Nanometer anti-cancer drug combining chemotherapy, photothermal therapy and radiotherapy, targeting carrier and preparation method

Abstract

The invention relates to a nano anti-cancer drug combining chemotherapy, photothermal therapy and radiotherapy, a targeting carrier and a preparation method of the nano anti-cancer drug, and the preparation method comprises the following steps: firstly, coupling Azo to Glu-PEG-NHS, and modifying carboxyl to obtain a Glu-PEG-Azo-COOH derivative; the preparation method comprises the following steps: respectively modifying a mitochondrial targeting group TPP and a photothermal agent IR808 on the surface of PAMAM to obtain Mito808; then, coupling the Glu-PEG-Azo conjugate with Mito808 to obtain a targeting carrier, and loading an anti-tumor drug and radionuclide through an electrostatic adsorption effect to obtain a nano anti-cancer drug; gLUT1 targeting is achieved, uptake of tumor cells is improved, meanwhile, positive charges carried by TPP can be shielded, the in-vivo circulation time can be prolonged, the in-vivo circulation stability can be enhanced, in the tumor hypoxia microenvironment, PEG derivatives can be removed, mitochondrial targeting groups TPP can be exposed, and the mitochondrial targeting effect can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted drugs, and specifically relates to a nano anticancer drug combined with chemotherapy, photothermal therapy and radiotherapy, a targeted carrier and a preparation method. Background Art

[0002] After the drug is taken up by tumor cells, how to achieve efficient mitochondrial targeting and release anti-tumor drugs is a key issue in inhibiting tumor growth and metastasis. In order to solve the problem of targeted release of tumor drugs, targeted drugs have emerged. The current tumor tissue targeted drugs are usually based on the "EPR" effect or modified with functional ligands on the surface of the carrier, and the recognition of receptors and ligands is used to enhance the targeting of tumor tissues. In addition, ligand modification can also provide in vivo stability of targeted drugs. For example, neutral substances such as polyethylene glycol (PEG) are modified on the surface of the carrier, which can shield the positive charge of the mitochondrial targeting ligand, thereby effectively avoiding being engulfed by the reticuloendothelial system, and achieving the effect of improving the in vivo stability of the mitochondrial targeted drug delivery system. However, the targeted drugs constructed in this way have the following disadvantages: 1) Due to the significant individual differences in tumor receptor expression, the targeting efficiency of this method is low; 2) After modifying PEG and tumor tissue targeting groups, the mitochondrial targeting groups are often shielded, affecting the mitochondrial targeting effect, thereby reducing the mitochondrial targeting efficiency.

[0003] Therefore, developing a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector with excellent targeting ability for use in nano-anticancer drugs combined with chemotherapy, photothermal therapy and radiotherapy is of great significance for achieving efficient targeted release of drugs. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a nano anticancer drug, a targeted carrier and a preparation method that combine chemotherapy, photothermal therapy and radiotherapy. The nano anticancer drug is mediated by the new blood vessel GLUT1 and has hypoxia responsiveness. It can not only achieve GLUT1 targeting and improve the uptake of tumor cells, but also shield the positive charge carried by TPP, prolong the circulation time in the body, and enhance the stability of the circulation in the body. In addition, in the hypoxic microenvironment of the tumor, the PEG derivative can be removed to expose the mitochondrial targeting group TPP to achieve mitochondrial targeting.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector comprises the following steps:

[0007] Step (1), coupling p-hydroxyazobenzene Azo to Glu-PEG-NHS and modifying the carboxyl group to obtain a Glu-PEG-Azo-COOH derivative; modifying the mitochondrial targeting group TPP and the photothermal agent IR808 on the surface of PAMAM to obtain a TPP-PAMAM-IR808 conjugate, i.e., Mito 808 ;

[0008] Step (2), Glu-PEG-Azo-COOH derivative and Mito 808 Esterification reaction occurred to obtain Glu-PEG-Azo / Mito 808 The conjugate is a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector.

[0009] As a further technical solution, in step (1), p-hydroxyazobenzene Azo is coupled to Glu-PEG-NHS, and the carboxyl group is modified to obtain a Glu-PEG-Azo-COOH derivative, comprising:

[0010] First, in solvent A, Glu-PEG-NHS reacts with p-hydroxyazobenzene Azo under the action of catalysts EDCI and HoBt at room temperature in the dark to generate Glu-PEG-Azo conjugate;

[0011] Then, in solvent B, the Glu-PEG-Azo conjugate and succinic anhydride undergo esterification reaction under the action of catalyst triethylamine at room temperature and in the dark to generate a bifunctional Glu-PEG-Azo-COOH derivative.

[0012] As a further technical solution, the solvent A and the solvent B are both dichloromethane.

[0013] As a further technical solution, in step 1, a photothermal agent IR 808 The surface of PAMAM was modified with triphenylphosphine TPP to obtain Mito 808 ,include:

[0014] First, in solvent C, triphenylphosphine TPP reacts with PAMAM under the action of catalysts EDCI and HoBt at room temperature to obtain a PAMAM-TPP conjugate;

[0015] Then, in solvent D, the PAMAM-TPP conjugate reacted with IR808 at room temperature in the dark to obtain Mito 808 .

[0016] As a further technical solution, the solvent C and the solvent D are both DMSO.

[0017] As a further technical solution, in step 2, the Glu-PEG-Azo-COOH derivative is reacted with Mito 808 Esterification reaction occurred to obtain Glu-PEG-Azo / Mito 808 The conjugate comprises the following steps:

[0018] In DMSO solvent, the bifunctional Glu-PEG-Azo-COOH derivative was reacted with Mito 808 Under the action of catalyst EDCI and HoBt, esterification reaction occurred at room temperature and in the dark to obtain Glu-PEG-Azo / Mito 808 Conjugate.

[0019] The GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector prepared by the preparation method.

[0020] A nano anticancer drug combining chemotherapy, photothermal therapy and radiotherapy, using the GLUT1-mediated and hypoxia-responsive mitochondrial targeting carrier Glu-PEG-Azo / Mito 808 The conjugate is loaded with anti-tumor drug Cel and radionuclide through electrostatic adsorption 177 Lu, obtain Glu-PEG-Azo / Mito-Cel 808 / 177 Lu is a nano anticancer drug that combines chemotherapy, photothermal therapy and radiotherapy functions.

[0021] As a further technical solution, the Glu-PEG-Azo / Mito-Cel 808 / 177 The particle size of Lu is 150-200 nm.

[0022] As a further technical solution, the Glu-PEG-Azo / Mito-Cel 808 / 177 The potential of Lu is - 12.6 ± 0.51mV

[0023] As a further technical solution, the Glu-PEG-Azo / Mito-Cel 808 / 177 The in vitro release behavior of Lu showed significant pH dependence.

[0024] As a further technical solution, the Glu-PEG-Azo / Mito-Cel 808 / 177 The temperature change of Lu under 808nm near-infrared light irradiation shows obvious concentration dependence and laser power dependence.

[0025] The method for preparing the nano anticancer drug combining chemotherapy, photothermal therapy and radiotherapy functions comprises:

[0026] First, Cel was stained with Glu-PEG-Azo / Mito 808 The conjugate is adsorbed to obtain the drug-loaded conjugate Glu-PEG-Azo / Mito-Cel 808 ;

[0027] Then, in sodium acetate buffer, at 37°C, 60 min metal bath conditions, 177Lu 8mCi was loaded with the drug-loaded conjugate Glu-PEG-Azo / Mito-Cel 808 Adsorption to obtain Glu-PEG-Azo / Mito-Cel 808 / 177 Lu.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention utilizes the characteristics of hypoxic microenvironment, which is commonly present in solid tumors but not in normal tissues, to construct a mitochondrial targeted drug delivery system mediated by GLUT1 and responding to hypoxia. Its targeting carrier can be transported through GLUT1, penetrate tumor neovascular endothelial cells into tumor tissue, and remove PEG in the hypoxic microenvironment of tumor tissue to activate mitochondrial targeting and improve the targeting effect on mitochondria. At the same time, mitochondria in a hypoxic environment are more sensitive to heat. The photothermal agent converts the absorbed light energy into heat energy, generates local high temperature, and promotes mitochondrial-mediated cell apoptosis; at the same time, high temperature can significantly increase the expression of tumor cell adhesion-related molecules and reduce the expression of degradation-related molecules, improve tumor cell adhesion and inhibit extracellular matrix degradation, thereby inhibiting the metastasis of tumor cells; combined with chemotherapy drugs, it can increase the sensitivity of tumor chemotherapy and produce a synergistic effect.

[0030] 2. The present invention uses GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector to target the “nuclear missile” - Lutetium 177 ( 177 Lu) is introduced into the body, specifically targeting tumor cells with precision, allowing the drug to play a triple role of biological therapy, photothermal therapy and intratumoral irradiation in tumor cells, thereby killing the tumor.

[0031] 3. The present invention adsorbs water-insoluble Cel onto the positively charged PAMAM conjugate through electrostatic adsorption, which greatly improves the solubility of Cel in water.

[0032] In summary, this patent starts from the perspective of mitochondrial metabolism, takes the changes in mitochondrial structure and function as the starting point, combines tumor angiogenesis and hypoxic microenvironment, and designs a mitochondrial targeted drug delivery system for anti-tumor metastasis. 808 and radionuclides 177 Lu was used as a model drug, and the nanocarrier was modified by functional ligands triphenylphosphine, glucose and p-hydroxyazobenzene to construct a mitochondrial targeted drug delivery system (Glu-PEG-Azo / Mito-Cel 808 / 177 Lu). After entering the body, the drug targets the tumor neovascularization GLUT1, and is transported across the vascular endothelium through GLUT1 to reach the tumor hypoxic microenvironment. In the hypoxic microenvironment, the nanocarrier structure changes, exposing the mitochondrial targeting group TPP, activating the mitochondrial targeting effect, thereby effectively delivering Celastrol (Cel) and the photothermal agent indocyanine green IR 808 and 177 Lu to mitochondria, Cel acts on the electron transfer chain complex I of the mitochondrial inner membrane, blocking the mitochondrial energy supply, and at the same time through IR 808 The high temperature and 177 Lu internal irradiation ablates mitochondria, enhances the intensity of the effect on mitochondria, triggers mitochondria-mediated cell apoptosis, and achieves the purpose of inhibiting tumor growth and metastasis. Therefore, the mitochondria-targeted nano anticancer drug (Glu-PEG-Azo / Mito-Cel) constructed by the present invention is mediated by tumor angiogenesis Glu transporter and hypoxia response. 808 / 177 Lu). It has high drug loading, good hypoxia sensitivity, pH-sensitive drug release, excellent photothermal conversion efficiency and internal irradiation efficiency. It has excellent cellular uptake and mitochondrial targeting in A549 cells overexpressing GLUT1 under hypoxic conditions, and has stronger cytotoxicity, apoptosis and metastasis inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a flow chart of the preparation of nano anticancer drugs in one embodiment of the present invention;

[0034] Figure 2 It is the H NMR spectrum of the conjugate;

[0035] Among them, a: PAMAM-TPP; b: TPP-PAMAM-IR808; c:

[0036] Glu-PEG-Azo / TPP-PAMAM-IR808; d: mPEG-Azo / TPP-PAMAM-IR808;

[0037] Figure 3 Figure 2 is a diagram showing the transport of different conjugates by A549 cells;

[0038] Figure 4 This is a diagram showing the localization of the conjugate in A549 cells under a laser confocal microscope;

[0039] Figure 5 This is a diagram showing the localization of the conjugate in A549 cells under hypoxic conditions under a laser confocal microscope.

[0040] Figure 6 The uptake of Glu-PEG-Azo / Mito808 at different concentrations by A549 cells;

[0041] Figure 7 The uptake of Glu-PEG-Azo / Mito808 by A549 cells at different incubation times. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0044] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0045] The present invention is described in further detail below with reference to the accompanying drawings.

[0046] In the present invention,

[0047] Glycosylated polyethylene glycol activated ester (Glu-PEG-NHS) was purchased from Beijing Jiankai Technology Co., Ltd.;

[0048] PAMAM used 4th generation polyamidoamine dendrimer (PAMAM G4), purchased from SIGMA ALDRICH;

[0049] Celastrol (Cel): purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0050] Indocyanine Green IR 808 : Purchased from Shanghai Kaiyulin Pharmaceutical Technology Co., Ltd.;

[0051] 177 Lu: purchased from Chengdu Newright Medical Technology Co., Ltd.;

[0052] p-Hydroxyazobenzene (Azo): purchased from J&K Technology Co., Ltd.;

[0053] Triphenylphosphine (TPP): purchased from ArkPharm.

[0054] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0055] Example 1

[0056] Nano anticancer drugs that combine chemotherapy, photothermal therapy and radiotherapy, such as Figure 1 As shown, the preparation method thereof specifically comprises the following steps:

[0057] Step 1: preparing a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector, specifically comprising:

[0058] Step 1-1, respectively prepare the bifunctional group Glu-PEG-Azo-COOH derivative and Mito 808 ,in,

[0059] A. Preparation of bifunctional Glu-PEG-Azo-COOH derivatives:

[0060] 1) Modification of Azo on Glu-PEG: Glu-PEG-NHS (400.0 mg, 0.2 mmol) and Azo (215.0 mg, 1.0 mmol) were accurately weighed, and dichloromethane was added to dissolve them respectively. After dissolution, they were added to the reaction bottle, and EDCI (192.0 mg, 1.0 mmol) and HoBt (135.0 mg, 1.0 mmol) were used as catalysts. The mixture was stirred for 24 h at room temperature in the dark. After the reaction was completed, the reaction solution was spin-dried, redissolved with water, and dialyzed for 24 h in the dark to remove unreacted Azo and catalyst. After the dialysis was completed, the dialyzate was centrifuged in a centrifuge (12000 rpm, 30 min), the lower precipitate was washed twice with pure water, and centrifuged under the above conditions. The supernatant was combined, filtered through a 0.22 μm microporous membrane, and lyophilized to obtain the Glu-PEG-Azo conjugate with a yield of about 48%.

[0061] 2) Introducing carboxyl groups into the Glu-PEG-Azo modified conjugate: Carboxyl groups of the Glu-PEG-Azo conjugate were modified by esterification reaction to synthesize bifunctional Glu-PEG-Azo-COOH derivatives, specifically:

[0062] Succinic anhydride (160.0 mg, 1.6 mmol) and Glu-PEG-Azo conjugate (352.0 mg, 0.16 mmol) were accurately weighed and dissolved in dichloromethane. Triethylamine (24.0 mg, 0.24 mmol) was used as a catalyst. The mixture was stirred at room temperature in the dark for 24 h. After the reaction, the reaction solution was spin-dried, redissolved in water, dialyzed in the dark for 24 h, filtered through a 0.22 μm microporous filter membrane, and freeze-dried to obtain a bifunctional Glu-PEG-Azo-COOH derivative with a yield of approximately 65%.

[0063] B. Preparation of Mito 808 :

[0064] 1) Preparation of PAMAM-TPP conjugate: Triphenylphosphine (TPP) (100.0 mg, 0.22 mmol) was accurately weighed and added to a reaction bottle, dissolved in DMSO, and EDCI (42.0 mg, 0.23 mmol) and HoBt (30.0 mg, 0.23 mmol) were used as catalysts. The mixture was stirred at room temperature for 2 h, and PAMAM (626.0 mg, 4.4 × 10 -2 mmol) methanol solution was redissolved in DMSO and added to the above reaction solution. The mixture was stirred at room temperature for 24 h. After the reaction, the reaction solution was dialyzed for 24 h to remove unreacted TPP and catalyst. The mixture was filtered through a 0.22 μm microporous membrane and freeze-dried to obtain PAMAM-TPP conjugate with a yield of about 76%.

[0065] 2) Preparation of TPP-PAMAM-IR808 conjugate: IR808 (137.0 mg, 0.18 mmol) was accurately weighed and dissolved in DMSO. The mixture was stirred at room temperature in the dark for 2 h according to the above reaction conditions. PAMAM-TPP (591.0 mg, 3.59×10 -2 mmol) was stirred at room temperature in the dark for 24 h. After the reaction was completed, the unreacted IR808 and catalyst were removed by dialysis under dark conditions, filtered through a 0.22 μm microporous membrane, and lyophilized to obtain TPP-PAMAM-IR808 (Mito 808 ) conjugate, the yield is about: 69%.

[0066] Step 1-2: Bifunctional Glu-PEG-Azo-COOH derivatives and Mito 808 Coupling to obtain Glu-PEG-Azo / Mito 808 The conjugate is a mitochondrial targeting vector mediated by GLUT1 and responding to hypoxia; specifically comprising:

[0067] The bifunctional Glu-PEG-Azo-COOH derivative (342.0 mg, 0.15 mmol) was accurately weighed into a reaction bottle, dissolved in DMSO, and EDCI (29.0 mg, 0.15 mmol) and HoBt (20.0 mg, 0.15 mmol) were used as catalysts. The mixture was stirred at room temperature in the dark for 2 h. Mito 808 (604.0 mg, 0.03 mmol), continued to stir at room temperature in the dark for 24 h, after the reaction was completed, the reaction solution was dialyzed for 24 h in the dark to remove the unreacted bifunctional Glu-PEG-Azo-COOH derivative and catalyst, filtered through a 0.22 μm microporous membrane, and freeze-dried to obtain Glu-PEG-Azo / Mito 808 Conjugate.

[0068] The structures of the conjugates prepared above were characterized. Figure 2 .

[0069] Step 2: Drug loading:

[0070] Accurately weigh an excess of Celastrol (100.0 mg, 0.220 mmol) into a reaction flask, add 10 mL of methanol to dissolve it, and then add Glu-PEG-Azo / Mito 808 (500.0 mg, 1.56 × 10 -2 mmol), stirred overnight at room temperature in the dark, the reaction solution was spin-dried, re-dissolved in water, centrifuged at 9000 rpm for 30 min, the supernatant was taken, filtered through a 0.22 μm microporous membrane, and freeze-dried.

[0071] Step 3, loading nuclide:

[0072] Take the drug-loaded conjugate Glu-PEG-Azo / Mito-Cel 808 (2Mm, 50μl in total) was cooled to room temperature, and 2μl (4nmol) was added to 40μl 0.5M NaAc buffer and mixed well. 4nmolGlu-PEG-Azo / Mito-Cel 808 Add 177Lu 8mCi and mix well. After 37℃ and 60min metal bath, Glu-PEG-Azo / Mito-Cel was obtained. 808 / 177 Lu, the labeling rate of the radionuclide conjugate was greater than 90% as determined by ultrafiltration centrifugation.

[0073] Example 2: Optimization test of process conditions of nano anticancer drugs

[0074] 1. Test method:

[0075] Based on the preparation method described in Example 1, the single factor method was used to investigate the effects of solvent type, solution pH and temperature on drug loading, encapsulation efficiency, particle size and potential. The results are shown in Tables 1-3.

[0076] 2. Test results

[0077] 1. Effect of solvent type on drug loading, encapsulation efficiency, particle size and potential in step 2 drug loading:

[0078] Table 1

[0079]

[0080]

[0081] 2. Effect of solution pH on drug loading, encapsulation efficiency, particle size and potential in step 2:

[0082] Table 2

[0083] Solution pH Drug loading% Encapsulation rate% Particle size (nm) Potential (mV) PBS (pH 5.6) 7.41 45.73 189.3 -12.0 PBS (pH 7.0) 11.53 65.75 185.6 -12.8 PBS (pH 8.0) 14.53 85.19 191.6 -12.6

[0084] 3. Effect of temperature on drug loading, encapsulation efficiency, particle size and potential in step 2 drug loading:

[0085] Table 3

[0086] Temperature(℃) Drug loading% Encapsulation rate% Particle size (nm) Potential (mV) 30 15.19 91.06 184.3 -12.8 35 15.03 89.76 192.5 -11.2 40 15.08 90.32 187.4 -12.3

[0087] As can be seen from Tables 1-3, the nano anticancer drug (Glu-PEG-Azo / Mito-Cel 808 / 177The optimal process conditions for the preparation of Lu) are: stirring with methanol as solvent at room temperature, with a drug loading of 16.13%, an encapsulation efficiency of 96.36%, a particle size of 192.6nm, and a potential of -12.3mV.

[0088] Example 3: Drug release test

[0089] 1. Test methods

[0090] Under hypoxic conditions, Glu-PEG-Azo / Mito-Cel 808 / 177 The hypoxic properties were investigated by observing the changes in the ultraviolet absorption spectrum of the Lu conjugate. The concentration dependence and power dependence of the photothermal effect were investigated under irradiation with 808nm near-infrared light from a fiber-coupled laser emitter. Different pH release media (neutral, acidic, and alkaline) were prepared to simulate the normal in vivo, tumor microenvironment, and mitochondrial matrix environment, respectively. The cumulative release rate of celastrol was determined, and the pH sensitivity of the release was investigated. The results are shown in Tables 4-6.

[0091] 2. Results and Analysis

[0092] 1. Concentration dependence:

[0093] Table 4

[0094]

[0095] 2. Power dependence:

[0096] Table 5

[0097] <![CDATA[Fiber Optic Coupled Laser Transmitter (W / CM 2 )]]> Temperature change (℃) power 0.5 18.4 1.0 21.6 1.5 33.4

[0098] 3. pH sensitivity of drug release

[0099] Table 6

[0100] pH of release medium Cumulative drug release rate 5.0 23.42％ 7.3 31.64％ 8.0 75.21％

[0101] From Table 4-5, it can be seen that the nano anticancer drug Glu-PEG-Azo / Mito-Cel 808 / 177 The temperature change of Lu under 808nm near-infrared light irradiation shows obvious concentration dependence and laser power dependence;

[0102] As shown in Table 6, the nano anticancer drug Glu-PEG-Azo / Mito-Cel 808 / 177 The in vitro release behavior of Lu showed significant pH dependence.

[0103] Comparative Example 1:

[0104] Mito 808 Preparation, the same as Mito in Example 1 808 preparation.

[0105] Comparative Example 2:

[0106] mPEG / Mito 808 Preparation: Same as Example 1, except that a carboxyl group was introduced into mPEG, and then mPEG-COOH and Mito 808 The coupling is carried out through esterification reaction under the action of catalyst EDCI and HoBt.

[0107] Comparative Example 3:

[0108] Glu-PEG / Mito 808 Preparation: Same as Example 1, except that a carboxyl group was introduced into Glu-PEG, and then Glu-PEG-COOH was reacted with Mito 808 The coupling is carried out through esterification reaction under the action of catalyst EDCI and HoBt.

[0109] Comparative Example 4

[0110] mPEG-Azo / Mito 808 Preparation: Same as Example 1, except that after Azo was modified on mPEG, a carboxyl group was introduced, and then mPEG-Azo-COOH was reacted with Mito 808 The coupling is carried out through esterification reaction under the action of catalyst EDCI and HoBt.

[0111] Effect example: Confocal laser scanning microscopy to evaluate GLUT1 targeting

[0112] 1. Test methods

[0113] A549 cells were cultured in 96-well plates at a concentration of 1 × 10 4 After 24 h, the cells were incubated with 100 μL of Mito 808 (from Comparative Example 1), mPEG / Mito 808 (from Comparative Example 2), Glu-PEG / Mito 808 (from Comparative Example 3), mPEG-Azo / Mito 808 (from Comparative Example 4) and Glu-PEG-Azo / Mito 808 The cells were incubated with complete medium containing the conjugate (derived from Example 1) (in terms of IR808, the final concentration was 6 μmol / L) for 8 h. After the incubation, the medium containing the conjugate was removed, and the cells were washed three times with pre-cooled PBS. Laser confocal microscopy was used to observe and photograph for qualitative analysis to examine the ability of different conjugates to be transported by GLUT1. The results are shown in FIG. Figure 3 To confirm that the increase in cellular uptake was due to the specific recognition of GLUT1 on the substrate Glu, the Glu-PEG-Azo / Mito 808 +Glu group, that is, before adding each conjugate, the cells were incubated with Glu at a concentration of 2.5mmol / L for 4h to block the transport of Glu on the conjugate by GLUT1. Similarly, the three different concentrations of Glu-PEG-Azo / Mito 808 The ability of the conjugates (calculated as IR808, the final concentrations were 1.5, 3, and 6 μmol / L, respectively) to be transported by GLUT1 was evaluated to evaluate the concentration dependence of GLUT1 transport in A549 cells. The results are shown in Figure 6 Cells were incubated with 6 μmol / L Glu-PEG-Azo / Mito 808 The conjugates were co-incubated for 2, 4, and 8 h, respectively, and photographed under a microscope to evaluate the time dependence of GLUT1 transport in A549 cells. The results are shown in Figure 7 .

[0114] 1) The ability of different conjugates to be transported by GLUT1 was investigated by laser confocal microscopy and the following findings were found: Figure 3 As shown in the figure, the conjugate modified with GLUT1 substrate Glu has a significantly increased uptake capacity by A549 cells compared with the conjugate without Glu modification, and the increased uptake can be blocked by pre-incubation with Glu. Therefore, it can be explained that the reason for the increased cellular uptake is the specific recognition and transport of Glu targeting ligand by GLUT1; while Mito 808 The conjugate also showed strong cellular uptake ability, which may be due to its strong positive charge, which can be taken up by cells through non-specific charge effects;

[0115] 2) By Figure 6 and Figure 7 A549 cells are sensitive to Glu-PEG-Azo / Mito 808 The uptake of 5-mercaptoethanol was concentration- and time-dependent, and the cellular uptake increased with the increase of co-incubation time and concentration.

[0116] Effect Example 2: Mitochondrial Targeting Evaluation

[0117] 1. Test methods

[0118] A549 cells were cultured in laser confocal microscopy at a concentration of 1 × 10 6 Cells were planted at a density of 100 μl / well. After 24 h, 3 mL of Mito 808 (from Comparative Example 1), mPEG / Mito 808 (from Comparative Example 2), Glu-PEG / Mito 808(From Comparative Example 3) The culture medium of the conjugate (in terms of IR808, the final concentration is 1.5 μmol / L) was incubated with the cells for 8 hours, and then the culture medium containing the conjugate was carefully removed with a pipette, and the cells were washed 3 times with pre-cooled PBS to remove the conjugate that was not taken up by the cells. Then, MitoTracker Green mitochondrial fluorescent probe reagent at a concentration of 50 nmol / L was added, and incubated in the dark for 30 minutes in an incubator; then 1 mL of Hoechst 33342 (10 μg / mL) nuclear fluorescent probe reagent was added, and the cells were incubated in the dark for another 20 minutes, and then the cells were washed 3 times with pre-cooled PBS to wash away the free dye in the culture medium. The distribution of different fluorescence in the cells was observed under a laser confocal microscope, and correlation analysis was performed. The results are shown in Figure 4 .

[0119] To evaluate hypoxic responsiveness, mPEG-Azo / Mito 808 +Hypoxia group (mPEG-Azo / Mito 808 Derived from Comparative Example 4) and Glu-PEG-Azo / Mito 808 +Hypoxia group (Glu-PEG-Azo / Mito 808 From Example 1), that is, the cells were incubated with the conjugate under hypoxic conditions for 8 h. Figure 5 .

[0120] 2. Test results and analysis

[0121] The intracellular localization of each conjugate after being taken into cells was observed by laser confocal microscopy. Figure 4-5 As shown in the figure, IR808 itself exhibits red fluorescence, so the red fluorescence represents the distribution of the conjugate; the cell nucleus is labeled with blue fluorescence by Hoechst 33342 nuclear fluorescent probe reagent; the mitochondria are labeled with green fluorescence by MitoTracker Green mitochondrial fluorescent probe reagent. When PAMAM is connected to the mitochondrial targeting group TPP, the conjugate mostly accumulates in the mitochondrial part after entering the cell, with obvious mitochondrial targeting; but when PAMAM is modified with PEG, it will obviously hinder the mitochondrial targeting effect of TPP, and it can be seen that mPEG-Azo / Mito 808 Conjugates and Glu-PEG-Azo / Mito 808 The conjugates are more localized in the nucleus, and under hypoxic conditions, mPEG-Azo / Mito 808 Conjugates and Glu-PEG-Azo / Mito 808The conjugate then aggregated in the mitochondrial area, indicating that the mitochondrial targeting effect is hypoxia-dependent. This is related to the fact that the azo bond can only be broken under hypoxic response conditions, thereby exposing the mitochondrial targeting group TPP and achieving mitochondrial targeting.

[0122] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector, characterized in that: The steps include: Step 1: conjugate p-hydroxyazobenzene Azo to Glu-PEG-NHS and modify the carboxyl group to obtain a Glu-PEG-Azo-COOH derivative; modify the mitochondrial targeting group TPP and the photothermal agent IR808 on the surface of PAMAM to obtain a TPP-PAMAM-IR808 conjugate, i.e., Mito 808 ; Step 2, Glu-PEG-Azo-COOH derivative and Mito 808 Esterification reaction occurred to obtain Glu-PEG-Azo / Mito 808 The conjugate is a GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector.

2. The preparation method according to claim 1, characterized in that In step 1, p-hydroxyazobenzene Azo is coupled to Glu-PEG-NHS and the carboxyl group is modified to obtain a Glu-PEG-Azo-COOH derivative, including: First, in solvent A, Glu-PEG-NHS reacts with p-hydroxyazobenzene Azo under the action of catalysts EDCI and HoBt at room temperature in the dark to generate Glu-PEG-Azo conjugate; Then, in solvent B, the Glu-PEG-Azo conjugate and succinic anhydride undergo esterification reaction under the action of catalyst triethylamine at room temperature and in the dark to generate a Glu-PEG-Azo-COOH derivative.

3. The preparation method according to claim 2, characterized in that: The solvent A and the solvent B are both dichloromethane.

4. The preparation method according to claim 1, characterized in that: In step 1, the photothermal agent IR 808 The surface of PAMAM was modified with triphenylphosphine TPP to obtain Mito 808 ,include: First, in solvent C, triphenylphosphine TPP reacts with PAMAM under the action of catalysts EDCI and HoBt at room temperature to obtain a PAMAM-TPP conjugate; Then, in solvent D, the PAMAM-TPP conjugate reacted with IR808 at room temperature in the dark to obtain Mito 808 .

5. The preparation method according to claim 4, characterized in that: The solvent C and the solvent D are both DMSO.

6. The preparation method according to claim 1, characterized in that: In step 2, the Glu-PEG-Azo-COOH derivative is reacted with Mito 808 Esterification reaction occurred to obtain Glu-PEG-Azo / Mito 808 The conjugate comprises the following steps: In DMSO solvent, Glu-PEG-Azo-COOH derivatives were reacted with Mito 808 Under the action of catalyst EDCI and HoBt, esterification reaction occurred at room temperature and in the dark to obtain Glu-PEG-Azo / Mito 808 Conjugate.

7. A GLUT1-mediated and hypoxia-responsive mitochondrial targeting vector prepared according to the preparation method of any one of claims 1 to 6.

8. A nano anticancer drug combining chemotherapy, photothermal therapy and radiotherapy, characterized in that: The GLUT1-mediated and hypoxia-responsive mitochondrial targeting carrier as claimed in claim 7 is used to load the anti-tumor drug Cel and the radionuclide 177 Lu, obtain Glu-PEG-Azo / Mito-Cel 808 / 177 Lu is a nano anticancer drug that combines chemotherapy, photothermal therapy and radiotherapy functions.

9. The nano anticancer drug combining chemotherapy, photothermal therapy and radiotherapy as claimed in claim 8, characterized in that: Glu-PEG-Azo / Mito-Cel 808 / 177 The particle size of Lu is 150 to 200 nm.

10. The method for preparing the nano anticancer drug combining chemotherapy, photothermal therapy and radiotherapy as claimed in claim 8, characterized in that: include: First, Cel was stained with Glu-PEG-Azo / Mito 808 The conjugate is adsorbed to obtain the drug-loaded conjugate Glu-PEG-Azo / Mito-Cel 808 ; Then, in sodium acetate buffer, at 37°C, 60 min metal bath conditions, 177Lu 8mCi was loaded with the drug-loaded conjugate Glu-PEG-Azo / Mito-Cel 808 Adsorption to obtain Glu-PEG-Azo / Mito-Cel 808 / 177 Lu.

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