CN114272393A - Light click reaction strategy based targeted nano diagnosis and treatment agent with glutathione activation and preparation method and application thereof - Google Patents
Light click reaction strategy based targeted nano diagnosis and treatment agent with glutathione activation and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a targeted nano diagnosis and treatment agent with glutathione activation characteristic based on a light click reaction strategy and a preparation method and application thereof, and the preparation method comprises the following steps: synthesizing a high molecular monomer HA-Cys-MA and a high molecular monomer HA-Lys-Tet; the high molecular monomer HA-Cys-MA, the high molecular monomer HA-Lys-Tet and the rifampicin form a nano diagnosis and treatment agent RIF @ HA-NG through a light spot biological orthogonal reaction; modifying the dye Cy5.5 to the nano diagnosis and treatment agent RIF @ HA-NG through amidation reaction to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with image tracing capability. The advantages of the invention include: the biocompatible and multifunctional composite nano diagnosis and treatment agent containing functional component substances such as hyaluronic acid, rifampicin and even Cy5.5 is constructed, and the biocompatible and multifunctional composite nano diagnosis and treatment agent can be used as an effective mycobacterium tuberculosis targeting nano material and is used for targeted chemotherapy of tuberculosis.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a tuberculosis medicine.
Background
Tuberculosis (TB) is highly contagious and has been a major public health problem worldwide, a chronic disease caused by Mycobacterium Tuberculosis (m.tb), most often affecting the lungs. Currently, surgery in combination with the combination of various anti-tuberculosis drugs is the conventional treatment modality for tuberculosis. Although Rifampicin (Rifampicin, RIF) and isoniazid have been the first choice for anti-tuberculosis drugs due to their excellent therapeutic effect and reasonable price, these chemotherapy drugs have increasingly prominent problems of short half-life in vivo and inevitable side effects, which bring much pain to patients and are of great interest to researchers in clinical medicine and researchers in the field of basic scientific research. Therefore, there is an urgent need to design and develop effective tuberculosis treatment strategies.
In recent years, the nano delivery system technology has made great progress in the diagnosis and treatment of many diseases, which not only provides a new idea for accurate diagnosis of diseases, but also has the possibility of significantly enhancing the treatment effect of different types of patients including cancer or tuberculosis. Among them, various rifampicin-coated nano drug delivery systems, such as chitosan-based hydrogel and liposome, have been widely studied and expected to improve the therapeutic effect of tuberculosis. Although these systems are beneficial to improving the effective dose of the drugs at the focus and relieving the toxic and side effects of the drugs, the conditions of drug leakage, too fast metabolism and the like often occur to hydrophilic drugs such as rifampicin and the like, so that the development of a nano delivery system is still far-reaching. In particular, most of the current delivery systems lack the functions of image tracing diagnosis and treatment monitoring of tuberculosis foci, so that the diagnosis and treatment of tuberculosis still remains a challenging scientific subject.
Disclosure of Invention
The invention aims to provide a targeted nano diagnosis and treatment agent based on a light click reaction strategy and having a glutathione activation characteristic, a preparation method thereof and application thereof in preparing a medicine for treating tuberculosis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a targeted nano diagnosis and treatment agent based on a light click reaction strategy and having a glutathione activation characteristic is selected from RIF @ HA-NG, RIF @ Cy5.5-HA-NG.
A preparation method of a targeted nano diagnosis and treatment agent based on a light click reaction strategy and having a glutathione activation characteristic comprises the following steps:
(1) synthesizing a high molecular monomer HA-Cys-MA and a high molecular monomer HA-Lys-Tet;
(2) the high molecular monomer HA-Cys-MA, the high molecular monomer HA-Lys-Tet and the rifampicin form a nano diagnosis and treatment agent RIF @ HA-NG through a light spot biological orthogonal reaction and an interaction between hydrophobic property and hydrophobic property;
(3) modifying the dye Cy5.5 to the nano diagnosis and treatment agent RIF @ HA-NG through amidation reaction to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with image tracing capability.
Preferably, the first and second electrodes are formed of a metal,
the step of synthesizing the high molecular monomer HA-Cys-MA in the step (1) comprises the following steps: di-tert-butyl dicarbonate and NH2-Cys-NH2Reacting to obtain Boc-Cys-NH2(ii) a Methacryloyl chloride and Boc-Cys-NH2Reacting to generate Boc-Cys-MA; the Boc-Cys-MA is stirred and reacted in methanol solution of trifluoroacetic acid to obtain NH2-Cys-MA, activated hyaluronic acid and NH2And reacting the-Cys-MA to generate HA-Cys-MA containing double bonds.
Preferably, the first and second electrodes are formed of a metal,
the detailed steps for synthesizing the high molecular monomer HA-Cys-MA in the step (1) comprise: di-tert-butyl dicarbonate (Boc) is first introduced2O, DiBoc) and NH2-Cys-NH2Dissolving in dimethyl formamide for reaction at room temp to obtain di-tert-butyl dicarbonate (Boc)2O) and NH2-Cys-NH2At a molar ratio of 0.5:1.0 or 1.0:1.0 or 1.5:1 for 1 or 6 or 12 hours, protecting the amino group at one end to obtain Boc-Cys-NH2；
Methacryloyl chloride and Boc-Cys-NH2Reacting in anhydrous dichloromethane at room temperature to generate Boc-Cys-MA, methacryloyl chloride and Boc-Cys-NH2The molar ratio and the time of the reaction are 1.5:1.0 and 2 hours respectively; the Boc-Cys-MA is stirred and reacted in methanol solution of trifluoroacetic acid to obtain NH2-Cys-MA；
Hyaluronic Acid (HA) and NH containing double bonds2-Cys-MA, EDC and NHS in a molar ratio of 1: 1: 1.5: 1.5 reaction for 6 hours at room temperature during which Hyaluronic Acid (HA) activated with EDC and NHS is reacted with NH2And (4) reacting-Cys-MA to obtain the light click reaction substrate HA-Cys-MA containing double bonds.
Preferably, the first and second electrodes are formed of a metal,
the di-tert-butyl dicarbonate (Boc)2O) and NH2-Cys-NH2The molar ratio of (A) to (B) was 1.0:1.0 for 6 hours.
Preferably, the first and second electrodes are formed of a metal,
the process for synthesizing the high molecular monomer HA-Lys-Tet in the step (1) comprises the following steps: activating hyaluronic acid with EDC and NHS and Boc-Lys-NH2Reacting at room temperature for 6h to generate HA-Lys-Boc; deprotection of Boc with methanol solution of trifluoroacetic acid at room temperature gives HA-Lys-NH2Then the activated tetrazole Tet is added into HA-Lys-NH2And performing amidation reaction for 6 hours, and performing amide bond coupling to obtain a monomer HA-Lys-Tet.
Preferably, the first and second electrodes are formed of a metal,
the operation process of the step (2) comprises the following steps: and (2) stirring and reacting the monomers HA-Cys-MA and HA-Lys-Tet prepared in the step (1) with rifampicin under the illumination conditions of a 350nm LED lamp, illumination power of 1.0W and illumination time of 15min, and forming the nano diagnosis and treatment agent RIF @ HA-NG through light spot biological orthogonal reaction induced by UV and interaction between hydrophobic property and hydrophobic property.
Preferably, the first and second electrodes are formed of a metal,
the operation process of the step (3) comprises the following steps: the nanometer diagnosis and treatment agent RIF @ HA-NG, EDC, NHS and Cy5.5 are mixed according to the molar ratio of 1: 1.5: 1.5: 2, reacting for 6 hours at room temperature, dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3000Da after the reaction is finished, and finally freeze-drying overnight to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with image tracing capability.
The invention also provides application of the targeted nano diagnosis and treatment agent with the glutathione activation characteristic in preparation of a tuberculosis treatment drug based on the light click reaction strategy.
The invention also provides application of the targeted nano diagnosis and treatment agent with the glutathione activation characteristic in image tracing diagnosis and treatment monitoring of tuberculosis lesions based on the light click reaction strategy.
The advantages of the invention include: a polymer pre-monomer with photosensitive functional groups of Methacryloyl (MA) and tetrazole (Tet) is designed and synthesized, a controllable nano drug delivery system is obtained, and as the tubercle part can generate vascular hyperplasia and the permeability becomes strong, when a nano diagnosis and treatment agent with the size of 20-200nm can be retained at the tubercle part through enhancing the permeation retention Effect (EPR), and in addition, under the triggering of high GSH content at the tubercle part, RIF is released from a nano carrier, so that the purpose of chemotherapy is achieved. The method can be applied and evaluated in anti-tuberculosis diagnosis and treatment research, so as to solve the problems that the conventional tuberculosis delivery system lacks an image tracing function, has short half-life and is cleared by organisms through too fast metabolism, and provide a tool with great potential for the diagnosis and treatment of tuberculosis in the future.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:
FIG. 1 shows the synthetic routes of HA-Cys-MA and HA-Lys-Tet.
FIG. 2 is a schematic diagram of the synthesis process and biological application of the nano-diagnostic agents RIF @ HA-NG and RIF @ Cy5.5-HA-NG.
FIG. 3A is a graph depicting dynamic light scattering characterization of RIF @ HA-NG nanoparticles.
FIG. 3B is a graph depicting zeta potential of RIF @ HA-NG nanoparticles.
FIG. 3C is a transmission electron microscopy characterization of the RIF @ Cy5.5-HA-NG nanoparticles.
FIG. 3D is a graphical representation of the release of rifampicin by RIF @ HA-NG nanoparticles in response to glutathione.
FIG. 4 is a graph showing the results of the measurement of the targeting ability of RIF @ Cy5.5-HA-NG nanoparticles.
FIG. 5A is a graph of the antimicrobial activity of RIF and RIF @ Cy5.5-HA-NG nanoparticles against M1 type macrophage intracellular bacteria.
FIG. 5B is a graph of the antimicrobial activity of RIF and RIF @ Cy5.5-HA-NG nanoparticles against M2 type macrophage intracellular bacteria.
FIG. 6A is a representation of the fluorescence image of the enrichment of the RIF @ Cy5.5-HA-NG nanoparticles at the tubercle sites.
FIG. 6B is a graph representing fluorescence images of the enrichment of RIF @ Cy5.5-HA-NG nanoparticles in different organs of mice.
FIG. 6C is a hematoxylin-eosin staining pattern of RIF @ Cy5.5-HA-NG nanoparticles for different organ injury conditions.
Detailed Description
The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.
Example 1: synthesis of RIF @ HA-NG and RIF @ Cy5.5-HA-NG nanoparticles
(1) Synthesizing a high-molecular monomer HA-Cys-MA with a photo-click reaction group and also with a double-bond group Methhacryloyl (MA) and glutathione-responsive cysteine (Cys), and a high-molecular monomer HA-Lys-Tet with a photo-click reaction group tetrazole (Tet) and lysine (Lys);
the specific steps for synthesizing HA-Cys-MA are as follows:
as shown in FIG. 1, di-tert-butyl dicarbonate (Boc) is first reacted2O, DiBoc) and NH2-Cys-NH2Dissolving in dimethyl formamide for reaction at room temperature to obtain di-tert-butyl dicarbonate (Boc)2O) and NH2-Cys-NH2The molar ratio of (1.0: 1.0) and the reaction time of 6 hours; protecting an amino group at one end to obtain Boc-Cys-NH2。
Methacryloyl chloride and Boc-Cys-NH2Reacting in anhydrous dichloromethane at room temperature to generate Boc-Cys-MA, methacryloyl chloride and Boc-Cys-NH2The molar ratio and time of reaction were 1.5:1.0 and 2 hours, respectively. Boc-Cys-MA in trifluoroacetic acid in methanol (TFA/CH)3OH) to obtain NH2-Cys-MA。
Hyaluronic Acid (HA) and NH containing double bonds2-Cys-MA, EDC and NHS in a molar ratio of 1: 1: 1.5: 1.5 reaction for 6 hours at room temperature during which Hyaluronic Acid (HA) activated with EDC and NHS is reacted with NH2And (4) reacting-Cys-MA to obtain the light click reaction substrate HA-Cys-MA containing double bonds.
Specific procedure for the synthesis of HA-Lys-Tet:
as shown in FIG. 1, Hyaluronic Acid (HA) activated with EDC and NHS was reacted with Boc-Lys-NH2The reaction was carried out at room temperature for 6h to yield HA-Lys-Boc. TFA/CH at room temperature3Deprotection of OH solution to Boc affords HA-Lys-NH2Then the activated tetrazole (Tet) is added into HA-Lys-NH2And performing amidation reaction for 6h, and performing amide bond coupling to obtain a monomer HA-Lys-Tet.
The activating mode of Hyaluronic Acid (HA) and tetrazole (Tet) refers to the activating mode in the synthesis step of HA-Cys-MA.
(2) The monomers HA-Cys-MA and HA-Lys-Tet prepared in the step (1) are stirred and react with Rifampicin (RIF) under the illumination condition of illumination power of 1.0W and illumination time of 15min by using a 350nm LED lamp, during the reaction, the high molecular monomers HA-Cys-MA and HA-Lys-Tet form a nano carrier through a light click biorthogonal reaction, a hydrophobic cavity is formed inside the nano carrier, then the water-insoluble rifampicin permeates into the hydrophobic cavity, the interaction force between the rifampicin and the hydrophobic cavity is the interaction force between hydrophobicity and hydrophobicity, and the process of forming the nano diagnosis and treatment agent RIF @ HA-NG through the UV-induced light click biorthogonal reaction and the interaction between hydrophobicity and hydrophobicity is shown as a step one in a picture 2;
(3) the method is characterized in that an anthocyanin near-infrared fluorescent dye Cy5.5 is modified to a nano-carrier RIF @ HA-NG through amidation reaction, and specifically comprises the following steps: the nanometer diagnosis and treatment agent RIF @ HA-NG, EDC, NHS and Cy5.5 are mixed according to the molar ratio of 1: 1.5: 1.5: 2, reacting at room temperature for 6 hours. After the reaction is finished, dialyzing for 24h by using a dialysis bag with the molecular weight cutoff of 3000Da, and finally freeze-drying overnight to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with the image tracing capability, wherein the process is shown as the step two in the figure 2; thus, the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG which HAs image tracing capability and can be used for tuberculosis diagnosis and treatment is obtained through the coupling mode of carboxyl and amino.
Example 2: characterization of RIF @ HA-NG, RIF @ Cy5.5-HA-NG nanoparticles
And (3) characterizing the particle size and the potential: the particle size distribution and zeta potential of RIF @ HA-NG were determined using a dynamic light scattering analyzer (DLS).
And (3) morphology analysis: morphology of RIF @ Cy5.5-HA-NG was characterized by TEM.
Determination of drug release rate: the drug release efficiency of RIF @ Cy5.5-HA-NG was determined by mass spectrometry with or without GSH addition.
As shown in FIG. 3A, the mean particle size of the RIF-supporting HA-NG was found to be around 100 nm.
Meanwhile, as shown in FIG. 3B, the zeta potentials are measured and found to be-27.5 mV and-32.5 mV before and after loading RIF respectively, which indicates that the nanoparticle RIF @ HA-NG HAs good stability.
As shown by TEM results in FIG. 3C, the nanoparticle RIF @ Cy5.5-HA-NG is a spherical particle having a particle size of about 100nm and good dispersibility.
To determine the drug delivery capacity of nanocarriers, we monitored RIF release, and set 2 experimental groups: the preparation method comprises the following steps of preparing a control group and a glutathione adding group, wherein the control group is PBS, the glutathione adding group is a solution (pH is 7.4) containing GSH, the nano diagnostic agent RIF @ HA-NG is filled in dialysis bags with the molecular weight cutoff of 1000Da, then the dialysis groups are respectively placed in the control group and the glutathione adding group for dialysis, dialysate of different time nodes in the time range of 0-70h is respectively taken, the dialysis amount is calculated through mass spectrum, the RIF amount responding to GSH release can be obtained, and then the release efficiency is obtained by dividing the total RIF amount. Results as shown in fig. 3D, the release of RIF may have two phases, within 10h, the release of RIF exhibits a time dependence; the RIF is released rapidly within 5h, then the release rate is reduced, the RIF release is close to saturation after 10h, the release rate of the group added with glutathione is higher than that of the control group, which shows that the glutathione GSH shears off disulfide bond-S-S-in the nano-carrier, so that the carrier collapses and releases RIF, the glutathione group can activate the RIF release in RIF @ HA-NG, and similarly, the glutathione group can also activate the RIF release in RIF @ Cy5.5-HA-NG.
Example 3: determination of targeting ability of RIF @ Cy5.5-HA-NG nanoparticles
In order to carry out image tracing in vivo and in vitro at the later stage, the nano diagnosis and treatment agent RIF @ HA-NG is subjected to functional modification, and a fluorescent dye Cy5.5 is linked to the surface of the nano diagnosis and treatment agent RIF @ HA-NG through a chemical bond to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG. This example uses confocal laser microscopy to evaluate the targeting ability of RIF @ Cy5.5-HA-NG.
In this example, macrophages containing mycobacterium tuberculosis were used for the experiment, and 3 experimental groups were set up: PBS, Cy5.5 (final concentration 10 mu M) and RIF @ Cy5.5-HA-NG (final concentration 10 mu M) are added into a control group, a Cy5.5 group and a RIF @ Cy5.5-HA-NG (final concentration 10 mu M) according to grouping requirements respectively, and are respectively incubated with macrophages containing mycobacterium tuberculosis for 1h, then the macrophages are washed for 3 times by PBS, then nuclear targeting dye DAPI (final concentration 10 mu M) and cytoskeleton Actin dye phalloidin (final concentration 10 mu M) are added, the incubation is carried out for 30min, and then the washing is carried out for 3 times by PBS for confocal microscopy imaging. As shown in FIG. 4, red fluorescence signals were detected in M.tuberculosis-containing cells of both Cy5.5 group and RIF @ Cy5.5-HA-NG group compared to the control group, indicating that Cy5.5 successfully modified RIF @ Cy5.5-HA-NG. In addition, clear red light (Cy5.5) can be observed in a cell area of the RIF @ Cy5.5-HA-NG group, which indicates that the RIF @ Cy5.5-HA-NG can be well recognized and phagocytized by phagocytic cells, and a nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG can be utilized to effectively release rifampicin RIF in response to a tubercle microenvironment with high-expression glutathione for killing mycobacterium tuberculosis, and the RIF @ HA-NG can also achieve the same effect.
Example 4: determination of antibacterial activity of RIF @ Cy5.5-HA-NG nanoparticles
To study the in vitro antibacterial activity of RIF and RIF @ Cy5.5-HA-NG, two intracellular bacteria (M1 type macrophage intracellular bacteria, M2 type macrophage intracellular bacteria) were first incubated with RIF and RIF @ Cy5.5-HA-NG, respectively, for various periods of time (1-6h), and then the cells were tested for viability by the MTT method to analyze the antibacterial effect of RIF and RIF @ Cy5.5-HA-NG.
As shown in FIG. 5A, after M1 type macrophage intracellular bacteria were treated with RIF and RIF @ Cy5.5-HA-NG for 1-6h, the cell viability of the RIF @ Cy5.5-HA-NG group was lower than that of the RIF group, indicating that the antibacterial activity of the RIF @ Cy5.5-HA-NG group was higher than that of the RIF group. After M2 type macrophage intracellular bacteria were treated with RIF and RIF @ Cy5.5-HA-NG for 1-6h, as shown in FIG. 5B, the antibacterial activity of the RIF @ Cy5.5-HA-NG group was higher than that of the RIF group for a certain period of time. These results indicate that RIF @ Cy5.5-HA-NG NPs (nanoparticles) have a high antibacterial effect on bacteria in vitro, and similarly, RIF @ HA-NG can also achieve the same effect.
Example 5: image tracing capability detection of RIF @ Cy5.5-HA-NG nano particles on living body level
Establishing a mouse mononuclear nodule model: mice models were established several days later by intravenous injection of mycobacterium tuberculosis through the mouse tail.
Detecting the tracing capacity of the in vivo image: mice were injected via tail vein with RIF @ Cy5.5-HA-NG NPs and tested using a small animal live imager at various time periods (0h, 6h, 12h, 24 h). Then dissecting the mouse, taking the liver, spleen, lung and kidney, and detecting the fluorescence signal by a small animal living body imager.
Evaluation of biological safety: mice injected with RIF @ cy5.5-HA-NG NPs were dissected to obtain major organs (liver, spleen, lung, kidney), fixed with 4% paraformaldehyde, and corresponding H & E stained sections were prepared and the tissue sections were observed by microscopy.
The in vivo image tracer capacity is shown in fig. 6A and 6B. After RIF @ Cy5.5-HA-NG nanoparticles are injected into tail veins, as shown in FIG. 6A, the fact that the fluorescence intensity of the RIF @ Cy5.5-HA-NG nanoparticles at tubercle positions is stronger and stronger after 6 hours, 12 hours and 24 hours shows that the enrichment concentration is higher and higher, and the nanoparticles can achieve the target effect of mouse tubercle. Furthermore, as shown in FIG. 6B, by ex vivo fluorescence imaging of mouse organs at the corresponding time nodes, we found that RIF @ Cy5.5-HA-NG NPs fluoresce significantly in mouse liver at 6h, then fluorescence decreased at 12h and 24h, indicating that these NPs can be metabolized by the liver. In addition, as shown in FIG. 6C, no pathological changes were observed in HE staining of each vital organ after RIF @ Cy5.5-HA-NG NPs injection at different times. These results indicate that RIF @ Cy5.5-HA-NG is a safe biomaterial that can be used in vivo and in vitro.
The advantages of the invention include: the composite material with biocompatibility and combined with functional medicines such as hyaluronic acid, rifampicin, Cy5.5 and the like is constructed, and the composite material can be used as an effective mycobacterium tuberculosis targeting nano material and is used for targeted chemotherapy of tuberculosis.
The drug is coated in the nano carrier, and can be released only in a GSH response state of a focus part through interaction between hydrophobicity and hydrophobicity, so that leakage in a normal tissue microenvironment state is prevented.
The GSH activated RIF @ Cy5.5-HA-NG HAs good targeting property and biocompatibility and HAs a good anti-tuberculosis effect. Therefore, the invention opens up a new way for developing the specific degradable nano material for the targeted imaging and treatment of tuberculosis.
Because the nano material with the particle size of 20-200nm has Enhanced osmotic retention Effect (EPR), the small molecule drug is prevented from being cleared by the rapid metabolism through the blood circulation.
The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.
Claims (10)
1. A targeted nano diagnosis and treatment agent based on a light click reaction strategy and having a glutathione activation characteristic is characterized in that:
it is selected from the group consisting of RIF @ HA-NG, RIF @ Cy5.5-HA-NG.
2. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 1, characterized in that:
the method comprises the following steps:
(1) synthesizing a high molecular monomer HA-Cys-MA and a high molecular monomer HA-Lys-Tet;
(2) the high molecular monomer HA-Cys-MA, the high molecular monomer HA-Lys-Tet and the rifampicin form a nano diagnosis and treatment agent RIF @ HA-NG through a light spot biological orthogonal reaction and an interaction between hydrophobic property and hydrophobic property;
(3) modifying the dye Cy5.5 to the nano diagnosis and treatment agent RIF @ HA-NG through amidation reaction to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with image tracing capability.
3. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 1, wherein the preparation method comprises the following steps:
the step of synthesizing the high molecular monomer HA-Cys-MA in the step (1) comprises the following steps: di-tert-butyl dicarbonate and NH2-Cys-NH2Reacting to obtain Boc-Cys-NH2(ii) a Methacryloyl chloride and Boc-Cys-NH2Reacting to generate Boc-Cys-MA; the Boc-Cys-MA is stirred and reacted in methanol solution of trifluoroacetic acid to obtain NH2-Cys-MA, activated hyaluronic acid and NH2-Cys-MA reaction to form HA-Cy containing double bonds-MA。
4. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 3, wherein the preparation method comprises the following steps:
the detailed steps for synthesizing the high molecular monomer HA-Cys-MA in the step (1) comprise: firstly, di-tert-butyl dicarbonate and NH are mixed2-Cys-NH2Dissolving the two components in dimethylformamide to react at room temperature, and reacting the two-tert-butyl dicarbonate and NH2-Cys-NH2At a molar ratio of 0.5:1.0 or 1.0:1.0 or 1.5:1 for 1 or 6 or 12 hours, protecting the amino group at one end to obtain Boc-Cys-NH2；
Methacryloyl chloride and Boc-Cys-NH2Reacting in anhydrous dichloromethane at room temperature to generate Boc-Cys-MA, methacryloyl chloride and Boc-Cys-NH2The molar ratio and the time of the reaction are 1.5:1.0 and 2 hours respectively; the Boc-Cys-MA is stirred and reacted in methanol solution of trifluoroacetic acid to obtain NH2-Cys-MA；
Hyaluronic acid and NH containing double bond2-Cys-MA, EDC and NHS in a molar ratio of 1: 1: 1.5: 1.5 reaction for 6 hours at room temperature during which Hyaluronic Acid (HA) activated with EDC and NHS is reacted with NH2And (4) reacting-Cys-MA to obtain the light click reaction substrate HA-Cys-MA containing double bonds.
5. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 4, wherein the preparation method comprises the following steps:
di-tert-butyl dicarbonate and NH2-Cys-NH2The molar ratio of (A) to (B) was 1.0:1.0 for 6 hours.
6. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 2, wherein the preparation method comprises the following steps:
the process for synthesizing the high molecular monomer HA-Lys-Tet in the step (1) comprises the following steps: activated with EDC and NHSHyaluronic acid with Boc-Lys-NH2Reacting at room temperature for 6h to generate HA-Lys-Boc; deprotection of Boc with methanol solution of trifluoroacetic acid at room temperature gives HA-Lys-NH2Then the activated tetrazole Tet is added into HA-Lys-NH2And performing amidation reaction for 6 hours, and performing amide bond coupling to obtain a monomer HA-Lys-Tet.
7. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 2, wherein the preparation method comprises the following steps:
the operation process of the step (2) comprises the following steps: and (2) stirring and reacting the monomers HA-Cys-MA and HA-Lys-Tet prepared in the step (1) with rifampicin under the illumination conditions of a 350nm LED lamp, illumination power of 1.0W and illumination time of 15min, and forming the nano diagnosis and treatment agent RIF @ HA-NG through UV induction of 'light spot biological orthogonal reaction' and interaction between hydrophobic property and hydrophobic property.
8. The preparation method of the targeted nano diagnostic and therapeutic agent with glutathione activation property based on the light click reaction strategy as claimed in claim 2, wherein the preparation method comprises the following steps:
the operation process of the step (3) comprises the following steps: the nanometer diagnosis and treatment agent RIF @ HA-NG, EDC, NHS and Cy5.5 are mixed according to the molar ratio of 1: 1.5: 1.5: 2, reacting for 6 hours at room temperature, dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3000Da after the reaction is finished, and finally freeze-drying overnight to obtain the nano diagnosis and treatment agent RIF @ Cy5.5-HA-NG with image tracing capability.
9. Use of the targeted nanopathology agent based on the light click reaction strategy and having glutathione activation properties according to claim 1 in the preparation of a medicament for treating tuberculosis.
10. The application of the targeted nano diagnosis and treatment agent based on the light click reaction strategy and having the glutathione activation characteristic in the image tracing diagnosis and treatment monitoring of tuberculosis focuses as claimed in claim 1.
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