Targeted anti-inflammatory nanoparticles for the treatment of radiation enteritis and methods of making the same

Abstract

This invention discloses targeted anti-inflammatory nanoparticles for treating radiation enteritis and their preparation method, belonging to the field of pharmaceutical technology. The nanoparticles comprise a three-layered composite structure of core, shell, and crown, with a particle size of 130-190 nm and a zeta potential of -12 to -6 mV. In this invention, the outer layer modified with an anti-γ-H2AX monoclonal antibody Fab fragment can specifically recognize the unique γ-H2AX protein marker in radiation-damaged intestinal epithelial cells in the lesion area, achieving localization of damaged cells; the RGD peptide can bind to the highly expressed αvβ3 integrin at the inflammatory site, forming a dual-locking effect on the inflammatory area; and the sodium alginate-grafted polyhistidine in the outer layer is protonated in the acidic inflammatory environment, forming electrostatic adhesion with the intestinal mucosa to achieve microenvironment anchoring and retention, constructing a highly efficient targeting mechanism.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to targeted anti-inflammatory nanoparticles for treating radiation enteritis and their preparation method. Background Technology

[0002] Radiation enteritis is a serious gastrointestinal complication that frequently occurs after radiotherapy for malignant tumors in the abdomen and pelvis. The incidence rate can reach over 50% when the total radiotherapy dose exceeds 45 Gy. Severe cases are accompanied by intestinal mucosal ulcers and perforation, which not only seriously affect the patient's quality of life but may also lead to the termination of radiotherapy and reduce the effectiveness of tumor treatment. Its core pathology involves radiotherapy-induced DNA damage and barrier disruption of the intestinal epithelium, which in turn activates inflammatory signaling pathways and induces a burst of reactive oxygen species (ROS), creating a vicious cycle.

[0003] Current clinical treatment primarily focuses on symptomatic support. Traditional anti-inflammatory drugs and mucosal protectants suffer from poor targeting, low bioavailability, and limited efficacy. To overcome this bottleneck, targeted anti-inflammatory nanoparticles have been widely studied due to their advantages of mucosal adhesion, sustained release, and local accumulation. These nanoparticles often utilize biocompatible materials such as PLGA and chitosan, and achieve inflammatory-responsive drug release through ligand targeting with RGD peptides or pH / ROS-sensitive design, demonstrating potential in the treatment of intestinal inflammation.

[0004] However, when existing targeted nanoparticles are applied to radiation enteritis, they only target general inflammatory targets such as αvβ3 integrin, ignoring the dual characteristics of radiation enteritis. The lesion area contains a large number of damaged intestinal epithelial cells expressing γ-H2AX (a DNA damage marker). Current technology does not utilize this specific target, leading to non-specific accumulation of nanoparticles in the normal intestine. The drug concentration at the lesion site is only 3-5 times that in normal tissue, still posing a risk of systemic side effects.

[0005] Furthermore, the intestinal mucus layer of patients with radiation enteritis thickens 2-3 times due to inflammation, and the negative charge density increases. Existing nanoparticles only use single modifications such as PEGylation or TAT peptides. The former can only reduce adsorption but cannot actively penetrate, while the latter is easily degraded by intestinal proteases, resulting in a mucus penetration rate that is generally less than 50%. The nanoparticles are blocked on the surface of the mucus and cannot reach the deep damaged mucosa.

[0006] Therefore, we propose targeted anti-inflammatory nanoparticles for the treatment of radiation enteritis and their preparation method to alleviate or solve the above problems.

[0007] The information disclosed above in this background section is only for enhancing the understanding of the background technology of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides targeted anti-inflammatory nanoparticles for treating radiation enteritis and their preparation method, thereby solving one of the problems in the prior art where targeted nanoparticles cannot meet the needs of precise treatment for radiation enteritis due to inaccurate targeting and difficulty in mucus penetration.

[0009] To achieve the above objectives, the present invention provides targeted anti-inflammatory nanoparticles for treating radiation enteritis. The nanoparticles comprise a three-layered composite structure of core, shell, and crown, with a particle size of 130-190 nm and a zeta potential of -12 to -6 mV. The specific composition is as follows:

[0010] The core carrier is a PLGA-PEG-Cystamine block copolymer, wherein the molar ratio of lactic acid to glycolic acid in PLGA is 50:50, the molecular weight of PEG is 2000 Da, and the core is loaded with recombinant human epidermal growth factor at a loading of 4-7 μg / mg nanoparticles.

[0011] The middle layer carrier is a pH-sensitive polyβ-amino ester with a pKa value of 5.0-5.5. The middle layer is loaded with a composite system of curcumin and celecoxib, in which the mass ratio of curcumin to celecoxib is 2:1 and the total loading is 14-19 μg / mg nanoparticles.

[0012] The outer layer is a trehalose-modified chitosan-sodium alginate composite membrane, wherein the sodium alginate is grafted with polyhistidine, and the outer surface is covalently bound to a dual-targeting ligand, which consists of a Fab fragment of an anti-γ-H2AX monoclonal antibody and an RGD peptide in a molar ratio of 1:2; the outer layer is loaded with hyaluronidase by electrostatic adsorption, wherein the loading amount of hyaluronidase is 3-5 U / mg nanoparticles.

[0013] Preferably, the PLGA-PEG-Cystamine block copolymer has a molecular weight of 22,000-25,000 Da and a purity of not less than 95%; in the modified chitosan-sodium alginate composite membrane, the mass ratio of chitosan to sodium alginate is 3:2, and the grafting rate of polyhistidine is 15-20%.

[0014] Preferably, the Fab fragment of the anti-γ-H2AX monoclonal antibody is prepared by papain digestion; the RGD peptide is a commercial standard containing arginine-glycine-aspartic acid in its sequence.

[0015] Preferably, the nanoparticles have sequential drug release characteristics, and in an inflammatory environment with pH 5.0-5.5 and high ROS, the release rate of celecoxib is not less than 58% within 1-2 hours, the release rate of curcumin is not less than 68% within 2-6 hours, and the release rate of recombinant human epidermal growth factor is not less than 78% within 6-24 hours.

[0016] Preferably, the middle layer poly-β-amino ester has a number average molecular weight of 5000-8000 Da and a terminal carboxyl modification rate of not less than 90%; the grafting rate of trehalose and chitosan is 10-15%.

[0017] Preferably, the total encapsulation efficiency of the nanoparticles is not less than 83%; when the concentration of the nanoparticles is not higher than 500 μg / mL, the survival rate of human intestinal epithelial cells is not less than 90%; the nanoparticles are formulated into enteric-coated capsules, each enteric-coated capsule containing 200 mg of nanoparticles, corresponding to 1.0 mg of recombinant human epidermal growth factor, 28 mg of curcumin, 14 mg of celecoxib, and 500 units of hyaluronidase.

[0018] Preferably, the drug concentration of the nanoparticles at the site of radiation enteritis lesions is 7-11 times that of the drug concentration in normal intestinal tissue, and the mucus penetration rate is not less than 82%.

[0019] The preparation method of targeted anti-inflammatory nanoparticles includes the following specific steps:

[0020] Step 1: Dissolve PLGA in dichloromethane, add DCC and DMAP for activation treatment, and then react with PEG-Cystamine under nitrogen protection and at 25°C for 12 hours. The reaction product is then precipitated, dialyzed, and freeze-dried to obtain the block copolymer.

[0021] Step 2: Using the double emulsion-solvent evaporation method, with recombinant human epidermal growth factor aqueous solution as the inner aqueous phase and PLGA-PEG-Cystamine dichloromethane solution as the oil phase, a W / O type primary emulsion is first prepared, and then the primary emulsion is dispersed in PVA aqueous solution to form a W / O / W type double emulsion. The double emulsion is obtained after solvent evaporation, centrifugation washing, and freeze drying.

[0022] Step 3: The core is dispersed in a poly-β-amino ester-curcumin-celecoxib dispersion and stirred at pH 6.0 to assemble a core-middle layer structure; the core-middle layer structure is then dispersed in a solution of trehalose-modified chitosan-sodium alginate composite membrane material and stirred at pH 5.5 to assemble core-shell structured nanoparticles. The core-shell structured nanoparticles are then centrifuged and washed for later use.

[0023] Step 4: Disperse the core-shell structured nanoparticles in MES buffer, activate them with EDC / NHS, add anti-γ-H2AXFab fragment and RGD peptide for covalent modification, load hyaluronidase through adsorption after modification, and finally obtain nanoparticles by centrifugation, washing and freeze-drying.

[0024] Preferably, in step 2, the ultrasonic parameters for colostrum preparation are: power 200W, single ultrasonic time 30 seconds, interval 10 seconds, repeated 3 times; the stirring speed for re-emulsion preparation is 10000rpm, and the stirring time is 30 minutes; the mass ratio of bovine serum albumin to recombinant human epidermal growth factor in the aqueous phase is 100:1, and the mass ratio of vitamin E to PLGA-PEG-Cystamine in the oil phase is 1:10.

[0025] Preferably, in step 1, the mass ratio of PLGA to PEG-Cystamine is 10:1, the molecular weight cutoff of the dialysis bag used for dialysis is 10000 Da, and the dialysis time is 48 hours; in step 3, the stirring time for the middle layer assembly is 4 hours, the stirring time for the outer layer assembly is 6 hours, the centrifugation speed for both assemblies is 10000 rpm, and the centrifugation time for both assemblies is 15 minutes.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] In this invention, the Fab fragment of the anti-γ-H2AX monoclonal antibody modified on the outer layer can specifically recognize the unique γ-H2AX protein marker in radiation-damaged intestinal epithelial cells in the lesion area, thereby achieving localization of damaged cells; the RGD peptide can bind to the αvβ3 integrin highly expressed at the inflammatory site, forming a dual-locking effect on the inflammatory area; and the polyhistidine grafted onto the outer layer of sodium alginate is protonated in the acidic environment of inflammation, forming electrostatic adhesion with the intestinal mucosa to achieve microenvironment anchoring and retention, thus constructing a highly efficient targeting mechanism.

[0028] In this invention, celecoxib is preferentially released within 1-2 hours to rapidly control acute inflammation, curcumin is released within 2-6 hours to clear oxidative stress damage, and rhEGF is slowly released within 6-24 hours to promote mucosal regeneration and repair, forming a treatment process that is highly matched with the pathological repair sequence, and significantly improving the mucosal healing effect.

[0029] In this invention, the synergistic design of three environmentally sensitive materials significantly improves the stability of nanoparticles, effectively controlling drug leakage in a simulated complex gastrointestinal environment, and exhibiting better stability than existing single-carrier nanoparticles.

[0030] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0031] Figure 1 This is a flowchart of the preparation method of the targeted anti-inflammatory nanoparticles of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only illustrative and not limiting.

[0033] Example 1: Preparation and performance testing of basic parameter-based targeted anti-inflammatory nanoparticles.

[0034] 1. Experimental Materials: PLGA (lactic acid / glycolic acid molar ratio 50:50, molecular weight 20000 Da, Sigma-Aldrich); PEG-Cystamine (PEG molecular weight 2000 Da, Shanghai Maclean Biochemical Technology Co., Ltd.); DCC, DMAP (purity ≥99%, Aladdin Reagent); recombinant human epidermal growth factor (rhEGF, purity ≥98%, Promega Biotechnology Co., Ltd.); pH-sensitive poly-β-amino ester (PBAE, pKa 5.2, number-average molecular weight 6000 Da, prepared in the laboratory by conventional ring-opening polymerization); curcumin (CUR, purity ≥98%, Xi'an Lvtian Biotechnology Co., Ltd.); celecoxib (CXB, purity ≥99%, Zhejiang Hisun Pharmaceutical Co., Ltd.); chitosan (CS, molecular weight 50000 Da, degree of deacetylation ≥90%, Sinopharm Chemical Reagent Co., Ltd.); sodium alginate (SA, molecular weight 800... 0.00 Da (Shanghai Yuanye Biotechnology Co., Ltd.); Trehalose (purity ≥99%, Sigma-Aldrich); Polyhistidine (molecular weight 10000 Da, Shanghai Hanhong Chemical Technology Co., Ltd.); Anti-γ-H2AX monoclonal antibody (derived from patent CN201980078563.2, Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.); RGD peptide (sequence containing arginine-glycine-aspartic acid, purity ≥98%, Jier Biochemical Co., Ltd.); Hyaluronidase (HAase, enzyme activity 1500 U / mg, Shanghai Sangon Biotech Co., Ltd.); PVA (molecular weight 10000 Da, Sinopharm Group); Bovine serum albumin (BSA, purity ≥98%, Sigma-Aldrich); Vitamin E (purity ≥98%, Aladdin Reagent); EDC, NHS, MES buffer (purity ≥99%, Shanghai Bide Pharmaceutical Technology Co., Ltd.); All other reagents were of analytical grade.

[0035] 2. Experimental Instruments: Dynamic light scattering instrument (Zetasizer Nano ZS90, Malvern Instruments Ltd.); High-performance liquid chromatograph (HPLC, Agilent 1260, Agilent Technologies Inc.); Ultrasonic cell disruptor (JY92-IIN, Ningbo Xinzhi Biotechnology Co., Ltd.); High-speed refrigerated centrifuge (Sigma 3-18K, Sigma GmbH, Germany); Freeze dryer (FD-1A-50, Beijing Boyikang Experimental Instrument Co., Ltd.); Thermostatic magnetic stirrer (DF-101S, Gongyi Yuhua Instrument Co., Ltd.); pH meter (PHS-3C, Shanghai Leici Instrument Factory).

[0036] 3. The preparation method of targeted anti-inflammatory nanoparticles, the specific steps are as follows:

[0037] Step 1: Dissolve 5.0 g PLGA in 20 mL dichloromethane, add 1.2 g DCC and 0.8 g DMAP, and activate by stirring at room temperature for 30 minutes; then add 0.5 g PEG-Cystamine, and react at 25 °C for 12 hours under nitrogen protection; after the reaction is complete, slowly pour the reaction solution into 100 mL ice-cold ether to precipitate, filter and collect the precipitate, dissolve the precipitate in deionized water, dialyze with a dialysis bag with a molecular weight cutoff of 10000 Da for 48 hours, changing the deionized water every 12 hours, and freeze-dry after dialysis to obtain PLGA-PEG-Cystamine block copolymer, which has a molecular weight of 23500 Da and a purity of 96.2%.

[0038] Step 2: Take 2 mL of rhEGF aqueous solution with a concentration of 1 mg / mL, add 0.1 g of BSA, and stir until completely dissolved; Oil phase preparation: Take 0.5 g of the above-synthesized PLGA-PEG-Cystamine block copolymer, dissolve it in 10 mL of dichloromethane, add 0.05 g of vitamin E, and stir until homogeneous; Slowly add the inner aqueous phase to the oil phase, and use an ultrasonic cell disruptor to ultrasonically emulsify at a power of 200W, with a single ultrasonication of 30 seconds, an interval of 10 seconds, and repeat 3 times to obtain a W / O type primary emulsion; Slowly drop the primary emulsion into 50 mL of 2% PVA aqueous solution, and stir at 10000 rpm for 30 minutes to form a W / O / W type double emulsion; Stir continuously at room temperature for 6 hours to volatilize the dichloromethane, then centrifuge at 8000 rpm for 15 minutes, collect the precipitate, wash it 3 times with deionized water, and freeze-dry it to obtain the core with a particle size of 98 nm.

[0039] Step 3: Dissolve 0.3 g PBAE in 20 mL of deionized water, add 0.03 g CUR and 0.015 g CXB, and sonicate at 150 W for 5 minutes to obtain a homogeneous poly-β-amino ester-curcumin-celecoxib dispersion; add 0.1 g of the above core to the dispersion, adjust the pH to 6.0, stir at room temperature for 4 hours, centrifuge at 10000 rpm for 15 minutes, collect the precipitate and wash twice to obtain a core-middle layer structure with a particle size of 128 nm;

[0040] 0.2 g of chitosan and sodium alginate in a mass ratio of 3:2 were dissolved in 20 mL of deionized water. 0.03 g of trehalose and 0.02 g of polyhistidine were added, and a trehalose-modified chitosan-sodium alginate composite membrane material solution was prepared by ester grafting reaction. The core-shell structure was added to the outer layer material solution, the pH was adjusted to 5.5, and the mixture was stirred at room temperature for 6 hours. The mixture was centrifuged at 10,000 rpm for 15 minutes, the precipitate was collected and washed 3 times to obtain core-shell structured nanoparticles with a particle size of 152 nm.

[0041] Step 4: Disperse the core-shell structured nanoparticles in 10 mL LME S buffer, pH 5.5, concentration 0.05 M, add 0.02 g EDC and 0.015 g NHS, and stir at room temperature for 30 minutes to activate. Then add 0.005 g Fab fragment of anti-γ-H2AX monoclonal antibody and 0.003 g RGD peptide, and stir at room temperature for 8 hours for covalent modification. After modification, add 0.01 g HAase, adsorb at room temperature for 2 hours, centrifuge at 12000 rpm for 20 minutes, collect the precipitate, wash 3 times with PBS buffer, and freeze-dry to obtain the targeted anti-inflammatory nanoparticles.

[0042] 4. Performance Testing

[0043] 4.1 Basic physicochemical properties were tested. Based on the principle of dynamic light scattering (DLS), the particle size was calculated by detecting the Brownian motion velocity of nanoparticles in the solution; the zeta potential was detected based on the principle of electrophoretic mobility.

[0044] Take 1 mg of nanoparticles and ultrasonically disperse them with deionized water at 100 W for 3 minutes to prepare a uniform dispersion of 0.1 mg / mL. Add 1 mL of the dispersion to the sample cell, set the test temperature to 25℃, and the equilibration time to 2 minutes. Repeat the test 3 times for each sample and take the average value as the final result.

[0045] The test results showed a particle size of 162 nm and a zeta potential of -9 mV.

[0046] 4.2 Drug loading and encapsulation efficiency test: The drug loaded in the nanoparticles was extracted by demulsification with organic solvent, and the drug concentration was detected by HPLC. The drug loading and encapsulation efficiency were calculated (drug loading = drug mass / total mass of nanoparticles × 100%; encapsulation efficiency = actual drug loading / theoretical drug loading × 100%).

[0047] Standard solutions of rhEGF (0.1-10 μg / mL), CUR (0.5-50 μg / mL), and CXB (0.5-50 μg / mL) were prepared and detected by HPLC (rhEGF: Ultimate C18 column, mobile phase water-acetonitrile = 90:10, flow rate 0.8 mL / min, detection wavelength 280 nm; CUR / CXB: Ultimate C18 column, mobile phase methanol-water = 70:30, flow rate 1.0 mL / min, detection wavelengths 428 nm and 262 nm, respectively), and concentration peak area standard curves were plotted.

[0048] Take 10 mg of nanoparticles, add 5 mL of methanol and sonicate to break up the emulsion at 150 W for 5 minutes. Centrifuge at 12000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm filter membrane. Detect the drug concentration by HPLC and calculate the drug mass by substituting it into the standard curve.

[0049] The test results showed that rhEGF was loaded with 5.3 μg / mg nanoparticles, CUR with 9.5 μg / mg nanoparticles, and CXB with 4.8 μg / mg nanoparticles, with a total encapsulation efficiency of 86.7%.

[0050] 4.3 Utilizing the semi-permeability of the dialysis bag, the drug inside the nanoparticles is released in a simulated environment. The drug concentration is measured at regular intervals, and the release curve is plotted.

[0051] Simulate a normal intestinal environment (pH 7.4 PBS buffer) and a simulated inflammatory environment (pH 5.2 PBS buffer, 100 μM H2O2, simulating a high ROS environment).

[0052] 20 mg of nanoparticles were dispersed in 5 mL of release medium, placed in a dialysis bag with a molecular weight cutoff of 10000 Da, sealed, and immersed in 45 mL of the corresponding release medium. The mixture was then incubated at 37°C with shaking at 100 rpm. Samples of 5 mL were taken at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 hours, with 5 mL of fresh release medium added simultaneously. The samples were filtered through a 0.22 μm filter membrane, and the drug concentration was determined by HPLC. The cumulative release rate was calculated.

[0053] In the test results, under inflammatory conditions, the release rate of CXB was 62% within 1.5 hours, the release rate of CUR was 72% within 4 hours, and the release rate of rhEGF was 83% within 18 hours; under normal intestinal conditions, the release rates of the three drugs were all less than 25% within 24 hours.

[0054] 4.4 The Transwell chamber was used to simulate the intestinal mucus barrier. A mixture of mucus and nanoparticles was added to the upper layer, and the permeated nanoparticles were collected in the lower layer. The permeation rate was calculated.

[0055] Colon tissue from SD rats was homogenized and centrifuged at 10,000 rpm for 30 minutes. The supernatant was collected as the natural mucus at a concentration of 5 mg / mL. 200 μL of mucus and 200 μL of nanoparticle dispersion were added to the upper layer of a Transwell chamber, and 600 μL of pH 5.2 PBS buffer was added to the lower layer. The chamber was incubated at 37°C for 2 hours. The lower layer was collected, and the drug concentration was determined by HPLC. The penetration rate was calculated (penetration rate = lower layer drug mass / total drug mass × 100%).

[0056] The test results showed a mucus penetration rate of 85%.

[0057] 4.5 WST-8 in CCK-8 reagent generates orange formazan under the action of cellular dehydrogenase. The amount of formazan is positively correlated with the number of live cells, and cell viability is reflected by detecting absorbance.

[0058] Caco-2 cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. The culture medium was discarded, and medium containing different concentrations of nanoparticles (10, 50, 100, 200, and 500 μg / mL) was added to each well, with three replicates per group. Cells were cultured for another 24 hours. A blank control group and a negative control group were also included. 10 μL of LCK-8 reagent was added to each well, and after incubation for 4 hours, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0059] The test results showed that at a concentration of 500 μg / mL, the cell viability was 93.2%.

[0060] 4.6 A rat radiation enteritis model was established. After drug administration, the drug concentration in the lesions and normal intestinal tissues was measured, and the concentration ratio was calculated.

[0061] SD rats underwent a single abdominal radiotherapy at a dose of 20 Gy, with an irradiation area of ​​3 cm × 3 cm. The model was confirmed to be successful 7 days after radiotherapy.

[0062] Model rats were orally administered enteric-coated nanoparticle capsules. Four hours after administration, the rats were sacrificed, and tissues from the colonic inflammatory lesions and normal small intestine were collected. After homogenization, 5 mL of methanol was added to break the emulsion, and the supernatant was collected by centrifugation. The drug concentration was determined by HPLC, and the lesion / normal tissue concentration ratio was calculated.

[0063] The test results showed that the drug concentration at the lesion site was 9.8 times that of normal intestinal tissue.

[0064] Example 2: Preparation and performance testing of low-grafting-rate, low-enzyme-loaded targeted anti-inflammatory nanoparticles

[0065] 1. The materials and instruments are the same as in Example 1, only the amount of polyhistidine and the HAase loading are adjusted.

[0066] 2. The preparation method of targeted anti-inflammatory nanoparticles, the specific steps are as follows:

[0067] The PLGA-PEG-Cystamine block copolymer was synthesized in the same manner as in Example 1, yielding a block copolymer with a molecular weight of 22800 Da and a purity of 95.5%.

[0068] The rhEGF-PLGA-PEG-Cystamine core was prepared in the same manner as in Example 1, yielding a core with a particle size of 92 nm.

[0069] The core-shell structure was prepared by layer-by-layer self-assembly, and the preparation of the middle layer dispersion was the same as in Example 1; the preparation of the core-middle layer structure was the same as in Example 1, resulting in a core-middle layer structure with a particle size of 121 nm.

[0070] Preparation of outer layer material solution: Take 0.2g of chitosan and sodium alginate, add 20mL of deionized water to dissolve, add 0.025g of trehalose and 0.015g of polyhistidine to prepare a trehalose-modified chitosan-sodium alginate composite membrane material solution; the preparation of core-shell structured nanoparticles is the same as in Example 1, and core-shell structured nanoparticles with a particle size of 145nm are obtained.

[0071] Core-shell structured nanoparticles were dispersed in 10 mL of LME S buffer and activated by EDC / NHS. Then, the same amount of anti-γ-H2AXFab fragment and RGD peptide were added for covalent modification as in Example 1. After modification, 0.006 g of HAase (loading 3 U / mg nanoparticles) was added and the nanoparticles were adsorbed at room temperature for 2 hours. The subsequent centrifugation, washing, and lyophilization steps were the same as in Example 1 to obtain the target nanoparticles.

[0072] 3. Performance Testing

[0073] In the basic physicochemical property tests, the particle size was 156 nm and the zeta potential was -11 mV.

[0074] In the drug loading and encapsulation efficiency tests, rhEGF was loaded with 4.7 μg / mg nanoparticles, CUR with 9.2 μg / mg nanoparticles, and CXB with 4.6 μg / mg nanoparticles, with a total encapsulation efficiency of 83.5%.

[0075] In the sequential drug release characteristic test, in an inflammatory simulation environment with pH 5.0 and high ROS, the CXB release rate was 59% within 1.8 hours, the CUR release rate was 69% within 5 hours, and the rhEGF release rate was 80% within 22 hours.

[0076] In the mucus penetration test, the in vitro mucus penetration rate was 82%.

[0077] In biocompatibility testing, when the nanoparticle concentration was 500 μg / mL, the survival rate of Caco-2 cells was 94.5%.

[0078] In the targeted enrichment effect test, in vivo experiments in a rat radiation enteritis model showed that the drug concentration at the lesion site was 7.6 times that of normal intestinal tissue.

[0079] Example 3: Preparation and performance testing of high-grafting-rate, high-enzyme-loaded targeted anti-inflammatory nanoparticles.

[0080] 1. Materials and Instruments

[0081] The materials and instruments were the same as in Example 1, except that the synthesis parameters of the PLGA-PEG-Cystamine block copolymer, the amount of polyhistidine, and the HAase loading were adjusted.

[0082] 2. Preparation steps

[0083] In the synthesis of PLGA-PEG-Cystamine block copolymer, 5.0g of PLGA was dissolved in 20mL of dichloromethane, 1.2g of DCC and 0.8g of DMAP were added and activated for 30 minutes, then 0.5g of PEG-Cystamine was added, and the reaction was carried out at 25°C for 12 hours under nitrogen protection. The subsequent precipitation, dialysis and lyophilization steps were the same as in Example 1, and a block copolymer with a molecular weight of 24200 Da and a purity of 95.8% was obtained.

[0084] The rhEGF-PLGA-PEG-Cystamine core was prepared in the same manner as in Example 1, yielding a core with a particle size of 105 nm.

[0085] The core-shell structure was prepared by layer-by-layer self-assembly. 0.3 g of PBAE was dissolved in 20 mL of deionized water, and 0.03 g of CUR and 0.015 g of CXB were added. After ultrasonic dispersion, a middle layer dispersion was obtained. 0.1 g of the core was added to the dispersion, and the mixture was stirred at pH 6.0 for 4 hours. After centrifugation and washing, a core-middle layer structure with a particle size of 132 nm was obtained. 0.2 g of chitosan and sodium alginate were dissolved in 20 mL of deionized water, and 0.035 g of trehalose and 0.025 g of polyhistidine were added to prepare a trehalose-modified chitosan-sodium alginate composite membrane material solution. The preparation of core-shell structured nanoparticles was the same as in Example 1, resulting in core-shell structured nanoparticles with a particle size of 160 nm.

[0086] The core-shell structured nanoparticles were dispersed in 10 mL of LME S buffer and activated by EDC / NHS. Then, the same amount of anti-γ-H2AXFab fragment and RGD peptide were added for covalent modification as in Example 1. After modification, 0.017 g of HAase was added and the nanoparticles were adsorbed at room temperature for 2 hours. The subsequent centrifugation, washing and lyophilization steps were the same as in Example 1 to obtain the target nanoparticles.

[0087] 3. Performance Testing

[0088] Among the basic physicochemical properties, the particle size is 178 nm and the zeta potential is -7 mV;

[0089] In terms of drug loading and encapsulation efficiency, rhEGF was loaded with 6.8 μg / mg nanoparticles, CUR with 9.8 μg / mg nanoparticles, and CXB with 4.9 μg / mg nanoparticles, with a total encapsulation efficiency of 87.2%.

[0090] In terms of sequential drug release characteristics, in an inflammatory simulation environment with pH 5.5 and high ROS, the CXB release rate was 65% within 1.2 hours, the CUR release rate was 75% within 3 hours, and the rhEGF release rate was 85% within 16 hours.

[0091] In terms of mucus penetration rate, the in vitro mucus penetration rate is 88%.

[0092] In terms of biocompatibility, when the nanoparticle concentration was 500 μg / mL, the survival rate of Caco-2 cells was 92.8%.

[0093] In the targeted enrichment effect, in vivo experiments in a rat radiation enteritis model showed that the drug concentration at the lesion site was 10.5 times that of normal intestinal tissue.

[0094] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0095] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A targeted anti-inflammatory nanoparticle for treating radiation enteritis, characterized in that, The nanoparticles comprise a three-layered composite structure consisting of a core, shell, and crown, with a particle size of 130-190 nm and a zeta potential of -12 to -6 mV. Their specific composition is as follows: The core carrier is a PLGA-PEG-Cystamine block copolymer, wherein the molar ratio of lactic acid to glycolic acid in PLGA is 50:50, the molecular weight of PEG is 2000 Da, and the core is loaded with recombinant human epidermal growth factor at a loading of 4-7 μg / mg nanoparticles. The middle layer carrier is a pH-sensitive polyβ-amino ester with a pKa value of 5.0-5.

5. The middle layer is loaded with a composite system of curcumin and celecoxib, in which the mass ratio of curcumin to celecoxib is 2:1 and the total loading is 14-19 μg / mg nanoparticles. The outer layer is a trehalose-modified chitosan-sodium alginate composite membrane, wherein the sodium alginate is grafted with polyhistidine, and the outer surface is covalently bound to a dual-targeting ligand, which consists of a Fab fragment of an anti-γ-H2AX monoclonal antibody and an RGD peptide in a molar ratio of 1:2; the outer layer is loaded with hyaluronidase by electrostatic adsorption, wherein the loading amount of hyaluronidase is 3-5 U / mg nanoparticles.

2. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The PLGA-PEG-Cystamine block copolymer has a molecular weight of 22,000-25,000 Da and a purity of not less than 95%; in the modified chitosan-sodium alginate composite membrane, the mass ratio of chitosan to sodium alginate is 3:2, and the grafting rate of polyhistidine is 15-20%.

3. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The Fab fragment of the anti-γ-H2AX monoclonal antibody was prepared by papain digestion; the RGD peptide is a commercial standard containing arginine-glycine-aspartic acid in its sequence.

4. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The nanoparticles have sequential drug release characteristics. In an inflammatory environment with pH 5.0-5.5 and high ROS, the release rate of celecoxib is not less than 58% within 1-2 hours, the release rate of curcumin is not less than 68% within 2-6 hours, and the release rate of recombinant human epidermal growth factor is not less than 78% within 6-24 hours.

5. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The middle layer poly-β-amino ester has a number average molecular weight of 5000-8000 Da and a terminal carboxyl modification rate of not less than 90%; the grafting rate of trehalose and chitosan is 10-15%.

6. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The total encapsulation efficiency of the nanoparticles is not less than 83%; when the concentration of the nanoparticles is not higher than 500 μg / mL, the survival rate of human intestinal epithelial cells is not less than 90%; the nanoparticles are formulated into enteric-coated capsules, each capsule containing 200 mg of nanoparticles, corresponding to 1.0 mg of recombinant human epidermal growth factor, 28 mg of curcumin, 14 mg of celecoxib, and 500 units of hyaluronidase.

7. The targeted anti-inflammatory nanoparticles according to claim 1, characterized in that, The drug concentration of the nanoparticles at the site of radiation enteritis lesions is 7-11 times that of normal intestinal tissue, and the mucus penetration rate is not less than 82%.

8. A method for preparing targeted anti-inflammatory nanoparticles as described in any one of claims 1-7, characterized in that, The specific steps are as follows: Step 1: Dissolve PLGA in dichloromethane, add DCC and DMAP for activation treatment, and then react with PEG-Cystamine under nitrogen protection and at 25°C for 12 hours. The reaction product is then precipitated, dialyzed, and freeze-dried to obtain the block copolymer. Step 2: Using the double emulsion-solvent evaporation method, with recombinant human epidermal growth factor aqueous solution as the inner aqueous phase and PLGA-PEG-Cystamine dichloromethane solution as the oil phase, a W / O type primary emulsion is first prepared, and then the primary emulsion is dispersed in PVA aqueous solution to form a W / O / W type double emulsion. The double emulsion is obtained after solvent evaporation, centrifugation washing, and freeze drying. Step 3: The core is dispersed in a poly-β-amino ester-curcumin-celecoxib dispersion and stirred at pH 6.0 to assemble a core-middle layer structure; the core-middle layer structure is then dispersed in a solution of trehalose-modified chitosan-sodium alginate composite membrane material and stirred at pH 5.5 to assemble core-shell structured nanoparticles. The core-shell structured nanoparticles are then centrifuged and washed for later use. Step 4: Disperse the core-shell structured nanoparticles in MES buffer, activate them with EDC / NHS, add anti-γ-H2AXFab fragment and RGD peptide for covalent modification, load hyaluronidase through adsorption after modification, and finally obtain nanoparticles by centrifugation, washing and freeze-drying.

9. The preparation method according to claim 8, characterized in that, In step 2, the ultrasonic parameters for colostrum preparation were: power 200W, single ultrasonic time 30 seconds, interval 10 seconds, repeated 3 times; the stirring speed for re-emulsion preparation was 10000rpm, and the stirring time was 30 minutes; the mass ratio of bovine serum albumin to recombinant human epidermal growth factor in the aqueous phase was 100:1, and the mass ratio of vitamin E to PLGA-PEG-Cystamine in the oil phase was 1:

10.

10. The preparation method according to claim 8, characterized in that, In step 1, the mass ratio of PLGA to PEG-Cystamine is 10:1, the molecular weight cutoff of the dialysis bag used for dialysis is 10000 Da, and the dialysis time is 48 hours; in step 3, the stirring time for the middle layer assembly is 4 hours, the stirring time for the outer layer assembly is 6 hours, the centrifugation speed for both assemblies is 10000 rpm, and the centrifugation time for both is 15 minutes.

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