Nano drug delivery system combined with chemical-photodynamic therapy as well as preparation method and application of nano drug delivery system

Abstract

The invention discloses a chemical-photodynamic therapy combined nano drug delivery system as well as a preparation method and application thereof. The preparation method comprises the following steps: synthesizing Zein-HPG from zein through anionic ring-opening polymerization reaction; then propiolic acid is combined with hydroxyl of the Zein-HPG through an esterification reaction, and a Zein-HPG-PA carrier is obtained; finally, curcumin is loaded to the Zein-HPG-PA carrier through a phenol-alkyne click reaction, and the nano drug delivery system Zein-HPG-PA (at) CUR is obtained. The nano drug delivery system disclosed by the invention has pH responsiveness, the loaded curcumin is released in a weak acid environment of tumors, and chemical / photodynamic combined treatment of tumors can be realized, so that a more ideal treatment effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a nano drug delivery system for combined chemical-photodynamic therapy and a preparation method and application thereof. Background Art

[0002] Cancer is one of the biggest threats to human health in the world. Over the past few decades, many treatment options have been developed, such as surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, phototherapy, and their combinations. Different cytotoxic small molecules have been used for chemotherapy, but these small molecules are widely distributed in different organs, resulting in poor bioavailability, unsatisfactory efficacy, severe adverse reactions, and systemic toxicity. Photodynamic therapy (PDT) is a light-based therapy for cancer treatment because it is less invasive than surgery, produces few adverse reactions, and requires a shorter treatment time. Despite this, their local therapeutic effect remains a major obstacle to the effective treatment of metastatic or recurrent tumors. In order to improve the therapeutic effect, the current method of combining photodynamic therapy and chemotherapy to treat tumors has attracted attention. However, due to the poor enhanced permeability and retention (EPR) effect and the nonspecific uptake of nanoparticles by the mononuclear phagocyte system (e.g., liver / spleen), resulting in low accumulation of nanoparticles in tumor tissues, the current clinical efficacy of the chemo-photodynamic combined chemotherapy method is not ideal. Therefore, it is necessary to further improve it. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nano drug delivery system for combined chemical-photodynamic therapy and a preparation method thereof. The nano drug prepared by the method has good biosafety, small size, good water solubility, high drug loading, and can release drugs in a pH-responsive manner. The present invention has important application value in the field of nano drug technology.

[0004] The present invention also provides an application of a nano drug delivery system combined with chemo-photodynamic therapy, through the chemotherapeutic effect and photodynamic effect of curcumin, chemo-photodynamic combined therapy can be achieved to enhance the anti-tumor effect.

[0005] To achieve the above technical objectives, the present invention adopts the following technical scheme: a nano drug delivery system for combined chemical-photodynamic therapy, wherein Zein-HPG is synthesized from zein (Zein) by anionic ring-opening polymerization; then propiolic acid is combined with the hydroxyl group of Zein-HPG by esterification reaction to obtain a Zein-HPG-PA carrier; finally, curcumin is loaded onto the Zein-HPG-PA carrier by a phenol-alkyne click reaction to obtain a nano drug delivery system Zein-HPG-PA@CUR.

[0007] The second aspect disclosed in the present invention is to provide a method for preparing a nano drug delivery system for combined chemical-photodynamic therapy, comprising the following steps:

[0008] Step 1, synthesis of Zein-HPG: dissolve zein, sodium hydride and 18-crown ether-6 in anhydrous DMF, stir ultrasonically to make them dispersed evenly, then heat to 75-85°C in an oil bath, add glycidol and react for 24-48h, dialyze and purify the product in water and ethanol, then concentrate and freeze-dry to obtain Zein-HPG;

[0009] Step 2, synthesis of Zein-HPG-PA carrier: Zein-HPG, propiolic acid and p-toluenesulfonic acid are added to CHCl3, the reaction mixture is heated to 75-85°C and refluxed for 16-24h, the reaction product is washed with saturated NaHCO3 solution, saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier;

[0010] Step 3. Synthesis of the nano drug delivery system Zein-HPG-PA@CUR: Dissolve the Zein-HPG-PA carrier and CUR in anhydrous THF, sonicate until uniformly dispersed, add DMAP and react in an anhydrous and vacuum environment. After the reaction, the product is dialyzed and purified in water and ethanol, and then concentrated and freeze-dried to obtain the nano drug delivery system Zein-HPG-PA@CUR.

[0011] Furthermore, in step 1, the mass ratio of glycidol to Zein is 1-4.

[0012] Furthermore, in step 2, the molar ratio of propiolic acid to p-toluenesulfonic acid is 2:1, and the mass ratio of propiolic acid to Zein-HPG is 2-4.

[0013] Furthermore, in step 3, the mass ratio of CUR to Zein-HPG-PA is 1-2.

[0014] The third aspect disclosed in the present invention is to provide a nano drug delivery system combined with chemical / photodynamic therapy for use in treating breast cancer.

[0015] The present invention has the following advantages:

[0016] 1. The present invention provides a preparation method and application of a nano drug delivery system for combined chemical / photodynamic therapy. In the nano drug delivery system, Zein-HPG is synthesized by Zein anion ring-opening polymerization, which greatly improves the disadvantage of poor water solubility of Zein. Then, propiolic acid (PA) is combined with the hydroxyl group of Zein-HPG through an esterification reaction to obtain an alkynyl-terminated Zein-HPG-PA carrier. This carrier can utilize the terminal acetylene functional group and a natural active product with a phenolic hydroxyl functional group similar to curcumin to achieve the loading of the natural active product through a phenol-acetylene click reaction, thereby obtaining a nano drug delivery system Zein-HPG-PA@CUR.

[0017] 2. The acid-responsive nano drug delivery system carrier Zein-HPG-PA of the present invention was verified by cell viability

[0019] It is also non-toxic at high concentrations, indicating that it has good biocompatibility.

[0020] 3. The acid-responsive nano drug delivery system Zein-HPG-PA@CUR of the present invention has good water dispersibility and a high drug loading capacity (31.5%). Due to the presence of acid-sensitive ester bonds, the nano drug delivery system can responsively release CUR in an acidic tumor environment and not release it in a neutral environment, thereby improving bioavailability while reducing the toxic side effects of the drug on normal tissues.

[0021] 4. The acid-responsive nano drug delivery system Zein-HPG-PA@CUR of the present invention has a good photodynamic effect, and combined with the anti-tumor effect of CUR itself, it can combine chemical-photodynamic therapy to treat breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The H NMR spectra of Zein (c), Zein-HPG (b), and Zein-HPG-PA@CUR (a) in Example 1, the NMR solvent is DMSO-d6;

[0023] Figure 2 The infrared spectra of Zein (e), Zein-HPG (d), Zein-HPG-PA (c), Zein-HPG-PA@CUR (b), and CUR (a) in Example 1;

[0024] Figure 3 The representative scanning electron microscope images of Zein-HPG (scale bar: 50 μm), Zein-HPG-PA (scale bar: 400 nm) and Zein-HPG-PA@CUR (scale bar: 300 nm) in Example 1;

[0025] Figure 4 Transmission electron microscope images and particle size distribution of Zein-HPG (A, D), Zein-HPG-PA (B, E) and Zein-HPG-PA@CUR (C, F) in Example 1, the transmission electron microscope scale is 500 nm;

[0026] Figure 5 The standard curve of CUR at ultraviolet 427nm in Example 1;

[0027] Figure 6 The release curve of CUR from Zein-HPG-PA@CUR at pH 5 and 7.4 in Example 1;

[0028] Figure 7 CLSM image of MDA-MB-231 cells incubated with Zein-HPG-PA@CUR and DAPI in Example 1 (scale bar: 15 μm);

[0029] Figure 8 The cell viability of MDA-MB-231 cells after incubation with Zein-HPG-PA for 24 hours in Example 1;

[0030] Fig. 9 The results are shown in Example 1. MDA-MB-231 cells were incubated with Zein-HPG-PA for 24 h under different light intensities (1 mW / cm 2 and 2mW / cm 2 ) The viability of MDA-MB-231 cells under irradiation for 3 min and the viability of MDA-MB-231 cells of Zein-HPG-PA@CUR without light irradiation (dark toxicity);

[0031] Fig.10 PBS, Zein-HPG-PA, Zein-HPG-PA@CUR, and Zein-HPG-PA@CUR+2mW / cm 2 Live and dead cell inverted fluorescence microscopy images of treated MDA-MB-231 cells (image scale: 60 μm);

[0032] Fig.11The green fluorescent probe DCFH-DA was selected in Example 1 to detect the total ROS production in the cells. (AC) are bright field photos of MDA-MB-231 cells under Zein-HPG-PA@CUR+DCFH light-proof conditions, DCFH+lighting, and Zein-HPG-PA@CUR+DCFH+lighting conditions; (A1-C1) are fluorescence photos of MDA-MB-231 cells under Zein-HPG-PA@CUR+DCFH light-proof conditions, DCFH+lighting, and Zein-HPG-PA@CUR+DCFH+lighting conditions. The concentration of DCFH is 20 μM, and the concentration of Zein-HPG-PA@CUR is 50 μg mL -1 , light intensity is 3mW / cm 2 , (image scale: 100μm);

[0033] Fig.12 In Example 1, the red fluorescent probe DHE was selected to detect the production of ·O2- in cells. (AC) are bright field photos of MDA-MB-231 cells under Zein-HPG-PA@CUR+DHE dark conditions, DHE+lighting, and Zein-HPG-PA@CUR+DHE+lighting; (A1-C1) are fluorescence photos of MDA-MB-231 cells under Zein-HPG-PA@CUR+DHE dark conditions, DHE+lighting, and Zein-HPG-PA@CUR+DHE+lighting. The concentration of DHE is 20μM, and the concentration of Zein-HPG-PA@CUR is 50μg mL -1 , light intensity is 3mW / cm 2 , (image scale: 100μm); DETAILED DESCRIPTION

[0034] In order to further understand the present invention, the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0035] Experimental instrument name and model:

[0036] Swiss Bruker AVANCE III HD 600MHz H NMR spectrum;

[0037] Nicolet 380, Fourier transform spectrometer, USA;

[0038] FEI Quanta 250 scanning electron microscope from the United States;

[0039] FEI Tecnai G 2Spirit TWIN transmission electron microscope from the United States;

[0040] Mastersizer 2000 Malvern particle size analyzer from the UK;

[0041] PerkinElmer Lambda 35 UV-visible spectrometer from the United States;

[0042] Andor Dragonfly laser confocal microscope from the UK;

[0043] Spark 20M microplate reader from Switzerland;

[0044] Nikon Ts2R fluorescence inverted microscope, Japan.

[0045] The present invention provides a nano drug delivery system for combined chemical-photodynamic therapy, wherein Zein-HPG is synthesized from zein by anionic ring-opening polymerization; propiolic acid is then combined with the hydroxyl group of Zein-HPG by esterification to obtain a Zein-HPG-PA carrier; finally, curcumin is loaded onto the Zein-HPG-PA carrier by a phenol-alkyne click reaction to obtain the nano drug delivery system Zein-HPG-PA@CUR.

[0046] The present invention provides a method for preparing a nano drug delivery system for combined chemical-photodynamic therapy, comprising the following steps:

[0047] Step 1, synthesis of Zein-HPG: dissolve zein, sodium hydride and 18-crown-6 in anhydrous DMF, stir ultrasonically for 20-30 min to make them evenly dispersed, then heat to 75-85° C. in an oil bath, add glycidol and react for 24-48 h, dialyze and purify the product in water and ethanol for 2-3 days, then concentrate and freeze-dry to obtain Zein-HPG; wherein the mass ratio of glycidol to Zein is 1-4;

[0048] Step 2, synthesis of Zein-HPG-PA carrier: Zein-HPG, propiolic acid and p-toluenesulfonic acid are added to CHCl3, the reaction mixture is heated to 75-85°C and refluxed for 16-24h, the reaction product is washed 2-4 times with saturated NaHCO3 solution, physiological saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier; wherein the molar ratio of propiolic acid to p-toluenesulfonic acid is 2:1, and the mass ratio of propiolic acid to Zein-HPG is 2-4;

[0049] Step 3. Synthesis of nano drug delivery system Zein-HPG-PA@CUR: Zein-HPG-PA and CUR were dissolved in anhydrous THF with a mass ratio of CUR to Zein-HPG-PA of 1-2. After ultrasonication for 10-30 min until uniform dispersion, DMAP was added and reacted in an anhydrous and vacuum environment for 3-6 h. After the reaction, the product was dialyzed and purified in water and ethanol for 1-2 days, and then concentrated and freeze-dried to obtain Zein-HPG-PA@CUR.

[0050] Example 1

[0051] A method for preparing a nano drug delivery system for combined chemical-photodynamic therapy comprises the following steps:

[0052] Step 1, synthesis of Zein-HPG: 0.5 g zein, 0.2 g sodium hydride and 0.5 g 18-crown ether-6 were dissolved in 20 mL anhydrous DMF, heated to 80°C after ultrasonic stirring, and then 1 mL glycidol was added to react for 24 h. After the reaction, the product was dialyzed and purified in water and ethanol for 2 days, and then concentrated and freeze-dried to obtain Zein-HPG;

[0053] Step 2, synthesis of Zein-HPG-PA carrier: 0.44 g Zein-HPG, 1.4 g propiolic acid and 1.77 g p-toluenesulfonic acid were added to 10 mL CHCl3, the reaction mixture was heated to 80°C and refluxed for 20 h, the reaction product was washed with saturated NaHCO3 solution, saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier;

[0054] Step 3. Synthesis of nano drug delivery system Zein-HPG-PA@CUR: 10 mg Zein-HPG-PA and 15 mg CUR were dissolved in 10 mL anhydrous THF, and after ultrasonication until uniformly dispersed, 2 mg DMAP was added and reacted in anhydrous and vacuum environment for 4 h. After the reaction, the product was dialyzed and purified in water and ethanol for 1 day, and then concentrated and freeze-dried to obtain Zein-HPG-PA@CUR.

[0055] Zein-HPG-PA@CUR (a), Zein-HPG (b), and Zein (c) were measured using nuclear magnetic resonance spectroscopy. Figure 1As can be seen in the hydrogen spectrum of Zein, peaks with chemical shifts of 0.9ppm and 1.35ppm can be found, which can be attributed to -CH and -CH2 / -CH3 respectively. The signal with a chemical shift of 3.3ppm should be attributed to H2O in Zein. It is worth noting that a small peak is also observed at about 7.2ppm, which can be attributed to the aromatic rings in some amino acids. Compared with the spectrum of Zein, the obvious change is in the spectrum of Zein-HPG. 1 A strong peak was observed between 3.2 and 3.8 ppm in the H NMR spectrum, which proved the presence of -OH and O-CH2-; the above comparison results proved that HPG was successfully grafted onto Zein. 1 In the H NMR spectrum, it can be noted that in addition to the strong signals at 3.2-3.8 ppm, there is a new peak around 4 ppm. The newly appeared signal can be attributed to the methyl ether group connected to the phenyl group in CUR. In addition, many small peaks between 5.5 and 8 ppm can be detected. These signals are due to C=C and the aromatic ring of CUR. Therefore, it can be concluded that CUR was successfully linked to the Zein-HPG-PA support via the phenol-alkyne click reaction.

[0056] The functional group information of CUR(a), Zein-HPG-PA@CUR(b), Zein-HPG-PA(c), Zein-HPG(d), and Zein(e) was tested by Fourier transform infrared spectroscopy. Figure 2 As can be seen in (a), 3500cm -1 The characteristic peak at 1600 cm-1 can be attributed to the stretching vibration of -OH in CUR. -1 The above peaks can be attributed to the stretching vibration of the aromatic ring and the C=C between the rings or the stretching vibration of the C=O between the rings. 1300-1100cm -1 The peaks between 1000 cm and 1000 cm can be attributed to the stretching vibration of the CO bond of the enol group coupled to C=C or the CO bond connected to the aromatic ring and the in-plane bending vibration of the CH in the aromatic ring. -1 The signal can be attributed to the out-of-plane bending vibration of CH in the aromatic ring. Zein's infrared spectrum has a peak at 3400-3200 cm -1 A small and broad peak is shown between the two, indicating the presence of free -NH2 or -OH in Zein. -1 The strong peak at about 3300 cm-1 can be attributed to the stretching vibration of C=O of amide in Zein. -1 、2900cm -1 and 1100cm -1Some new peaks or peak intensities increase at 2143 cm-1, which can be attributed to the stretching vibrations of -OH, -CH2 and CO, respectively. Therefore, it can be concluded that HPG is successfully grafted onto Zein. -1 A small peak was detected at 3400-3200cm, which can be attributed to the stretching vibration of C≡C. At the same time, in the infrared spectrum of Zein-HPG-PA, the signal intensity associated with the terminal -OH group was weakened accordingly. The above comparison results clearly confirmed the successful coupling of PA with Zein-HPG. Compared with the infrared spectrum of Zein-HPG-PA, the peak at 3400-3200cm -1 The peaks related to -OH reappeared between the two groups, which can be attributed to the enolic hydroxyl group of CUR. In addition, many peaks at 1600-1100 cm -1 The changes in the infrared signals indicated that CUR was loaded on Zein-HPG-PA via the phenol-alkyne click reaction.

[0057] The morphological characteristics of Zein-HPG, Zein-HPG-PA, and Zein-HPG-PA@CUR were observed by scanning electron microscopy. Figure 3 Scanning electron micrographs of Zein-HPG, Zein-HPG-PA, and Zein-HPG-PA@CUR are shown. Many pores were observed in the Zein-HPG photograph, and the size of these pores was even larger than 10 μm. The morphology of Zein-HPG was significantly different from that of Zein-HPG. The sizes of Zein-HPG-PA nanoparticles were uneven, connections between nanoparticles could also be observed, and the surfaces of these nanoparticles were quite smooth. The reason for the formation of nanoparticles by Zein-HPG-PA can be attributed to the coupling of hydrophobic PA with Zein-HPG. And the formation of self-assembled nanoparticles of Zein-HPG-PA@CUR can also be observed. However, compared with the morphology of Zein-HPG-PA, the particles were more irregular and the surface of the nanoparticles became rough. The morphological changes between the two samples also indicate that CUR was successfully loaded with Zein-HPG-PA.

[0058] The size and morphology of Zein-HPG, Zein-HPG-PA, and Zein-HPG-PA@CUR were characterized by transmission electron microscopy and Malvern particle size analyzer. Figure 4 As shown in Figure 2, nanoparticles can be found in all samples. Figure 4Some irregular aggregates with a size less than 500 nm and low contrast were observed in A. This is mainly due to the lack of conjugated structure in Zein-HPG. In addition, many particles with smaller sizes were detected, indicating that Zein-HPG is difficult to assemble into nanoparticles because its surface is covered with hydrophilic polymers. The DLS results also reflect the poor self-assembly phenomenon of Zein-HPG ( Figure 4 D). The size distribution of Zein-HPG is divided into two parts, one of which is centered at about 100nm and the other is centered at about 500nm. Based on the DLS results, the size distribution of Zein-HPG is 482nm, and the PDI and Zeta potential are 0.488 and -16.9mV, respectively. The introduction of terminal alkynes will increase the hydrophobicity of Zein-HPG-PA, thus facilitating the self-assembly of Zein-HPG-PA. Many irregular nanoparticles with higher contrast can be seen in the figure. From the transmission electron microscopy images, it can also be observed that there are some irregular small particles in Zein-HPG-PA, and the particle size of Zein-HPG-PA is reduced to a certain extent ( Figure 4 B). In good agreement with these changes, the average particle size of Zein-HPG-PA is approximately 388.2 nm, the PDI is 0.497, and the Zeta potential is -35.5 mV ( Figure 4 E). The better self-assembly behavior can be attributed to the introduction of hydrophobic PA at the end of HPG. Figure 4 C shows the TEM image of Zein-HPG-PA@CUR. Smaller and uniform spherical nanoparticles were also observed in the figure. In addition, due to the introduction of CUR with a conjugated structure, the image contrast also increased accordingly. The size of Zein-HPG-PA@CUR is approximately 290.4nm, the PDI is 0.154, and the Zeta potential is -40.3mV ( Figure 4 F). Based on the TEM images and DLS results, it can be concluded that PA is linked to Zein-HPG via an ester bond and CUR can be loaded onto Zein-HPG-PA via a phenol-alkyne click reaction. The introduction of these hydrophobic components can enhance their self-assembly ability and form a stable dispersion in pure aqueous solution, providing the possibility for the delivery of CUR under physiological conditions.

[0059] Through the ultraviolet absorption spectrum of CUR, the absorbance value at 427nm can be calculated to monitor the amount of CUR loaded on Zein-HPG-PA. According to the standard curve of CUR ( Figure 5), the drug loading and encapsulation efficiency of the drug delivery system were calculated to be 31.5% and 46%, respectively; this is a fairly high loading efficiency. In addition, the drug loading can be adjusted by changing the parameters for synthesizing Zein-HPG and Zein-HPG-PA. Drug release behavior is crucial for drug delivery systems. Next, the release behavior of CUR from Zein-HPG-PA@CUR in PBS solutions at 37°C and pH 5.0 and 7.4 was studied. The release curves at pH 5.0 and 7.4 are shown in Figure 2. Figure 6 As shown. It can be clearly observed from the figure that the release of CUR from Zein-HPG-PA@CUR is greatly related to the pH value. In a pH 7.4 solution, almost no drug release was detected at 72h, while in an acidic environment of pH 5.0, the release percentage of CUR reached 20% within the first 30min. After 12h, CUR basically stopped releasing, with a release rate of about 32%. The overall low release may be due to the fact that CUR is covalently linked to the carrier through a phenol-alkyne reaction and is stable at neutral pH. Therefore, it can effectively overcome the initial drug release for storage. It is well known that the ester bond breakage between PA and Zein-HPG is pH-dependent and is generally easier to break under strong acid and strong base conditions. This is very consistent with the release behavior, indicating that the drug delivery system can remain stable in the blood without release, which is conducive to the intracellular delivery of CUR.

[0060] The uptake behavior of drugs into cells was obtained by confocal microscopy. After cell recovery, MDA-MB-231 cells were inoculated into culture dishes and placed in an incubator at 37°C, 5% CO2 and 95% humidity. DMEM medium containing 10% fetal bovine serum was used for culture, and the medium was replaced the next day. When the cells adhere to the wall and grow to a density of 70-80%, they can be used for cell laying and other cell experiments. 24h before the experiment, 1mL of cells were plated at 1×10 5 mL -1 The density of Zein-HPG-PA@CUR was inoculated into the confocal culture dish. After 24 hours, Zein-HPG-PA@CUR (50 μg mL -1 ) were added to the cell culture dish and incubated for 4 h in the dark. The culture medium was removed and washed three times with PBS buffer solution to remove the remaining culture medium. Then 1 mL of Hoechst 33342 staining solution was added to each confocal culture dish and incubated at 37 ° C for 20-30 min. The staining solution was aspirated and washed 2-3 times with PBS. Fluorescence images were acquired by confocal microscopy under 405 nm and 488 nm laser excitation. Figure 7 A is the laser confocal imaging image under 488nm laser excitation after incubation of cells and Zein-HPG-PA@CUR, from which obvious green fluorescence signals can be seen. Figure 7B is the laser imaging under 405nm laser excitation, in which obvious blue fluorescence of DAPI can be seen, and the location of the blue fluorescence is the location of the cell nucleus. Figure 7 C is a laser confocal bright field image. It can be seen that after incubation with Zein-HPG-PA@CUR, the cells still maintain a good cell morphology and are well attached to the cell culture dish, indicating that under this concentration and incubation time condition, Zein-HPG-PA@CUR has no obvious cytotoxicity. Figure 7 D is a combined diagram of AC, and it can be clearly seen that Zein-HPG-PA@CUR can be taken up by cells and is mainly distributed in the cytoplasm. Since the intracellular environment is an acidic environment, when Zein-HPG-PA@CUR is taken up by cells, the intracellular delivery and release of CUR can be achieved by breaking the pH-responsive ester bond between the drug and the carrier, and the anti-tumor effect of CUR can be exerted. The construction of a CUR drug delivery system by the above method can effectively solve the water solubility problem of CUR. The formed nanoscale assembly can exist stably in the blood without releasing drugs. After these drug-loaded nanoassemblies are targeted to the tumor site through the EPR effect, they can break the ester bond and release CUR through cell endocytosis to exert their effects.

[0061] The dark toxicity of Zein-HPG-PA was determined by CCK-8. MDA-MB-231 cells were plated at 5 × 10 3 The cells were inoculated into 96-well plates at a density of 10 cells / mL and incubated for 24 h. Then, different concentrations of the test samples (0 μg mL -1 , 1.56 μg·mL -1 , 3.125 μg·mL -1 , 6.25 μg·mL -1 , 12.5 μg·mL -1 , 25 μg·mL -1 , 50μg·mL -1 , 100 μg·mL -1 After 24 h of incubation, 100 μL of freshly prepared CCK-8 solution (0.5 mg mL -1 ) was added to each well and further incubated for 2 h. The absorbance of CCK-8 at 450 nm was measured using a microplate reader. Cell viability was expressed as the ratio of the absorbance of cells cultured with the test sample to cells cultured with the medium alone. Six replicate wells were set for each concentration of each microplate, and three separate experiments were performed. The results are shown as mean ± standard deviation. Figure 8 It can be seen that when the material concentration reaches 100 μg·mL -1At a concentration of , the cell survival rate was still over 90%, indicating that Zein-HPG-PA has good biocompatibility and can be used as a drug carrier.

[0062] The phototoxicity of Zein-HPG-PA@CUR was determined by CCK-8. MDA-MB-231 cells were plated at 5×10 3 The cells were inoculated into 96-well plates at a density of 10 cells / mL and incubated for 24 h. -1 , 0.39 μg·mL -1 , 0.78 μg·mL -1 , 1.56 μg·mL -1 , 3.125 μg·mL -1 , 6.25 μg·mL -1 , 12.5 μg·mL -1 , 25 μg·mL -1 , 50 μg·mL -1 , 100 μg·mL -1 After 4 h of incubation, cells were treated with a test sample at an optical density of 1 mW / cm 2 and 2mW / cm 2 The cells were irradiated with a 450nm laser for 3min and incubated for another 20h. CCK-8 reagent was then added and incubated for a further 2h. Cell viability was measured using a microplate reader at 450nm absorbance. The cell viability in the photodynamic experiment was calculated by using the absorbance ratio between cells cultured with the sample under light and cells cultured only in culture medium. Six replicate wells were set for each concentration of each microplate, and three independent experiments were performed. The results are shown as the mean ± standard deviation. In order to compensate for the weaker antitumor activity of Zein-HPG-PA@CUR, the phototoxicity of Zein-HPG-PA@CUR under laser irradiation was investigated. After Zein-HPG-PA@CUR and cells were incubated for 4h, the cells were illuminated with a 450nm laser (laser intensities were 1mW / cm 2 , 2mW / cm 2 ) Cell viability after 3 min of irradiation. Fig. 9 It can be clearly seen that compared with the non-illumination group, the Zein-HPG-PA@CUR concentration was 100 μg mL -1 , light intensity is 1mW / cm 2 When the laser is increased to 2mW / cm 2 100 μg·mL -1Zein-HPG-PA@CUR killed almost all cells. The above results show that the strong photodynamic effect of CUR can enhance its ability to kill triple-negative breast cancer cells. It proves that Zein-HPG-PA@CUR has the potential for combined chemotherapy and PDT tumor treatment.

[0063] Live and dead cells were stained with Calcein AM and PI. MDA-MB-231 cells were plated at 4 × 10 5 The cells were seeded into 6-well plates at a density of 100 cells and incubated for 24 h. -1 ) was added to the cell culture dish and incubated for 4 h in the dark. 2 Irradiate with 450nm laser for 3min. After washing three times with PBS, add 2μM Calcein AM stock solution and incubate in the dark at 37℃ for 20-25min. Then add 4μM PI stock solution and incubate in the dark for 5min. Finally, wash the cell culture dish twice with PBS. Fig.10 The results of live cell and dead cell imaging show that, under non-illumination conditions, Zein HPG-PA and the blank PBS group both emit strong AM green fluorescence and almost no PI red fluorescence, indicating that the cells are in good condition, further demonstrating the good biocompatibility of Zein HPG-PA. -1 After incubation with Zein-HPG-PA@CUR for 24 hours, the green fluorescence of the cells decreased significantly, and the red fluorescence did not appear significantly, indicating that Zein-HPG-PA@CUR has a certain toxicity to the cells, but its killing effect on tumor cells is limited. 2 After laser irradiation, it was found that there was no green fluorescence in the entire field of view, and most cells were dyed red, indicating that most cells were dead. Consistent with the results of cell viability evaluation, the live-dead cell staining results also proved that light can significantly enhance the anti-tumor effect of Zein-HPG-PA@CUR.

[0064] The green fluorescent probe DCFH-DA and the red fluorescent probe DHE were used to detect the total ROS and O2 in the cells. - MDA-MB-231 cells were used as model cells. DCFH-DA indicator was also used to detect the production of ROS in cells under light. Samples (50 μg mL -1) and cells were incubated for 4 h, the culture medium was removed, and the cells were washed three times with PBS buffer solution. DCFH-DA and dihydroethidium (DHE) (20 μM) were then added to the culture medium, and the cells were incubated at 37°C for 30 min, and then carefully washed three times with PBS. After adding 100 μL PBS buffer solution, the cells were placed under a light intensity of 3 mW / cm 2 The cells were irradiated with a 450 nm laser for 3 min. Finally, an inverted fluorescence microscope was used to obtain cell fluorescence images. Fig.11 , Fig.12 The results showed that the ROS produced by Zein-HPG-PA@CUR breast cancer cells had no obvious fluorescence in pure DCFH and pure DHE, indicating that the ROS in the original cells was very low; and no obvious fluorescence was observed after Zein-HPG-PA@CUR and cells were incubated without light, indicating that CUR itself did not produce many free radicals. However, at 3mW / cm 2 After illumination, Zein-HPG-PA@CUR can be seen with obvious green and red fluorescence, indicating that Zein-HPG-PA@CUR has a strong ability to produce ROS under illumination conditions. In addition, it was found that the green fluorescence is stronger than the red fluorescence, indicating that CUR is a type II photosensitizer, and the reason for the red fluorescence may be the mutual conversion of free radicals in cells.

[0065] Example 2

[0066] A method for preparing a nano drug delivery system for combined chemical-photodynamic therapy comprises the following steps:

[0067] Step 1, synthesis of Zein-HPG: 0.5 g zein, 0.2 g sodium hydride and 0.5 g 18-crown-6 were dissolved in 20 mL anhydrous DMF, heated to 80°C with ultrasonic stirring, and then 0.5 mL glycidol was added to react for 24 h. After the reaction, the product was dialyzed and purified in water and ethanol for 2 days, and then concentrated and freeze-dried to obtain Zein-HPG;

[0068] Step 2, synthesis of Zein-HPG-PA carrier: 0.7 g Zein-HPG, 1.4 g propiolic acid and 1.77 g p-toluenesulfonic acid were added to 10 mL CHCl3, the reaction mixture was heated to 80°C and refluxed for 20 h, the reaction product was washed with saturated NaHCO3 solution, saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier;

[0069] Step 3. Synthesis of nano drug delivery system Zein-HPG-PA@CUR: 10 mg Zein-HPG-PA and 10 mg CUR were dissolved in 10 mL anhydrous THF, and after ultrasonication until uniformly dispersed, 2 mg DMAP was added and reacted in anhydrous and vacuum environment for 4 hours. After the reaction, the product was dialyzed and purified in water and ethanol for 1 day, and then concentrated and freeze-dried to obtain Zein-HPG-PA@CUR.

[0070] Example 3

[0071] A method for preparing a nano drug delivery system for combined chemical-photodynamic therapy comprises the following steps:

[0072] Step 1, synthesis of Zein-HPG: 0.5 g zein, 0.2 g sodium hydride and 0.5 g 18-crown-6 were dissolved in 20 mL anhydrous DMF, heated to 80°C with ultrasonic stirring, and then 2 mL glycidol was added to react for 24 h. After the reaction, the product was dialyzed and purified in water and ethanol for 2 days, and then concentrated and freeze-dried to obtain Zein-HPG;

[0073] Step 2, synthesis of Zein-HPG-PA carrier: 0.35 g Zein-HPG, 1.4 g propiolic acid and 1.77 g p-toluenesulfonic acid were added to 10 mL CHCl3, the reaction mixture was heated to 80°C and refluxed for 20 h, the reaction product was washed with saturated NaHCO3 solution, saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier;

[0074] Step 3. Synthesis of nano drug delivery system Zein-HPG-PA@CUR: 10 mg Zein-HPG-PA and 20 mg CUR were dissolved in 10 mL anhydrous THF, and after ultrasonication until uniformly dispersed, 2 mg DMAP was added and reacted in anhydrous and vacuum environment for 4 h. After the reaction, the product was dialyzed and purified in water and ethanol for 1 day, and then concentrated and freeze-dried to obtain Zein-HPG-PA@CUR.

[0075] In summary, the nano drug delivery system of the present invention has stable properties, good biocompatibility, high drug loading, and pH-responsive release. Curcumin is both a chemotherapeutic drug and a photosensitizer, achieving a combined chemical-photodynamic therapy effect and having a better therapeutic effect on breast cancer. It has a good application prospect in the research of natural active anti-tumor drugs.

Claims

1. A nano drug delivery system for combined chemo-photodynamic therapy, characterized in that: Zein-HPG was synthesized by anionic ring-opening polymerization of zein protein; then propiolic acid was combined with the hydroxyl group of Zein-HPG through esterification reaction to obtain Zein-HPG-PA carrier; finally, curcumin was loaded onto the Zein-HPG-PA carrier through phenol-alkyne click reaction to obtain the nano drug delivery system Zein-HPG-PA@CUR.

2. A method for preparing the nano drug delivery system according to claim 1, characterized in that: The following steps are involved: Step 1, synthesis of Zein-HPG: dissolve zein, sodium hydride and 18-crown ether-6 in anhydrous DMF, stir ultrasonically to make them dispersed evenly, then heat to 75-85°C in an oil bath, add glycidol and react for 24-48h, dialyze and purify the product in water and ethanol, then concentrate and freeze-dry to obtain Zein-HPG; Step 2, synthesis of Zein-HPG-PA carrier: Zein-HPG, propiolic acid and p-toluenesulfonic acid are added to CHCl3, the reaction mixture is heated to 75-85°C and refluxed for 16-24h, the reaction product is washed with saturated NaHCO3 solution, saline and ultrapure water, dried over anhydrous magnesium sulfate overnight, concentrated by rotary evaporation and freeze-dried to obtain Zein-HPG-PA carrier; Step 3. Synthesis of the nano drug delivery system Zein-HPG-PA@CUR: Dissolve the Zein-HPG-PA carrier and CUR in anhydrous THF, sonicate until uniformly dispersed, add DMAP and react in an anhydrous and vacuum environment. After the reaction, the product is dialyzed and purified in water and ethanol, and then concentrated and freeze-dried to obtain the nano drug delivery system Zein-HPG-PA@CUR.

3. The method for preparing the nano drug delivery system for combined chemical-photodynamic therapy according to claim 2, characterized in that: The mass ratio of glycidol to zein is 1-4.

4. The method for preparing the nano drug delivery system for combined chemical-photodynamic therapy according to claim 2, characterized in that: The molar ratio of propiolic acid to p-toluenesulfonic acid is 2:

1.

5. The method for preparing a nano drug delivery system for combined chemical-photodynamic therapy according to claim 2, characterized in that: The mass ratio of propiolic acid to Zein-HPG is 2-4.

6. The method for preparing the nano drug delivery system for combined chemical-photodynamic therapy according to claim 2, characterized in that: The mass ratio of CUR to Zein-HPG-PA carrier is 1-2.

7. The method for preparing a nano drug delivery system for combined chemo-photodynamic therapy according to claim 2, characterized in that: In step 1, ultrasonic stirring is performed for 20-30 min.

8. The method for preparing a nano drug delivery system for combined chemical-photodynamic therapy according to claim 2, characterized in that: In step 3, ultrasonic stirring is performed for 10-30 min.

9. Use of the nano drug delivery system as claimed in claim 1 in inhibiting breast cancer cells in vitro.

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