Milk exosome-coated PLGA (poly (lactic-co-glycolic acid)) nano antituberculosis drug system as well as preparation and application thereof

Abstract

The invention provides a milk exosome coated PLGA (poly (lactic-co-glycolic acid)) nano antituberculosis drug system as well as preparation and application thereof. The structure of the system is as follows: anti-tuberculosis drug-milk exosome-coated PLGA-anti-tuberculosis drug. The antituberculous drug is selected from one or a combination of more of aminoquinolones, benzothiazoles, aryl quinoline, nitrobenzamides and benzothiazinone antituberculous drugs. According to the nano antituberculosis drug system, the circulation time of nano particles in blood is prolonged, and the bioavailability of the drug is improved. The cellular uptake efficiency of the nanoparticles is enhanced, and the drug delivery effect is improved. The immunological rejection and the potential toxicity are reduced, and the treatment safety is improved.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a milk exosome-coated PLGA nano anti-tuberculosis drug system and its preparation and application. Background Art

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The cell wall of M. tuberculosis is known for its complexity, impermeability and hydrophobicity, thus acting as an insurmountable barrier that makes Mtb naturally resistant to many antibiotics. The key enzyme in cell wall synthesis (Decaprenylphosphoryl-β-D-ribose 2′epimerase, DprE1) is a flavoenzyme located in the intercellular compartment of the Mtb cell wall. In mycobacteria and Corynebacterium, DprE1 catalyzes the conversion of Decaprenylphosphoryl-D-ribose (DPR) to Decaprenylphosphoryl-2-Ketoribose (DPX), which is subsequently converted to Decaprenylphosphoryl-β-D-arabinose (DPA), which is the sole source of arabinose and a basic monomer required for the synthesis of arabinogalactose and cell wall formation. This makes DprE1 a crucial and attractive drug target. The lack of DprE1 in the human body further emphasizes that this enzyme can be a suitable drug target. AmBeed closely follows the development of research and development, and actively reserves active molecular compounds required in the field of hot target research and development. Existing DprE1 target drugs under development are also the direction followed by AmBeed.

[0003] Currently, there are several new candidate drugs for tuberculosis, including aminoquinolones, benzothiazoles, quinolines, nitrobenzamides, and benzothiazinones (BTZs) (Chikhale, et al. 2018).

[0004] PBTZ169, also known as macozinone, is a benzothiazinone anti-tuberculosis drug, which was synthesized by optimizing the structure with BTZ043 as the lead compound. As a very promising new drug for drug-resistant tuberculosis, PBTZ-169 is in Phase IIa clinical trials and has shown 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 under acidic conditions of pH = 2, the solubility is 0.90 mg / mL. Its solubility in DMSO is also only 5 mg / mL. PBTZ-169 also does not meet Lipinski's "rule of five" (ClogP = 5.06). Due to its special physicochemical properties, its drugability is poor, and the oral bioavailability of PBTZ-169 is low, making the efficacy in in vivo studies inferior to that 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. It was approved by the FDA in 2012. However, BDQ has low polarity, high lipid solubility (logP=6.37), and poor drugability. BDQ has more serious QT interval prolongation and other adverse reactions, such as hyperuricemia, nausea, joint pain, liver damage, etc. BDQ may cause arrhythmias by prolonging the QT interval. In addition, there are also reports that the high lipophilicity of BDQ is the cause of cardiac toxicity.

[0007]

[0008] Milk exosomes (MEs) have been shown to survive the highly 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 oral anti-tuberculosis drugs prepared by using milk exosomes (Milkexosome, ME) to coat PLGA-NPs (ME-PLGANPs). Based on the prior art, the present invention has found through research that the oral anti-tuberculosis drugs prepared by using milk exosomes (Milkexosome, ME) to coat PLGA-NPs (ME-PLGANPs) for anti-tuberculosis drugs by using specific methods and techniques have unexpected technical effects. Summary of the invention

[0010] One object of the present invention is to provide a nano anti-tuberculosis drug system of milk exosomes coated with PLGA;

[0011] Another object of the present invention is to provide a method for preparing the milk exosome-coated PLGA nano anti-tuberculosis drug system;

[0012] Another object of the present invention is to provide the use of the milk exosome-coated PLGA nano anti-tuberculosis drug system.

[0013] To achieve the above-mentioned purpose, on the one hand, the present invention provides a milk exosome-coated PLGA nano anti-tuberculosis drug system, wherein the system structure is:

[0014] Anti-tuberculosis drugs-milk exosomes@PLGA-anti-tuberculosis drugs.

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

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

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

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

[0019] According to some specific embodiments of the present invention, the PLGA molecular formula is [C 3 H 4 O 2 ] x [C 2 H 2 O 2 ] y .

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

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

[0022] According to some specific embodiments of the present invention, the PLGA molecule is produced by Aladdin Reagents Company.

[0023] The milk exosomes described in the present invention are milk exosomes obtained by separation and purification from fresh milk according to existing methods. Any commercially available milk exosomes or milk exosomes that meet commercial standards can be used in the present invention and achieve the effects of the present invention. According to some specific embodiments of the present invention, the diameter of the milk exosomes is 30-200nm.

[0024] According to some specific embodiments of the present invention, the milk exosomes are produced by Shanghai Noning Company.

[0025] On the other hand, the present invention also provides a method for preparing the nano anti-tuberculosis drug system of milk exosomes coated with PLGA according to the present invention, wherein the method comprises using anti-tuberculosis drugs, PLGA and milk exosomes as main raw materials to prepare the nano anti-tuberculosis drug system of milk exosomes coated with PLGA

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

[0027] Preparation of oil phase: dissolving anti-tuberculosis drugs in an organic solvent of PLGA as the oil phase;

[0028] Preparation of aqueous phase: Double distilled water is used as the aqueous phase for later use;

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

[0030] Preparation of nano anti-tuberculosis drugs: milk exosomes and the obtained precipitate are mixed in a mass ratio of (1:2)-(1:1), and after co-incubation, the milk exosome-coated PLGA nano anti-tuberculosis drug system is obtained.

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

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

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

[0034] In the preparation step of the precipitation, the mass ratio of PVA in the aqueous phase to the anti-tuberculosis drug in the oil phase is (5:1)-(15:1).

[0035] According to some specific embodiments of the present invention, the step of preparing the oil phase includes: dissolving the anti-tuberculosis drug in an organic solvent of PLGA, and ultrasonically vortexing for 5-10 minutes to obtain a mixed solution of the anti-tuberculosis drug and PLGA as the oil phase.

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

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

[0038] According to some specific embodiments of the present invention, the step of preparing the aqueous phase further includes the use of a surfactant, including: adding the surfactant into double distilled water to obtain a uniform solution as the aqueous phase.

[0039] According to some specific embodiments of the present invention, the step of preparing the aqueous phase includes: dissolving a surfactant in double distilled water, heating and stirring until uniform after swelling, and cooling to room temperature for use as the aqueous phase.

[0040] According to some specific embodiments of the present invention, the preparation step of the aqueous phase includes: dissolving the surfactant in double distilled water, stirring and swelling at room temperature for 1-2 hours, heating to 100-200°C and stirring for 1.5-3 hours until uniform, and cooling to room temperature as the aqueous phase for standby use.

[0041] According to some specific embodiments of the present invention, the step of preparing the aqueous phase includes: dissolving the surfactant in double distilled water, stirring and swelling at room temperature for 1 hour, heating to 140°C and stirring for 2 hours until uniform, and cooling to room temperature as the aqueous phase for standby use.

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

[0043] According to some specific embodiments of the present invention, the preparation step of the aqueous phase includes: dissolving the surfactant in double distilled water, stirring and swelling at a speed of 260 rpm at room temperature, heating and stirring at a speed of 260 rpm until uniform, and cooling to room temperature as the aqueous phase for standby use.

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

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

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

[0047] According to some specific embodiments of the present invention, the double distilled water is a Milli-Q aqueous solution.

[0048] According to some specific embodiments of the present invention, the step of preparing the precipitate further includes a step of removing the organic solvent from the homogenized emulsion, and then centrifuging the remaining solution to obtain the precipitate.

[0049] According to some specific embodiments of the present invention, the method, in the step of preparing the precipitate, comprises removing the organic solvent by rotary evaporation or overnight stirring.

[0050] According to some specific embodiments of the present invention, in the step of preparing the precipitate, the rotary evaporation is carried out 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 specific embodiments of the present invention, in the step of preparing the precipitate, the rotary evaporation is carried out at 32° C., under a pressure of 0.09 mPa, and at a rotation speed of 90 rpm.

[0052] According to some specific embodiments of the present invention, the overnight stirring includes stirring at room temperature overnight to remove the organic solvent.

[0053] According to some specific embodiments of the present invention, the overnight stirring includes stirring at 200-500 rpm at room temperature overnight to remove the organic solvent.

[0054] According to some specific embodiments of the present invention, the overnight stirring includes stirring overnight with a magnetic stirrer at room temperature to remove the organic solvent.

[0055] According to some specific embodiments of the present invention, the overnight stirring comprises stirring at 25° C.-30° C. overnight to remove the organic solvent.

[0056] According to some specific embodiments of the present invention, in the step of preparing the precipitate, after the centrifugation is completed and the supernatant is removed, the precipitate is washed 1 to 3 times, and then the precipitate is collected to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system.

[0057] According to some specific embodiments of the present invention, in the step of preparing the precipitate, after the centrifugation is completed and the supernatant is removed, water is added to the precipitate, washing and resuspending it 1 to 3 times, and then the precipitate is collected to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system.

[0058] According to some specific embodiments of the present invention, in the step of preparing the precipitate, the water added is ultrapure water.

[0059] According to some specific embodiments of the present invention, in the preparation of the precipitate, the oil phase is added dropwise to the water phase under ice bath conditions, and ultrasonic emulsification is performed during the dropping process to obtain a homogeneous emulsion. During the ultrasonic emulsification process, the temperature of the solution is controlled to be lower than 20°C throughout the process.

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

[0061] According to some specific embodiments of the present invention, in the preparation of the precipitate, ultrasonic emulsification is performed at an amplitude of 60%-90% for 10-30 minutes during the dropwise addition process to obtain a homogeneous emulsion.

[0062] According to some specific embodiments of the present invention, in the preparation of the precipitate, ultrasonic emulsification is performed at an amplitude of 80% for 15 minutes during the dropwise addition process to obtain a homogeneous emulsion.

[0063] According to some specific embodiments of the present invention, in the preparation of the precipitate, the ultrasonic wave is paused for 8-20 seconds every 40-60 seconds until a homogeneous emulsion is obtained.

[0064] According to some specific embodiments of the present invention, in the preparation of the precipitate, the ultrasonic treatment is performed for 50 seconds and then paused for 10 seconds until a homogeneous emulsion is obtained.

[0065] According to some specific embodiments of the present invention, in the preparation of the precipitate, the centrifugal rotation speed is 6000-10000 rpm; preferably 8000-9000 rpm.

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

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

[0068] According to some specific embodiments of the present invention, in the step of preparing the precipitate, after washing and resuspension, the precipitate is collected by centrifugation to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system.

[0069] According to some specific embodiments of the present invention, in the step of preparing the precipitate, the rotation speed of the centrifuge after washing and resuspension is 6000-10000 rpm; preferably 8000-9000 rpm.

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

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

[0072] Transmission electron microscope images show that the precipitate obtained by the method of the present invention is spherical particles (vesicles) with uniform shapes and uniform particle sizes.

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

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

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

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

[0077] According to some specific embodiments of the present invention, the preparation step of the nano anti-tuberculosis drug includes: mixing the milk exosomes with the obtained precipitate, incubating them together by ultrasound, and then centrifuging and discarding the supernatant to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system.

[0078] According to some specific embodiments of the present invention, the preparation steps of the nano anti-tuberculosis drug include: mixing the milk exosomes with the obtained precipitate, incubating them together by ultrasound, and then repeatedly extruding them using a liposome extruder, and 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 specific embodiments of the present invention, in the step of preparing the nano anti-tuberculosis drug, the milk exosomes are mixed with the obtained precipitate at room temperature.

[0080] According to some specific embodiments of the present invention, in the step of preparing the nano anti-tuberculosis drug, the milk exosomes are mixed with the obtained precipitate in 0.9% saline or PBS solution.

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

[0082] According to some specific embodiments of the present invention, in the preparation step of the nano anti-tuberculosis drug, the time of ultrasonic co-incubation is 3-10 min, preferably 5-7 min.

[0083] According to some specific embodiments of the present invention, in the step of preparing the nano anti-tuberculosis drug, extrusion using a liposome extruder is through a 350-500nm polycarbonate membrane; preferably a 400-450nm polycarbonate membrane.

[0084] According to some specific embodiments of the present invention, in the step of preparing the nano anti-tuberculosis drug, the number of extrusions using a liposome extruder is 5-10 times, preferably 8-9 times.

[0085] According to some specific embodiments of the present invention, in the step of preparing the nano anti-tuberculosis drug, the centrifugal rotation speed is 6000-10000 rpm; preferably 8000-9000 rpm.

[0086] According to some specific embodiments of the present invention, in the preparation step of the nano anti-tuberculosis drug, the centrifugation time is 5-20 min, preferably 10-15 min.

[0087] According to some specific embodiments of the present invention, in the preparation step of the nano anti-tuberculosis drug, the temperature of the extruded solution during centrifugation is -10°C to 0°C; preferably -6°C to -2°C; more preferably -4°C.

[0088] According to some specific embodiments of the present invention, the particle size of the milk exosome-coated PLGA nano anti-tuberculosis drug system particles obtained by the method of the present invention is 230-272 nm.

[0089] According to some specific embodiments of the present invention, wherein the anti-tuberculosis drug is PBTZ169, the particle size of the nano anti-tuberculosis drug system particles of milk exosomes coated with PLGA obtained by the method of the present invention is 200-400 nm.

[0090] According to some specific embodiments of the present invention, wherein the anti-tuberculosis drug is PBTZ169, the particle size of the nano anti-tuberculosis drug system particles of milk exosomes coated with PLGA obtained by the method of the present invention is 236 nm.

[0091] According to some specific embodiments of the present invention, wherein the anti-tuberculosis drug is BDQ, the particle size of the nano anti-tuberculosis drug system particles of milk exosomes coated with PLGA obtained by the method of the present invention is 200-400 nm.

[0092] According to some specific embodiments of the present invention, wherein the anti-tuberculosis drug is BDQ, the particle size of the nano anti-tuberculosis drug system particles of milk exosomes coated with PLGA obtained by the method of the present invention is 270.82 nm.

[0093] On the other hand, the present invention also provides a nano anti-tuberculosis drug system of milk exosomes coated with PLGA prepared by the above-mentioned preparation method of the present invention.

[0094] In yet another aspect, the present invention further provides a pharmaceutical composition, comprising a therapeutically effective amount of any of the above-described milk exosome-coated PLGA nano anti-tuberculosis drug systems of the present invention, and a pharmaceutically acceptable carrier.

[0095] According to some specific embodiments of the present invention, the pharmaceutical composition is in the form of tablets, capsules, granules, pills, powders, pastes or oral liquids.

[0096] In another aspect, the present invention also provides the use of any of the above-mentioned milk exosomes-coated PLGA nano anti-tuberculosis drug systems or drug compositions in the preparation of drugs for treating tuberculosis.

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

[0098] The present invention uses a technique of ultrasonic incubation followed by liposome extrusion membrane extrusion to coat PLGA nanoparticles with exosomes. The method can make the size of the synthesized sample more uniform, stable, and better dispersed.

[0099] In particular, the present invention is the first to use PLGA nanoparticles to coat PBTZ169 and BDQ, thereby improving their solubility. It is also the first to use milk exosomes to coat PLGA-PBTZ169 and PLGA-BDQ NPs, thereby overcoming the inherent disadvantages of PLGA:

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

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

[0102] 3. Traditional nanoparticles may trigger immune responses. Milk exosomes are natural endogenous substances with high biocompatibility and low immunogenicity. They reduce immune rejection and potential toxicity, and improve the safety of treatment.

[0103] Traditional PLGA NPs are difficult to achieve targeted therapy due to material limitations. Targeted delivery to organs or tissues can be achieved through specific modification of the surface of exosomes. The concentration of drugs in specific sites is increased, the therapeutic effect is enhanced, and the side effects on other non-targeted sites are reduced. And it has unique biological properties and can be easily absorbed by the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 , A is the SEM image of PLGA-PBTZ169 NPs, and B is the local magnified image.

[0105] Figure 2 , A is the transmission electron microscopy image of naked milk exosomes (ME) without PLGA NPs, with scale bars of 1 μm and 100 nm, and B is a local magnified image.

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

[0107] Figure 4 , A is the SEM image of PLGA-BDQ NPs, and B is the local magnified image.

[0108] Figure 5 , A is the transmission electron microscopy image of naked milk exosomes (ME) without PLGA NPs, with scale bars of 1 μm and 100 nm, and B is a local magnified image.

[0109] Figure 6 , A is the transmission electron microscopy image of ME-PLGA-BDQ NPs after coating with PLGA-NPs, with scale bars of 1 μm and 200 nm, and B is a local magnified image. DETAILED DESCRIPTION

[0110] The implementation process of the present invention and the beneficial effects produced are described in detail below through specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention, and is not intended to limit the scope of implementation of the present invention.

[0111] Example 1

[0112] Preparation and structural characterization of milk exosome-encapsulated PLGA nano-drug delivery system, active ingredient: PBTZ169

[0113] The preparation method is as follows:

[0114] Step 1, synthesis of PLGA nanoparticles by emulsification solvent evaporation method:

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

[0116] (2) Preparation of aqueous phase: Weigh 2500 mg of PVA powder (molecular weight 20,000-50,000) and dissolve it in 50 mL of Milli-Q aqueous solution. After swelling for 1 hour at room temperature with magnetic stirring (260 rpm), heat at 140° C. with magnetic stirring (260 rpm) for 2 hours until uniform. During the heating period, wrap the bottle mouth with aluminum foil to prevent evaporation. Then cool to room temperature as the aqueous phase.

[0117] (3) The oil phase prepared in step (1) was added dropwise to the aqueous phase. During this process, the mixed solution was emulsified for 15 minutes at 80% amplitude using probe ultrasound (130w, 20kHz). To avoid excessive heat generation, the emulsification was carried out in an ice bath throughout the process, with ultrasound 50s and paused for 10s to obtain a homogeneous emulsion. The dichloromethane in the emulsion was removed by rotary evaporation at 32°C, 90rpm and 0.09mPa pressure in a water bath for 45min. The emulsion obtained after rotary evaporation was centrifuged (8000rpm, -4°C, 10min) and the supernatant was removed. Ultrapure water was added, and the mixture was washed and resuspended three times to remove unencapsulated free drugs and PVA. Finally, the bottom precipitate (PLGA-PBTZ169 NPs) was collected by centrifugation (8000rpm, -4°C, 10min).

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

[0119] The milk exosomes stored at -80°C (Shanghai Noning Company, milk exosome extract, 1 g / L, NW3311) were thawed at room temperature, and ME and PLGA-PBTZ169 NPs (the carrier solution was 0.9% saline) were mixed in a mass concentration ratio of 1:1 at room temperature of 25°C, incubated in a water bath ultrasonically at 32°C for 5 minutes, and then extruded 9 times using a liposome extruder (through a 400nm polycarbonate membrane), and then centrifuged (8000rpm, -4°C, 10min) and the supernatant was discarded to remove the uncoated ME, thereby obtaining ME-PLGA-PBTZ169 NPs coated with PLGA-PBTZ169NPs.

[0120] Structural characterization

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

[0122] Transmission electron microscopy (TEM): TEM images show the morphology and nanosize of PLGA-PBTZ169 NPs. Figure 1 A is a scanning electron microscope image of PLGA-PBTZ169 NPs, showing spherical particles of uniform size and shape.

[0123] Figure 1 Scanning electron microscopy images of PLGA-PBTZ 169NPs. The scale bars are 1 μm, 500 nm and 200 nm.

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

[0125] Table 1A shows the particle size and PDI index of PLGA-blank NPs and PLGA-PBTZ169 NPs, where 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 PBTZ169 content in PLGA-PBTZ169 NPs was directly determined using UV spectrophotometry. First, the standard curve of PBTZ169 in DMSO solution was determined to be 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% based on 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-169NPs 48±6.72％ 16.63±1.92％

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

[0133] name Contact angle (°) PLGA-blank NPs 73.34±3.74 PLGA-PBTZ169NPs 61.92±1.48 PBTZ169 97.35±2.25

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

[0135] (1) Transmission electron microscopy (TEM): TEM images show the morphology and nanosize of ME and ME-PLGA-PBTZ169 NPs. Figure 2 and Figure 3 These are the scanning electron microscopy images of ME and ME-PLGA-PBTZ169 NPs, respectively, showing vesicles of uniform size and shape.

[0136] Figure 2 Transmission electron microscopy images of naked milk exosomes (ME) without PLGA NPs encapsulation, with scale bars of 1 μm and 100 nm.

[0137] Figure 3 Transmission electron microscopy images of ME-PLGA-PBTZ 169NPs after coating with PLGA-NPs. The scale bars are 1 μm and 200 nm.

[0138] (2) Nano Brook 90 Plus PALS nanoparticle size analyzer / zeta potential detector (company: Brookhaven Instruments, serial number: Serial NO: 250385) was used to measure the hydrated particle size distribution and zeta potential values ​​of ME, ME-PLGA-blank NPs, and ME-PLGA-PBTZ169 NPs. The results are shown in Table 1D.

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

[0140]

[0141] Figure 2 , Figure 3 As shown in Table 1D , the particle size of ME-PLGA-PBTZ169 NPs encapsulated in ME is about 236 nm, which is much larger than that of bare ME, whose particle size is only about 111 nm.

[0142] Cytotoxicity assay

[0143] The MTT method was used to detect the toxic effects of PBTZ169 API, 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 values ​​of ME-PLGA-PBTZ169 NPs containing the same concentration of raw material drug PBTZ169 were 50 The value is lower than that of the raw material drug. In addition, PLGA-blank NPs carriers and ME-PLGA-blank NPs carriers have strong safety and biocompatibility.

[0145] Table 1E. IC50 results of original 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 antimicrobial activity assay

[0149] The MABA method was used to determine the MIC of the raw material drug PBTZ169, PLGA-blank NPs carrier, PLGA-PBTZ169NPs, ME-PLGA-blank NPs carrier, and ME-PLGA-PBTZ169 NPs against the standard strain of Mycobacterium tuberculosis H37Rv (see Table 1F for specific data).

[0150] Table 1F, Minimum inhibitory concentrations of PBTZ169, PLGA-PBTZ169 NPs, ME-PLGA-PBTZ169 NPs and two blank carrier materials against the standard strain of Mycobacterium tuberculosis H37Rv in vitro

[0151] sample MIC (μg / mL) PBTZ169 0.00007 PLGA-PBTZ169NPs 0.00014 ME-PLGA-PBTZ169NPs 0.00015 PLGA-blank NPs carrier >1.56 ME-PLGA-blank NPs carrier >1.56

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

[0153] In vivo PK experiment:

[0154] BALB / c mice were randomly divided into three groups: PBTZ169, PLGA-PBTZ169 NPs, and ME-PLGA-PBTZ169NPs. The PBTZ169 dose was 10 mg / kg and was administered orally orally. There were 18 mice in each group. LC-MS was used to determine the drug concentration distribution of PBTZ169 in the blood, lungs, and spleen of the three groups of mice at different time points.

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

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

[0157]

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

[0161]

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

[0163] Embodiment 2:

[0164] Preparation and structural characterization of milk exosome-encapsulated PLGA nano-drug delivery system, active ingredient: BDQ

[0165] The preparation method is as follows:

[0166] Step 1: Synthesis of PLGA nanoparticles by emulsification solvent evaporation method:

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

[0168] (2) Preparation of aqueous phase: Weigh 2500 mg of PVA (molecular weight 20,000-50,000) powder and dissolve it in 50 mL of Milli-Q aqueous solution. After swelling at room temperature with magnetic stirring (260 rpm) for 1 hour, heat at 140°C with magnetic stirring (260 rpm) for 2 hours until uniform. During heating, wrap the bottle mouth with aluminum foil to prevent evaporation. Then cool to room temperature as the aqueous phase.

[0169] (3) The oil phase prepared in step (1) was added dropwise to the aqueous phase prepared in step (2). During this process, the mixed solution was emulsified for 15 minutes at 80% amplitude using probe ultrasound (130w, 20kHz). To avoid excessive heat generation, the emulsification was carried out in an ice bath throughout the process, with ultrasound 50s and paused for 10s to obtain a homogeneous emulsion. The dichloromethane in the emulsion was removed by rotary evaporation at 32°C, 90rpm and 0.09mPa pressure in a water bath for 45min. The emulsion obtained after rotary evaporation was centrifuged (8000rpm, -4°C, 10min) and the supernatant was removed. Ultrapure water was added and the mixture was washed and resuspended three times to remove the unencapsulated free drug and PVA. Finally, the bottom precipitate was collected by centrifugation (8000rpm, -4°C, 10min).

[0170] Step 2: Ultrasonic method - milk exosomes (ME, Milk-exosome) coated PLGA-BDQ NPs:

[0171] The milk exosomes stored at -80°C were thawed at room temperature, and ME and PLGA-BDQ NPs (the carrier solution was 0.9% saline) were mixed at a mass concentration ratio of 1:1 at room temperature of 25°C, incubated in a water bath with ultrasound at 32°C for 5 minutes, and then extruded 9 times using a liposome extruder (through a 400nm polycarbonate membrane), and then centrifuged (8000rpm, -4°C, 10min) and the supernatant was discarded to remove the uncoated ME, thus obtaining ME-PLGA-BDQ NPs coated with PLGA-BDQ NPs.

[0172] Structural characterization

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

[0174] Transmission electron microscopy (TEM): TEM images show the morphology and nanosize of PLGA-BDQ NPs. Figure 4 A is a scanning electron microscope image of PLGA-BDQ NPs, showing spherical particles of uniform size and shape.

[0175] Figure 4 Scanning electron microscopy image of PLGA-BDQ NPs, scale bar is 500nm and 200nm

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

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

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

[0179]

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

[0181] The encapsulation efficiency of PLGA-BDQ NPs was determined to be 42.67±4.11% and the drug loading was 13.76±1.32% based on 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 BDQ drug increased after encapsulation into PLGA core, the contact angle comparison of raw drug BDQ and PLGA-BDQ NPs was measured. (θ < 90° is hydrophilic, θ > 90° is hydrophobic) It can be seen that PLGA-BDQ NPs have increased hydrophilicity compared with raw drug BDQ (see Table 2C).

[0185] Table 2C. Contact angle measurements of raw drug BDQ, PLGA-blank NPs, and 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 the milk exosomes (ME) synthesized in this example and the ME-PLGA-BDQ NPs coated therewith:

[0188] (1) Transmission electron microscopy (TEM): TEM images show the morphology and nanosize of ME and ME-PLGA-BDQ NPs. Figure 5 and Figure 6 The following are the scanning electron microscopy images of ME and ME-PLGA-BDQ NPs, respectively, showing vesicles of uniform size and shape.

[0189] Figure 5 Transmission electron microscopy images of naked milk exosomes (ME) without PLGA NPs encapsulation, with scale bars of 1 μm and 100 nm.

[0190] Figure 6 Transmission electron microscopy images of ME-PLGA-BDQ NPs after coating with PLGA-NPs. The scale bars are 1 μm and 200 nm.

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

[0192] Table 2 Particle size and PDI index of D, ME, ME-PLGA-blank NPs, and ME-PLGA-BDQ NPs, n=3

[0193]

[0194] Figure 5 , Figure 6 As shown in Table 2D , the particle size of ME-PLGA-BDQ NPs encapsulated in ME is about 270.82 nm, which is much larger than that of bare ME, whose particle size is only about 111 nm.

[0195] Cytotoxicity assay

[0196] The MTT method was used to detect the toxic effects of BDQ API, 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 shown in Table 2E, the IC values ​​of PLGA-BDQ NPs containing the same concentration of raw drug BDQ were 50 The value is lower than that of the raw material drug, and ME-PLGA-BDQ NPs is equivalent to the raw material drug. In addition, PLGA-blank NPs carriers and ME-PLGA-blank NPs carriers have strong safety and biocompatibility.

[0198] Table 2E. IC of original drug BDQ, PLGA-blank NPs carrier, PLGA-BDQ NPs, ME-PLGA-blank NPs carrier, and ME-PLGA-BDQ NPs on J774A.1 cells 50 Measurement results

[0199]

[0200]

[0201] In vitro antimicrobial activity assay

[0202] The MABA method was used to determine the MIC of the raw material drug BDQ, PLGA-blank NPs carrier, PLGA-BDQ NPs, ME-PLGA-blank NPs carrier, and ME-PLGA-BDQ NPs against the standard strain of Mycobacterium tuberculosis H37Rv. (See Table 2F for specific data)

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

[0204] sample MIC (μg / 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] The data in Table 2F show that the two blank carrier materials did not show obvious antibacterial activity, and the BDQ loaded by the carrier showed better antibacterial activity in vitro than the raw drug.

Claims

1. A milk exosome-coated PLGA nano anti-tuberculosis drug system, wherein: The system structure is: Anti-tuberculosis drugs-milk exosomes@PLGA-anti-tuberculosis drugs.

2. The milk exosome-coated PLGA nano anti-tuberculosis drug system according to claim 1, wherein: The anti-tuberculosis drug is selected from one or more combinations of aminoquinolones, benzothiazoles, arylquinolines, nitrobenzamides, and benzothiazinones (preferably, the anti-tuberculosis drug is selected from PBTZ169 and / or bedaquiline).

3. The milk exosome-coated PLGA nano anti-tuberculosis drug system according to claim 1 or 2, wherein: The PLGA molecular formula is [C3H4O2] x [C2H2O2] y , molecular mass Mw is 38000-54000 (preferably lactide: glycolide = (30:70) - (50:50)).

4. The method for preparing the nano anti-tuberculosis drug system of milk exosomes coated with PLGA according to any one of claims 1 to 3, wherein: The method comprises the steps of using anti-tuberculosis drugs, PLGA and milk exosomes as main raw materials to prepare the milk exosome-coated PLGA nano anti-tuberculosis drug system.

5. The preparation method according to claim 4, wherein The method comprises the following steps: Preparation of oil phase: dissolving anti-tuberculosis drugs in an organic solvent of PLGA as the oil phase; Preparation of aqueous phase: using double distilled water as the aqueous phase for standby use (preferably adding a surfactant (preferably PVA) into double distilled water to obtain a uniform solution as the aqueous phase); Preparation of precipitate: add the oil phase dropwise into the water phase to obtain a homogeneous emulsion, and remove the supernatant by centrifugation to obtain a precipitate; Preparation of nano anti-tuberculosis drugs: milk exosomes and the obtained precipitate are mixed in a mass ratio of (1:2)-(1:1), and the milk exosomes-coated PLGA nano anti-tuberculosis drug system is obtained after co-incubation.

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

7. The preparation method according to claim 5 or 6, wherein: The step of preparing the precipitate also includes a step of removing the organic solvent from the homogenized emulsion (preferably by rotary evaporation), and then centrifuging to obtain the precipitate.

8. The preparation method according to any one of claims 5 to 7, wherein: In the step of preparing the precipitate, after the centrifugation is completed and the supernatant is removed, the precipitate is washed 1 to 3 times, and then the precipitate is collected to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system.

9. The preparation method according to any one of claims 5 to 8, wherein: In the preparation of the precipitate, the oil phase is added dropwise to the water phase under ice bath conditions, and ultrasonic emulsification is performed during the dropping process to obtain a homogeneous emulsion. During the ultrasonic emulsification process, the temperature of the solution is controlled to be lower than 20°C throughout the entire process.

10. The preparation method according to any one of claims 5 to 9, wherein: The preparation steps of the nano anti-tuberculosis drug include: mixing the milk exosomes with the obtained precipitate, incubating them together by ultrasound, and then centrifuging and discarding the supernatant to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system; or The preparation steps of the nano anti-tuberculosis drug include: mixing the milk exosomes with the obtained precipitate, incubating them together by ultrasound, and then repeatedly extruding them with a liposome extruder, and then centrifuging the extruded solution and discarding the supernatant to obtain the milk exosome-coated PLGA nano anti-tuberculosis drug system; Preferably, the ultrasonic co-incubation is carried out at a temperature of 25-40° C.; more preferably, the ultrasonic co-incubation time is 3-10 min.

11. The nano anti-tuberculosis drug system of milk exosomes coated with PLGA prepared by the preparation method according to any one of claims 5 to 10.

12. A pharmaceutical composition comprising a therapeutically effective amount of the milk exosome-coated PLGA nano anti-tuberculosis drug system according to claims 1 to 3 or claim 11, and a pharmaceutically acceptable carrier.

13. Use of the milk exosome-coated PLGA nano anti-tuberculosis drug system according to claims 1 to 3 or claim 11 or the pharmaceutical composition according to claim 12 in the preparation of drugs for treating tuberculosis.

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