Targeting nano drug delivery system and application thereof in chemotherapy-photothermal combined treatment of breast cancer

Abstract

The invention discloses a targeted nano drug delivery system and application thereof in chemotherapy-photothermal combined treatment of breast cancer, and belongs to the technical field of tumor treatment. According to the targeted nano drug delivery system, oxidized mesoporous carbon nanospheres are used as carriers, P6 peptides specifically combined with RON receptors are modified on the surfaces of the carriers, and cytotoxic drugs are loaded in the carriers. The drug loading rate of the nano drug delivery system can reach 83.5%, and the nano drug delivery system has good biocompatibility, can specifically target breast cancer cells, has good photo-thermal performance, realizes a chemotherapy-photo-thermal therapy combined treatment effect, and provides a new thought for breast cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor treatment, and particularly relates to a targeted nano-drug delivery system and its application in the combined chemo-photothermal therapy of breast cancer. Background Art

[0002] Breast cancer is the most common malignant tumor in women, accounting for about one-third of all female tumor incidences, and its five-year survival rate is extremely low while the mortality rate remains high. Clinical studies have shown that the main reasons for the low cure rate of breast cancer are as follows: the inability to specifically deliver therapeutic drugs to the tumor site, resulting in poor efficacy after systemic administration and high toxicity to normal tissues; breast cancer cells are prone to develop drug resistance to chemotherapeutic drugs, seriously affecting the anti-tumor effect of single chemotherapeutic drugs. Therefore, an effective means to control breast cancer is to enable therapeutic drugs to specifically recognize and kill breast cancer cells and achieve targeted combined therapy.

[0003] In terms of targeted recognition, the ligand-receptor system-mediated targeted drug delivery strategy has become a hot topic in tumor treatment research. In recent years, reports on breast cancer-targeted drug delivery systems mediated by receptors have emerged in an endless stream. Common ones include related studies on targeted drug delivery systems mediated by folate receptor, integrin receptor, membrane heat shock protein 70, gastrin-releasing peptide receptor, vasoactive intestinal peptide receptor, etc. However, so far, there has been little further progress in these studies, and the limiting factors may be attributed to the following two points: one is that specific receptors are only expressed in some breast cancer tissues. For example, studies have shown that only 29% of breast cancer tissues express folate receptor and 26% of breast cancer tissues express integrin receptor; the other is that some receptors are overexpressed not only in breast cancer tissues but also in normal breast tissues. For example, gastrin-releasing peptide receptor and vasoactive intestinal peptide receptor. These factors make the corresponding targeted drug delivery systems prone to serious off-target effects in vivo, resulting in little therapeutic effect and large side effects. Therefore, selecting a receptor specifically expressed in breast cancer tissues and finding a suitable ligand for this receptor are the key factors for achieving targeted drug delivery.

[0004] Some studies have shown that up to 50% of breast cancer tissues overexpress the Recepteur d’Origine Nantais (RON) tyrosine kinase receptor, while normal breast tissues express less, suggesting that the RON receptor has the potential to be a site for active targeting of breast cancer. Next, a targeted drug delivery system constructed by finding a suitable ligand for the RON receptor is expected to achieve a more effective breast cancer targeting effect.

[0005] Macrophage-stimulating protein (MSP), as the natural ligand of the RON receptor, can specifically bind to the RON receptor. This binding can activate multiple signaling pathways, including phosphatidylinositol 3-kinase (PI3K) / AKT, mitogen-activated protein kinase, c-Jun N-terminal kinase, and β-catenin signaling pathways, and is involved in cell proliferation, division, and metastasis, playing an important role in the proliferation and metastasis of breast cancer cells. Therefore, MSP is not suitable as a targeting ligand for the nano-drug delivery system.

[0006] Clinical studies have shown that single chemotherapy has limited therapeutic effects on breast cancer, while the combined effect of chemotherapy and photothermal therapy can significantly improve the anti-tumor efficacy. This is mainly because photothermal therapy helps chemotherapeutic drugs enter tumor cells while killing tumor cells, thereby enhancing the cytotoxicity of chemotherapeutic drugs. Oxidized mesoporous carbon nanospheres (OMCN) are nanospheres with a three-dimensional structure, simple structure, stable properties, good biocompatibility, and large specific surface area. While having ordered mesopores on the surface, they also maintain the photothermal conversion performance of carbon materials and can be used as a carrier for loading chemotherapeutic drugs and a material for photothermal therapy. Given that OMCN can be used as both a carrier to deliver chemotherapeutic drugs and a photothermal therapy reagent, it has great application value in the nano-drug delivery system. Summary of the Invention

[0007] Based on the above analysis, an object of the present invention is to provide a nano-drug delivery system targeting breast cancer tissue. The system uses OMCN as a carrier and internally loads anti-tumor drugs. The photothermal conversion performance of OMCN enables it to help anti-tumor drugs enter tumor cells while performing photothermal therapy, strengthening the cytotoxic effect of anti-tumor drugs. In addition, OMCN has rich functional groups (such as -COOH, etc.) on its surface, which are easy to chemically modify. Targeting groups can be modified on the surface of OMCN by chemical coupling methods to form a targeted delivery system with the combined therapeutic effect of chemotherapy and photothermal therapy. The second object of the present invention is to provide a preparation method of the nano-drug delivery system and its application in the preparation of drugs for treating breast cancer.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] In the first aspect, the present invention provides an application of a nano-drug delivery system in the preparation of drugs for treating breast cancer, characterized in that the nano-drug delivery system uses oxidized mesoporous carbon nanospheres (OMCN) as a carrier, and a polypeptide that specifically binds to the RON receptor is modified on the surface. The polypeptide is P6 peptide, and the amino acid sequence of the P6 peptide is FEHSLYKEMTHL.

[0010] The P6 peptide described in the present invention is a polypeptide obtained by phage display technology. It specifically binds to the RON receptor with high affinity. Moreover, the binding site of the P6 peptide on the RON receptor is different from that of MSP, and it will not competitively inhibit with MSP and does not affect the physiological function of MSP itself. Therefore, the technical personnel of the present invention judge that the P6 peptide is a potential novel breast cancer targeting ligand and can be used to mediate the selective enrichment of the nano-drug delivery system in breast cancer tissues. Based on this, the nano-drug delivery system creatively constructed with the P6 peptide as a ligand in the present invention is expected to achieve the effect of specifically targeting breast cancer.

[0011] The present invention does not limit the way of modifying the P6 peptide onto the surface of the oxidized mesoporous carbon nanosphere carrier. Since the surface of the oxidized mesoporous carbon nanosphere has abundant functional groups such as -COOH, those skilled in the art can choose to modify it by directly coupling the P6 peptide with the oxidized mesoporous carbon nanosphere, or choose to use a linker as a bridge to connect the P6 peptide with the oxidized mesoporous carbon nanosphere.

[0012] In a specific embodiment of the present invention, the way of modifying the P6 peptide on the surface of the oxidized mesoporous carbon nanosphere carrier is selected from using a bifunctional PEG to connect the P6 peptide and the oxidized mesoporous carbon nanosphere. The bifunctional PEG is amino-polyethylene glycol-maleimide (NH 2 -PEG-MAL, with a molecular weight of 3500 Da). The N-terminus of the P6 peptide is coupled with its maleamide group through cysteine containing -SH, and the -COOH on the surface of the oxidized mesoporous carbon nanosphere is coupled with its -NH 2 group.

[0013] Furthermore, the nano-drug delivery system further includes loading a cytotoxic drug inside the oxidized mesoporous carbon nanosphere carrier.

[0014] The cytotoxic drug is selected from all drugs commonly used in the field of tumor chemotherapy, including but not limited to doxorubicin, epirubicin, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, SN-38, cisplatin drugs, vinblastine, vincristine, docetaxel, gemcitabine.

[0015] In some embodiments of the present invention, the cytotoxic drug is selected from doxorubicin, camptothecin or paclitaxel.

[0016] In a specific embodiment of the present invention, the cytotoxic drug is doxorubicin.

[0017] In the present invention, the cytotoxic drug, especially doxorubicin, is loaded into the mesopores of the oxidized mesoporous carbon nanosphere through π-π conjugation.

[0018] The drug loading rate of the nano-drug delivery system targeting breast cancer tissue prepared by the present invention can reach 83.5%, and it has good biocompatibility. In an environment with a pH of 7.4, the release rate of cytotoxic drugs is low and the stability is good. Under an acidic environment with a pH of 5.0 and / or under near-infrared laser irradiation, the release of cytotoxic drugs is triggered. Based on the above characteristics, the nano-drug delivery system provided by the present invention can rapidly release cytotoxic drugs in the acidic microenvironment of breast cancer, achieve targeted chemotherapy, reduce damage to normal tissues, improve the treatment effect and reduce side effects. Further, near-infrared laser irradiation is implemented at the tumor lesion site, and the nano-drug delivery system has photothermal conversion performance to achieve the photothermal treatment effect at the tumor site. Secondly, the technical personnel of the present invention found that near-infrared laser irradiation can accelerate the release of cytotoxic drugs, enrich the drugs in the tumor area, and the photothermal performance of the nano-drug delivery system under light irradiation can increase the permeability of the tumor cell membrane and promote drug penetration, further enhancing the chemotherapy effect.

[0019] In the present invention, the technical personnel unexpectedly found that the release time and release site of cytotoxic drugs can be regulated by pH and / or near-infrared laser irradiation. In a preferred embodiment of the present invention, the near-infrared laser irradiation method is an alternating cycle of laser activation (Laser on) and laser inactivation (Laser off). In each cycle, the laser activation time is 5 min, and the laser inactivation time is 115 min or 235 min, and 7-8 cycles are performed within 24 h. The photothermal effect of the nano-drug delivery system prepared by the present invention is concentration-dependent and laser intensity-dependent on the nano-drug delivery system. Therefore, the present invention does not specifically limit the laser wavelength and intensity, and those skilled in the art can obtain the optimal laser wavelength and laser intensity through debugging in routine use. In a specific embodiment of the present invention, the laser wavelength is 808 nm, and the laser intensity is 3 W / cm 2 。

[0020] In a specific embodiment of the present invention, the application of the nano-drug delivery system in the preparation of drugs for treating breast cancer includes administering a therapeutically effective amount of the nano-drug delivery system described in the present invention alone to a subject in need, or administering it in combination with other medicaments or other treatment methods.

[0021] Specifically, the other medicaments include, but are not limited to, immune checkpoint inhibitors and chemotherapeutic agents. The immune checkpoint inhibitors include antibodies, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies. The other treatment methods include, but are not limited to, radiotherapy.

[0022] In a second aspect, the present invention provides a preparation method of a nano-drug delivery system, which is characterized in that the method includes the following steps:

[0023] (1) Prepare oxidized mesoporous carbon nanospheres (OMCN);

[0024] (2) Stir the oxidized mesoporous carbon nanospheres and the cytotoxic drug in a buffer solution in proportion for 6 - 24 h, centrifuge, discard the supernatant, and obtain the oxidized mesoporous carbon nanospheres loaded with the drug;

[0025] (3) Activate the oxidized mesoporous carbon nanospheres loaded with the drug, add amino - polyethylene glycol - maleimide for coupling, centrifuge, discard the supernatant, and obtain the oxidized mesoporous carbon nanospheres - PEG complex loaded with the drug;

[0026] (4) Add P6 peptide and the oxidized mesoporous carbon nanospheres - PEG complex loaded with the drug to a buffer solution system, stir and react for 6 - 24 h, centrifuge, discard the supernatant, and obtain the nano - drug delivery system.

[0027] In a specific embodiment of the present invention, the cytotoxic drug in step (2) is selected from doxorubicin, and the mass ratio of doxorubicin to oxidized mesoporous carbon nanospheres is (1 - 2):1.

[0028] In a third aspect, the present invention provides a nano - drug delivery system for combined chemo - photothermal therapy of breast cancer. It is characterized in that the nano - drug delivery system uses oxidized mesoporous carbon nanospheres as a carrier, the carrier internally loads a cytotoxic drug, and the surface of the carrier is modified with P6 peptide.

[0029] Preferably, the method of surface - modifying P6 peptide is selected from using amino - polyethylene glycol - maleimide (NH 2 -PEG - MAL) to connect P6 peptide and oxidized mesoporous carbon nanospheres.

[0030] The cytotoxic drug is selected from all drugs conventionally used in the field for tumor chemotherapy, including but not limited to doxorubicin, epirubicin, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, SN - 38, cisplatin - type drugs, vinblastine, vincristine, docetaxel, gemcitabine.

[0031] In some embodiments of the present invention, the cytotoxic drug is selected from doxorubicin, camptothecin or paclitaxel.

[0032] In a specific embodiment of the present invention, the cytotoxic drug is doxorubicin.

[0033] In the present invention, the cytotoxic drug, especially doxorubicin, is loaded into the mesopores of oxidized mesoporous carbon nanospheres through π - π conjugate action.

[0034] In a fourth aspect, the present invention provides a pharmaceutical composition, which is characterized in that the pharmaceutical composition includes an effective dose of the nano - drug delivery system described in the third aspect of the present invention.

[0035] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0036] In a preferred embodiment of the present invention, the pharmaceutical composition is suitable for parenteral administration, such as by intravenous, intramuscular, intradermal, and subcutaneous routes. Therefore, according to the applicable dosage form, the pharmaceutically acceptable excipients are selected from antioxidants, buffers, bacteriostatic agents, suspending agents, solubilizers, or solutes that render the formulation isotonic with the subject's blood.

[0037] In a specific embodiment of the present invention, the pharmaceutical composition is an injection and is administered to the subject by intravenous injection.

[0038] The present invention constructs a novel polypeptide-modified nanodrug delivery system to clarify the possibility of the RON receptor as an active targeting site for breast cancer and the ability of polypeptide P6 as a breast cancer targeting ligand. Using this nanodrug delivery system for targeted recognition and chemo-photothermal therapy of breast cancer under fluorescence imaging guidance provides a new method to improve the low cure rate of breast cancer and enhance the therapeutic effect of breast cancer.

[0039] The technical solution provided by the present invention has the following advantages:

[0040] 1) The nanodrug delivery system OMCNPPD provided by the present invention has a simple structure, is easy to prepare, and has a high drug loading capacity;

[0041] 2) It has been preliminarily confirmed to have good photothermal ability, showing the potential of dual stimuli-triggered drug release by photothermal and pH, achieving precise controlled release of drugs at specific times and specific sites, and improving the bioavailability and safety of drugs;

[0042] 3) It can specifically target breast cancer cells, realizing the combined treatment effect of chemo-photothermal therapy, and providing new ideas for the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Characterization results of the nanodrug delivery system; (A) TEM image of OMCN, Bar = 50 nm; (B) particle size distribution and (C) Zeta potential of OMCN, OMCNP, and OMCNPP; (D) fluorescence spectra of free DOX, OMCN / DOX, OMCNPPD, and OMCNPP; (E) fluorescence images of free DOX, OMCNPP, and OMCNPPD solutions at different DOX concentrations (0, 5, 10, 20, 40, 60 μg / mL).

[0044] Figure 2 Photothermal effect of OMCNPP; (A) 808-nm laser (3.0 W / cm 2Thermal imaging pictures of OMCNPP aqueous solutions with different concentrations (0, 15, 30, 45, 60, 75, and 100 μg / mL) after 5 min of irradiation; (B) Photothermal pictures of 45 μg / mL OMCNPP aqueous solution after 5 min of 808-nm laser irradiation at 1 W / cm 2 , 2 W / cm 2 , and 3 W / cm 2

[0045] Figure 3 Drug release of OMCNPPD; (A) Cumulative release curves of DOX from OMCNPPD at pH 7.4 and pH 5.0 (n = 3); (B) At pH 7.4 and pH 5.0, before each preset time point, the OMCNPPD solution was irradiated with NIR laser at a power density of 3 W / cm 2 for 5 min, and the cumulative release curve of DOX released from OMCNPPD was recorded (n = 3).

[0046] Figure 4 Results of Western blot assay; (A) Protein expression of RON receptor and its α-subunit in MDA-MB-231 cells and MCF-10A cells; (B) Densitometric analysis of RON receptor protein level normalized to β-actin level (n = 3).

[0047] Figure 5 Cell uptake of OMCNPD and OMCNPPD; (A-I) Fluorescence pictures of MDA-MB-231 cells incubated with OMCNPD, OMCNPPD (pre-incubated with P6 peptide), or OMCNPPD; (J-O) Fluorescence pictures of MCF-10A cells incubated with OMCNPD or OMCNPPD, Bar = 100 μm.

[0048] Figure 6 Representative 2D fluorescence pictures of each confocal microscope scanning layer and corresponding 2.5D fluorescence pictures in the middle of the z-plane of MDA-MB-231 tumor spheres incubated with OMCNPD or OMCNPPD for 12 h.

[0049] Figure 7 In vitro anti-tumor ability; (A) Live-dead assay and (B) CCK-8 assay of MDA-MB-231 cells treated with normal saline, free DOX, OMCNPP + NIR, OMCNPPD, and OMCNPPD + NIR, Bar = 100 μm. Detailed implementation methods

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all of them. 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.

[0051] 1. Research methods

[0052] 1.1 Preparation and characterization of the nano-drug delivery system OMCNPPD

[0053] S1: Synthesize OMCN using the low-concentration hydrothermal-oxidation method

[0054] Weigh 0.6 g of phenol, add 2.1 mL of formaldehyde solution (37%) and 15 mL of sodium hydroxide solution (0.1 M), and stir at 70 °C for 30 min. Weigh 0.96 g of the triblock copolymer Pluronic F127 and dissolve it in 15 mL of distilled water, then add it to the above mixture. After continuous stirring for 2 h, add 50 mL of distilled water. Stir rapidly for 18 h, add 56 mL of distilled water for dilution, transfer the solution to a reaction kettle, and then continue to heat and react in a high-temperature blast drying oven at 130 °C for 24 h. Collect the product, wash it with water and freeze-dry it. During the carbonization process, under an environment of high-purity nitrogen, the temperature is programmed to rise from 25 °C to 350 °C in 1 h, from 350 °C to 550 °C in 2 h, from 550 °C to 750 °C in 2 h, and maintained at 750 °C for 1 h to synthesize carbon spheres. Then, mix concentrated sulfuric acid and concentrated nitric acid according to a volume ratio of 3:1, add the carbon spheres, sonicate for 3 h, and stir in a water bath at 60 °C for 4 h. Dilute with distilled water and wash by repeated centrifugation until the pH of the supernatant is neutral, collect and freeze-dry to obtain OMCN.

[0055] S2: Load DOX into OMCN to obtain OMCN / DOX

[0056] Mix 1 mL of OMCN (1 mg / mL) and DOX in a ratio of 1:(1 - 2) and stir in PBS buffer (pH 7.4) for 24 h, then centrifuge and discard the supernatant to obtain OMCN / DOX.

[0057] S3: Couple the linker to the surface of OMCN / DOX to obtain OMCNPD

[0058] Immediately add MES buffer (pH 6.0) to disperse OMCN / DOX. Weigh 50 mg of EDC and 90 mg of NHS and add them to the solution, stir for 2 h, centrifuge and discard the supernatant. Subsequently, dissolve the bifunctional PEG (NH 2 -PEG-MAL) with MES buffer (pH 6.0) and add it to the activated OMCN / DOX, stir for 4 h, check the pH, centrifuge and discard the supernatant to obtain OMCNPD.

[0059] S4: OMCNPD is connected to P6 polypeptide to obtain OMCNPPD

[0060] Add 2 mL of P6 polypeptide (1 mg / mL) to OMCNPD, stir in PBS buffer (pH 7.0) for 24 h, centrifuge, discard the supernatant, and finally obtain OMCNPPD.

[0061] The OMCNP refers to a nano-delivery carrier formed by conjugating a bifunctional PEG (NH 2 -PEG-MAL) on the surface of OMCN. Its difference from OMCNPPD is that OMCN does not encapsulate the cytotoxic drug DOX, and the surface of OMCN is not modified with P6 peptide.

[0062] The OMCNPP refers to a nano-delivery carrier formed by conjugating a bifunctional PEG (NH 2 -PEG-MAL) on the surface of OMCN and then modifying it with P6 peptide. Its difference from OMCNPPD is that OMCN does not encapsulate the cytotoxic drug DOX.

[0063] Characterize this nano-drug delivery system by means of morphology, particle size, Zeta potential, drug loading, fluorescence spectrum, fluorescence image, and drug release.

[0064] 1.2 Photothermal imaging effect

[0065] Evaluate its photothermal performance by measuring the temperature fluctuation of the OMCNPP solution under 808 nm laser irradiation. Take appropriate amounts of OMCNPP solutions with different concentrations (0, 15, 30, 45, 60, 75, and 100 μg / mL) and place them in EP tubes. Use a near-infrared laser at 808 nm (power density of 1 W / cm 2 , 2 W / cm 2 and 3 W / cm 2 ) to irradiate continuously for 5 min, and use an infrared thermal imager to record the change of temperature over time.

[0066] 1.3 Drug release

[0067] Adopt the dialysis bag technique to evaluate the release behavior of the drug under in vitro stimulation. Put the same volume of OMCNPPD solution into a dialysis bag, and soak it in different release media (PBS with pH values of 5.0 and 7.4 respectively) under light protection at 37 °C and shake gently. Extract samples of the release medium at regular time intervals for analysis, and supplement an equal amount of fresh release medium. For the groups containing photothermal therapy, before extracting the release medium, irradiate the solution with an NIR laser at a power density of 3 W / cm 2 for 5 min. Use an enzyme-linked immunosorbent assay reader to measure and calculate the release amount of DOX.

[0068] 1.4 Expression level of RON receptor on specific cells

[0069] Appropriately add cell lysate into the culture plates of MDA-MB-231 cells and CHO cells, place on ice bath for 30 min, centrifuge the collected cell lysate at 12,000 rpm for 10 min at 4 °C, take the supernatant, which is the total protein solution, and measure its concentration using the BCA method.

[0070] Prepare an SDS-PAGE gel, load the protein samples, and perform electrophoresis. After the electrophoresis is terminated, transfer the membrane. Block the transferred membrane with 5% skim milk (prepared with 0.5% TBST) at room temperature for 1 h.

[0071] Add appropriate diluted RON primary antibody and β-actin primary antibody, incubate overnight at 4 °C, wash 3 times with TBST at room temperature, 5 min each time. Add horseradish peroxidase-conjugated secondary antibody, incubate at room temperature for 30 min, then wash 3 times with TBST at room temperature, 5 min each time. Use the Enhanced Chemiluminescence Detection System (ECL) to observe the protein expression level.

[0072] 1.5 In vitro targeting evaluation of OMCNPPD nanodrug delivery system

[0073] MDA-MB-231 cells and MCF-10A cells are respectively inoculated into confocal dishes at a concentration of 1×10 5 cells / dish, and are used for experiments after adhering for 24 h. Add 1.5 mL of medium containing OMCNPPD or OMCNPD (DOX concentration is 50 μg / mL) to the two types of cells respectively, and incubate at 37 °C for 2 h. Another group of MDA-MB-231 cells are pre-incubated with an excessive amount of P6 peptide before adding OMCNPPD, and incubated at 37 °C for 20 min. After incubation, wash twice with PBS, add 4% paraformaldehyde solution for fixation, remove the paraformaldehyde, wash again with PBS, obtain fluorescence pictures, and evaluate the targeting of the nanodrug delivery system through the cell uptake situation.

[0074] Compared with the two-dimensional monolayer cell culture model, three-dimensional tumor spheres can more truly reflect the structural characteristics of solid tumors in vivo. Therefore, in this study, in vitro MDA-MB-231 tumor spheres are constructed to further evaluate the targeting ability of the nanodrug delivery system.

[0075] The construction process of MDA-MB-231 tumor spheres is as follows: Add an appropriate amount of low melting point agarose powder to the basal medium, sterilize it using an autoclave and heat to completely dissolve the powder to make a 0.02 g / mL agarose solution, and then add it to a 48-well plate while it is still hot (150 μL per well). Wait for the solution to completely cool and become gelatinous, and then add MDA-MB-231 cells to the plate. After about one week, the MDA-MB-231 tumor spheres can be used for experiments. Add cell culture medium containing the OMCNPD or OMCNPPD nano-drug delivery system to the wells containing MDA-MB-231 tumor spheres, incubate for 12 h, then wash, fix, and obtain fluorescence images.

[0076] 1.6 Evaluation of the in vitro therapeutic effect of the OMCNPPD nano-drug delivery system

[0077] Inoculate MDA-MB-231 cells into a cell culture plate. After they adhere, discard the culture medium and add the corresponding drugs in groups. The drug groups to be used are as follows:

[0078] 1○ Saline group: Saline

[0079] 2○ Free DOX group: free DOX

[0080] 3○ PTT (photothermal therapy) group: OMCNPP + NIR

[0081] 4○ Chemo (chemotherapy group): OMCNPPD

[0082] 5○ Chemo-PTT (combined chemotherapy-photothermal therapy) group: OMCNPPD + NIR

[0083] After adding the drugs, incubate for 6 h. For the groups containing photothermal therapy, at the end of the incubation, irradiate with an 808-nm near-infrared laser for 5 min (3 W / cm 2 ), discard the drugs, replace with fresh cell culture medium, and continue to incubate. After the culture is completed, aspirate the drug solution, wash twice with PBS, add 100 μL of fresh culture medium and 10 μL of CCK-8 reagent to each well, and incubate at 37 °C for 1 h. Use an enzyme-linked immunosorbent assay (Synergy2, Bio-tek, USA) to detect the absorbance at a wavelength of 450 nm. Calculate the cell viability (%) using the following formula: Cell viability = (A sample / A control ) × 100%, where A sample and A control represent the absorbances of the experimental group and the blank control group, respectively. Verify the in vitro therapeutic effect of the nano-drug delivery system by comparing the survival rates of breast cancer cells after single therapy and dual therapy.

[0084] The in vitro efficacy qualitative experiment was detected using a Live-dead kit, and fluorescence images were obtained using a confocal microscope.

[0085] 2. Research Results

[0086] 2.1 Preparation and Characterization of the OMCNPPD Nanodrug Delivery System

[0087] 2.1.1 Morphology, Particle Size, and Zeta Potential

[0088] The transmission electron microscopy results showed that OMCN had a consistent, approximately spherical porous structure and was uniformly distributed in aqueous solution ( Figure 1 A). The particle sizes of OMCN, OMCNP, and OMCNPP measured using a Malvern potentiometer / particle size analyzer were 140.5 nm, 171.3 nm, and 202.1 nm, respectively ( Figure 1 B), and the potentials were -46.1 ± 3.5 mV, -30.6 ± 4.1 mV, and -25.5 ± 3.6 mV, respectively ( Figure 1 C). The gradual increase in particle size was mainly caused by surface modification. OMCN itself was negatively charged, and due to the shielding effect of PEGylation and the weak positive charge of P6 peptide, the charge increased slightly.

[0089] 2.1.2 Fluorescence Spectrum

[0090] To investigate the loading of DOX in the OMCNPPD nanodrug delivery system, the fluorescence spectra of free DOX, the OMCN-bound state (OMCN / DOX), OMCNPPD, and OMCNPP were measured at an excitation wavelength of 488 nm ( Figure 1 D). The results showed that under the same concentration condition of DOX, the fluorescence intensities of OMCNPPD and OMCN / DOX were significantly weaker than that of free DOX. This fluorescence quenching might be due to the quenching of DOX fluorescence by OMCN during aggregation at high concentrations.

[0091] In addition, Figure 1 E presents the fluorescence images of free DOX, OMCNPP, and OMCNPPD solutions obtained using an IVIS spectral imaging device. Under the conditions of an excitation wavelength of 488 nm and an emission spectrum scanning range of 510 - 610 nm, the OMCNPP solution did not show obvious fluorescence emission. Due to the fluorescence quenching effect of OMCN and the fluorescence quenching induced by the aggregation of DOX in OMCN, OMCNPPD presented a relatively weak fluorescence signal. And when the DOX concentration was in the range of 0 - 60 μg / mL, the fluorescence intensity of free DOX was significantly higher than that of OMCNPPD. The above results indicate that DOX was successfully loaded into OMCNPP.

[0092] 2.1.3 Drug Loading Capacity

[0093] In the OMCNPPD nanodrug delivery system, the carbon core of OMCN promotes the loading of the aromatic anti-tumor drug DOX through π-π conjugation. The drug loading capacity (DLC, %) and encapsulation efficiency (EE, %) of OMCNPPD measured by fluorescence spectroscopy reach about 83.5%, which is significantly better than most other carriers.

[0094] 2.2 Investigation of photothermal ability

[0095] By measuring the temperature changes of OMCNPP aqueous solutions with different concentrations under NIR laser irradiation, the in vitro photothermal ability of OMCNPP was evaluated. The temperature distribution and photothermal images are as Figure 2 shown. The results show that the strength of the near-infrared laser photothermal effect mainly depends on the OMCNPP concentration and the laser power density. The temperature increase of pure water (0 μg / mL OMCNPP) after 5 min of irradiation is the smallest. However, at the same power density, when the concentration increases from 15 μg / mL to 100 μg / mL, the temperature of the OMCNPP aqueous solution increases rapidly ( Figure 2 A). At the same time, the temperature of the OMCNPP aqueous solution will gradually increase with the increase of the power density ( Figure 2 B). In addition, under the action of NIR laser with a power density of 3 W / cm 2 , the temperature of the OMCNPP aqueous solution (45 μg / mL) can exceed 50 °C, reaching the biological limit of cell thermal lethality. These findings indicate that OMCNPP can be used as an effective photothermal agent for photothermal therapy.

[0096] 2.3 In vitro NIR- and pH-sensitive DOX release

[0097] To investigate the NIR- and pH-sensitive DOX release effect, two kinds of PBS buffers with neutral and acidic pH were selected in this study to monitor the cumulative release of DOX in the buffer within 48 hours. As Figure 3 shown in A, in PBS at pH 7.4, the cumulative release rate of DOX within 48 hours is only 18.8% ± 0.61%, which confirms that under standard physiological conditions, DOX can be firmly loaded into OMCNPP. While in the buffer at pH 5.0, the drug cumulative release rate is 73.3% ± 0.8%, showing a pH-sensitive release effect. This phenomenon may be due to the protonation of the amino functional group of DOX under acid induction, increasing the electrostatic repulsion between DOX and the OMCN graphite core.

[0098] In addition, through a series of alternating NIR activation (Laser on) and inactivation (Laser off) cycles, this study deeply explored the drug release characteristics of OMCNPPD under NIR activation conditions. Under NIR laser irradiation, the release of DOX showed an obvious accelerating trend, demonstrating a rapid release characteristic, while in the absence of NIR laser irradiation, the release of DOX was relatively slow, showing a continuous and gentle release behavior( Figure 3 B). Under the condition of pH 5.0, after seven NIR laser irradiation cycles within 24 hours, the cumulative release amount of DOX could reach 64.7% ± 0.7%. However, under the condition of pH 7.4, the release of DOX in the control group showed a significantly limited and slow release trend, with a cumulative release rate of approximately 24.9% ± 1.0%. In summary, we can precisely control the drug release by adjusting the pH value of the solution and the irradiation of NIR laser.

[0099] 2.4 Expression level of RON receptor on specific cells

[0100] The expression levels of RON receptor on MDA-MB-231 cells (human breast cancer cells) and MCF-10A cells (human normal mammary epithelial cells) were evaluated by Western blot assay. Densitometric studies showed that the RON receptor and its α-subunit (located extracellularly and mainly responsible for ligand binding) were highly expressed on MDA-MB-231 cells( Figure 4 ), which was consistent with the results reported in previous studies. This result indicated that MDA-MB-231 cells could be an ideal model for breast cancer targeted delivery and treatment. The expression of RON receptor on MCF-10A cells was relatively low and could be used as a negative control for detecting the function of the targeted RON receptor. Therefore, this study used MDA-MB-231 cells and MCF-10A cells to evaluate the targeting of the OMCNPPD nanodrug delivery system.

[0101] 2.5 In vitro targeting evaluation

[0102] 2.5.1 Cellular level

[0103] To prove that the P6 peptide-modified nanodrug delivery system can actively target cells with high expression of RON receptor, this study used the targeted (OMCNPPD) and non-targeted nanodrug delivery systems (OMCNPD) to verify the uptake of breast cancer MDA-MB-231 cells and healthy mammary epithelial MCF-10A cells, respectively. After the cells were incubated with OMCNPPD or OMCNPD containing the same DOX concentration for a certain time, the uptake of DOX in the cells was observed using a confocal microscope( Figure 5), where DOX shows red fluorescence and the cell nucleus shows blue fluorescence after being stained with DAPI. The experimental results show that the red fluorescence intensity in MDA-MB-231 cells in the OMCNPPD group is significantly higher than that in the OMCNPD group, indicating that the uptake of OMCNPPD in MDA-MB-231 cells is more than that of OMCNPD. However, the uptake of both nano-drug delivery systems in MCF-10A cells is low and there is no significant difference. These results suggest that the surface-modified P6 polypeptide can significantly enhance the targeting ability of the nano-drug delivery system to MDA-MB-231 cells. In addition, after pre-incubating MDA-MB-231 cells with excessive free P6 peptide, the cellular uptake of OMCNPPD was significantly inhibited( Figure 5 D-F), and the fluorescence intensity was much weaker than that of the normal incubation group. This obvious difference may be due to the fact that the specific binding of free P6 peptide to the RON receptor on the surface of MDA-MB-231 cells competitively inhibits the uptake of OMCNPPD.

[0104] 2.5.2 Tumor sphere level

[0105] Compared with the two-dimensional monolayer cell culture model, three-dimensional tumor spheres can more truly reflect the structural characteristics of solid tumors in vivo. Therefore, in this study, MDA-MB-231 tumor spheres were constructed in vitro to further evaluate the targeting ability of the nano-drug delivery system. As Figure 6 shown, the fluorescence intensity of tumor spheres after incubation with OMCNPPD is significantly higher than that of OMCNPD, and there is an obvious difference in the depth of penetration of the two nano-drug delivery systems into the tumor spheres, indicating that the modification of P6 peptide can not only increase the targeting accumulation ability of the nano-drug delivery system in tumor spheres, but also improve the penetration ability of the nano-drug delivery system into tumor spheres.

[0106] 2.6 In vitro anti-tumor effect

[0107] In this study, the in vitro anti-tumor activity of different nano-drug delivery systems was qualitatively evaluated by the Live-dead experiment. In this study, live cells stained with Calcein-AM emit green fluorescence, while dead cells stained with PI emit red fluorescence. As Figure 7 shown in A, cells treated with physiological saline showed the deepest green fluorescence, indicating good cell viability. The cell mortality rate in the chemo-photothermal therapy group (OMCNPPD + NIR group) was significantly higher than that in the single chemotherapy and hyperthermia groups (OMCNPPD group and OMCNPP + NIR group), which indicates that the anti-breast cancer effect of the chemo-photothermal therapy dual therapy is significantly better than that of the single therapy. Further, the anti-tumor activity of the nano-drug delivery system was quantitatively verified by the CCK-8 experiment. As Figure 7As shown in Figure B, the MDA-MB-231 cell viability in the chemotherapy-photothermal therapy combination treatment group was the lowest, and the cytotoxicity was 5.21 times and 2.82 times that of the single chemotherapy group (OMCNPPD group) and the single hyperthermia group (OMCNPP+NIR group), respectively. Generally speaking, the research results indicate that the chemotherapy-photothermal therapy combination treatment shows obvious anti-tumor effects in vitro, which may be due to the targeting effect of P6 peptide on breast cancer cells and the synergistic effect of different therapies.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of a nano drug delivery system in the preparation of a drug for treating breast cancer, characterized in that: The nano drug delivery system uses oxidized mesoporous carbon nanospheres as carriers, and the surface is modified with a polypeptide that specifically binds to the RON receptor, wherein the polypeptide is a P6 peptide, and the amino acid sequence of the P6 peptide is FEHSLYKEMTHL.

2. The use according to claim 1, characterized in that: The nano drug delivery system further comprises loading cytotoxic drugs inside the oxidized mesoporous carbon nanosphere carrier.

3. The use according to claim 2, characterized in that: The cytotoxic drug is selected from doxorubicin, epirubicin, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, SN-38, cisplatin drugs, vinblastine, vincristine, docetaxel or gemcitabine.

4. The use according to any one of claims 2 to 3, characterized in that: The release of cytotoxic drugs is regulated by pH and / or near-infrared laser irradiation. The near-infrared laser irradiation method is an alternating laser activation and laser deactivation cycle. In each cycle, the laser activation time is 5 minutes, the laser deactivation time is 115 minutes or 235 minutes, and 7-8 cycles are performed within 24 hours.

5. A method for preparing a nano drug delivery system, characterized in that: The method comprises the following steps: (1) preparing oxidized mesoporous carbon nanospheres; (2) stirring the oxidized mesoporous carbon nanospheres and the cytotoxic drug in a buffer solution according to a certain ratio for 6-24 hours, centrifuging, and discarding the supernatant to obtain the oxidized mesoporous carbon nanospheres loaded with the drug; (3) activating the drug-loaded oxidized mesoporous carbon nanospheres, adding amino-polyethylene glycol-maleimide for coupling, centrifuging, discarding the supernatant, and obtaining the drug-loaded oxidized mesoporous carbon nanosphere-PEG complex; (4) P6 peptide and drug-loaded oxidized mesoporous carbon nanosphere-PEG complex were added to the buffer system, stirred for reaction for 6-24 hours, centrifuged, and the supernatant was discarded to obtain a nano drug delivery system.

6. A nano drug delivery system, which is used for chemotherapy-photothermal combined treatment of breast cancer, characterized in that: The nano drug delivery system uses oxidized mesoporous carbon nanospheres as carriers, the interior of the carriers is loaded with cytotoxic drugs, and the surface of the carriers is modified with P6 peptides.

7. The nano drug delivery system according to claim 6, characterized in that: The surface modification method of the P6 peptide is selected from the group consisting of using amino-polyethylene glycol-maleimide to connect the P6 peptide and oxidized mesoporous carbon nanospheres.

8. The nano drug delivery system according to claim 6, characterized in that: The cytotoxic drug is selected from doxorubicin, epirubicin, camptothecin, camptothecin derivatives, paclitaxel, paclitaxel derivatives, SN-38, cisplatin drugs, vinblastine, vincristine, docetaxel or gemcitabine.

9. The nano drug delivery system according to claim 8, characterized in that: The cytotoxic drug is selected from adriamycin, camptothecin or paclitaxel, and the cytotoxic drug is loaded into the mesopores of the oxidized mesoporous carbon nanospheres through π-π conjugation.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective dose of the nano drug delivery system according to any one of claims 6 to 9, and pharmaceutically acceptable excipients.

Images