has good biosafety.
[0045] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.
Claims
1. A method for preparing a bionic nanosystem loaded with amphotericin B, characterized in that: The steps include: Isolation and purification of vaginal epithelial cell membranes; AmB was encapsulated into PLGA nanoparticles, AmB is amphotericin B, and PLGA is poly(lactic-co-glycolic acid); The cell membrane of vaginal epithelial cells was wrapped onto the surface of PLGA nanoparticles containing AmB; The steps of encapsulating AmB into PLGA nanoparticles are as follows: Dissolve 0.67 dL / g of PLGA with a viscosity of 50:50 and carboxyl-terminally modified in acetone to 7.5 mg / mL for later use; Mix 0.65 mL of 4.6 mg / mL AmB in DMSO with 2 mL of PLGA solution; Then, 5 mL of 0.5% w / v polyvinyl alcohol aqueous solution was quickly added to the mixture, and after stirring to volatilize the organic solvent, the mixture was centrifuged at 13300 rpm for 30 min to remove excess AmB and DMSO, and AmB@PLGA nanoparticles were collected; The steps of wrapping the cell membrane of vaginal epithelial cells onto the surface of PLGA nanoparticles containing AmB are as follows: AmB@PLGA nanoparticles were mixed with vaginal epithelial cell membranes in a mass ratio of PLGA to vaginal epithelial cell membrane protein of 2:1, and the vaginal epithelial cell membranes were coated on the surface of AmB@PLGA nanoparticles using water bath ultrasound at 120W for 5 min to construct the AmB@PLGA@VEM bionic nanosystem.
2. The preparation method according to claim 1, characterized in that: Cell membranes were isolated and purified by hypotonic and mechanical disruption methods.
3. The preparation method according to claim 1, characterized in that: Vaginal epithelial cells were also cultured prior to isolation and purification.
4. A composition loaded with amphotericin B biomimetic nanosystem, characterized in that: The composition is obtained by any one of the preparation methods of claims 1 to 3.
5. An application of the composition of the amphotericin B-loaded bionic nanosystem as claimed in claim 4, characterized in that: The composition is used for preparing nanomedicine for treating Candida albicans infection.