Preparation method of lung-targeted hemoglobin nanoparticles, drug-loaded hemoglobin nanoparticle solution and application of drug-loaded hemoglobin nanoparticle solution

Abstract

The invention discloses a preparation method of lung-targeted hemoglobin nanoparticles, a drug-loaded hemoglobin nanoparticle solution and application, and relates to the technical field of nano medicine. The hemoglobin with a lung targeting function is used as a drug carrier, and the hemoglobin is improved to have a drug loading function and can be used for loading drug active substances to act on the lung, so that the drug active substances can realize accurate lung targeting therapy, the bioavailability of the drug is improved, and the dosage is reduced. The drug-loaded hemoglobin nanoparticle solution provided by the invention belongs to an antibiotic-hemoglobin nanoparticle compound drug delivery system, and has the advantages of increasing the distribution of antibiotics in targeted lung tissues and improving the availability. The preparation method of the lung-targeted hemoglobin nanoparticles provided by the invention is simple and good in repeatability, and industrial production is easy to realize.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a preparation method of lung-targeted hemoglobin nanoparticles, a drug-loaded hemoglobin nanoparticle solution and applications. Background Art

[0002] Existing antibiotics such as tigecycline and polymyxin lack tissue distribution specificity in the human body, so increased dosage is required to increase drug concentration in lung tissue. At the same time, due to the evolutionary expression of efflux pump resistance genes, drugs that enter the cells of Klebsiella pneumoniae are excreted, limiting the effectiveness of the drugs. More drugs need to be delivered into the bacteria to achieve a bactericidal / inhibitory effect.

[0003] Hemoglobin nanoparticles are nanoscale particles developed using the properties of hemoglobin (Hb). They have broad application prospects in the fields of medicine and biotechnology, for example, by using hemoglobin nanoparticles for oxygen delivery, biomedical imaging, drug carriers or biosensors.

[0004] In the field of nanomedicine, the application trend of protein nanoparticles is developing continuously, among which hemoglobin nanoparticles are one of the research hotspots. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a lung-targeted hemoglobin nanoparticle, a preparation method and a lung-targeted drug.

[0006] In order to solve the above problems, the present invention proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing lung-targeted hemoglobin nanoparticles, comprising the following steps: weighing a preset mass of hemoglobin, adding ultrapure water, stirring evenly and then ultrasonicating to obtain a solution containing lung-targeted hemoglobin nanoparticles of a preset concentration.

[0008] A further technical solution is that in the solution containing lung-targeted hemoglobin nanoparticles, the concentration of hemoglobin nanoparticles is 2.5-80 μM.

[0009] A further technical solution is that the particle size of the hemoglobin nanoparticles is 150-300nm.

[0010] A further technical solution is that the polydispersity index of the solution containing lung-targeted hemoglobin nanoparticles is less than 0.3.

[0011] A further technical solution is that the ultrasonic condition is 330-400W for 2-8min.

[0012] It can be understood that in other technical solutions, the above preparation method also includes filtering with a water filter after ultrasound to obtain a solution containing lung-targeted hemoglobin nanoparticles.

[0013] In a second aspect, the present invention also provides the use of the lung-targeted hemoglobin nanoparticles prepared by the preparation method described in the first aspect in the preparation of drugs for treating lung infectious diseases.

[0014] In a third aspect, the present invention provides a method for preparing a drug-loaded hemoglobin nanoparticle solution, comprising the following steps:

[0015] Obtaining a solution containing lung-targeted hemoglobin nanoparticles according to the preparation method described in the first aspect;

[0016] Dissolving the active pharmaceutical ingredient in ultrapure water and mixing well to obtain a pharmaceutical solution of a preset concentration;

[0017] The solution containing the lung-targeted hemoglobin nanoparticles is evenly mixed with the drug solution, subjected to ultrasound, filtered with a water filter, and subjected to ultrafiltration centrifugation using an ultrafiltration tube to obtain a drug-loaded hemoglobin nanoparticle solution.

[0018] A further technical solution is that the pharmaceutically active substance includes tigecycline, polymyxin or other pharmaceutically active substances that can act on Klebsiella.

[0019] A further technical solution is that the ultrasonic condition is 330-400W for 2-8min.

[0020] A further technical solution is that the pore size of the water filter head is less than 0.6 μm, preferably 0.45 μm.

[0021] A further technical solution is that the molecular weight cutoff value of the ultrafiltration tube is 10 kDa.

[0022] A further technical solution is that the ultrafiltration centrifugation conditions are 4800-5200 rpm, 45-75 min.

[0023] In a fourth aspect, the present invention also provides a drug-loaded hemoglobin nanoparticle solution prepared by the preparation method of the third aspect.

[0024] In a fifth aspect, the present invention also provides the use of the drug-loaded hemoglobin nanoparticle solution prepared by the preparation method of the third aspect, or the drug-loaded hemoglobin nanoparticle solution provided by the fourth aspect in the preparation of drugs for treating lung infectious diseases.

[0025] A further technical solution is that the pulmonary infectious disease includes Klebsiella pneumoniae infection.

[0026] Compared with the prior art, the technical effects achieved by the present invention include:

[0027] The present invention utilizes hemoglobin with lung targeting function as a drug carrier, and improves it to have the function of loading drugs. It can be used to load active pharmaceutical ingredients to act on the lungs, so that the active pharmaceutical ingredients can achieve precise lung targeting treatment, improve the bioavailability of the drug, and reduce the usage.

[0028] The drug-loaded hemoglobin nanoparticle solution provided by the present invention belongs to an antibiotic-hemoglobin nanoparticle complex drug delivery system, and has the advantages of increasing the distribution of antibiotics in targeted lung tissues and improving utilization.

[0029] The preparation methods of the lung-targeted hemoglobin nanoparticles and the drug-loaded hemoglobin nanoparticle solution provided by the present invention are simple and have good repeatability, and are easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The zeta potential diagram of HBNP and TIG-HBNP prepared in Example 1 of the present invention.

[0031] Figure 2 This is the particle size distribution diagram of HBNP and TIG-HBNP prepared in Example 1 of the present invention.

[0032] Figure 3 This is a transmission electron microscope (TEM) image of TIG-HBNP prepared in Example 1 of the present invention (scale bar: 1000 / 200 nm).

[0033] Figure 4 The stability test results of TIG-HBNP prepared in Example 1 of the present invention are shown in the figure on the left; the stability results at 4°C and the stability results at 37°C are shown in the figure on the right, and n=3.

[0034] Figure 5 The results of the targeting study of hemoglobin nanoparticles and serum protein nanoparticles in Example 3 of the present invention in the lung tissue of pneumonia model mice; in the figure: ICG-HBNP refers to hemoglobin nanoparticles labeled with ICG; ICG-BSANP refers to serum protein nanoparticles labeled with ICG; ICG-Free refers to free ICG; ns, no significant difference; **, P<0.01; ****, P<0.0001.

[0035] Figure 6 This is a comparison diagram of various organs of mice in each group in the experiment of Example 4 of the present invention.

[0036] Figure 7 This is a comparison chart of the number of colony counts in the lung tissues of each group of mice in the experiment of Example 4 of the present invention.

[0037] Figure 8This is the evaluation result of the colony count of lung tissue of each group of mice in the experiment of Example 4 of the present invention. In the figure, +++ indicates strong positive (colony count > 10^3), ++ indicates moderate positive (10^3 > colony count > 10^1), + indicates positive (10^1 > colony count > 1), and - indicates negative. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0040] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0041] Example 1 Preparation of drug-loaded hemoglobin nanoparticle solution

[0042] (1) Screening the concentration of hemoglobin nanoparticles in the drug-loaded hemoglobin nanoparticle solution

[0043] Six hemoglobin concentration gradient groups in the range of 2.5μM-80μM were set up to prepare hemoglobin nanoparticles (HBNP). The specific operation is as follows: 0.161, 0.322, 0.645, 1.29, and 5.16 mg of hemoglobin (molecular mass 64.5kDa) were weighed respectively, added to 1 mL of ultrapure water, stirred evenly, and ultrasonicated (360W) for 5 minutes, filtered with a water filter (pore size 0.45μm), and the particle size and dispersion (PDI) were determined. The success of the preparation method was preliminarily judged by measuring the particle size and dispersion (PDI) of HBNP. From the experimental results, when the concentration of the hemoglobin solution was 10μM, the obtained HBNP particle size was about 200nm and the polydispersity index (PDI) was less than 0.3 (see Table 1), which was consistent with the expected experimental goals.

[0044] Table 1 Effects of different concentrations of hemoglobin (HB) on the particle size and polydispersity index of hemoglobin nanoparticles

[0045]

[0046]

[0047] (2) Screening of the feed ratio in the drug-loaded hemoglobin nanoparticle solution

[0048] Six feed ratio groups ranging from 10% to 60% (mass percentage of hemoglobin to drug) were set up to prepare tigecycline-hemoglobin nanoparticles (TIG-HBNP).

[0049] The specific operation is as follows: weigh 12.9 mg of hemoglobin, add 18 mL of ultrapure water, stir evenly and then ultrasonicate (360 W) for 5 min. Subsequently, weigh tigecycline (1.29, 2.58, 3.87, 5.16, 6.45, 7.74 mg) and dissolve them in 2 mL of ultrapure water. Mix the above two liquids evenly and then ultrasonicate (360 W) for 5 min. Filter with a water filter (pore size 0.45 μm), and use an ultrafiltration tube (20 mL, molecular weight cutoff value 10 kDa) for ultrafiltration centrifugation (5000 rpm) for 1 h to remove unencapsulated free drugs and obtain a drug-loaded hemoglobin solution.

[0050] The drug concentration in the lower layer of liquid after ultrafiltration is the free drug (the concentration of tigecycline in the medium is measured using an ultraviolet spectrophotometer), which is converted to the drug concentration in the drug-loaded hemoglobin solution, thereby calculating the drug encapsulation rate. The formula for calculating the encapsulation rate and drug loading is as follows:

[0051] Encapsulation efficiency = (W drug - W free) / W drug × 100%;

[0052] Drug loading = (W drug - W free) / [(W drug - W free) + W total)] × 100%;

[0053] W drug: total amount of drug input;

[0054] W free: amount of unentrapped drug;

[0055] Wtotal: The total amount of carrier input.

[0056] The success of the preparation method was preliminarily determined by measuring the particle size and dispersion (PDI) of TIG-HBNP. From the experimental results, when the feed ratio was 40% (TIG / HB), the obtained TIG-HBNP particle size was about 200nm, the encapsulation efficiency reached about 60%, and the drug loading was close to 20% (see Table 2), which was consistent with the expected experimental goals.

[0057] Table 2 Effects of different feed ratios (TIG / HB) on particle size, encapsulation efficiency and drug loading of tigecycline-hemoglobin nanoparticles

[0058]

[0059] Through the above process screening and optimization, the optimal preparation process of tigecycline-hemoglobin nanoparticles was determined as follows:

[0060] ① Preparation of HBNPs: Prepare a 10 μM hemoglobin nanoparticle solution and sonicate (360 W) for 5 min;

[0061] ② Preparation of TIG-HBNP: Prepare tigecycline solution, mix the hemoglobin nanoparticle solution and the tigecycline solution evenly according to the feed ratio of 40% (TIG / HB), ultrasonicate (360W) for 5min, filter with a water filter (pore size 0.45μm), and then ultrafiltration centrifuge (5000g, molecular weight cutoff 10kDa) for 1h to remove unencapsulated free drugs to obtain tigecycline-hemoglobin nanoparticle (TIG-HBNP) solution.

[0062] The specific operation of preparing the drug-loaded hemoglobin nanoparticle solution in an optimal embodiment of the present invention is as follows: weigh 12.9 mg of hemoglobin, add 18 mL of ultrapure water, stir evenly and ultrasonicate (360 W) for 5 min. Subsequently, weigh 5.2 mg of tigecycline and dissolve it in 2 mL of ultrapure water. Mix the above two liquids evenly and ultrasonicate (360 W) for 5 min. Filter with a water filter (pore size 0.45 μm), and use an ultrafiltration tube (20 mL, molecular weight cutoff value 10 kDa) for ultrafiltration centrifugation (5000 rpm) for 1 hour to remove the unencapsulated free drug and obtain a drug-loaded hemoglobin nanoparticle solution, hereinafter referred to as "tigecycline-hemoglobin nanoparticle (TIG-HBNP) solution".

[0063] The following examples all use the tigecycline-hemoglobin nanoparticles prepared by the above-mentioned preparation process for characterization and related research.

[0064] Example 2: Characterization of Tigecycline-Hemoglobin Nanoparticles

[0065] The prepared tigecycline-hemoglobin nanoparticle solution was characterized, and the particle size, polydispersity index (PDI) and Zeta potential of the tigecycline-hemoglobin nanoparticles in the tigecycline-hemoglobin nanoparticle solution were analyzed using a Malvern laser particle size analyzer. The results showed that the particle size was 192.82±7.13nm, the PDI value was 0.266±0.011, and the Zeta potential was -8.69±0.94mV (see Table 3, Figure 1 , Figure 2 ); The morphology of tigecycline-hemoglobin nanoparticles was observed using transmission electron microscopy (TEM) (see Figure 3At the same time, the storage stability of tigecycline-hemoglobin nanoparticles at 4°C for 7 days and 37°C for 24 hours was evaluated by examining the change in particle size (see Figure 4 ).

[0066] from Figure 4 The results show that the particle size of the tigecycline-hemoglobin nanoparticles in the tigecycline-hemoglobin nanoparticle solution prepared by the optimal preparation process is relatively stable during the test, indicating that the prepared tigecycline-hemoglobin nanoparticles have good stability.

[0067] Table 3 Particle size, polydispersity index and Zeta potential distribution of hemoglobin nanoparticles (HBNP) and tigecycline-hemoglobin nanoparticles (TIG-HBNP)

[0068]

[0069] Example 3: Evaluation of the targeting effect of hemoglobin nanoparticles in animal lung tissue by whole body fluorescence imaging system (FOBI)

[0070] To confirm the specific distribution of the hemoglobin nanoparticles prepared in the embodiment of the present invention to the lesion tissue of Klebsiella pneumoniae, the present embodiment prepares serum protein nanoparticles with the same preparation process as a control, and adopts indocyanine green (ICG) to mark hemoglobin (HB) and serum albumin (BSA) respectively, according to the above-mentioned optimal preparation process: weigh 12.9 mg of hemoglobin, add 18 mL of ultrapure water, stir evenly, and then ultrasonically (360 W) 5 min) to make hemoglobin nanoparticles, and add indocyanine green to mark. The preparation method of serum protein nanoparticles is the same.

[0071] The ICG-HBNP group, ICG-BSANP group, and ICG-Free group were injected into pneumonia model mice through the tail vein, and the mice were dissected 6 hours later. The fluorescence signal accumulation in the lung tissue was imaged and analyzed using the whole body fluorescence imaging system (FOBI). The results showed that the fluorescence accumulation in the ICG-HBNP group was more, and was significantly higher than that in the ICG-BSANP group and the ICG-Free group; the higher the degree of infection in the lung tissue, the higher the fluorescence intensity in the ICG-HBNP group (see Figure 5 ). This indicates that the hemoglobin nanoparticles prepared in the embodiment of the present invention have a strong lung tissue targeting ability against Klebsiella pneumoniae in vivo.

[0072] Example 4: Lung-targeted therapeutic effect of tigecycline-hemoglobin nanoparticles in treating Klebsiella pneumoniae-infected mice

[0073] Thirty mice were randomly divided into five groups (n=6 in each group): control group (control), model group (model), tigecycline treatment group (ITG), hemoglobin nanoparticle treatment group (HBNP), and tigecycline-hemoglobin nanoparticle treatment group (ITG-HBNP). All mice were anesthetized by inhalation of chloroform. After anesthesia, the mice were kept in a standing position with their heads up. 40 μL of Klebsiella pneumoniae suspension (concentration of 3×10^7 CFU·mL^-1) was inoculated into the lungs by nasal instillation; the control group mice received an equal volume of PBS buffer in the same way. After inoculation, the mice continued to keep the head-up position for 20 seconds, and then the mice were rotated. After waking up, the mice were placed in cages and allowed to eat freely. One hour after modeling, the control group and model group were given 200 μL PBS buffer per mouse through the tail vein. The tigecycline treatment group (ITG) and the tigecycline-hemoglobin nanoparticle treatment group (ITG-HBNP) were given 10 mg / kg (volume of about 200 μL) according to the tigecycline content, and the hemoglobin nanoparticle treatment group (HBNP) was given an equal volume of PBS buffer. The mice were observed for 7 consecutive days, and the criterion for successful modeling was based on the appearance of the following clinical symptoms: after inoculation of the bacterial solution, the mice showed varying degrees of increased respiratory rate, decreased activity, erect or messy hair, and increased secretions around the eyes. These symptoms reflect the successful modeling of mice infected with Klebsiella pneumoniae.

[0074] The mice were treated for 7 consecutive days and observed daily. The dead mice were dissected and the right lung tissue was taken for colony counting experiment. The mice that did not die after 7 days of treatment were euthanized and the right lung tissue was taken for colony counting experiment. The specific operation steps of the lung tissue colony counting experiment were as follows: sterile thoracotomy was performed, the lung tissue was removed and rinsed with sterile saline; the right lung tissue was weighed after absorbing the surface water, and it was placed in 1mL PBS for homogenization, and 100μL was taken and smeared on MacConkey agar plates, and evenly spread with glass beads; the plates were incubated at 37℃ for 24h, and then the bacteria were counted.

[0075] Dissection shows that Figure 6 As shown in the figure, Klebsiella pneumoniae infection caused severe lung tissue damage, obvious swelling, and severe bleeding in the model group mice. The lung tissue damage of mice in the tigecycline group and the tigecycline-hemoglobin nanoparticle group was significantly alleviated. At the same time, the results of lung tissue colony counts showed that Figure 7 As shown, the number of colonies in the lung tissue of mice in the model group was significantly higher than that in the control group and the tigecycline treatment group, and the infection was severe. The number of colonies in the lung tissue of mice in the tigecycline-hemoglobin nanoparticle group was significantly lower than that in the tigecycline group and the hemoglobin nanoparticle group, which was consistent with the control group. The results show that the tigecycline-hemoglobin nanoparticles prepared by the present invention can improve the therapeutic effect of tigecycline, such as Figure 8As shown, the number of colonies in mouse lung tissue was reduced.

[0076] It should be noted that the above examples use tigecycline-hemoglobin nanoparticles as an example to explore the therapeutic effect of the drug-loaded hemoglobin nanoparticles prepared by the present invention on lung targeting. The hemoglobin nanoparticles of the present invention are used to encapsulate other drug active substances targeting the lungs for use in therapeutic drugs for the lungs, which are all within the scope of protection of the present invention.

[0077] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0078] The above is a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed by the present invention, and these modifications or replacements should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A method for preparing lung-targeted hemoglobin nanoparticles, characterized in that: The method comprises the following steps: weighing a preset mass of hemoglobin, adding ultrapure water, stirring evenly and then ultrasonicating to obtain a solution containing lung-targeted hemoglobin nanoparticles with a preset concentration.

2. The preparation method according to claim 1, characterized in that In the solution containing lung-targeted hemoglobin nanoparticles, the concentration of hemoglobin nanoparticles is 2.5-80 μM.

3. The preparation method according to claim 1, characterized in that: The particle size of the hemoglobin nanoparticles is 150-300nm.

4. The preparation method according to claim 1, characterized in that: The polydispersity index of the solution containing lung-targeted hemoglobin nanoparticles is less than 0.

3.

5. The preparation method according to claim 1, characterized in that: The ultrasonic conditions are 330-400W, 2-8min.

6. Use of the lung-targeted hemoglobin nanoparticles prepared by the preparation method according to any one of claims 1 to 5 in the preparation of drugs for treating lung infectious diseases.

7. A method for preparing a drug-loaded hemoglobin nanoparticle solution, characterized in that: The following steps are involved: A solution containing lung-targeted hemoglobin nanoparticles is obtained according to any one of the preparation methods of claims 1 to 5; Dissolving the active pharmaceutical ingredient in ultrapure water and mixing well to obtain a pharmaceutical solution of a preset concentration; The solution containing the lung-targeted hemoglobin nanoparticles is evenly mixed with the drug solution, subjected to ultrasound, filtered with a water filter, and subjected to ultrafiltration centrifugation using an ultrafiltration tube to obtain a drug-loaded hemoglobin nanoparticle solution.

8. The preparation method according to claim 7, characterized in that: The pharmaceutically active substances include tigecycline, polymyxin or other pharmaceutically active substances that can act on Klebsiella.

9. A drug-loaded hemoglobin nanoparticle solution, characterized in that: Prepared by the preparation method described in claim 7 or 8.

10. Use of the drug-loaded hemoglobin nanoparticle solution prepared by the preparation method according to claim 7 or 8, or the drug-loaded hemoglobin nanoparticle solution according to claim 9 in the preparation of a drug for treating pulmonary infectious diseases, wherein the pulmonary infectious diseases include Klebsiella pneumoniae infection.

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