Nano prodrug for reversing multidrug resistance in NIR-II photothermal therapy and preparation method and application thereof

Abstract

The invention discloses a nano prodrug for reversing multidrug resistance in NIR-II photothermal therapy and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The preparation method comprises the following steps: preparing a procyanidine-gold conjugate nano composite material solution from procyanidine by a one-pot method; or preparing a gold nanorod solution by using a hydroquinone seed-free growth method; preparing a doxorubicin prodrug monomer which is encapsulated by lipidosome and modified by a disulfide bond; and preparing the procyanidine-gold conjugated nano prodrug or the hydroquinone-gold conjugated nano prodrug. The prepared nano prodrug is uniform in particle size distribution and high in DOX drug loading capacity, presents pH-GSH-NIR-II responsive drug release, remarkably improves the targeting sensitivity of drug-resistant cells, improves the sensitivity of the drug-resistant cells through the synergistic effect of chemotherapy / photothermal therapy, and achieves the efficient and low-toxicity tumor inhibition effect.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and specifically relates to a nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy, and a preparation method and application thereof. Background Art

[0002] The small molecule chemotherapeutic drug doxorubicin hydrochloride (DOX·HCl) is usually accompanied by significant cardiotoxic side effects and the risk of P-glycoprotein (P-gp)-dependent multidrug resistance. Research has found that by means such as transmembrane transport through liposome encapsulation and responsive nano-prodrug modification, drug leakage can be reduced. However, at normal body temperature, the liposome membrane presents a densely arranged liquid crystalline state, and hydrophilic drugs are difficult to diffuse out through the liposome membrane. Research has found that thermosensitive liposomes can be transformed into a controllable fluid at local high temperatures, and the thermal motion of drug molecules is promoted by high-temperature induction to promote drug release. However, local high temperatures easily induce rapid heat stress in cells, resulting in reduced chemotherapy efficiency. How to reduce the expression of P-gp through mild hyperthermia so as to reverse the drug resistance side effects is still the key to the current design of thermosensitive liposome prodrugs.

[0003] Gold nanoparticles exhibit excellent photothermal stability due to their special surface plasmon resonance effect. However, current common preparation methods such as the template method, photochemical method, electrochemical method, seed-mediated method, one-step chemical oxidation method, and seedless growth method still need to solve problems such as complex operations, long reaction times, template toxicity, and low photothermal conversion efficiency. Therefore, it is urgent to develop green synthesis, high photothermal conversion efficiency, and near-infrared second region (NIR-II)-responsive gold nanomaterials.

[0004] Proanthocyanidins (OPC) are naturally occurring polyphenolic compounds, which can reduce oxidative stress damage, prevent cancer progression, and promote apoptosis by downregulating molecular content, scavenging free radicals, and inhibiting signal pathways. Based on the reducibility and stability of polyphenols, hydrothermal methods and one-pot methods can be used to design polyphenol-gold conjugate nanoparticles.

[0005] Therefore, how to provide a nano-prodrug that can achieve mild photothermal under NIR-II light irradiation, improve the photothermal conversion efficiency, reduce the expression of P-gp, thereby improving the targeting sensitivity of drugs to drug-resistant cells and reversing multidrug resistance, as well as a preparation method and application thereof, is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy, and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A preparation method of a nano - prodrug for reversing multidrug resistance in NIR - Ⅱ photothermal therapy, comprising the following steps:

[0009] (1) Prepare an OPC - Au NPs (procyanidin - gold conjugate nanocomposite) solution: Stir and heat an aqueous solution of chloroauric acid to boiling, then add an aqueous solution of procyanidin and heat - stir to obtain an OPC - Au NPs solution. Centrifuge to collect the precipitate and make up the volume for storage;

[0010] Or, prepare an Au NRs (gold nanorods) solution: Sequentially add an aqueous solution of chloroauric acid, an aqueous solution of silver nitrate, hydrochloric acid, and an aqueous solution of hydroquinone to an aqueous solution of cetyltrimethylammonium bromide and stir, then add an aqueous solution of sodium borohydride and stir again. Age to obtain an Au NRs solution. Centrifuge to collect the precipitate and make up the volume for storage;

[0011] (2) Prepare Lipid (lipid) DOX - ss (liposome - encapsulated doxorubicin prodrug monomer modified with a disulfide bond): Dissolve DSPE - PEG 2000 - NHS (1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - N - hydroxysuccinimide - polyethylene glycol 2000) and DOX - ss (doxorubicin prodrug monomer modified with a disulfide bond) in DMSO (dimethyl sulfoxide), add triethylamine and stir. Dialyze the stirred sample and freeze - dry to obtain Lipid DOX - ss;

[0012] (3) Prepare OPC - Au / DOX - ss LNPs (procyanidin - gold conjugate nano - prodrug) or Au / DOX - ss LNRs (hydroquinone - gold conjugate nano - prodrug): Dissolve Lipid DOX - ss, DPPC (dipalmitoylphosphatidylcholine), DSPE - PEG 2000 - SH (1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - polyethylene glycol 2000 - mercapto), and cholesterol in a mixed solution of chloroform and methanol, perform rotary evaporation and vacuum drying, then add a diluted solution of the OPC - Au NPs solution or the Au NRs solution obtained in step (1) and stir. Centrifuge and collect the precipitate to obtain the above - mentioned nano - prodrug for reversing multidrug resistance in NIR - Ⅱ photothermal therapy, denoted as OPC - Au / DOX - ss LNPs and Au / DOX - ss LNRs.

[0013] Furthermore, in step (1), when preparing the OPC - Au NPs solution, the molar ratio of chloroauric acid to procyanidin is 4.53:1.

[0014] Even further, in step (1), when preparing the OPC - Au NPs solution, the concentration of the chloroauric acid aqueous solution is 0.24 mg / mL, and the concentration of the procyanidin aqueous solution is 2 mg / mL.

[0015] Further, in step (1), in the preparation of the AuNRs solution, the molar ratio of cetyltrimethylammonium bromide, chloroauric acid, silver nitrate, hydrogen chloride, hydroquinone, and sodium borohydride is 1000:40:23:100:540:1.

[0016] Furthermore, in step (1), in the preparation of the AuNRs solution, the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1 mol / L, the concentration of the chloroauric acid aqueous solution is 10 mmol / L, the concentration of the silver nitrate aqueous solution is 100 mmol / L, the concentration of hydrochloric acid is 1 mol / L, the concentration of the hydroquinone aqueous solution is 0.1 mol / L, and the concentration of the NaBH4 aqueous solution is 0.1 mmol / L.

[0017] Further, in step (1), the chloroauric acid aqueous solution is heated to boiling, and then procyanidins are added and heated with stirring until the solution changes from light yellow to dark red; the chloroauric acid aqueous solution, silver nitrate aqueous solution, hydrochloric acid, and hydroquinone aqueous solution are sequentially added to the cetyltrimethylammonium bromide aqueous solution and stirred until the color of the solution changes from orange to light yellow and gradually becomes clear and transparent, and then sodium borohydride is added and stirred again for 15 s, with a stirring speed of 400 rpm; the aging temperature is 25 °C, the aging time is 24 h, after centrifugation once, the centrifugation speed is 12000 rpm, the centrifugation time is 40 min, and the precipitate is diluted to a certain concentration and stored at 4 °C as a storage solution.

[0018] Furthermore, in step (1), the chloroauric acid aqueous solution is heated to boiling, and then procyanidins are added and heated with stirring until the solution changes from light yellow to dark red, the heating temperature is 110 °C, the stirring time is 10 min, and the stirring speed is 400 rpm; the chloroauric acid aqueous solution, silver nitrate aqueous solution, hydrochloric acid, and hydroquinone aqueous solution are sequentially added to the cetyltrimethylammonium bromide aqueous solution and stirred until the color of the solution changes from orange to light yellow and gradually becomes clear and transparent, the stirring speed is 400 rpm, the stirring time is 15 min, and then the sodium borohydride aqueous solution is added and stirred again for 15 s, with a stirring speed of 400 rpm.

[0019] Further, in step (2), the molar ratio of DSPE-PEG 2000-NHS and DOX-ss is 1:3.14, and the mass-volume ratio of DSPE-PEG2000-NHS, DOX-ss, DMSO, and triethylamine is 67 mg:20 mg:10 mL:12 μL.

[0020] Further, in step (2), triethylamine is added and stirred for 24 h, with a stirring speed of 600 rpm, the stirred sample is dialyzed and freeze-dried, the molecular weight cut-off of the dialysis membrane used for dialysis is 2000 Da, the dialysis time is 48 h, the freeze-drying temperature is -40 °C, the vacuum degree is 200 mTorr, and the freeze-drying time is 12 h.

[0021] Furthermore, in step (3), the mass-volume ratio of Lipid DOX-ss, DPPC, DSPE-PEG 2000-SH, cholesterol, chloroform, methanol and the dilution of OPC-Au NPs solution or the dilution of Au NRs solution is 50 mg: 25 mg: 18.5 mg: 12.5 mg: 7.5 mL: 2.5 mL: 18 mL. In the dilution of OPC-Au NPs solution or the dilution of Au NRs solution, the Au concentration is 0.1 mg / mL.

[0022] Furthermore, in step (3), the temperature of rotary evaporation is 40 °C, the time of rotary evaporation is 30 min, and the rotation rate is 100 r / min; the temperature of vacuum drying is 40 °C, the time of vacuum drying is 12 h, and the vacuum degree is -0.08 MPa; the stirring speed is 400 rpm, and the stirring time is 24 h; centrifuge twice. After the first centrifugation, take the precipitate and centrifuge again. Take the precipitate. The centrifugation speed is 12,000 rpm, and the centrifugation time for each time is 15 min.

[0023] The present invention also provides a nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy prepared by the above method. The particle size of the above OPC-Au / DOX-ss LNPs is 141.8 nm, and the particle size of the above Au / DOX-ss LNRs is 122.4 nm.

[0024] The present invention also provides an application of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy in the preparation of anti-tumor drugs.

[0025] Advantages of the present invention: The present invention synthesizes procyanidin-gold conjugate nanocomposites OPC-AuNPs by using procyanidin as a reducing agent in a one-pot method, and synthesizes gold nanorods AuNRs by using hydroquinone in a seedless growth method. Subsequently, doxorubicin prodrug monomer DOX-ss modified with disulfide bonds is encapsulated by liposomes, and then modified with thermosensitive liposomes prepared based on dipalmitoylphosphatidylcholine DPPC. The preparation method is simple. Among them, hydroquinone as a reducing agent can significantly increase the reduction rate of gold ions and reduce the non-uniformity of gold nanorods during the synthesis process; procyanidin OPC is a polyphenolic compound mainly present in fruits, vegetables, flowers, wines, nuts, beans and seeds. As a natural antioxidant, OPC has good pharmacological activities in protecting cells from oxidative damage, and also has protective effects on neurodegenerative diseases such as cancer, cardiovascular diseases and Alzheimer's disease. The pharmacological properties of OPC have been proven to have low toxicity and no potential genotoxicity, and are expected to become natural multidrug resistance reagents for combined use with traditional chemotherapy drugs; OPC-AuNPs and AuNRs exhibit NIR-II responsiveness. Compared with NIR-I light, NIR-II light has high light transmittance, low background signal and high safety limit; the sensitivity of disulfide bonds to reducing agents such as GSH (glutathione) enables them to quickly break in tumor cells. In the tumor microenvironment, the concentration of GSH is much higher than that in normal cells. Therefore, disulfide bonds can achieve selective drug release in tumor cells, thereby reducing damage to normal cells; DPPC has excellent biodegradability, drug encapsulation and thermosensitivity. Thermosensitive liposomes based on DPPC, as a drug delivery system with stimulus-responsive characteristics combined with hyperthermia, can increase the vascular permeability and interstitial transport of drugs in the diseased area, while minimizing the delivery of drugs to key normal tissues. On the other hand, hyperthermia can enhance the permeability of the lipid bilayer, thereby promoting the release of the payload.

[0026] In the present invention, gold nanoparticles AuNRs and OPC-Au NPs are synthesized by using hydroquinone in a seedless growth method and procyanidin in a one-pot method respectively. By using Au-S covalent coupling, near-infrared responsive gold nanodrug Au / DOX-ss LNRs and OPC-Au / DOX-ss LNPs are synthesized through encapsulation with thermosensitive liposomes. The system of the present invention can achieve pH-GSH-NIR responsive drug release, and improve the targeting sensitivity to drug-resistant cells through the synergistic treatment of chemotherapy / photothermal therapy, achieving a highly efficient and low-toxic tumor suppression effect, thereby providing a theoretical basis for the treatment of breast cancer drug resistance.

[0027] OPC-Au NPs and Au NRs exhibit NIR-II responsiveness; the disulfide bond endows doxorubicin with GSH responsiveness; secondly, the encapsulation of thermosensitive liposomes can improve the drug encapsulation efficiency and drug loading rate, reduce the toxic side effects of DOX, enhance the targeting sensitivity of DOX, and under near-infrared irradiation, the thermosensitive liposomes can minimize the delivery of drugs to normal tissues and enhance the release of drugs at the tumor site; the nano-prodrugs prepared in the present invention have a uniform particle size distribution and stable physicochemical properties. The encapsulation efficiency and drug loading rate of the procyanidin-gold conjugate nano-prodrug OPC-Au / DOX-ss LNPs and the hydroquinone-gold conjugate nano-prodrug Au / DOX-ss LNRs for DOX are 84.8%, 29.66% and 94.80%, 26.70% respectively.

[0028] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) Au NRs synthesized with hydroquinone as a reducing agent and OPC-Au NPs synthesized with procyanidin as a reducing agent exhibit NIR-II responsiveness. Compared with visible light and NIR-I light, NIR-II light can effectively penetrate biological tissues and induce apoptosis of tumor cells.

[0030] (2) The disulfide bond in the doxorubicin prodrug monomer is sensitive to GSH, and the NIR-II-GSH cascade response can achieve selective drug release in tumor cells and reduce damage to normal cells.

[0031] (3) The thermosensitive liposomes prepared based on DPPC can control the temperature change through the mild photothermal response of NIR-II, regulate the drug release, significantly improve the therapeutic effect of the drug, and reverse multi-drug resistance. Description of the Drawings

[0032] Figure 1 For the chemical property characterization of OPC-Au NPs in Example 1, wherein, Figure A is the particle size, Figure B is the Zeta potential, Figure C is the ultraviolet absorption spectrum, and Figure D is the TEM image;

[0033] Figure 2 For the particle size analysis of Au NRs synthesized under different doses of NaBH4, wherein, Figure A is Comparative Example 1, Figure B is Comparative Example 2, Figure C is Comparative Example 3, and Figure D is Comparative Example 4;

[0034] Figure 3 For the particle size analysis of Au NRs synthesized under different doses of HCl and AgNO3, wherein, Figure A is Comparative Example 5, Figure B is Comparative Example 6, Figure C is Comparative Example 7, and Figure D is Comparative Example 8;

[0035] Figure 4Potential diagrams and ultraviolet absorption spectra of Au NRs synthesized under different conditions. Among them, diagrams A - C are potential diagrams of Au NRs synthesized in Comparative Examples 1 - 8, and diagrams D - F are ultraviolet absorption spectra of Au NRs synthesized in Comparative Examples 1 - 8;

[0036] Figure 5 Chemical property characterization of OPC - Au / DOX - ss LNPs in Example 1. Among them, diagram A is the particle size, diagram B is the Zeta potential, diagram C is the ultraviolet absorption spectrum, and diagram D is the FT - IR;

[0037] Figure 6 Chemical property characterization of Au / DOX - ss LNRs in Example 2. Among them, diagram A is the particle size, diagram B is the Zeta potential, diagram C is the ultraviolet absorption spectrum, and diagram D is the FT - IR;

[0038] Figure 7 In vitro drug release of OPC - Au / DOX - ss LNPs in Application Example 1;

[0039] Figure 8 In vitro drug release of Au / DOX - ss LNRs in Application Example 1;

[0040] Figure 9 Hemolysis analysis diagrams of OPC - Au / DOX - ss LNPs and Au / DOX - ss LNRs in Application Example 2. Among them, diagram A is the hemolysis experiment of OPC - Au / DOX - ss LNPs, diagram B is the hemolysis experiment of Au / DOX - ss LNRs, diagram C is the hemolysis rate of OPC - Au / DOX - ss LNPs, and diagram D is the hemolysis rate of Au / DOX - ss LNRs;

[0041] Figure 10 Photothermal conversion cyclic curves of each carrier and drug - loaded nanoparticles in Application Example 3. Among them, diagram A is OPC - AuNPs, diagram B is OPC - Au / DOX - ss LNPs, diagram C is Au NRs, and diagram D is Au / DOX - ss LNRs;

[0042] Figure 11 Cell activities after the blank carrier and polyphenol - gold nanodrug - prodrug act on L - 02 and MCF - 7 / ADR cells for 24 h in Application Example 4. Among them, diagram A is the cell activity after the blank carrier acts on L - 02 cells for 24 h, diagram B is the cell activity after the blank carrier acts on MCF - 7 / ADR cells for 24 h, diagram C is the cell activity after the polyphenol - gold nanodrug - prodrug acts on L - 02 cells for 24 h, and diagram D is the cell activity after the polyphenol - gold nanodrug - prodrug acts on MCF - 7 / ADR cells for 24 h;

[0043] Figure 12Cell viability after 48 h of the blank carrier, polyphenol-gold nanomedicine acting on L-02 and MCF-7 / ADR cells in Application Example 4. Among them, Figure A shows the cell viability after 48 h of the blank carrier acting on L-02 cells, Figure B shows the cell viability after 48 h of the blank carrier acting on MCF-7 / ADR cells, Figure C shows the cell viability after 48 h of the polyphenol-gold nanomedicine acting on L-02 cells, and Figure D shows the cell viability after 48 h of the polyphenol-gold nanomedicine acting on MCF-7 / ADR cells;

[0044] Figure 13 For Application Example 4, the half-lethal concentration (IC 50 value) of the polyphenol-gold nanomedicine after incubating with MCF-7 / ADR cells for 24 h and 48 h.

[0045] Figure 14 Analysis of the expression level of P-gp protein after culturing various drug-loaded nanoparticles with MCF-7 / ADR cells for 24 h in Application Example 5. Among them, Figure A shows the P-gp protein band detected by Western blot, and Figure B shows the quantitative analysis of the P-gp protein. Detailed implementation manners

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0047] Preparation of DOX-ss in the embodiments of the present invention: 2.64 mL of 2-hydroxyethyl disulfide, 4.20 mL of triethylamine and 100 mL of tetrahydrofuran were successively added to a three-necked flask. After purging with N2 and cooling to 0 °C in an ice bath, 50 mL of a tetrahydrofuran solution containing 2.04 mL of methacryloyl chloride was added dropwise to the three-necked flask under vigorous stirring at a stirring speed of 600 rpm. Stir magnetically at 25 °C for 24 h with a stirring speed of 600 rpm, then filter by suction through a 0.22 μm filter membrane. The filtrate was rotary evaporated at 40 °C for 1 h with a rotation rate of 100 r / min to obtain ethyl 2-(2-hydroxyethyl) disulfonyl methacrylate. Then, 58 mg of DOX·HCl and 48 mg of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane. Under N2 protection, 274 mg of triphosgene was added, and the mixture was stirred at 25 °C for 30 min with a stirring speed of 600 rpm. Subsequently, 20 μL of ethyl 2-(2-hydroxyethyl) disulfonyl methacrylate was added dropwise, and the mixture was stirred at 25 °C for 24 h with a stirring speed of 600 rpm. After rotary evaporation at 40 °C for 30 min with a rotation rate of 100 r / min, DOX-ss was obtained by freeze-drying. The freeze-drying temperature was -40 °C, the vacuum degree was 200 mTorr, and the time was 12 h.

[0048] Example 1

[0049] A preparation method of a procyanidin-gold conjugate nanomedicine for reversing multidrug resistance in NIR-II photothermal therapy, comprising the following steps:

[0050] (1) Preparation of OPC-Au NPs solution: 25 mL of an aqueous solution of chloroauric acid with a concentration of 0.24 mg / mL was added to a round-bottom flask and magnetically stirred at a stirring speed of 400 rpm. After heating to boiling at 110 °C, 1 mL of an aqueous solution of procyanidin with a concentration of 2 mg / mL was quickly added for a reduction reaction. (The aqueous solution of procyanidin was filtered through a 0.22 μm filter head.) Heat and stir magnetically at 110 °C for 10 min until the solution changed from light yellow to dark red with a stirring speed of 400 rpm to obtain the OPC-Au NPs solution. After centrifuging at 12,000 rpm for 40 min, the obtained precipitate was made up to 4 mL with water and stored at 4 °C;

[0051] The content of gold element was detected by inductively coupled plasma optical emission spectrometer (ICP-OES). The PC-Au NPs solution after volume fixation was put into a microwave digestion tank or a polytetrafluoroethylene crucible containing acid for digestion until the digestion was completed. According to the element to be measured, standard curves were prepared according to different concentration gradients, and then the test solution was taken for on-machine testing. The content of gold element was 623.0 mg / L, and the gold recovery rate was 83%.

[0052] Add 4 mL of the OPC-Au NPs solution after volume determination to 20 mL of water to dilute the solution to a gold element concentration of 0.1 mg / mL, obtaining a diluted solution of the OPC-Au NPs solution for step (3);

[0053] (2) Preparation of Lipid DOX-ss: Dissolve 67 mg of DSPE-PEG 2000-NHS and 20 mg of DOX-ss in 10 mL of DMSO, add 12 μL of triethylamine, and stir magnetically at 25 °C for 24 h with a stirring speed of 600 rpm. Dialyze the stirred sample and freeze-dry it to obtain Lipid DOX-ss. The molecular weight cut-off of the dialysis membrane used for dialysis is 2000 Da, the dialysis time is 48 h, the freeze-drying temperature is -40 °C, the vacuum degree is 200 mTorr, and the freeze-drying time is 12 h;

[0054] (3) Preparation of OPC-Au / DOX-ss LNPs: Dissolve 50 mg of Lipid DOX-ss obtained in step (2), 25 mg of DPPC, 18.5 mg of DSPE-PEG 2000-SH, and 12.5 mg of cholesterol in a mixed solution of 10 mL of chloroform and methanol (chloroform:methanol volume ratio = 3:1). Rotate and evaporate at 40 °C for 30 min with a rotation rate of 100 r / min, and dry in vacuum at 40 °C for 12 h with a vacuum degree of -0.08 MPa. Then add 18 mL of the diluted solution of the OPC-Au NPs solution (Au concentration is 0.1 mg / mL), stir magnetically at 25 °C for 24 h with a stirring speed of 400 rpm, centrifuge twice. After the first centrifugation, take the precipitate and centrifuge again, with a centrifugation speed of 12000 rpm and a centrifugation time of 15 min each time. Take the precipitate and resuspend the procyanidin-gold conjugate nanomedicine to 5 mL, denoted as OPC-Au / DOX-ss LNPs (the procyanidin-gold conjugate nanomedicine contains water, gold is stored in an aqueous solution, gold nanomaterials are thermally sensitive during drying, prone to aggregation, and their properties change after drying and cannot be resuspended back to the dispersed state. To avoid aggregation, the final product does not need to be dried and is used as a stock solution for the next reaction).

[0055] The particle size of OPC-Au / DOX-ss LNPs is 141.8 nm. The gold element content is measured to be 40 mg / L by inductively coupled plasma optical emission spectrometer (ICP-OES), and the gold recovery rate is 11.45%.

[0056] Example 2

[0057] A preparation method of hydroquinone-gold conjugate nanomedicine for reversing multidrug resistance in NIR-II photothermal therapy, comprising the following steps:

[0058] (1) Preparation of Au NRs solution: Add 10 mL of an aqueous solution of cetyltrimethylammonium bromide with a concentration of 0.1 mol / L into a round-bottom flask, place it on a magnetic stirrer, and sequentially add 0.4 mL of an aqueous solution of chloroauric acid with a concentration of 10 mmol / L, 28 μL of an aqueous solution of silver nitrate with a concentration of 100 mmol / L, 30 μL of hydrochloric acid with a concentration of 1 mol / L, and 0.54 mL of an aqueous solution of hydroquinone with a concentration of 0.1 mol / L. Stir magnetically at 25 °C for 15 min until the color of the solution changes from orange to light yellow and gradually becomes clear and transparent. The stirring speed is 400 rpm. Then add 10 μL of an aqueous solution of sodium borohydride with a concentration of 0.1 mmol / L (prepared freshly before use) and stir for another 15 s. The stirring speed is 400 rpm. After aging in a 25 °C water bath for 24 h, the obtained Au NRs solution is centrifuged at 12000 rpm for 40 min. The resulting precipitate is made up to 1 mL and stored at 4 °C;

[0059] Use an inductively coupled plasma optical emission spectrometer (ICP-OES) to detect the content of gold element in the Au NRs synthesized under the optimal conditions. Put the made-up Au NRs solution into a microwave digestion tank or a polytetrafluoroethylene crucible containing acid for digestion until the digestion is completed. According to the element to be measured, prepare a standard curve according to different concentration gradients, and then take the test solution to be measured for on-machine testing. Take the Au NRs synthesized under the optimal conditions, and the content of gold element detected by the ICP-OES method is 622.8 mg / L, and the gold recovery rate is 79.05%.

[0060] Add 5 mL of water to the made-up 1 mL AuNRs solution to dilute the solution until the concentration of gold element is 0.1 mg / mL, and obtain a diluted solution of AuNPs solution for step (3);

[0061] Step (3) requires 18 mL of the diluted Au NRs solution. Therefore, repeat operation step (1) three times to obtain 18 mL of the diluted AuNRs solution;

[0062] (2) Preparation of Lipid DOX-ss: Dissolve 67 mg of DSPE-PEG 2000-NHS and 20 mg of DOX-ss in 10 mL of DMSO, add 12 μL of triethylamine, and stir magnetically at 25 °C for 24 h. The stirring speed is 600 rpm. Dialyze the stirred sample and freeze-dry it to obtain Lipid DOX-ss. The molecular weight cut-off of the dialysis bag used for dialysis is 2000 Da, the dialysis time is 48 h, the freeze-drying temperature is -40 °C, the vacuum degree is 200 mTorr, and the freeze-drying time is 12 h;

[0063] (3) Preparation of Au / DOX-ss LNRs: Dissolve 50 mg of Lipid DOX-ss obtained in step (2), 25 mg of DPPC, 18.5 mg of DSPE-PEG 2000-SH, and 12.5 mg of cholesterol in 10 mL of a mixed solution of chloroform and methanol (chloroform:methanol volume ratio = 3:1), perform rotary evaporation at 40 °C for 30 min, vacuum dry at 40 °C for 12 h with a vacuum degree of -0.08 MPa, then add 18 mL of a diluted Au NRs solution (Au concentration 0.1 mg / mL), stir magnetically at 25 °C for 24 h with a stirring speed of 400 rpm, centrifuge twice. After the first centrifugation, take the precipitate and centrifuge again with a centrifugation speed of 12,000 rpm and a centrifugation time of 15 min each time. Take the precipitate and resuspend the hydroquinone-gold conjugate nanomedicine precursor to 5 mL, denoted as Au / DOX-ss LNRs (the hydroquinone-gold conjugate nanomedicine precursor contains water, gold is stored in an aqueous solution, gold nanomaterials are thermally sensitive when dried, prone to aggregation, and their properties change after drying and cannot be resuspended back to a dispersed state. To avoid aggregation, the final product does not need to be dried and is used as a stock solution for the next reaction).

[0064] The particle size of Au / DOX-ss LNRs is 122.4 nm. The content of gold element measured by inductively coupled plasma optical emission spectrometer (ICP-OES) is 39.5 mg / L, and the gold recovery rate is 11.34%.

[0065] Comparative Example 1: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 10 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0066] Comparative Example 2: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 20 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0067] Comparative Example 3: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 30 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0068] Comparative Example 4: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 40 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0069] Comparative Example 5: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 20 μL, and the volume of the sodium borohydride aqueous solution is 10 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0070] Comparative Example 6: Except that the volume of the silver nitrate aqueous solution is 23 μL, the volume of hydrochloric acid is 30 μL, and the volume of the sodium borohydride aqueous solution is 10 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0071] Comparative Example 7: Except that the volume of the silver nitrate aqueous solution is 28 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 10 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0072] Comparative Example 8: Except that the volume of the silver nitrate aqueous solution is 33 μL, the volume of hydrochloric acid is 10 μL, and the volume of the sodium borohydride aqueous solution is 10 μL, all other operation steps and parameters are exactly the same as those in Example 2.

[0073] Figure 1 For the chemical property characterization of OPC-AuNPs in Example 1, where Figure A is the particle size, Figure B is the Zeta potential, Figure C is the ultraviolet absorption spectrum, and Figure D is the TEM image. As Figure 1 shown in Figure A, the particle size of OPC-Au NPs is 78.8 nm. As Figure 1 shown in Figure B, the Zeta potential is -23.5 mV. As Figure 1 shown in Figure C, a characteristic absorption peak appears at 541 nm and presents an asymmetric narrow shape, and a weak absorption peak appears at 1088 nm. This is because proanthocyanidins are composed of monomer units flavan-3-ol (+)-catechin and (-)-epicatechin, rich in hydroxyl structures, with reducibility, which can reduce gold ions to gold atoms, and some additional hydroxyl groups interact with the gold surface, ultimately forming a shell of gold nanoparticles, that is, proanthocyanidin-gold conjugate nanocomposites, as Figure 1 shown in Figure D. It is found that small-sized nanoparticles can diffuse deep into tumor tissues and enter tumor cells through endocytosis. In addition, positively charged carriers are easily captured by macrophages in the blood circulation and bind to immune proteins, causing inflammation and tissue damage, while negatively charged or uncharged nano-drugs can achieve long circulation in the body.

[0074] Figure 2 For the particle size analysis of Au NRs synthesized under different doses of NaBH4, where Figure A is Comparative Example 1, Figure B is Comparative Example 2, Figure C is Comparative Example 3, and Figure D is Comparative Example 4. As Figure 2As can be seen from Figures A - D, as the dosage of NaBH4 increases, the particle size of Au NRs decreases from the initial 91.3 nm to 50.8 nm, and the aspect ratio gradually decreases, but the proportion of smaller gold nanoparticles increases gradually. This is because NaBH4 is a strong reducing agent that forms nuclei by the LaMer effect, and then forms crystal nuclei through rapid random attachment and particle ripening. However, the randomness of the nucleation process leads to the generation of gold nanoparticles of various sizes. High - concentration NaBH4 rapidly induces smaller AuNRs, thus affecting the uniformity of AuNRs.

[0075] Figure 3 Particle size analysis of Au NRs synthesized under different dosages of HCl and AgNO3. Among them, Figure A is Comparative Example 5, Figure B is Comparative Example 6, Figure C is Comparative Example 7, and Figure D is Comparative Example 8. From Figure 2 Figure A of Figure 3 As can be seen from Figure A and Figure B of Figure 2 Figure A of Figure 3 As can be seen from Figure B and Figure C of

[0076] Figure 4 Potential diagrams and ultraviolet absorption spectra of Au NRs synthesized under different conditions. Among them, Figures A - C are potential diagrams of Au NRs synthesized in Comparative Examples 1 - 8, and Figures D - F are ultraviolet absorption spectra of Au NRs synthesized in Comparative Examples 1 - 8. From Figure 4 As can be seen from Figure A and Figure D of Figure 4 As can be seen from Figure B and Figure E of Figure 4 As can be seen from Figure C and Figure F of+ Only affects the lateral deposition of gold nanorods, Ag + The ion strength effect of has a weak influence on the lateral resonance absorption peak of gold nanorods.

[0077] Figure 5 For the chemical property characterization of OPC-Au / DOX-ss LNPs in Example 1, where Figure A is the particle size, Figure B is the Zeta potential, Figure C is the ultraviolet absorption spectrum, and Figure D is the FT-IR. After OPC-AuNPs are coated with liposomes, they are denoted as OPC-Au LNPs, and from Figure 1 Figure A of Figure 5 Figure A of, it can be seen that its particle size increases significantly, from the initial 78.8 nm to 105.7 nm. After further loading Lipid DOX-ss, its particle size further increases to 141.8 nm; from Figure 5 Figure B of, it can be seen that the potential changes from -23.50 mV to -44.07 mV, and after further loading Lipid DOX-ss, its potential is -48.4 mV. From Figure 5 Figure C of, it can be seen that the ultraviolet absorption peak of OPC-Au / DOX-ss LNPs blue-shifts to 957 nm compared with OPC-Au NPs. This indicates the successful preparation of the polyphenol-gold nanomedicine OPC-Au / DOX-ss LNPs, and the negative charge is beneficial for the drug-loaded nanoparticles to enter cells and play a role.

[0078] Figure 5 Figure D of is the infrared spectrum of OPC-Au / DOX-ss LNPs. The broad and strong absorption peak at 3392 cm -1 corresponds to the stretching vibration peak of the hydroxyl group on the aromatic ring of OPC. The peaks at 1614 cm -1 , 1523 cm -1 and 1446 cm -1 correspond to the skeletal vibrations of the aromatic rings on the benzopyran ring respectively. Compared with OPC, the peak of OPC-Au shifts to about 3430 cm -1 , and the peak shape becomes narrower. This indicates that the phenolic hydroxyl groups of procyanidins can undergo redox reactions with Au 3+ . The skeletal vibration peaks of the aromatic rings on the benzopyran ring of OPC-Au shift to 1635 cm -1 , 1523 cm -1 and 1401 cm -1 , which is due to the redox reaction of the phenolic hydroxyl groups. Due to the enhanced electron-donating ability of the oxygen anion to the benzene ring, the positions and types of the absorption peaks on the benzene ring framework change. The above results indicate the successful synthesis of OPC-Au NPs. The characteristic absorption peaks at 805 and 1220 cm -1 are related to DOX, and the peaks at 1285 and 1412 cm-1 The peaks at this position are the C-OH and C-C characteristic peaks of DOX; 1614 cm -1 The peak at this position is the stretching vibration peak of the double carbonyl group of DOX; at 539 cm -1 The stretching vibration peak of S-S appears at this position, proving that the disulfide bond is successfully connected to DOX. 1739 cm -1 The peak at this position is the stretching vibration peak of the carbonyl group of DSPE-PEG2000, 2918 cm -1 and 2854 cm -1 The peaks at this position are the CH alkyl characteristic peaks of DSPE-PEG2000. These results indicate that the liposome successfully encapsulates DOX-ss. 765 cm -1 The characteristic absorption peak of the Au-S bond appears at this position, indicating that OPC-Au is connected to Lipid DOX-ss, and OPC-Au / DOX-ss LNPs are successfully synthesized.

[0079] Figure 6 For the chemical property characterization of Au / DOX-ss LNRs in Example 2, where Figure A is the particle size, Figure B is the Zeta potential, Figure C is the ultraviolet absorption spectrum, and Figure D is the FT-IR. After AuNRs are coated with liposomes, they are denoted as Au LNRs. As can be seen from Figure 6 Figure A of, the major axis and minor axis increase to 105.7 nm and 18.2 nm respectively, and after further encapsulating Lipid DOX-ss, the particle size further increases to 122.4 nm. As can be seen from Figure 6 Figure B of, its potential changes from 31.4 mV to -11.23 mV, and after further encapsulating Lipid DOX-ss, the potential is -13.9 mV. As can be seen from Figure 6 Figure C of, the ultraviolet absorption peak of Au / DOX-ss LNRs blue-shifts to 1068 nm compared with Au NRs and still lies in the NIR-II region. This indicates that Au / DOX-ss LNRs are successfully synthesized.

[0080] Figure 6 Figure D of is the infrared spectrum of Au / DOX-ss LNRs. The characteristic absorption peaks at 805 and 1220 cm -1 are related to DOX. The peaks at 1285 and 1412 cm -1 are the C-OH and C-C characteristic peaks of DOX; 1614 cm -1 The peak at this position is the stretching vibration peak of the double carbonyl group of DOX; at 539 cm -1 The stretching vibration peak of S-S appears at this position, proving that the disulfide bond is successfully connected to DOX. 1739 cm -1 The peak at this position is the stretching vibration peak of the carbonyl group of DSPE-PEG2000, 2918 cm -1 and 2854 cm -1The peak at this position is the characteristic peak of the CH alkyl group of DSPE-PEG2000. These results indicate that the liposomes have successfully encapsulated DOX-ss. 765 cm -1 The peak at this position is the characteristic absorption peak of the Au-S bond, indicating that Au NRs are connected to LipidDOX-ss, and Au / DOX-ss LNRs have been successfully synthesized.

[0081] The products of Example 1 and Example 2 were used in Application Examples 1-5:

[0082] Verification experiments were carried out on the pH response, NIR response, and GSH-responsive drug release of the drug-loaded nanoparticle samples in Application Example 1

[0083] The in vitro drug release of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs under different pH and GSH conditions was investigated respectively. 3 mg of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs were dispersed in 3 mL of PBS buffer solution under the corresponding conditions. The above liquids were respectively placed into dialysis bags with a molecular weight cut-off of 500 Da, and then placed into 30 mL of PBS buffer solution under the corresponding conditions, and continuously oscillated in a constant temperature shaker at 37 °C. 3 mL of the release solution was taken at regular intervals and 3 mL of PBS buffer solution under the corresponding conditions was added. The absorbance of the sample at an absorption wavelength of 480 nm was measured. According to the standard curve of DOX under different pH conditions, the cumulative release rate of DOX was calculated, and a graph was plotted with time as the abscissa and the cumulative release rate as the ordinate.

[0084] Figure 7 and Figure 8 The in vitro drug release of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs is shown respectively. As shown in the figure, after 48 h, the release rate of the drug tended to be stable, and both OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs showed significant pH-GSH-NIR responsiveness.

[0085] The release rate of OPC-Au / DOX-ss LNPs at pH 5.5 (58.18%) was about 1.65 times that at pH 7.4 (35.08%), which is beneficial to reducing drug release in normal tissues. The release rate under the condition of pH5.5+GSH (81.28%) was about 2.31 times that under pH7.4. The release rate under the condition of pH5.5+NIR (70.91%) was about 2.02 times that under pH 7.4. The release rate under the condition of pH5.5+GSH+NIR (92.09%) was about 2.63 times that under pH 7.4.

[0086] The release rate of Au / DOX-ss LNRs at pH 5.5 (62.74%) is about 1.66 times that at pH 7.4 (37.78%), which is beneficial to reducing drug release in normal tissues. The release rate under the condition of pH5.5 + GSH (87.73%) is about 2.32 times that under the condition of pH 7.4. The release rate under the condition of pH5.5 + NIR (76.03%) is about 2.01 times that under the condition of pH 7.4. The release rate under the condition of pH5.5 + GSH + NIR (97.57%) is about 2.58 times that under the condition of pH 7.4.

[0087] The pH-GSH-NIR responsiveness of the OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs system is beneficial to drug release in tumor tissues and reduces the toxic and side effects on normal tissues.

[0088] Example 2 verifies the biocompatibility of the nanocarrier and the drug-loaded nanoparticles through experiments.

[0089] Take 5 mL of mouse blood and place it in a centrifuge tube containing sodium heparin to prevent blood coagulation. After centrifuging at 4°C and 1000 rpm for 5 min, pour off the supernatant and wash it with normal saline until the supernatant is colorless. Subsequently, configure the red blood cells into a 2% cell suspension with normal saline, store it at 4°C, and perform hemolytic analysis. Use normal saline to configure different concentrations of OPC-Au / DOX-ssLNPs and Au / DOX-ss LNRs (1, 10, 100, 250, 500 μg / mL) as the experimental groups, normal saline as the negative control (0% hemolysis), and 1% TritonX-100 as the positive control (100% hemolysis). Add 250 μL of different concentrations of OPC-Au / DOX-ssLNPs, Au / DOX-ss LNRs, normal saline, and 1% TritonX-100 to 250 μL of the red blood cell suspension, mix well, place it in a constant temperature incubator at 37°C for 2 h. After incubation, centrifuge the samples at 1000 rpm for 5 min in a high-speed centrifuge, take 100 μL of the supernatant into a 96-well plate, detect its absorbance value at a wavelength of 540 nm, and calculate the hemolysis rate according to formula (1).

[0090] Hemolysis rate (%) = (A 实验组 - A 阴性对照 ) / (A 阳性对照 - A 阴性对照 ) × 100% (1)

[0091] A 实验组 —Absorbance of the experimental group at 540 nm; A 阴性对照 —Absorbance of the negative control at 540 nm; A 阳性对照—Absorbance of the positive control at 540 nm.

[0092] Figure 9 It is the hemolysis analysis chart of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs. When the hemolysis rate is lower than 5%, it indicates that the test sample has good biocompatibility. From the hemolysis experiment, within the test concentration range, the hemolysis rates of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs are both lower than 5%, indicating that both have good biocompatibility.

[0093] Experimental verification of photothermal conversion performance in Application Example 3

[0094] Take OPC-Au NPs solution, OPC-Au / DOX-ss LNPs, Au NRs solution and Au / DOX-ss LNRs, use a laser with a wavelength of 1064 nm, and irradiate and heat up for 5 min under the NIR of 1 W / cm 2 , and record the temperature change every 30 s. Subsequently, remove the NIR light source and let the sample cool naturally for 5 min, and record the temperature change every 30 s. Through 4 heating-cooling cycle periods, evaluate the thermal stability and photothermal conversion performance of the sample. Subsequently, draw the temperature change curves of the samples under the irradiation of 1064 nm NIR light respectively, and study their photothermal conversion performance in 4 cycles, and calculate their photothermal conversion efficiency according to formulas (2)(3)(4)(5).

[0095] θ=(T - T0) / (T max - T0) (2)

[0096] Where T is the temperature of the solution at different times in the natural cooling state, T is the highest temperature that the solution can reach in the equilibrium state under laser irradiation, and T0 is the initial temperature of the solution.

[0097] Then, plot with time t on the y-axis and -lnθ on the x-axis, and calculate the system heat transfer time constant (τ s ) from the slope of the fitted straight line, so as to calculate the hS value.

[0098] hS = Σ i m i C p,i / τ s (3)

[0099] Where h is the heat transfer coefficient, S is the surface area of the container, m i is the mass of the component, and C p,i is the specific heat capacity of the component.

[0100] Calculate the energy (Q0) released by the absorption of light by an equal amount of pure aqueous solution.

[0101] Q0 = cmΔT / t (4)

[0102] Where c is the specific heat capacity of water, m is the mass of water, ΔT is the temperature drop value of the pure aqueous solution after turning off the laser, and t is the heat dissipation time of pure water.

[0103] Based on the calculated values of hS and Q0, the photothermal conversion efficiency η is calculated.

[0104] η = [hS(T max - T0)] / [I(1 - 10 -A )] (5)

[0105] Where A is the ultraviolet absorption value of the nanomaterial at 808 nm and 1064 nm, and I is the power.

[0106] Figure 10 are the photothermal conversion cycle curves of each carrier and drug-loaded nanoparticles. Among them, Figure A is OPC-Au NPs, Figure B is OPC-Au / DOX-ss LNPs, Figure C is Au NRs, and Figure D is Au / DOX-ss LNRs. As Figure 10 shown in Figures A - D, during four NIR irradiation cycles, the photothermal curves of OPC-Au NPs, OPC-Au / DOX-ss LNPs, Au NRs, and Au / DOX-ss LNRs did not change significantly, indicating that they all have good photostability. The photothermal conversion efficiencies of OPC-Au NPs and OPC-Au / DOX-ss LNPs under 1064 nm NIR light irradiation are 47.10% and 59.22% respectively; the photothermal conversion efficiencies of Au NRs and Au / DOX-ss LNRs under 1064 nm NIR light irradiation are 66.53% and 55.34% respectively. The above data show that compared with NIR-I light, NIR-II light can produce a higher photothermal effect through a deeper penetration depth in biological tissues.

[0107] Application Example 4 Verification of Cytotoxicity Experiment

[0108] The cytotoxicity of the nanocarriers and drug-loaded nanoparticles was investigated by the MTT method respectively. Normal hepatocytes (L-02) and breast cancer drug-resistant cells (MCF-7 / ADR) in the logarithmic growth phase were collected, digested and counted, and then seeded at a density of 10 4Inoculate into 96-well plates through holes. After culturing at 37°C for 12 h, remove the old culture medium. Add empty vectors and drug-loaded nanoparticles at different concentrations and culture for 24 h and 48 h respectively. Then, add 100 μL of 20% MTT to the 96-well plates. After incubating at 37°C for 4 h, aspirate the MTT. Add 100 μL of DMSO to each well, shake for 10 min, and measure the absorbance of each well at a wavelength of 490 nm. Compare with the blank control group, and calculate the cell survival rate according to formula (6):

[0109] Cell survival rate (%) = (A 实验组 - A 对照组 ) / (A 未处理组 - A 对照组 ) × 100% (6)

[0110] A 实验组 — Absorbance of cells at 490 nm after incubation with nano-drug carriers;

[0111] A 对照组 — Absorbance of the culture medium at 490 nm;

[0112] A 未处理组 — Absorbance of untreated cells at 490 nm.

[0113] Figure 11 Figure 30 shows the cell viability of the blank carrier and polyphenol-gold nanomedicine prodrug after 24 h of incubation with L-02 and MCF-7 / ADR cells in Application Example 4. Among them, Figure A shows the cell viability of the blank carrier after 24 h of incubation with L-02 cells, Figure B shows the cell viability of the blank carrier after 24 h of incubation with MCF-7 / ADR cells, Figure C shows the cell viability of the polyphenol-gold nanomedicine prodrug after 24 h of incubation with L-02 cells, and Figure D shows the cell viability of the polyphenol-gold nanomedicine prodrug after 24 h of incubation with MCF-7 / ADR cells; Figure 12 Figure 32 shows the cell viability of the blank carrier and polyphenol-gold nanomedicine prodrug after 48 h of incubation with L-02 and MCF-7 / ADR cells in Application Example 4. Among them, Figure A shows the cell viability of the blank carrier after 48 h of incubation with L-02 cells, Figure B shows the cell viability of the blank carrier after 48 h of incubation with MCF-7 / ADR cells, Figure C shows the cell viability of the polyphenol-gold nanomedicine prodrug after 48 h of incubation with L-02 cells, and Figure D shows the cell viability of the polyphenol-gold nanomedicine prodrug after 48 h of incubation with MCF-7 / ADR cells; As shown in Figures A-B of Figure 11 and Figures A-B of Figure 12 , after culturing OPC-AuNPs, Au NRs, and Lipid empty carriers with L-02 cells and MCF-7 / ADR cells for 24 h and 48 h, their cell survival rates are all above 80%, showing good biosafety. From Figure 11 Figure C of Figure 12As can be seen from Figure C, DOX has greater toxicity to L-02 cells, and the cell survival rate after 48 h is 62.10% (DOX: 2 μg / mL). After prodrug design and liposome coating, its toxicity is significantly reduced, and the cell survival rate of Lipid DOX-ss is significantly increased to 86.68%. The cell survival rates of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs are slightly lower than that of Lipid DOX-ss, but still above 80%, showing low toxicity to normal cells and good biosafety.

[0114] As can be seen from Figure 11 Figure D and Figure 12 Figure D, after incubating DOX with MCF-7 / ADR cells for 48 h, the cell survival rate is 71.71%. After encapsulation with Lipid, the cell survival rate is 90.98%, which is attributed to the good biocompatibility of the liposome, reducing the toxicity of DOX. The cell survival rates of MCF-7 / ADR cells in the OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs groups are reduced to 55.96% and 62.67% respectively. Compared with Au / DOX-ss LNRs, OPC-Au / DOX-ss LNPs can enhance the sensitivity of MCF-7 / ADR cells to doxorubicin more effectively. After NIR light irradiation, compared with the Lipid DOX-ss group, the cell survival rates of MCF-7 / ADR cells in the OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs groups are reduced by 1.72 times and 1.53 times respectively.

[0115] Figure 13 are the half-lethal concentrations (IC 50 values) after incubating the polyphenol-gold nanodrug with MCF-7 / ADR cells for 24 h and 48 h. After 48 h, the IC 50 values of free DOX, Lipid DOX-ss, OPC-Au / DOX-ss LNPs, Au / DOX-ss LNRs, and OPC-Au / DOX-ss LNPs+NIR, Au / DOX-ss LNRs+NIR against MCF-7 / ADR are 36.41, 126.1, 3.285, 4.13, and 3.03, 3.91 μg / mL respectively. Compared with Lipid DOX-ss, the IC 50 values of OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs against MCF-7 / ADR are reduced by 38.39 times and 30.53 times respectively. Therefore, the polyphenol-gold nanodrug can significantly improve the sensitivity of MCF-7 / ADR cells.

[0116] Experimental Verification of P-gp Protein Expression Analysis by Western Blot in Application Example 5

[0117] (1) Protein extraction: Seed MCF-7 / ADR cells in a 6-well plate (10 5 / well). After 12 h, remove the old culture medium. Prepare DOX, Lipid DOX-ss, Au / DOX-ssLNRs, Au / DOX-ss LNRs+NIR, OPC-Au / DOX-ss LNPs, and OPC-Au / DOX-ss LNPs+NIR using RPMI 1640 complete medium and RPMI 1640 complete medium containing 1 μg / mL DOX. Add each drug-loaded nanoparticle to the 6-well plate and incubate for 24 h. Then, digest the cells with trypsin, collect them, add cell lysis buffer, and lyse the cells on ice for 15 min. After 30 min in a 37 °C metal bath, collect the protein samples.

[0118] (2) Gel preparation: Assemble the glass plates and the gel maker. Prepare the separating gel according to the ratio, add the separating gel to the glass plates, slowly add methanol for liquid sealing. After the separating gel solidifies, pour off the methanol. Prepare the stacking gel according to the ratio, add the stacking gel to the glass plates, insert the comb teeth. After the stacking gel solidifies, remove the comb teeth.

[0119] (3) Protein sample treatment: Mix the protein treatment solution with the protein samples collected in step (1) according to the ratio, incubate in a 37 °C metal bath for 5 min, and then keep on ice for later use.

[0120] (4) Loading and electrophoresis: Slowly remove the comb teeth, add 5 μL of Marker as an indicator, and add 20 μL of protein samples to each of the remaining wells. Set the voltage to 80 V. After the protein runs through the stacking gel, set the voltage to 120 V. Turn off the power when the protein reaches the edge of the separating gel.

[0121] (5) Gel cutting: After electrophoresis, take out the protein gel and place it in the wet transfer buffer. Cut the gel at the position of the target band with reference to the Marker, and cut the PVDF membrane according to the size of the cut protein gel.

[0122] (6) Wet transfer: Stack the cut protein gel into the wet transfer cassette in order, set the current to 220 mA for wet transfer, and transfer the protein bands separated by electrophoresis from the gel to the PVDF membrane.

[0123] (7) Blocking and membrane washing: Prepare a 5% skim milk blocking solution with TBST, put the PVDF membrane into it for blocking for 1 h. After blocking, wash the membrane three times with TBST solution.

[0124] (8) Primary antibody incubation: Incubate with the primary antibody against the target protein at 4 °C for 16 h, then wash 3 times with TBST solution to rinse off the unbound antibody.

[0125] (9) Secondary antibody incubation: Incubate with a fluorescent secondary antibody for 1 h at room temperature in the dark, then wash 3 times with TBST solution and rinse to remove unbound antibodies.

[0126] (10) Development: Place the PVDF membrane in an infrared laser imaging system for development and analyze the relative expression level of the target protein.

[0127] The expression level of P-gp in cells was detected by Western blot to investigate the change in the drug resistance of MCF-7 / ADR cells to DOX, and Image Studio software was used for quantitative analysis of the expression of P-gp glycoprotein.

[0128] Figure 14 For the analysis of the expression level of P-gp protein after culturing MCF-7 / ADR cells with each drug-loaded nanoparticle in Application Example 5, where Figure A is the Western blot detection of the P-gp protein band and Figure B is the quantitative analysis of the P-gp protein. It can be seen from Figure 14 that the change in the expression level of P-gp protein in MCF-7 / ADR cells in the Au / DOX-ss LNRs group was not significant. After adding procyanidins, the expression level of P-gp protein in the procyanidin-gold conjugate nanomedicine OPC-Au / DOX-ss LNPs decreased by 3.1-fold, indicating that the drug-loaded nanoparticles can effectively inhibit the expression of P-gp protein, thereby reducing the drug resistance of MCF-7 / ADR cells and improving the anti-tumor effect. This is mainly attributed to the fact that the natural polyphenol - procyanidin (OPC) can scavenge free radicals, reduce oxidative stress damage, inhibit signal pathways, prevent cancer progression, and promote apoptosis.

[0129] In summary, the present invention constructs NIR-II responsive procyanidin-gold conjugate nanomedicine and hydroquinone-gold conjugate nanomedicine. First, Au NRs and OPC-AuNPs were synthesized. Based on DOX-ss, the gold nanomedicines Au / DOX-ss LNRs and OPC-Au / DOX-ss LNPs obtained by Au-S covalent coupling and encapsulation with thermosensitive liposomes have high drug loading, good biocompatibility, and exhibit pH-GSH-NIR-II responsive drug release, significantly improving the targeting sensitivity of drug-resistant cells. Therefore, Au / DOX-ss LNRs and OPC-Au / DOX-ss nanomedicines are an excellent and efficient nanodrug delivery system.

[0130] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy, characterized in that, It includes the following steps: (1) Prepare the OPC-AuNPs solution: After stirring and heating the chloroauric acid aqueous solution to boiling, add the procyanidin aqueous solution and heat and stir to obtain the OPC-Au NPs solution. Centrifuge to collect the precipitate and make up the volume for storage; Or, prepare the Au NRs solution: Sequentially add the chloroauric acid aqueous solution, silver nitrate aqueous solution, hydrochloric acid, and hydroquinone aqueous solution to the cetyltrimethylammonium bromide aqueous solution and stir, then add the sodium borohydride aqueous solution and stir again. Age to obtain the AuNRs solution. Centrifuge to collect the precipitate and make up the volume for storage; (2) Prepare Lipid DOX-ss: Dissolve DSPE-PEG 2000-NHS and DOX-ss in DMSO, add triethylamine and stir. Dialyze the stirred sample and freeze-dry to obtain Lipid DOX-ss; (3) Prepare OPC-Au / DOX-ss LNPs or Au / DOX-ss LNRs: Dissolve the Lipid DOX-ss, DPPC, DSPE-PEG 2000-SH, and cholesterol obtained in step (2) in a mixed solution of chloroform and methanol, rotary evaporate, vacuum dry, and then add the dilution of the OPC-AuNPs solution or the dilution of the AuNRs solution obtained in step (1) and stir. Centrifuge to collect the precipitate to obtain the nano-prodrug for reversing multidrug resistance in the NIR-II photothermal therapy, denoted as OPC-Au / DOX-ss LNPs and Au / DOX-ss LNRs.

2. The preparation method of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, wherein, In step (1), when preparing the OPC-AuNPs solution, the molar ratio of chloroauric acid to procyanidin is 4.53:

1.

3. The preparation method of the nano prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, characterized in that, In step (1), when preparing the AuNRs solution, the molar ratio of cetyltrimethylammonium bromide, chloroauric acid, silver nitrate, hydrogen chloride, hydroquinone, and sodium borohydride is 1000:40:23:100:540:

1.

4. The preparation method of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, wherein, In step (1), after stirring and heating the chloroauric acid aqueous solution to boiling, add procyanidin and heat and stir until the solution changes from light yellow to dark red; Sequentially add the chloroauric acid aqueous solution, silver nitrate aqueous solution, hydrochloric acid, and hydroquinone aqueous solution to the cetyltrimethylammonium bromide aqueous solution and stir until the color of the solution changes from orange to light yellow and gradually becomes clear and transparent, then add the sodium borohydride aqueous solution and stir again for 15 s, the stirring speed is 400 rpm, the aging temperature is 25 °C, the aging time is 24 h, centrifuge once, the centrifugation speed is 12,000 rpm, the centrifugation time is 40 min, dilute the precipitate to a certain concentration, and store it at 4 °C as the stock solution.

5. The preparation method of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, wherein, In step (2), the molar ratio of DSPE-PEG 2000-NHS to DOX-ss is 1:3.14, and the mass-volume ratio of DSPE-PEG2000-NHS, DOX-ss, DMSO, and triethylamine is 67 mg:20 mg:10 mL:12 μL.

6. The preparation method of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, characterized in that, In step (2), triethylamine was added and stirred for 24 h at a stirring speed of 600 rpm. The stirred sample was dialyzed and freeze-dried. The molecular weight cut-off of the dialysis bag used for dialysis was 2000 Da, the dialysis time was 48 h, the freeze-drying temperature was -40 °C, the vacuum degree was 200 mTorr, and the freeze-drying time was 12 h.

7. The preparation method of the nano-prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, wherein, In step (3), the mass-volume ratio of Lipid DOX-ss, DPPC, DSPE-PEG 2000-SH, cholesterol, chloroform, methanol and the dilution of OPC-AuNPs solution or the dilution of AuNRs solution was 50 mg: 25 mg: 18.5 mg: 12.5 mg: 7.5 mL: 2.5 mL: 18 mL. In the dilution of OPC-AuNPs solution or the dilution of AuNRs solution, the Au concentration was 0.1 mg / mL.

8. The preparation method of the nano prodrug for reversing multidrug resistance in NIR-II photothermal therapy according to claim 1, wherein In step (3), the temperature of rotary evaporation was 40 °C, the time of rotary evaporation was 30 min, and the rotation rate was 100 r / min; the temperature of vacuum drying was 40 °C, the vacuum drying time was 12 h, and the vacuum degree was -0.08 MPa; the stirring speed was 400 rpm, and the stirring time was 24 h; centrifugation was performed twice. After the first centrifugation, the precipitate was taken and centrifuged again. The precipitate was taken, the centrifugation speed was 12000 rpm, and the centrifugation time for each time was 15 min.

9. A nano-prodrug for reversing multi-drug resistance in NIR-II photothermal therapy prepared by the method according to any one of claims 1-8, characterized in that, The particle size of the OPC-Au / DOX-ss LNPs was 141.8 nm, and the particle size of the Au / DOX-ss LNRs was 122.4 nm.

10. Use of the nano-prodrug for reversing multidrug resistance in the NIR-II photothermal therapy according to claim 9 in the preparation of an anti-tumor drug.

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