Milk exosome-coated plga nanometer anti-tuberculosis drug system and preparation and application thereof

Abstract

The application provides a milk exosome coated PLGA nanometer anti-tuberculosis drug system and preparation and application. The system structure is: anti-tuberculosis drug-milk exosome@PLGA-anti-tuberculosis drug. The anti-tuberculosis drug is selected from a combination of one or more of aminoquinolones, benzothiazoles, arylquinolines, nitrobenzamide and benzothiazinone anti-tuberculosis drugs. The nanometer anti-tuberculosis drug system prolongs the circulation time of nanoparticles in blood, improves the bioavailability of the drug, enhances the cell uptake efficiency of nanoparticles, improves the drug delivery effect, reduces the immune rejection reaction and potential toxicity, and improves the safety of treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to a kind of milk exosome coated PLGA nanometer antituberculous drug system and preparation and application. BACKGROUND

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The cell wall of Mycobacterium tuberculosis is known for its complexity, impermeability and hydrophobicity, thus acting as an insurmountable barrier, making Mtb naturally resistant to many antibiotics. Decaprenylphosphoryl-β-D-ribose 2' epimerase (DprE1), a flavin enzyme located in the intercellular space of the cell wall of Mtb, catalyzes the conversion of Decaprenylphosphoryl-D-ribose (DPR) to Decaprenylphosphoryl-2-Ketoribose (DPX) in Mycobacterium and Corynebacterium, which is subsequently converted to Decaprenylphosphoryl-β-D-arabinose (DPA), the only source of arabinose, a basic monomer required for the synthesis of arabinogalactan and cell wall formation. This makes DprE1 a crucial and attractive drug target. The lack of DprE1 in humans further emphasizes that this enzyme can serve as a suitable drug target. AmBeed closely follows the research and development dynamics and proactively reserves active molecular compounds required in the research and development field of hot target points. The existing DprE1 target drug under research is also the direction followed by AmBeed.

[0003] Currently, several new TB candidate drugs include aminoquinolones, benzothiazoles, quinolizolines, nitrobenzamides, benzothiazinones (BTZs) and several other categories (Chikhale, et al. 2018).

[0004] PBTZ169, also known as macozinone, is a benzothiazinone anti-tuberculosis drug, which is synthesized after structural optimization of the lead compound BTZ043. As a very promising anti-drug-resistant tuberculosis new drug, PBTZ-169 is in phase IIa clinical trials, showing great potential. However, the high lipid solubility and poor solubility of PBTZ-169 limit its clinical application. In PBS buffer at pH = 7.4, the solubility of PBTZ-169 is only 0.01 μg / mL, and in acidic conditions at pH = 2, the solubility is 0.90 mg / mL. Its solubility in DMSO is only 5 mg / mL. PBTZ-169 also does not meet Lipinski's "five rules" (ClogP = 5.06). Due to its special physicochemical properties, its drugability is poor, and the low oral bioavailability of PBTZ-169 makes the in vivo study show less efficacy than in vitro. The hepatotoxicity and gastrointestinal reactions of PBTZ-169 cannot be ignored.

[0005]

[0006] Bedaquiline (BDQ) is an anti-drug-resistant tuberculosis drug with a new mechanism, which was approved for marketing by FDA in 2012. However, BDQ has poor drugability due to its small polarity and high lipid solubility (logP = 6.37). BDQ has more serious QT interval prolongation and other adverse reactions, such as hyperuricemia, nausea, joint pain, liver damage, etc. BDQ can cause arrhythmia by prolonging the QT interval. In addition, it has also been reported that the high lipophilicity of BDQ is the cause of cardiotoxicity.

[0007]

[0008] It has been proved that milk exosomes (MEs) can survive in the strong acidic conditions in the stomach and the degradation conditions in the intestine, and their ability to cross the gastrointestinal barrier makes them a promising oral drug delivery tool.

[0009] So far, there has been no report on the use of milk exosomes (MEs) to coat PLGA-NPs (ME-PLGA NPs) to prepare oral anti-tuberculosis drugs. Based on the prior art, the present application found that the use of a specific method and technology to coat anti-tuberculosis drugs with milk exosomes (Milk exosome, ME) to prepare ME-PLGA NPs (ME-PLGA NPs) into oral anti-tuberculosis drugs has unexpected technical effects. SUMMARY

[0010] One purpose of the present application is to provide a milk exosome coated PLGA nanometer anti-tuberculosis drug system.

[0011] Another purpose of the present application is to provide a preparation method of the milk exosome coated PLGA nanometer anti-tuberculosis drug system.

[0012] Still another purpose of the present application is to provide the use of the milk exosome coated PLGA nanometer anti-tuberculosis drug system.

[0013] To achieve the above purposes, in one aspect, the present application provides a milk exosome coated PLGA nanometer anti-tuberculosis drug system, wherein the system structure is:

[0014] Anti-tuberculosis drug-milk exosome@PLGA-anti-tuberculosis drug.

[0015] According to some specific embodiments of the present application, the anti-tuberculosis drug is selected from the combination of one or more of the aminoquinolones, benzothiazoles, arylquinolines, nitrobenzamides, and benzothiazinones anti-tuberculosis drugs.

[0016] According to some specific embodiments of the present application, the anti-tuberculosis drug is selected from PBTZ169 and / or bedaquiline.

[0017] According to some specific embodiments of the present application, the anti-tuberculosis drug is selected from PBTZ169.

[0018] The PLGA molecule of the present application is a polylactic acid-glycolic acid copolymer.

[0019] According to some specific embodiments of the present application, the molecular formula of the PLGA is [C3H4O2] x [C2H2O2] y .

[0020] According to some specific embodiments of the present application, the molecular weight Mw of the PLGA is 38000-54000.

[0021] According to some specific embodiments of the present application, the ratio of lactide:glycolide in the PLGA molecule is (30:70)-(50:50).

[0022] According to some specific embodiments of the present application, the PLGA molecule is produced by Aldrich Reagent Company.

[0023] The milk exosome according to the present application is a milk exosome obtained by separation and purification from fresh milk according to the existing method, and any commercially available or commercially standard milk exosome can be used in the present application and achieve the effect of the present application, and according to some specific embodiments of the present application, wherein the diameter of the milk exosome is 30-200nm.

[0024] According to some specific embodiments of the present application, wherein the milk exosome is produced by Shanghai Nuning Company.

[0025] In another aspect, the present application also provides a preparation method of the PLGA-coated nano anti-tuberculosis drug system of the milk exosome according to the present application, wherein the method comprises using an anti-tuberculosis drug, PLGA and a milk exosome as main raw materials to prepare the PLGA-coated nano anti-tuberculosis drug system of the milk exosome.

[0026] According to some specific embodiments of the present application, wherein the method comprises the following steps:

[0027] Preparation of oil phase: dissolve the anti-tuberculosis drug in the organic solvent of PLGA as the oil phase;

[0028] Preparation of water phase: use double distilled water as the water phase for standby;

[0029] Preparation of precipitate: add the oil phase to the water phase to obtain a homogeneous emulsion, and centrifuge to remove the supernatant to obtain the precipitate;

[0030] Preparation of nano anti-tuberculosis drug: mix the milk exosome and the obtained precipitate at a mass ratio of (1:2)-(1:1), and after incubation together, obtain the PLGA-coated nano anti-tuberculosis drug system of the milk exosome.

[0031] According to some specific embodiments of the present application, wherein the organic solvent used in the preparation step of the oil phase is selected from one or more of dichloromethane, chloroform and acetone.

[0032] According to some specific embodiments of the present application, wherein,

[0033] The mass ratio of the anti-tuberculosis drug to PLGA in the preparation step of the oil phase is (1:5)-(1:10);

[0034] The mass ratio of PVA in the water phase to the anti-tuberculosis drug in the oil phase in the preparation step of the precipitate is (5:1)-(15:1).

[0035] According to some specific embodiments of the present application, wherein the preparation step of the oil phase comprises: dissolving the anti-tuberculosis drug in the organic solvent of PLGA, ultrasonic vortexing for 5-10min to obtain a mixed solution of the anti-tuberculosis drug and PLGA as the oil phase.

[0036] According to some embodiments of the present application, the frequency of the ultrasound in the preparation step of the oil phase is 50-100 Hz.

[0037] According to some embodiments of the present application, the rotation speed of the vortex in the preparation step of the oil phase is 1000-3000 rpm.

[0038] According to some embodiments of the present application, the preparation step of the water phase further comprises the use of a surfactant, comprising: adding the surfactant to the double distilled water, and obtaining a uniform solution as the water phase.

[0039] According to some embodiments of the present application, the preparation step of the water phase comprises: dissolving the surfactant in the double distilled water, swelling after heating and stirring to uniform, cooling to room temperature as the water phase for use.

[0040] According to some embodiments of the present application, the preparation step of the water phase comprises: dissolving the surfactant in the double distilled water, stirring at room temperature for 1-2 h after swelling, heating to 100-200℃ and stirring for 1.5-3 h to uniform, cooling to room temperature as the water phase for use.

[0041] According to some embodiments of the present application, the preparation step of the water phase comprises: dissolving the surfactant in the double distilled water, stirring at room temperature for 1 h after swelling, heating to 140℃ and stirring for 2 h to uniform, cooling to room temperature as the water phase for use.

[0042] According to some embodiments of the present application, the preparation step of the water phase comprises: dissolving the surfactant in the double distilled water, stirring at room temperature after swelling at a rotation speed of 200-500 rpm, heating and stirring at a rotation speed of 200-500 rpm to uniform, cooling to room temperature as the water phase for use.

[0043] According to some embodiments of the present application, the preparation step of the water phase comprises: dissolving the surfactant in the double distilled water, stirring at room temperature after swelling at a rotation speed of 260 rpm, heating and stirring at a rotation speed of 260 rpm to uniform, cooling to room temperature as the water phase for use.

[0044] According to some embodiments of the present application, the surfactant is selected from one or more of the mixtures of PVA, sodium dodecyl sulfate, poloxamer and Tween.

[0045] According to some embodiments of the present application, the poloxamer is poloxamer 188 and / or poloxamer 407.

[0046] According to some embodiments of the present application, the Tween is Tween 80.

[0047] According to some embodiments of the application, wherein the double distilled water is a Milli-Q water solution.

[0048] According to some embodiments of the application, wherein in the preparation step of the precipitate, further comprising the step of removing the organic solvent from the homogenized emulsion, and then centrifuging the remaining solution to obtain the precipitate.

[0049] According to some embodiments of the application, wherein in the preparation step of the precipitate, the method comprises removing the organic solvent by rotary evaporation or overnight stirring.

[0050] According to some embodiments of the application, wherein in the preparation step of the precipitate, the rotary evaporation is performed at 30-40°C (preferably in a water bath) at a pressure of 0.05-0.1 mPa, and at a rotation speed of 30-100 rpm.

[0051] According to some embodiments of the application, wherein in the preparation step of the precipitate, the rotary evaporation is performed at 32°C at a pressure of 0.09 mPa, and at a rotation speed of 90 rpm.

[0052] According to some embodiments of the application, wherein the overnight stirring comprises stirring overnight at room temperature to remove the organic solvent.

[0053] According to some embodiments of the application, wherein the overnight stirring comprises stirring overnight at room temperature at a rotation speed of 200-500 rpm to remove the organic solvent.

[0054] According to some embodiments of the application, wherein the overnight stirring comprises stirring overnight at room temperature using a magnetic stirrer to remove the organic solvent.

[0055] According to some embodiments of the application, wherein the overnight stirring comprises stirring overnight at a temperature of 25-30°C to remove the organic solvent.

[0056] According to some embodiments of the application, wherein in the preparation step of the precipitate, after removing the supernatant at the end of centrifugation, further comprising washing the obtained precipitate 1-3 times, and then collecting the precipitate to obtain the milk exosome-coated PLGA nanosystem of anti-tuberculosis drugs.

[0057] According to some embodiments of the application, wherein in the preparation step of the precipitate, after removing the supernatant at the end of centrifugation, further comprising adding water to the precipitate, washing the resuspension 1-3 times, and then collecting the precipitate to obtain the milk exosome-coated PLGA nanosystem of anti-tuberculosis drugs.

[0058] According to some embodiments of the application, wherein in the preparation step of the precipitate, the water added is ultrapure water.

[0059] According to some embodiments of the present application, in the preparation of the precipitation, the oil phase is added dropwise into the water phase under ice bath condition, and the homogeneous emulsion is obtained by ultrasonic emulsification during the dropwise addition, and the temperature of the solution is controlled below 20°C during the ultrasonic emulsification.

[0060] According to some embodiments of the present application, in the preparation of the precipitation, the parameters of the ultrasonic during the dropwise addition are 100-200W, 20-30kHz; preferably 130w, 20kHz.

[0061] According to some embodiments of the present application, in the preparation of the precipitation, the homogeneous emulsion is obtained by ultrasonic emulsification at 60%-90% amplitude for 10-30min during the dropwise addition.

[0062] According to some embodiments of the present application, in the preparation of the precipitation, the homogeneous emulsion is obtained by ultrasonic emulsification at 80% amplitude for 15min during the dropwise addition.

[0063] According to some embodiments of the present application, in the preparation of the precipitation, the pause is 8-20s for each 40-60s of ultrasonic until the homogeneous emulsion is obtained.

[0064] According to some embodiments of the present application, in the preparation of the precipitation, the pause is 10s for each 50s of ultrasonic until the homogeneous emulsion is obtained.

[0065] According to some embodiments of the present application, in the preparation of the precipitation, the speed of the centrifugation is 6000-10000rpm; preferably 8000-9000rpm.

[0066] According to some embodiments of the present application, in the preparation of the precipitation, the time of the centrifugation is 5-20min; preferably 10-15min.

[0067] According to some embodiments of the present application, in the preparation of the precipitation, the temperature of the solution during the centrifugation is -10°C to 20°C; preferably -6°C to 20°C; more preferably -4-20°C.

[0068] According to some embodiments of the present application, in the preparation step of the precipitation, after the washing and resuspension, the precipitate is collected by centrifugation to obtain the milk exosome coated PLGA nanosystem of antituberculosis drugs.

[0069] According to some embodiments of the present application, in the preparation step of the precipitation, after the washing and resuspension, the speed of the centrifugation is 6000-10000rpm; preferably 8000-9000rpm.

[0070] According to some embodiments of the present application, in the preparation of the precipitate, the time for centrifugation after washing and resuspension is 5-20 min; preferably 10-15 min.

[0071] According to some embodiments of the present application, in the preparation of the precipitate, the temperature of the solution during centrifugation after washing and resuspension is -10℃ to 0℃; preferably -6℃ to -2℃; more preferably -4℃.

[0072] Transmission electron microscopy images show that the precipitate obtained by the method of the present application is a uniform spherical particle (vesicle) in shape, and the particle size is uniform.

[0073] According to some embodiments of the present application, the PDI index of the precipitate particles is less than or equal to 0.3.

[0074] According to some embodiments of the present application, the encapsulation efficiency of the precipitate particles is 38%-55%, and the drug loading is 12%-20%.

[0075] According to some embodiments of the present application, the anti-tuberculosis drug is PBTZ169, and the encapsulation efficiency of the precipitate particles is 48±6.72%, and the drug loading is 16.63±1.92%.

[0076] According to some embodiments of the present application, the anti-tuberculosis drug is BDQ, and the encapsulation efficiency of the precipitate particles is 42.67±4.11%, and the drug loading is 13.76±1.32%.

[0077] According to some embodiments of the present application, the preparation steps of the nano-anti-tuberculosis drug include: mixing the milk exosome with the obtained precipitate, ultrasonic incubation, then centrifugation to discard the supernatant, to obtain the milk exosome coated PLGA nano-anti-tuberculosis drug system.

[0078] According to some embodiments of the present application, the preparation steps of the nano-anti-tuberculosis drug include: mixing the milk exosome with the obtained precipitate, ultrasonic incubation, then repeatedly extruding with a liposome extrusion instrument, then centrifuging the extruded solution and discarding the supernatant, to obtain the milk exosome coated PLGA nano-anti-tuberculosis drug system.

[0079] According to some embodiments of the present application, in the preparation steps of the nano-anti-tuberculosis drug, the milk exosome is mixed with the obtained precipitate at room temperature.

[0080] According to some embodiments of the present application, in the preparation steps of the nano-anti-tuberculosis drug, the milk exosome is mixed with the obtained precipitate in 0.9% physiological saline or PBS solution.

[0081] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the ultrasonic incubation is performed at a temperature of 25-40°C; preferably 32-35°C.

[0082] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the ultrasonic incubation is performed for 3-10 minutes; preferably 5-7 minutes.

[0083] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the extrusion by the liposome extruder is performed through a 350-500 nm polycarbonate membrane; preferably a 400-450 nm polycarbonate membrane.

[0084] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the extrusion by the liposome extruder is performed 5-10 times; preferably 8-9 times.

[0085] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the centrifugation is performed at a speed of 6000-10000 rpm; preferably 8000-9000 rpm.

[0086] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the centrifugation is performed for 5-20 minutes; preferably 10-15 minutes.

[0087] According to some specific embodiments of the present application, in the preparation of the nano-antituberculosis drug, the extrusion solution is centrifuged at a temperature of -10°C to 0°C; preferably -6°C to -2°C; more preferably -4°C.

[0088] According to some specific embodiments of the present application, the nano-antituberculosis drug system particle coated with milk exosome-PLGA obtained by the method of the present application has a particle size of 230-272 nm.

[0089] According to some specific embodiments of the present application, the anti-tuberculosis drug is PBTZ169, and the nano-antituberculosis drug system particle coated with milk exosome-PLGA obtained by the method of the present application has a particle size of 200-400 nm.

[0090] According to some specific embodiments of the present application, the anti-tuberculosis drug is PBTZ169, and the nano-antituberculosis drug system particle coated with milk exosome-PLGA obtained by the method of the present application has a particle size of 236 nm.

[0091] According to some specific embodiments of the present application, the anti-tuberculosis drug is BDQ, and the nano-antituberculosis drug system particle coated with milk exosome-PLGA obtained by the method of the present application has a particle size of 200-400 nm.

[0092] According to some specific embodiments of the present application, wherein the anti-tuberculosis drug is BDQ, the particle size of the milk exosome-coated PLGA nanometer anti-tuberculosis drug system obtained by the method of the present application is 270.82 nm.

[0093] In another aspect, the present application also provides a milk exosome-coated PLGA nanometer anti-tuberculosis drug system prepared by the preparation method of the present application.

[0094] In another aspect, the present application also provides a pharmaceutical composition containing a therapeutically effective amount of the milk exosome-coated PLGA nanometer anti-tuberculosis drug system of any of the preceding aspects of the present application, and a pharmaceutically acceptable carrier.

[0095] According to some specific embodiments of the present application, the pharmaceutical composition is a tablet, a capsule, a granule, a pill, a powder, a paste or an oral liquid.

[0096] In another aspect, the present application also provides the use of the milk exosome-coated PLGA nanometer anti-tuberculosis drug system or the pharmaceutical composition of any of the preceding aspects in the preparation of a drug for treating tuberculosis.

[0097] In summary, the present application provides a milk exosome-coated PLGA nanometer anti-tuberculosis drug system, and its preparation and application. The technical solution of the present application has the following advantages:

[0098] The present application uses ultrasonic incubation and then uses the technology of liposome membrane extrusion to coat the exosome on the PLGA nanoparticles. This method can make the size of the synthesized sample more uniform, stable and better dispersed.

[0099] In particular, the present application first uses PLGA nanoparticles to coat PBTZ169 and BDQ, which improves their solubility, and first uses milk exosome to coat PLGA-PBTZ169 and PLGA-BDQ NPs, which overcomes the inherent shortcomings of PLGA:

[0100] 1. Traditional PLGA NPs are easily recognized and removed by the reticuloendothelial system (RES) due to their hydrophobicity, resulting in a short blood circulation time. Exosomes have a lipid bilayer structure and decorated transmembrane proteins, which can avoid being recognized and removed by the RES system. This prolongs the circulation time of the nanoparticles in the blood and improves the bioavailability of the drug.

[0101] 2. The surface of traditional PLGA NPs is negatively charged, which hinders the endocytosis of cells. Milk exosomes have transmembrane proteins (such as CD9, CD63, CD81) on their surface, which can promote the endocytosis of cells. This enhances the cellular uptake efficiency of the nanoparticles and improves the delivery effect of the drug.

[0102] 3. Traditional nanoparticles can trigger immune responses. Milk exosomes are natural endogenous substances in vivo, with high biocompatibility and low immunogenicity. Reducing immune rejection and potential toxicity, improving the safety of treatment.

[0103] Traditional PLGA NPs are difficult to achieve targeted therapy due to the limitations of the material. Through specific modification of the surface of exosomes, targeted delivery of organs or tissues can be achieved. It improves the concentration of drugs in specific sites, enhances the therapeutic effect, and at the same time reduces the side effects on other non-targeted sites. And it has unique biological properties, can be easily absorbed by the gastrointestinal tract. BRIEF DESCRIPTION OF DRAWINGS

[0104] Figure 1 , A is a scanning electron microscope image of PLGA-PBTZ169 NPs, and B is a local enlarged view.

[0105] Figure 2 , A is a transmission electron microscope image of naked milk exosomes (ME) without wrapping PLGA NPs, scale bar 1 μm and 100 nm, B is a local enlarged view.

[0106] Figure 3 , A is a transmission electron microscope image of ME-PLGA-PBTZ 169 NPs after coating PLGA-NPs, scale bar 1 μm and 200 nm, B is a local enlarged view.

[0107] Figure 4 , A is a scanning electron microscope image of PLGA-BDQ NPs, B is a local enlarged view.

[0108] Figure 5 , A is a transmission electron microscope image of naked milk exosomes (ME) without wrapping PLGA NPs, scale bar 1 μm and 100 nm, B is a local enlarged view.

[0109] Figure 6 , A is a transmission electron microscope image of ME-PLGA-BDQ NPs after coating PLGA-NPs, scale bar 1 μm and 200 nm, B is a local enlarged view. DETAILED DESCRIPTION

[0110] The following will be described in detail by specific examples The implementation process and beneficial effects produced by the present application are intended to help the reader better understand the essence and characteristics of the present application, and are not limited to the scope of the present application.

[0111] Example 1

[0112] Preparation of milk exosome coated PLGA nanoparticle drug delivery system, active ingredient: PBTZ169

[0113] Preparation method as follows:

[0114] Step 1, emulsion solvent evaporation method to synthesize PLGA nanoparticles:

[0115] (1) Oil phase preparation: 50 mg of PBTZ169 drug was dissolved in 500 mg of dichloromethane solution containing PLGA (lactide: glycolide = 30:70-50:50, Mw 30000-60000) carrier material, ultrasonic vortex for 5 min, to get the dichloromethane mixed solution of PLGA and PBTZ169 with concentration of 50 mg / mL and 5 mg / mL, as oil phase.

[0116] (2) Water phase preparation: 2500 mg of PVA powder (molecular weight 20000-50000) was dissolved in 50 mL of Milli-Q water solution, and was swelled for 1 hour at room temperature with magnetic stirring (260 rpm), and then was heated at 140℃ with magnetic stirring (260 rpm) for 2 hours until uniform, and the bottle opening was wrapped with aluminum foil during heating to prevent water evaporation, and then was cooled to room temperature as water phase for use.

[0117] (3) The oil phase prepared in step (1) was added dropwise to the water phase, and in this process, the mixed solution was emulsified with a probe ultrasonic (130w, 20kHz) at 80% amplitude for 15 min, in order to avoid generating too much heat, the whole emulsification process was carried out in ice bath, ultrasonic 50s pause 10s, to get homogeneous emulsion. 32℃ 90rpm with 0.09mPa pressure water bath rotary evaporation for 45min to remove dichloromethane in the emulsion. After rotary evaporation, the emulsion was centrifuged (8000rpm, -4℃, 10min) to remove the supernatant, and then was washed with ultrapure water for three times to remove the free drug and PVA, and finally was centrifuged (8000rpm, -4℃, 10min) to collect the bottom precipitate (PLGA-PBTZ169 NPs).

[0118] Step 2, ultrasonic method-milk exosome (ME, Milk-exosome) coated PLGA-PBTZ169 NPs:

[0119] The milk exosome stored at -80°C (Shanghai Nuoning Company, milk exosome extract, 1 g / L, NW3311) was thawed at room temperature. ME and PLGA-PBTZ169 NPs (carrier solution was 0.9% physiological saline) were mixed at a mass concentration ratio of 1:1 at room temperature, and then incubated in a 32°C water bath for 5 min. Then the liposome extrusion instrument was used to extrude 9 times (through a 400 nm polycarbonate membrane), and then centrifuged (8000 rpm, -4°C, 10 min) to discard the supernatant to remove the uncoated ME, i.e. to obtain ME-PLGA-PBTZ169 NPs coated with PLGA-PBTZ169 NPs.

[0120] Structural characterization

[0121] The synthesized PLGA-PBTZ169 NPs were characterized as follows:

[0122] Transmission electron microscopy (TEM): The transmission electron microscopy image represents the morphology and nanosize of the PLGA-PBTZ169 NPs, Figure 1 Figure A is a scanning electron microscopy image of the PLGA-PBTZ169 NPs, which can be seen to be uniform in size and uniform spherical particles.

[0123] Figure 1 Figure A is a scanning electron microscopy image of the PLGA-PBTZ169 NPs, which can be seen to be uniform in size and uniform spherical particles.

[0124] Nano Brook 90Plus PALS nanoparticle size analyzer / zeta potential detector (company: Brookhaven Instruments Serial NO: 250385) was used to determine the hydrated particle size distribution and zeta potential value of the PLGA-blank NPs (PLGA nanoparticles without loading antituberculosis drugs) and PLGA-PBTZ169 NPs.

[0125] Table 1A is the particle size and PDI index determination of the PLGA-blank NPs and PLGA-PBTZ169 NPs, wherein the PDI index is less than 0.3, indicating that the distribution is relatively uniform.

[0126] Table 1A, particle size, PDI index and zeta potential of PLGA-blank NPs and PLGA-PBTZ169 NPs, n = 3

[0127]

[0128] To quantify how much PBTZ169 drug was encapsulated into the PLGA core, the content of PBTZ169 in PLGA-PBTZ169 NPs was directly determined by UV spectrophotometry. First, the standard curve of PBTZ169 in DMSO solution was determined as y = 12.401x + 0.0032, R 2 = 1.

[0129] The encapsulation efficiency of PLGA-PBTZ169 NPs was determined to be 48 ± 6.72% and the drug loading was 16.63 ± 1.92% by the standard curve. As shown in Table 1B.

[0130] Table 1B, encapsulation efficiency and drug loading of PLGA-PBTZ169 NPs, n = 3

[0131] Name Encapsulation efficiency Drug loading PLGA-PBTZ-169 NPs 48±6.72％ 16.63±1.92％

[0132] To determine whether the hydrophilicity of PBTZ169 drug increases after being encapsulated into the PLGA core, the contact angles of the raw drug PBTZ169 and PLGA-PBTZ169 NPs were determined. (θ < 90° is hydrophilic, θ > 90° is hydrophobic) It can be seen that the hydrophilicity of PLGA-PBTZ169 NPs increases compared with the raw drug PBTZ169 (see Table 1C). Table 1C, contact angle determination of raw drug PBTZ169, PLGA-blank NPs, PLGA-PBTZ169 NPs, n = 3

[0133] Name Contact angle (°) PLGA-Blank NPs 73.34±3.74 PLGA-PBTZ169 NPs 61.92±1.48 PBTZ169 97.35±2.25

[0134] Characterization of milk exosomes (ME) synthesized in this example and its coated ME-PLGA-PBTZ169 NPs:

[0135] (1) Transmission electron microscopy (TEM): TEM images represent the morphology and nanosize of ME and ME-PLGA-PBTZ169 NPs, Figure 2 and Figure 3 Scanning electron micrographs of ME and ME-PLGA-PBTZ169 NPs, respectively, can be seen uniform size, uniform shape vesicles.

[0136] Figure 2 Transmission electron micrograph of naked milk exosomes (ME) without PLGA NPs coating, scale bar is 1 μm and 100 nm.

[0137] Figure 3 Transmission electron micrograph of ME-PLGA-PBTZ169 NPs after coating with PLGA-NPs, scale bar is 1 μm and 200 nm.

[0138] (2) Nano Brook 90Plus PALS Nanoparticle Size Analyzer / Zeta Potential Detector (Company: Brookhaven Instruments Serial NO: 250385) was used to determine the hydrated particle size distribution and zeta potential value of ME, ME-PLGA-Blank NPs, and ME-PLGA-PBTZ169 NPs. The results are shown in Table 1D.

[0139] Table 1D, Particle size and PDI index of ME, ME-PLGA-Blank NPs, and ME-PLGA-PBTZ169 NPs, n = 3

[0140]

[0141] Figure 2 、 Figure 3 and Table 1D shows that PLGA-NPs are coated in ME, and the particle size of ME-PLGA-PBTZ169 NPs is about 236 nm, which is much larger than that of bare ME, which is only about 111 nm.

[0142] Cytotoxicity test

[0143] MTT method was used to detect the toxic effect of PBTZ169 raw material, PLGA-Blank NPs carrier, PLGA-PBTZ169 NPs, ME-PLGA-Blank NPs carrier, and ME-PLGA-PBTZ169 NPs on J774A.1 macrophages. The specific results are shown in Table 1E.

[0144] As shown in Table 1E, the IC 50 value of ME-PLGA-PBTZ169 NPs with the same concentration of raw drug PBTZ169 is lower than that of the raw drug, in addition, PLGA-Blank NPs carrier and ME-PLGA-Blank NPs carrier have strong safety and biocompatibility.

[0145] Table 1E, IC50 determination results of raw drug PBTZ169, PLGA-Blank NPs carrier, PLGA-PBTZ169 NPs, ME-PLGA-Blank NPs carrier, and ME-PLGA-PBTZ169 NPs on J774A.1 cells

[0146]

[0147]

[0148] In vitro antibacterial activity determination

[0149] The MIC of PBTZ169, PLGA-blank NPs carrier, PLGA-PBTZ169 NPs, ME-PLGA-blank NPs carrier, ME-PLGA-PBTZ169 NPs against M. tuberculosis standard strain H37Rv was determined using MABA method (see Table 1F for specific data).

[0150] Table 1F: In vitro minimum inhibitory concentration of PBTZ169, PLGA-PBTZ169 NPs, ME-PLGA-PBTZ169 NPs and two blank carrier materials against M. tuberculosis standard strain H37Rv

[0151] Sample MIC (pg / mL) PBTZ169 0.00007 PLGA-PBTZ169 NPs 0.00014 ME-PLGA-PBTZ169 NPs 0.00015 PLGA-Blank NPs carrier >1.56 ME-PLGA-Blank NPs carrier >1.56

[0152] Table 1F data showed that the two blank carrier materials did not show significant antibacterial activity, and the antibacterial activity of PBTZ169 loaded by the carrier in vitro did not show significant difference from the raw drug.

[0153] In vivo PK experiment:

[0154] BALB / c mice were randomly divided into 3 groups, PBTZ169, PLGA-PBTZ169 NPs, ME-PLGA-PBTZ169 NPs, containing PBTZ169 dose of 10 mg / kg, oral gavage administration, 18 in each group, using LC-MS to determine the drug concentration distribution of PBTZ169 in blood and lung and spleen of three groups of mice at different time points.

[0155] The results are shown in Table 1G and Table 1H, Table 1I as follows:

[0156] Table 1G: Pharmacokinetic parameters of PBTZ169 in lung tissue after oral administration of the same dose of different dosage forms of PBTZ169 in mice (Mean ± SD, n = 3)

[0157]

[0158] Table 1H: Pharmacokinetic parameters of PBTZ169 in blood after oral administration of the same dose of different dosage forms of PBTZ169 in mice (Mean ± SD, n = 3)

[0159]

[0160] Table 1I: Pharmacokinetic parameters of PBTZ169 in spleen tissue after oral administration of the same dose of different dosage forms of PBTZ169 in mice (Mean ± SD, n = 3)

[0161]

[0162] From the above results, it can be seen that the bioavailability of mice administered with the new dosage form is significantly increased by more than three times in blood, lungs and spleen, and the peak concentration is also significantly increased by more than three times.

[0163] Example 2:

[0164] Preparation of milk exosome coated PLGA nanodrug delivery system, active ingredient: BDQ

[0165] The preparation method is as follows:

[0166] Step one, emulsion solvent evaporation method to synthesize PLGA nanoparticles:

[0167] (1) Preparation of oil phase: 50 mg of BDQ drug was dissolved in 500 mg of dichloromethane solution containing PLGA (lactide: glycolide = 30:70-50:50, Mw 30000-60000) carrier material, ultrasonic vortex for 5 min, to get a dichloromethane mixed solution with PLGA and BDQ concentration of 50 mg / mL and 5 mg / mL respectively, as oil phase.

[0168] (2) Preparation of water phase: 2500 mg of PVA (molecular weight 20000-50000) powder was dissolved in 50 mL of Milli-Q water solution, and after swelling for 1 hour at room temperature with magnetic stirring (260 rpm), it was heated at 140℃ with magnetic stirring (260 rpm) for 2 hours to be uniform. During heating, the bottle opening was wrapped with aluminum foil to prevent water evaporation. After cooling to room temperature, it was used as water phase.

[0169] (3) The oil phase prepared in step (1) was added dropwise to the water phase prepared in step (2), and in this process, the mixed solution was emulsified with a probe ultrasonic (130w, 20kHz) at 80% amplitude for 15min. To avoid generating too much heat, the whole emulsification process was carried out in an ice bath, and the ultrasonic was stopped for 10s after 50s. To obtain a homogeneous emulsion. 32℃ 90rpm with 0.09mPa pressure water bath rotary evaporation for 45min to remove dichloromethane in the emulsion. After rotary evaporation, the emulsion was centrifuged (8000rpm, -4℃, 10min) to remove the supernatant, and then washed with ultrapure water and resuspended three times to remove the free drug and PVA that was not coated. Finally, centrifugation (8000rpm, -4℃, 10min) was performed to collect the bottom precipitate.

[0170] Step two, ultrasonic method-milk exosome (ME, Milk-exosome) coated PLGA-BDQ NPs:

[0171] The bovine milk exosomes stored at -80°C were thawed at room temperature. The ME and PLGA-BDQ NPs (carrier solution was 0.9% normal saline) were mixed at a mass concentration ratio of 1:1 at 25°C, and then incubated in a 32°C water bath for 5 min. Then the liposome extrusion instrument was used to extrude 9 times. (Through a 400 nm polycarbonate membrane), and then centrifuged (8000 rpm, -4°C, 10 min) to discard the supernatant to remove the uncoated ME, and the ME-PLGA-BDQ NPs coated with PLGA-BDQ NPs were obtained.

[0172] Structural characterization

[0173] The synthesized PLGA-BDQ NPs were characterized as follows:

[0174] Transmission electron microscopy (TEM): The transmission electron microscopy images represent the morphology and nanosize of the PLGA-BDQ NPs, Figure 4 Figure 1A is a scanning electron microscopy image of the PLGA-BDQ NPs, which shows uniform size and uniform spherical particles.

[0175] Figure 4 Figure 1A is a scanning electron microscopy image of the PLGA-BDQ NPs, which shows uniform size and uniform spherical particles.

[0176] The Nano Brook 90Plus PALS nanoparticle size analyzer / zeta potential detector (company: Brookhaven Instruments Serial NO: 250385) was used to determine the hydrated particle size distribution and zeta potential value of the PLGA-blank NPs and PLGA-BDQ NPs.

[0177] Table 2A is the particle size and PDI index determination of the PLGA-blank NPs and PLGA-BDQ NPs, wherein the PDI index is less than 0.3, indicating that the distribution is relatively uniform.

[0178] Table 2A, the particle size, PDI index and zeta potential of the PLGA-blank NPs and PLGA-BDQ NPs, n = 3

[0179]

[0180] To quantify how much BDQ drug is encapsulated into the PLGA core, the BDQ content in the PLGA-BDQ NPs was directly determined by ultraviolet spectrophotometry. First, the standard curve of BDQ in DMSO solution was determined as y = 15.59x + 0.06516, R 2 = 0.9938.

[0181] The encapsulation efficiency of PLGA-BDQ NPs was 42.67 ± 4.11% and the drug loading was 13.76 ± 1.32% as determined by the standard curve. As shown in Table 2B.

[0182] Table 2B, encapsulation efficiency and drug loading of PLGA-BDQ NPs, n = 3

[0183] Name Encapsulation efficiency Drug loading PLGA-BDQ NPs 42.67±4.11％ 13.76±1.32％

[0184] To determine whether the hydrophilicity of the BDQ drug increases after being encapsulated into the PLGA core, the contact angles of the raw drug BDQ and the PLGA-BDQ NPs were determined. (θ < 90° is hydrophilic, θ > 90° is hydrophobic) It can be seen that the hydrophilicity of PLGA-BDQ NPs increases compared with the raw drug BDQ (see Table 2C).

[0185] Table 2C, contact angle determination of raw drug BDQ, PLGA-blank NPs, PLGA-BDQ NPs, n = 3

[0186] Name Contact angle (°) PLGA-Blank NPs 73.34±3.74 PLGA-BDQ NPs 65.33±3.18 BDQ 95.39±0.42

[0187] Characterization of milk exosomes (ME) synthesized in this example and its coated ME-PLGA-BDQ NPs:

[0188] (1) Transmission electron microscopy (TEM): The transmission electron microscopy images represent the morphology and nanosize of ME and ME-PLGA-BDQ NPs, Figure 5 and Figure 6 are the scanning electron microscopy images of ME and ME-PLGA-BDQ NPs, respectively, which can be seen that the size is uniform and the shape of the vesicles is uniform.

[0189] Figure 5 is the transmission electron microscopy image of naked milk exosomes (ME) without PLGA NPs, scale bar is 1 μm and 100 nm.

[0190] Figure 6 is the transmission electron microscopy image of ME-PLGA-BDQ NPs after coating PLGA-NPs, scale bar is 1 μm and 200 nm.

[0191] (2) Nano Brook 90Plus PALS nanoparticle size analyzer / zeta potential detector (company: Brookhaven Instruments Serial NO: 250385) was used to determine the hydrated particle size distribution and zeta potential value of ME, ME-PLGA-blank NPs, ME-PLGA-BDQ NPs. The results are shown in Table 2D.

[0192] Table 2D, ME, ME-PLGA-Blank NPs, ME-PLGA-BDQ NPs particle size and PDI index, n = 3

[0193]

[0194] Figure 5 、 Figure 6 Table 2D shows that the PLGA-NPs coated in ME, the particle size of ME-PLGA-BDQ NPs is about 270.82 nm, which is much larger than the naked ME, and its particle size is only about 111 nm.

[0195] Cytotoxicity test

[0196] The MTT method was used to detect the toxic effect of BDQ raw material, PLGA-Blank NPs carrier, PLGA-BDQ NPs, ME-PLGA-Blank NPs carrier, and ME-PLGA-BDQ NPs on J774A.1 macrophages. The specific results are shown in Table 2E.

[0197] As can be seen from the results in Table 2E, under the condition of containing the same concentration of raw drug BDQ, the IC 50 of PLGA-BDQ NPs is lower than that of the raw drug, and the IC of ME-PLGA-BDQ NPs is equivalent to that of the raw drug. In addition, the PLGA-Blank NPs carrier and the ME-PLGA-Blank NPs carrier have strong safety and biocompatibility.

[0198] Table 2E, raw drug BDQ, PLGA-Blank NPs carrier, PLGA-BDQ NPs, ME-PLGA-Blank NPs carrier, and ME-PLGA-BDQ NPs IC 50 determination results of J774A.1 cells

[0199]

[0200]

[0201] In vitro antibacterial activity determination

[0202] The MABA method was used to determine the MIC of raw drug BDQ, PLGA-Blank NPs carrier, PLGA-BDQ NPs, ME-PLGA-Blank NPs carrier, and ME-PLGA-BDQ NPs on Mycobacterium tuberculosis standard strain H37Rv. (The specific data are shown in Table 2F)

[0203] Table 2F, BDQ, PLGA-BDQ NPs, ME-PLGA-BDQ NPs, and two blank carrier materials in vitro minimum inhibitory concentration of Mycobacterium tuberculosis standard strain H37Rv

[0204] Sample MIC (pg / mL) BDQ 0.049 PLGA-BDQ NPs 0.007 ME-PLGA-BDQ NPs 0.008 PLGA-Blank NPs carrier >10 ME-PLGA-Blank NPs carrier >10

[0205] Table 2F data shows that two blank carrier materials do not exhibit significant antibacterial activity, and BDQ loaded by the carrier exhibits better antibacterial activity in vitro than the raw drug.

Claims

1. An oral milk exosome coated PLGA nanotuberculosis drug system, wherein, The system structure is: The anti-tuberculosis drug is a combination of one or more selected from the group consisting of aryl quinoline and benzothiazinone anti-tuberculosis drugs; The PLGA has a molecular formula of [C3H4O2] x [C2H2O2] y and a molecular mass Mw of 38000-54000; The anti-tuberculosis drug is selected from PBTZ169 and / or bedaquiline; The preparation method of the milk exosome-coated PLGA nanometer anti-tuberculosis drug system comprises using an anti-tuberculosis drug, PLGA and milk exosome as main raw materials to prepare the milk exosome-coated PLGA nanometer anti-tuberculosis drug system, and comprises the following steps: Preparation of the oil phase: dissolving the anti-tuberculosis drug in the organic solvent of PLGA as the oil phase; Preparation of the water phase: using double-distilled water as the water phase for standby; Preparation of the precipitate: dropping the oil phase into the water phase to obtain a homogeneous emulsion, and removing the supernatant by centrifugation to obtain the precipitate; Preparation of the nanometer anti-tuberculosis drug: mixing the milk exosome and the obtained precipitate at a mass ratio of (1:2)-(1:1), and incubating together to obtain the milk exosome-coated PLGA nanometer anti-tuberculosis drug system.

2. The milk exosome coated PLGA nanocarrier antitubercular drug system as claimed in claim 1, wherein, The PLGA molecule is composed of 30-50% lactide and 70-50% glycolide.

3. The method of claim 1 or 2, wherein the preparation of the PLGA-coated nanosize anti-tuberculosis drug system of the milk exosome comprises the steps of: The preparation method of the milk exosome-coated PLGA nanometer anti-tuberculosis drug system comprises using an anti-tuberculosis drug, PLGA and milk exosome as main raw materials to prepare the milk exosome-coated PLGA nanometer anti-tuberculosis drug system; and comprises the following steps: Preparation of the oil phase: dissolving the anti-tuberculosis drug in the organic solvent of PLGA as the oil phase; Preparation of the water phase: using double-distilled water as the water phase for standby; Preparation of the precipitate: dropping the oil phase into the water phase to obtain a homogeneous emulsion, and removing the supernatant by centrifugation to obtain the precipitate; Preparation of the nanometer anti-tuberculosis drug: mixing the milk exosome and the obtained precipitate at a mass ratio of (1:2)-(1:1), and incubating together to obtain the milk exosome-coated PLGA nanometer anti-tuberculosis drug system.

4. The production method according to claim 3, wherein The preparation step of the water phase comprises adding a surfactant into double-distilled water to obtain a uniform solution as the water phase.

5. The production method according to claim 4, wherein The surfactant is PVA.

6. The preparation method according to claim 3, wherein, The mass ratio of the anti-tuberculosis drug to PLGA in the preparation step of the oil phase is (1:5)-(1:10); The mass ratio of PVA in the water phase to the anti-tuberculosis drug in the oil phase in the preparation step of the precipitate is (5:1)-(15:1).

7. The preparation method according to claim 3, wherein, In the preparation step of the precipitate, the step of removing the organic solvent from the homogeneous emulsion is further included before centrifugation to obtain the precipitate.

8. The production method according to claim 7, wherein In the preparation step of the precipitate, the step of removing the organic solvent comprises rotary evaporation.

9. The preparation method according to claim 3, wherein, In the preparation step of the precipitate, after removing the supernatant at the end of centrifugation, the obtained precipitate is further washed for 1-3 times, and then the precipitate is collected to obtain the milk exosome-coated PLGA nanometer anti-tuberculosis drug system.

10. The preparation method according to claim 3, wherein, In the preparation of the precipitate, the oil phase is dropped into the water phase under ice bath condition, and the homogeneous emulsion is obtained by ultrasonic emulsification during the dropping process, and the temperature of the solution is controlled below 20℃ during the whole ultrasonic emulsification process.

11. The preparation method of any one of claims 3-10, wherein the preparation of the nano-antituberculosis drug comprises: mixing the milk exosome with the obtained precipitate, and then ultrasonically incubating together, and then centrifuging, and discarding the supernatant to obtain the milk exosome-coated PLGA nano-antituberculosis drug system. The preparation of the nano-antituberculosis drug comprises: mixing the milk exosome with the obtained precipitate, and then ultrasonically incubating together, and then repeatedly extruding with a liposome extrusion instrument, and then centrifuging the extruded solution, and discarding the supernatant to obtain the milk exosome-coated PLGA nano-antituberculosis drug system. The ultrasonic incubation is performed at a temperature of 25-40℃.

12. The method of making according to claim 11, wherein, The ultrasonic incubation is performed for 3-10 min.

13. The method of making according to claim 11, wherein, 14. The milk exosome-coated PLGA nano-antituberculosis drug system prepared by the preparation method of any one of claims 3-13.

15. A pharmaceutical composition comprising a therapeutically effective amount of the milk exosome-coated PLGA nano-antituberculosis drug system of any one of claims 1-2 or 14, and a pharmaceutically acceptable carrier.

16. Use of the milk exosome-coated PLGA nano-antituberculosis drug system of any one of claims 1-2 or 14 or the pharmaceutical composition of claim 15 in the preparation of a drug for treating tuberculosis. ​

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