A polymyxin b-loaded nano-formulation, its preparation method and use thereof

Abstract

The application provides a polymyxin B-loaded nano preparation and a preparation method and application thereof, and belongs to the field of biological nanotechnology.The method comprises the following steps: (1) preparing a water-in-oil emulsion: mixing a water solution of polymyxin B and a dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a water-in-oil emulsion; (2) preparing an ethanol aqueous solution of hyaluronic acid; (3) mixing the water-in-oil emulsion and the ethanol aqueous solution of hyaluronic acid, and stirring to obtain a nano dispersion; (4) vacuum rotary evaporation of the nano dispersion, collection of a colloidal dispersion, and dispersion of the colloidal dispersion in water to obtain a nano particle solution.The nano preparation prepared by the application does not need a chemical crosslinking agent, and thus effectively avoids problems caused by the introduction of chemical crosslinking.The preparation process is simple and easy to control, has good safety, has good biocompatibility, and provides a basis for the approval of clinical trials and industrial development of atomization drug delivery of antibiotics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological nanotechnology, and particularly relates to a polymyxin B-loaded nano-preparation and a preparation method and application thereof. BACKGROUND

[0002] At present, the treatment of pulmonary infection by inhalation of antibiotics, especially in patients with chronic pulmonary infection, has attracted more and more attention. As a superior method, aerosolized antibiotics can deliver high-concentration drugs directly to the infection site, reduce the side effects of drugs, and improve the bioavailability of existing antimicrobial drugs. Among them, the concentration / persistence of antibiotics at the infection site and the degree of drug penetration into the lung lining fluid, alveoli and bronchial mucosa are important factors affecting the clinical results when aerosolized antibiotics are used.

[0003] Among the preparation methods developed for delivering drugs to the lungs, nanoparticles are most promising for antibiotic inhalation. In fact, only nanoparticles small enough can avoid the spatial inhibition of dense mucin fiber mesh. Moreover, the delivery efficiency of nanoparticles by inhalation in the lungs needs to be further improved. SUMMARY

[0004] The present application provides a polymyxin B (PMB)-loaded nano-preparation and a preparation method thereof. The method encapsulates water-soluble PMB in polylactic acid-glycolic acid copolymer (PLGA) in the form of water-in-oil, and complexes hydrophilic hyaluronic acid (HA) on the surface of PLGA, giving PLGA-PMB good hydrophilicity and negative charge, and improving the mucus penetration properties of the overall nano-delivery system.

[0005] The present application provides a preparation method of a polymyxin B-loaded nano-preparation, comprising the following steps:

[0006] (1) preparing a water-in-oil emulsion: mixing a water solution of polymyxin B and a dichloromethane solution of polylactic acid-glycolic acid copolymer to obtain a water-in-oil emulsion;

[0007] (2) preparing an ethanol aqueous solution of hyaluronic acid;

[0008] (3) mixing the above water-in-oil emulsion and the ethanol aqueous solution of hyaluronic acid, and stirring to obtain a nano-dispersion;

[0009] (4) vacuum rotary evaporation of the above nano-dispersion, collecting a colloidal dispersion, and dispersing the obtained colloidal dispersion in water to obtain a nanoparticle solution.

[0010] Further, the mass ratio of polymyxin B in the aqueous solution of polymyxin B, polylactic acid-glycolic acid copolymer in the dichloromethane solution of polylactic acid-glycolic acid copolymer, and hyaluronic acid in the aqueous ethanol solution of hyaluronic acid is 1:5-40:1-10.

[0011] Preferably, the mass ratio of polymyxin B in the aqueous solution of polymyxin B, polylactic acid-glycolic acid copolymer in the dichloromethane solution of polylactic acid-glycolic acid copolymer, and hyaluronic acid in the aqueous ethanol solution of hyaluronic acid is 1:5-20.

[0012] Further, in step (1), the concentration of the aqueous solution of polymyxin B is 0.5-5 mg / 100 μL; preferably, the concentration of the aqueous solution of polymyxin B is 1 mg / 100 μL.

[0013] Further, in step (1), the concentration of the dichloromethane solution of polylactic acid-glycolic acid copolymer is 5-20 mg / mL; preferably, the concentration of the dichloromethane solution of polylactic acid-glycolic acid copolymer is 5 mg / mL; wherein the mass-volume concentration of the dichloromethane solution is 0.5%.

[0014] Further, in step (2), the mass-volume ratio of hyaluronic acid to the aqueous ethanol solution is 5 mg:25 ml.

[0015] The molecular weight of hyaluronic acid is 8000 Da.

[0016] The volume fraction of the aqueous ethanol solution is 50%.

[0017] Further, in step (3), the stirring is at room temperature at a rotation speed of 300-500 r / min for 1-10 min.

[0018] Preferably, the stirring is at room temperature at a rotation speed of 500 r / min for 10 min.

[0019] Further, in step (4), the temperature of vacuum rotary evaporation is 20-40℃.

[0020] Preferably, the temperature of vacuum rotary evaporation is 30℃.

[0021] The present application also provides a nanoscale preparation of polymyxin B prepared by any of the above preparation methods.

[0022] The present application also provides the use of any of the above nanoscale preparations in the treatment of lung infection by antibiotic nebulization inhalation.

[0023] The present application has the following advantages:

[0024] (1) The preparation method of the polynyxin B loaded nano-preparation provided by the present application first prepares a water-in-oil system of PLGA-PMB, and then uses an auxiliary polymer HA to complex on the surface of the PLGA to prepare HA@PLGA-PMB nanoparticle delivery. The method is simple to operate, and the raw materials are easy to obtain. After the surface modification of HA, the surface has good hydrophilicity, and the surface required for the lung delivery of the nanoparticle is endowed with negative charge and mucosal permeability, which is beneficial to improve the mucus permeability efficiency, reduce the toxicity of polynyxin, and improve the bioavailability of polynyxin, thereby providing a new preparation method for the lung delivery system of antibiotics.

[0025] (2) The polynyxin B loaded nano-preparation obtained by the present application has good biocompatibility and is easy to realize clinical transformation. The toxicity experiment of the HA@PLGA-PMB nanoparticle in vitro using lung epithelial cells shows that when the lung epithelial cells are treated with PMB, the cell viability decreases to below 70% when the concentration of PMB is 250 μg / ml. In contrast, when the cells are treated with the HA@PLGA-PMB nanoparticle solution, more than 95% of the cells still survive when the concentration range is from 15.63 to 250 μg / ml, which proves the good cell compatibility.

[0026] (3) The polynyxin B loaded nano-preparation obtained by the present application can release polynyxin B slowly, which avoids the direct contact of high-concentration polynyxin with the lung parenchyma during lung delivery, and reduces the toxic side effects of polynyxin (PMB). The drug release experiment shows that the in vitro release study results under physiological pH and temperature (pH=7.2 and T=37℃) show that the PMB-loaded nanoparticles exhibit two-stage release characteristics in each case, an initial rapid release stage (i.e. burst release), followed by a regulated and gradual release of PMB lasting more than 7 days. BRIEF DESCRIPTION OF DRAWINGS

[0027] The drawings constituting a part of the present application are used to provide a further understanding of the present application, and the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0028] Figure 1 A schematic diagram for the preparation of HA@PLGA-PMB nanoparticles (HA modified water-in-oil structure of PLGA-PMB system);

[0029] Figure 2Figure 6. Characterization of the fluid particle size and zeta potential of HA@PLGA- PMB nanoparticles with different ratios. A) Size fluid particle size; B) zeta potential; all data are expressed as mean ± SD (n = 3 independent experiments); ****P < 0.0001; *P < 0.03 compared with the HA@PLGA (1:1)-PMB nanoparticle solution group;

[0030] Figure 3 Figure 7. Transmission electron microscopy image of HA@PLGA (1:1)-PMB nanoparticles.

[0031] Figure 4 Figure 8. Release kinetics of PMB in different ratios of HA@PLGA-PMB nanoparticles.

[0032] Figure 5 Figure 9. Cell toxicity assay of nanoparticles on HPAEpiC human alveolar epithelial cells; ***P < 0.0002; **P < 0.001; *P < 0.02 compared with the HA@PLGA-PMB nanoparticle solution group.

[0033] Figure 6 Figure 10. Mucus penetration experiment of HA@PLGA-PMB nanoparticles; A) Visual inspection of the penetration of various samples through the artificial mucus layer; B) Absorbance of the gelatin layer at 595 nm after incubation with HA@PLGA-PMB nanoparticle solution; ***P < 0.0009; *P < 0.02 compared with the PLGA-PMB nanoparticle group.

[0034] Figure 7 Figure 11. In vivo aerosol antibacterial experiment of HA@PLGA-PMB nanoparticle solution in mice. A) Lung index of different aerosol administration groups; B) Bacterial count of different aerosol administration groups; ****P << 0.0001; **P < 0.0045 compared with the Model group.

[0035] Figure 8 Figure 12. Effect of different PLGA addition amounts on the dispersion of the fluid particle size of nanoparticles.

[0036] Figure 9 Figure 13. Effect of different stirring times on the dispersion of the fluid particle size of nanoparticles. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. In the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0038] An embodiment of the present application provides a preparation method of a polymyxin B loaded nano-preparation, which comprises the following steps:

[0039] (1) preparing a water-in-oil emulsion: mixing a polymyxin B (PMB) aqueous solution and a polylactic acid-glycolic acid copolymer (PLGA) dichloromethane solution to obtain the water-in-oil emulsion;

[0040] (2) preparing a hyaluronic acid (HA) ethanol aqueous solution;

[0041] (3) mixing the water-in-oil emulsion and the hyaluronic acid ethanol aqueous solution and stirring to obtain a nano-dispersion;

[0042] (4) vacuum rotary evaporation of the nano-dispersion, collecting a colloidal dispersion, and dispersing the colloidal dispersion in water to obtain a nano-particle solution.

[0043] The preparation method of the PMB loaded nano-preparation (HA@PLGA-PMB nano-particle) provided by the embodiment of the present application adopts a water-in-oil mode to wrap the water-soluble PMB in the PLGA, and hyaluronic acid is used to complex on the surface of the PLGA in order to make the nano-particle more easily penetrate the mucus tissue, and the HA@PLGA-PMB nano-particle delivery system is prepared. Since the hyaluronic acid HA is a linear anion, has strong hydrophilicity, can target the CD44 receptor on the cell surface, has good biocompatibility and other advantages. After the hyaluronic acid is complexed by the method of the present application, the nano-particle has negative electricity, can effectively avoid the electrostatic attraction between the positive electricity PMB and the negative electricity mucus tissue, makes the PLGA-PMB surface have good hydrophilicity, avoids the interaction between the nano-particle and the hydrophobic region of the mucin molecules in the mucus tissue, and thus the mucus penetration efficiency of the HA@PLGA-PMB nano-particle is enhanced, and the bioavailability of the atomization drug delivery is improved. 44

[0044] The nano-preparation prepared by the present application does not need a chemical crosslinking agent, and thus the problems caused by the introduction of the chemical crosslinking are effectively avoided. The preparation process is simple and easy to control, has good safety, the polymyxin B loaded nano-preparation can release the polymyxin B slowly, avoids the direct contact between the high concentration polymyxin and the lung parenchyma during the lung delivery, reduces the toxic side effects of the polymyxin (PMB), and has good biocompatibility, which provides a basis for the approval of the atomization drug delivery clinical test of the antibiotic and the industrialized development.

[0045] In the embodiment of the present application, the mass ratio of the polymyxin B in the polymyxin B aqueous solution, the polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer dichloromethane solution and the hyaluronic acid in the hyaluronic acid ethanol aqueous solution is 1:5-40; 1-10.

[0046] ​Preferably, the mass ratio of polymyxin B in the aqueous solution of polymyxin B, polylactic-glycolic acid in the dichloromethane solution of polylactic-glycolic acid, and hyaluronic acid in the aqueous ethanol solution of hyaluronic acid is 1:5-20.

[0047] In the embodiment of the present application, when the amount of PLGA added exceeds 20 mg, blocky precipitates are produced, which is not conducive to the formation of nanodispersion. This is mainly because PLGA has certain adhesion, and a large amount of addition will make the nanodispersion unstable, and thus blocky precipitates are produced. The experiment determines the HA@PLGA-PMB nanoparticles with the best stability by optimizing the mass ratio of different HA and PLGA.

[0048] In one embodiment of the present application, in step (1), the concentration of the aqueous solution of polymyxin B is 0.5-5 mg / 100 μL; preferably, the concentration of the aqueous solution of polymyxin B is 1 mg / 100 μL.

[0049] In step (1), the concentration of the dichloromethane solution of polylactic-glycolic acid is 5-20 mg / mL; preferably, the concentration of the dichloromethane solution of polylactic-glycolic acid is 5 mg / mL. The mass-volume concentration of the dichloromethane solution is 0.5%. The unit of mass-volume concentration is kg / L.

[0050] In one embodiment of the present application, in step (2), the mass-volume ratio of hyaluronic acid to the aqueous ethanol solution is 5 mg:25 ml. In step (2), the molecular weight of hyaluronic acid is 8000 Da. In step (2), the volume fraction of the aqueous ethanol solution is 50%.

[0051] In one embodiment of the present application, in step (3), the stirring is carried out at room temperature at a speed of 300-500 r / min for 1-10 min; preferably, the stirring is carried out at room temperature at a speed of 500 r / min for 10 min. The experiment shows that when the stirring speed is 500 r / min, stirring for more than 10 min will also produce blocky precipitates, because the extension of stirring time will make the nanodispersion unstable, and thus blocky precipitates are produced, so the conditions of magnetic stirring also need to be controlled.

[0052] In one embodiment of the present application, in step (4), the temperature of vacuum rotary evaporation is 20-40℃. Preferably, in step (4), the temperature of vacuum rotary evaporation is 30℃. By vacuum rotary evaporation, the organic solvent is removed, and most of the water is also evaporated. The temperature of vacuum rotary evaporation is 30℃.

[0053] In step (4), the obtained colloidal dispersion is dispersed in ultrapure water.

[0054] The present application will be described in detail below with reference to the embodiments.

[0055] The raw materials and reagents used in the present application are as follows:

[0056] Polylactic acid-glycolic acid copolymer (PLGA: 1-20,000 Da Shanghai Yuanye Biotechnology Co., Ltd.), sodium hyaluronate (HA: 8000 Da QuFu Guanglong Biotechnology Co., Ltd.), polymyxin (PMB: Beijing Solaybao Technology Co., Ltd.), anhydrous ethanol and dichloromethane were purchased from Beijing Chemical Plant, mucin (pig gastric mucus Merck Chemical Technology (Shanghai) Co., Ltd.), DTPA (diethylene triamine pentaacetic acid Merck Chemical Technology (Shanghai) Co., Ltd.), DNA (salmon sperm Merck Chemical Technology (Shanghai) Co., Ltd.), RPMI medium (Thermo Fisher Scientific), 50% egg yolk emulsion (Qingdao Haibo Biotechnology Co., Ltd.), gelatin (Tianjin Xinsuo Optics and Electronics Technology Co., Ltd.).

[0057] Example 1 The preparation method of the HA@PLGA-PMB nanoparticle solution comprises the following steps:

[0058] (1) Prepare an ethanol aqueous solution of hyaluronic acid: dissolve the hydrophilic polymer hyaluronic acid HA (5 mg) in water, and then mix with 25 ml of anhydrous ethanol to prepare a 50% ethanol aqueous solution; wherein the volume fraction of the ethanol aqueous solution is 50% (v / v);

[0059] (2) Prepare a water-in-oil emulsion: add 1 mg of polymyxin (PMB) to 100 microliters of water to dissolve it in water, then add 1 milliliter of a PLGA-dichloromethane solution (0.5% w / v) containing 5 mg of PLGA, and mix uniformly to obtain a water-in-oil emulsion;

[0060] (3) Add the water-in-oil emulsion to the ethanol aqueous solution of hyaluronic acid (50% v / v) obtained in step (1) to generate an antibiotic precipitate;

[0061] (4) After stirring the obtained antibiotic precipitate dispersion at room temperature for 10 minutes (at a speed of 500 r / min), remove the residual organic solvent by vacuum rotary evaporation at 30°C, collect the obtained colloidal dispersion, and disperse it in ultrapure water to a final volume of 5 ml.

[0062] Examples 2-4 The preparation method of the HA@PLGA-PMB nanoparticle solution comprises the following steps:

[0063] The same as Example 1, except that in step (2) of Examples 2-4, 10 mg, 15 mg, and 20 mg of PLGA were added, respectively.

[0064] The HA@PLGA-PMB nanoparticles with different ratios of HA to PLGA were prepared in Examples 1-4. The specific experimental conditions can be referred to Table 1.

[0065] Table 1 Preparation of HA@PLGA-PMB nanoparticle with different ratio of raw materials

[0066]

[0067]

[0068] Comparative Example 1 The preparation method of HA@PLGA-PMB nanoparticle solution includes the following steps:

[0069] The same as Example 1, except that the amount of PLGA added is increased to 30 mg and 40 mg.

[0070] In order to verify the influence of the amount of PLGA added on the dispersion of the fluid particle size of the nanoparticle, the dispersion of the nanoparticle solution when the amount of PLGA added is 20 mg, 30 mg and 40 mg is measured, and the results are shown in Table 2. Figure 8 Referring to Table 2, when the amount of PLGA added is 20 mg, the fluid particle size of the nanoparticle is less than 1000 nm; when the amount of PLGA added is 30 mg and 40 mg, most of the fluid particle size of the nanoparticle is between 1000 nm and 3000 nm, and the fluid particle size of the particle is large, which is not conducive to the absorption of the lung after atomization. Figure 8

[0071] Comparative Example 2 The preparation method of HA@PLGA-PMB nanoparticle solution includes the following steps:

[0072] The same as Example 1, except that the stirring time is 20 minutes and 30 minutes, and the influence of different stirring times on the dispersion of the nanoparticle is verified. The dispersion of the nanoparticle solution when the stirring time is 10 min, 20 min and 30 min is measured, and the results are shown in Table 3. Figure 9 Referring to Table 3, when the stirring time is 10 min, the fluid particle size of the nanoparticle is less than 1000 nm; when the stirring time is 20 min or 30 min, most of the fluid particle size of the nanoparticle is dispersed in 1000-2000 nm, therefore, the longer the stirring time, the larger the fluid particle size of the nanoparticle, and the stirring time is too long, which is not conducive to the preparation of the nanoparticle with appropriate size. Figure 9

[0073] Characterization of HA@PLGA-PMB nanoparticle Test Example 1 (1) Measurement of fluid particle size and surface potential

[0074]

[0075] ​​The size and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS) equipment at 25°C, equipped with a helium-neon laser (633 nm). The nanoparticle aqueous dispersions were appropriately diluted in ultrapure water and analyzed in an electrophoretic cell at a fixed potential of ± 150 mV. The hydrodynamic size of the HA@PLGA-PMB nanoparticles of different mass ratios of HA to PLGA (1:1-1:4) prepared in Example 1-4 were measured by dynamic light scattering (DLS), and the results are shown in Figure 2 A. See Figure 2 A, the HA@PLGA-PMB nanoparticles with a mass ratio of HA to PLGA of 1:1 showed a smaller hydrodynamic size (D h = 223.2 ± 1.5 nm). The complex modification of HA made the HA@PLGA-PMB nanoparticles show a very strong negative zeta potential, and the results are shown in Figure 2 B. See Figure 2 B, the HA@PLGA(1:1)-PMB nanoparticles had the largest |ζ|, indicating that they had strong stability.

[0076] (2) Transmission electron microscopy characterization

[0077] The nanoparticle morphology was evaluated by transmission electron microscopy (TEM) (CM 12 TEM microscope, Philips). The nanoparticle solution (10 μl; 2 mg / ml) was dropped onto a copper sheet, and after 10 min the droplet was absorbed, and the nanoparticles were deposited on the copper sheet. Detection was performed by transmission electron microscopy (TEM), and the results are shown in Figure 3 . See Figure 3 , the HA@PLGA-PMB nanoparticles had a regular and well-defined spherical morphology with a maximum size of about 500 nm.

[0078] Test Example 2 In vitro release kinetics of HA@PLGA-PMB nanoparticles

[0079] HA@PLGA-PMB nanoparticles were further characterized for in vitro release kinetics of PMB. Release studies were performed by diluting nanoparticle solutions to 1.5 mg / ml in phosphate buffer (120 mM NaCl, 2.7 mM KC1, 10 mM phosphate) at pH = 7.2 (PBS). The diluted dispersions were incubated in a 37 °C water bath. At predetermined time intervals, samples were ultrafiltered using ultrafiltration tubes with a molecular weight cut-off of 3000 Da. 0.5 ml of the outer medium was removed and replaced with an equal volume of fresh PBS. The PMB content in the filtered medium was analyzed by spectrophotometry (215 nm). Experiments were performed in triplicate for each time point of the release kinetics. Results are expressed as the percentage of PMB released from nanoparticles ± SD over time. All PMB-loaded nanoparticles exhibited a two-stage release profile characterized by an initial burst release phase followed by a regulated and gradual release of PMB over more than 7 days. The rate of PMB release from HA-modified HA@PLGA-PMB nanoparticles depends on the ratio of HA to PLGA used, and the results are shown in Figure 4 . In particular, the rate of PMB release from the HA@PLGA(1 : 1)-PMB nanoparticle group was much higher than the other experimental groups. On the other hand, HA-modified nanoparticle formulations showed very similar PMB release profiles regardless of the PLGA type used. While the PMB release from HA@PLGA(1 : 1)-PMB nanoparticles was more than 50% at 3 days, the amount of PMB released decreased with increasing PLGA ratio. The PMB release from HA@PLGA(1 : 1)-PMB nanoparticles reached 99% at 10 days.

[0080] Test Example 3 In vitro compatibility of HA@PLGA-PMB nanoparticles

[0081] Pulmonary epithelial cell (HAEpic) toxicity of nanoparticles. HAEpic cells were cultured, passaged and plated at 5000 cells / well in 4 replicates per sample, and incubated for 24 h before adding diluted HA@PLGA-PMB nanoparticle solutions (concentration range from 15.63 to 250 pg / ml). Cell viability after different treatments was further quantified by CCK-8 analysis, and the results are shown in Figure 5 . A typical dose-dependent cytotoxicity was observed from the PMB group. With increasing PMB dose, the viability of different cells gradually decreased. In contrast, when cells were treated with HA@PLGA or HA@PLGA(1 : 1)-PMB nanoparticles at a concentration range from 15.63 to 250 pg / ml, more than 95% of the cells were still viable, demonstrating their good cell compatibility.

[0082] Test Example 4Mucus penetration experiment of HA@PLGA-PMB nanoparticles

[0083] Prepare 50 mL of artificial mucus by adding 500 mg DNA, 250 μL sterile egg yolk emulsion, 250 mg mucin, 0.295 mg DTPA, 250 mg NaCl, 110 mg KCl, and 1 mL RPMI to 50 mL of water. Stir the dispersion until a homogeneous mixture is obtained. Prepare a 10% (w / v) gelatin solution in hot water. Place one mL of gelatin solution in several identical glass vials, allow to harden at room temperature, and store at 4 °C until use.

[0084] Place 1.5 ml of artificial mucus on the hardened gelatin. Stain 500 μL of the sample stained with Coomassie Brilliant Blue with Coomassie Brilliant Blue and deposit it on the surface of the artificial mucus layer, then maintain at 37°C. (See attached image) Figure 6 Over time, HA@PLGA-PMB nanoparticles were observed to rapidly penetrate the mucus layer in Figure A. The permeability of the mucus was quantitatively determined by analyzing the absorbance of the gelatin layer after penetration.

[0085] After depositing HA@PLGA-PMB nanoparticles onto the artificial mucus layer, increased permeability was observed (as shown in the attached image). Figure 6 A) indicates that HA-modified nanoparticles have better mucus permeability than bare nanoparticles, which is also verified by the absorbance of the gelatin layer (see attached). Figure 6 (B) The absorbance of the gelatin layer was measured to be greater for HA-modified nanoparticles. The modified HA molecules are hydrophilic, which can better promote the penetration of HA@PLGA-PMB nanoparticles through the mucus layer.

[0086] Test Example 5 Study on the antibacterial effect of HA@PLGA-PMB nanoparticles delivered to mice via atomization

[0087] To evaluate the antibacterial activity of HA@PLGA-PMB nanoparticles against acute pulmonary infection. Approximately 10 nanoparticles were delivered via catheter. 6 CFU pathogens were directly instilled into the lungs to construct a mouse model of acute lung infection. Figure 7 A) The dosage of nanoparticles in mice is determined based on the amount of bacteria inoculated, with the mice receiving either two doses of nanoparticle solution (equivalent to 4 mg kg⁻¹ of PMB) or free PMB (4 mg kg⁻¹), administered via a small nebulizer. The nebulization concentration in mice is generally determined by the amount of bacteria inoculated, but typically does not exceed 10 mg kg⁻¹.

[0088] The first dose was instilled at the time of infection and the second dose was instilled 4 hours post-infection. Mice were observed 24 hours after the last dose, their lungs were collected after asphyxiation and photographed, and the lung index was calculated for each group by weighing the lungs Figure 7 B) To confirm the effect of PLGA-PMB and HA@PLGA-PMB nanoparticles, we homogenized the lungs in PBS and counted the number of bacteria in them. The number of bacteria in the mice treated with HA@PLGA-PMB nanoparticles was significantly reduced, and the lung index was lower, which was comparable to the free PMB group, due to the effective mucus penetration ability and sustained drug release ability of HA@PLGA-PMB nanoparticles. The antibacterial effect of PLGA-PMB was worse than that of HA@PLGA-PMB, and the number of bacteria was more than that of HA@PLGA-PMB, mainly because the PLGA-PMB was not modified by the hydrophilic HA, resulting in poor mucus penetration ability, and the aerosol administration could not be effectively delivered to the infection site, resulting in more bacteria and poor antibacterial effect in vivo after aerosol administration. Therefore, it is proved that the hydrophilically modified HA@PLGA-PMB nanoparticles solve the problem of low mucus penetration efficiency during aerosol administration, and they have more advantages in lung aerosol administration.

[0089] The test examples of the present application respectively characterize the particle size, potential and other particle properties of HA@PLGA-PMB nanoparticles; verify the biocompatibility of the optimized nanoparticle preparation with lung cells. Verify the mucus penetration ability of HA@PLGA-PMB nanoparticles in vitro.

[0090] The above is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a polymyxin B-loaded nanoformulation for administration by nebulization, comprising the following steps: (1) preparing a water-in-oil emulsion by mixing a polymyxin B aqueous solution and a polylactic acid-glycolic acid copolymer dichloromethane solution to obtain a water-in-oil emulsion; (2) preparing a hyaluronic acid ethanol aqueous solution; (3) mixing the water-in-oil emulsion and the hyaluronic acid ethanol aqueous solution to obtain a nano dispersion; wherein the stirring is at a speed of 300-500 r / min at room temperature for 1-10 min; (4) vacuum rotary evaporation of the nano dispersion to obtain a colloidal dispersion, and dispersing the colloidal dispersion in water to obtain a nanoparticle solution; wherein the mass ratio of polymyxin B in the polymyxin B aqueous solution, polylactic acid-glycolic acid copolymer in the polylactic acid-glycolic acid copolymer dichloromethane solution, and hyaluronic acid in the hyaluronic acid ethanol aqueous solution is 1:5-20:

5. 2.The method of claim 1, wherein in step (1), the concentration of the polymyxin B aqueous solution is 0.5-5 mg / 100 μL. 3.The method of claim 2, wherein in step (1), the concentration of the polymyxin B aqueous solution is 1 mg / 100 μL. (3) mixing the water-in-oil emulsion with an ethanol aqueous solution of hyaluronic acid, stirring to obtain a nanodispersion; wherein, 4.The method of claim 1, wherein in step (1), the concentration of the polylactic acid-glycolic acid copolymer dichloromethane solution is 5-20 mg / mL, and the mass concentration of the dichloromethane solution is 0.5%. 5.The method of claim 1, wherein in step (2), the mass-volume ratio of hyaluronic acid to the hyaluronic acid ethanol aqueous solution is 5 mg:25 ml; the molecular weight of the hyaluronic acid is 8000 Da; and the volume fraction of the hyaluronic acid ethanol aqueous solution is 50%. 6.The method of claim 1, wherein in step (3), the stirring is at a speed of 500 r / min at room temperature for 10 min. 7.The method of claim 1, wherein in step (4), the temperature of the vacuum rotary evaporation is 20-40℃. 8.The method of claim 7, wherein in step (4), the temperature of the vacuum rotary evaporation is 30℃. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

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