CN111603565A - Anti-inflammatory nano-drug carrier, pharmaceutical composition thereof, preparation method and application - Google Patents

Anti-inflammatory nano-drug carrier, pharmaceutical composition thereof, preparation method and application Download PDF

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CN111603565A
CN111603565A CN202010464905.XA CN202010464905A CN111603565A CN 111603565 A CN111603565 A CN 111603565A CN 202010464905 A CN202010464905 A CN 202010464905A CN 111603565 A CN111603565 A CN 111603565A
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王云兵
马博轩
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Abstract

The invention discloses an anti-inflammatory nano-drug carrier, a drug composition thereof, a preparation method and application, wherein the nano-drug carrier is a double-block polymer formed by choline polyphosphate and poly (methylthio ethyl acrylate), the drug composition comprises the nano-drug carrier and diagnosis and treatment compound molecules carried in the nano-drug carrier, and the diagnosis and treatment compound molecules are formed by connecting fluorescent molecules with two-photon aggregation induced emission effects with drugs with anti-inflammatory functions through dicarbonyl bonds. The pharmaceutical composition has gradual active oxygen responsiveness, and the poly (methyl ethyl acrylate) block in the nano-drug carrier can be sensitively converted into hydrophilic to trigger carrier dissociation; the dicarbonyl bond in the diagnosis and treatment compound molecule can also be sensitively broken, so that the released fluorescent molecule and the anti-inflammatory drug respectively play a role in diagnosis and treatment, and have good diagnosis and treatment effects on acute and chronic inflammation, such as atherosclerosis, arthritis, pneumonia and other diseases.

Description

Anti-inflammatory nano-drug carrier, pharmaceutical composition thereof, preparation method and application
Technical Field
The invention belongs to the technical field of nano-drug carriers, and particularly relates to an anti-inflammatory nano-drug carrier, a pharmaceutical composition thereof, a preparation method and application.
Background
Inflammation is widely recognized as a natural protective mechanism of the human body against trauma, including processes of penetration of tissues by microorganisms, instruction and scheduling of cells, removal of microorganisms and infected cells, liquefaction of surrounding tissues, and healing of damaged tissues. During inflammation, inflammation can also lead to tissue damage if accurate destruction and assisted repair is not possible. Therefore, rational strategies for treating inflammatory lesions, including long-term inflammation such as atherosclerosis, arthritis, and short-term inflammation such as pneumonia, have been extensively studied. In general, glucocorticoids and non-steroidal anti-inflammatory drugs are commonly used in the treatment of inflammation. However, various side effects of these drugs are also of concern, such as osteoporosis, gastrointestinal bleeding, and chronic kidney disease. In order to reduce adverse reactions and improve therapeutic effects, various nano-drug carriers such as polymeric micelles, liposomes, and the like have been developed in recent years for the treatment of inflammation.
The systemically constructed drug delivery nanosystems have demonstrated selective accumulation in inflamed tissues, where the leaky blood vessels of edematous tissues provide an extravasation pathway for nanoparticles. Therefore, nano-drug carriers are widely used in the treatment of inflammation. However, the nano-carrier needs to further realize intelligent and accurate release after drug accumulation. Along with the inflammation process, the over-expressed active oxygen is found in the inflammatory tissue, and a target point is provided for accurate drug delivery in the treatment of inflammation. Therefore, a series of active oxygen response structures are introduced into the nanoparticles to realize accurate and efficient release of the inflammation treatment drug.
Furthermore, in addition to rational nano-drug treatment strategies, the in vivo visualization of nanoparticles should be accurately monitored throughout the circulation, accumulation and distribution of sites of inflammation. In recent years, fluorescence imaging has proven to be a powerful tool for high-resolution observation of biological microstructures. However, the conventional fluorophores have serious defects of aggregation quenching effect after accumulation, background fluorescence interference and the like. A fluorophore with aggregation-induced emission effect is a novel molecule, and has weak fluorescence emission in a dilute solution and strong fluorescence in an aggregation state. In addition, two-photon excitation fluorescence microscopy can provide clearer spatial resolution with less autofluorescence and deeper tissue penetration than conventional single photon fluorescence imaging. Therefore, fluorescent molecules with two-photon excitation and aggregation-induced emission effects would be a suitable strategy to address nanomedical tracking and spatial fluorescence imaging of inflamed tissues
Therefore, the diagnosis and treatment nano-drug carrier for acute and chronic inflammations such as atherosclerosis, arthritis, pneumonia and the like plays an important role in inflammation diagnosis and treatment.
Disclosure of Invention
The invention aims to: in view of the above problems in the prior art, the present invention provides an anti-inflammatory nano-drug carrier, a pharmaceutical composition thereof, a preparation method thereof, and applications thereof, for diagnosis and treatment of acute and chronic inflammations such as atherosclerosis, arthritis, and pneumonia, wherein the nano-drug carrier has sensitivity to a high active oxygen environment and a fluorescence function of two-photon aggregation-induced luminescence, can be accurately and efficiently administered to inflammation sites such as atherosclerotic plaques, arthritis, and pneumonia injury sites, and simultaneously, realizes accurate tracking and positioning of drugs in vivo and in cells.
The technical scheme adopted by the invention is as follows:
an anti-inflammatory nano-drug carrier comprises a choline polyphosphate block and a poly (methyl-thioethyl acrylate) block; the structural formula of the nano-drug carrier is shown as follows:
Figure BDA0002510777190000021
wherein the polymerization degree of the choline polyphosphate is 1-100; the polymerization degree of the poly (methylthio ethyl acrylate) is 1-100; preferably, the polymerization degree of the choline polyphosphate is 1 to 50; the polymerization degree of the poly (methylthioethyl acrylate) is 1-50.
The preparation method of the anti-inflammatory nano-drug carrier comprises the following steps:
s1, mixing methylthioethyl acrylate, 4-cyanovaleric acid dithiobenzoic acid, azobisisobutyronitrile and tetrahydrofuran, sequentially performing three cycles of freezing-pumping-thawing, reacting for 12-36h at 65-75 ℃ under the protection of argon, dialyzing the reactant in ionized water for 12-36h, and freeze-drying to obtain the poly (methylthioethyl acrylate);
s2, dissolving the poly (methylthioethyl acrylate) obtained in the step S1 and the methacryloyl phosphorylcholine in tetrahydrofuran, adding azodiisobutyronitrile, reacting for 24-28h at 65-75 ℃ after freezing and removing oxygen, dialyzing, and freeze-drying to obtain the product.
Further, the preparation method specifically comprises the following steps:
s1, mixing methylthioethyl acrylate, 4-cyanovaleric acid dithiobenzoic acid, azobisisobutyronitrile and tetrahydrofuran, sequentially performing three cycles of freezing-pumping-thawing, reacting at 70 ℃ for 24 hours under the protection of argon, dialyzing the reactant in ionized water (MCWO 1000) for 24 hours, and freeze-drying to obtain the poly (methylthioethyl acrylate);
s2, dissolving the poly (methylthioethyl acrylate) obtained in the step S1 and the methacryloyl phosphorylcholine in tetrahydrofuran, adding azodiisobutyronitrile, reacting at 70 ℃ for 26 hours after freezing and removing oxygen, and then dialyzing and freeze-drying to obtain the product.
Further, the mass ratio of methylthioethyl acrylate, 4-cyanovaleric acid dithiobenzoic acid and azobisisobutyronitrile in step S1 is 3500-7000: 100-200: 25-50; preferably 5000:150: 30.
Further, the mass ratio of the poly (methyl ethyl methacrylate), the methacryloyl phosphorylcholine and the azobisisobutyronitrile in the step S2 is 1500-: 3000-6000: 12-25; preferably 2000:4500: 20.
A pharmaceutical composition comprises the nano-drug carrier and a diagnosis and treatment compound encapsulated in the nano-drug carrier.
Further, the diagnosis and treatment compound is formed by connecting fluorescent molecules with two-photon aggregation induced emission effect with drugs with anti-inflammatory function through dicarbonyl bonds, and is as follows:
Figure BDA0002510777190000031
further, the diagnosis and treatment compound is prepared by the following method:
dissolving the fluorescent molecular group, dropwise adding into oxalyl chloride solution, stirring at room temperature for 10-15h, removing residual oxalyl chloride and solvent under reduced pressure, adding medicine and triethylamine, reacting at room temperature for 12-36h, and purifying to obtain the final product; wherein the molar ratio of the fluorescent molecular group, oxalyl chloride, triethylamine and the medicine is 1-10: 3-30: 3-30: 1-10.
Further, the diagnosis and treatment compound is prepared by the following method:
dissolving the fluorescent molecular group, dropwise adding the fluorescent molecular group into an oxalyl chloride solution, stirring at room temperature for 12h, removing residual oxalyl chloride and solvent under reduced pressure, adding a medicament and triethylamine, reacting at room temperature for 24h, and carrying out chromatographic purification to obtain the fluorescent molecular group; wherein the molar ratio of the fluorescent molecular group to the oxalyl chloride to the triethylamine to the medicine is 5: 20: 15: 5.
the preparation method of the pharmaceutical composition comprises the following steps:
dissolving the nano-drug carrier and the diagnosis and treatment compound in a mixed solvent of tetrahydrofuran and dimethyl sulfoxide, stirring for 1-2h to obtain a mixed solution, adding the mixed solution into normal saline or ultrapure water with the volume 2-20 times that of the mixed solution, stirring for 1-4h, and removing the organic solvent to obtain the compound preparation;
or dissolving the nano-drug carrier and the diagnosis and treatment compound in tetrahydrofuran, performing rotary evaporation at 40-60 ℃ for 0.5-2h, removing the organic solvent, adding normal saline or ultrapure water, and fully oscillating for hydration to obtain the compound.
Further, the volume ratio of the tetrahydrofuran to the dimethyl sulfoxide is 1-2: 1-2; preferably 1: 1.
Use of the nano-drug carrier or the pharmaceutical composition of claim 5 in the preparation of a medicament for diagnosing and/or treating acute and chronic inflammation.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, the poly (methyl-thioethyl acrylate) block can be sensitively converted into hydrophilic to initiate the dissociation of the carrier; the dicarbonyl bond in the diagnosis and treatment compound molecule can also be broken sensitively, has the functions of triggering the drug release characteristic by active oxygen sensitivity, inducing luminescence by two-photon aggregation and the like, can be accurately and efficiently administered at inflammatory parts such as atherosclerotic plaques, arthritis and pneumonia injury parts, and simultaneously realizes the accurate tracking and positioning of the drug in vivo and cells;
2. in the invention, the poly phosphorylcholine block is used as a carrier hydrophilic shell layer to provide excellent biocompatibility for a nano carrier, and the particle size of the prepared drug-loaded micelle is distributed between 10 and 200 nm;
3. compared with free drugs at the in vitro cell level and the in vivo animal level, the drug-loaded micelle prepared by the invention has obviously improved drug effect, biological safety and circulation time, and can realize high-efficiency and low-toxicity drug delivery;
4. the drug-loaded micelle prepared by the invention has good two-photon imaging effect on inflammatory tissues in the process of atherosclerosis, arthritis and pneumonia, can realize efficient anti-inflammation on a cell level, and has excellent inflammation treatment effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a polymer carrier;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the diagnosis and treatment compound;
FIG. 3 is a transmission electron microscope image of a distribution diagram of the particle size of the diagnostic micelle;
FIG. 4 is a graph showing the results of the variation of the particle size of micelles under different active oxygen conditions;
FIG. 5 is a fluorescence emission spectrum of a micelle;
FIG. 6 is a graph showing the results of in vitro drug release of micelles under different conditions;
FIG. 7 is a graph showing the results of inhibition of inflammatory factors of macrophages by micelles;
FIG. 8 is a graph of the results of micelle imaging and treatment of pneumonia in animal experiments;
FIG. 9 is a graph of the results of imaging and treatment of arthritis with micelles in an animal experiment;
figure 10 is a graph of the results of imaging and treatment of atherosclerosis with micelles in animal experiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The invention provides an anti-inflammatory nano-drug carrier and a pharmaceutical composition thereof, wherein the pharmaceutical composition comprises the nano-drug carrier and a diagnosis and treatment compound encapsulated in the nano-drug carrier. The nano-drug carrier is a double-block polymer formed by choline polyphosphate and poly (methylthio ethyl acrylate), and the diagnosis and treatment compound is formed by connecting fluorescent molecules with two-photon aggregation induced emission effects with drugs with anti-inflammatory functions through dicarbonyl bonds.
The preparation method of the nano-drug carrier specifically comprises the following steps:
(1) methylthioethylacrylate (3.5g), 4-cyanovaleric dithiobenzoic acid (0.1g) and azobisisobutyronitrile (25mg) were added to a Schlenk flask of tetrahydrofuran. After three cycles of freezing-pumping-unfreezing procedures, the reaction was carried out at 70 ℃ under the protection of argon for 24 hours. The resulting solution was dialyzed against ionized water (MCWO 1000) for 24 hours and then freeze-dried to give polythioethyl acrylate;
(2) dissolving the product (0.3g) obtained in the step (1) and methacryloyl phosphorylcholine (0.6) in tetrahydrofuran, then adding azobisisobutyronitrile (2.5mg), reacting at 70 ℃ for 26h after freezing and removing oxygen, then dialyzing, and finally freeze-drying to obtain a nano-drug carrier; wherein the polymerization degree of the choline polyphosphate block is 30; the polymerization degree of the poly (methyl ethyl acrylate) block is 30;
(3) fluorophore (0.2g) was dissolved and added dropwise to a solution of oxalyl chloride (0.032 mL). After stirring at room temperature for 12 hours, excess oxalyl chloride and solvent were removed under reduced pressure. The drug (0.09g) and triethylamine (0.11mL) were dissolved and added. After reacting for 24h at room temperature, purifying by chromatography to obtain the diagnosis and treatment compound.
The nuclear magnetic resonance hydrogen spectrogram of the prepared nano-drug carrier is shown in figure 1.1H NMR(DMSO-d6/CD3OD＝1/1,400MHz):＝2.10(m,100H),2.71(m,102H),3.33(m,900H),3.66(m,100H),3.94(m,100H),4.11(m,100H),4.20(m,100H)ppm.
The NMR spectrum of the compound is shown in FIG. 2.1H NMR(DMSO-d6,400MHz):＝0.87(m,3H),1.23(s,3H),1.39(s,2H),1.64(m,2H),2.01(m,1H),2.30(m,1H),3.89(d,1H),4.54(d,2H),4.89(s,2H),7.04(t,4H),7.36(t,6H),7.74(d,4H),7.92(d,2H),8.18(t,2H),8.59(t,2H),9.06(d,2H)ppm.
The peak position and integral proportion of the nano-drug carrier and the diagnosis and treatment compound are known to be successfully synthesized.
Example 2
On the basis of example 1, the preparation method of the pharmaceutical composition specifically comprises the following steps:
weighing 20mg of the nano-drug carrier prepared in the example 1 and 10mg of the diagnosis and treatment compound, dissolving the nano-drug carrier and the diagnosis and treatment compound in 4mL of mixed solution of dimethyl sulfoxide and tetrahydrofuran, wherein the volume ratio of the dimethyl sulfoxide to the tetrahydrofuran is 1:1, then dropwise adding the mixed solution into 10mL of ultrapure water, stirring for 4 hours, and dialyzing to remove the organic solvent to prepare the drug composition micelle.
The particle size diagram and the transmission electron microscope diagram of the prepared micelle of the pharmaceutical composition are shown in fig. 3, and the diagram shows that the carrier prepared by the invention can coat the diagnosis and treatment compound in water, and the micelle with a core-shell structure is formed by self-assembly, and has uniform size and regular shape.
Test example 1
Study of stability and sensitivity of micelles
The micelle obtained in example 2 was exposed to 37 ℃ and hydrogen peroxide concentrations (0,0.01,0.1,1,10mM) at different concentrations, and the change in particle size was measured at regular intervals by a dynamic light scattering instrument DLS, and the results of the change in particle size are shown in FIG. 4.
As can be seen from FIG. 4, the micelle prepared by the present invention shows good stability without hydrogen peroxide, and the micelle rapidly expands under the hydrogen peroxide condition with the concentration of 1mM or more, and the particle size of the micelle is finally stabilized at about 160 nm.
Test example 2
Study on micelle aggregation-induced emission characteristics
The micelle prepared in example 2 was added to dimethylsulfoxide, aqueous hydrogen peroxide or pure water, and the polymer drug-loaded micelle was excited at 410nm to observe the fluorescence emission, which is shown in fig. 5.
As can be seen from fig. 5, the micelle fluorescence intensity disappears along with the dissolution of the two-photon fluorophore in dmso, and the polymer drug-loaded micelle is dissolved in water or hydrogen peroxide solution, and has stronger fluorescence due to its more compact aggregation state.
Test example 3
Micellar drug release behavior in vitro
The micelle prepared in example 2 was prepared into a solution having a concentration of 1mg/mL, 2mL of the solution was transferred into a dialysis bag having a molecular weight cutoff of 3500, the dialysis bag was sealed and placed in 100mL of PBS or 1mM hydrogen peroxide solution, and 2mL of the solution was taken out at 37 ℃ with constant shaking in the dark, and the amount of drug released was measured by high performance liquid chromatography, and the results are shown in fig. 6.
As can be seen from fig. 6, the micelle achieves rapid drug release under active oxygen environment.
Test example 4
Anti-inflammatory effects of micelles on macrophages
The micelle prepared in example 2, or the free drug, was co-cultured with lipopolysaccharide-activated macrophages for 48h, and the supernatant was taken to measure the content of inflammatory molecules (TNF-. alpha., IL-1. beta., MPO), the results of which are shown in FIG. 7.
As can be seen from fig. 7, the micelle prepared by the present invention can achieve highly efficient anti-inflammatory activity at the cell level, compared to the free drug.
Test example 5
Animal experiments verify the imaging ability and anti-inflammatory effect of the micelle on inflammatory tissues
Freeze drying micelle with known drug loading rate, re-dissolving with physiological saline to prepare stock solution with certain concentration, and preparing free drug stock solution with the same drug concentration. An inflammation model is established by taking an atherosclerosis mouse, an arthritis mouse and a pneumonia mouse as animal models. Free drug, micelles or equal volume of saline was injected separately from the tail vein. The drug dosage is 10mg/Kg mouse. The imaging ability of the micelle is observed by a two-photon laser confocal microscope, various inflammation indexes are detected, and the experimental results are respectively shown in fig. 8, fig. 9 and fig. 10.
As can be seen from fig. 8, 9 and 10, the micelle has excellent two-photon imaging ability, and strong fluorescence emitted by the enrichment of the micelle can be clearly seen at inflammatory lesions such as atherosclerosis, arthritis and pneumonia. In addition, micelles have a better inflammatory inhibitory effect than free drugs.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. An anti-inflammatory nano-drug carrier is characterized by comprising a choline polyphosphate block and a poly (methyl-thioethyl acrylate) block; the structural formula of the nano-drug carrier is shown as follows:
Figure FDA0002510777180000011
wherein the polymerization degree of the choline polyphosphate is 1-100; the polymerization degree of the poly (methylthioethyl acrylate) is 1-100.
2. The method for preparing an anti-inflammatory nano-drug carrier according to claim 1, comprising the steps of:
s1, mixing methylthioethyl acrylate, 4-cyanovaleric acid dithiobenzoic acid, azobisisobutyronitrile and tetrahydrofuran, sequentially performing three cycles of freezing-pumping-unfreezing, reacting for 12-36h at 65-75 ℃ under the protection of argon, dialyzing reactants in ionized water, and finally performing freeze drying to obtain the poly (methylthioethyl acrylate);
s2, dissolving the poly (methylthioethyl acrylate) obtained in the step S1 and the methacryloyl phosphorylcholine in tetrahydrofuran, adding azodiisobutyronitrile, reacting for 24-28h at 65-75 ℃ after freezing and removing oxygen, dialyzing, and freeze-drying to obtain the product.
3. The method for preparing an anti-inflammatory nano-drug carrier as claimed in claim 2, wherein the mass ratio of the methylthioethylacrylate, the 4-cyanovaleric dithiobenzoic acid and the azobisisobutyronitrile in the step S1 is 3500-7000: 100-200: 25-50.
4. The method for preparing an anti-inflammatory nano-drug carrier as claimed in claim 2, wherein the mass ratio of the poly (methyl ethyl acrylate), the methacryloyl phosphorylcholine and the azobisisobutyronitrile in the step S2 is 1500-: 3000-6000: 12-25.
5. A pharmaceutical composition comprising the nano-drug carrier of claim 1 and the encapsulated diagnostic complex thereof.
6. The pharmaceutical composition of claim 5, wherein the diagnostic complex is prepared by linking a fluorescent molecule with two-photon aggregation-induced emission effect to a drug with anti-inflammatory function through a dicarbonyl bond.
7. The pharmaceutical composition according to claim 6, wherein said diagnostic complex is prepared by:
dissolving the fluorescent molecular group, dropwise adding into oxalyl chloride solution, stirring at room temperature for 10-15h, removing residual oxalyl chloride and solvent under reduced pressure, adding medicine and triethylamine, reacting at room temperature for 12-36h, and purifying to obtain the final product; wherein the molar ratio of the fluorescent molecular group, oxalyl chloride, triethylamine and the medicine is 1-10: 3-30: 3-30: 1-10.
8. A process for the preparation of a pharmaceutical composition according to any one of claims 5 to 7, comprising the steps of:
dissolving the nano-drug carrier and the diagnosis and treatment compound in a mixed solvent of tetrahydrofuran and dimethyl sulfoxide, stirring for 1-2h to obtain a mixed solution, adding the mixed solution into normal saline or ultrapure water with the volume 2-20 times that of the mixed solution, stirring for 1-4h, and removing the organic solvent to obtain the compound preparation;
or dissolving the nano-drug carrier and the diagnosis and treatment compound in tetrahydrofuran, performing rotary evaporation at 40-60 ℃ for 0.5-2h, removing the organic solvent, adding normal saline or ultrapure water, and fully oscillating for hydration to obtain the compound.
9. The method of claim 8, wherein the volume ratio of tetrahydrofuran to dimethylsulfoxide is 1-2: 1-2.
10. Use of the nano-drug carrier of claim 1 or the pharmaceutical composition of any one of claims 5 to 7 for the preparation of a medicament for the diagnosis and/or treatment of acute and chronic inflammation.
CN202010464905.XA 2020-05-08 2020-05-27 Anti-inflammatory nano-drug carrier, pharmaceutical composition thereof, preparation method and application Pending CN111603565A (en)

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