CN112472687A - Rhein-loaded lactoferrin nano-particle and preparation method and application thereof - Google Patents

Rhein-loaded lactoferrin nano-particle and preparation method and application thereof Download PDF

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CN112472687A
CN112472687A CN202011510888.5A CN202011510888A CN112472687A CN 112472687 A CN112472687 A CN 112472687A CN 202011510888 A CN202011510888 A CN 202011510888A CN 112472687 A CN112472687 A CN 112472687A
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lactoferrin
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CN112472687B (en
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高飞
罗锐锋
林美斯
章津铭
廖婉
石金凤
张晨
陈其艳
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Chengdu University of Traditional Chinese Medicine
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The invention provides rhein-loaded lactoferrin nanoparticles and a preparation method and application thereof, belonging to the technical field of biomedical materials. Specifically provides drug-loaded lactoferrin nanoparticles which are nanoparticles having a three-layer structure; the three-layer structure comprises a pectin layer, a hyaluronic acid layer and a drug-loaded lactoferrin layer from outside to inside. The drug is preferably rhein. The drug-loaded dual-targeting drug delivery nanoparticle has good biocompatibility and stability, can be orally administered, and has good targeting capability. Wherein, the rhein-loaded nano particles have good colon lesion inflammation and colon injury targeting capability. The nano particle can keep stability in stomach and small intestine when being orally taken, releases the medicine slowly, and can release the medicine continuously after targeting to the colon lesion part, thereby having good effect on treating colitis, in particular ulcerative colitis. The nano particles have excellent treatment effect and good application prospect.

Description

Rhein-loaded lactoferrin nano-particle and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a rhein-loaded lactoferrin nano particle, a preparation method and application thereof.
Background
Ulcerative Colitis (UC) is a common inflammatory disease in the colon, with an increasing trend in incidence and prevalence worldwide, and is more likely to become diseased in young people, suggesting that it is a global disease. UC is characterized by disruption of the epithelial barrier and an imbalance in inflammatory homeostasis. Thus, the primary goal of clinical UC treatment is to accelerate colon healing and reduce inflammation. At present, first-line therapy for UC is mainly focused on anti-inflammatory drugs, immunomodulators and biologicals. However, adverse reactions have been reported in patients with the emergence of various clinically approved drugs, such as nephrotoxicity, neurotoxicity, and opportunistic infections. Screening natural bioactive compounds has become a promising alternative strategy in order to improve therapeutic efficacy and reduce adverse effects.
Rhein (RH) is an active ingredient isolated from rhubarb, Tanggute rhubarb, and other Chinese herbal medicines. Modern pharmacological research proves that rhein has good anti-inflammatory effect. In recent years, studies have shown that RH can inhibit inflammation in a range of diseases through a variety of inflammatory pathways (NF-. kappa.B, TLR4-MyD 88-NF-. kappa.B, MAPK, JAK/STAT, PI3K-AKT-mTOR, etc.). In particular, NF- κ B is one of the well-recognized signaling molecules mediating the anti-inflammatory effects of RH in vitro, suggesting that RH may be a promising active compound for the treatment of UC through the TLR 4-linked NF- κ B signaling pathway. On the other hand, RH has been reported to have a protective effect on the intestinal mucosal barrier in a range of diseases such as UC and IgA nephropathy. The two clear effects of RH on UC correspond exactly to the clinical treatment of UC in anti-inflammatory and repair of colonic lesions.
However, Rhein (RH) has some problems in use, such as: low water solubility, low bioavailability and poor colon targeting ability. These problems have greatly limited the clinical application of RH, and overcoming these problems would be advantageous for the clinical application of RH.
Disclosure of Invention
The invention aims to provide a rhein-loaded lactoferrin nano particle and a preparation method and application thereof.
The invention provides a drug-loaded lactoferrin nanoparticle, which is a nanoparticle with a three-layer structure; the three-layer structure comprises a pectin layer, a hyaluronic acid layer and a drug-loaded lactoferrin layer from outside to inside.
Further, the pectin layer is a pectin layer crosslinked by calcium ions.
Further, the drug-loaded lactoferrin nanoparticles are prepared from the following raw materials in parts by weight: 50-100 parts of lactoferrin, 1-10 parts of a medicine, 10-50 parts of hyaluronic acid, 10-50 parts of pectin and 10-50 parts of calcium chloride;
preferably, the feed additive is prepared from the following raw materials in parts by weight: 50-60 parts of lactoferrin, 5-10 parts of a medicine, 10-20 parts of hyaluronic acid, 10-20 parts of pectin and 10-20 parts of calcium chloride;
more preferably, the composition is prepared from the following raw materials in parts by weight: 50 parts of lactoferrin, 5 parts of a medicine, 20 parts of hyaluronic acid, 10 parts of pectin and 10 parts of calcium chloride.
Further, the medicine is rhein, curcumin, chlorogenic acid, emodin or geniposide;
preferably, the drug is rhein.
Further, the preparation method of the drug-loaded lactoferrin nanoparticles comprises the following steps:
(1) dissolving lactoferrin in a water phase, dissolving a drug in an oil phase, and then mixing the water phase and the oil phase to obtain drug-loaded lactoferrin;
(2) adding hyaluronic acid into the drug-loaded lactoferrin to enable HA to be adhered to the surface of the drug-loaded lactoferrin so as to obtain the drug-loaded lactoferrin with hyaluronic acid;
(3) and (3) adding pectin in the step (2), then adding calcium chloride for cross-linking reaction, centrifuging, and collecting the precipitate to obtain the pectin.
Further, the air conditioner is provided with a fan,
in the step (1), the water phase is deionized water;
and/or in the step (1), the mass-to-volume ratio of the lactoferrin to the water phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the oil phase is DMSO;
and/or in the step (1), the mass-volume ratio of the medicine to the oil phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the water phase and the oil phase are mixed and then subjected to ultrasonic treatment, and then dialysis and filtration are carried out;
and/or in the step (2), adding hyaluronic acid into the lactoferrin carrying the medicine, and then shaking for 2-5 hours at room temperature;
and/or in the step (3), the crosslinking reaction time is 2-5 hours;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the mass-to-volume ratio of the lactoferrin to the aqueous phase is 5 mg: 2 mL;
and/or in the step (1), the mass volume ratio of the medicine to the oil phase is 5 mg: 1 mL;
and/or, in step (1), the ultrasonic treatment is ultrasonic treatment at 50% amplitude power to form an emulsion.
The invention also provides a method for preparing the drug-loaded lactoferrin nanoparticles, which comprises the following steps:
(1) dissolving lactoferrin in a water phase, dissolving a drug in an oil phase, and then mixing the water phase and the oil phase to obtain drug-loaded lactoferrin;
(2) adding hyaluronic acid into the drug-loaded lactoferrin to enable HA to be adhered to the surface of the drug-loaded lactoferrin so as to obtain the drug-loaded lactoferrin with hyaluronic acid;
(3) and (3) adding pectin in the step (2), then adding calcium chloride for cross-linking reaction, centrifuging, and collecting the precipitate to obtain the pectin.
Further, the air conditioner is provided with a fan,
in the step (1), the water phase is deionized water;
and/or in the step (1), the mass-to-volume ratio of the lactoferrin to the water phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the oil phase is DMSO;
and/or in the step (1), the mass-volume ratio of the medicine to the oil phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the water phase and the oil phase are mixed and then subjected to ultrasonic treatment, and then dialysis and filtration are carried out;
and/or in the step (2), adding hyaluronic acid into the lactoferrin carrying the medicine, and then shaking for 2-5 hours at room temperature;
and/or in the step (3), the crosslinking reaction time is 2-5 hours;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the mass-to-volume ratio of the lactoferrin to the aqueous phase is 5 mg: 2 mL;
and/or in the step (1), the mass volume ratio of the medicine to the oil phase is 5 mg: 1 mL;
and/or, in step (1), the ultrasonic treatment is ultrasonic treatment at 50% amplitude power to form an emulsion.
The invention also provides the application of the drug-loaded lactoferrin nano-particle in the preparation of anti-inflammatory drugs; the drug in the drug-loaded lactoferrin nanoparticles is rhein.
Further, the medicament is a medicament for treating colitis;
preferably, the medicament is a medicament for the treatment of ulcerative colitis.
The present invention studies a drug delivery system with Calcium Pectate (CP) coating and Hyaluronic Acid (HA) modified NPs, which HAs dual targeting ability and can be orally treated. The drug delivery system has a well-controlled particle size, a narrow particle size distribution, and a negatively charged surface. The nanostructure is specifically designed for the pharmacological effects of RH in anti-inflammatory and colonic lesion repair. CP/HA/RH-NPs can be divided into three layers from outside to inside, namely CP, HA and LF:
(1) and (3) CP layer: according to the results of in vitro studies on the evaluation of gastric stability and release profile, CP/HA/RH-NPs showed good gastric stability with the help of CP and controlled release of RH was performed under stimulation of rat cecum containing abundant intestinal flora. Importantly, in vivo targeted delivery studies, the results indicate that CP on the surface of CP/HA/IR780-NPs can help LF not to prematurely expose around the normal intestinal segment and further focus the exposure and targeting of colonic lesions, while the other two focus the exposure and targeting of colonic lesions due to unprotected and exposed LF, i.e. confer CP/HA/RH-NPs colon targeting ability.
(2) HA layer: HA-modified nanoparticles can target macrophages via the CD 44-mediated endocytic pathway, thereby increasing macrophage uptake rates, which is demonstrated in absorption assessment studies in qualitative and quantitative assays. In addition, the macrophage target design can effectively promote the anti-inflammatory effect of RH through a TLR4/MYD 88/NF-kB way, and has the in-vivo anti-UC treatment effect.
(3) And (3) LF layer: LF as a common nano-carrier has abundant sources, good drug-loading capacity and high RH encapsulation efficiency (95.08%). In addition, this study demonstrates that LF can increase cellular uptake and enhance rhein repair.
Compared with other oral NDDs, the lactoferrin rhein nano-particles also have two advantages: first, all materials in NPs, such as pectin, LF, and HA, are food grade and environmentally benign, and are much less toxic than other synthetic chemical nanomaterials. Moreover, the preparation process of nanoparticles is relatively simple and easy to scale up, and drugs and polymers are relatively cheap and available in large quantities. Secondly, researches show that RH can inhibit the expression levels of proinflammatory cytokines such as TNF-alpha, IL-6, IL-1 beta, iNOS and the like, improve the DSS-induced colitis of mice, and firstly prove that the anti-inflammatory effect of RH is realized through the TLR4/MYD 88/NF-kappa B pathways in the researches. The invention uses the nanotechnology to enhance the RH targeting ability and pharmacological action in UC treatment, provides an active UC-resistant natural compound RH, and designs an environment-friendly nano-carrier specially used for RH applied clinical treatment.
In conclusion, the invention provides a drug-loaded dual-targeting drug delivery nanoparticle which has good biocompatibility, good stability, can be orally administered and has good targeting capability. The invention mainly provides rhein-loaded dual-targeting drug delivery nanoparticles, which have good biocompatibility and stability, can be orally administered and have good targeting capability on colon lesion inflammation and colon injury. The nano particle can keep stability in stomach and small intestine when being orally taken, releases the medicine slowly, and can release the medicine continuously after targeting to the colon lesion part, thereby having good effect on treating colitis, in particular ulcerative colitis. The nano particle has better treatment effect than that of single-use rhein and rhein-loaded nano particles with other structures, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the particle size distribution and Zeta potential measurements of RH-NPs, HA/RH-NPs and CP/HA/RH-NPs according to the present invention.
Fig. 2 shows the physical and chemical characteristics characterization results of the lactoferrin rhein nanoparticle of the present invention (n is 3): a is a schematic diagram of the preparation process of the lactoferrin rhein nano-particles; b is the Zeta potential detection results of RH-NPs, HA/RH-NPs and CP/HA/RH-NPs; c is a transmission electron microscope picture and particle size distribution of the lactoferrin rhein nano-particles; d is an X-ray diffraction pattern of different samples; e is an infrared spectrogram of different samples; f is the simulated gastric stability results of HA/RH-NPs and CP/HA/RH-NPs; g is the in vitro release profile of RH from free RH, HA/RH-NPs, CP/HA/RH-NPs in PBS without rat cecal content and RH from CP/HA/RH-NPs in PBS with rat cecal content at 37 ℃; in the diagrams D and E, a is raw material RH, b is LF, c is pectin, D is HA, E is blank NPs, f is CP/HA/RH-NPs, and g is a mixture of raw material RH and blank NPs.
FIG. 3 is a graph of the use of Caco-2 cell line and Raw264.7 macrophages on various C6Quantitative assessment of cellular uptake of the preparation: a is the amount of HA/C taken up by Caco-2 cells after incubation for 4 hours at various concentrations (6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL and 100ng/mL)6-NPs; b is at C6HA/C uptake by Caco-2 cells was quantitatively measured at different incubation time points (0h, 0.25h, 0.5h, 1h, 2h and 4h) at a concentration of 100ng/mL6-NPs; c is at C6After 4 hours incubation at a concentration of 100ng/mL, the different formulations were measured quantitatively (control, free C6，C6-NPs and HA/C6-uptake by Caco-2 cells in NPs); d is a quantitative measure with C in the presence or absence of LF6-NPs and HA/C6-uptake of Caco-2 cells incubated with NPs; e is the quantitative measurement of HA/C uptake by Raw264.7 macrophages after incubation for 4 hours at various concentrations (0ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, and 100ng/mL)6-NPs; f is at C6HA/C uptake by Raw264.7 macrophages was measured quantitatively at different incubation time points (0h, 0.25h, 0.5h, 1h, 2h and 4h) at a concentration of 100ng/ml6-NPs; g is at C6After incubation for 4 hours at a concentration of 100ng/mL, the different formulations (control, free C)6，C6-NPs and HA/C6-NPs) Raw264.7 macrophage uptake; h is in the presence or absence of HA with C6-NPs and HA/C6Quantitative measurement of Raw264.7 macrophage uptake incubated with NPs. Data are presented as mean ± SD (n ═ 3). P < 0.05; p < 0.01.
FIG. 4 is a graph of the use of Caco-2 cell line and Raw264.7 macrophages on various C6Qualitative assessment of cellular uptake of the preparations: a is the reaction with free C in the presence or absence of LF6，C6-NPs or HA/C6-results of qualitative analysis of Caco-2 cellular uptake incubated with NPs; b is the reaction of free C with or without HA6，C6-NPs or HA/C6-qualitative analysis of Raw264.7 macrophage uptake incubated with NPs.
FIG. 5 is the in vivo biodistribution of NPs: a is a fluorescence image of ulcerative colitis mice after oral delivery of different formulations at 3h, 6h, 12h and 24 h; b is a histogram of fluorescence signal analysis of ulcerative colitis mice after oral delivery of different formulations at 3h, 6h, 12h and 24 h; c is a fluorescence image of the small intestine and colon after different preparations are adopted at the final point; d is a histogram of fluorescence signal analysis of mouse small intestine and colon by each preparation at the final point; e is a frozen section of post-administration colitis tissue and green is C6And blue is Hoechst 33342 (nucleus). Data are presented as mean ± SD (n ═ 6). P<0.05vs free IR 780; p<0.01vs free IR 780; # p<0.01vs IR780-NPs；▲▲p<0.01vs CP/HA/IR780-NPs。
FIG. 6 is a graph of the in vivo therapeutic effect of CP/HA/RH-NPs in the treatment of ulcerative colitis: a is a curve graph of the change of the weight of the mice in each preparation along with time, and is normalized to the percentage of the weight of the mice in zero day; b is a graphical analysis of the DAI scores in each formulation; c is a photograph of the colon of a rat after oral treatment with different RH formulations; d is histogram analysis of colon length between different preparations; e is histogram analysis of spleen weight between different preparations; f is histogram analysis of mouse colon MPO expression in six formulations; g is H & E staining of colon sections in each preparation. Data are expressed as mean ± SD (n ═ 6); p <0.01, p < 0.01.
FIG. 7 is H & E staining of heart, liver, spleen, lung and kidney sections of mice receiving different formulations.
FIG. 8 is the results of changes in colonic inflammatory cytokines and protein expression following treatment of ulcerative colitis with CP/HA/RH-NPs: a is histogram analysis of changes in murine colonic inflammatory cytokines (IL-1. beta., iNOS, TNF-. alpha., and IL-6) in each formulation; b are the Western blot results and histogram analysis of murine colonic protein expression (MyD88, TLR4, NF-. kappa.Bp 65 and GAPDH) in each formulation. Data are expressed as mean ± SD (n ═ 6); p <0.01, p < 0.01.
FIG. 9 is a graph of the effect of NPs on tight junction protein expression in DSS-induced experimental colitis; a is an immunohistochemical analysis of zona-occludin 1(ZO-1) and Claudin-1; b is the average area density of ZO-1 and Claudin-1 in the distal colon; c is the Western blot results and histogram analysis of murine colonic Claudin-1 (ZO-1, Claudin-1 and β -actin) tight junction protein expression in each formulation. Data are expressed as mean ± SD (n ═ 6); p <0.01, p < 0.01.
Detailed description of the preferred embodiments
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products. The main materials are as follows:
animal experiments were approved by the ethical committee of the university of Chinese medicine of Chengdu (CDUTCM, approved CDU2019S121), and all animal experiments were performed strictly according to the guidelines for nursing and using experimental animals of the department of scientific and technological sciences of China. Male BALB/c mice (22-25g) were obtained from SPF (Beijing) Biotechnology, Inc. (Beijing, China). Mice were housed under standard conditions and provided food and distilled water.
Human colon adenocarcinoma cell line (Caco-2 cells) and murine leukemia mononuclear macrophage cell line (raw264.7 macrophages) were obtained from the american type culture collection (manassas usa).
IR780, pectin and Hyaluronic Acid (HA) were purchased from Sigma-Aldrich Company (St. Louis, Mo.). Dextran sulfate sodium (DSS, molecular weight: 36-50kDa) was purchased from MP Biomedicals Inc. (California, USA). Rhein (RH) was provided by Dalian plum biotechnology limited (Dalian in China). Calcium chloride and coumarin 6 (C) were obtained from Aladdin reagent Inc. (Shanghai, China)6). Hoechst 33342 is provided by Suzhou Yuchang Biotechnology, Inc. (Suzhou, China). Lactoferrin (LF) was purchased from glycarbo. Myeloperoxidase (MPO) kit, interleukin 1 beta (IL-1 beta) kit, tumor necrosis factor-alpha (TNF-alpha) kit, interleukin 6(IL-6) kit and anti-myeloid differentiation factor 88(MyD88), anti-Claudin-1, anti-ZO-1, anti-nuclear factor kappa-B (NF-kappa B) p65 and anti-Toll-like receptor 4(TLR4) antibodies were provided by the Multi-science (Union) Biotechnology Co., Ltd., Hangzhou, China. Inducible Nitric Oxide Synthase (iNOS) kits were purchased from treasure biotechnology limited (wuhan, china). All solvents (including acetone, acetonitrile and methanol) were of chromatographic grade and used without further modification.
Example 1 preparation of Lactoferrin-rhein nanoparticles of the invention
Adopting dialysis technique to prepare lactoferrin rhein nano-particles (rhein-loaded lactoferrin nano-particles). Lactoferrin (LF)50mg was dissolved in 20mL deionized water to prepare an LF solution. Meanwhile, Rhein (RH)5mg was completely dissolved in 1mL of DMSO to obtain a RH solution. The RH solution described above was added dropwise to the LF solution under constant magnetic stirring. The mixture was then placed in an ice bath and sonicated using a probe Sonicator (sonic XL, Misonix, Melville, NY, USA) at 50% amplitude power for 5 minutes to form an emulsion. The emulsion was then dialyzed (molecular weight cut-off 1000Da) in deionized water for 6h to remove DMSO. Subsequently, it was filtered with a 0.45 μm filter to remove dust and free drug, and RH-NPs were obtained after the filtration.
Thereafter, Hyaluronic Acid (HA)20mg was added to the RH-NPs, and shaken at room temperature for 2 hours to electrostatically adhere HA to the surface of the RH-NPs to form HA/RH-NPs.
Finally, 10mg of pectin was added to HA/RH-NPs under constant magnetic stirring and after stirring for 30 minutes, 10mg of calcium chloride was added to perform a crosslinking reaction for 2 hours. Finally, the crosslinked system is centrifuged at 15000rpm for 15 minutes, and the precipitate is collected to obtain the lactoferrin rhein nano-particles (CP/HA/RH-NPs). Washing the precipitate for 3 times, and freeze drying to obtain dried CP/HA/RH-NPs. The final dried CP/HA/RH-NPs were stored at-20 ℃ until use.
The advantageous effects of the present invention are demonstrated by specific test examples below.
The data in the experimental examples were statistically analyzed, i.e., all data were expressed as mean ± Standard Deviation (SD), and all experimental results were confirmed in at least three independent separate experiments under the same conditions (i.e., n ≧ 3). Statistical comparisons were performed using student's t-test. p <0.05 is considered statistically significant.
Experimental example 1 physicochemical characteristics of Lactoferrin-rhein nanoparticles of the present invention
First, test method
(1) Measurement of particle diameter and Zeta potential
RH-NPs, HA/RH-NPs and CP/HA/RH-NPs were prepared according to the method of example 1, and the mean hydrodynamic particle diameter, polydispersity index (PDI) and Zeta potential thereof were measured using Dynamic Light Scattering (DLS) and particle analyzer Litesizer 500 (Oseltamipal), respectively. Each set of NPs was 3 replicates (n-3).
(2) TEM inspection
Dry CP/HA/RH-NPs were prepared according to the method of example 1, and their surface structure and shape were analyzed using a transmission electron microscope (TEM, JEM 1200X, JEOL, Japan). A drop of the diluted sample was deposited on a carbon-coated copper mesh, after 5 minutes the excess sample was removed with filter paper, and the NPs on the copper mesh were negatively stained with uranyl acetate and then analyzed by TEM.
(3) XRD detection
The raw materials rhein (namely free rhein, RH), Lactoferrin (LF), Hyaluronic Acid (HA), pectin and different NPs (blank NPs, mixture of CP/HA/RH-NPs, RH and blank NPs prepared according to the method of the embodiment 1) are respectively detected by an X-ray diffractometer (D8 Advance, BRUKER, Germany) to obtain an X-ray diffraction (XRD) spectrum. Detection conditions for X-ray diffraction: the measurements were made at a speed of 6, scanning from 5 ° to 90 °, 6 ° per minute, and operating at 40kV and 40 mA.
The preparation method of the blank NPs comprises the following steps:
lactoferrin (LF)50mg was dissolved in 20mL deionized water to prepare an LF solution. Under constant magnetic stirring, 1mL DMSO was added dropwise to the LF solution. The mixture was then placed in an ice bath and sonicated using a probe Sonicator (sonic XL, Misonix, Melville, NY, USA) at 50% amplitude power for 5 minutes to form an emulsion. The emulsion was then dialyzed (molecular weight cut-off 1000Da) in deionized water for 6h to remove DMSO. Subsequently, filtration was performed with a 0.45 μm filter to remove dust, and NPs (blank NPs) were obtained after filtration.
(4) Infrared detection
The starting rhein (i.e., free rhein, RH), Lactoferrin (LF), Hyaluronic Acid (HA), pectin and various NPs (blank NPs, CP/HA/RH-NPs prepared according to the method of example 1) were taken and tested using a Fourier transform infrared spectrometer (IR Tracer-100, Shimadzu, Japan). The wave number range was set to 400-4000cm-1And in the range of 4cm-1The spectra were recorded with 16 scans on average. The sample was mixed with KBr and the resulting mixture was then pressed further into a pancake shape before measurement. The blank NPs were prepared as described in "(3) XRD assay".
(5) Loading efficiency and encapsulation efficiency
Both RH Loading Efficiency (LE) and Encapsulation Efficiency (EE) were determined using High Performance Liquid Chromatography (HPLC) (LC-45202-46, SHIMADZU, Japan) equipped with a C18 column (250X 4.6 mm). The mobile phase was methanol/0.1% phosphoric acid (85: 15, v/v), the detection wavelength was 254nm, the flow rate was 1mL/min, and the column temperature was 25 ℃. Prior to HPLC analysis, NPs structure was disrupted and RH was dissolved using 1mL methanol. EE and LE were calculated using formulas I and II, respectively:
encapsulation efficiency: EE (%) ═ loading of RH/addition amount of RH × 100% formula I
Loading efficiency: LE (%). RH Loading/Total amount of material from which NPs are made × 100% formula II
(6) Simulated gastric fluid stability evaluation
CP/HA/RH-NPs and HA/RH-NPs were prepared according to the method of example 1, and the stability of CP/HA/RH-NPs and HA/RH-NPs was evaluated in simulated gastric fluid. 1mL of freshly prepared CP/HA/RH-NPs or HA/RH-NPs was added to 9mL of simulated gastric fluid (SGF, pH2, containing 1mg/mL pepsin) and incubated at 37 ℃ for 2 hours before measuring TEM, particle size and PDI.
(7) NPs release profile evaluation
The RH and free RH release profiles of different NPs (CP/HA/RH-NPs and HA/RH-NPs prepared according to example 1) were examined using a dialysis method. 3mL of deionized water to dissolve free RH, HA/RH-NPs and CP/HA/RH-NPs (RH concentration 190. mu.g/mL) were added separately to the dialysis bag (molecular weight cut-off 1000 Da). The bags containing free RH, HA/RH-NPs, CP/HA/RH-NPs were immersed in 30mL of PBS medium (pH7.4) containing 1% Tween-80 for release profile evaluation. Also, the release profile of CP/HA/RH-NPs was evaluated with 30ml of PBS with a cecal content of 5% (pH 7.4): the same day rats were sacrificed and the cecal contents (polysaccharide degrading enzymes) were collected in a nitrogen chamber and added to PBS. Four sets of samples were shaken at 37 ℃ at 100rpm and 1mL of medium was collected at different predetermined time points (1, 2, 4, 8, 12, 16, 20, 24h) and supplemented with equal amounts of fresh dissolution medium. The concentration of drug in the resulting filtrate was determined by HPLC.
Second, test results
The electrostatic adsorption and crosslinking reaction are the process mechanism of the invention for forming three layers of CP/HA/RH-NPs.
FIG. 1 shows the particle size distribution and Zeta potential measurements of RH-NPs, HA/RH-NPs and CP/HA/RH-NPs. From the results shown in FIG. 1, it can be seen that: LF encapsulated RH (RH-NPs) has a hydrodynamic particle size of about 172nm, an absolute positive Zeta potential (21.50 + -0.31 mV) and a relatively low polydispersity index (PDI) value (0.206 + -0.009). After shaking with HA, the absolute Zeta potential value decreased from about 21.50mV to 3.52mV, demonstrating that HA was successfully electrostatically adsorbed to the second layer of NPs. Furthermore, the particle size of the HA/RH-NPs increased slightly from about 172 to 190 and the PDI increased to about 0.285, indicating that HA may cause the system to become relatively inhomogeneous. The Zeta potential (Zeta potential) of CP/HA/RH-NPs changes from positive (3.52 + -0.90 mV) to highly negative (-24.9 + -0.95 mV), confirming that calcium pectinate is formed on the nanoparticle surface; at this time, the PDI value was decreased to 0.163. + -. 0.062, and the uniformity of NPs was improved. The test result shows that the lactoferrin rhein nano-particle is successfully prepared.
FIG. 2 is the physical and chemical characteristics characterization result of the lactoferrin rhein nanoparticle of the present invention. Wherein, the figure A is a schematic diagram of the preparation process of the lactoferrin rhein nano-particles; FIG. B shows Zeta potential detection results of RH-NPs, HA/RH-NPs and CP/HA/RH-NPs; FIG. C is a transmission electron microscope image and particle size distribution of the lactoferrin rhein nanoparticles of the present invention; panel D is an X-ray diffraction pattern of the starting materials emodin (RH), Lactoferrin (LF), pectin, Hyaluronic Acid (HA), nanoparticles blanc (NPs), CP/HA/RH-NPs of example 1, and mixtures of NPs blanc and RH; panel E is an infrared spectrum of RH, LF, pectin, HA, blank NPs and CP/HA/RH-NPs; FIG. F shows simulated gastric fluid stability results for HA/RH-NPs and CP/HA/RH-NPs; FIG. G is an in vitro release profile of RH from free RH, HA/RH-NPs, CP/HA/RH-NPs in phosphate buffer (pH7.4) with and without rat cecal contents containing 1% Tween-80 at 37 ℃.
As can be seen from fig. 2: the average particle size distribution of CP/HA/RH-NPs was 284.37 + -2.70 nm, and the zeta potential was-24.9 + -0.95 mV. As mentioned previously, CP/HA/RH-NPs have extremely high absolute negative zeta potentials, indicating good stability and also avoiding particle aggregation. In addition, EE (%) and LE (%) of CP/HA/RH-NPs were 95.08% and 6.17%, respectively, which satisfied the requirements of NPs. Morphologically, CP/HA/RH-NPs TEM images showed uniform spherical shapes with smooth surfaces (FIG. 2C).
To determine whether RH HAs been encapsulated in CP/HA/RH-NPs, the corresponding XRD and FTIR patterns of CP/HA/RH-NPs were studied, which also contained raw materials RH, LF, HA, pectin, blank NPs and a mixture of free RH and blank NPs. As shown in FIG. 2D, a representative XRD diffraction pattern of the starting RH (free RH) showed many sharp peaks, indicating that it isHas a crystalline nature. In contrast, LF, HA, pectin, blank NPs and CP/HA/RH-NPs powders did not have these representative peaks, indicating that no crystalline complex was formed between RH and its LF matrix. These results provide clear evidence that crystalline RH HAs been converted to an amorphous molecule in the LF matrix, indicating that RH HAs been encapsulated in CP/HA/RH-NPs. As shown in FIG. 2E, in the FTIR spectrum of RH, strong signals appeared at 1629cm-1、1452cm-1And 764cm-1To (3). The first two peaks are attributable to the skeletal vibration of C ═ C in the benzene ring, and the latter peak corresponds to the flexural vibration of C — H in the benzene ring. While these three peaks were not observed in CP/HA/RH-NPs, indicating that hydrophobic interactions between RH and LF may exist. In LF spectrum, amide I is at about 1652cm-1The characteristic LF absorption peak at (A) is caused by C ═ O stretching vibration of the peptide group, while the amide II is at about 1537cm-1The characteristic LF absorption peak at (a) is due to NH bending. It was observed that LF has positively charged amino (-NH)3 +) Bending vibration (1537 cm)-1) And HA negatively charged COO- (1619 cm)-1) The electrostatic interaction of (2) shifts the peak of amide II to a higher wavenumber (1545/1547 cm)-1). 3431cm of CP-1The peak of (a) is ascribed to the polar band in the hydroxyl group. Furthermore, 3200-3500cm was found in the blank NPs and CP/HA/RH-NPs after the addition of CP-1The peak shape of (A) was sharp as compared with LF powder, and the peak was shifted to 3431cm-1Indicating that CP and LF may form new hydrogen bonds and conjugates in NP.
The gastric barrier stability of NPs is an essential feature of oral NPs, which ensures the intact structure and pharmacological action of NPs. The present invention evaluates the stability of HA/RH-NPs and CP/HA/RH-NPs in simulated gastric fluid with low pH gastric enzymes. As shown in FIG. 2F, after 2 hours of incubation under gastric conditions, two peaks appeared for the HA/RH-NPs group with particle size greater than 350nm and PDI > 0.3. Meanwhile, TEM images of HA/RH-NPs show non-uniform shapes. However, under the same conditions, the particle size of CP/HA/RH-NPs was slightly increased (341.54. + -. 0.88nm), the corresponding PDI was low (0.213. + -. 0.004), and TEM images of CP/HA/RH-NPs showed uniform spherical shape with smooth surface. Thus, there is evidence that pectin is a promising protectant that may stabilize NPs and prevent the damaging effects on the gastric environment after oral administration.
Continuous release of drugs from NPs is an important parameter of drug delivery systems. Thus, the release profiles of free RH, HA/RH-NPs and CP/HA/RH-NPs were evaluated in buffer at pH 7.4. As shown in FIG. 2G, HA/RH-NPs and CP/HA/RH-NPs were released more slowly than free RH. In particular, at about 4 hours of release, the free RH was almost completely released. However, after 4 hours of incubation, only about 30% of the RH was released from the HA/RH-NPs, while 87% of the accumulated RH was released within 24 hours. After addition of pectin and calcium chloride, the slow release of RH can be effectively controlled in CP/HA/RH-NP, RH is only continuously released by about 15% in the first 4 hours, and the cumulative RH release is only over 56% in 24 hours. It is clear that pectin coated CP/HA/RH-NPs give good control of RH release with timed release of coated RH over 24 hours.
With the help of the intestinal flora, the Calcium Pectin (CP) layer may be degraded and the encapsulated drug is rapidly released to the target site. Thus, the release profile of CP/HA/RH-NPs in a buffer with a cecal content of 5% was also investigated. The average percentage of RH accumulation was higher in the CP/HA/RH-NPs group with the caecal content buffer than in the CP/HA/RH-NPs group without the caecal content buffer than in the HA/RH-NPs group without the caecal content buffer, and the release rate was slower than in the HA/RH-NPs group without the caecal content buffer (FIG. 2G). All experimental studies showed that CP can significantly reduce the rate of NPs release.
Test example 2 cell culture and evaluation of cellular uptake
First, test method
Caco-2 cells and Raw264.7 cells were cultured in MEM/DMEM medium supplemented with 1% streptomycin and penicillin (Thermo Fisher Scientific, USA) and 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Billerica, USA), respectively. For in vitro experiments, free RH was dissolved in DMSO and diluted more than 1000-fold. The drug was diluted with incomplete medium (supplemented with 0.5% FBS and 1% streptomycin and penicillin).
Cellular uptake is an important indicator for in vitro nano-drug assessment. Cellular uptake was measured using Flow Cytometry (FCM) (ACEA NovoCytet, san Diego, USA) and Confocal Laser Scanning Microscopy (CLSM) (LEICA TCS SP8 SR, Weztlar, Germany). Since RH did not show any significant fluorescence, HA/C6-NPs and C6-NPs were prepared by the above-mentioned preparation method using C6 instead of RH as a fluorescent probe. Both Caco-2 cells and Raw264.7 macrophages were used for cellular uptake assessment.
FCM is used to quantify cellular uptake of NPs. Caco-2 cells and Raw264.7 macrophages were seeded into six-well plates, cultured separately for 12 hours until adhesion, and then different C6-loaded formulations were added to each well and incubated at 37 ℃ for further evaluation. For the difference in cellular uptake between the different C6 loaded formulations, cells were incubated with HA/C6-NPs, C6-NPs and free C6(100ng/mL C6) for 4 hours. For concentration-dependent studies of HA/C6-NPs, cells were incubated with HA/C6-NPs at different C6 concentrations (6.25, 12.5, 25, 50 and 100ng/mL) for 4 hours at 37 ℃. For time-dependent studies of HA/C6-NPs, cells were incubated with HA/C6-NPs (C6 concentration 100ng/mL) at different time points (0.25, 0.5, 1, 2 and 4 h). For LF receptor competitive inhibition studies, free LF (2.5mg/mL) serum-free medium was added to six-well plates of Caco-2 cells, pre-incubation for 0.5h was performed, and HA/C6-NPs and C6-NPs were added for additional incubation for 4h, respectively. For the HA receptor competitive inhibition studies, free HA (5mg/mL) serum-free medium was added to six-well plates with Raw264.7 macrophages, preincubated for 0.5h, and HA/C6-NPs were added for an additional 4h incubation. In all experiments, untreated Caco-2 cells and Raw264.7 macrophages were used as controls.
CLSM was used to qualitatively analyze cellular uptake of NPs. Caco-2 cells and Raw264.7 macrophages were seeded into confocal petri dishes separately and cultured for 12 hours until adhesion, and then different C6 load formulations were added for further evaluation. For the absorption differences between the different C6 loaded formulations, both Caco-2 cells and Raw264.7 macrophages were incubated with HA/C6-NPs, C6-NPs and free C6(100ng/mL C6) for 4 hours. Meanwhile, LF preincubated Caco-2 cells and HA preincubated Raw264.7 macrophages were incubated with HA/C6-NPs and C6-NPs at the same concentration of C6(100ng/mL) for 4 hours. After incubation, cells were washed thoroughly with PBS to eliminate excess formulation, then fixed in 4% paraformaldehyde for 10 min and stained with Hoechst 33342 for later CLSM imaging analysis.
Second, test results
Efficient cellular uptake is a major requirement for the therapeutic efficacy of RH loaded NPs. To study the uptake properties of HA/LF functionalized NPs, two opposing cells, Raw264.7 macrophages and Caco-2 cells, respectively, were used as models. Since RH did not fluoresce significantly, C6 was used as a fluorescent probe for tracking. The results of the in vitro NPs release studies showed that the CP layer of CP/HA/C6-NPs is first degraded by the intestinal flora and then the released HA/C6-NPs are taken up by the cells. Thus, in vitro uptake assays were performed only in the HA/C6-NPs, C6-NPs and free C6 groups, but not in the CP/HA/C6-NPs group, in the absence of gut flora interaction.
The NPs were qualitatively analyzed by FCM to confirm their absorption effect and to evaluate the absorption pattern of the NPs. For Caco-2 cells, as shown in FIGS. 3A and 3B, the fluorescence intensity of C6 in HA/C6-NPs was significantly increased with the concentration from 6.25 to 100ng/mL and the time from 0 to 4h, demonstrating that HA/C6-NPs are concentration-dependent and time-dependent in Caco-2 cells. As shown in FIG. 3C, after incubation of Caco-2 cells with free C6, C6-NPs or HA/C6-NPs at a concentration of 100ng/mL for 4 hours, the fluorescence intensity of C6-NPs or HA/C6-NPs was significantly stronger than that of free C6(p <0.01) by FCM assay. However, there was no significant difference in fluorescence intensity between C6-NPs and HA/C6-NPs. In contrast, when exposed to C6-NPs or HA/C6-NPs pre-incubated with free LF Caco-2 cells that had previously bound to Caco-2 cell membrane surface receptor sites, the intracellular fluorescence was significantly reduced (FIG. 3D, p < 0.01). These results indicate that LF can promote intracellular absorption of therapeutic agents. In addition, the same concentration dependence and time dependence was also observed in Raw264.7 macrophages (fig. 3E, fig. 3F). In contrast, as shown in FIG. 3G, HA/C6-NPs showed the highest fluorescence evident after 4 hours of incubation with free C6, C6-NPs and HA/C6-NPs, respectively, followed by C6-NPs and free C6(p < 0.01). The results indicate that HA increases the HA/C6-NPs uptake by Raw264.7 macrophages. FIG. 3H shows that the fluorescence intensity of HA/C6-NPs decreased rapidly (p <0.01) after pre-incubation of Raw264.7 macrophages with free HA compared to the group of HA/C6-NPs that were not pre-incubated with free HA. All results indicate that surface functionalization of LF or HA can promote cellular uptake efficiency of NPs by Caco-2 cells and raw264.7 macrophages, respectively.
To further characterize the uptake of cells by different agents, CLSM was also performed in this study. The intensity of green fluorescence in the image (FIG. 4) indirectly reflects the absorption of C6 by Caco-2 cells and Raw264.7 macrophages. Similarly, Caco-2 cells and Raw264.7 macrophages were incubated with free C6, C6-NPs or HA/C6-NPs at a concentration of 100ng/mL C6 for 4 hours, and then fluorescence intensity was measured using CLSM. Studies showed that free C6 showed weak green fluorescence in Caco-2 cells, while higher C6 intensity was detected in cells treated with C6-NPs or HA/C6-NPs (FIG. 4A), indicating that LF plays an important role in the efficiency of C6 uptake by Caco-2 cells. To further verify whether LF induced a higher therapeutic effect, competitive cellular uptake was investigated. As shown in FIG. 4A, C6-NPs and HA/C6-NPs showed moderate green fluorescence in the absence of LF in the medium. However, when LF was introduced into the medium, the fluorescence intensity dropped dramatically. This result is similar to the FCM study, which demonstrated that the increase in cellular uptake efficiency in both the C6-NPs and HA/C6-NPs groups was mediated by LF receptors. On the other hand, to further investigate whether the HA molecules could maintain their macrophage targeting ability, the cellular uptake efficiency of free C6, C6-NPs and HA/C6-NPs was qualitatively compared with Raw264.7 macrophages. As shown in FIG. 4B, free C6 showed slightly weaker fluorescence than C6-NPs, and HA/C6-NPs showed the highest fluorescence signal in all reference groups. To confirm the HA receptor mediated cellular uptake of HA functionalized NPs, the cellular uptake efficiency of Raw264.7 macrophages on HA-RH-NPs in medium supplemented with free HA that competed with the group of C6-NPs or HA/C6-NPs was also examined. As shown in FIG. 4B, cellular uptake of HA/C6-NPs was significantly reduced in the presence of free HA, but no significant change in fluorescence intensity of C6-NPs was observed when HA was introduced in the culture medium. These results indicate that HA/C6-NPs are internalized into cells due to HA receptors on the surface of Raw264.7 macrophages.
In general, both qualitative and qualitative analyses confirmed that LF molecules could maintain their Caco-2 cell targeting ability and HA molecules could maintain their macrophage targeting ability.
Test example 3 evaluation of in vivo biodistribution
First, test method
In vivo biodistribution assessment of NPs was performed on the UC mouse model by an in vivo imaging system (PerkinElmer, MA, usa). In male BALB/C mice, the UC mouse model was induced by feeding DSS (3%, w/v) to them in their drinking water over a period of 7 days. IR780, a NIR dye with maximum absorbance at 780nm, was encapsulated in NPs (named IR780-NPs, HA/IR780-NPs and CP/HA/IR780-NPs) instead of RH. UC mice were first randomized into four groups (n ═ 6 per group) and the same dose (1.5 mg/kg dose) of free IR780, IR780-NPs, HA/IR780-NPs or CP/HA/IR780-NPs was administered orally to each group once. After oral administration, mice were imaged at four different time points (3, 6, 12 and 24 hours). At the final end of dosing, mice were euthanized and the small intestine and distal colon were separated and fluorescence was detected immediately without washing.
To describe the colon targeting potential of NPs, healthy and DSS-induced UC mice received a single oral dose of the same dose (2 mg/kg dose) of free C6, C6-NPs, HA/C6-NPs and CP/HA/C6-NPs. CLSM was used to qualitatively study the accumulation of C6 in different groups of colitis tissues. All mice were sacrificed by euthanasia 24 hours after dosing. The colons of the mice were collected in a dark environment. Subsequently, the tissues were embedded in optimal cutting temperature compounds, sectioned, and further stained with Hoechst 33342 to detect the accumulation of C6 by CLSM.
Second, test results
Selective accumulation of therapeutic agents in inflamed colon tissue is critical for the oral administration of therapeutic agents to treat UC. To assess the efficacy of NPs targeting inflammatory colon tissue, IVIS and confocal microscopy were employed. Mice with UC induced by DSS were evaluated and fluorescence images were taken by in vivo imaging system at each time point after oral administration and statistically analyzed. As shown in FIGS. 5A and 5B, there was no significant difference in the mean fluorescence intensity of the entire NPs group after 3h or 6h of tube feeding. After 12 hours, the mean fluorescence intensity of CP/HA/IR780-NPs was strongest, followed by HA/IR780-NPs, IR780-NPs and free IR780, with statistical differences (p <0.01), and these differences persisted to the 24 hour end point. Furthermore, 24 hours after intragastric administration, colon and small intestine were collected and the resulting fluorescence images and histograms are shown in FIG. 5C and FIG. 5D, the intensity of the IR780-NPs and HA/IR780-NPs groups was significantly higher than that of the free IR780 and CP/HA/IR780-NPs (p <0.01), indicating that unprotected and exposed LF of IR780-NPs and HA/IR780-NPs could be targeted to the small intestine by LF ligands. However, in the CP/HA/IR780-NPs group, the fluorescence intensity around the small intestine was weak and highest near the colon (p <0.01), indicating that CP at the surface of CP/HA/IR780-NPs could successfully avoid premature LF exposure around the normal small intestine and digest by intestinal flora until CP further releases HA/IR780-NPs around the colon lesion, then target the colon by the exposed LF. Thus, the CP layer contributes to the specific target ability of LF to colonic lesions.
Observation of NPs absorbed by the target colon tissue directly reflects the delivery status and targeting ability. Thus, the efficacy of CP/HA/C6-NPs for drug delivery at specific sites in inflamed colon was assessed by cryosectioning technique and CLSM using C6 loaded NPs. As shown in FIG. 5E, a clear green fluorescence was seen in colitis tissue sections of CP/HA/C6-NPs treatment group, indicating that NPs can effectively accumulate in colitis tissue after oral administration. The green fluorescence signal of the HA/C6-NPs treated group was much stronger than that of the C6-NPs treated group, indicating that HA enhanced the accumulation of NPs in the colitis tissue.
Taken together, the ex vivo luminescence images indicate that CP/HA/IR780-NPs may accumulate and remain in the inflamed areas of the colon for a longer period of time. Thus, the nanoparticles of the present invention can effectively target the colonic inflamed area.
Test example 4 evaluation of in vivo therapy
First, test method
In vivo treatment evaluation was performed with DSS-induced UC mouse model. Mice were randomly divided into six groups (each group n ═ 6): (1) normal group, (2) model group, (3) free RH group, (4) RH-NPs group, (5) HA/RH-NPs group, (6) CP/HA/RH-NPs group. Mice were treated (orally) with 25mg/kg RH (dose per unit body weight) from day 3 to day 10 after molding. Body weight, visible changes in fecal consistency and fecal bleeding were assessed daily throughout the treatment period. Disease Activity Index (DAI) is defined as the sum of weight loss (0-4 points), fecal consistency status (0-4 points) and fecal bleeding (0-4 points) (the lower the score, the healthier the mouse). On the tenth day of the endpoint, mice were anesthetized and euthanized, and then the colon and major organs (heart, liver, spleen, lung and kidney) were collected. Organs and colon were fixed in 4% formalin, embedded in paraffin, cut into thin slices (5 μm), and H & E stained. Formalin-fixed paraffin-embedded colon sections were immunohistochemically stained for claudin-1 and ZO-1 by immunohistochemistry using immunohistochemical kits including rabbit anti-claudin-1 and rabbit anti-ZO-1. Subsequently, the expression levels of claudin-1 and ZO-1 in the sample were shown by a chemochromic reaction. IL-1 β, IL-6, iNOS and TNF-. alpha.cytokines in the colon were evaluated using a commercially available ELISA kit according to the manufacturer's instructions. Western blot analysis was performed to detect the expression levels of the colonic proteins Claudin-1, ZO-1, MyD88, TLR4 and NF-. kappa.Bp 65. Colon samples were collected and homogenized using radioimmunoprecipitation assay (RIPA) lysis buffer. Primary antibodies against claudin-1, ZO-1, MyD88, TLR4 and NF-. kappa.Bp 65 were then incubated overnight at 4 ℃. Beta-actin and GAPDH antibodies were used as internal controls to determine the equal loading of the protein. The chemiluminescence signals obtained were analyzed with Image J software.
Second, test results
1. Effect of NPs on the severity of DSS-induced colitis in mice
The DSS-induced mouse model of UC is an easily generated and highly reproducible model, similar to human UC (e.g., weight loss, bloody stools and diarrhea). Therefore, the study used DSS-induced UC mice to evaluate the in vivo therapeutic effect of CP/HA/RH-NPs.
With the changes in body weight of FIG. 6A, the mice treated with CP/HA/RH-NPs were nearly identical to the original body weight, but slightly changed, while the other four drug-treated groups showed different degrees of weight loss. Similar differences were observed in fig. 6B with respect to DAI scores. The DAI score for the CP/HA/RH-NPs group was lower than that of the other four groups from day 6 to day 10. In particular, the DAI score for CP/HA/RH-NPs treated mice was 0.67. + -. 1.03 at the end point, similar to that of healthy normal mice.
The colon of the mouse was removed at the final point, then photographed and measured. As shown in fig. 6C and 6D, the colon length was significantly longer in the CP/HA/RH-NPs group than in the free RH, RH-NPs and HA/RH-NPs groups (. p. <0.01), indicating that CP/HA/RH-NPs had a good ameliorating effect on the typical colon shortening observed in DSS treatment. In addition, spleen is an important organ of the human immune system, and an increase in spleen weight is used as a marker of the severity of inflammation. As shown in FIG. 6E, CP/HA/RH-NPs treated mice (average spleen weight of 102.2. + -. 4.9mg) showed lower average spleen weight than HA/RH-NPs (average spleen weight of average spleen weight) or RH-NPs (average spleen weight of 149.4. + -. 4.0mg), indicating a greater improvement of colitis by CP/HA/RH-NPs. To assess the effect of CP/HA/RH-NPs on inflammatory infiltration of experimental colitis, MPO activity and histopathological changes in colon tissue were determined. MPO activity is a marker of neutrophil infiltration. As shown in FIG. 6F, the CP/HA/RH-NPs group showed the lowest colonic MPO activity in all treatment groups, with a significant difference compared to the model group (p < 0.01). Furthermore, as shown in fig. 6G, the DSS group showed evidence of inflammation compared to the normal group, as indicated by the destruction of the colonic epithelium, infiltration of various inflammatory cells, and depletion of mucus secreting goblet cells. The crypts of the mice in the RH-NPs and CP/HA/RH-NPs groups were more intact relative to the DSS group, indicating that both RH-NPs and CP/HA/RH-NPs improve signs and symptoms of colonic inflammation. Importantly, major organ sections of all mouse groups showed no histopathological evidence of lesions after treatment (fig. 7), indicating excellent biocompatibility and low toxicity of CP/HA/RH-NPs.
2. Anti-inflammatory effects of NPs through inhibition of TLR 4-associated NF- κ B signaling pathway
Activation of macrophages, accompanied by neutrophil infiltration in the inflamed colonic region, leads to the secretion of pro-inflammatory cytokines, including iNOS and cytokines (e.g., IL-1 β, iNOS, TNF- α, and IL-6) that are recognized as key indicators related to the degree of inflammation. Thus, the therapeutic efficacy of CP/HA/RH-NPs on the colon of mice was evaluated using an ELISA kit. As shown in FIG. 8A, the levels of IL-1. beta., iNOS, TNF-. alpha.and IL-6 in the DSS group were significantly increased (p <0.01) as compared with the healthy group. Meanwhile, CP/HA/RH-NPs inhibit the expression of IL-1 beta, iNOS, TNF-alpha and IL-6 more significantly than other treatment groups, which indicates that CP/HA/RH-NPs vector is the best and efficient way for delivering RH to inflamed colon to enhance the curative effect of UC.
The TLR4 signaling pathway is a pathway commonly referred to as classical, involves a series of proteins and cytokines, mediates inflammatory responses, and plays a key role in the pathogenesis of UC. However, there is no evidence that RH can treat UC through the TLR 4/NF-. kappa.B signaling pathway. Thus, based on DSS-induced NF- κ B activation associated with TLR 4-induced colon shortening, the efficacy of RH in this pathway was studied, and NPs were further studied by evaluating typical proteins (e.g., TLR4, MyD88 and NF- κ B) using Western Blotting analysis. As shown in FIG. 8B, total protein expression of TLR4, MyD88 and NF-. kappa.Bp 65 was significantly increased in colon tissue of the model group compared to the normal group (p < 0.01). However, CP/HA/RH-NPs significantly reduced the expression of these three proteins compared to the other groups (. p.0.05;. p.0.01). In conclusion, HA/RH-NPs and CP/HA/RH-NPs have good inhibition effects on TLR 4/NF-kB signal channels, and the inhibition effects of CP/HA/RH-NPs are obviously better than that of HA/RH-NPs.
3. NPs treatment can protect the colonic barrier of UC mice
Reduction of claudin increases intestinal permeability and leads to colonic barrier dysfunction. Intestinal epithelial cells linked together by TJs proteins (including ZO-1 and Claudin-1) prevent free passage of ions and small molecules through the space between the two epithelial cells. The expression of ZO-1 and Claudin-1 was detected by Western Blotting and immunohistochemical methods, respectively. As shown in FIG. 9A, in the immunohistochemical staining, ZO-1 and Claudin-1 were more immunoreactive in the normal group and CP/HA/RH-NPs group than in the model group. Meanwhile, the average area densities of ZO-1 and Claudin-1 were significantly increased in the CP/HA/RH-NPs group compared to the model group, free RH, RH-NPs group (p <0.01, FIG. 9B). Furthermore, the same results can be found in FIG. 9C, where the expression of ZO-1 and Claudin-1 in the CP/HA/RH-NPs group is significantly higher than in the free RH, RH-NPs group (p < 0.01). The result shows that the CP/HA/RH-NPs group can effectively protect the intestinal barrier of UC mice, and the effect is better than that of the RH-NPs group and free RH.
In conclusion, the present invention HAs studied a delivery system of CP-coated and HA-modified NPs (CP/HA/RH-NPs) with dual targeting capability for oral treatment of UC. CP/HA/RH-NPs have well-controlled particle size, narrow particle size distribution, negatively charged surface. The nanostructure is specifically designed for the pharmacological effects of RH in anti-inflammatory and colonic lesion repair. CP/HA/RH-NPs can be divided into three layers from outside to inside, namely CP, HA and LF:
(1) and (3) CP layer: according to the results of in vitro studies on the evaluation of gastric stability and release profile, CP/HA/RH-NPs showed good gastric stability with the help of CP and controlled release of RH was performed under stimulation of rat cecum containing abundant intestinal flora. Importantly, in vivo targeted delivery studies, the results indicate that CP on the surface of CP/HA/IR780-NPs can help LF not prematurely expose around the normal bowel segment, but further concentrate exposure and target colonic lesions. Namely, the colon targeting capability of CP/HA/RH-NPs is endowed.
(2) HA layer: HA-modified nanoparticles can target macrophages via the CD 44-mediated endocytic pathway, thereby increasing macrophage uptake rates, which is demonstrated in absorption assessment studies in qualitative and quantitative assays. In addition, the macrophage target design can effectively promote the anti-inflammatory effect of RH through a TLR4/MYD 88/NF-kB way, and has the in-vivo anti-UC treatment effect.
(3) And (3) LF layer: LF as a common nano-carrier has abundant sources, good drug-loading capacity and high RH encapsulation efficiency (95.08%). In addition, this study demonstrates that LF can increase cellular uptake and enhance rhein repair.
Compared with other oral NDDs, the lactoferrin rhein nano-particles also have two advantages: first, all the nanomaterials in NPs, such as CP, LF and HA, are food grade and environmentally benign and are much less toxic than other synthetic chemical nanomaterials. Moreover, the preparation process of nanoparticles is relatively simple and easy to scale up, and drugs and polymers are relatively cheap and available in large quantities. Secondly, researches show that RH can inhibit the expression levels of proinflammatory cytokines such as TNF-alpha, IL-6, IL-1 beta, iNOS and the like, improve the DSS-induced colitis of mice, and firstly prove that the anti-inflammatory effect of RH is realized through the TLR4/MYD 88/NF-kappa B pathways in the researches. The invention uses the nanotechnology to enhance the RH targeting ability and pharmacological action in UC treatment, provides an active UC-resistant natural compound RH, and designs an environment-friendly nano-carrier specially used for RH applied clinical treatment.
In conclusion, the invention provides the drug-loaded dual-targeting drug delivery nanoparticle which has good biocompatibility and stability, can be orally administered and has good targeting capability. The invention mainly provides rhein-loaded dual-targeting drug delivery nanoparticles, which have good biocompatibility and stability, can be orally administered and have good targeting capability on colon lesion inflammation and colon injury. The nano particle can keep stability in stomach and small intestine when being orally taken, releases the medicine slowly, and can release the medicine continuously after targeting to the colon lesion part, thereby having good effect on treating colitis, in particular ulcerative colitis. The nano particle has better treatment effect than that of single-use rhein and rhein-loaded nano particles with other structures, and has good application prospect.

Claims (10)

1. A lactoferrin nanoparticle carrying medicine is characterized in that: it is a nanoparticle with a three-layer structure; the three-layer structure comprises a pectin layer, a hyaluronic acid layer and a drug-loaded lactoferrin layer from outside to inside.
2. The drug-loaded lactoferrin nanoparticles of claim 1, wherein: the pectin layer is a pectin layer crosslinked by calcium ions.
3. Drug-loaded lactoferrin nanoparticles according to claim 1 or 2, characterized in that: the composition is prepared from the following raw materials in parts by weight: 50-100 parts of lactoferrin, 1-10 parts of a medicine, 10-50 parts of hyaluronic acid, 10-50 parts of pectin and 10-50 parts of calcium chloride;
preferably, the feed additive is prepared from the following raw materials in parts by weight: 50-60 parts of lactoferrin, 5-10 parts of a medicine, 10-20 parts of hyaluronic acid, 10-20 parts of pectin and 10-20 parts of calcium chloride;
more preferably, the composition is prepared from the following raw materials in parts by weight: 50 parts of lactoferrin, 5 parts of a medicine, 20 parts of hyaluronic acid, 10 parts of pectin and 10 parts of calcium chloride.
4. The drug-loaded lactoferrin nanoparticles of claim 3, wherein: the medicine is rhein, curcumin, chlorogenic acid, emodin or geniposide;
preferably, the drug is rhein.
5. The drug-loaded lactoferrin nanoparticles of claim 4, wherein: the preparation method of the drug-loaded lactoferrin nanoparticles comprises the following steps:
(1) dissolving lactoferrin in a water phase, dissolving a drug in an oil phase, and then mixing the water phase and the oil phase to obtain drug-loaded lactoferrin;
(2) adding hyaluronic acid into the drug-loaded lactoferrin to enable HA to be adhered to the surface of the drug-loaded lactoferrin so as to obtain the drug-loaded lactoferrin with hyaluronic acid;
(3) and (3) adding pectin in the step (2), then adding calcium chloride for cross-linking reaction, centrifuging, and collecting the precipitate to obtain the pectin.
6. The drug-loaded lactoferrin nanoparticles of claim 5, wherein:
in the step (1), the water phase is deionized water;
and/or in the step (1), the mass-to-volume ratio of the lactoferrin to the water phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the oil phase is DMSO;
and/or in the step (1), the mass-volume ratio of the medicine to the oil phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the water phase and the oil phase are mixed and then subjected to ultrasonic treatment, and then dialysis and filtration are carried out;
and/or in the step (2), adding hyaluronic acid into the lactoferrin carrying the medicine, and then shaking for 2-5 hours at room temperature;
and/or in the step (3), the crosslinking reaction time is 2-5 hours;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the mass-to-volume ratio of the lactoferrin to the aqueous phase is 5 mg: 2 mL;
and/or in the step (1), the mass volume ratio of the medicine to the oil phase is 5 mg: 1 mL;
and/or, in step (1), the ultrasonic treatment is ultrasonic treatment at 50% amplitude power to form an emulsion.
7. A process for the preparation of drug-loaded lactoferrin nanoparticles of any one of claims 1 to 6, wherein: it comprises the following steps:
(1) dissolving lactoferrin in a water phase, dissolving a drug in an oil phase, and then mixing the water phase and the oil phase to obtain drug-loaded lactoferrin;
(2) adding hyaluronic acid into the drug-loaded lactoferrin to enable HA to be adhered to the surface of the drug-loaded lactoferrin so as to obtain the drug-loaded lactoferrin with hyaluronic acid;
(3) and (3) adding pectin in the step (2), then adding calcium chloride for cross-linking reaction, centrifuging, and collecting the precipitate to obtain the pectin.
8. The method of claim 7, wherein:
in the step (1), the water phase is deionized water;
and/or in the step (1), the mass-to-volume ratio of the lactoferrin to the water phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the oil phase is DMSO;
and/or in the step (1), the mass-volume ratio of the medicine to the oil phase is (1-10) mg: (1-10) mL;
and/or, in the step (1), the water phase and the oil phase are mixed and then subjected to ultrasonic treatment, and then dialysis and filtration are carried out;
and/or in the step (2), adding hyaluronic acid into the lactoferrin carrying the medicine, and then shaking for 2-5 hours at room temperature;
and/or in the step (3), the crosslinking reaction time is 2-5 hours;
preferably, the first and second electrodes are formed of a metal,
in the step (1), the mass-to-volume ratio of the lactoferrin to the aqueous phase is 5 mg: 2 mL;
and/or in the step (1), the mass volume ratio of the medicine to the oil phase is 5 mg: 1 mL;
and/or, in step (1), the ultrasonic treatment is ultrasonic treatment at 50% amplitude power to form an emulsion.
9. Use of the drug-loaded lactoferrin nanoparticles of any one of claims 1 to 6 in the preparation of an anti-inflammatory drug; the drug in the drug-loaded lactoferrin nanoparticles is rhein.
10. Use according to claim 9, characterized in that: the medicament is used for treating colitis;
preferably, the medicament is a medicament for the treatment of ulcerative colitis.
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