CN114939171B

CN114939171B - Nanometer medicine carrying system and preparation method and application thereof

Info

Publication number: CN114939171B
Application number: CN202210465094.4A
Authority: CN
Inventors: 梅恒; 胡豫; 陈钊钊; 唐宇翔
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2023-05-30
Anticipated expiration: 2042-04-29
Also published as: CN114939171A

Abstract

The invention belongs to the technical field of biochemistry, and mainly discloses a nano medicine carrying system and a preparation method and application thereof. Application of HES-CH compound shown in formula I in preparing anti-inflammatory targeted medicine. The preparation method of the inflammation endothelial active targeting nanoparticle comprises the steps of obtaining HES-CH; preparing TPCA-1@HCNPs: slowly adding an oil phase solution of TPCA-1 into the HES-CH solution, performing rotary evaporation, and extracting TPCA-1@HCNPs from supernatant; mAb-TPCA-1@HCNPs were prepared: mixing the TPCA-1@HCNPs solution with DSC, dialyzing, adding PECAM-1 4G6mAb, and reacting to obtain mAb-TPCA-1@HCNPs. The invention provides a preparation method of novel drug-loaded nanoparticles and provides novel anti-inflammatory drugs. Meanwhile, a targeted treatment mode is provided for the lung diseases, such as targeted treatment for solving sepsis lung injury.

Description

Nanometer medicine carrying system and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a nano medicine carrying system and a preparation method and application thereof.

Background

The nano carrier is an important part formed by a nano medicine carrying system, the nano carrier carries medicines to obtain a medicine preparation of between 0 and 1000 nanometers, and the accumulation of the medicines at tumor positions can be increased by enhancing the retention and permeation effects, so that the effects of synergism and toxicity reduction are realized on the medicines; meanwhile, the blood stability of the medicine is improved, the medicine is prevented from being excreted by the kidney too fast, and the half life of the loaded medicine is prolonged, so that the bioavailability of the medicine is improved.

There are several reports of anti-inflammatory targeted delivery systems for animal models, such as using hyaluronic acid as an anti-inflammatory targeted delivery system, to improve atherosclerosis by using hyaluronic acid nanoparticles to investigate atherosclerosis-related inflammation. In another example, PLGA is encapsulated with erythrocyte and platelet membranes as an anti-inflammatory targeted delivery system. It follows that most of the anti-inflammatory targeted delivery systems currently use synthetic materials or require chemical modification, and the process is relatively complex, and biochemical reactions and degradation products involved in the body are often not well-defined, which are challenges for clinical transformation. Thus, the potential risk to human health is greater. Thus, when anti-inflammatory targeting systems modified or associated with chemicals are used in humans, the safety of their use is also a major concern.

Acute lung injury (acute lung injury, ALI) is a systemic inflammatory response syndrome characterized by refractory hypoxia and respiratory distress caused by various intrapulmonary pathogenic factors. Common causes of acute lung injury include sepsis, trauma, shock, etc. Sepsis (sepis) is its most common cause. Because of the complex etiology and pathogenesis of the acute lung injury, the death rate is high, and no exact and effective medicine for controlling the death rate is clinically available at present. Common treatments are mechanical ventilation, β2 receptor agonists, anti-inflammatory and antioxidant, anti-coagulant thrombolytics, surfactants, and the like. Pneumonia is caused by the invasion of pathogens into the lung parenchyma and overgrowth in the lung parenchyma beyond the host's defenses, resulting in the appearance of exudates in the alveolar space, which causes pneumonia. The occurrence and severity of pneumonia is primarily determined by a balance between pathogen and host factors.

Disclosure of Invention

Aiming at the problems, the invention provides a nano drug-loading system, a preparation method and application thereof, and mainly aims to solve the problems of deficiency of the existing anti-inflammatory targeted drugs and deficiency of some drugs for treating pulmonary diseases.

In order to solve the problems, the invention adopts the following technical scheme:

application of HES-CH compound in preparing anti-inflammatory targeted medicine, wherein HES-CH has a structure shown in formula I,

wherein n is between 1 and 100.

In some embodiments, the use is the use of a HES-CH compound in the manufacture of a medicament for treating pulmonary inflammation or injury.

In some aspects, the pulmonary inflammation or injury is sepsis pulmonary inflammation or sepsis pulmonary injury.

A method for preparing active targeting nano-particles of inflammatory endothelium,

acquiring HES-CH;

preparing TPCA-1@HCNPs:

slowly adding an oil phase solution of TPCA-1 into the HES-CH solution, performing rotary evaporation, and extracting TPCA-1@HCNPs from supernatant;

mAb-TPCA-1@HCNPs were prepared:

mixing the TPCA-1@HCNPs solution with DSC, dialyzing, adding PECAM-14G6 mAb, and reacting to obtain mAb-TPCA-1@HCNPs.

In some embodiments, mAb-TPCA-1@HCNPs are prepared: adding sufficient DSC into the TPCA-1@HCNPs aqueous solution, stirring overnight, then dialyzing, adding PECAM-14G6 mAb, and stirring at room temperature for reaction to obtain mAb-TPCA-1@HCNPs nanoparticle aqueous solution.

Use of mAb-TPCA-1@hcnps in the preparation of in vitro HUVEC, raw264.7 cell activity inhibitor non-diagnostic therapies.

The application of mAb-TPCA-1@HCNPs in preparing anti-inflammatory drugs.

The application of mAb-TPCA-1@HCNPs in preparing a lung targeting therapeutic drug.

Use of mAb-TPCA-1@hcnps in the preparation of a medicament for treating pulmonary inflammation or injury.

In some embodiments, the mAb-TPCA-1@hcnps are at least shown to reduce infiltration of T cells, macrophages, and active oxygen production in pulmonary inflammation or injury.

A preparation method of HES-CH compound shown in a formula I,

fully mixing HES and enough bromopropylamine hydrobromic acid, adding into NaOH solution, heating to react under constant-temperature oil bath stirring environment, adding concentrated hydrochloric acid, regulating pH to neutrality, and removing impurities to obtain HES-NH ₂ ；

Dispersing CH in a pyridine solution system, adding succinic anhydride, heating the mixture for reaction, then performing rotary evaporation, and extracting a precipitate to obtain CH-COOH;

HES-NH ₂ And (3) dissolving the polymer and CH-COOH in a DMSO solution together, then adding EDCI and HOBt, heating to react, thereby obtaining HES-CH, removing impurities from the obtained polymer, and purifying to obtain HES-CH.

In some embodiments, HES-NH is prepared ₂ In the method, the temperature rise reaction temperature is 45 ℃, and the mass concentration of concentrated hydrochloric acid is 37%;

in the preparation of CH-COOH, the temperature of the reaction is raised to 70 ℃, the precipitate is extracted by alcohol, and succinic anhydride and CH are added in equal quantity;

in the preparation of HES-CH, the temperature of the reaction is raised to 80 ℃, and EDCI and HOBt are added in equal amounts.

In some embodiments, HES-NH ₂ Is synthesized by the following steps: fully mixing HES and a sufficient amount of bromopropylamine hydrobromic acid, adding into a NaOH solution, reacting at 45 ℃ under the condition of constant-temperature oil bath stirring, adding concentrated hydrochloric acid, titrating and adjusting the pH value to 7, fully dialyzing by a dialysis bag to remove impurities, and finally obtaining a purified product, namely HES-NH ₂ ；

Synthesis of CH-COOH: dispersing CH in a pyridine system, adding succinic anhydride with equal amount, reacting the mixture in a constant-temperature oil bath stirring environment at 70 ℃, then performing rotary evaporation, extracting the precipitate with alcohol, performing rotary evaporation again to remove the alcohol, thus obtaining CH-COOH, and finally performing freeze-drying to obtain CH-COOH powder;

synthesis of HES-CH: HES-NH ₂ Dissolving the HES-CH and CH-COOH powder in DMSO solution, adding EDCI and HOBt, reacting at 80deg.C under constant temperature under stirring, dialyzing the obtained polymer in dialysis bag overnight, and lyophilizing the dialyzed solution to obtain HES-CH powder.

The beneficial effects of the invention are as follows:

the nanoparticle has stable structure and good biocompatibility. Provides a new preparation method of drug-loaded nano-particles and provides a new anti-inflammatory drug. Meanwhile, a targeted treatment mode is provided for the lung diseases, such as targeted treatment for solving sepsis lung injury. The invention can obviously improve the lung injury degree of mice with sepsis lung injury, inflammatory cell infiltration and active oxygen generation.

Drawings

FIG. 1A is a schematic diagram of a synthetic route for HES-CH materials;

FIG. 1B is HES-NH ₂ A chemical structural formula of (a);

FIG. 1C is a chemical formula of CH-COOH;

FIG. 1D is a chemical formula of HES-CH;

FIG. 1E is a schematic representation of the preparation of mAb-TPCA-1@HCNPs;

FIG. 1F is a chemical formula of TPCA-1;

FIG. 2 is a graph of characterization results of mAb-TPCA-1@HCNPs;

FIG. 3 shows drug release and blood compatibility of mAb-TPCA-1@HCNPs, (A) drug release of free TPCA-1 and mAb-TPCA-1@HCNPs, (B) ordinary photographs after 2h centrifugation of gradient concentration mAb-TPCA-1@HCNPs co-incubated with red blood cell suspensions, (C) erythrocyte hemolysis rate curves, (D) photographs of red blood cells under ordinary light microscope (scale: 50 μm);

FIG. 4 shows toxicity evaluation of mAb-TPCA-1@HCNPs to HUVEC and Raw264.7 by (A) and (B) mAb-TPCA- @ HCNPs, respectively;

FIG. 5 is a graph showing the results of in vitro intervention, (A) evaluation of antioxidant capacity, (B) evaluation of inhibition of macrophage M1 polarization effect, (C) evaluation of inhibition of macrophage NO production, and (D) to (F) are statistical analyses of (A) to (C), respectively;

FIG. 6 is a graph showing in vitro anti-inflammatory effects of (A) and (B) respectively, nano-drugs reducing secretion of IL-6 and TNF-alpha by macrophages;

FIG. 7 is a confocal laser image of binding of (A) - (B) mAb-C6@HCNPs to HUVEC and MAEC after inflammatory stimuli, respectively, (10 μm and 20 μm on panels A and B, respectively) nanoparticles binding to inflammatory endothelial cells and in macrophages, (C) mAb-TPCA- @ HCNPs in time-dependent cellular monitoring and co-localization analysis of macrophages (20 μm on panels);

FIG. 8 is a quantitative analysis of dose-dependent entry and flow cytometry, with (A) dose-dependent entry monitoring (scale 20 μm) and (B) - (C) flow cytometry for quantitative detection of time-dependent and dose-dependent entry, respectively;

FIG. 9 is a pharmacokinetic study of in vivo pharmacokinetic behavior of DIR@HCNPs and mAb-DIR@HCNPs for (A) and (C), respectively, and of fluorescence quantification of (A) and (C), respectively;

FIG. 10 shows in vivo lung targeting studies of mAb-TPCA-1@HCNPs in sepsis ALI model, (A) in vivo primary organ biodistribution of different treatment groups, (B) quantitative and statistical analysis of Panel A, (C) lung tissue section immunofluorescence (scale 200 μm);

FIGS. 11-12 are drug efficacy studies of nanomedicines, (A) staining of H & E in lung tissue sections of different treatment groups, (B) to (C) flow cytometry loop gate strategy of T cells and macrophages respectively, (D) to (E) flow charts of T cells and macrophages of mouse lung tissues of different groups respectively, (F) scoring of lung injury degree of different treatment groups, (G) to (H) statistical analysis of graphs (D) to (E) respectively, (I) staining fluorescent charts of active oxygen DHE in lung tissue sections of different treatment groups (scale 200 μm);

FIG. 13 shows in vivo biosafety assessment, (A) change in body weight within 14 days after nanoparticle injection in normal mice, (B) blood routine analysis, (C) change in biochemical index of blood, (D) H & E staining of major organs of mice treated differently (scale: 200 μm).

Detailed Description

The first section introduces the new use of HES-CH:

firstly, the compound HES-CH is applied to the preparation of anti-inflammatory targeted drugs, wherein the HES-CH has a structure shown in a formula I (shown in figure 1D), and n is between 1 and 100.

Wherein other compounds are modified on the basis of formula I by simple substitution groups, but should equally be within the scope of the invention without affecting the main properties. It can be used for preparing medicines for targeting inflammatory endothelium. In some aspects, the anti-inflammatory targeting drug is pulmonary targeting.

HES-CH (amphiphilic hydroxyethyl starch coupled cholesterol polymer) may be prepared as disclosed in CN113105566B, but other preparable methods may also be used.

HES-CH is used as a carrier of targeted drugs in drugs and is used for loading the drugs. HES-CH has better lung focus targeting; especially has better endothelial targeting capability.

Secondly, the HES-CH is applied to the preparation of medicines for treating lung inflammation or injury.

Pneumonia is an infectious inflammation of alveoli, distal airways and pulmonary interstitium, which can be caused by infection with bacteria, viruses and other pathogens, among which bacterial and viral pneumonia are most common; and may also be induced by other conditions.

Among these, in some cases, lung inflammation or injury is primarily sepsis-induced lung disease. The lung inflammation or injury is sepsis lung inflammation or sepsis lung injury. The human body has lower immunity under the state of sepsis symptom, and is easy to cause lung infection. Sepsis lung injury is mainly sepsis-induced acute lung injury.

The second section describes the preparation of mAb-TPCA-1@HCNPs nanoparticles:

the preparation method of the nanoparticle comprises the following steps:

preparing TPCA-1@HCNPs: slowly adding an oil phase solution of TPCA-1 into the HES-CH solution, performing rotary evaporation, and extracting TPCA-1@HCNPs from supernatant;

mAb-TPCA-1@HCNPs were prepared: mixing the TPCA-1@HCNPs solution with DSC, dialyzing, adding PECAM-14G6 mAb, and reacting to obtain mAb-TPCA-1@HCNPs.

Wherein, in some examples, mAb-TPCA-1@hcnps are prepared: adding sufficient DSC into the TPCA-1@HCNPs aqueous solution, stirring overnight, then dialyzing, adding PECAM-14G6 mAb, and stirring at room temperature for reaction to obtain mAb-TPCA-1@HCNPs nanoparticle aqueous solution; preferably, the aqueous solution of TPCA-1@HCNPs has a concentration of 1mg/ml.

Specific reactant addition ratios can be seen in the following examples, and the specific ratios in this section are not excessively limited without affecting the final results.

The third section describes the novel use of mAb-TPCA-1@HCNPs:

mAb-TPCA-1@HCNPs nanoparticle. Which is the nanoparticle prepared in the second part. The results of analytical characterization of the nanoparticles obtained in the second fraction also enabled the characterization of mAb-TPCA-1@HCNPs of this fraction. But is not limited to, it must be prepared using the foregoing procedure.

Firstly, the mAb-TPCA-1@HCNPs are applied to the preparation of in vitro HUVEC and Raw264.7 cell activity inhibitor non-diagnostic treatment. It is mainly directed to the use in some agents for non-therapeutic purposes for modulating cellular activity.

Secondly, the mAb-TPCA-1@HCNPs are applied to the preparation of anti-inflammatory medicaments. Since mAb-TPCA-1@HCNPs have a targeted modulating effect on the endothelium after inflammatory stimuli, they can be used in anti-inflammatory drugs. Pulmonary inflammation is one of these. mAb-TPCA-1@HCNPs also have corresponding anti-inflammatory effects in vitro experiments, so that the mAb-TPCA-1@HCNPs can be used in anti-inflammatory drugs for other diseases.

Thirdly, the mAb-TPCA-1@HCNPs are applied to preparing a lung targeting therapeutic drug.

Wherein, the mAb-TPCA-1@HCNPs is applied to the preparation of medicines for treating lung inflammation or injury in medicines for targeted treatment of lung focus.

The nanoparticle has the active targeting capability of inflammatory endothelium, and can perform targeted treatment on endothelial cells. It is capable of effectively binding endothelial cells after inflammatory stimuli.

More particularly, in some application scenarios, the lung inflammation or injury is sepsis lung inflammation or sepsis lung injury; and/or, mAb-TPCA-1@HCNPs are at least effective in reducing infiltration of T cells and macrophages in lung tissue and reducing one of the production of reactive oxygen species in lung inflammation or injury.

The amphiphilic polymer synthesized by coupling hydroxyethyl starch and cholesterol is used as a nano-carrier, hydrophobic anti-inflammatory drug TPCA-1 is entrapped to form nano particles by self-assembly, and a 4G6 monoclonal antibody targeting the breaking point of the IgD6 region of the extracellular section of PECAM-1 is modified on the surface to construct mAb-TPCA-1@HCNPs.

mAb-TPCA-1@HCNPs may be prepared by the second part of the process or by other processes.

The fourth section is further described in connection with specific research projects:

scheme design

(1) The synthesis of amphiphilic hydroxyethyl starch coupled cholesterol polymerization (HES-CH) was characterized and verified by NMR, FTIR, mass spectrometry.

(2) TPCA-1@HCNPs were prepared by Pickering emulsion solvent evaporation and mAb-TPCA-1@HCNPs were characterized by TEM, DLS, zeta potential, XPS.

(3) Detecting the drug loading rate and the encapsulation efficiency of mAb-TPCA-1@HCNPs by a DMSO membrane rupture method; the drug release behavior was monitored by dialysis and the haemocompatibility was tested by erythrocyte hemolysis experiments.

(4) The mice with sepsis ALI model were established by intraperitoneal injection of LPS.

(5) In vitro experiments: detecting cytotoxicity of the nanoparticle by a CCK8 method; detecting the in vitro antioxidant capacity and the intervention effect on macrophage M1 polarization and NO production by using flow cytometry; ELISA is used for detecting the level of the nano-drug interfering with the secretion of TNF-alpha and IL-6 by macrophages stimulated by LPS; the endothelial targeting was observed by laser confocal microscopy and the cell entry behavior at macrophages was monitored, while quantified by flow cytometry.

(6) In vivo pharmacokinetic studies: mAb-DIR@HCNPs nanoparticles are constructed, injected into normal mice, blood is taken from the back of the orbit at a plurality of time points, and the fluorescence intensity of the blood is detected and quantified through a living animal imaging system.

(7) In vivo biodistribution study: mAb-DIR@HCNPs are injected into a model mouse body through tail veins, main tissues are taken out after 12 hours, and the fluorescence intensity is measured by a small animal living body imaging system; in addition, mAb-C6@HCNPs nanoparticles were constructed, injected into model mice, and distributed in the lungs by immunofluorescence.

(8) In vivo efficacy study: mAb-TPCA-1@HCNPs are injected into a model mouse, infiltration degree of lung tissue T cells and macrophages is measured through a flow cytometer, and damage degree is observed through ROS tissue fluorescence and H & E staining.

(9) Biological safety assessment: mAb-TPCA-1@HCNPs are injected into a normal mouse, the weight change of the mouse is monitored within 14 days, indexes such as blood convention, liver function, kidney function and the like are detected by taking peripheral blood after two weeks, and finally, whether the main organs are subjected to morphological change and damage is observed through H & E staining.

The results illustrate:

(1) The amphiphilic HES-CH polymer has good self-assembly behavior, can encapsulate hydrophobic anti-inflammatory drugs and is subjected to surface modification of a 4G6 antibody, and the prepared mAb-TPCA-1@HCNPs has good stability, long circulation effect, low cytotoxicity and good biosafety.

(2) mAb-TPCA-1@HCNPs can effectively bind to the extracellular segment breakpoint of PECAM-1, and have excellent inflammatory endothelium and inflammatory lung targeting in vitro and in vivo.

(3) mAb-TPCA-1@HCNPs have excellent anti-inflammatory capability in vitro and in vivo, and can remarkably improve the injury and inflammatory cell infiltration of the lung of the sepsis ALI model.

Experimental details

Preparation of nanoparticles

Synthesis and characterization of HES-CH：

The material synthesis route is schematically shown in FIG. 1A.

The specific experimental steps of the polymer chemical synthesis are as follows:

(1)HES-NH ₂ is synthesized by the following steps: the chemical structural formula is shown in figure 1B.

(2) Synthesis of CH-COOH: the chemical structural formula is shown in figure 1C.

(3) Synthesis of HES-CH: the chemical structural formula (formula I) is shown in figure 1D, wherein n is between 1 and 100.

(4)HES-NH ₂ Characterization verification of CH-COOH, HES-CH: (1) nuclear magnetic resonance spectroscopy sample preparation: HES, HES-NH ₂ HES-CH powder is respectively dissolved in deuterated DMSO, the final concentration is 1mg/ml, and the hydrogen spectrum is scanned 256 times; (2) FTIR preparation: the sample is firstly placed in a baking oven for full baking, and a certain amount of HES, CH-COOH and HES-NH are taken ₂ The HES-CH was placed in a milling bowl with twice the amount of potassium bromide as the sample, respectively, and ground sufficiently to a homogeneously mixed powder. Uniformly spreading the powder on a tabletting groove, pressing for 10s at 200kPa by a tablet press to obtain uniform slices, translating the uniform slices to a sample hole by a blade, and placing the uniform slices in a sample cell to obtain the final product And (5) starting detection.

(5) Mass spectrometer: the well lyophilized CH-COOH samples were directly checked on the machine.

Preparation and characterization of mAb-TPCA-1@HCNPs

The nanoparticle assembly and preparation schematic is shown in fig. 1E.

1) Assembly of TPCA-1@HCNPs: dissolving 50mg of HES-CH in 50ml of deionized water, ultrasonically dissolving for 10min (with the frequency of 50Hz, exceeding 2s and stopping for 1 s) by using an ultrasonic crusher to obtain 1mg/ml of HES-CH aqueous solution, slowly dropwise adding 5ml of oil phase solution of TPCA-1 (1 mg/ml dissolved in chloroform) into the solution, simultaneously ultrasonically processing for 5min by using the ultrasonic crusher to obtain milky uniform oil/water mixed solution, fully steaming by using a rotary evaporator at 45 ℃ to remove chloroform, centrifuging (5000 rpm,10 min) the obtained aqueous solution of TPCA-1@HCNPs, discarding precipitation, dialyzing supernatant overnight to remove non-entrapped free TPCA-1, and fully freeze-drying to obtain TPCA-1@HCNPs nanoparticle powder. Likewise, HES-CH NPs (HCNPs), DIR@HCNPs and C6@HCNPs nanoparticles, which were not entrapped with the drug, were prepared as described above. Wherein the chemical structural formula of the hydrophobic TPCA-1 is shown in figure 1F.

2) Preparation of mAb-TPCA-1@HCNPs: adding sufficient DSC into 1mg/ml of TPCA-1@HCNPs aqueous solution at room temperature, stirring overnight, dialyzing for 24 hours, adding 25ug of PECAM-14G6 mAb, and stirring at room temperature for reaction for 3 hours to finally obtain mAb-TPCA-1@HCNPs nanoparticle aqueous solution; similarly, mAb-DIR@HCNPs and mAb-C6@HCNPs nanoparticles were prepared as described above;

3) Characterization experiments: detecting particle size distribution and Zeta potential of the TPCA-1@HCNPs and the mAb-TPCA-1@HCNPs aqueous solution by a laser particle size analyzer, respectively placing the nanoparticle dispersion liquid in PBS, 10% FBS and RMPI1640 for 7 days at 37 ℃, and monitoring particle size change of the mAb-TPCA-1@HCNPs every day; a drop of the solution of TPCA-1@HCNPs, mAb-TPCA-1@HCNPs was dropped onto the copper mesh and air dried at room temperature, followed by counterstaining with a 0.2% (w/w) phosphotungstic acid solution for 60s, and the morphology was observed by TEM.

4) Drug Load (DLC) and Encapsulation Efficiency (EE) determination: a DMSO solution of a gradient concentration of TPCA-1 was prepared at a concentration of 125. Mu.g/ml, 100. Mu.g/ml, 50. Mu.g/ml, 25. Mu.g/ml, 12.5. Mu.g/ml, and standard curve was obtained by HPLC using a DMSO solution blank. Then, the TPCA-1@HCNPs is broken by DMSO (standing for 30min to fully break the membrane, wherein the concentration of the nano-drug is 100 mug/ml), and the obtained sample is subjected to HPLC detection and substituted into standard curve, so that the concentration and the quality of the TPCA-1 are obtained.

DLC＝(Wt(TPCA-1))/(Wt(TPCA-1@HCNPs))×100％，

EE＝(Wt(TPCA-1))/(Wt(Total TPCA-1))×100％，

Wherein Wt (TPCA-1) is the mass of TPCA-1 in TPCA-1@HCNPs, wt (TPCA-1@HCNPs) is the mass of TPCA-1@HCNPs nanoparticles, and Wt (Total TPCA-1) is the Total mass of the TPCA-1 charged when preparing the nanoparticles.

Drug release and hemocompatibility of mAb-TPCA-1@HCNPs nanoparticles

1) Drug release: 3ml of mAb-TPCA-1@HCNPs solution (concentration is 1 mg/ml) is placed in a dialysis bag with molecular retention of 10000Da, the control group is equal volume of free TPCA-1 solution, the control group is respectively sealed and put into a blue mouth bottle containing 5ml of PBS, PBS is used as a release medium, the solution is incubated for 24 hours in a desktop constant temperature shaker with the speed of 300rpm/min at 37 ℃, 1ml of release medium is respectively taken at each time point of 2 hours, 4 hours, 8 hours, 12 hours and 24 hours, and equal volume of PBS is added into the solution, and the concentration of TPCA-1 is detected by HPLC (high performance liquid chromatography).

2) Haemocompatibility: 1ml of PBS and 1% Triton X-100 solution were prepared in advance, and a series of equal volume (1 ml) gradient concentrations (5. Mu.g/ml, 10. Mu.g/ml, 25. Mu.g/ml, 50. Mu.g/ml, 100. Mu.g/ml, 250. Mu.g/ml, 500. Mu.g/ml) of mAb-TPCA-1@HCNPs solution were prepared using PBS as a solvent.

Taking peripheral blood of a Bal/bc mouse by an eyeball picking method, collecting the peripheral blood by an anticoagulant tube, slowly cleaning the peripheral blood by PBS in an ultra-clean workbench, performing gentle action, then centrifuging at a low speed of 1500rpm/min for 10min until the supernatant is clear and transparent, sucking erythrocyte sediment, and slightly oscillating to obtain a uniformly dispersed erythrocyte suspension. Adding an equal volume of erythrocyte suspension into the prepared solution, sufficiently oscillating for 2 hours in a desk-top constant temperature oscillator with the temperature of 37 ℃ and the rotating speed of 300rpm/min, taking out a sample, centrifuging (1500 rpm,10 min), photographing, finally sucking supernatant, detecting the absorbance OD value of hemoglobin by an enzyme-labeling instrument, taking a 1% Triton X-100 solution group as a positive control and a PBS group as a negative control, setting three compound holes for each sample, detecting the wavelength to be 541nm, and calculating the erythrocyte Hemolysis ratio (HR, hemolysisis ratio) according to the OD value, wherein the calculation formula is as follows:

Wherein ODt is the OD of each experimental group, ODn is the OD of a PBS negative control group, and ODp is the OD of a 1% Triton X-100 solution positive control group.

In vitro cell experiments

1) Determination of cytotoxicity by CCK8 method: setting each gradient concentration group of the nano material: 250. 200, 150, 100, 50, 0 μg/ml, with zeroing holes set, 5 complex holes per group.

(1) Taking log-phase growing cells, conventionally digesting the cells, centrifuging the cells, and collecting precipitates;

(2) re-suspending cells by using a complete cell culture medium, blowing and uniformly mixing to prepare single cell suspension;

(3) cell density was adjusted to 5X 10 ⁴ /ml；

(4) Planting 1X 104 cells in each hole, and placing the cells in a 37 ℃ cell incubator for incubation for 24 hours;

(5) after cell adhesion, adding gradient concentration mAb-TPCA-1@HCNPs drug (taking complete cell culture medium as solvent) into each hole;

(6) after incubation in a 37℃cell incubator for 24h, 10 μl of CCK8 solution was added per well;

(7) after incubating for 1-4 hours in a cell incubator at 37 ℃, selecting 450nm wavelength and measuring an OD value by an enzyme-labeling instrument;

(8) results analysis, cell relative activity:

Relative Cell viability＝(ODt-ODb)/(ODc-ODb)×100％

wherein ODt is the OD value of each gradient concentration well, ODb is the OD value of the zeroing well, and ODc is the OD value of the blank group.

2) Evaluation of anti-inflammatory ability in vitro: setting groups: blank, LPS, LPS+HCNPs, LPS+TPCA-1, LPS+TPCA-1@HCNPs, LPS+mAb-TPCA-1@HCNPs. The log phase growth raw264.7 cell line was taken and seeded at 2 x 105/well in six well plates with three duplicate wells per group. Incubate overnight, until it adheres. The cell culture supernatant was discarded the next time, rinsed with PBS, and then pre-stimulated with 100ng/ml LPS for 2h, without any treatment in the blank. Thereafter, each group was added with HCNPs, TPCA-1 (0.02. Mu.g/ml), TPCA-1@HCNPs and mAb-TPCA-1@HCNPs, which were equivalent to the drug loading of 0.02. Mu.g/ml TPCA-1, and PBS group was rinsed without any drug. After 24h incubation in a 37℃cell incubator, the cell culture supernatant was centrifuged at 1000 Xg for 10min to remove the precipitate, and the supernatant was assayed for IL-6 and IL-1. Beta. By ELISA in the same manner as 6-1).

3) Evaluation of antioxidant ability under in vitro inflammatory stimulus: grouping was set, blank, LPS, LPS+HCNPs, LPS+TPCA-1, LPS+TPCA-1@HCNPs, LPS+mAb-TPCA-1@HCNPs.

(1) Log phase growing cells were taken and seeded at 2 x 105/well in six well plates with three wells per group. Incubate overnight in cell culture incubator at 37 ℃. The following day was pre-stimulated with 100ng/ml LPS for 2h without any treatment in the blank group. Adding HCNPs, TPCA-1, TPCA-1@HCNPs, mAb-TPCA-1@HCNPs and PBS into each group, washing, adding a complete cell culture medium, and adding no medicine;

(2) DCFH-DA (ROS fluorescent probe) was diluted to a final concentration of 10. Mu. Mol/L. After the cells are incubated for 24 hours in a 37 ℃ cell incubator, cell culture supernatant is discarded, then probes are loaded in situ in each hole, 1ml of diluted DCFH-DA solution is added, and the cells are incubated for 20 minutes in the 37 ℃ cell incubator in a dark place;

(3) in a light-shielding environment, discarding the probe diluent, washing the cells for 3 times by using PBS (phosphate buffer solution) to sufficiently remove DCFH-DA probes which are dissociated outside the cells;

(4) the cells were collected, gently swirled and mixed to form a single cell suspension, and the flow cytometer was on-line and detected with FITC channels.

4) Evaluation of macrophage NO production intervention ability under in vitro inflammatory stimulus: grouping was set, blank, LPS, LPS+HCNPs, LPS+TPCA-1, LPS+TPCA-1@HCNPs, LPS+mAb-TPCA-1@HCNPs.

(1) Log phase growing cells were taken and seeded at 2 x 105/well in six well plates in a 37 ℃ cell incubator overnight. The following day was pre-stimulated with 100ng/ml LPS for 2h without any treatment in the blank group. Adding HCNPs, TPCA-1, TPCA-1@HCNPs, mAb-TPCA-1@HCNPs and PBS into each group, washing, adding a complete cell culture medium, and adding no medicine;

(2) the NO fluorescent probe DAF-FM DA was diluted with the diluent at a final concentration of 10. Mu. Mol/L at 1:1000. After the cells are incubated for 24 hours in a 37 ℃ cell incubator, cell culture supernatant is discarded, then probes are loaded in situ in each hole, 1ml of diluted DAF-FM DA solution is added, and the cells are incubated for 20 minutes in the 37 ℃ cell incubator in a dark place; (3) in a light-shielding environment, discarding the probe diluent, washing the cells for 3 times by using PBS (phosphate buffer solution) to sufficiently remove the DAF-FM DA probe which is dissociated outside the cells;

5) Macrophage M1 polarization intervention ability evaluation under in vitro inflammatory stimulus: setting groups: blank, LPS, LPS+HCNPs, LPS+TPCA-1, LPS+TPCA-1@HCNPs, LPS+mAb-TPCA-1@HCNPs.

(1) Cells grown in log phase were grown in six well plates at 2X 105/well and incubated overnight in a 37℃cell incubator until the cells attached. The following day was pre-stimulated with LPS for 2h (LPS concentration 100 ng/ml), the blank group was not treated at all. Adding HCNPs, TPCA-1, TPCA-1@HCNPs, mAb-TPCA-1@HCNPs and PBS into each group, washing, adding a complete cell culture medium, and adding no medicine;

(2) After 24h, cells were collected with flow tubes, spun down (800 rpm,10 min) after PBS rinse and resuspended as single cell suspension, 1 μl FcR blocker was added per tube followed by incubation in a refrigerator at 4 ℃ for 10min;

(3) mu.l of Mouse PE-Anti-CD80 streaming antibody is added to each tube, and incubated for 30min at room temperature in a dark place;

(4) after washing 3 times with PBS, the single cell suspension obtained by re-suspending after centrifugation was put on a flow cytometer and detected by a PE channel.

6) And (3) evaluating targeting ability of the nano drug carrying system and performing cell entry monitoring:

(1) targeting ability evaluation: setting groups: blank and LPS groups. Cell lines of HUVEC, MAEC and Raw264.7 growing in log phase were taken and planted in confocal dishes, 1×105 cells were planted in each dish, and incubated overnight in a 37℃cell incubator. The cell culture supernatant was discarded the next time, the LPS-stimulated group was stimulated with 100ng/ml LPS for 24 hours, while the blank group was not treated. After 24h, the supernatant was discarded and washed 3 times with PBS followed by addition of 20 μg/ml mAb-c6@hcnps nanoparticles, incubation for 1h followed by three washes with PBS to remove free nanomaterials, followed by fixation with paraformaldehyde (15 min), removal of paraformaldehyde and addition of 1ml DAPI staining solution for 15min, followed by removal of DAPI, rinsing 3 times with PBS, followed by observation by a rotating disc laser confocal microscope;

(2) Monitoring cell entering behavior: raw264.7 was seeded into petri dishes at 1X 105 cells/dish, stimulated with 100ng/ml LPS for 24h after cell attachment, and mAb-C6@HCNPs solution was added after supernatant was discarded.

Cell entry test for monitoring confocal laser: a. for dose-dependent cell entry monitoring experiments, nano-drugs at gradient concentrations: 100. Mu.g/ml, 50. Mu.g/ml, 20. Mu.g/ml, 10. Mu.g/ml, 5. Mu.g/ml, 1. Mu.g/ml, respectively, were co-incubated with cells in a confocal dish for 1h, followed by removal of mAb-C6@HCNPs solution, rinsing 3 times with PBS, fixation with 4% paraformaldehyde for 15min, addition of DAPI staining solution for 15min, rinsing 3 times with PBS, and finally observation by a rotary disc laser confocal microscope; b. for time-dependent cell entry monitoring experiments, cells were co-incubated with 1. Mu.g/ml mAb-C6@HCNP solution in confocal dishes for 24h, 12h, 8h, 4h, 2h, 0.5h, respectively. The Lyso-Tracker Red lysosome Red fluorescent probe was diluted with cell culture medium according to 1:1:3:3:3:3 to give a Lyso-Tracker Red working solution, and pre-warmed in a 37 ℃ cell incubator. After the incubation at each time point is finished, removing the cell culture medium, adding a Lyso-Tracker Red working solution, then placing the cells in a 37 ℃ cell incubator for incubation for 30-120 min, removing the working solution, rinsing 3 times with PBS, fixing for 15min with 4% paraformaldehyde, adding a DAPI staining solution for dying the nuclei for 15min, rinsing 3 times with PBS, and finally observing the cells by a turntable laser confocal microscope And (5) inspecting.

For flow cytometry detection of cell entry experiments: a. for dose-dependent entry detection, the nanomedicine at gradient concentrations: 100. Mu.g/ml, 50. Mu.g/ml, 20. Mu.g/ml, 10. Mu.g/ml, 5. Mu.g/ml, 1. Mu.g/ml, respectively, were incubated with cells in six well plates for 1h, followed by removal of mAb-C6@HCNPs solution, rinsing 3 times with PBS, loading onto a flow cytometer, detection by FITC channels; b. for time-dependent cell entry monitoring experiments, cells were co-incubated with 1. Mu.g/ml mAb-C6@HCNP solution in confocal dishes for 24h, 12h, 8h, 4h, 2h, 0.5h, respectively. After incubation at each time point, the cell culture medium was removed, rinsed 3 times with PBS, and the flow cytometer was turned on and detected by FITC channels. In vivo pharmacokinetic studies: setting experiment groups: DIR@HCNPs group, mAb-DIR@HCNPs group, 3 mice per group. Wherein the DIR administration dose is 2mg/kg body weight. The dir@hcnps and mAb-dir@hcnps solutions of comparable drug loading were injected into normal mice via tail vein, respectively, at various time points: 0h, 2h, 4h, 8h, 12h, 24h and 48h of retroorbital blood is taken, 50 μl of blood is paved in a 96-well plate each time, and finally the fluorescence intensity is detected by a small animal imager and quantitatively analyzed (DIR: ex/Em:748/780 nm).

In vivo pharmacokinetic studies

Setting experiment groups: DIR@HCNPs group, mAb-DIR@HCNPs group, 3 mice per group. Wherein the DIR administration dose is 2mg/kg body weight. The dir@hcnps and mAb-dir@hcnps solutions of comparable drug loading were injected into normal mice via tail vein, respectively, at various time points: 0h, 2h, 4h, 8h, 12h, 24h and 48h of retroorbital blood is taken, 50 μl of blood is paved in a 96-well plate each time, and finally the fluorescence intensity is detected by a small animal imager and quantitatively analyzed (DIR: ex/Em:748/780 nm).

In vivo biodistribution studies

(1) Detecting the distribution condition of the main tissue and organs by a small animal living body imager: setting experiment groups: mAb-dir@hcnps sepsis group, mAb-dir@hcnps healthy group, isotype IgG-dir@hcnps sepsis group, free DIR sepsis group. Wherein the DIR administration dose is 2mg/kg body weight. Injecting 8mg/kg LPS into mice to induce sepsis ALI model, injecting mAb-DIR@HCNPs, isotype IgG-DIR@HCNPs and free DIR containing equivalent DIR into model mice via tail vein respectively after 12h, injecting mAb-DIR@HCNPs into healthy mice, anesthetizing and dissecting the mice after 12h, taking out organs such as heart, liver, spleen, lung and kidney, rinsing with normal saline to wash off blood stain on the surface, absorbing water stain on the surface with water-absorbing paper, detecting fluorescence intensity by a small animal imager, and performing quantitative analysis (DIR: ex/Em:748/780 nm).

(2) Detecting the distribution of the nanoparticles in the lung by tissue immunofluorescence: setting experiment groups: mAb-c6@hcnps sepsis group, mAb-c6@hcnps healthy group, isotype IgG-c6@hcnps sepsis group, free C6 sepsis group. Wherein, the dosage of C6 is 2mg/kg body weight. Injecting 8mg/kg LPS into a mouse body to induce sepsis ALI model in an intraperitoneal mode, injecting mAb-C6@HCNPs, isotype IgG-C6@HCNPs and free C6 containing equivalent amounts of C6 into the model mouse body through tail veins respectively after 12 hours, injecting mAb-C6@HCNPs into a healthy mouse body, anesthetizing and dissecting the mouse after 12 hours, taking out the lung, rinsing with normal saline to wash out blood stains on the surface, absorbing and fixing water stains, detecting macrophages in the lung tissue through tissue immunofluorescence, and detecting the distribution of nanoparticles in inflammatory lung under a microscope, wherein one antibody is an Anti-CD68 antibody through tissue immunofluorescence experimental step as 1.1.4.6- (3).

In vivo efficacy study

Setting experiment groups: healthy group, LPS+PBS group, LPS+HCNPs group, LPS+TPCA-1 group, LPS+Isotype IgG-TPCA-1@HCNPs group, LPS+mAb-TPCA-1@HCNPs group, 5 mice per group. Wherein, the dosage of TPCA-1 is 1mg/kg.

8mg/kg LPS is injected into mice in an abdominal cavity to induce an ALI model of sepsis, healthy mice are injected into an equal amount of PBS in an abdominal cavity, and then corresponding medicines are injected into the mice in each group through tail veins. After 12h, peripheral blood of the mice was collected by eyeball-picking, and plasma was collected by centrifugation (1000 Xg, 10 min) at 4 ℃. The mice were dissected, the lung tissue removed, rinsed with ice-cold PBS to flush out surface residual blood traces or impurities, and rubbed dry with absorbent paper. A tissue block is sheared from the lung, weighed, recorded and sheared, so that fragments are as small as possible, and the tissue block is convenient to sufficiently homogenize. The tissue was placed in a milling bowl and added with liquid nitrogen, the lung tissue was rapidly and repeatedly milled, a certain amount of lysate (Cocktail: PMSF: RIPA: =1:1:100) was added, followed by homogenization (3000 rpm, 3-4 s apart, 5 repetitions) in a homogenizer, and the lung tissue homogenate was centrifuged at 4 ℃ (13000 rpm,10 min) and the supernatant was taken.

(1) The lung tissue H & E staining protocol was the same as 1.2.4- (2), and lung tissue injury scoring was performed, 3 lung tissue sections were randomly extracted from each group of mice, 3 fields were randomly selected for each section, 4 injury manifestations of alveolar hemorrhage, congestion, alveolar wall thickening and leukocyte infiltration were observed, and scores were based on the degree of injury, wherein no abnormality was scored as 0 score, mild scored as 1 score, moderate scored as 2 score, severe scored as 3 score, severe scored as 4 score, and the average of the 3 scores obtained was used as the final score for the group of lung injuries.

(2) Flow cytometry detects inflammatory cell infiltration of lung tissue:

1) Pre-preparing tissue digestion solution: each tube was filled with 3ml of system digests (2.7 ml RMPI 1640 cell culture medium, 0.3ml FBS), 16mg type I collagenase and 0.25mg DNase were added, and vortexed to dissolve well.

2) Taking grain-size lung tissue, fully cutting, placing in tissue digestion solution, then placing in a table-type constant-temperature oscillator at 37 ℃ for incubation for 2 hours, filtering with a 40-mu m filter screen for 3 times to obtain uniform single-cell suspension, then adding 3 times of erythrocyte lysate, placing on ice for 40 minutes, centrifuging (450 Xg, 10 minutes), discarding supernatant, adding 1 mu l of FcR blocker into each tube after PBS (phosphate buffer solution) resuspension, incubating for 10 minutes in a refrigerator at 4 ℃, adding Fixable Viability Stain 780 (live dead cell dye), incubating for 10 minutes at room temperature in a dark place, and washing with PBS for 3 times after centrifugation. For macrophage flow assay, 1 μl of each of the following flow-through antibodies was added: FITC-Anti-CD45, PE-Anti-CD11b, APC-Anti-F4/80; for T cell flow assays, 1 μl of each of the following flow antibodies was added: FITC-Anti-CD45, APC-Anti-CD3. Incubate for 30min at room temperature in the absence of light, then centrifuge and wash 3 times with PBS, fix with 4% paraformaldehyde, and finally check on-machine by flow cytometry.

(3) Tissue ROS fluorescence assay procedure:

1) Tissue embedding: taking mouse lungs fixed in 4% paraformaldehyde solution, fully flushing surface blood trace with ultrapure water, sequentially taking ethanol with different concentrations (75% ethanol 30min, 85% ethanol 30min, 95% ethanol 60min, and 100% ethanol 60min 2 times) as a dehydrating agent, removing water in tissues, and then embedding the lungs in xylene. Wax-soaking the tissue at 60 ℃ for 2 hours, solidifying the surface layer of the wax liquid at room temperature, and rapidly putting the tissue into cold water overnight;

2) Paraffin section: cutting the tissue wax block into slices by using a slicing machine, putting the cut tissue slices into warm water, unfolding and ironing, fully sucking water by using filter paper, transferring the water to the center of an anti-drop slide glass, and putting the slide glass into a 60 ℃ oven for drying for 4 hours;

3) Dewaxing to water: sequentially placing the slices into xylene I for 15 minutes, xylene II for 15 minutes, xylene III for 15 minutes, alcohol I with the concentration of 99.57 percent for 5 minutes, alcohol II with the concentration of 99.57 percent for 5 minutes, alcohol with the concentration of 5 percent for 5 minutes and alcohol with the concentration of 85 percent for 5 minutes, and flushing with distilled water;

4) And (5) circling: the histochemical pen was circled around the tissue and incubated with DHE dye at 37 ℃ in the dark for 40min, the DHE dye was thrown off and washed 3 times with PBS.

5) Nuclear dyeing: the slides were placed in PBS and shaken on a shaker for 5min, and the washing procedure was repeated 3 times. And (3) dripping DAPI (DAPI) into the slices after the slices are slightly dried, and incubating for a certain time in a dark place at room temperature.

6) Sealing piece: the slides were placed in PBS and shaken on a shaker for 5min, and the washing procedure was repeated 3 times. Anti-quenching agent is added to the slice, thereby sealing the slice.

7) The images were observed under a fluorescence microscope and scanned.

Biosafety assessment

Setting experiment groups: PBS group, nanomaterial group, 5 mice each. 200 μl of 1mg/ml mAb-TPCA-1@HCNPs solution was injected into normal Bal/bc mice via the tail vein, and the control group was injected with an equal amount of PBS.

(1) The body weight of the mice was measured every 2 days, and the change of the body weight was continuously monitored for 14 days;

(2) Peripheral blood of mice was collected by eye-drop method. For routine blood detection, a whole blood sample is sampled and placed in an anticoagulant tube, and the whole blood sample is oscillated upside down for several times to fully mix the blood and the anticoagulant, and then indexes such as RBC, WBC, PLT, HGB, HCT are detected by a routine blood analyzer. For biochemical detection of blood, whole blood samples are placed in a promoting tube after being sampled, and are centrifuged (3000 rpm,15 min) after being placed at room temperature for 2 hours, and the supernatant is taken and then indexes such as ALT, AST, ALB, BUN, CREA, UREA and the like are detected by a biochemical analyzer;

(3) The mice were dissected and stained for H & E, and the experimental procedure was the same as 1.2.4- (2), and the presence or absence of substantial damage to the organs was detected.

Experimental results

Characterization of mAb-TPCA-1@HCNPs

HES-CH Polymer System HES-NH ₂ And CH-COOH, on the one hand, carries a hydrophilic group HES and on the other hand carries a hydrophobic lipophilic group CH, and self-assembly can occur in an oil-water (O/W) interface in a chloroform and water mixed solution which is fully oscillated by ultrasound, so as to form spherical nanoparticles. As shown in FIG. 2A, TPCA-1@HCNPs are relatively regular spherical nanoparticles, the particle size of the nanoparticles is about 110nm, the boundary is clear, and the surface is smooth. After the surface modification of mAb, the particle size was slightly increased, the size was about 120nm, the morphology was similar to sphere, and the surface became relatively rough, as shown in FIG. 2B. Further detected by a laser particle analyzer, the particle size of HCNPs is 99.68+/-2.449 nm, PDI is 0.2947 +/-0.0163, and Zeta potential value is 27.70+/-1.570 mV; after the TPCA-1 is entrapped, the grain diameter is 112+/-0.7521 nm, the PDI value is 0.2955 +/-0.004324, and the Zeta potential is 27.83+/-0.8452 mV; the particle size was found to be 123.3.+ -. 1.301nm, PDI was 0.2902.+ -. 0.004568, zeta potential was 19.37.+ -. 0.2186mV after further modification of the mAb on the nanoparticle surface, as shown in FIGS. 2C-E. The above results show that the empty carrier nanoparticles of HCNPs without entrapped drugs have smaller particle size, and the nanoparticles are positively charged due to the large amount of amino residues on HES groups, while the particle size of the nanoparticles is increased after the encapsulation of TPCA-1 by Pickering emulsion volatilization, the nanoparticles are still positively charged, the Zeta potential value is similar to that of HCNPs, which indicates that the entrapment of hydrophobic drugs does not affect the surface potential, and finally, after mAb is modified on the surface, the particles The diameter is further increased, the Zeta potential is greatly reduced, and the difference is obvious from the Zeta potential, because DSC reacts with part of amino groups on the surface of the nanoparticle, so that the quantity of the amino groups on the surface is reduced, and mAb is taken as a protein, and the negatively charged positive charge also has a partial neutralization effect. In addition, it is generally believed that the polydispersity index PDI<3, the three nanoparticle aqueous solutions PDI are distributed uniformly, which shows that the distribution is uniform. The nanoparticle is used as a drug delivery system, the stability of the nanoparticle is crucial, and the stability of mAb-TPCA-1@HCNPs is continuously monitored in PBS, RMPI 1640 and 10% FBS respectively, so that the particle size of the nanoparticle is not more than 130nm in PBS and FBS within 7 days, and the particle size of the nanoparticle is not more than 160nm in 10% FBS even if plasma proteins are adsorbed and neutralized, and the nanoparticle drug delivery system is not easy to depolymerize and agglomerate and has better stability, as shown in figure 2F. The presence of mAb on the nanoparticle surface was indirectly demonstrated above by TEM, particle size distribution and Zeta potential. The antibody contains a large amount of N element as a protein. The XPS full spectrum found that the peak at N1S was relatively high for mAb-TPCA-1@HCNPs compared to TPCA-1@HCNPs and TPCA-1@HCNPs+mAb, demonstrating that mAb was successfully coupled to the nanoparticle surface, whereas no antibody was added to the TPCA-1@HCNPs group, no DSC chain bridge was added to TPCA-1@HCNPs+mAb, so neither mAb could be coupled.

mAb-TPCA-1@HCNPs have drug slow release effect and good blood compatibility

The study researches the drug release behavior of the mAb-TPCA-1@HCNPs nano drug delivery system by a dialysis method, as shown in a in figure 3A, the accumulated drug release rate of free TPCA-1 in 24 hours exceeds 80%, and the mAb-TPCA-1@HCNPs is less than 35%, which fully proves the slow release effect of the nano drug delivery system. TPCA-1 is a small molecular drug, can easily pass through the pores of the dialysis bag, while nanoparticles cannot pass through the pores of the dialysis bag due to larger particle size and molecular weight, wherein the entrapped TPCA-1 can only be slowly released. The biocompatibility of an effective nano medicine carrying system is not neglected. After entering the peripheral blood, the nanoparticles are in direct contact with various blood cells, so that the blood compatibility of the nanoparticles is necessary to be examined. As shown in FIG. 3B, after incubation of gradient concentration mAb-TPCA-1@HCNPs with the erythrocyte suspension for 2 hours, the supernatant remained clear by visual observation, and the color was close to that of the PBS negative control group, and forms a huge contrast with the maximum positive group of 1% Triton X-100. While the hemolysis rate was measured at each concentration, it was found that the hemolysis rate was less than 1% (0.65.+ -. 0.063%) even at a concentration of 500. Mu.g/ml, and the morphology of each group of erythrocyte pellet was further observed under a common optical microscope (as shown in FIG. 3, C, D), it was found that the erythrocytes of the 1% Triton X-100 most positive group had been ruptured and no erythrocytes were seen, while the erythrocytes of the HCNPs group, mAb-TPCA-1@HCNPs group and PBS group still had a complete morphology. As described above, mAb-TPCA-1@HCNPs have lower blood cytotoxicity and better blood compatibility.

In vitro cell experiments

(1) The mAb-TPCA-1@HCNPs have better cell compatibility: as shown in FIG. 4A-B, the activities of the endothelial cell line HUVEC and the macrophage line Raw264.7 decrease with increasing concentration of the nano-drug, but even under the condition of the highest concentration of 250 mug/ml, the relative cell activities of HUVEC are 71.23 +/-1.388%, and Raw264.7 is 77.02 +/-3.956%, both keep higher activities, which indicates that the nano-drug delivery system has lower cytotoxicity and better cell compatibility.

(2) mAb-TPCA-1@HCNPs have good antioxidation effect: as shown in fig. 5, A, D, the relative ROS production of the control group was 18.77±1.255%, increased to 51.13 ± 6.709% (P < 0.0001) upon LPS stimulation,

the group of LPS+HCNPs is 48.63 + -2.170% (P= 0.9640), and has no obvious difference from the group of LPS, which indicates that the HCNPs serving as nano-carriers have no antioxidation effect. However, after dry prognosis of TPCA-1, TPCA-1@HCNPs and mAb-TPCA-1@HCNPs, respectively, under inflammatory stimulus, the relative ROS production was reduced to 23.93+ -1.576% (P < 0.001), 23.73+ -0.6119 (P < 0.001) and 11.87+ -1.257% (P < 0.0001), respectively, which suggests that the nano-drug coated with the nanocarrier still retains a strong antioxidant effect compared with free TPCA-1, and the nano-drug modified by the mAb surface has a stronger effect, which may be related to the binding of the cleavage site of the extracellular segment of PECAM-1 more favorable for promoting the entry of the nanoparticles into cells.

(3) mAb-TPCA-1@HCNPs are effective in inhibiting macrophage M1 polarization under inflammatory stimuli: as shown in fig. 5, B, E, the control group has a polarization ratio of only 49.27±1.317%, and increases to 91.30 ±1.457% after stimulation by LPS, the lps+hcnps group has a M1-type ratio of 85.73±1.417% (p= 0.2070), and the M1-type ratio is significantly reduced compared with the LPS group when the TPCA-1, TPCA-1@hcnps and mAb-TPCA-1@hcnps are added, by 59.17 ±2.034%, 57.03 ± 3.309% and 60.20± 0.6658%, respectively, the P values are all <0.0001, and the significant difference is seen. Therefore, both free TPCA-1 and nanocarrier-entrapped macrophages strongly inhibited M1 polarization upon inflammatory stimuli.

(4) mAb-TPCA- @ HCNPs inhibit macrophage production of NO: as shown in FIG. 5, C, F, the relative NO production of the control group was only 6.240 + -0.5327%, and increased to 20.37 + -4.161 after LPS stimulation, and was similarly not effective (19.97+ -2.038%) after HCNPs intervention, and decreased to 11.24+ -1.335%, 11.10+ -1.429% and 10.77+ -1.886% after treatment with TPCA-1, TPCA-1@HCNPs and mAb-TPCA-1@HCNPs, respectively, and P value was <0.05. The results show that mAb-TPCA-1@HCNPs have the effect of inhibiting NO generation by macrophages under inflammatory stimulus.

(5) mAb-TPCA- @ HCNPs are effective in inhibiting IL-6 and TNF- α secretion by macrophages upon inflammatory stimulation: as shown in FIG. 6 at A, B, the control group had IL-6 and TNF-. Alpha.secretion levels of 48.65.+ -. 4.772pg/ml and 504.20.+ -. 18.60pg/ml, respectively, and significantly increased to 153.30.+ -. 6.143pg/ml and 1038.+ -. 73.68pg/ml after LPS stimulation, whereas the LPS+HCNPs group had no efficacy (170.40.+ -. 9.383pg/ml and 1014.+ -. 51.86pg/ml, with P values of 0.4136 and 0.9988, respectively), and IL-6 and TNF-. Alpha.concentrations were reduced by the drying of TPCA-1, TPCA-1.+ -. HCNPs and mAb-TPCA-1.+ -. HCNPs, respectively, 68.04.+ -. 8.303pg/ml (P < 0.0001) and 685.70.+ -. 38.34pg/ml (P < 0.01), 64.17.+ -. 9.334pg/ml (P < 0.0001) and 662.20.+ -. 66.45pg/ml (P < 0.001), 54.27.+ -. 89 pg/ml (P < 0.0001), and P < 28.15 >.001. Thus, mAb-TPCA-1@HCNPs are effective in inhibiting the secretion of pro-inflammatory cytokines by macrophages upon inflammatory stimuli.

(6) mAb-TPCA-1@HCNPs bind efficiently to endothelial cells following inflammatory stimuli: as shown in fig. 7 a-B, mAb-c6@hcnps bound to cells significantly increased in both endothelial cell lines HUVEC and MAEC, compared to the control group, and emitted intense green fluorescence, and showed a decrease in green fluorescence after blocking with sufficient 4g6 mAb. The endothelial targeting ability of mAb-TPCA-1@HCNPs was fully demonstrated above. The control and blocking groups also have some degree of green fluorescence, which may be due to spontaneous cleavage of the extracellular segment of PECAM-1 leading to exposure of the binding site and low dose uptake by endothelial endocytosis.

(7) mAb-TPCA-1@hcnps enter the cell in a time and dose dependent manner, and lysosomal escape begins to occur at 8 h: as shown in fig. 7C, after LPS stimulation, the green fluorescence of the cells gradually increased over time, indicating that the uptake of mAb-c6@hcnps by macrophages gradually increased, with time dependence. In addition, the green fluorescence overlapped with the red fluorescence of the lysosome probe to a greater extent (Pearson correlation coefficients all > 0), indicating that cells ingest mAb-c6@hcnps via the classical endosomal pathway. Analysis of the co-localization of green and red fluorescence using Image J found that Pearson correlation coefficient gradually increased to 0.8356 before 8h followed by a decrease, suggesting that mAb-c6@hcnps had the greatest degree of co-localization with lysosomes via cellular uptake at 8h, followed by gradual migration and turnover of nanoparticles from lysosomes, i.e. macrophages began to undergo lysosomal escape at 8 h. Further, over the same incubation time, the green fluorescence of the cells increased with increasing concentration of the nanomaterials, suggesting that their uptake was dose dependent, as shown in fig. 8A. Finally, the time and dose dependent uptake of the nanomedicine by macrophages was quantified by flow cytometry, the trend of which was consistent with the confocal pictures described above, as shown in fig. 8B-C.

mAb-TPCA-1@HCNPs have long circulating effects in vivo

As shown in FIGS. 9A-B, the fluorescence intensity of DIR@HCNPs gradually decreased with time, and was almost completely lost when 48 hours had elapsed. After surface modification of the mAb, mAb-dir@hcnps had similar pharmacokinetic effects as dir@hcnps, as shown in fig. 9C-D. The nano drug carrying system can circulate for 48h in a normal mouse body, and a very small amount of drug still remains in peripheral blood after 2 days, which proves that the nano drug carrying system has a good long circulation effect in peripheral blood.

mAb-TPCA-1@HCNPs have good lung targeting in sepsis ALI mice

mAb-TPCA-1@HCNPs are used as a drug delivery system, and focus targeting is extremely important. As shown in fig. 10 a-B, mAb-dir@hcnps were significantly enriched in lung (P < 0.0001) and emitted intense fluorescence, as well as enhanced fluorescence in liver and spleen, compared to healthy mice, while Isotype IgG-dir@hcnps lacking inflammatory endothelial cell targeting had only slight accumulation in sepsis lung (P < 0.05). Free DIR is neither targeted by mAb nor delivered by nanocarriers, and thus the degree of fluorescence in inflammatory lungs is weak (P < 0.01). In each of the above groups, the fluorescence of the heart and kidneys is weaker, which may be related to its tissue densification, and less endothelial damage and leakage under inflammatory stimuli. Tissue immunofluorescence was similar to the results of the in vivo distribution above, as shown at C in fig. 10. In conclusion, mAb-TPCA-1@HCNPs are known to have excellent inflammatory lung targeting.

mAb-TPCA-1@HCNPs can effectively reduce lung inflammation and injury of sepsis ALI mice

The nanoparticle can be used as a carrier for drug delivery while targeting the focus to exert curative effect. As shown in fig. 11-12, A, F, the lps+mab-TPCA-1@hcnps group showed significantly improved pulmonary thrombosis and congestion, significantly reduced infiltration of inflammatory cells, thinning of the mouse alveolar wall and alveolar septum, reduced extent of pulmonary interstitial swelling, and significantly reduced lung injury score (lps.vs lps+mab-TPCA-1@HCNPs15.00 ±0.5477.vs.5.20± 0.3742) compared to the LPS group. Free TPCA-1 and IgG-TPCA-1@HCNPs also improved the extent of inflammation and injury, but had limited efficacy (lung injury score: LPS.VS.TPCA-1:15.00.+ -. 0.5477.VS.10.60.+ -. 0.5099, LPS.VS.IgG-TPCA-1@HCNPs:15.00.+ -. 0.5477.VS.8.20.+ -. 0.3742). The effect of mAb-TPCA-1@HCNPs intervention is better than that of IgG-TPCA-1@HCNPs and TPCA-1 (mAb-TPCA-1@HCNPs.VS.IgG-TPCA-1@HCNPs:5.20 + -0.3742. VS.8.20+ -0.3742, mAb-TPCA-1@HCNPs.VS.TPCA-1:5.20+ -0.3742. VS.10.60+ -0.5099) with the addition of active targeting effect.

The infiltration of T cells and macrophages in lung tissue was further quantitatively studied by flow cytometry, as shown in FIGS. 11B-E and 12G-H. The study found that mAb-TPCA-1@HCNPs significantly reduced infiltration of sepsis ALI mice lung tissue T cells and macrophages (T cells: LPS.VS.LPS+mAb-TPCA-1@HCNPs:32.67 + -2.356%. VS.7.933+ -0.2612%, macrophages: LPS+mAb-TPCA-1@HCNPs:53.27 + -2.817%. VS.21.53% + -0.8762%) compared to LPS groups after intervention. The free TPCA-1 and IgG-TPCA-1@HCNPs are improved, but the effect is inferior to that of mAb-TPCA-1@HCNPs (T cells: LPS.VS.TPCA-1:32.67+ -2.356%. VS.18.33+ -0.7219%, LPS.VS.IgG-TPCA-1@HCNPs: TPCA-1:32.67+ -2.356%. VS.14.37+ -0.2603%, macrophages: LPS.VS.TPCA-1:53.27+ -2.817%. VS.42.70+ -0.4509%, LPS.VS.IgG-TPCA-1@HCs: TPCA-1:53.27+ -2.817%. VS.32.17+ -1.288%). Furthermore, as shown in fig. 12I, ROS fluorescence of lung tissue sections of different treatment groups showed that the fluorescence intensity was significantly reduced compared to that of LPS group after mAb-TPCA-1@hcnps treatment, demonstrating a significant reduction in ROS production, but the effects were general in both TPCA-1 group and IgG-TPCA-1@hcnps.

The mAb-TPCA-1@HCNPs have good biocompatibility and safety

The present study also systematically evaluated the in vivo biosafety of mAb-TPCA-1@HCNPs. As shown in fig. 13 a, the body weight change profile of the nanoparticle injected into the normal mice in 14 was substantially no different from that of the PBS control group, and the body weight of both were slightly increased after 14 days, indicating that the nano-drug was not systematically toxic to the normal mice. In addition, detection indexes such as blood routine (RBC, WBC, PLT, HGB and HCT) and blood biochemistry (AST, ALT, ALB, BUN, CREA and UREA) are also in the normal range, which suggests that mAb-TPCA-1@HCNPs have no toxic and side effects, as shown in B, C in FIG. 13. Similarly, the major organs (heart, liver, spleen, lung and kidney) of the two groups of mice were H & E stained to investigate their potential toxicity, and the results showed that each organ exhibited normal morphology, and no substantial damage was found under the microscope, as shown by D in fig. 13. Taken together, these preliminary data suggest that mAb-TPCA-1@hcnps have no toxic response to normal mice and have excellent biocompatibility.

It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the general spirit and concepts of the invention. Which fall within the scope of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims

1. A method for preparing nanoparticles for treating pulmonary inflammation or injury, characterized by:

mAb-TPCA-1@HCNPs were prepared: mixing the TPCA-1@HCNPs solution with DSC, dialyzing, adding PECAM-14G6 mAb, and reacting to obtain mAb-TPCA-1@HCNPs;

wherein the HES-CH has a structure shown in formula I

In formula 1: n is between 1 and 100.

2. The method for preparing nanoparticles according to claim 1, wherein:

when mAb-TPCA-1@HCNPs are prepared: adding sufficient DSC into the TPCA-1@HCNPs aqueous solution, stirring overnight, then dialyzing, adding PECAM-14G6 mAb, and stirring at room temperature for reaction to obtain mAb-TPCA-1@HCNPs nanoparticle aqueous solution.

3. The method of preparing nanoparticles according to claim 2, wherein: the concentration of the TPCA-1@HCNPs aqueous solution is 1mg/ml.

4. A mAb-TPCA-1@hcnps nanoparticle obtained by the method of any one of claims 1-3.

5. The use of mAb-TPCA-1@hcnps of claim 4 in the manufacture of a medicament for treating pulmonary inflammation or injury.

6. The use according to claim 5, wherein the lung inflammation or injury is sepsis lung inflammation or sepsis lung injury; and/or, the mAb-TPCA-1@HCNPs exhibit at least one of reduced T cells in lung tissue, reduced infiltration of macrophages, reduced reactive oxygen species formation in lung inflammation or injury.