CN115068629A - Drug-loaded nanoparticle based on curcumin and cerium oxide, and preparation method and application thereof - Google Patents

Drug-loaded nanoparticle based on curcumin and cerium oxide, and preparation method and application thereof Download PDF

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CN115068629A
CN115068629A CN202210769968.5A CN202210769968A CN115068629A CN 115068629 A CN115068629 A CN 115068629A CN 202210769968 A CN202210769968 A CN 202210769968A CN 115068629 A CN115068629 A CN 115068629A
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curcumin
aqueous solution
cerium oxide
mannose
chitosan
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CN115068629B (en
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李学明
杨婧
任浩
王永禄
孟政杰
王琪玥
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Nanjing Tech University
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Abstract

The invention provides a drug-loaded nanoparticle based on curcumin and cerium oxide and a preparation method thereof. The drug-loaded nanoparticles can be orally taken, the drug reaches an inflammation part through the macrophage targeting effect of mannose, the drug is released, the polarization of the macrophage is regulated, the oxidation resistance is realized, the iron death is inhibited, and the effect of balancing intestinal flora by adding beneficial bacteria and inhibiting harmful bacteria is realized, so that the effect of more effectively treating the intestinal inflammation is achieved.

Description

Drug-loaded nanoparticle based on curcumin and cerium oxide, and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to drug-loaded nanoparticles based on curcumin and cerium oxide, and a preparation method and application thereof.
Background
Inflammatory Bowel Disease (IBD) is a chronic relapsing and remitting inflammatory disease of the small intestine and colon. The existing IBD treatment schemes mostly adopt anti-inflammatory drugs aiming at inflammatory symptoms, can relieve the symptoms of IBD patients in short time generally, but have unsatisfactory curative effect in long term due to the limitation of the anti-inflammatory drugs. Anti-inflammatory drugs such as aminosalicylates, biologicals (i.e., anti-TNF monoclonal antibodies), corticosteroids and immunosuppressants are currently used extensively to treat IBD. The aminosalicylic acid preparation belongs to the medicines which are used most at the earliest and have the least side effect, but the side effect of the medicines is not ignored, the headache, the vomiting and the diarrhea, the skin anaphylactic reaction and the like are the most common, and in addition, the aminosalicylic acid preparation also has toxic action on the kidney.
Through the investigation of inflammatory bowel disease pathological microenvironment, the inflammatory colon is found to have the characteristics of positive charge protein enrichment and high Reactive Oxygen Species (ROS) overexpression at a focus part, however, among a plurality of known substances for resisting oxidation, scavenging ROS or carrying negative charges, substances capable of generating a relieving effect on IBD are rare, and although individual antioxidant substances develop IBD therapeutic agents, the effects and various limitations in application are not satisfactory.
Chinese patent publication No. CN111773243A discloses a drug for inflammatory bowel disease, its preparation method and use, the drug comprises ceria-based nanoenzyme and/or metal element-doped ceria-based nanoenzyme, the ceria-based nanoenzyme is an enzyme mimic with activities of superoxide dismutase and catalase, and removing hydroxyl radical, etc., and the enzyme mimic eliminates high active oxygen free radical at pathological part of enteritis by this enzyme catalysis to achieve anti-inflammatory effect. The oral preparation can eliminate local inflammation and repair ulcer through the target contact with pathological enteritis parts with positive charges, and importantly, the oral preparation can avoid the potential toxic and side effects of the cerium oxide-based nano enzyme absorbed by organisms to the greatest extent, and finally achieves the aim of safely treating enteritis. However, the drug can only treat inflammatory enteritis by a single mechanism, the treatment effect is limited, and the small size of the drug enables the drug to be rapidly absorbed through gastrointestinal walls, thereby increasing the systemic exposure and causing adverse side effects, and the ideal treatment effect cannot be achieved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a drug-loaded nanoparticle based on curcumin and cerium oxide, which can regulate macrophage polarization, eliminate overhigh active oxygen free radicals at enteritis pathological parts and inhibit iron death so as to achieve the aim of anti-inflammation, and can regulate intestinal flora, stabilize the intestinal environment, provide favorable microenvironment for anti-inflammation and improve the anti-inflammation effect.
According to the first aspect of the invention, the drug-loaded nanoparticles based on curcumin and cerium oxide are formed by taking chitosan modified by mannose as a carrier and coating the curcumin-cerium oxide nanoparticles in the carrier.
Preferably, the average particle size of the curcumin-cerium oxide nanoparticles is 100-140 nm.
Preferably, the average particle size of the drug-loaded nanoparticles is 325-480 nm.
Preferably, curcumin is combined with human serum albumin and then combined with cerium ions under alkaline conditions to form the curcumin-cerium oxide nanoparticles.
According to a second aspect of the invention, the application of the drug-loaded nanoparticles based on curcumin and cerium oxide in preparing drugs for preventing and treating inflammatory enteritis and regulating intestinal flora is provided.
Preferably, macrophage polarization is regulated through curcumin-cerium oxide nanoparticles, excessive active oxygen free radicals at enteritis pathological parts are eliminated, iron death is inhibited, the anti-inflammatory purpose is achieved, and meanwhile, the favorable microenvironment is provided for anti-inflammation through regulating intestinal flora and stabilizing the intestinal environment, so that the anti-inflammatory effect is improved.
According to a third aspect of the invention, a preparation method of the drug-loaded nanoparticles based on curcumin and cerium oxide is provided, which comprises the following steps:
s1, adding the curcumin solution into a human serum albumin aqueous solution, and stirring for reaction to obtain a curcumin nanoparticle aqueous solution;
s2, taking the curcumin nanoparticle aqueous solution obtained in the step S1, adding an aqueous solution containing a cerium source into the curcumin nanoparticle aqueous solution to obtain a first mixed solution, adjusting the first mixed solution to be alkaline, and then stirring for reaction to obtain a curcumin-cerium oxide nanoparticle aqueous solution;
s3, taking the curcumin-cerium oxide nanoparticle aqueous solution obtained in the step S2, dropwise adding the curcumin-cerium oxide nanoparticle aqueous solution into the chitosan aqueous solution modified by mannose, stirring for reaction, and dialyzing and purifying to obtain the mannose-chitosan coated curcumin-cerium oxide nanoparticles.
Preferably, the curcumin solution is ethanol, propanedione or acetone solution of curcumin, and the concentration is 1-3 mg/mL; the concentration of the human serum albumin aqueous solution is 5-20 mg/mL; the mass ratio of curcumin to human serum albumin is (1:10) - (1: 50).
Preferably, the specific process of step S2 is:
adding a cerium source-containing aqueous solution into a curcumin nanoparticle aqueous solution, stirring for the first time to obtain a first mixed solution, adjusting the pH value of the first mixed solution to be 12-13, continuing stirring for the second time, and after stirring is finished, obtaining the curcumin-cerium oxide nanoparticle aqueous solution.
Preferably, the concentration of the aqueous solution containing the cerium source is 0.1-1M, and the cerium source is Ce (NO) 3 ) 3 ·6H 2 O; the mass ratio of the human blood albumin to the cerium ions in the curcumin nanoparticles is (19:1) - (78: 1);
the reaction conditions are as follows: the reaction temperature of the system is 37-80 ℃, the time of the first stirring is 5-15min, the time of the second stirring is 0.25-2h, and the stirring speed is 400-600 rmp.
Preferably, in the step S3, the volume ratio of the curcumin-cerium oxide nanoparticle aqueous solution to the mannose-modified chitosan aqueous solution is (1:1) - (1: 4);
the aqueous solution of chitosan modified by mannose is prepared by dissolving chitosan modified by mannose in an aqueous solution containing acid, wherein the concentration of the chitosan modified by mannose is 0.5-2% w/v, and the aqueous solution containing acid is an aqueous solution of hydrochloric acid, formic acid, acetic acid or lactic acid with 1-10% v/v.
Preferably, the mannose-modified chitosan is prepared as follows:
dropwise adding the chitosan solution into an aqueous solution containing d-mannose and NaBH (OAc)3, stirring, dialyzing after stirring, and freeze-drying to obtain mannose-chitosan powder.
Preferably, the chitosan solution is prepared by dissolving chitosan in acid-containing aqueous solution and adjusting the pH value to 5-6, wherein the concentration of the chitosan is 0.3-1% w/v, and the acid-containing aqueous solution is 1-10% v/v of hydrochloric acid, formic acid, acetic acid or lactic acid;
containing d-mannose and NaBH (OAc) 3 In an aqueous solution of (2), the concentration of d-mannose is 0.05M, NaBH (OAc) 3 The concentration of (A) is 0.01M;
the chitosan solution contains d-mannose and NaBH (OAc) 3 The volume ratio of the aqueous solution of (1:1) to (1: 2).
Compared with the prior art, the invention has the beneficial effects that:
1. the drug-loaded nano particles based on curcumin and cerium oxide are successfully prepared, the drug-loaded nano particles can be orally taken for administration, targeted macrophages are taken to achieve fixed-point administration at intestinal inflammation parts through mannose-chitosan shells, after the nano particles reach the focus, the phenotype of the macrophages can be promoted to be converted from an inflammatory M1 phenotype to another activated anti-inflammatory M2 phenotype, so that the anti-inflammatory purpose is achieved, and meanwhile, the drug-loaded nano particles can inhibit iron death at the inflammation parts, eliminate overhigh active oxygen free radicals at enteritis lesion parts, reduce damage to cells and mucosa barriers at the inflammation parts, inhibit proinflammatory activation of the macrophages, provide good microenvironment for inflammation resistance, improve the anti-inflammatory efficiency and strengthen the anti-inflammatory effect.
On the other hand, the drug-loaded nano particle also has the function of regulating intestinal flora, and can balance the flora in the intestinal tract by killing harmful bacteria in the intestinal tract and increasing beneficial bacteria, so that the intestinal microorganisms are in a physiological steady state, the damage to cells and mucosal barriers at inflammatory parts is further reduced, and the infiltration of inflammatory cells such as macrophages and the secretion of inflammatory cytokines are prevented from being changed due to the disturbance of the intestinal microorganisms, thereby increasing the susceptibility to IBD inflammation, providing a good microenvironment for anti-inflammation, improving the anti-inflammation efficiency and enhancing the anti-inflammation effect.
Therefore, the drug-loaded nano particle provided by the invention can be used for eliminating overhigh active oxygen free radicals at enteritis pathological change parts and inhibiting iron death by regulating macrophage polarization, so that the anti-inflammatory purpose is achieved, and meanwhile, the intestinal flora can be regulated, the intestinal environment is stabilized, a favorable microenvironment is provided for anti-inflammation, and the anti-inflammatory effect is improved.
2. The drug-loaded nanoparticles can improve the compliance of patients and solve a plurality of technical problems of preparation by preparing an oral drug-loaded nano-system, and curcumin-cerium oxide nanoparticles are coated by taking mannose-modified chitosan as a carrier, so that the irritation of drugs to gastrointestinal tracts is reduced, and the effect of directional release can be achieved through structural modification.
3. The drug-loaded nanoparticles are simple to prepare and easy to operate, and can realize high loading capacity and good bioavailability of incompatible drugs.
Drawings
FIG. 1 is a TEM image of Cur-Ce @ MCS NPs obtained by an example of the present invention.
FIG. 2A is a graph showing the particle sizes of Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in examples of the present invention.
FIG. 2B is a graph showing the particle sizes of Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in the example of the present invention.
FIG. 3A is a graph showing the colon length of mice treated with Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in accordance with an embodiment of the present invention, and a control group.
FIG. 3B is a graph of Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained by an embodiment of the present invention, and DAI after a control treatment.
FIG. 4 is a graph showing the staining patterns of the 4-HNE immunohistochemistry after the treatment of the control group and the Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in the example of the present invention.
FIG. 5 is a graph showing the staining patterns of the CD206 immunohistochemistry after the control group treatment, and the Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in the example of the present invention.
FIG. 6 is a graph showing the MDA content of the treated tissue homogenate of Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs obtained in the examples of the present invention, and the control.
FIG. 7 is a graph of the horizontal colony distribution of Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, and Cur-Ce @ MCS NPs obtained in accordance with an embodiment of the present invention, and a control treatment backgate.
Detailed Description
In order to better understand the technical content of the present invention, specific embodiments are described below with reference to the accompanying drawings.
In this disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. Embodiments of the present disclosure are not necessarily intended to include all aspects of the invention. It should be appreciated that the various concepts and embodiments described above, as well as those described in greater detail below, may be implemented in any of numerous ways.
The drug-loaded nanoparticles based on curcumin and cerium oxide can be orally administered, the drug reaches an inflammation part through macrophage targeting effect of mannose, the drug is released, macrophage polarization is regulated, oxidation resistance is realized, iron death is inhibited, beneficial bacteria are added, the effect of harmful bacteria on balancing intestinal flora is inhibited, and the effect of effectively treating intestinal inflammation is achieved.
In an exemplary embodiment of the invention, a drug-loaded nanoparticle based on curcumin and cerium oxide is provided, chitosan modified by mannose is used as a carrier, and the curcumin-cerium oxide nanoparticle is coated in the carrier to form the drug-loaded nanoparticle.
In a preferred embodiment, the average particle size of the curcumin-cerium oxide nanoparticles is 100-140 nm.
In a preferred embodiment, the average particle size of the drug-loaded nanoparticles is 325-480 nm.
In a preferred embodiment, curcumin is combined with human serum albumin and then combined with cerium ions under alkaline conditions to form the curcumin-cerium oxide nanoparticles.
In another exemplary embodiment of the invention, the application of the drug-loaded nanoparticles based on curcumin and cerium oxide in preparing drugs for preventing and treating inflammatory enteritis and regulating intestinal flora is provided.
In a preferred embodiment, macrophage polarization is regulated through the curcumin-cerium oxide nanoparticles, excessive active oxygen free radicals at enteritis lesion sites are eliminated, iron death is inhibited, so that the anti-inflammatory purpose is achieved, and meanwhile, the favorable microenvironment is provided for anti-inflammatory through regulating intestinal flora and stabilizing the intestinal environment, so that the anti-inflammatory effect is improved.
The invention also provides a preparation method of the curcumin-cerium oxide nanoparticles coated with mannose coupling chitosan, which comprises the steps of preparing curcumin nanoparticles by a desolvation method, generating curcumin-cerium oxide nanoparticles by self-assembly, combining mannose modified chitosan on the surfaces of the curcumin-cerium oxide nanoparticles by electrostatic adsorption, dialyzing, purifying, freezing, drying and collecting.
In another exemplary embodiment of the invention, there is provided a preparation method of the drug-loaded nanoparticles based on curcumin and cerium oxide, comprising the following steps:
s1, adding the curcumin (Cur) solution into a Human Serum Albumin (HSA) aqueous solution, and stirring for reaction to obtain a curcumin nanoparticle (CurNPs) aqueous solution;
s2, taking the curcumin nanoparticle aqueous solution obtained in the step S1, adding an aqueous solution containing a cerium source into the curcumin nanoparticle aqueous solution to obtain a first mixed solution, adjusting the first mixed solution to be alkaline, and then carrying out stirring reaction to obtain a curcumin-cerium oxide nanoparticle (Cur-Ce NPs) aqueous solution;
s3, taking the curcumin-cerium oxide nanoparticle aqueous solution obtained in the step S2, dropwise adding the curcumin-cerium oxide nanoparticle aqueous solution into a chitosan (M-CS) aqueous solution modified by mannose, stirring for reaction, and dialyzing and purifying to obtain mannose-chitosan coated curcumin-cerium oxide nanoparticles (Ce @ MCS NPs).
In a preferred embodiment, the curcumin solution is ethanol, propanedione or acetone solution of curcumin with a concentration of 1-3 mg/mL; the concentration of the human serum albumin aqueous solution is 5-20 mg/mL; the mass ratio of curcumin to human serum albumin is (1:10) - (1: 50).
In a preferred embodiment, the specific process of step S2 is as follows:
adding a cerium source-containing aqueous solution into a curcumin nanoparticle aqueous solution, stirring for the first time to obtain a first mixed solution, adjusting the pH value of the first mixed solution to be 12-13, continuing stirring for the second time, and after stirring is finished, obtaining the curcumin-cerium oxide nanoparticle aqueous solution.
In a preferred embodiment, the concentration of the aqueous solution containing the cerium source is 0.1-1M, and the cerium source is Ce (NO) 3 ) 3 ·6H 2 O; the mass ratio of the human blood albumin to the cerium ions in the curcumin nanoparticles is (19:1) - (78: 1);
the reaction conditions are as follows: the reaction temperature of the system is 37-80 ℃, the time of the first stirring is 5-15min, the time of the second stirring is 0.25-2h, and the stirring speed is 400-600 rmp.
In a preferred embodiment, in step S3, the volume ratio of the curcumin-cerium oxide nanoparticle aqueous solution to the mannose-modified chitosan aqueous solution is (1:1) - (1: 4);
the aqueous solution of chitosan modified by mannose is prepared by dissolving chitosan modified by mannose in an aqueous solution containing acid, wherein the concentration of the chitosan modified by mannose is 0.5-2% w/v, and the aqueous solution containing acid is an aqueous solution of hydrochloric acid, formic acid, acetic acid or lactic acid with 1-10% v/v.
In a preferred embodiment, the mannose-modified chitosan is prepared as follows:
the chitosan solution was added dropwise to a solution containing d-mannose and NaBH (OAc) 3 Stirring, dialyzing after stirring, and freeze-drying to obtain mannose-chitosan powder.
In a preferred embodiment, the chitosan solution is prepared by dissolving chitosan in an acid-containing aqueous solution with concentration of 0.3-1% w/v, and adjusting pH value to 5-6, wherein the acid-containing aqueous solution is 1-10% v/v hydrochloric acid, formic acid, acetic acid or lactic acid aqueous solution;
containing d-mannose and NaBH (OAc) 3 In an aqueous solution of (2), the concentration of d-mannose is 0.05M, NaBH (OAc) 3 The concentration of (A) is 0.01M;
the chitosan solution contains d-mannose and NaBH (OAc) 3 The volume ratio of the aqueous solution of (1:1) to (1: 2).
The preparation of the drug-loaded nanoparticles and their effects are exemplified and compared below with specific examples and tests. Of course, the embodiments of the invention are not limited thereto.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents, and the like used in the following embodiments are commercially available unless otherwise specified.
[ example 1 ]
Preparation of mannose-chitosan
600mg of 10w chitosan was dissolved in 200mL of 1% aqueous acetic acid, and the pH of the solution was maintained at 5.5 using 1M NaOH.
1801.6mg d-mannose and 423.88mg NaBH (OAc) 3 200mL of the aqueous solution was prepared and 200mL of the chitosan solution was added dropwise to 200mL of d-mannose and NaBH (OAc) 3 While stirring at 100-. The resulting solution was incubated for 48 hours to successfully obtain a mannose chitosan polymer.
The polymer was then dialyzed in double distilled water for 72 hours using a dialysis membrane with a molecular weight cut-off of 10kDa, and the purified solution was freeze-dried to give mannose-chitosan.
[ example 2 ]
Preparation of curcumin nanoparticles
First, 10mL HSA (5mg/mL, pH 9.0) was added to 1mL Cur (3mg/mL), and the mixture was stirred and mixed at 37 ℃ for 1 hour to obtain curcumin nanoparticles (CurNPs).
Preparation of cerium oxide nanoparticles
To 10mL HSA (5mg/mL, pH 9.0) was added 0.44mL 43.422mg/mL Ce (NO) 3 ) 3 ·6H 2 Stirring the O aqueous solution for 15 minutes, quickly dripping 0.1mL of 40mg/mL NaOH into the solution, reacting and continuously stirring for 15 minutes to obtain cerium oxide nanoparticles (Ce NPs).
Preparation of curcumin-cerium oxide nanoparticles
First, 10mL HSA (5mg/mL, pH 9.0) was added to 1mL Cur (3mg/mL), and after stirring and mixing at 37 ℃ for 1 hour, 0.44mL 43.422mg/mLCe (NO) was added 3 ) 3 ·6H 2 The aqueous O solution was stirred for 15 minutes.
Then, 0.1mL of 40mg/mL NaOH solution is quickly added into the solution dropwise to react for further stirring for 15 minutes, so that curcumin-cerium oxide nanoparticles (Cur-Ce NPs) are obtained.
[ example 3 ]
Preparation of mannose-chitosan coated curcumin nano-particle
The obtained 50mg of mannose-chitosan of example 1 was dissolved in 5mL of 1% glacial acetic acid to obtain a mannose-chitosan solution.
5mL of curcumin nanoparticles (Cur NPs) obtained in example 2 are added into 5mL of 10mg/mL mannose-chitosan solution, and stirred for 1h at normal temperature to obtain mannose-chitosan coated curcumin nanoparticle solution.
And subsequently, dialyzing for 24h by a 100w dialysis membrane to purify to obtain mannose-chitosan coated curcumin nanoparticles (Cur @ MCS NPs).
Preparation of mannose-chitosan coated cerium oxide nano particle
The obtained 50mg of mannose-chitosan of example 1 was dissolved in 5mL of 1% glacial acetic acid to obtain a mannose-chitosan solution.
5mL of the cerium oxide nanoparticles (Ce NPs) obtained in example 2 were added to 5mL of a 10mg/mL mannose-chitosan solution, and the mixture was stirred at room temperature for 1 hour to obtain a mannose-chitosan-coated cerium oxide nanoparticle solution.
Subsequently, the mixture is purified by dialysis for 24h through a 100w dialysis membrane, and cerium oxide nanoparticles (Ce @ MCS NPs) coated with mannose-chitosan are obtained.
Preparation of mannose-chitosan coated curcumin-cerium oxide nanoparticles
The obtained 50mg of mannose-chitosan of example 1 was dissolved in 5mL of 1% glacial acetic acid to obtain a mannose-chitosan solution.
And (3) adding 5mL of the curcumin-cerium oxide nanoparticles obtained in the example 2 into 5mL of 10mg/mL mannose-chitosan solution, and stirring at normal temperature for 1h to obtain a mannose-chitosan coated curcumin-cerium oxide nanoparticle solution.
And subsequently, dialyzing for 24h by a 100w dialysis membrane to purify to obtain mannose-chitosan coated curcumin-cerium oxide nanoparticles (Cur-Ce @ MCS NPs).
[ example 4 ]
Preparation of chitosan coated curcumin-cerium oxide nanoparticles
10w of 50mg of chitosan was dissolved in 5mL of 1% glacial acetic acid to obtain a chitosan solution.
And (3) adding 5mL of the curcumin-cerium oxide nanoparticles obtained in the example 2 into 5mL of 10mg/mL chitosan solution, and stirring at normal temperature for 1h to obtain a chitosan-coated curcumin-cerium oxide nanoparticle aqueous solution.
And subsequently, dialyzing for 24h by a 100w dialysis membrane to purify to obtain the curcumin-cerium oxide nanoparticle water (Cur-Ce @ CS NPs) coated with chitosan.
The samples used in the following tests, all from examples 1 to 4
[ example 5 ]
TEM test
The Cur-Ce @ MCS NPs samples were dispersed with water and then placed on a copper grid for TEM characterization, with the results shown in figure 1.
As can be seen from the figure, Cur-Ce @ MCS NPs have nearly monodisperse particles with spherical morphology, and curcumin-cerium oxide nanoparticles are coated within mannose-chitosan.
Therefore, the method can prove that the mannose-chitosan coated curcumin-cerium oxide nanoparticles are successfully prepared.
[ example 6 ]
Particle size and potential testing
The particle size and zeta potential of the nanoparticles prepared were determined by Zetasizer Nano ZS-90(Malvern Instruments, Malvern, UK).
As shown in FIG. 2A, the average particle size of Cur-Ce @ CS NPs is 359nm, while the average particle size of Cur-Ce @ MCS NPs is 443.4nm, because mannose modification enhances the hydrophilicity of the polymer and enlarges the hydration layer of the NPs.
As shown in FIG. 2B, the Zeta potential of Cur-Ce @ CS NPs is 53.6mV, and the Zeta potential of Cur-Ce @ MCS NPs is 48.9 mV. The decrease in zeta potential and sharper peak are due to chitosan binding to the CHO group of mannose.
From the above, it can be further proved that the mannose-chitosan coated curcumin-cerium oxide nanoparticles are successfully prepared by the method.
[ example 7 ]
Test of drug efficacy
Male Balb/c mice (18-20g) were kept under 12 hours light, 12 hours dark conditions and given sufficient standard mouse food and water. After one week of acclimatization, weighed and randomly divided into 7 groups of 7-9, treated as follows:
(1) blank Control group (Control group) without any treatment
(2) Positive control group for colitis Induction with DSS (DSS group)
(3) For the group of Cur @ MCS NPs and DSS-induced colitis (Cur @ MCS NPs group)
(4) Experiment groups for colitis Induction by Ce @ MCS NPs and DSS (Ce @ MCS NPs group)
(5) To the group of Cur-Ce @ CS NPs and DSS-induced colitis (group of Cur-Ce @ CS NPs)
(6) For the group of Cur-Ce @ MCS NPs and DSS-induced colitis (Cur-Ce @ MCS NPs group)
The specific procedure for the experimental group was to administer 0.2mL of the drug to the mice 7 times every other day by gavage. On day 7, 5% (w/v) DSS (36-50kDa) was initially added to the drinking water to induce acute colitis. Mice were measured daily for body weight and observed for clinical signs of colitis. After 14 days, all mice were sacrificed by cervical dislocation.
Colon shortening is a key feature of DSS-induced colitis mice, and we measured colon length in colitis mice treated with Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, Cur-Ce @ MCS NPs. As shown in figure 3A, the mean colon length of DSS-induced mice was reduced by approximately 31% compared to uninduced mice. Oral administration of Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, and Cur-Ce @ MCS NPs all resulted in an increase in colon length. In all treatment groups, mice treated with Cur-Ce @ MCS NPs had the least decrease in mean colon length and the best therapeutic effect.
The DAI index is a typical indicator of UC. As shown in FIG. 3B, the inflammation and immunosuppression induced by DSS treatment decreased the body weight and increased the DAI level in mice compared to the Control group, however, both oral Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, and Cur-Ce @ MCS NPs decreased the DAI level compared to the DSS group, and the DAI treatment in mice treated with Cur-Ce @ MCS NPs was closest to the Control group and was most effective.
[ example 8 ]
Test for inhibiting iron death
Iron death is a form of Regulated Cell Death (RCD) that is morphologically, biochemically, and genetically distinct from other types of RCD, such as apoptosis, necroptosis, and autophagy. The iron metabolism and lipid peroxidation pathways are central mediators of the iron death process. Excess iron regulates iron death by producing lethal Reactive Oxygen Species (ROS) via fenton's reaction, while reduction of Glutathione (GSH) consumption and/or inhibition of glutathione peroxidase 4(Gpx4) triggers iron death by accumulation of intracellular lipid ROS and overwhelming lipid peroxidation. In addition, ROS attack polyunsaturated fatty acids (PUFAs) of the lipid membrane, producing large amounts of lipid peroxides and leading to membrane damage and cell death. Excess iron in the gut generates ROS through the Fenton reaction, thereby initiating oxidative stress. Lipid peroxidation occurs procedurally and induces iron-dead cell death. Thus, the intestinal epithelial cells are disrupted and the intestinal mucosal barrier is compromised resulting in IBD.
In order to prove the protection effect of Cur-Ce @ MCS NPs on intestinal iron death, embedded slices with the thickness of 5 mu m are prepared to be sealed in goat serum. Sections were incubated with 4-Hydroxyneal (4-HNE) primary antibody overnight at 4 ℃ and then held with HRP-conjugated goat anti-mouse antibody for 60 minutes at 37 ℃. Sections were then visualized using DAB solution to stop the immune response and counterstained with hematoxylin.
As shown in FIG. 4, in the colonic epithelial tissues of the colitis mice treated with Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs, the levels of 4-HNE (a marker of lipid peroxidation) were inhibited compared with the DSS group, indicating that the treatment with Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs inhibits iron death in the colonic epithelial tissues, while the Cur-Ce @ MCS NPs of the present invention has the most significant inhibitory effect on 4-HNE and the best inhibitory effect on iron death.
[ example 9 ]
Macrophage polarization regulating assay
Intestinal macrophages are members of the innate immune system of the intestine, are key cells for maintaining intestinal homeostasis, and belong to the largest macrophage population of the body. In the gut, macrophages differentiate between harmless antigens and potential pathogens, eliminating bacteria and apoptotic cells, among others, to maintain immune homeostasis. Intestinal macrophages capture and fight harmful bacteria using mucins and antimicrobial peptides secreted by goblet cells and Pan cells in the intestinal epithelium. As an antigen presenting cell, intestinal macrophages can produce and secrete a plurality of immunoregulatory factors, and further activate other intestinal immune cells. In addition, dysfunction of intestinal macrophages can lead to loss of their tolerance to commensal bacteria and food antigens. Macrophages of type M1 also cause intestinal barrier dysfunction, further contributing to intestinal inflammation. M2 type macrophages were able to reduce intestinal inflammation in mice. M2 type macrophages have the effects of protecting intestinal tissues and resisting inflammation in various IBD mouse models. In general, both in mouse models and in IBD patients, intestinal macrophages and their polarization state play an important role in the intestinal inflammatory response, and macrophages may therefore be a potential target for IBD treatment.
To demonstrate the anti-inflammatory effect of Cur-Ce @ MCS NPs on intestinal inflammation, embedded sections with a thickness of 5 μm were prepared for occlusion in goat serum. Sections were incubated with CD206 primary antibody overnight at 4 ℃ and then held with HRP-conjugated goat anti-mouse antibody for 60 minutes at 37 ℃. Sections were then visualized using DAB solution to stop the immune response and counterstained with hematoxylin.
CD206 as a phenotypic marker of M2, as shown in FIG. 5, the number of M2 cells in the DSS group was the least, and the number of M2 cells in the Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, and Cur-Ce @ MCS NPs groups was the most, wherein the number of M2 cells in the Cur-Ce @ MCS NPs group was the most.
Therefore, the drug-loaded nanoparticles of the invention can be proved to be more effective in regulating the polarization of macrophages to the M2 phenotype.
[ example 10 ]
Active oxygen free radical (antioxidation) test capable of eliminating overhigh lesion part of enteritis
Studies have shown that overproduction of Reactive oxygen and nitrogen species (ROS/RNS) and their associated oxidative stress and redox regulation are important in connection with inflammatory bowel disease
In the IBD model, ROS/RNS expression is increased in the colonic mucosa, with common products being superoxide, hydrogen peroxide, hypochlorous acid, peroxynitrite, and the like, and associated with disease progression. While endogenous antioxidants such as glutathione and Copper, zinc superoxide dismutase (Cu/ZnSOD) levels are reduced, biomarkers of oxidative stress such as lipid peroxidation products are increased.
Oxidative stress in IBD not only produces excessive ROS/RNS to damage cellular lipids and other components, leading to mucosal damage, dysfunction and inflammation, directly leading to intestinal tract injury, but also causes redox signal imbalance, activates NF-xB and other signal pathways, and promotes over-expression of inflammatory factors and adhesion molecules.
The test comprises the steps of cleaning colon tissues by precooling PBS, removing contents, placing the colon tissues in a culture dish, cutting the colon tissues into small pieces by surgical scissors, washing the small pieces twice by the PBS, centrifuging to obtain precipitates, adding a proper amount of tissue protein lysate into the obtained tissue pieces, placing the tissue pieces in a glass grinding tube for repeated grinding, stopping grinding when the whole process is carried out on ice, centrifuging at 10000rpm for 20min at 4 ℃ for 20min when the solution is not transparent and is not too viscous, collecting supernatant, adding a proper amount of protease inhibitor, and storing the supernatant for later use at-80 ℃.
To assess the level of oxidative stress in colon tissue, MDA of mouse colon tissue was determined. The experimental procedures were performed exactly as described in the kit instructions.
As shown in FIG. 6, the Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs and Cur-Ce @ MCS NPs all significantly reduced the high level of MDA (a lipid peroxide) caused by the DSS treatment, with the Cur- @ Ce MCS NPs group treatment providing the most reduced MDA and the best results.
Therefore, Cur-Ce @ MCS NPs can eliminate the over-high active oxygen free radicals at the enteritis pathological part, have the antioxidation effect, reduce the damage to the cell and mucosa barrier of the inflammatory part, inhibit the proinflammatory activation of macrophages, and provide a good microenvironment for anti-inflammation.
[ example 11 ]
Intestinal flora regulating experiment
Intestinal microorganisms are a large group of microorganisms that colonize the mucosal surfaces and cavities of the intestine for a long time. The intestinal microorganisms play an important role in UC. Once the epithelial barrier is compromised, intestinal microbes translocate into the lamina propria, over-activating immune cells, causing inflammation, inducing IBD, exacerbating the immune response and the loss of imbalance and diversity of the intestinal flora. This may be associated with the intestinal microorganisms having the effect of inducing a regulatory immune response and a protective immunity. The down-regulation of short-chain fatty acids (SCFAs) in IBD can lead to increased intestinal pH, adversely affecting flora and exacerbating the disease. The microorganism can also act with pathogen and immune cell, and inhibit infection of pathogen by means of competing or inducing AMPs to enhance epithelial barrier function and transmit immune signal.
In the test, 200mg of the fecal sample in colon tissues of each group of mice is taken respectively, and the total DNA of the fecal sample of the mice is extracted by strictly referring to the steps listed in the TIANGEN kit specification. Detecting the extracted fecal genome DNA by agarose gel electrophoresis, measuring the purity and concentration of mouse fecal DNA by a spectrophotometer, and storing the extracted DNA at-20 deg.C for use.
Synthesizing related primers based on a 16S rRNA gene V3-V4 region of the intestinal flora, and carrying out Miseq PCR amplification, wherein the amplified primer sequences are as follows:
338F:5'-ACTCCTACGGGAGGCAGCA-3'；
806R:5'-GGACTACHVGGGTWTCTAAT-3'
after the PCR amplification product is subjected to quantitative detection, mixing is carried out according to the requirement of the minimum sequencing quantity required by a sample in a corresponding proportion, and the related library construction and Miseq high-throughput sequencing process are completed by Migi biology company.
High throughput sequencing will yield thousands of sequences, and the raw data must first be filtered to remove chimeric sequences, thereby ensuring high quality of the sequences, while for ease of bioinformatics analysis, the resulting sequences are classified into multiple subgroups, usually clustered into Operable Taxa (OTU) at a 97% similarity level.
On the basis of the classification into OTU results, the composition of the flora structure can be analyzed from different levels of biological classification (phylum to genus).
As shown in FIG. 7, the results of the DSS group Bacteroides significantly increased, the firmicutes significantly decreased, and the ratio of the abundance of firmicutes to the abundance of Bacteroides (F/B) decreased, which is an important indicator of the intestinal microbial imbalance. In contrast, Cur @ MCS NPs, Ce @ MCS NPs, Cur-Ce @ CS NPs, and Cur-Ce @ MCS NPs treatment decreased the relative abundance of Bacteroides and increased the relative abundance of firmicutes, indicating its regulatory effect on gut microbiota compared to the DSS group. It is emphasized that Cur-Ce @ MCS NPs are most significantly regulated in the firmicutes and Bacteroides of colitis mice, even towards normal levels.
The result proves that the Cur-Ce @ MCS NPs can reduce harmful bacteria in the intestinal tract and increase beneficial bacteria in the intestinal tract, so that the balance of intestinal microorganisms is adjusted, the damage to cells and mucosa barriers at inflammatory parts is further reduced, and the infiltration of inflammatory cells such as macrophages and the like and the secretion of inflammatory cytokines are prevented from being changed due to the disturbance of the intestinal microorganisms, so that the susceptibility to IBD inflammation is increased, a good microenvironment is provided for anti-inflammation, the anti-inflammation efficiency is improved, and the anti-inflammation effect is enhanced.
In conclusion, it can be proved that the drug-loaded nanoparticle of the invention achieves the purpose of anti-inflammation by combining the regulation of macrophage polarization, elimination of excessive active oxygen free radicals at the pathological part of enteritis and inhibition of iron death, simultaneously has the effects of antioxidation and inhibition of iron death, provides a good microenvironment for anti-inflammation, further promotes macrophage polarization to M2 phenotype, further stabilizes the intestinal environment by regulating intestinal flora, further reduces damage to cells and mucosal barriers at the inflammatory part, and avoids the change of infiltration of inflammatory cells such as macrophages and secretion of inflammatory cytokines due to intestinal microbial disturbance, thereby increasing susceptibility to IBD inflammation, providing a good microenvironment for anti-inflammation, improving anti-inflammation efficiency and strengthening anti-inflammation effect.
Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims (13)

1. The drug-loaded nanoparticle is characterized in that chitosan modified by mannose is used as a carrier, and the curcumin-cerium oxide nanoparticle is coated in the carrier to form the drug-loaded nanoparticle.
2. The drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 1, wherein the average particle size of the curcumin-cerium oxide nanoparticles is 100-140 nm.
3. The drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 1, wherein the average particle size of the drug-loaded nanoparticles is 325-480 nm.
4. The drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 1, wherein curcumin is combined with human serum albumin and then combined with cerium ions under alkaline conditions to form the curcumin-cerium oxide nanoparticles.
5. Use of the drug-loaded nanoparticles based on curcumin and cerium oxide according to any one of claims 1 to 4 for the preparation of a medicament for the prevention and treatment of inflammatory bowel inflammation and for the regulation of intestinal flora.
6. The application of the curcumin-cerium oxide nanoparticle composition disclosed by claim 5, wherein macrophage polarization is regulated through the curcumin-cerium oxide nanoparticle composition, excessive active oxygen free radicals at enteritis lesion parts are eliminated, iron death is inhibited, so that the anti-inflammatory purpose is achieved, and meanwhile, the intestinal flora is regulated, the intestinal environment is stabilized, a favorable microenvironment is provided for anti-inflammation, so that the anti-inflammatory effect is improved.
7. A preparation method of drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in any one of claims 1 to 4, characterized by comprising the following steps:
s1, adding the curcumin solution into a human serum albumin aqueous solution, and stirring for reaction to obtain a curcumin nanoparticle aqueous solution;
s2, taking the curcumin nanoparticle aqueous solution obtained in the step S1, adding an aqueous solution containing a cerium source into the curcumin nanoparticle aqueous solution to obtain a first mixed solution, adjusting the first mixed solution to be alkaline, and then stirring for reaction to obtain a curcumin-cerium oxide nanoparticle aqueous solution;
s3, taking the curcumin-cerium oxide nanoparticle aqueous solution obtained in the step S2, dropwise adding the curcumin-cerium oxide nanoparticle aqueous solution into the chitosan aqueous solution modified by mannose, stirring for reaction, and dialyzing and purifying to obtain the mannose-chitosan coated curcumin-cerium oxide nanoparticles.
8. The method for preparing drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 7, wherein in step S1, the curcumin solution is ethanol, propanedione or acetone solution of curcumin with concentration of 1-3 mg/mL; the concentration of the human serum albumin aqueous solution is 5-20 mg/mL; the mass ratio of curcumin to human serum albumin is (1:10) - (1: 50).
9. The method for preparing drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 7, wherein the specific process of step S2 is as follows:
adding a cerium source-containing aqueous solution into a curcumin nanoparticle aqueous solution, stirring for the first time to obtain a first mixed solution, adjusting the pH value of the first mixed solution to be 12-13, continuing stirring for the second time, and after stirring is finished, obtaining the curcumin-cerium oxide nanoparticle aqueous solution.
10. Curcumin and cerium oxide based according to claim 9The preparation method of the drug-loaded nano particle is characterized in that the concentration of the aqueous solution containing the cerium source is 0.1-1M, and the cerium source is Ce (NO) 3 ) 3 ·6H 2 O; the mass ratio of the human blood albumin to the cerium ions in the curcumin nanoparticles is (19:1) - (78: 1);
the reaction conditions are as follows: the reaction temperature of the system is 37-80 ℃, the time of the first stirring is 5-15min, the time of the second stirring is 0.25-2h, and the stirring speed is 400-600 rmp.
11. The method for preparing drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 7, wherein in step S3, the volume ratio of the curcumin-cerium oxide nanoparticle aqueous solution to the mannose-modified chitosan aqueous solution is (1:1) - (1: 4);
the aqueous solution of chitosan modified by mannose is prepared by dissolving chitosan modified by mannose in an aqueous solution containing acid, wherein the concentration of the chitosan modified by mannose is 0.5-2% w/v, and the aqueous solution containing acid is an aqueous solution of hydrochloric acid, formic acid, acetic acid or lactic acid with 1-10% v/v.
12. The method for preparing drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 11, wherein the preparation of mannose modified chitosan is as follows:
the chitosan solution was added dropwise to a solution containing d-mannose and NaBH (OAc) 3 Stirring, dialyzing after stirring, and freeze-drying to obtain mannose-chitosan powder.
13. The method for preparing drug-loaded nanoparticles based on curcumin and cerium oxide as claimed in claim 12, wherein the chitosan solution is prepared by dissolving chitosan in acid-containing aqueous solution with pH value adjusted to 5-6, wherein the concentration of chitosan is 0.3-1% w/v, and the acid-containing aqueous solution is 1-10% v/v of hydrochloric acid, formic acid, acetic acid or lactic acid aqueous solution;
containing d-mannose and NaBH (OAc) 3 In an aqueous solution of (2), the concentration of d-mannose is 0.05M, NaBH (OAc) 3 The concentration of (A) is 0.01M;
the chitosan solution contains d-mannose and NaBH (OAc) 3 The volume ratio of the aqueous solution of (1:1) to (1: 2).
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