Nano-micelle responding to inflammation microenvironment as well as preparation method and application of nano-micelle

Abstract

The invention provides a nano-micelle responding to an inflammation microenvironment as well as a preparation method and application of the nano-micelle. According to the nano-micelle, polyethylene glycol (PEG)-poly L-glutamic acid (PLG) is grafted with 4-hydroxymethyl phenylboronic acid (PBA), and the drug loading capacity of a hydrophobic anti-inflammatory drug is improved based on a hydrophobic effect and a borate bond formed by the PBA and a 1, 3-diol or 1, 2-diol structure of dexamethasone, curcumin and luteolin. The nano-micelle (PPDex micelle) has reversible splitting and rapid release behaviors under an inflammatory condition (the ROS level is increased or the pH value is reduced). The nano-micelle has a proper particle size, can effectively relieve inflammatory cell infiltration and pulmonary edema in the lung of an acute lung injury (ALI) mouse after pulmonary administration, reduces pro-inflammatory cytokines in the lung, inhibits oxidative stress, promotes polarization of macrophages from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype, and finally inhibits the progress of ALI. The invention provides a novel effective anti-inflammatory strategy.

Description

Technical Field

[0001] The present invention relates to the field of new biomedical materials, and in particular to an inflammatory microenvironment-responsive nanomicelle and a preparation method and application thereof. Background Art

[0002] Acute lung injury (ALI) is a severe, acute, progressive respiratory disease characterized by diffuse alveolar edema, progressive hypoxemia, and easy progression to systemic respiratory distress syndrome (ARDS), with high mortality and poor prognosis. Currently, the treatment methods for ALI mainly include ventilator-assisted support and glucocorticoid shock therapy, but ventilator-associated infection and systemic side effects of large amounts of glucocorticoids are also problems that cannot be ignored.

[0003] More and more studies have shown that reactive oxygen species (ROS) play an important role in the initiation and progression of ALI. Excessive ROS leads to an imbalance of pro-inflammatory / anti-inflammatory factors, and even leads to inflammatory factor storms, which seriously affect the structure and function of the lungs. With the development of nanotechnology, nanomaterials have shown unique advantages in disease treatment, such as controllable size, good stability, and surface modification (see Chen, Ting, et al. Inhaled curcumin mesoporous polydopamine nanoparticles against radiation pneumonitis. Acta Pharmaceutica Sinica B, 2022, 12, 2522-2532.). However, some nanomaterials cannot specifically identify the lesion site, and continuously release drugs during transport in the body, so it may cause insufficient drug dosage in the lesion site and damage normal tissues. High levels of ROS and microacidity are unique features of the ALI lesion microenvironment. In recent years, researchers have designed drug delivery systems specific to the inflammatory environment (see Muhammad, Wali, et al. ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment. Acta biomaterialia, 2022, 148, 258-270.), but the low drug loading limits its clinical application. Therefore, it is necessary to design a stable nanomaterial with a high drug loading and specific to the inflammatory microenvironment, which can be responsively released in the ALI lesion environment and continuously exert anti-inflammatory effects. Summary of the invention

[0004] In order to overcome the above-mentioned deficiencies of the prior art, the present disclosure provides an inflammatory microenvironment-responsive nanomicelle and a preparation method and application thereof.

[0005] According to the first aspect of the present disclosure, a nanomicelle responsive to an inflammatory microenvironment is provided, characterized in that the nanomicelle is composed of polyethylene glycol PEG-poly L-glutamic acid PLG, i.e. PLG-g-PEG, grafted with 4-hydroxymethylphenylboronic acid PBA to form a hydrophilic and hydrophobic polymer carrier PPBA, wherein the hydrophilic and hydrophobic polymer carrier PPBA comprises a hydrophilic end and a hydrophobic end, wherein the hydrophilic end is PEG and PLG, and the hydrophobic end is PBA; the hydrophilic and hydrophobic polymer carrier utilizes the hydrophobic effect and the boric acid ester bond formed by PBA and the 1,3-diol or 1,2-diol structure of the hydrophobic anti-inflammatory drug, and the loaded hydrophobic anti-inflammatory drug is dexamethasone Dex;

[0006] Furthermore, the molecular weight of PEG is 2000-10000Da, the mass ratio of PEG to PLG is 1-2:1, the mass ratio of PLG-g-PEG to PBA is set at 1-2:1; the mass ratio of the hydrophilic and hydrophobic polymer carrier PPBA to the hydrophobic anti-inflammatory drug is set at 0.5-2:1;

[0007] Furthermore, the supported hydrophobic anti-inflammatory drug may also be curcumin Cur or luteolin Lut;

[0008] Furthermore, the specific preparation steps of the polymer carrier are as follows: (1) 500 mg of PEG with molecular weights of 2000, 5000, and 10000 Da and 500 mg of PLG are respectively selected and stirred in 5-10 mL of dimethyl sulfoxide (DMSO) for 48-72 h, and then dialyzed in deionized water and freeze-dried to obtain a powdered product; (2) 300 mg of the PLG-g-PEG is stirred and reacted with 300 mg and 150 mg of PBA in 8 mL of DMSO for 48-72 h, and then dialyzed in deionized water and freeze-dried to construct the hydrophilic and hydrophobic polymer carrier PPBA;

[0009] According to a second aspect of the present disclosure, a method for preparing nanomicelles responsive to an inflammatory microenvironment is provided, characterized in that the nanomicelles are prepared by three consecutive steps: (1) polyethylene glycol PEG is grafted onto poly-L-glutamic acid PLG to obtain PLG-g-PEG; (2) hydrophobic end 4-hydroxymethylphenylboronic acid PBA is grafted onto the PLG-g-PEG to obtain a hydrophilic and hydrophobic polymer carrier PPBA; (3) the hydrophobic anti-inflammatory drug carried by the hydrophilic and hydrophobic polymer carrier PPBA is dexamethasone Dex, curcumin Cur or luteolin Lut;

[0010] Furthermore, the hydrophilic and hydrophobic polymer carrier PPBA and Dex, Cur, and Lut are stirred in 2 to 3 mL of dimethyl sulfoxide (DMSO) at a mass ratio of 0.5 to 2:1 overnight, and then dialyzed in deionized water to obtain the nanomicelles;

[0011] According to a third aspect of the present disclosure, there is provided a use of an inflammatory microenvironment-responsive nanomicelle in acute lung injury (ALI) lesions;

[0012] Furthermore, transpulmonary administration releases anti-inflammatory drugs in the inflammatory environment of the lungs in acute lung injury (ALI).

[0013] The technical effects of the present invention are:

[0014] The nanomicelles in the present invention carry the hydrophobic drug Dex through the combined action of the hydrophobic effect and the phenylboronic acid ester bond, reach the inflammatory site of the lung after pulmonary administration, and release Dex in a high ROS / low pH environment, thereby achieving the effects of reducing pro-inflammatory factors and alleviating pulmonary edema; secondly, the phenylboronic acid ester bond in the nanomicelles consumes excessive ROS in the inflammatory site, can reduce the oxidative stress level in the lungs of ALI mice, and reduce tissue damage caused by excessive ROS and the inflammatory factor storm induced by it; in addition, the nanomicelles play an immunoregulatory role in the ALI lungs by reducing the level of M1 macrophages in the inflammatory site, increasing the level of M2 macrophages, and balancing the M1 / M2 cell levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. The accompanying drawings are used to better understand the present solution and do not constitute a limitation of the present disclosure. In the accompanying drawings, the same or similar reference numerals represent the same or similar elements, among which:

[0016] Figure 1 This is a scanning electron microscope image of the inflammatory microenvironment responsive nanomicelles obtained in an example of the present invention. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0018] According to the present invention, a nano-micelle responsive to the microenvironment of inflammatory lesions is provided, wherein the hydrophilic and hydrophobic polymer carrier of the nano-micelle is composed of PLG-g-PEG grafted with PBA, and then the hydrophobic drug is loaded by hydrophobic interaction and phenylboronic acid ester bond. The nano-micelle is prepared by a continuous three-step preparation process: (1) PEG grafted with PLG; (2) hydrophobic end PBA grafted with PLG-g-PEG; (3) loading the hydrophobic anti-inflammatory drugs Dex, Cur, and Lut.

[0019] According to the present invention, in the preparation process of PLG-g-PEG, the molecular weight of PEG is preferably 2000-10000Da, more preferably 2000 and 5000Da, the mass ratio of PEG to PLG is (1-2):1, the volume of DMSO is preferably 5-10mL, more preferably 5-8mL. The reaction time is preferably 24-72h, more preferably 72h.

[0020] According to the present invention, in the preparation process of the hydrophobic end PBA grafted PLG-g-PEG, the reaction mass ratio of PLG-g-PEG to PBA is preferably 1:1 and 2:1, more preferably 1:1, and the stirring reaction time in DMSO is preferably 48 to 72 hours, more preferably 48 hours.

[0021] According to the present invention, in the preparation of the nanomicelles, the mass ratio of the hydrophilic and hydrophobic polymer carrier to the hydrophobic anti-inflammatory drug is preferably (0.5-2):1, more preferably 1:1, and the nanomicelles are obtained after stirring in DMSO overnight and dialyzing in deionized water.

[0022] In the embodiments of the present invention, the following is performed:

[0023] 1) Cell culture: In the present invention, RAW264.7 cell line is selected, and the cells are preferably cultured in DMEM medium containing 10% fetal bovine serum. The culture conditions are preferably carried out in an incubator with a volume fraction of 5% carbon dioxide, and the culture temperature is preferably 37°C.

[0024] 2) Cytotoxicity: In the present invention, the RAW264.7 cell line was selected for cytotoxicity evaluation. The RAW264.7 cell density was 1×10 4 / well were planted in a 96-well plate and cultured overnight. After adding different concentrations of nano-micelles to the well plate and incubating with the cells for 24 hours, the nanoparticles were washed with PBS and fresh culture medium was added. Finally, 20 μL of MTT solution was added to each well. After further incubation for 4 hours, 150 μL of DMSO was added to each well and the well plate was gently shaken. The absorbance value of each well was detected by an enzyme reader at 492 nm. The cell survival rate was calculated by the following formula.

[0025] Cell survival rate (%) = (A sample / A blank) × 100

[0026] 3) Anti-inflammatory treatment at the cellular level: In the present invention, the RAW264.7 cell line is preferred for anti-inflammatory treatment at the cellular level. The RAW264.7 cell density is 1×10 4 / well were planted in a 96-well plate and cultured overnight. The model group and the drug treatment group were stimulated with lipopolysaccharide (LPS) at a concentration of 1-5 μg / mL, and drug treatment was given 6-12 hours later. The LPS concentration was preferably 1 μg / mL, and the stimulation time was preferably 6 hours.

[0027] 4) Animal model: In the present invention, the ALI model induced by LPS was used in the experiment, and Balb / c mice were selected. LPS was injected into the mice intratracheally, with a dose of (5-20) mg / kg, preferably 5 mg / kg, and a volume of 20 μL. The stimulation time was 6-12 hours, and the drug treatment experiment for different groups was preferably started after 6 hours.

[0028] The reagents used in the following examples are all commercially available products.

[0029] Examples 1 to 36

[0030] First, PEG and PLG with preferred molecular weights of 2000 and 5000 were dissolved in 5 mL of DMSO at a mass ratio of (1-2):1, stirred for 72 h, dialyzed in deionized water, and freeze-dried to obtain white powdery PLG-g-PEG; (2) PLG-g-PEG and PBA were dissolved in DMSO at a ratio of (1-2):1 and stirred for 48 h, dialyzed in deionized water, and freeze-dried to complete the grafting of PBA at the hydrophobic end; (3) The hydrophilic and hydrophobic polymers were loaded with hydrophobic anti-inflammatory drugs at a ratio of (0.5-2):1, and the two were stirred overnight. After dialyzed in deionized water, nanomicelles responsive to the microenvironment of inflammatory lesions were obtained. The molecular weight of PEG, the ratio of PEG to PLG, the ratio of PLG-g-PEG to PBA, and the ratio of hydrophilic and hydrophobic polymers PPBA to hydrophobic anti-inflammatory drugs are shown in Table 1.

[0031] Table 1 Mass ratio of different raw material dosages in Examples 1 to 12

[0032]

[0033]

[0034] The inflammatory microenvironment-responsive nanomicelles prepared in Example 14 were analyzed using a scanning electron microscope to obtain a scanning electron microscope image, as shown in FIG. Figure 1 As shown, the results show that the size of the nanoparticles prepared in Example 14 is between 200 and 220 nm.

[0035] Embodiment 37

[0036] Anti-inflammatory effect of nano-micelles responsive to inflammatory microenvironment at the cellular level. RAW264.7 cells were cultured at a density of 1×10 4 / well were planted in a 96-well plate and cultured overnight. The model group and the drug treatment group were stimulated with lipopolysaccharide (LPS) at a concentration of 1μg / mL, and then given drug treatment 6 hours later. After 24 hours of co-incubation with the drug, the pro-inflammatory factors TNF-α, IL-6, and IL-1β in the cell supernatant of the nanomicelle group were significantly lower than those in the model group.

[0037] Embodiment 38

[0038] The effect of nano-micelles responding to the inflammatory microenvironment on the treatment of ALI in vivo was studied. Balb / C mice weighing about 20 g were selected and the ALI model was established 6 hours after intratracheal inoculation of LPS. The nano-micelles of the present invention were administered to the lungs at 12 mg / mL, 40 μL. After 24 hours of treatment, compared with the lung tissue of the model group mice, HE staining showed that the infiltration of inflammatory cells in the lungs was reduced and the destruction of the alveolar structure was alleviated; WB showed that pro-inflammatory factors such as IL-6 and IL-1β were downregulated.

[0039] According to the present invention, the inflammatory microenvironment responsive nanomicelles have the advantages of easy operation, high drug loading, good specificity, etc.; they have a suitable nanosize and can be enriched in the diseased lung tissue through pulmonary administration; the phenylboronic acid ester bond is cleaved in a high ROS / low pH environment, and the drug is released at the lesion site to exert a sustained anti-inflammatory effect; the phenylboronic acid ester bond consumes excessive ROS at the inflammatory site, reducing tissue damage caused by excessive ROS and the possible induction of inflammatory factor storms; by balancing the M1 / M2 cell levels, it exerts an immune regulatory effect in the ALI lungs.

[0040] The inflammatory microenvironment responsive nano-micelles provided by the present invention have the advantages of simple operation, high drug loading, good specificity, etc.; the size is 200-220nm, and can be enriched in the lesion lung tissue through pulmonary administration; the phenyl boronate ester bond is cleaved in a high ROS / low pH environment, and the drug is released at the lesion site to exert a sustained anti-inflammatory effect; the phenyl boronate ester bond consumes excessive ROS at the inflammatory site, reducing tissue damage caused by excessive ROS and the inflammatory factor storm that may be induced; by balancing the M1 / M2 cell level, it exerts an immune regulation effect in the ALI lung. Compared with the untreated group and the free drug group, the nano-micelle group has a better effect in treating ALI.

[0041] Although the embodiments of the present disclosure have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. For those skilled in the art, the present disclosure may have various modifications and variations. Any changes, modifications, substitutions, combinations, and simplifications made to the present disclosure shall be equivalent replacement methods and shall be included in the protection scope of the present disclosure.

Claims

1. A nanomicelle responsive to an inflammatory microenvironment, characterized in that: The nano micelle is made of polyethylene glycol PEG-poly L-glutamic acid PLG, i.e. PLG-g-PEG, grafted with 4-hydroxymethylphenylboronic acid PBA to form a hydrophilic and hydrophobic polymer carrier PPBA, wherein the hydrophilic and hydrophobic polymer carrier PPBA comprises a hydrophilic end and a hydrophobic end, wherein the hydrophilic end is PEG and PLG, and the hydrophobic end is PBA; the hydrophilic and hydrophobic polymer carrier uses the hydrophobic effect and the boric acid ester bond formed by PBA and the 1,3-diol or 1,2-diol structure of the hydrophobic anti-inflammatory drug to carry the hydrophobic anti-inflammatory drug dexamethasone Dex.

2. The nano micelle according to claim 1, characterized in that The molecular weight of the PEG is 2000-10000Da, the mass ratio of the PEG to the PLG is 1-2:1, the mass ratio of the PLG-g-PEG to the PBA is set at 1-2:1; the mass ratio of the hydrophilic and hydrophobic polymer carrier PPBA to the hydrophobic anti-inflammatory drug is set at 0.5-2:

1.

3. The nano micelle according to claim 1, characterized in that The supported hydrophobic anti-inflammatory drug may also be curcumin Cur or luteolin Lut.

4. The nano micelle according to claim 2, characterized in that The specific preparation steps of the hydrophilic and hydrophobic polymer carrier are as follows: (1) 500 mg of PEG and 500 mg of PLG with molecular weights of 2000, 5000, and 10000 Da are respectively selected and stirred in 5-10 mL of dimethyl sulfoxide (DMSO) for 48-72 hours, and then dialyzed in deionized water and freeze-dried to obtain a powdered product; (2) 300 mg of the PLG-g-PEG is stirred and reacted with 300 mg and 150 mg of the PBA in 8 mL of DMSO for 48-72 hours, and then dialyzed in deionized water and freeze-dried to construct the hydrophilic and hydrophobic polymer carrier PPBA.

5. The method for preparing the inflammatory microenvironment-responsive nanomicelles according to claim 1, characterized in that: The nanomicelles are prepared by three consecutive steps: (1) polyethylene glycol PEG is grafted onto poly-L-glutamic acid PLG to obtain PLG-g-PEG; (2) hydrophobic end 4-hydroxymethylphenylboronic acid PBA is grafted onto the PLG-g-PEG to obtain a hydrophilic and hydrophobic polymer carrier PPBA; (3) the hydrophobic anti-inflammatory drug loaded on the hydrophilic and hydrophobic polymer carrier PPBA is dexamethasone Dex, curcumin Cur or luteolin Lut.

6. The method for preparing the inflammatory microenvironment-responsive nanomicelles according to claim 4, characterized in that: Also includes: The hydrophilic and hydrophobic polymer carrier PPBA and Dex, Cur and Lut are stirred in 2 to 3 mL of dimethyl sulfoxide (DMSO) at a mass ratio of 0.5 to 2:1 overnight, and then dialyzed in deionized water to obtain the nano micelles.

7. Use of the inflammatory microenvironment-responsive nanomicelles according to any one of claims 1 to 3 in acute lung injury (ALI) lesions.

8. Use of the inflammatory microenvironment-responsive nanomicelles according to claim 7 in acute lung injury lesions, characterized in that: Pulmonary administration releases anti-inflammatory drugs in the inflammatory environment of the lungs in acute lung injury (ALI).

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