Polyphenol / amine complex, lung-targeted delivery system and construction method and application thereof

Abstract

The application discloses a polyphenol / amine complex, a lung-targeted delivery system and a construction method and application thereof, and belongs to the technical field of medicines.The complex is prepared by condensation of polyphenol and amine and aldehyde, has good biocompatibility, and a nano delivery system constructed from the complex or a pharmaceutically acceptable form thereof can load various hydrophilic and hydrophobic functional molecules, such as drugs, tracers and fluorescent probes; when the polyphenol / amine complex is applied to guest molecule delivery, the polyphenol / amine complex has excellent lung targeting and high in-vivo delivery efficiency, can efficiently deliver drugs, probes and other guest molecules to the lung, significantly improves the signal-to-noise ratio of a lung disease detection probe and the treatment effect of drugs, and has important application value for precise diagnosis and treatment of lung diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to polyphenol / amine complexes, lung-targeted delivery systems, their construction methods, and applications. Background Technology

[0002] Respiratory diseases impose a significant health burden worldwide. The accurate diagnosis and treatment of lung diseases remain extremely challenging due to the non-specific distribution of therapeutic agents such as tracers and drugs within the body. The development of nanotechnology and nanomedicine offers a promising approach to addressing these issues. Targeted modification of nanoparticles can enhance the accumulation of tracers or drugs in the lungs, thereby improving the diagnostic signal-to-noise ratio and therapeutic efficacy. However, once these targeted nanoparticles enter the body, the targeting efficiency is significantly reduced because the targeting ligand is masked by the body's protein corona.

[0003] By modulating the chemical composition of nanodelivery systems, endogenous lung targeting can be achieved, avoiding off-target effects caused by ligand masking. In recent years, various lipid-based nanodelivery material formulations with lung-targeting capabilities have been developed, demonstrating significant advantages in the field of lung-targeted delivery. However, current research mainly focuses on the modification of LNP-based formulations, with nucleic acid drugs being the primary delivery guest molecules.

[0004] Breaking through LNP formulations and expanding the range of guest molecules, developing novel lung-targeted delivery systems capable of loading multiple guest molecules for the diagnosis and treatment of lung diseases is of great significance. Summary of the Invention

[0005] One of the objectives of this invention is to provide a polyphenol / amine complex to solve the above-mentioned problems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a polyphenol / amine complex, wherein the polyphenol / amine complex is obtained by condensation reaction of polyphenols, amines and aldehydes, and then precipitation from the aqueous phase, wherein the amine is selected from glutathione and penicillamine.

[0007] The polyphenols mentioned, according to CN111888481A, can be tea polyphenols, phloroglucinol, anthocyanins, tannic acid, etc., and the tea polyphenols can be epicatechin, gallate, catechin, epicatechin gallate, epigallocatechin gallate, epigallocatechin and gallocatechin gallate, etc. The aldehydes mentioned, according to CN110623937A, can be formaldehyde, acetaldehyde or glutaraldehyde, or aldehydes produced by the decomposition of other polymers during the preparation process, such as compounds containing aldehyde groups produced by the decomposition of paraformaldehyde or hexamethylenetetramine.

[0008] This invention utilizes the preparation methods disclosed in Chinese patent applications CN110623937A and CN111888481A to obtain polyphenol / amine complexes and methods for preparing nano-formulations using them as delivery systems. It has discovered chemical components with lung-targeting properties, which have broad application prospects in the precise diagnosis and treatment of lung diseases.

[0009] A second objective of this invention is to provide a lung-targeted delivery system constructed from the aforementioned polyphenol / amine complex, wherein the lung-targeted delivery system is constructed by loading the polyphenol / amine complex with a drug, probe, or tracer.

[0010] As a preferred technical solution, the drug is selected from one of cisplatin, carboplatin, pemetrexed, etoposide, gemcitabine, docetaxel, paclitaxel, pembrolizumab, bevacizumab, streptomycin, amikacin, capreomycin, ceftazidime, penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, and dexamethasone.

[0011] As a preferred technical solution, the probe is selected from one of the DCFH-DA probe, Rod-Cl, and Rod-Br probe.

[0012] As a preferred technical solution, the tracer is selected from indolecyanate green and Cy5.

[0013] A third objective of this invention is to provide a method for constructing the aforementioned lung-targeted delivery system, wherein when the polyphenol / amine complex is loaded with a lipid-soluble drug, probe, or tracer, the construction method is as follows:

[0014] The polyphenol / amine complex is prepared into a solution using an organic solvent, and a lipid-soluble drug or probe is prepared into a solution using an organic solvent. The two solutions are thoroughly mixed and then rapidly mixed with the assembly solution using microfluidic technology, a pipette, or an extruder at a volume ratio of 1:1 to 1:10. After mixing, the mixture is centrifuged and washed with water to obtain a lung-targeting nanomedicine, a lung-targeting probe, or a lung-targeting tracer. The assembly solution is water, physiological saline, or a glucose solution for injection.

[0015] The fourth objective of this invention is to provide a method for constructing the above-mentioned lung-targeted delivery system, wherein when the polyphenol / amine complex is loaded with a water-soluble drug, probe, or tracer, the construction method is as follows:

[0016] The water-soluble drug, probe, or tracer is dissolved in the assembly solution, and then the polyphenol / amine complex solution and the assembly solution are rapidly mixed using microfluidic technology, a pipette, or an extruder. The mixture is then centrifuged and washed with water to obtain a lung-targeting nanodrug, a lung-targeting probe, or a lung-targeting tracer. The assembly solution is water, physiological saline, or a glucose solution for injection.

[0017] Alternatively, the water-soluble drug, probe, or tracer can be added during the synthesis of the polyphenol / amine complex. After the reaction is complete, the mixture is centrifuged and washed with water to obtain a lung-targeting nanodrug, a lung-targeting probe, or a lung-targeting tracer.

[0018] The fifth objective of this invention is to provide the application of the above-mentioned lung-targeted delivery system in the preparation of drugs for targeted treatment of lung diseases, wherein the amine in the lung-targeted delivery system is selected from one of valine, cysteine, glutathione, asparagine, penicillamine, glutamine, methionine, and at least one of polypeptides or polymers containing the above-mentioned amines.

[0019] As a preferred technical solution, the lung disease is one of the following: infectious / non-infectious pneumonia, lung cancer, pulmonary tuberculosis, pulmonary fibrosis, COPD, pulmonary hypertension, or asthma.

[0020] The sixth objective of this invention is to provide the application of the above-mentioned lung-targeted delivery system in the preparation of diagnostic reagents targeting the lungs, wherein the amine in the lung-targeted delivery system is selected from one of valine, cysteine, glutathione, asparagine, penicillamine, glutamine, and methionine, as well as at least one of polypeptides and polymers containing the above-mentioned amines.

[0021] The lung-targeting polyphenol / amine complex in this invention has a negatively charged surface, solving the potential toxicity problem caused by the positive charge of cationic liposomes in existing lung-targeting LNP formulations. Furthermore, the lung-targeting polyphenol / amine complex of this invention can efficiently load a variety of clinically commonly used respiratory drugs, while the types of nucleic acid drugs delivered by LNP delivery systems currently approved for clinical use are extremely limited, and there are currently no nucleic acid drugs on the market for lung diseases. Compared to lung-targeting LNP delivery systems, the lung-targeting polyphenol / amine complex of this invention has a wider range of applications and greater potential for translational applications.

[0022] Compared with the prior art, the advantages of the present invention are as follows: When the polyphenol / amine complex provided by the present invention is applied to the delivery of guest molecules such as drugs, probes, and tracers, it has excellent lung targeting and high in vivo delivery efficiency. It can efficiently deliver guest molecules such as drugs and probes to the lungs, significantly improve the signal-to-noise ratio of lung disease detection probes, improve the therapeutic effect of drugs, and has good biocompatibility, no toxic side effects on organs or the circulatory system, and high safety. It has important application value for the accurate diagnosis and treatment of lung diseases. Attached Figure Description

[0023] Figure 1 Images A, B, and C are transmission electron microscope (TEM) images of nanoparticles assembled from polyphenol / amine complexes synthesized from valine, cysteine, and glutathione, respectively.

[0024] Figure 2 The particle size distribution and polydispersity index (PDI) of the EGCG / cysteine ​​nano-ICG prepared in Example 3 are shown.

[0025] Figure 3 The image shows the morphology of the EGCG / cysteine ​​nano-ICG prepared in Example 3.

[0026] Figure 4 Distribution diagrams of ICG alone and lung-targeting ICG prepared in Example 3 in different organs of mice;

[0027] Figure 5 Figure A shows the simple ICG, and Figure B shows the quantitative distribution of lung-targeted ICG prepared in Example 3 in different organs of mice.

[0028] Figure 6 The effect of different experimental groups on mouse body weight;

[0029] Figure 7 The lung-targeting ROS probe prepared in Example 7 exhibits luminescence response in different organs of mice with acute lung injury.

[0030] Figure 8 The effect of lung-targeted chemotherapy drugs in Example 8 on the treatment of lung metastases in mice. Detailed Implementation

[0031] The present invention will now be described in more detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The preparation of polyphenol / amine complexes, and the preparation of polyphenol / amine complexes loaded with drugs, probes, or tracers, are all based on the methods disclosed in CN110623937A and CN111888481A.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] The mice used in this invention are male Balb / c mice (Jicui Yaokang), which are well known in the art.

[0035] Example 1

[0036] Synthesis of different polyphenol / amine complexes

[0037] In this embodiment, epigallocatechin gallate (EGCG) is used as the polyphenol raw material, formaldehyde is used as the aldehyde raw material, and different polyphenol / amine complexes are prepared using different amines. The specific method is as follows:

[0038] EGCG aqueous solution and amine aqueous solution were prepared separately. The two solutions were mixed with formaldehyde solution and then sealed and stirred. The raw material ratio and process parameters are shown in Table 1. The mass ratio of polyphenol:amine:formaldehyde is 10:4:4. After stirring and reacting at room temperature for 12 hours, the mother liquor after reaction was centrifuged at high speed (10,000 rpm, 10 minutes) for solid-liquid separation. The solid product, namely polyphenol / amine complex, was washed and collected.

[0039] Table 1. Raw materials, proportions, and processes for different composites

[0040]

[0041] Example 2

[0042] Characterization of nanoparticles constructed from polyphenol / amine complexes

[0043] The particle size and polydispersity index (PDI) of the polyphenol / amine complex prepared in Example 1 were measured using a nanolaser particle size analyzer (Malvin, UK, ZEN3690), and the results are shown in Table 2. The morphology of the nanoparticles assembled from the polyphenol / amine complex prepared with valine, cysteine, and glutathione was characterized using transmission electron microscopy (TEM, JEOL, Japan). Figure 1 . Figure 1 In the table, A, B, and C represent the morphologies of nanoparticles assembled from polyphenol / amine complexes synthesized from valine, cysteine, and glutathione, respectively. It can be seen that all three complexes produce spherical nanoparticles after assembly, with particle sizes consistent with the hydrated particle sizes measured in Table 2.

[0044] Table 2. Particle size and PDI of different polyphenol-amine complexes

[0045]

[0046] Example 3

[0047] Preparation of polyphenol / amine nanotracers by loading near-infrared fluorescent molecules onto polyphenol / amine complexes

[0048] The preparation of polyphenol / amine nanotracers using indolecyanine green (ICG), a clinically used near-infrared fluorescent molecule, as the guest molecule is as follows:

[0049] (1) The ten different polyphenol / amine complexes prepared in Example 1 were dissolved in ethanol with a mass concentration of 30% to form a complex solution with a concentration of 20 mg / mL, and an ICG aqueous solution with a water concentration of 5 mg / mL was prepared by deionization.

[0050] (2) Dilute the ICG solution 3-15 times with water as the assembly solution, mix it quickly and evenly at room temperature and pressure according to the mass ratio of polyphenol / amine complex to ICG of 10:1, let it stand for 0.5 hours, centrifuge at high speed (10000 rpm, 10 min), wash and remove unassembled complex, free ICG molecules and ethanol to obtain ten different polyphenol / amine nano ICG.

[0051] Example 4

[0052] Characterization of polyphenol / cysteine ​​nano-ICG

[0053] Taking the polyphenol / amine complex synthesized from cysteine ​​as an example (i.e., EGCG / cysteine ​​nano-ICG), the particle size distribution and polydispersity index (PDI) of the EGCG / cysteine ​​nano-ICG were detected using a nano-laser particle size analyzer (Malvin, UK, ZEN3690). The results are as follows: Figure 2 , Figure 2 The particle size was 73.42 nm, and the PDI was 0.159. Its morphology was observed using transmission electron microscopy (TEM, JEOL, Japan), and the results are as follows. Figure 3 Compared to Figure 1 The EGCG / cysteine ​​nanoparticles, after being loaded with ICG, showed a significant reduction in particle size and a more irregular morphology.

[0054] Example 5

[0055] In vivo delivery efficiency of polyphenol / amine nano-ICG

[0056] Healthy Balb / c mice were selected and injected intravenously via the tail vein with lung-targeting nano-ICG prepared in Example 3 at a dose of 1 mg / kg. The control group received the same dose of ICG, meaning the ICG was not loaded with a polyphenol / amine complex. Twelve hours later, the mice were euthanized, and their major organs—heart, liver, kidney, lung, and spleen—were dissected. Chemiluminescence imaging of the major organs was performed using the IVIS small animal in vivo imaging system (PerkinElmer). The distribution of different polyphenol / amine complexes as delivery materials for near-infrared fluorescent ICG molecules in various organs after intravenous injection into mice showed significant differences. The average fluorescence intensity distribution in each organ is shown in Table 3. ICG not loaded with a polyphenol / amine complex was mainly distributed in the liver, followed by the kidney. The ratio of the average fluorescence intensity of the liver to that of the lung (liver / lung) was 2.31. Using polyphenol / amine complexes as delivery materials altered the distribution of ICG in vivo. Complexes synthesized from valine, cysteine, aspartic acid, glutamine, methionine, penicillamine, and glutathione increased ICG fluorescence intensity in the lungs and decreased it in the liver. The cysteine-based delivery material showed the highest lung targeting efficiency, with a liver / lung ratio of 0.43. The liver / lung ratios for phenylalanine and alanine were similar to those for ICG, at 2.50 and 2.34, respectively. Isoleucine, however, increased the liver / lung ratio of ICG to 3.47.

[0057] It is evident that not all polyphenol / amine complexes synthesized in Example 1 exhibit lung-targeting properties; only the complexes synthesized from valine, cysteine, asparagine, glutamine, methionine, penicillamine, and glutathione exhibit lung-targeting properties.

[0058] Table 3. Mean fluorescence intensity ratio of liver / lung for different complexes

[0059]

[0060] Taking the lung-targeting nano-ICG prepared in Example 3 as an example, its lung-targeting efficiency was tested. ICG signal intensity in different organs was observed using a small animal in vivo imaging system 12, 24, and 48 hours after injection. The results are as follows: Figure 4 As shown in the figure, the relative fluorescence signal in the lungs of mice in the lung-targeted nano-ICG group was enhanced, while the relative fluorescence intensity in the liver was weakened. The statistical results of the average fluorescence intensity percentage of each organ in the mice are shown below. Figure 5 As shown. Figure 5 Figure A shows the quantitative distribution of ICG in different organs of mice. The average fluorescence intensity of ICG in the liver was 44.81%, and the average fluorescence intensity in the lungs was 19.41%. The average fluorescence intensity ratio of the lungs to the liver was 0.433. Figure 5Figure B shows the quantitative distribution of lung-targeted ICG prepared in Example 3 in different organs of mice. In Figure B, the lung-targeted delivery system reduced the average fluorescence intensity of ICG in the liver to 35.02% and increased the average fluorescence intensity in the lungs to 44.10%, which is more than twice the 19.41%. The average fluorescence intensity ratio of the lungs to the liver increased to 1.26, which is nearly three times the 0.433, demonstrating its highly efficient lung-targeted delivery.

[0061] Example 6

[0062] Effects of lung-targeting polyphenol / glutamine complex on body weight changes in mice

[0063] Eight-week-old male Balb / c mice were randomly divided into three groups (n=5): a control group (glucose group), a polyphenol group (EGCG group), and a lung-targeted EGCG / glutamine group (lung-targeted polyphenol / amine group). The control group was injected with glucose solution, while the polyphenol and lung-targeted polyphenol / glutamine complex groups were injected with polyphenol and polyphenol / glutamine nanoparticles, respectively. The dosage of polyphenol was 25 mg / kg. The safety of polyphenol / glutamine nanoparticles was initially assessed by intravenous injection. Injections were administered via tail vein every 3 days, on days 1, 4, 7, and 10, for a total of 4 injections. Mouse weight data were recorded. Results are as follows: Figure 6 As shown, there was no significant difference in weight change among the three groups, demonstrating that lung-targeted EGCG / glutamine nanoparticles have good biocompatibility and no toxic side effects on organs or the circulatory system.

[0064] Example 7

[0065] Application of Lung-Targeted Polyphenol / Glutamine Nanodelivery System in the Detection of Acute Lung Injury

[0066] The lung-targeting polyphenol / glutamine complex prepared in Example 1 was used as a delivery system loaded with a ROS-responsive fluorescent probe for ROS detection in a mouse model of acute lung injury.

[0067] The ROS probe used in this embodiment is the Rod-Br probe, which was prepared by the inventors with reference to the literature "Monitoring Endoplasmic Reticulum Peroxynitrite Fluctuations in Primary Tendon-Derived Stem Cells and Insights into Tendinopathy, ACS Sens. 2024, 9, 12, 6750–6758".

[0068] The loading and performance testing steps for the ROS probe are as follows:

[0069] (1) Dissolve the EGCG / glutamine complex solution with 30% ethanol to form a concentration of 20 mg / mL. Prepare a ROS probe solution with a concentration of 5 mg / mL using ethanol solvent. Mix the two solutions in a volume ratio of 10:1, then quickly mix with the assembly solution water. After standing for 0.5 hours, centrifuge and wash with water to obtain the lung-targeting ROS probe.

[0070] (2) Prepare a 2.5 mg / mL LPS solution with PBS, anesthetize mice with isoflurane, and then drip 50 μL LPS solution into the trachea to construct a mouse acute lung injury model;

[0071] (3) 24 hours after the establishment of the mouse acute lung injury model, the lung-targeting ROS probe prepared in step (1) was injected intravenously, and the ROS probe molecule was used as a control.

[0072] (4) Mice were euthanized 1 hour and 24 hours after ROS probe injection. Liver, lungs, kidneys, spleen, and heart were dissected and used for imaging. The results are as follows: Figure 7 .

[0073] from Figure 7 As can be seen, in the acute lung injury model, the signal of the lung-targeted ROS probe is stronger in the lung than in the liver, while the signal intensity of the probe molecules is stronger in the liver than in the lung. This indicates that the lung-targeted delivery system increases the accumulation ratio of ROS probes in the lung / liver and that the release of probes in the lung reacts with high levels of ROS in the inflammatory environment to produce luminescence, thereby improving the signal-to-noise ratio of pneumonia detection.

[0074] Example 8

[0075] Application of Lung-Targeted Polyphenol / Cysteine ​​Nanodelivery System in Lung Cancer Treatment

[0076] The lung-targeting EGCG / cysteine ​​complex prepared in Example 1 was used as a delivery system loaded with paclitaxel chemotherapy drug for the treatment of lung metastases in mice. The steps are as follows:

[0077] (1) Dissolve the EGCG / cysteine ​​complex solution with 30% ethanol to form a solution with a concentration of 20 mg / mL. Prepare a paclitaxel solution with a concentration of 20 mg / mL using ethanol solvent. Mix the two solutions in a 1:1 volume ratio, then quickly mix with the assembly solution water. After standing for 0.5 hours, centrifuge and wash with water to obtain lung-targeted paclitaxel.

[0078] (2) Mouse breast cancer cells 4T1-luc in the logarithmic growth phase were injected into mice via the tail vein at a dose of 5 × 10⁻⁶. 6 200 μL / mouse were used to construct a mouse lung metastasis model;

[0079] (3) One week after modeling, the mice were randomly divided into three groups (n=3 per group): a blank control group (i.e., the "glucose" group), a paclitaxel group, and a lung-targeted paclitaxel group. The blank control group was injected with glucose, the paclitaxel group was injected with a paclitaxel solution prepared with polyoxyethylene castor oil, and the lung-targeted paclitaxel group was injected with the lung-targeted paclitaxel prepared in step (1). The drugs were administered via the tail vein every three days. The experiment was terminated after four treatments. The growth of the tumor was recorded during the treatment. The results are as follows: Figure 8 .

[0080] In a mouse model of lung metastases, lung-targeted paclitaxel was more effective than the paclitaxel drug molecule, indicating that the lung-targeted delivery system enhances the anti-tumor effect of the drug by increasing the accumulation concentration of the drug in the lungs.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a lung-targeted delivery system in the preparation of drugs for targeted treatment of lung diseases or in the preparation of diagnostic reagents targeting the lungs, characterized in that, The lung-targeted delivery system is constructed by loading a drug, probe, or tracer onto a polyphenol / amine complex. The polyphenol / amine complex is obtained by condensing polyphenols, amines, and aldehydes through a condensation reaction and then precipitating them from an aqueous phase. The polyphenol is epigallocatechin gallate, and the amine is selected from one of glutathione, asparagine, penicillamine, and glutamine.

2. The application according to claim 1, characterized in that, The drug is selected from one of the following: cisplatin, carboplatin, pemetrexed, etoposide, gemcitabine, docetaxel, paclitaxel, pembrolizumab, bevacizumab, streptomycin, amikacin, capreomycin, penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, and dexamethasone.

3. The application according to claim 2, characterized in that, The cephalosporin antibiotic in question is ceftazidime.

4. The application according to claim 1, characterized in that, The probe is selected from one of the following: DCFH-DA probe, Rod-Cl probe, and Rod-Br probe.

5. The application according to claim 1, characterized in that, The tracer is selected from indocyanine green and Cy5.

6. The application according to claim 1, characterized in that, The lung disease mentioned is one of the following: infectious or non-infectious pneumonia, lung cancer, pulmonary tuberculosis, pulmonary fibrosis, COPD, pulmonary hypertension, or asthma.

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