CN115957197B - Siveliroxostat coupled ROS sensitive albumin nanoparticle coated with anti-inflammatory drug, and preparation method and application thereof - Google Patents
Siveliroxostat coupled ROS sensitive albumin nanoparticle coated with anti-inflammatory drug, and preparation method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
An anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle and a preparation method and application thereof belong to the technical field of biological medicine. The preparation method specifically comprises a Sivelslat-ROS sensitive albumin conjugate and an anti-inflammatory drug entrapped by the Sivelslat-ROS sensitive albumin conjugate, wherein the Sivelslat-ROS sensitive albumin conjugate is prepared by sequentially coupling and synthesizing Sivelslat, PEG, ROS sensitive bonds and albumin. The anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle is prepared by a solvent-free method. The prepared anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle prolongs the circulation time of the anti-inflammatory drug and Siveliroxostat, improves the stability, can highly target an inflammation part and release the drug under the condition of high ROS of the inflammation part, and reduces the accumulation and systemic toxicity of non-target parts, thereby realizing the efficient treatment of various inflammatory diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and discloses a Sivelslata-coupled ROS sensitive albumin nanoparticle for entrapping an anti-inflammatory drug, and a preparation method and application thereof.
Background
Inflammatory diseases have long been known. In the case of rheumatoid arthritis (Rheumatoid arthritis, RA), RA is a chronic autoimmune disease characterized by synovitis, cartilage destruction, bone erosion and periarticular decalcification, which ultimately results in impairment of joint function (George G, shei G L, raghu K.Current and novel therapeutic targets in the treatment of rheumatoid arthritis [ J ]. Informammophily, 2020,28 (6): 1457-1476. Guo Q, wang Y, xu D, et al. Rheumatoid ar-travel: pathological mechanisms and modern pharmacologic therapies [ J ]. Bone research,2018,6 (1): 1-14.). Genetic factors and environmental factors are two major risk factors for the development and progression of rheumatoid arthritis. RA affects about 1% of the population worldwide (Ariyo O, victor H.Estimizing Maternal Mortality: surveillance methods and agenda for improvement [ J ]. The Routledge Handbook of African Demography, 2022:787-799.). During the development of inflammation, neutrophils (neutrophilis) are activated and migrate first into the joint cavity, playing a role in the inflammation process. There is established evidence that neutrophil extracellular traps (Neutrophil extracellular traps, NETs) are involved in the production of anti-citrullinated protein autoantibodies, triggering an inflammatory immune response. The NETs produced by inflammatory neutrophils affect other immune cells through various cytokines, thereby allowing the inflammatory state of inflammation to persist. Thus, modulation of the inflammatory microenvironment by inhibition of NETs formation would provide a new strategy for inflammatory treatment. Neutrophil elastase (Neutrophil elastase, NE) is a proteolytic enzyme stored in neutrophil azurin granules and is named because it is able to degrade the extracellular matrix protein elastin. There is evidence that NE affects the formation of NETs (Nishinaka Y, arai T, adachi S, et al Singlet oxygen is essential for neutrophil extracellular trap formation [ J ]. Biochemical and biophysical research communications,2011,413 (1): 75-79.Okeke E B,Louttit C,Fry C,et al.Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock[J ]. Biomaterials,2020, 238:119836.).
Sivelestat (ST) is a specific inhibitor of NE and is used in clinical applications in patients with acute lung injury associated with systemic inflammatory response. However, ST has relatively poor bioavailability due to extensive first pass metabolism (Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock [ J ]. Biomaterials,2020, 238:119836.) and is readily hydrolyzed to inactive metabolites in vitro. Therefore, improving the stability and bioavailability of the ciliristat is beneficial to improving the treatment effect of the ciliristat on inflammatory diseases. However, given that inflammation is mediated by a large number of cytokines, strategies that inhibit only a single or a small number of cytokines have difficulty controlling and even reversing the progression of the disease [ McInnes IB, schett G. Cytokines in the pathogenesis of rheumatoid arthritis [ J ]. Nature Reviews Immunology,2007,7 (6): 429-442.Zhang Q,Dehaini D,Zhang Y,et al.Neutrophil membrane-coated nanoparticles inhibit synovial inflammation and alleviate joint damage in inflammatory arthritis [ J ]. Nature nanotechnology,2018,13 (12): 1182-1190 ]. In addition, the modulation of a single cytokine may disrupt cytokine balance in vivo and produce side effects. Therefore, to improve the therapeutic effect on inflammatory diseases, multifunctional nano-drug delivery platforms are widely developed.
Disclosure of Invention
The invention provides a Sivelesta coupling ROS sensitive albumin nanoparticle coated with anti-inflammatory drugs, and a preparation method and application thereof. The coated cilomilast coupled ROS sensitive albumin nanoparticle can improve the stability of cilomilast and the coated anti-inflammatory drug and prolong the circulation time. And through specific targeting of inflammatory neutrophils, the cilvelistat and the anti-inflammatory drug are released in a responsive way under the condition of high ROS concentration of the inflammatory neutrophils, so that the preparation has strong potential in the aspects of realizing drug delivery and inflammatory treatment.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention relates to a Sivelslat-ROS sensitive albumin conjugate, which is prepared by sequentially coupling Sivelslat, PEG, ROS sensitive bond and albumin.
Further, the albumin is preferably mammalian albumin, more preferably human serum albumin or bovine serum albumin.
Sivelesta in molar ratio: albumin= (1-15): 1.
the structural formula of the PEG is COOH-PEG-NH 2 The molecular weight is 200-10000kDa, more preferably 1000kDa.
The ROS sensitive bond is selected from one of a compound containing a thioketal bond, a selenium-containing polymer, a tellurium-containing polymer and an oxalate polymer; more preferred are compounds containing a thioketal bond.
The preparation method of the Siveliroxostat-ROS sensitive albumin conjugate comprises the following steps:
activating the cilexetil by adopting an EDC-NHS system, and adding PEG to perform amidation reaction to obtain COOH-PEG-cilexetil;
activating COOH-PEG-cilovidone and ROS sensitive bond by EDC-NHS system, and carrying out amidation reaction to obtain ROS-PEG-cilovidone;
and carrying out amidation reaction on the ROS-PEG-cilexetil and albumin under EDC-NHS condition to obtain the cilexetil-ROS sensitive albumin conjugate (BSA-POS-PEG-ST).
When the ROS sensitive bond is a compound containing a thioketal bond, the BSA-POS-PEG-ST of the invention is BSA-TK-PEG-ST, and the synthetic route is as follows:
when the ROS sensitive bond is oxalate polymer, the BSA-POS-PEG-ST of the invention is BSA-OE-PEG-ST, and the synthetic route is as follows:
the invention relates to a ciliristat-ROS-conjugated reactive albumin nanoparticle for encapsulating an anti-inflammatory drug, which comprises a ciliristat-ROS-conjugated albumin and the anti-inflammatory drug, and more particularly relates to the ciliristat-ROS-conjugated albumin for encapsulating the anti-inflammatory drug.
According to the mass ratio, the anti-inflammatory medicament comprises the following components: sivelestat-ROS sensitive albumin conjugate = 1: (15 to 40), preferably 1: (20-35), more preferably 1:25.
The anti-inflammatory drug is selected from one of glucocorticoid anti-inflammatory drugs, non-steroidal anti-inflammatory drugs or antirheumatic drugs, wherein the glucocorticoid anti-inflammatory drugs are preferably dexamethasone palmitate, the non-steroidal anti-inflammatory drugs are preferably ibuprofen, and the antirheumatic drugs are preferably methotrexate.
The invention relates to a preparation method of anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle, which is a desolvation method, and specifically comprises the following steps:
dissolving the Siveliroxostat-ROS sensitive albumin conjugate in PBS buffer solution, adding an ethanol solution of an anti-inflammatory drug, precipitating albumin, and then adding glutaraldehyde to crosslink albumin to prepare the Siveliroxostat conjugated ROS sensitive albumin nanoparticle coated with the anti-inflammatory drug.
The invention relates to application of a Sivelesta coupling ROS sensitive albumin nanoparticle coated with an anti-inflammatory drug in a drug for treating inflammatory diseases.
The invention relates to a Sivelesta coupling ROS sensitive albumin nanoparticle for encapsulating anti-inflammatory drugs, and a preparation method and application thereof, and has the beneficial effects that:
the anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle can greatly improve the stability of Siveliroxostat and the entrapped anti-inflammatory drug and prolong the circulation time. The Siveliroxostat coupled ROS sensitive albumin nanoparticle coated with the anti-inflammatory drug can specifically target inflammatory neutrophils, and responds to ROS to release Siveliroxostat and the anti-inflammatory drug under the condition of high concentration of the inflammatory neutrophils, so that the treatment of inflammatory diseases and the regulation of inflammatory microenvironment are realized, the anti-inflammatory curative effect is greatly enhanced, the accumulation and systemic toxicity of non-target parts are reduced, and the high-efficiency treatment of various inflammatory diseases is realized. Has important significance for realizing the in vivo targeted delivery and treatment of the ciliristat and the anti-inflammatory drug.
The invention adopts albumin to wrap the hydrophobic medicine. Albumin has a unique spatial structure and can encapsulate drugs in a physical encapsulation or chemical bond coupling manner. And albumin can increase the solubility of the hydrophobic drug in blood plasma, and has better protective effect on the easily oxidized drug.
Drawings
FIG. 1 is a schematic diagram of the assembly of anti-inflammatory drug-entrapped Siveliroxostat-conjugated ROS-sensitive albumin nanoparticle of the present invention;
FIG. 2 is a diagram of a coomassie brilliant blue dye for the structural confirmation of BSA-TK-PEG-ST synthesis, 1 for BSA-TK-PEG-ST,2 for BSA-OE-PEG-ST, and 3 for BSA;
FIG. 3 shows a schematic representation of a synthesized structure of BSA-TK-PEG-ST, 1 representing BSA-TK-PEG-ST,2 representing BSA-OE-PEG-ST, and 3 representing BSA;
FIG. 4 shows a BSA-TK-PEG-ST synthetic structure-confirmed infrared spectrogram;
FIG. 5 shows a BSA-OE-PEG-ST synthetic structure-confirmed infrared spectrum;
fig. 6 is DP: change in encapsulation efficiency for BTST (BSA-TK-PEG-ST) at different ratios;
fig. 7 is IBU: change in encapsulation efficiency for BTST (BSA-TK-PEG-ST) at different ratios;
fig. 8 is MTX: change in encapsulation efficiency for BTST (BSA-TK-PEG-ST) at different ratios;
fig. 9 is DP: a plot of change in encapsulation efficiency for BOST (BSA-OE-PEG-ST) at different ratios;
fig. 10 is IBU: a plot of change in encapsulation efficiency for BOST (BSA-OE-PEG-ST) at different ratios;
fig. 11 is MTX: a plot of change in encapsulation efficiency for BOST (BSA-OE-PEG-ST) at different ratios;
FIG. 12 is a particle size distribution diagram of BTST (BSA-TK-PEG-ST) nanoparticles;
FIG. 13 is a graph showing the particle size distribution of DP/BTST nanoparticles;
FIG. 14 is a particle size distribution plot of IBU/BTST nanoparticles; (particle diameter of ibuprofen-entrapped nanoparticle)
FIG. 15 is a graph showing the particle size distribution of MTX/BTST nanoparticles; (particle diameter of methotrexate-entrapped nanoparticle)
FIG. 16 is a graph showing the particle size distribution of BOST (BSA-OE-PEG-ST) nanoparticles;
FIG. 17 is a graph showing the particle size distribution of DP/BOST nanoparticles;
FIG. 18 is a particle size distribution plot of IBU/BOST nanoparticles; (particle diameter of ibuprofen-entrapped nanoparticle)
FIG. 19 is a particle size distribution plot of MTX/BOST nanoparticles; (particle diameter of methotrexate-entrapped nanoparticle)
FIG. 20 is a transmission electron microscope image of BTST nanoparticles;
FIG. 21 is a transmission electron microscope image of DP/BTST nanoparticles;
FIG. 22 is a transmission electron microscope image of IBU/BTST nanoparticles;
FIG. 23 is a transmission electron microscope image of MTX/BTST nanoparticles;
FIG. 24 is a transmission electron microscope image of BOST nanoparticles;
FIG. 25 is a transmission electron microscope image of the DP/BOST nanoparticle;
FIG. 26 is a transmission electron microscope image of IBU/BOST nanoparticles;
FIG. 27 is a transmission electron microscope image of MTX/BOST nanoparticles;
FIG. 28 is a plot of foot swelling volume change in arthritic mice, normal representing Normal mice, N.S. representing arthritic mice given saline groups, DP/BTST NPs representing arthritic mice given DP/BTST nanoparticle treatment.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
The present example provides a ciliristat-conjugated ROS-sensitive albumin nanoparticle entrapped with an anti-inflammatory drug, said nanoparticle being composed of ciliristat (Siv)elestat, ST) -ROS-sensitive albumin (BSA) conjugate (BSA-TK-PEG 2K -ST) entrapped anti-inflammatory drug formation, the method for preparing the sivelestat-ROS sensitive albumin conjugate comprises the steps of:
(1) Sivelslastat 10mg is weighed and dissolved in 2ml DMSO, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) (1.1 eq.) and N-hydroxysulfosuccinimide (NHS) (1.1 eq.) are added for complete dissolution and reaction at room temperature for 2h; adding COOH-PEG-NH 2 38mg (0.9 eq.) are completely dissolved, triethylamine (2.0 eq.) is added and reacted for 4h at room temperature; the reaction solution was precipitated by adding a large amount of glacial ethyl ether: methanol=3: 1 purifying for three times, filtering and collecting a product, and vacuum drying to obtain the COOH-PEG-cilexetil. In this example, COOH-PEG-NH was used 2 Has a molecular weight of 2000kDa.
(2) The COOH-PEG-cilexetil 20mg was weighed out and dissolved in 2ml DMSO, EDC (1.2 eq.) and NHS (1.2 eq.) were added and reacted for 2h at room temperature. The ROS sensitive bond (NH) is weighed 2 -TK-NH 2 ) 2.5mg (2.0 eq.) of dissolved in 0.5ml of methanol, triethylamine (2.0 eq.) was added and the activated COOH-PEG-cilexetil was slowly added dropwise to NH 2 -TK-NH 2 In the solution, the reaction is carried out at room temperature overnight, the reaction solution is added into a large amount of glacial ethyl ether for precipitation, and the glacial ethyl ether is used for precipitation: methanol=3: 1 purifying for three times, filtering and collecting the product, and vacuum drying to obtain NH 2 -TK-PEG-cilvistasat.
(3) Weighing NH 2 -TK-PEG 2K 20mg of cilomilast is dissolved in 0.5ml of chloroform, succinic anhydride (2.0 eq.) and triethylamine (2.0 eq.) are added and dissolved completely, and reacted for 2h at room temperature; adding the reaction solution into a large amount of glacial diethyl ether for precipitation, filtering and collecting a product, and drying in vacuum to obtain COOH-TK-PEG-cilexetil.
(4) 10mg of COOH-TK-PEG-cilexetil is weighed and dissolved in 0.5ml of DMSO, EDC (1.2 eq.) and NHS (1.2 eq.) are added for complete dissolution and reaction at room temperature for 2h; 135mg (0.5 eq.) of BSA is weighed and dissolved in 5ml of PBS buffer solution with pH of 7.4, activated COOH-TK-PEG-cilexetil is added into the BSA solution, the reaction is carried out for 4 hours at room temperature, the reaction solution is transferred to a dialysis bag (molecular weight cut-off 3500 Da), dialysis is carried out for 12 hours in pure water, and the dialysate is collected and frozen and dried to obtain the BSA-TK-PEG-ST.
Example 2
The embodiment provides a Sivelestat coupling ROS sensitive albumin nanoparticle which is coated with an anti-inflammatory drug, wherein the nanoparticle is formed by coating the anti-inflammatory drug with a Sivelestat (ST) -ROS sensitive albumin (BSA) conjugate (BSA-OE-PEG-ST), and the preparation method of the Sivelestat-ROS sensitive albumin conjugate comprises the following steps:
(1) Sivelslastat 10mg is weighed and dissolved in 2ml DMSO, EDC (1.1 eq.) and NHS (1.1 eq.) are added for complete dissolution and reaction at room temperature for 2h; adding COOH-PEG-NH 2 38mg (0.9 eq.) are completely dissolved, triethylamine (2.0 eq.) is added and reacted for 4h at room temperature; the reaction solution was precipitated by adding a large amount of glacial ethyl ether: methanol=3: 1 purifying for three times, filtering and collecting a product, and vacuum drying to obtain the COOH-PEG-cilexetil. In this example, COOH-PEG-NH was used 2 Has a molecular weight of 2000kDa.
(2) The COOH-PEG-cilexetil 20mg was weighed out and dissolved in 2ml DMSO, EDC (1.2 eq.) and NHS (1.2 eq.) were added and reacted for 2h at room temperature. Weighing NH 2 -OE-NH 2 2.5mg (2.0 eq.) of dissolved in 0.5ml of methanol, triethylamine (2.0 eq.) was added and the activated COOH-PEG-cilexetil was slowly added dropwise to NH 2 -OE-NH 2 In the solution, the reaction is carried out at room temperature overnight, the reaction solution is added into a large amount of glacial ethyl ether for precipitation, and the glacial ethyl ether is used for precipitation: methanol=3: 1 purifying for three times, filtering and collecting the product, and vacuum drying to obtain NH 2 -OE-PEG-cilovitrestat.
(3) Weighing NH 2 20mg of OE-PEG-cilexetil is dissolved in 0.5ml of chloroform, succinic anhydride (2.0 eq.) and triethylamine (2.0 eq.) are added and completely dissolved, and reacted for 2h at room temperature; adding the reaction solution into a large amount of glacial diethyl ether for precipitation, filtering and collecting a product, and vacuum drying to obtain COOH-OE-PEG-cilexetil.
(4) 10mg of COOH-OE-PEG-cilexetil is weighed and dissolved in 0.5ml DMSO, EDC (1.2 eq.) and NHS (1.2 eq.) are added for complete dissolution and reaction at room temperature for 2h; 135mg (0.5 eq.) of BSA is weighed and dissolved in 5ml of PBS buffer solution with pH of 7.4, the activated COOH-OE-PEG-cilexetil is added into the BSA solution for reaction for 4 hours at room temperature, the reaction solution is transferred to a dialysis bag (molecular weight cut-off 3500 Da), dialysis is carried out for 12 hours in pure water, and the dialysate is collected and freeze-dried to obtain the BSA-OE-PEG-ST.
Example 3
This example provides structural confirmation of BSA-TK-PEG-ST and BSA-OE-PEG-ST:
(1) Coomassie brilliant blue staining
See FIG. 2, band 1 is BSA-TK-PEG-ST, band 2 is BSA-OE-PEG-ST, and band 3 is BSA. The molecular weight of BSA is 66kDa, and the electrophoresis band accords with theory; BSA-TK-PEG-ST and BSA-OE-PEG-ST have molecular weights greater than BSA, proving successful synthesis of BSA-TK-PEG-ST and BSA-OE-PEG-ST.
(2) PEG color development
See FIG. 3, where band 1 is BSA-TK-PEG-ST, band 2 is BSA-OE-PEG-ST, and band 3 is BSA in FIG. 3. BSA molecular weight 66kDa, BSA developed without PEG, BSA-TK-PEG-ST and BSA-OE-PEG-ST developed with PEG, demonstrating successful synthesis of BSA-TK-PEG-ST and BSA-OE-PEG-ST.
(3) Infrared spectrum
The structure of BSA-TK-PEG-ST was confirmed by IR spectroscopy, the IR spectrum of which is shown in FIG. 4, and the result of FIG. 4 shows that BSA-TK-PEG 2K ST successful synthesis. The structure of BSA-OE-PEG-ST was confirmed by infrared spectroscopy, the infrared spectrum of which is shown in FIG. 5, and the results of FIG. 5 indicate that BSA-OE-PEG-ST was successfully synthesized.
Example 3
The assembly schematic diagram of the anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle is shown in figure 1, the anti-inflammatory drug-entrapped Siveliroxostat coupled ROS sensitive albumin nanoparticle is formed by entrapping an anti-inflammatory drug by BSA-TK-PEG-ST, the anti-inflammatory drug is selected from dexamethasone palmitate (Dexamethasone palmitate, DP) as an example, and the ratio of DP to BSA-TK-PEG-ST is screened by the encapsulation rate (Encapsulate efficiency, EE%) of DP:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; protein was precipitated by adding 4mL of Dexamethasone Palmitate (DP) in absolute ethanol, DP: the mass ratio of BSA-TK-PEG-ST is 1 (15-35); 100 mu L glutaraldehyde (the concentration is 2%) is added dropwise at the rate of 0.3mL/min, the crosslinked nanoparticles are fully stirred, absolute ethyl alcohol and redundant glutaraldehyde solution are removed by centrifugation at a high rotating speed (12000 rpm/min), and the DP/BTST nanoparticles are obtained by resuspension with PBS.
As shown in FIG. 6, the encapsulation efficiency of DP was highest and could be up to (90.2.+ -. 6.2)% when the mass ratio of DP to BTST was 1:25. Wherein, when the mass ratio of DP to BTST is 1:15-1:40, the encapsulation rate reaches more than 50%.
TABLE 1
Example 4
The present embodiment provides a sivelestat coupling ROS sensitive albumin nanoparticle coated with an anti-inflammatory drug, formed by BSA-TK-PEG-ST coated with an anti-inflammatory drug, for example, ibuprofen (IBU) is selected as the anti-inflammatory drug, and the ratio of IBU to BSA-TK-PEG-ST is selected by EE% of IBU:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; adding 4mL of IBU absolute ethanol solution to precipitate protein, IBU: the mass ratio of BSA-TK-PEG-ST is 1 (25-35); 100 mu L glutaraldehyde (the concentration is 2%) is added dropwise at the rate of 0.3mL/min, the crosslinked nanoparticles are fully stirred, absolute ethyl alcohol and redundant glutaraldehyde solution are removed by centrifugation at a high rotating speed (12000 rpm/min), and the IBU/BTST nanoparticles are obtained by re-suspending with PBS. As can be seen from FIG. 7, the encapsulation efficiency of IBU is highest and can reach (78.0.+ -. 2.2)% when the mass ratio of IBU to BTST is 1:25. When the mass ratio of IBU to BTST is 1:20-1:35, the encapsulation efficiency is more than 50%.
Example 5
The present embodiment provides an anti-inflammatory drug-entrapped sivelestat-coupled ROS-sensitive albumin nanoparticle formed by BSA-TK-PEG-ST entrapping an anti-inflammatory drug, which is exemplified by Methotrexate (MTX), the ratio of MTX to BSA-TK-PEG-ST being screened by EE% of MTX:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; protein was precipitated by adding 4mL of an absolute ethanol solution of MTX: the mass ratio of BSA-TK-PEG-ST is 1 (20-40); 100 mu L glutaraldehyde (the concentration is 2%) is added dropwise at the rate of 0.3mL/min, the crosslinked nanoparticles are fully stirred, absolute ethyl alcohol and redundant glutaraldehyde solution are removed by centrifugation at a high rotating speed (12000 rpm/min), and the MTX/BTST nanoparticles are obtained by re-suspending with PBS. As shown in FIG. 8, the encapsulation efficiency of MTX was highest at a mass ratio of MTX to BTST of 1:25, and it was found that the encapsulation efficiency was 80.0.+ -. 2.9%. When the mass ratio of MTX to BTST is 1:20-1:40, the entrapment rate is more than 50%.
Example 6
The present embodiment provides an anti-inflammatory drug-entrapped sivelestat-conjugated ROS-sensitive albumin nanoparticle formed by entrapping an anti-inflammatory drug with BSA-OE-PEG-ST, and the anti-inflammatory drug is selected from dexamethasone palmitate (Dexamethasone palmitate, DP), and the ratio of DP to BSA-OE-PEG-ST is selected by the encapsulation ratio of DP (Encapsulate efficiency, ee%) screening:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; protein was precipitated by adding 4mL of Dexamethasone Palmitate (DP) in absolute ethanol, DP: the mass ratio of BSA-OE-PEG-ST is 1 (15-40); slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain the DP/BOST nanoparticles. As shown in FIG. 9, the encapsulation efficiency of DP was highest and reached (87.8.+ -. 3.2)% when the mass ratio of DP to BOST was 1:25. When the mass ratio of DP to BOST is 1:15-1:40, the entrapment rate is more than 50%.
Example 7
The embodiment provides a Sivelslastat coupling ROS sensitive albumin nanoparticle coated with anti-inflammatory drugs, which is formed by coating anti-inflammatory drugs with BSA-OE-PEG-ST, wherein the anti-inflammatory drugs are Ibuprofen (IBU), and the ratio of the IBU to the BSA-OE-PEG-ST is screened by the encapsulation rate (Encapsulate efficiency, EE%) of the IBU:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; adding 4mL of IBU absolute ethanol solution to precipitate protein, IBU: the mass ratio of BSA-OE-PEG-ST is 1 (20-35); slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethyl alcohol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain IBU/BOST nanoparticles. As can be seen from FIG. 10, the encapsulation efficiency of IBU is highest and can reach (83.2.+ -. 2.7)% when the mass ratio of IBU to BOST is 1:30. When the mass ratio of IBU to BOST is 1:20-1:35, the entrapment rate is more than 50%.
Example 8
The present embodiment provides an anti-inflammatory drug-entrapped sivelestat-conjugated ROS-sensitive albumin nanoparticle formed by BSA-OE-PEG-ST entrapping an anti-inflammatory drug, and the anti-inflammatory drug is Methotrexate (MTX), and the ratio of MTX to BSA-OE-PEG-ST is selected by EE% of MTX:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; protein was precipitated by adding 4mL of an absolute ethanol solution of MTX: the mass ratio of BSA-OE-PEG-ST is 1 (20-40); slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain MTX/BOST nanoparticles. As shown in FIG. 11, the encapsulation efficiency of MTX was highest at a mass ratio of MTX to BOST of 1:30, and it was found that the encapsulation efficiency was 86.2.+ -. 3.0%. When the mass ratio of MTX to BOST is 1:20-1:40, the entrapment rate is more than 50%.
Example 9
The embodiment provides a cevelirst coupling ROS sensitive albumin nanoparticle of an entrapped anti-inflammatory drug, wherein the nanoparticle is formed by BSA-TK-PEG-ST entrapped anti-inflammatory drug Dexamethasone Palmitate (DP), and preferably, the mass ratio of DP to BTST is 1:25, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; protein was precipitated by adding 4mL of Dexamethasone Palmitate (DP) in absolute ethanol, DP: the mass ratio of BSA-TK-PEG-ST is 1:25; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain the DP/BTST nanoparticles.
Example 10
The embodiment provides a Sivelestat coupling ROS sensitive albumin nanoparticle for encapsulating anti-inflammatory drugs, wherein the nanoparticle is formed by BSA-TK-PEG-ST encapsulating anti-inflammatory drug Ibuprofen (IBU), preferably, the IBU/BTST mass ratio is 1:25, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; adding 4mL of IBU absolute ethanol solution to precipitate protein, IBU: the mass ratio of BSA-TK-PEG-ST is 1:25; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethyl alcohol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain IBU/BTST nanoparticles.
Example 11
The embodiment provides a Sivelestat coupling ROS sensitive albumin nanoparticle for encapsulating anti-inflammatory drugs, wherein the nanoparticle is formed by BSA-TK-PEG-ST encapsulating anti-inflammatory drug Methotrexate (MTX), preferably, taking MTX/BTST mass ratio of 1:25 as an example, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-TK-PEG-ST in 1mL PBS buffer solution at 25deg.C, and stirring at 300rpm/min; protein was precipitated by adding 4mL of an absolute ethanol solution of MTX: the mass ratio of BSA-TK-PEG-ST is 1:25; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain MTX/BTST nanoparticles.
Example 12
The embodiment provides a sivelestat coupling ROS sensitive albumin nanoparticle coated with an anti-inflammatory drug, wherein the nanoparticle is formed by BSA-OE-PEG-ST coated with the anti-inflammatory drug Dexamethasone Palmitate (DP), and preferably, the mass ratio of DP to BOST is 1:25, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; protein was precipitated by adding 4mL of Dexamethasone Palmitate (DP) in absolute ethanol, DP: the mass ratio of BSA-OE-PEG-ST is 1:25; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain the DP/BOST nanoparticles.
Example 13
The embodiment provides a Siveliroxostat coupling ROS sensitive albumin nanoparticle for encapsulating anti-inflammatory drugs, wherein the nanoparticle is formed by BSA-OE-PEG-ST encapsulating anti-inflammatory drug Ibuprofen (IBU), preferably, the mass ratio of IBU to BOST is 1:25, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; adding 4mL of IBU absolute ethanol solution to precipitate protein, IBU: the mass ratio of BSA-OE-PEG-ST is 1:30; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethyl alcohol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain IBU/BOST nanoparticles.
Example 14
The embodiment provides a Siveliroxostat coupling ROS sensitive albumin nanoparticle coated with an anti-inflammatory drug, wherein the nanoparticle is formed by BSA-OE-PEG-ST coated with the anti-inflammatory drug Methotrexate (MTX), preferably, the mass ratio of MTX to BOST is 1:25, and the preparation method of the nanoparticle comprises the following steps:
dissolving BSA-OE-PEG-ST in 1mL PBS buffer solution at 25deg.C, stirring to dissolve, stirring at 300rpm/min; protein was precipitated by adding 4mL of an absolute ethanol solution of MTX: the mass ratio of BSA-OE-PEG-ST is 1:30; slowly dripping 100 mu L glutaraldehyde (with concentration of 2%) at 0.3mL/min, fully stirring to crosslink the nanoparticles, centrifuging at high rotation speed (12000 rpm/min) to remove absolute ethanol and redundant glutaraldehyde solution, and re-suspending with PBS to obtain MTX/BOST nanoparticles.
Example 15
The embodiment provides a Siveliroxostat coupled ROS sensitive albumin nanoparticle coated with an anti-inflammatory drug, and the particle size of the nanoparticle is detected by using a dynamic light scattering method and a transmission electron microscope. As can be seen from fig. 12, 13, 14 and 15, the particle diameters of BTST nanoparticle (fig. 12), DP/BTST nanoparticle (fig. 13), IBU/BTST nanoparticle (fig. 14) and MTX/BTST nanoparticle (fig. 15) were (124.8±1.5), (125.3±1.2), (123.6±2.4) and (122.8±1.6) nm, respectively. As can be seen from FIGS. 16, 17, 18 and 19, the particle diameters of the BOST nanoparticle (FIG. 16), the DP/BOST nanoparticle (FIG. 17), the IBU/BOST nanoparticle (FIG. 18) and the MTX/BOST nanoparticle (FIG. 19) were (125.5.+ -. 2.5), (126.6.+ -. 2.7), (123.8.+ -. 3.2) and (128.4.+ -. 3.6) nm, respectively. The transmission electron microscope results are shown in FIGS. 20 to 27. All the nanoparticles are in a spherical or spheroid-like structure and have uniform size.
Example 16
The present example investigated the therapeutic effect of DP/BTST nanoparticles on rheumatoid arthritis mice.
C57BL/6RA rheumatoid arthritis (Rheumatoid arthritis, RA) mice were used to assess the arthritic efficacy of BTS NPs, DP/BTS NPs, IBU/BTS NPs, MTX/BTS NPs, BOST NPs, DP/BOST NPs, IBU/BOST NPs, MTX/BOST NPs in vivo. CIA mice were randomly divided into 3 groups (n=6). Saline (n.s.), BTST NPs, DP/BTST NPs, IBU/BTST NPs, MTX/BTST NPs, BOST NPs, DP/BOST NPs, IBU/BOST NPs, and MTX/BOST NPs were injected intravenously into each group. After the end of the treatment, the extent of the swelling of the feet of the mice was observed and recorded by photographing (see fig. 28).
The graph 28 shows that after the nanoparticle treatment of each group is given, the foot swelling volume of RA mice is obviously reduced, which indicates that the Sivelestat coupling ROS sensitive albumin nanoparticle coated with the anti-inflammatory drug of the invention responds to ROS to release Sivelestat and the anti-inflammatory drug under the condition of high ROS concentration of inflammatory neutrophils, thereby realizing the treatment of inflammatory diseases and the regulation of inflammatory microenvironment and greatly enhancing the anti-inflammatory curative effect.
Claims (8)
1. The Siveliroxostat-ROS sensitive albumin conjugate is characterized in that Siveliroxostat, PEG, ROS sensitive bond and albumin are sequentially coupled and synthesized;
the ROS sensitive bond is selected from one of a compound containing a thioketal bond and an oxalate polymer;
the preparation method of the Siveliroxostat-ROS sensitive albumin conjugate specifically comprises the following steps:
activating the cilexetil by adopting an EDC-NHS system, and adding PEG to perform amidation reaction to obtain COOH-PEG-cilexetil;
activating COOH-PEG-cilovidone and ROS sensitive bond by EDC-NHS system, and carrying out amidation reaction to obtain ROS-PEG-cilovidone;
carrying out amidation reaction on ROS-PEG-cilexetil and albumin under EDC-NHS condition to obtain the cilexetil-ROS sensitive albumin conjugate.
2. The sivelestat-ROS sensitive albumin conjugate of claim 1, wherein said albumin is mammalian albumin.
3. The sivelestat-ROS sensitive albumin conjugate of claim 1, wherein, in molar ratio, sivelestat: albumin= (1-15): 1.
4. the sivelestat-ROS sensitive albumin conjugate of claim 1, wherein said PEG has the structural formula COOH-PEG-NH 2 The molecular weight is 200-10000 kDa.
5. A cevelirst-ROS-conjugated albumin nanoparticle entrapped with an anti-inflammatory drug, comprising the cevelirst-ROS-conjugated albumin conjugate of any one of claims 1-4 and an anti-inflammatory drug;
according to the mass ratio, the anti-inflammatory medicament comprises the following components: sivelestat-ROS sensitive albumin conjugate = 1: (15-40).
6. The anti-inflammatory drug-entrapped sivelestat conjugated ROS-sensitive albumin nanoparticle of claim 5, wherein the anti-inflammatory drug is selected from one of a glucocorticoid anti-inflammatory drug, a non-steroidal anti-inflammatory drug, or an antirheumatic drug.
7. The method for preparing the anti-inflammatory drug-entrapped sivelestat coupled ROS sensitive albumin nanoparticle according to claim 5 or 6 is a desolvation method, and is characterized by comprising the following steps:
dissolving the Siveliroxostat-ROS sensitive albumin conjugate in PBS buffer solution, adding an ethanol solution of an anti-inflammatory drug, precipitating albumin, and then adding glutaraldehyde to crosslink albumin to prepare the Siveliroxostat conjugated ROS sensitive albumin nanoparticle coated with the anti-inflammatory drug.
8. Use of the anti-inflammatory drug-entrapped sivelestat conjugated ROS sensitive albumin nanoparticle of claim 5 or 6 for use in a medicament for treating inflammatory diseases.
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