CN114569740A - Novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by targeting immune germinal center, preparation method and application - Google Patents

Novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by targeting immune germinal center, preparation method and application Download PDF

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CN114569740A
CN114569740A CN202110792109.3A CN202110792109A CN114569740A CN 114569740 A CN114569740 A CN 114569740A CN 202110792109 A CN202110792109 A CN 202110792109A CN 114569740 A CN114569740 A CN 114569740A
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msn
spacdex
tac
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沈佳
张宏博
严芃芃
刘昌�
黄洪锋
王仁定
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Zhejiang University ZJU
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Abstract

The invention discloses a novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by a targeted immune germinal center, a preparation method and application.

Description

Novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by targeting immune germinal center, preparation method and application
Technical Field
The invention particularly relates to a novel nano slow-release drug-carrying material for inhibiting antibody-mediated rejection by a targeted immune germinal center, a preparation method and application thereof.
Background
Nanoscale Drug Carriers (Nanoscale Drug Carriers) are a submicron Drug carrier delivery system that belongs to the Nanoscale micro-category. The drug is encapsulated in submicron particles, so that the release speed can be adjusted, the permeability of a biological membrane is increased, the distribution in a body is changed, the bioavailability is improved, and the like. Nanoparticles (nanoparticles), also known as nanoparticles, are solid colloidal particles of 10-1000 nm in size, generally composed of natural or synthetic polymeric substances, and can be used as carriers for conducting or delivering drugs.
However, the existing nano-scale drug carrier has some disadvantages, such as lack of focus characteristic targeting, lack of controllable release function, and peripheral and systemic drug damage, so that a novel nano-drug-carrying material needs to be provided.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides a novel nano slow-release drug-carrying material for inhibiting antibody-mediated rejection by targeting immune germinal centers, a preparation method and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by targeting immune germinal centers is prepared by the following steps:
(1) preparing mesoporous silica nanospheres: firstly, 20-40mL of aqueous solution of CTAC with the weight percent of 25 and 0.18g of TEA are added into 36mL of water and stirred for 1 hour at the temperature of 60 ℃; then 20mL of 10 v/v% TEOS cyclohexene solution was added to the above mixture and reacted overnight in a 60 ℃ oil bath with magnetic stirring at 150rpm, the resulting product was collected by centrifugation and the collected product was dispersed in 0.6 wt% ammonium nitrate ethanol solution;
(2) synthesis of partially oxidized dextran: dissolving 1g of dextran having an average molecular weight of 9000-11000 in 4mL of water, adding 0.22g of sodium periodate to the above solution and stirring at room temperature for 5 hours to produce partially oxidized dextran; the partially oxidized dextran was further purified by dialysis against distilled water several times through a regenerated cellulose membrane MW 3500 Da; lyophilizing to obtain white, partially oxidized dextran powder;
(3) synthesis of partially oxidized acetylated dextran, AcDEX: adding 1.0g partially oxidized dextran into 10ml anhydrous DMSO, stirring to dissolve completely, adding 0.062mmol pyridinium p-toluenesulfonate and 37mmol 2-methoxypropene, and sealing in N2Under protection; after 3 hours, 1mL TEA was added to stop the reaction and 100mL distilled water was added to precipitate the modified partially oxidized dextran; the product was isolated by centrifugation at 10000rpm for 10 minutes and the resulting precipitate was washed with deionized water, followed by centrifugation and removal of the supernatant; obtaining the AcDEX by lyophilization;
(4) synthesis of spermine-modified Oxyacetylated dextran (SpAcDEX): 1.0g of partially oxidized AcDEX was dissolved in 10mL of anhydrous DMSO and mixed with 4.0g of spermine and reacted at 50 ℃ for 24 h; 2.0g of NaBH4Adding the mixture and reacting at room temperature for 18 hours; precipitating by adding 40mL of deionized water to the above solution, and separating by centrifuging at 10000rpm for 10 minutes; washing the obtained precipitate with deionized water for several times, and collecting SpAcDEX after freeze drying;
(5) preparation of SpAcDEX-encapsulated MSN (i.e., MSN @ SpAcDEX): 1.0 mg. multidot.mL-1MSN and 5 mg. mL- 1SpAcDEX was mixed in ethanol as the internal phase of the microfluidics and 1% PVA was usedThe aqueous solution is used as an external phase; connecting the two-phase solution to a polyethylene tube of a syringe, precipitating the MSN @ SpAcDEX obtained after mixing the two-phase solution, collecting in a 0.1% PVA solution, and stirring at 200 rpm; subsequently, it was centrifuged at 10000rpm for 5 minutes and washed several times with deionized water.
Further, the preparation of the drug-loaded material also comprises the following steps:
adding 5-10mg of nanoparticle MSN @ SpAcDEX to 2ml of 1mM bis sulfosuccinate diimidate PBS reagent; the mixture was stirred with magnetic beads at room temperature for 6 h; after washing with PBS, 5-10mg/ml BS 3-bound nanoparticles were incubated with 0.15mg/ml anti-mouse CD3e antibody and stirred at 4 ℃ overnight; CD3eAb conjugated nanoparticles were washed with PBS, centrifuged at 10,000g for 5 minutes, and repeated three times.
The application of the drug-loaded material in treating the inflammation of the transplanted organ is disclosed.
The invention has the beneficial effects that:
the novel nano drug-loaded material has targeting property, can slowly release drugs, can be used for treating inflammatory infiltration of transplanted organs, and has small toxicity (low neurotoxicity and renal toxicity) and small side effect.
Drawings
FIG. 1 is a schematic diagram of the synthesis of SpAcDEX.
In fig. 2, a is a transmission electron micrograph of MSN nanoparticles; b is a transmission electron microscope photo of a tacrolimus @ MSN @ SpAcDEX core-shell structure; c is the release profile of MSN @ SpAcDEX tacrolimus at different pH values.
Figure 3 is a graphical representation of the results of the cytotoxicity study after nanoparticle loading with drug.
FIG. 4 is a graph showing the results of in vivo toxicity evaluation of drugs, wherein A is a statistical graph of mouse body weight; b is a statistical chart of the results of the three indexes of the kidney function of the mouse: b1 is a 24-hour urinary protein measurement result statistical chart, B2 is a serum creatinine measurement result statistical chart, and B3 is a serum urea nitrogen measurement result statistical chart; c is a statistical chart of the detection result of the mouse lipid metabolism index: c1 is a statistical chart of the detection results of serum total glycerol fatty acid, C2 is a statistical chart of the detection results of serum total cholesterol,c3 is a statistical chart of the results of low-density cholesterol measurement, C4 is a statistical chart of the results of high-density cholesterol measurement, and C5 is a liver oil red O staining picture (Bar is 50 μm, the horizontal line in the figure is a scale, and represents 50 μm); d is a statistical chart of the test results of the single-plank bridge: d1 is a passing time statistic chart, D2 is a toe sliding frequency statistic; data presented in x ± SD form<0.01,***P<0.001,****P<A group of 0.0001vs Control,#P<0.05,##P<0.001,###P<0.001,####P<group 0.0001 vs. Tac;
Figure BDA0003161446550000031
vs. Tac-NP group.
Fig. 5 is a graph showing the results of HE staining of lung, liver, spleen, kidney, and brain (hippocampus and striatum) tissues in each group of mice (Bar is 100 μm, horizontal line in the graph is a scale, and represents 100 μm).
Fig. 6 is a graph showing Masson staining results of heart, lung, liver, spleen and kidney tissues of each group of mice (Bar is 100 μm, horizontal line in the graph is a scale, and represents 100 μm).
Fig. 7 is a graph showing the results of nanoparticle targeting cytology, with CD3eAb-FITC as the FITC (green) signal, cy5.5 NP as the cy5.5 (red) signal, and nuclei as the blue signal (Bar 10 μm, with the horizontal line being a scale representing 10 μm).
FIG. 8 is a graphical representation of nanoparticle targeting in-vivo IVIS results; b is a graph showing fluorescence staining results of mouse spleen tissues, with CD3 ebab as FITC (green) signal, cy5.5 NP as cy5.5 (red) signal, and nuclei as blue signal (Bar 25 μm indicates 25 μm with scale on the horizontal line).
Fig. 9 is a graph showing the results of transplanted kidney function in mice, wherein a is a graph showing the results of PAS staining of transplanted kidney tissues in mice (Bar 25 μm, horizontal line in the graph is a scale, and represents 25 μm); b is a statistical chart of the measurement results of three indexes of the kidney function of the mice (24-hour urine protein, serum creatinine and serum urea nitrogen), and the data are presented in a form of x +/-SD<0.05，**P<0.01,***P<0.001,****P<The 0.0001 vs. Isograft group,##P<0.001,####P<a set of 0.0001 vs. allogradient,
Figure BDA0003161446550000041
the set of vs. Tac is,
Figure BDA0003161446550000046
vs. Tac-NP group.
Fig. 10 is a graph showing the results of measurement of complement activation and DSA level of transplanted mouse kidney, wherein a is CD31 (green) and C4d (red) fluorescence staining results of transplanted mouse kidney tissue (Bar 25 μm, indicating that the horizontal line in the graph is a scale and represents 25 μm); b is a graph of the measurement result of the abundance of the mouse serum anti-donor antigens IgM and IgG; data presented in x ± SD form<0.01,***P<0.001,****P<A set of 0.0001 vs. allogradient,#P<0.05,##P<the 0.001 vs. Tac group,
Figure BDA0003161446550000042
vs. Tac-NP group.
FIG. 11 is a graph showing the results of measurement of the level of activation of rejection in the center of development after kidney transplantation in mice, wherein A is a typical flow chart of the activated Tfh cell (CXCR5+/Bcl-6+) population and plasma cell (CD138+/CD19mid + low) population in the spleen cells; b is a statistical plot of the proportion of activated Tfh cell (CXCR5+/Bcl-6+) populations to T cells and the proportion of plasma cell (CD138+/CD19mid + low) populations to B cells in spleen cells; c is a schematic diagram of spleen hyperplasia of each group, C1 is a picture of spleen size of each group, C2 is a statistical diagram of spleen/body weight ratio of each group; data presented in x ± SD form, P<0.05，**P<0.01,***P<0.001,****P<The 0.0001 vs. Isograft group,###P<0.001,####P<a set of 0.0001vs allogradient,
Figure BDA0003161446550000043
Figure BDA0003161446550000044
the set of vs. Tac is,
Figure BDA0003161446550000045
vs Tac group.
Fig. 12 is a schematic view of the structure of the microfluidic device.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention, wherein 1M is 1 mol/L.
First, the synthetic process
1. Preparation of mesoporous silica nanospheres
The MSN is continuously grown by a one-pot two-phase layering method by taking a cationic surfactant CTAC (cetyltrimethylammonium chloride) as a template, TEOS (tetraethyl orthosilicate) as a silicon dioxide source, TEA (triethylamine) as a catalyst and cyclohexene as an emulsifier.
The synthesis method comprises the following steps: first 20-40mL (25 wt%) CTAC aqueous solution and 0.18g TEA were added to 36mL water and stirred slowly at 60 ℃ for 1 hour. Then 20mL (10 v/v%) of TEOS cyclohexene solution was added to the above mixture of water, CTAC and TEA and reacted for 8-12h in a 60 ℃ oil bath with magnetic stirring at 150rpm, the resulting product was collected by centrifugation at 10000rpm for 10 minutes and washed several times with ethanol to remove the residual reactant, and all the product collected in the above reaction was dispersed in a volume of 10mL of 0.6% (w/v) ammonium nitrate (NH/v)4NO3) In ethanol solution. The resulting MSN was stored in 0.6% (w/v) ethanolic ammonium nitrate and stored at 4 ℃ for subsequent experiments.
Sulfo-Cy5.5-NHS-tagged MSN (MSN-Cy5.5)
The preparation of the MSN-Cy5.5 nano-structure adopts the synthesis process similar to the MSN, and 10 mu L of 1.0mg/mL Sulfo-Cy5.5-NHS is added into CTAC aqueous solution.
3. Synthesis of spermine-modified oxidized acetyl dextran (SpAcDEX)
3.1 Synthesis of partially oxidized Glucan
1g of dextran having an average molecular weight of 9000-11000 was first dissolved in 4mL of water, 0.22g of sodium periodate was added to the above solution, and stirred at room temperature for 5 hours to produce partially oxidized dextran. The partially oxidized dextran was further purified by multiple dialysis against distilled water through a regenerated cellulose membrane (MW 3500 Da). After lyophilization, a white, partially oxidized dextran powder was obtained.
3.2 Synthesis of partially oxidized acetylated Glucan (AcDEX)
1.0g partially oxidized dextran was added to 10mL anhydrous DMSO, stirred to dissolve completely, pyridinium p-toluenesulfonate (15.6mg, 0.062mmol) and 2-methoxypropene (3.4mL, 37mmol) were added and sealed in N2Under protection to prevent evaporation of 2-methoxypropene. After 3 hours, the reaction was terminated by adding 1mL of TEA, and the modified partially oxidized dextran was precipitated by adding 100mL of distilled water. The product was isolated by centrifugation at 10000rpm for 10 minutes, and the resulting precipitate was washed thoroughly with deionized water by ultrasonic dispersion and then centrifuged at 10000rpm for 10 minutes to remove the supernatant. A white powder of partially oxidized acetal dextran obtained by lyophilization.
3.3 Synthesis of spermine-modified Oxyacetylated Glucan (SpAcDEX)
1.0g of partially oxidized AcDEX was dissolved in 10mL of anhydrous DMSO and mixed with 4.0g of spermine and reacted at 50 ℃ for 24 h. 2.0g of NaBH4Added to the mixture and reduced at room temperature for an additional 18 hours. Precipitation was performed by adding 40mL of deionized water to the above solution, and separation was performed by centrifugation at 10000rpm for 10 minutes. The resulting precipitate was washed several times with deionized water, after freeze-drying, the SpAcDEX was collected and stored at-20 ℃. FIG. 1 is a schematic diagram of the synthesis of SpAcDEX.
4. Manufacture of three-dimensional microfluidic co-flow device
Borosilicate glass capillaries of different parameters were used and assembled on glass slides: one end of a cylindrical capillary tube (inner diameter and outer diameter of 580 and 1000 μm, respectively, World Precision Instruments, inc., USA) used as an inner tube was drawn using a micropipette puller (P-97, Sutter USA Instruments) to form a micro-bore capillary tube; the orifice diameter was then enlarged to 80 μm using sandpaper. The fabricated inner tube was then inserted into another thicker cylindrical capillary outer tube (inner and outer diameters 1100 and 1500 μm, respectively, Vitrocom, Inc.) and assembled into a coaxial configuration. The outer capillary was terminated by two hypodermic needles (Warner Instruments, USA). The assembly was sealed using a clear epoxy (5 min Epoxi, Devcon) if necessary. The two miscible liquids were injected separately into the microfluidic device at a constant flow rate through polyethylene tubing connected to a syringe. The flow rates of the various liquids were controlled by pumps (PHD 2000, Harvard Apparatus, USA).
Preparation of SpAcDEX Encapsulated MSN (MSN @ SpAcDEX)
To encapsulate MSN into SpAcDEX polymers, 1.0 mg. mL was first introduced-1MSN and 5 mg. mL-1SpAcDEX was mixed in ethanol as the internal phase of the microfluidics and 1% (v/v) aqueous PVA (polyvinyl alcohol) was used as the external phase. The biphasic solution was passed through a polyethylene tube connected to a syringe with an internal phase of 2 mL. h-1And outer phase 40 mL. h-1The schematic diagram of the microfluidic device is shown in fig. 12, and the flow rate is controlled by the microfluidic pump. The two-phase solution was mixed in the microfluidic pump line to obtain a MSN @ SpAcDEX precipitate, which was collected at the output in a 0.1% volume fraction PVA solution in water and gently stirred at 200 rpm. Subsequently, it was centrifuged at 10000rpm for 5 minutes and washed several times with deionized water. MSN @ SpAcDX was stored in deionized water at 4 ℃.
6. Characterization of the nanocomposites
The structure of the synthesized MSN and MSN @ SpAcDEX was evaluated by transmission electron microscopy (TEM, JEOL 1400Plus, usa) at an acceleration voltage of 80 kV. The surface zeta potential of MSN @ SpAcDX was measured using Zetasizer Nano ZS by using disposable folded capillary cells (DTS1070, Malvern, UK).
7. MSN (or Tac-NP) was loaded with tacrolimus.
For tacrolimus encapsulation, it was loaded into MSN nanoparticles using the impregnation method.
First, 5.0 mg. multidot.mL of the suspension was prepared-1And 1.0mg of MSN was resuspended in the solution at a mass ratio of 1:5(MSN: tacrolimus ═ 1: 5). The mixture was stirred slowly at room temperature for 24 hours to reach saturation of the MSN loading. Thereafter, the precipitate was obtained by centrifugation at 10000rpm for 5 minutes, washed 3 times with ethanol, and then tacrolimus-loaded MSN (tacrolimus @ MSN) was obtained. All tacrolimus in the supernatant was carefully collected and performed according to a standard curve of tacrolimus in ethanol solutionAnd (4) quantifying. The loading capacity is calculated according to the following formula: loading% (% tacrolimus in MSN/total MSN 100).
8. In vitro drug release test
The in vitro release of tacrolimus from the nanocomposite was evaluated in phosphate buffered saline solution, ph7.4 and 5.0 were used to simulate the extracellular and intracellular environment, respectively. Specific release profiles were performed by placing drug-loaded nanocomposites (500 μ g) into the respective buffer solutions and shaking at 100rpm and 37 ± 1 ℃. Free tacrolimus was used as control. Due to the low solubility of tacrolimus, amphiphilic F-127 (5%, w/v) was added to all release buffers. Samples were first centrifuged (10000rpm, 5min) at different time points, 100 μ L of supernatant was aspirated, an equal volume of pre-heated RPMI1640 medium was added instead of the aspirated volume, and then the drug concentration in the supernatant was quantified by HPLC.
9. Binding of nanoparticles to CD3e antibodies
To bind empty Nanoparticles (NP) and drug-loaded nanoparticles (Tac-NP) to CD3eAb, 5mg of nanoparticles (based on spadex mass) were added to 2ml of freshly prepared 1mM disulfosuccinic diimidate (BS3) PBS reagent (1mM, pH 7.4). The mixture was stirred at room temperature with magnetic beads (200Hz) for 6 h. After washing twice with PBS (1mM, pH 7.4), BS 3-bound nanoparticles (concentration of BS 3-bound nanoparticles in PBS of 5mg/ml) were incubated with anti-mouse CD3e antibody (0.15mg/ml, clone number 145-2C11, BD Pharmingen, CA, USA) and stirred at 4 ℃ for 8-12 h. Free BS3 was blocked with 50mM glycine and stirred at room temperature for 2 h. CD3eAb conjugated nanoparticles (NP-CD3eAb) were washed with PBS, centrifuged at 10,000g for 5 minutes, and repeated three times.
Bound antibody concentrations were detected electrophoretically on Bis-Tris gels (NP0326BOX, ThermoFisher, Calif., USA), stained with a gradient bovine serum albumin standard (Cat.23208, ThermoFisher) in gel using Coomassie brilliant blue, and the areas of bands displayed were quantified using ImageJ and diluted to 100. mu.g/ml with PBS. 1ml of fresh PBS was used to resuspend the targeting nanoconjugate and stored at 4 ℃.
Second, toxicity study
1. In vitro: and (3) taking primary splenocytes to determine the toxicity of the nanoparticles. The water soluble tetrazolium salt (WST-1) analysis was performed according to the manufacturer's instructions.
Briefly, an acute isolated spleen single cell suspension (5X 104/100. mu.l/well) was plated in 96-well microplates and incubated with 0, 10, 15, 20, 30, 50, 100. mu.g/ml tacrolimus (Tac) or nanoparticles of the same drug loading (Tac-NP) for 8 hours. 10. mu.l/well of WST-1 reagent (cat.: Cellpro-ro, Roche) was added and incubated at 37 ℃ for 4 hours. Absorbance at 450nm and 600nm was measured using a multifunctional microplate reader (Infine M1000 Pro).
2. In vivo:
the animal-related experimental protocol was approved by the research ethics committee of the first subsidiary hospital of the university of Zhejiang medical school (number: 2019-. Male Balb/C and C57BL/6 mice (body weight: 23. + -.2 g) at 8 weeks of age were provided by the university of Zhejiang laboratory animals center. Animals were free to obtain water and standard rodent chow and housed in an environment at 25 ℃ for 12 hours light/dark cycles.
C57BL/6 mice were injected intravenously with PBS, tacrolimus, Tac-NP, or Tac-NP-CD3eAb every 3 days for 4 weeks. The dose of tacrolimus in the Tac, Tac-NP and Tac-NP-CD3eAb groups corresponds to 1mgTac/kg of mice. Body weight was monitored weekly. At the end of 4 weeks, mice were evaluated for neurotoxicity by the monologue test, renal toxicity by the metabolic cage study, and sacrificed for serum and multi-organ access.
2.1 neurotoxicity assessment
The neurotoxicity of the drug to each group of mice was assessed using the striga bridge experiment. Driving the mice to walk on a wooden stick with the length of 1m and the width of 0.5cm, recording the crossing time and the number of hind limb slippage, and repeatedly walking each mouse three times;
2.2 evaluation of renal toxicity
Collecting 24h urine of a mouse, and measuring the level of the urine protein; collecting mouse serum, and measuring serum creatinine (Cr) and Blood Urea Nitrogen (BUN) levels in a biochemical analyzer (DRI-CHEM 7000i, Fujifilm) using FUJI DRI-CHEM reagent tablet;
2.3 evaluation of lipid metabolism
Mouse sera were collected and analyzed for serum Triglycerides (TG), cholesterol (TCHO) and High Density Lipoprotein (HDL) in a biochemical analyzer using FUJI DRI-CHEM reagent tablets.
2.4 histological examination
Mouse kidney, heart, liver, spleen, lung and brain tissues were fixed with 4% paraformaldehyde by mass fraction, paraffin-embedded, cut into 3 μm-thick sections, and stained with hematoxylin and eosin (H & E), Periodic Acid Schiff (PAS) and/or Masson trichrome. The liver tissue was partially frozen at a thickness of 15 μm and stained with oil red O to observe neutral lipids, and the stained images were taken using an optical microscope (DM4000, Leica).
Thirdly, targeting research
1. Separation in vitro: spleen C57BL/C mouse spleen was incubated with collagenase type I (1mg/ml, Cat.17018029, ThermoFisher, MA, USA) for 15 min at 37 deg.C, the digested suspension was passed through a 40 μm cell strainer (Cat.431750, Corning, NY, USA), centrifuged at 300g for 5min, and resuspended to 5X10 in 10% volume fraction heat-inactivated (Cat.10100139C, Gibco) RPMI1640 medium (Cat.11835030, Gibco, NY, USA)6Cells/ml. The CD3eAb label FITC conjugation kit was kept ready for use by the FITC conjugation kit (ab 102884).
To assess the binding specificity of the NP-CD3eAb, 1/10 volumes of Cy5.5-NP or Cy5.5-NP-CD3eAb were added to the prepared cell suspension and incubated at 37 ℃ for 1 hour, and the cell suspension to which Cy5.5-NP-CD3eAb was added was divided into two portions, one of which was previously incubated with 10. mu.g/ml of CD3eAb for 1 hour for antigen blocking. The combined cells were washed with PBS, pelleted at 350g for 8min, resuspended in 1ml PBS solution, and then assessed for targeting ability by fluorescence image analysis.
2. In vivo: Cy5.5-NP-CD3eAb or Cy5.5-NP (5mg/kg, 200. mu.l) was injected intravenously into C57BL/6 mice after homo-or hetero-renal transplantation, 24 hours later, mice were anesthetized with isoflurane and subjected to in vivo spectral imaging (IVIS). Then, the mice were sacrificed with an excess of sodium pentobarbital (250mg/kg, east China medicine) to collect spleens for organ IVIS, tissue Immunofluorescence (IF), and Transmission Electron Microscopy (TEM) analysis.
2.1IVIS
Spectroscopic imaging was performed using IVIS Lumina LT series III (Perkin Elmer, MA, USA) with a Cy5.5 filter set (excitation 675nm, emission 720nm, exposure time 1s) to obtain a Cy5.5 signal for NP. All images were acquired and analyzed using the Living Image version 4.5 software (Perkin Elmer) and displayed on the same fluorescence intensity scale, with the fluorescence emission normalized to photons per second per square centimeter per steradian (p/s/cm 2/sr).
2.2IF
Frozen sections of the spleen at 15 μm were blocked with 0.5% normal goat serum at room temperature for 2h, incubated with CD3e antibody at 4 ℃ overnight, and labeled with FITC. Nuclei were stained with Hoechst (Cat.62249, Thermo Fisher) and fluorescence images were taken using a Leica TCS-SP8 confocal laser scanning microscope.
2.3TEM
Spleen tissue was fixed overnight in a volume fraction of 2.5% glutaraldehyde solution, carefully washed 3 times with PBS (1mM, pH 7.4); fixing with 1% (w/v) osmium tetroxide water solution for 1 hr, washing with pure water for 3 times; staining with 2% (w/v) aqueous uranium acetate solution for 30 minutes, gradually dehydrating in 50% -100% ethanol and 100% acetone by volume, infiltrating into the resin and acetone mixture (1:1, v/v) at room temperature for 2 hours, and then embedding into the resin. Embedded spleen specimens were cut at 70nm thickness using a microtome (Leica UC7) and observed and photographed at 80KV intensity in an electron microscope (Tecnai G2 Spirit TEM, Thermo Fisher).
Fourth, study of therapeutic effects
ABMR disease model and drug treatment
C57BL/6 recipient mice received a 1cm diameter skin graft from Balb/C donor mice to the dorsal area for presensitization after intraperitoneal injection of sodium pentobarbital (50mg/kg) anesthesia. Two weeks after skin transplantation, kidneys from Balb/C (allograft, Allo) or C57BL/6 (allograft, Iso) donor mice were transplanted into C57BL/6 recipient mice, which had renal resection.
After transplantation, Allo recipient mice were treated daily with intravenous injection of sterile PBS, tacrolimus (Tac group), Tac-NP (Tac-NP group), Tac-NP-CD3eAb (Tac-NP-CD3eAb group), and Iso mice were treated with intravenous injection of sterile PBS. In each group containing the drug treatment, the dosage equivalent of tacrolimus is 1 mg/kg/d. 5 days after the kidney transplantation, the mice were sacrificed by intraperitoneal injection of excess pentobarbital sodium, and samples were taken for corresponding detection.
1.1DSA assay
Balb/C donor mouse spleen cells were incubated with C57BL/6 receptor mouse serum for 30 minutes at room temperature and washed with PBS. Use of anti-mouse IgM Alexa
Figure BDA0003161446550000111
647(Cat. ab150123, Abcam) and anti-mouse IgG
Figure BDA0003161446550000112
488(cat. ab96879, Abcam) labeled surface binds to DSA and the fluorescence intensity of the DSA signal is measured by CytoFLEX LX flow cytometer.
1.2C4d deposition
Frozen sections of transplanted kidneys of 15 μm were blocked with 0.5% normal goat serum at room temperature for 2h, incubated overnight at 4 ℃ with anti-mouse C4d antibody, and stained with FITC-fluorescently labeled secondary antibody. Nuclei were stained with Hoechst (Cat.62249, Thermo Fisher) and fluorescence images were taken using a Leica TCS-SP8 confocal laser scanning microscope.
1.3 histological examination
Transplanted kidneys were sectioned in 3 μm paraffin, stained by PAS and photographed using an optical microscope (DM4000, Leica).
Fifth, statistical analysis
All statistical analyses were performed using GraphPad Prism 8(GraphPad Inc, CA, USA). Results are shown as mean ± Standard Deviation (SD) unless otherwise indicated. Tukey post-variance analysis was used for the multiple comparisons. P <0.05 was statistically significant.
Results section
First, the physical and chemical properties of the nanoparticles
Representative Transmission Electron Microscope (TEM) image results of MSN showed an average particle size of about 80 nm (fig. 2A). zeta potential measurement shows a surface charge of-15.1+ -0.5 mV. The nanoparticles were then treated with 0.6 wt% ammonium Nitrate (NH) at 60.0 deg.C4NO3) The ethanol solution was stirred for 6 hours, and the stirring was repeated twice to remove the MSN synthesis template cetyltrimethylammonium chloride (CTAC).
In order to track the uptake process of the nanoparticles by cells, a red fluorescent dye Sulfo-Cyanine 5.5NHS ester (Cy5.5) is wrapped in MSN (Cy5.5-MSN), namely, a certain amount of Cy5.5 is mixed with a silicon dioxide synthesis precursor in the process of synthesizing the MSN. Tacrolimus is used as a model drug, mixed in ethanol solution under stirring and loaded in MSN pore channels, and after incubation for 24 hours, the loading capacity of the tacrolimus is measured to be up to 438.2 mg/g.
During the loading process, since tacrolimus and SpAcDEX are insoluble in water, an amount of SpAcDEX was added to the loaded tacrolimus @ MSN ethanol solution with stirring. Polymers are deposited on nanoparticles by changing the solubility of solutes in miscible solutions with the aid of microfluidic technology.
Firstly, the mixed solution of the drug-loaded nanoparticles and the polymer is set as a microfluidic internal phase, and the aqueous solution is used as an external phase. The two-phase solution was attached to a microfluidic chip, and the flow rate was controlled by a microfluidic pump, and the two-phase solution was mixed to obtain a precipitate of tacrolimus @ MSN @ SpAcDEX (i.e., Tac @ MSN @ SpAcDEX) (fig. 2B). The core/shell structure of tacrolimus @ MSN @ SpAcDEX (i.e., Tac @ MSN @ SpAcDEX) is clearly seen in the figure because of the difference in electron permeability between the core and the polymer shell. This new system combines the easy synthesis of microfluidic technology with the biocompatibility of polymers, with the additional advantages of controlled payload release sensitive to physiologically relevant acidic conditions.
Acid sensitive systems have particularly desirable properties because drug release can be triggered in response to acidification of lysosomes upon cellular uptake. The in vitro release profile of tacrolimus (figure 2C) from Tac @ MSN @ SpAcDEX nanocomposites was evaluated in Phosphate Buffered Saline (PBS) according to a standard curve, with PBS solutions at pH7.4 and 5.0 to simulate extracellular and intracellular environments, respectively. At pH7.4, the drug concentration remained constant for several hours with little drug release. In contrast, at pH 5.0, the rapid release of tacrolimus is consistent with the degradation of SpAcDEX at high acidity. The system showed rapid release behavior with a cumulative release of 68.9% over 48 hours. Tacrolimus is released in a controlled mode after endocytosis, which provides a way for regulating the release flux of tacrolimus to achieve the expected biological effect.
Second, toxicity study
1. In vitro results:
the WST-1 result (figure 3) shows that the growth curve result of tacrolimus in the concentration range of 0-30 mug/ml has no significant difference from that of a drug-free control group, which indicates that the tacrolimus has no significant damage to spleen cells in the concentration range, and in the concentration range of 50-100 mug/ml, the growth curve value is significantly lower than that of the drug-free control group, which shows the drug toxicity of the tacrolimus to the spleen cells; the growth curve result of the drug-loaded nanoparticles is not obviously different from that of a drug-free control group within the concentration range of 0-50 mu g/ml, which indicates that the drug-loaded nanoparticles do not obviously damage spleen cells within the concentration range, and the growth curve is obviously reduced compared with that of the drug-free control group but is still higher than that of a tacrolimus group at the concentration of 100 mu g/ml.
2. In vivo results:
2.1 Overall toxicity assessment
Body weight measurements were performed weekly for each group of mice, and the results showed that no significant difference in body weight was observed between each group of mice and the PBS-injected control group within 4 weeks of administration (fig. 4A).
2.2 evaluation of renal toxicity
Serum creatinine (Cr) in the PBS injection group was 39.8 + -3.1 μmol/L at the end of the experiment; the Tac group Cr values increased significantly to 78.3 ± 14.9 μmol/L (P <0.01vs PBS) after 4 weeks of tacrolimus injection; the Cr values of the Tac-NP and Tac-NP-CD3eAb are 52.3 +/-10.8 mu mol/L and 48.8 +/-14.2 mu mol/L respectively, and have no significant difference from the PBS group (P >0.05) and are both significantly lower than the Tac group (P <0.05vs Tac-NP and P <0.05vs Tac-NP-CD3 eAb). The serum BUN levels of the PBS group, the TAC group, the Tac-NP group and the Tac-NP-CD3eAb group are 34.6 +/-3.8 mg/dL, 28.8 +/-0.7 mg/dL, 28.6 +/-2.2 mg/dL and 28.6 +/-1.8 mg/dL respectively, and no significant difference (P >0.05) exists among the groups. Urine protein gel electrophoresis showed that TAC and TAC-NP injections significantly increased protein urine levels, while TAC-NP-CD3eAb administration decreased urine protein up-regulation levels (fig. 4B).
2.3 evaluation of lipid metabolism
Serological test results (fig. 4C) showed no significant difference between serum TG and TCHO for each group (P >0.05), but oil red O staining results showed a significant increase in lipid droplet deposition in the hepatocytes of the Tac group 4 weeks after tacrolimus injection; after tacrolimus loading with NP, lipid droplet deposition was significantly reduced in hepatocytes of the Tac-NP group and the Tac-NP-CD3eAb group compared to the Tac group; treatment with Tac-NP-CD3eAb even increased the serum level of HDL cholesterol, termed "good" cholesterol (2.67 ± 0.16mmol/L vs 2.00 ± 0.16in Control group, p < 0.001; 2.67 ± 0.16mmol/L vs 2.14 ± 0.17 in the Tac group and 2.12 ± 0.14 in the Tac-NP group, p < 0.01).
2.4 neurotoxicity assessment
Mice motor function deficits and coordination were reflected by the single-plank bridge (Beam-Walking) test (fig. 4D). The time for the mice to pass through the single-plank bridge is not obviously different among all groups; the number of times of toe sliding in the crossing process is as follows: 5.9 +/-0.7 times of PBS injection group, 10.4 +/-0.8 times of Tac group, 6.8 +/-1.0 times of Tac-NP group and 5.6 +/-0.4 times of Tac-NP-CD3eAb group, statistics show that the foot sliding frequency of the mice in the Tac group is obviously increased compared with that of the mice in the other three groups (P <0.01vs PBS, P <0.05vs Tac-NP and P <0.01vs Tac-NP-CD3eAb), but no obvious difference exists among the other three groups, which indicates that the neurotoxicity of the Tac at least has obviously reduced influence on the fine motor ability after the nano particles are wrapped.
2.5 histological examination
Histological evaluation of brain (hippocampus and striatum), lung, heart, liver, spleen and kidney using hematoxylin and eosin (H & E) staining was performed, and no significant structural differences were observed within each group (fig. 5); however, the perivascular collagen deposition in the heart, liver and kidney of mice in the Tac group was relatively increased compared to those in the PBS, Tac-NP and Tac-NP-CD3eAb groups.
Thirdly, targeting research
1. In vitro: the results of cellular fluorescence (FIG. 7) show that only green fluorescence of CD3eAb-FITC is detected in the blank group and NP group, while green and red fluorescence can be detected on the same cell surface by the administration of Cy5.5-NP-CD3eAb and FITC-CD3 eAb; if CD3eAb is used for blocking CD3e antigen on the surface of the cells in advance, and Cy5.5-NP-CD3eAb and FITC-CD3eAb are given to incubate the cells, only very weak red and green fluorescence is generated. Thus, only CD3 eAb-labeled NPs (NP-CD3eAb) can specifically bind to CD3e + cells, while the CD3+ cell-targeted binding ability of NPs-CD3eAb can be competitively inhibited by the cognate antigen. Fig. 7 is a graph of the results of nanoparticle targeting cytology, showing CD3eAb-FITC as the FITC (green) signal, cy5.5 NP as the cy5.5 (red) signal, and nuclei as the blue signal (Bar 10 μm).
2. In vivo: the IVIS results showed that the spleen cy5.5 signal was strong in mice 24 hours after iv injection of NPs-CD3eAb, with a further increase of approximately 3.7-fold in the spleen of allogeneic kidney transplant mice, whereas the signal aggregation of cy5.5 could not be detected in the NP iv group. The IF results showed that there was a high coincidence of the red (Cy.5.5) fluorescence signal with the green Fluorescence (FITC) signal in spleen tissue of the Cy5.5-NPs-CD3 eAb-injected group, whereas in the Cy5.5-NPs-injected group, the red signal was very small and did not overlap with the green fluorescence signal; TEM results showed that NP signals were detectable in cytoplasm of spleens of NPs-CD3eAb group, while no NP signals were detectable in spleens of NP group. This suggests that the targeting specificity of NPs in vivo also depends on the affinity of the linking antibody, and that signal density is positively correlated by target abundance. Fig. 8 is a graph a of nanoparticle targeting in experience versus IVIS results in vivo; B. as a result of fluorescent staining of mouse spleen tissue, FITC (green) signal was CD3eAb, Cy5.5 (red) signal was Cy5.5-NP, and blue signal was nuclei (Bar 25 μm).
Fourth, therapeutic study
NP reduction of DSA levels
PAS staining results (fig. 9A) showed no other significant damage to kidney tissue in the Iso group; in the Allo group, renal histopathology presented glomerulonephritis, tubular epithelial swelling, interstitial inflammation and interstitial edema, as typical acute ABMR phenotypes; mild interstitial inflammation and glomerulitis of renal tissue were present in the Tac group; the Tac-NP group has remarkable tubulitis and glomerulitis phenotypes; there was mild tubular epithelial cell shedding in the NP-Tac-CD3e group, but no ABMR pathological phenotype. Urinary protein, serum creatinine and urea nitrogen increased sharply in the Allo group at 24 hours, were significantly down-regulated in the Tac group, and were back-regulated in Tac-NP, with each index being the lowest among the allograft groups in the NP-Tac-CD3eAb group. The mice had 24 hours of proteinuria, serum creatinine, serum urea nitrogen significantly increased in the Allo group; the Tac group was significantly downregulated due to the effect of tacrolimus; all Tac-NP groups had some callbacks, which indicated that the drug release was retarded; all indices in NP-Tac-CD3eAb were minimal, lower than tacrolimus alone, and no significant difference was seen in Iso group (fig. 9B).
NP reduction of C4d deposition and pathological changes in transplanted Kidney tissue
Results of immunofluorescence co-localization to C4d and CD31 (fig. 10A) show: in the Iso group, there was only a weak C4d signal in peri-renal capillary vessels transplanted, which was associated with ischemia-reperfusion injury in the transplant; in the Allo group, there was strong C4d signal deposition in peri-transplanted renal tubular capillaries, suggesting a strong ABMR response; in the Tac group, C4d signal was significantly inhibited, indicating significant down-regulation of complement activation levels; the NP-Tac group shows a descending trend compared with the Allo group, but a high-intensity C4d signal can still be observed, which indicates that the NP-Tac group releases a small amount of tacrolimus and has the effect of reducing ABMR; the C4d signal in the NP-CD3e-Tac group is weak, which indicates that the complement activation level is maintained at an extremely low level, and the ABMR is well inhibited.
Serum DSA levels of mice were measured by flow cytometry 5 days after renal transplantation after skin graft sensitization (fig. 10B). The IgG and IgM in the Iso group are kept at low abundance level, and have no obvious difference with those in the non-transplanted group; IgM in the Allo group was significantly elevated compared to the Iso group; the use of tacrolimus reduced IgM in the Tac group to a level that was not significantly different from the Iso group; there was a slight decrease in IgM in the NP-TAC group, but still significantly higher than in the Iso and Tac groups; the NP-CD3-TAC group significantly down-regulated IgM levels, and did not differ significantly from the Tac group.
NP inhibition of spleen Effect Tfh cell activation and plasma cell production
Flow cytometry was used to detect the production of plasma cells in the spleen and the activation level of Tfh (fig. 11A, B), with the results showing: the proportion of plasma cells (CD138+) in the Allo group was significantly increased and the proportion of activated Tfh cells (CXCR5+/Bcl-6+) was also significantly increased compared to the Iso group; the proportion of these two populations of cells in the Tac group was significantly lower than in the Allo group (P < 0.001); the proportion of plasma cells and activated Tfh cells in the Tac-NP group is slightly reduced, but is still obviously higher than that in the Iso group; the plasma cell and Tfh cell population in the NP-CD3e-Tac group is further reduced compared with that in the Tac group, and better inhibition effect is presented. On the other hand, gross examination of spleen tissue (FIG. 11C) revealed that spleen tissue volume and weight were significantly increased in the Allo group compared to the Iso group, and in the Tac group and NP-CD3e-Tac group, spleen tissue volume and weight were similar to the Iso group, thus reflecting the high proliferation and mobilization of spleen lymphocytes in the course of ABMR pathology (Allo group) and significantly decreased by Takemus or targeted drug administration.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (3)

1.A novel nano slow-release drug-loaded material for inhibiting antibody-mediated rejection by a targeting immune germinal center is characterized by being prepared by the following steps:
(1) preparation of mesoporous silica nanospheres: firstly, 20-40mL of aqueous solution of CTAC with the weight percent of 25 and 0.18g of TEA are added into 36mL of water and stirred for 1 hour at the temperature of 60 ℃; then 20mL of 10 v/v% TEOS cyclohexene solution was added to the above mixture and reacted overnight in a 60 ℃ oil bath with magnetic stirring at 150rpm, the resulting product was collected by centrifugation and the collected product was dispersed in 0.6 wt% ammonium nitrate ethanol solution;
(2) synthesis of partially oxidized dextran: dissolving 1g of dextran having an average molecular weight of 9000-11000 in 4mL of water, adding 0.22g of sodium periodate to the above solution and stirring at room temperature for 5 hours to produce partially oxidized dextran; the partially oxidized dextran was further purified by dialysis against distilled water several times through a regenerated cellulose membrane MW 3500 Da; lyophilizing to obtain white, partially oxidized dextran powder;
(3) synthesis of partially oxidized acetylated dextran, AcDEX: 1.0g of partially oxidized dextran was added to 10ml of anhydrous DMSO,stirring until completely dissolved, adding 0.062mmol of pyridinium p-toluenesulfonate and 37mmol of 2-methoxypropene, and sealing in N2Under protection; after 3 hours, 1mL TEA was added to stop the reaction and 100mL distilled water was added to precipitate the modified partially oxidized dextran; the product was isolated by centrifugation at 10000rpm for 10 minutes and the resulting precipitate was washed with deionized water, followed by centrifugation and removal of the supernatant; obtaining the AcDEX by lyophilization;
(4) synthesis of spermine-modified Oxyacetylated dextran (SpAcDEX): 1.0g of partially oxidized AcDEX was dissolved in 10mL of anhydrous DMSO and mixed with 4.0g of spermine and reacted at 50 ℃ for 24 h; 2.0g of NaBH4Adding the mixture and reacting at room temperature for 18 hours; precipitating by adding 40mL of deionized water to the above solution, and separating by centrifuging at 10000rpm for 10 minutes; washing the obtained precipitate with deionized water for several times, and collecting SpAcDEX after freeze drying;
(5) preparation of SpAcDEX-encapsulated MSN (i.e., MSN @ SpAcDEX): 1.0 mg. multidot.mL-1MSN and 5 mg. mL-1SpAcDEX was mixed in ethanol as the internal phase of the microfluidics and 1% aqueous PVA was used as the external phase; connecting the two-phase solution to a polyethylene tube of a syringe, precipitating the MSN @ SpAcDEX obtained after mixing the two-phase solution, collecting in a 0.1% PVA solution, and stirring at 200 rpm; subsequently, it was centrifuged at 10000rpm for 5 minutes and washed several times with deionized water.
2. The novel nano slow-release drug-loaded material for the targeting of the immune germinal center to inhibit the antibody-mediated rejection reaction according to claim 1, which is characterized in that the preparation of the drug-loaded material further comprises the following steps:
adding 5-10mg of nanoparticle MSN @ SpAcDEX to 2ml of 1mM bis sulfosuccinate diimidate PBS reagent; the mixture was stirred with magnetic beads at room temperature for 6 h; after washing with PBS, 5-10mg/ml BS 3-bound nanoparticles were incubated with 0.15mg/ml anti-mouse CD3e antibody and stirred at 4 ℃ overnight; CD3eAb conjugated nanoparticles were washed with PBS, centrifuged at 10,000g for 5 minutes, and repeated three times.
3. Use of a drug-loaded material for the treatment of inflammation of a transplanted organ, wherein the material is a drug-loaded material according to any one of claims 1-2.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115737797A (en) * 2022-10-28 2023-03-07 山东大学 High-affinity Siglec-1 ligand-modified glucan nano vaccine and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100196280A1 (en) * 2006-06-08 2010-08-05 Katrin Claudia Fischer Functionalized solid polymer nanoparticles for diagnostic and therapeutic applications
CN111208223A (en) * 2020-01-13 2020-05-29 浙江大学 Metabolite combination for pre-operation early warning of transplanted kidney delayed recovery of donated recipient after cardiac death and screening method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100196280A1 (en) * 2006-06-08 2010-08-05 Katrin Claudia Fischer Functionalized solid polymer nanoparticles for diagnostic and therapeutic applications
CN111208223A (en) * 2020-01-13 2020-05-29 浙江大学 Metabolite combination for pre-operation early warning of transplanted kidney delayed recovery of donated recipient after cardiac death and screening method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JESSICA L. COHEN ET.AL.: "Acid-Degradable Cationic Dextran Particles for the Delivery of siRNA Therapeutics", 《BIOCONJUGATE CHEM.》, vol. 22, no. 6, pages 2 - 4 *
SHRESTHA, BRAJESH: "Synthesis of CuS@MSN@SpAcDex for Tacrolimus delivery to treat End Stage Renal Disease", HTTPS://URN.FI/URN:NBN:FI-FE2021070541161, pages 2 - 3 *
THOMAS FEHR ET.AL.: "Antibody-mediated kidney allograft rejection: therapeutic options and their experimental rationale", 《TRANSPLANT INTERNATIONAL》, vol. 25, pages 628 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115737797A (en) * 2022-10-28 2023-03-07 山东大学 High-affinity Siglec-1 ligand-modified glucan nano vaccine and preparation method and application thereof

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