Anti-tumor nanoparticles as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to an anti-tumor nanoparticle as well as a preparation method and application thereof. The invention claims to protect an anti-tumor nanoparticle which comprises a compound PTer N25, and the nanoparticle is prepared by simultaneously entrapping a camptothecin compound and the compound PTer N25 with phospholipid, the structural formula of the compound PTer N25 is # imgabs0 #. According to the invention, cRGD and camptothecin compounds are used for assisting an organic conjugated polymer material to prepare a dual-targeting mediated chemotherapy and photothermal therapy combined treatment composite system, so that targeted recognition of cancer cells by cRGD is realized; under a lesion environment, time-space light dual stimulation response controllable drug release, chemotherapy and photothermal therapy are synergistically treated and integrated, so that the toxic and side effects of the drug are remarkably reduced while the anti-tumor effect is ensured.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to anti-tumor nanoparticles and a preparation method and application thereof. Background Art

[0002] Cancer is a disease that seriously threatens human health. Although chemotherapy is currently one of the main treatment methods, its toxic side effects, poor targeting and drug resistance limit its efficacy. In order to improve the effect of cancer treatment, researchers have been conducting research and exploring a variety of new treatment methods. Among them, photothermal therapy, radiotherapy, photodynamic therapy and gene therapy have received widespread attention and research. These treatment methods use light, radiation and genes to destroy cancer cells and have achieved certain research results. However, a single treatment method often cannot completely eliminate tumors. Therefore, researchers have begun to devote themselves to the development of functional combined treatment systems, that is, combining multiple treatment methods to improve the treatment effect. These combined treatment systems can act on cancer cells in different ways, increase the targeting and efficacy of treatment, and reduce the side effects of single treatment methods.

[0003] Photothermal therapy (PTT) is a minimally invasive treatment method that has been widely studied and paid attention to. At present, researchers have successfully developed nanoreagents of various materials that can exhibit dual-mode functions of PTT and photodynamic therapy (PDT) through laser excitation of specific wavelengths. These materials include metal nanoparticles such as gold, silver, and palladium, semiconductor nanoparticle materials such as copper, and carbon nanomaterials such as graphene and carbon tubes. However, the biological metabolism and long-term toxicity problems of some metal nanoparticles, as well as the biological toxicity of carbon nanomaterials, limit their application in photothermal therapy. In contrast, organic photothermal molecules containing chromophores and having light absorption ability in the infrared region have attracted much attention from researchers due to their good biocompatibility, optical stability, and high photothermal conversion efficiency. However, there are still some problems with pure organic small molecules in photothermal therapy, such as poor chemical stability, easy photobleaching, and poor targeting in vivo. These problems result in the drug not being able to stay in the tumor site for a long enough time. After intravenous administration, the drug is quickly metabolized and cleared, and an effective PTT concentration cannot be reached. In contrast, organic conjugated polymers have become one of the most promising PTT / PDT agents due to their adjustable absorption range, high absorption coefficient, excellent photostability and biocompatibility. Near-infrared (NIR) light has high penetrability in the body, and researchers have developed NIR polymers with high PTT / PDT effects and photostability. In order to improve the therapeutic effect, combined therapy systems have become a hot topic of research. Therefore, there is an urgent need to develop a chemical-photothermal combined therapy system nanosystem to achieve the simultaneous delivery of chemotherapeutic drugs and photothermal agents to the tumor site for combined cancer treatment.

[0004] Chemotherapy is an important component of the combined treatment system. In addition to surgery, radiotherapy and immunotherapy, it is one of the main cancer treatment methods. Although these treatments have achieved relatively satisfactory results, they also have many disadvantages, such as poor pharmacokinetics, nonspecific drug biodistribution and low targeting ability. The poor solubility and specificity of drugs are considered to be the main obstacles to their application in cancer treatment. Therefore, there is an urgent need to overcome these shortcomings and improve the therapeutic effect on tumors. Targeted delivery of toxic chemotherapeutic drugs to cancer cells is expected to improve the effectiveness of drugs and reduce side effects. Camptothecin compounds are a type of DNA topoisomerase I inhibitor with strong antitumor activity and are a commonly used chemotherapeutic drug. However, their inherent high toxicity, poor water solubility and structural instability limit their clinical application. To meet clinical needs, researchers have made a lot of efforts to solve this problem. For example, irinotecan and topotecan, as derivatives of CPT, have been widely used in the clinical treatment of tumor diseases. However, they still have problems with poor circulation and targeting ability. Therefore, it is necessary to further study and develop new strategies for combined treatment to overcome the limitations of chemotherapeutic drugs and achieve precise delivery to tumor cells. This will help improve the effectiveness of the treatment and reduce damage to healthy tissue.

[0005] Nowadays, we attach great importance to precision medicine. In the field of cancer treatment, targeted identification plays a vital role in cancer cells. Considering that angiogenesis is one of the main characteristics of tumor development, it provides nutrients for the growth, invasion and metastasis of tumor cells. Integrin is one of the main regulators of angiogenesis, especially α v β3 integrin is most abundantly expressed in neovascular endothelial cells during angiogenesis and tumor progression, but is rarely found in normal endothelial cells. v The β3 integrin receptor is highly specific, making it an ideal targeting ligand for drug delivery applications. The smaller size of peptide lipid nanoparticles results in a higher surface density, which in turn enhances the affinity with the receptor. Through this host-guest interaction, the valve on the targeted nanoparticle can be destroyed under specific pathological environmental conditions (such as hypoxia, weak acid, and thermodynamic conditions), achieving real-time targeted drug release. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-tumor nanoparticle and a preparation method and application thereof, and to improve the killing effect on tumor cells by combining phototherapy with chemotherapy.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] An anti-tumor nanoparticle includes a compound PTe N25, wherein the nanoparticle is phospholipid-encapsulated with a camptothecin compound and the compound PTe N25; the structural formula of the compound PTe N25 is:

[0009]

[0010] Preferably, the camptothecin compound includes any one or more of camptothecin, hydroxycamptothecin and irinotecan.

[0011] Preferably, the phospholipid is any one of natural phospholipid, synthetic phospholipid and PEGylated synthetic phospholipid or a combination thereof.

[0012] Preferably, the natural phospholipid is soybean lecithin; and the synthetic phospholipid is hydrogenated soybean lecithin.

[0013] PEGylated synthetic phospholipids refer to synthetic phospholipids modified with one or more polyethylene glycols.

[0014] Preferably, the PEGylated synthetic phospholipids include PEGylated synthetic phospholipids modified with RGD peptide and its analogs.

[0015] The RGD peptide analogs include the terminal groups of the RGD peptide being substituted with any one or more C1-C6 alkyl groups.

[0016] Preferably, the PEGylated synthetic phospholipid modified with the RGD peptide and its analogs is DSPE-PEG2000-cRGD.

[0017] Preferably, the PEGylated synthetic phospholipids include DSPE-PEG2000-cRGD and DSPE-PEG2000.

[0018] Preferably, the RGD peptide is a linear polypeptide, cyclic peptide or peptidomimetic compound with an arginine-glycine-aspartic acid (RGD) tripeptide sequence as the active center.

[0019] Among RGD peptides, especially cRGD peptide, v The β3 integrin receptor is highly specific, making it an ideal targeting ligand for drug delivery applications. The small size of the peptide allows for a higher density on the surface of the nanoparticle, thus providing a higher affinity for the receptor.

[0020] Preferably, the RGD peptide is a cyclic peptide compound with an arginine-glycine-aspartic acid (RGD) tripeptide sequence as the active center.

[0021] RGD cyclic peptide can be purchased commercially or prepared in-house. The commercial manufacturer is Xi'an Ruixi Biotechnology Co., Ltd.

[0022] Preferably, the chemical name of the RGD peptide is as follows: Cyclo(Arg-Gly-Aspart-Tyr-Lys), and its chemical structure is as follows:

[0023]

[0024] The present invention also claims a method for preparing the anti-tumor nanoparticles, comprising:

[0025] Weigh phospholipids, camptothecin compounds and PTe N25, add solvent to dissolve, and perform ultrasound; then remove the solvent by rotary evaporation, and perform ultrafiltration and centrifugation to obtain anti-tumor nanoparticles.

[0026] Preferably, the method for preparing the anti-tumor nanoparticles comprises: weighing phospholipids, camptothecin compounds and PTeN25, adding them to a solvent to dissolve, then mixing PTe N25, phospholipids and CPT solution, adding them to 9 ml of ultrapure water at one time, and ultrasonicating for 10 minutes. Then placing in a rotary evaporation flask, and removing the solvent by rotary evaporation under reduced pressure. Then ultrafiltration centrifugation (7000rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain a liposome nanoparticle solution. After freeze drying, the product is obtained and subjected to relevant tests and characterizations.

[0027] Preferably, the mass ratio of phospholipid, camptothecin compound and PTe N25 is (2-5):(0.2-0.7):1.0.

[0028] Preferably, the mass ratio of phospholipid, camptothecin compound and PTe N25 is 3:0.5:1.0.

[0029] Preferably, the phospholipids include DSPE-PEG2000-cRGD and DSPE-PEG2000.

[0030] Preferably, the mass ratio of DSPE-PEG2000:DSPE-PEG2000-cRGD:camptothecin compound:PTer N25 is (2-5):(0.2-0.7):(0.2-0.7):1.0.

[0031] The ratio of phospholipids, camptothecin compounds and PTe N25 is closely related to the size, stability and tumor inhibition effect of the prepared nanoparticles. Too large or too small a ratio will have an adverse effect and cannot balance the three properties.

[0032] Preferably, the mass ratio of DSPE-PEG2000:DSPE-PEG2000-cRGD:camptothecin compound:PTer N25 is 2.5:0.5:0.5:1.0.

[0033] Preferably, the parameters of the ultrasonic treatment step are: ultrasonic time 15 min, ultrasonic 3 s, stop 3 s, ultrasonic power 150 W.

[0034] The tumor is one or more of breast cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, melanoma and soft tissue sarcoma.

[0035] The present invention is further described below:

[0036] The present invention combines the concept of spatiotemporal targeting with the concept of cRGD targeted recognition to achieve dual targeted therapy. This complementary approach greatly improves the efficiency of cancer treatment. This strategy can accurately identify cancer cells and accurately release therapeutic drugs to the target location, thereby improving the effectiveness of treatment and reducing damage to normal tissues.

[0037] The present invention uses cRGD and camptothecin compounds as auxiliary organic conjugated polymer materials to prepare a dual-targeted mediated chemotherapy-photothermal therapy combined treatment composite system, which realizes cRGD targeted recognition of cancer cells, controllable drug release in spatiotemporal light / cRGD targeted dual stimulation response under the pathological environment, and integration of chemotherapy and photothermal therapy synergistic treatment, while ensuring the anti-tumor effect, significantly reducing the toxic side effects of the drug.

[0038] In the present invention, the organic conjugated polymer PTe N25 is copolymerized with an aromatic thiophene unit D unit to form PSMA using an organic small molecule compound (SMA) with a low band gap electron-withdrawing unit-electron-donating unit-electron-withdrawing unit (Acceptor-Donor-Acceptor; ADA) structure as the A1 unit. A special advantage of polymerized PSMA is its strong absorption in the near-infrared region, which is due to its SMA unit. NDI derivatives are introduced as the second A2 unit in these ternary polymers to copolymerize with the aromatic thiophene unit D. By copolymerizing the A2 unit and the aromatic D thiophene unit, the electronic energy level of PSMA can be easily adjusted. The resulting PTe N25 has a D-A1-D-A2 structure that has high electron affinity, excellent electron mobility, good thermal stability, oxidative stability, and excellent photothermal conversion efficiency. In addition, the carbonyl and cyano groups contained in the PTe N25 organic conjugated polymer act as electron-withdrawing groups, which can effectively lower the LUMO energy level. Therefore, the D-A1-D-A2 push-pull electronic structure in the molecule has strong and broad absorption in the near-infrared range, and induces intramolecular charge transfer, effectively reducing the fluorescence quantum efficiency.

[0039] The effects of the cRGD recognition and light dual-targeting mediated chemotherapy-phototherapy combined cancer treatment composite system prepared by the present invention are mainly manifested in the following aspects:

[0040] 1) PTe N25 nanoparticles with good biocompatibility and excellent photothermal conversion ability give the entire system synergistic and efficient photothermal therapy capabilities. In addition, PTe N25 nanoparticles are simple to modify with cRGD, easy to functionalize and have high drug loading capacity. The synergistic effect of cRGD and camptothecin compounds assisting PTe N25 gives the entire system ideal chemotherapy-phototherapy capabilities.

[0041] 2) Based on the host-guest π-π interaction, the PTe N25 and CPT system constructed a quasi-synergistic machine, which achieved controllable drug release under the stimulation of temperature factors under external laser conditions, synergistically achieved a targeted therapeutic effect and improved the treatment efficiency.

[0042] 3) cRGD targeting cancer cells and laser spatiotemporal irradiation complement each other, giving the entire treatment system dual targeting capabilities, greatly improving treatment efficiency, and providing a new idea for improving treatment efficiency for precision cancer treatment.

[0043] 4) The camptothecin compound CPT is released for chemotherapy and downregulates the expression of HSP70, greatly enhancing the sensitivity of the tumor to PTe N25-mediated PTT, and can kill almost all cancer cells at extremely low CPT dosage concentrations. Under normal circumstances, after laser irradiation of PTe N25, cancer cells actively upregulate the expression of HSP70 protein, which will reduce the effect of photothermal therapy. Camptothecin compounds, as chemotherapy drugs, can not only downregulate the expression of HSP70 protein, but also play a role of metronomic chemotherapy in the form of low concentrations in diseased tissues. The use of PTe N25 also reduces the dosage of camptothecin compounds, so PTe N25 and camptothecin compounds play a synergistic role.

[0044] Compared with the prior art, the beneficial effects of the present invention mainly include the following aspects:

[0045] 1. The dual-targeted combined therapy system of the present invention is highly flexible in design and easy to prepare. The structural design of the targeted nanoparticles can be flexibly replaced according to treatment needs, which expands the ideas for the design and preparation of future multifunctional cancer treatment systems.

[0046] 2. The PTe N25 nanostructure is convenient for post-modification and gives the entire composite drug delivery system good phototherapeutic ability with few side effects.

[0047] 3. In the present invention, cRGD cancer cell targeting molecules and light control are combined with PTe N25 and CPT nanoparticles at the same time, and work synergistically, integrating dual targeted recognition of cancer cells, photothermal therapy and chemotherapy. At the same time, CPT is released for chemotherapy, and the expression of HSP70 is downregulated to enhance the sensitivity of tumors to PTe N25-mediated PTT. The design of this new system provides a new idea for the future medical combination of lipid nanoparticles and the development of their functional composite materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0049] Figure 1 This is the synthetic route of cRGD-PTer-N25 / CPT NPs lipid nanoparticles.

[0050] Figure 2 Photos, spectral test results, particle size and photodynamic performance test results of cRGD-PTer-N25 / CPT NPs in THF and PBS, respectively.

[0051] Figure 3 The photothermal performance test results of cRGD-PTer-N25 / CPT NPs.

[0052] Figure 4 Cellular uptake results of cRGD-PTer-N25 / CPT NPs.

[0053] Figure 5 The results of the MTT assay for the toxicity of nanomaterials to breast cancer 4T1 cells.

[0054] Figure 6 Intracellular ROS generation detection and cell apoptosis results of cRGD-PTer-N25 / CPT NPs.

[0055] Figure 7 The nanomaterial cRGD-PTer-N25 / CPT NPs induced HSP70 inhibition in vitro.

[0056] Figure 8 In vivo near-infrared fluorescence imaging evaluation of the nanomaterial cRGD-PTer-N25 / CPT NPs.

[0057] Fig. 9In vivo photothermal imaging of the nanomaterial cRGD-PTer-N25 / CPT NPs.

[0058] Fig.10 is the tumor size of the nanomaterial-treated group.

[0059] Fig.11 is the average tumor volume of the nanomaterial-treated group.

[0060] Fig.12 is the body weight of mice in the nanomaterial treatment group.

[0061] Fig.13 H&E stained sections of tumors in the nanomaterial treatment group. DETAILED DESCRIPTION

[0062] The technical scheme of the present invention is further described in detail below by specific embodiments and in conjunction with the accompanying drawings, which is only used as an explanation of the present invention, but is by no means a limitation of the present invention, and the protection content of the present invention is not limited to the following embodiments. In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0063] Example 1

[0064] Preparation of cRGD-PTer-N25 / CPT NPs lipid nanoparticles

[0065] according to Figure 1 The synthetic route shown was used to synthesize cRGD-PTer-N25 / CPT NPs lipid nanoparticles.

[0066] Example 1: Weigh 1 mg of PTe N25, 2 mg of DSPE-PEG2000 and 0.5 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, 0.2 mg of CPT and dissolve them in 1 mL of DMSO solution, then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure water at one time, and ultrasonicate for 10 minutes. Then place it in a rotary evaporation flask and evaporate the THF solvent under reduced pressure. Then ultrafiltration centrifuge (MW 3500) and ultrafiltration centrifuge (7000rpm) for 15 minutes to obtain a liposome nanoparticle solution. After freeze drying, the product is obtained and related tests and characterizations are performed.

[0067] Comparative Example 1: Weigh 3 mg of PTe N25, 3 mg of DSPE-PEG2000 and 0.5 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, and 0.5 mg of CPT and dissolve them in 1 mL of DMSO solution. Then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure water at one time, and ultrasonicate for 10 minutes. Then place it in a rotary evaporation flask and evaporate the THF solvent by rotary evaporation under reduced pressure. Then ultrafiltration centrifuge tube (MW 3500) ultrafiltration centrifuge (7000rpm) for 15 minutes to obtain liposome nanoparticle solution. The product is obtained after freeze drying and related tests and characterizations are performed.

[0068] Example 2: Weigh 0.6 mg of PTe N25, 4 mg of DSPE-PEG2000, and 0.6 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, and 0.6 mg of CPT and dissolve them in 1 mL of DMSO solution. Then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure water at one time, and ultrasonicate for 10 minutes. Then place it in a rotary evaporation flask and evaporate the THF solvent under reduced pressure. Then ultrafiltration centrifuge tube (MW 3500) ultrafiltration centrifuge (7000rpm) for 15 minutes to obtain liposome nanoparticle solution. After freeze drying, the product is obtained and relevant tests and characterizations are performed.

[0069] Example 3: Weigh 1 mg of PTe N25, 5 mg of DSPE-PEG2000 and 0.7 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, and 0.7 mg of CPT and dissolve them in 1 mL of DMSO solution. Then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure water at one time, and ultrasonicate for 10 minutes. Then place it in a rotary evaporation flask and evaporate the THF solvent under reduced pressure. Then ultrafiltration centrifuge (MW 3500) and ultrafiltration centrifuge (7000rpm) for 15 minutes to obtain a liposome nanoparticle solution. After freeze drying, the product is obtained and related tests and characterizations are performed.

[0070] Comparative Example 2: Weigh 1 mg of PTe N25, 3 mg of DSPE-PEG2000 and 0.5 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, 0.5 mg of doxorubicin (DOX) and dissolve them in 1 mL of DMSO solution, then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure at one time, and ultrasonicate for 10 min. Then place it in a rotary evaporation flask, and evaporate the THF solution under reduced pressure and rotary evaporation. Then ultrafiltration centrifuge tube (MW 3500) ultrafiltration centrifuge (7000rpm) for 15 min to obtain liposome nanoparticle solution. The product is obtained after freeze drying and related tests and characterizations are performed.

[0071] Comparative Example 3: Weigh 1 mg of PTe N25, 3 mg of DSPE-PEG2000 and 0.5 mg of DSPE-PEG2000-cRGD and dissolve them in 1 mL of tetrahydrofuran, 0.5 mg of paclitaxel (PTX) and dissolve them in 1 mL of DMSO solution, then mix the PTe N25, DSPE-PEG2000 and CPT solutions and add them to 9 ml of ultrapure at one time, and ultrasonicate for 10 minutes. Then place it in a rotary evaporation flask, and evaporate the THF solution under reduced pressure and rotary evaporation. Then ultrafiltration centrifuge tube (MW 3500) ultrafiltration centrifuge (7000rpm) for 15 minutes to obtain liposome nanoparticle solution. The product is obtained after freeze drying and related tests and characterizations are performed.

[0072] Example 2

[0073] The nanomaterials obtained in the examples and comparative examples were characterized by the following tests:

[0074] a) The particle size and distribution of the co-loaded CPT and PTeN25 nanoparticles were measured using a particle size and zeta potential analyzer (Zetasizer Nano ZS 90; Alvern Co., Ltd., Malvern. UK), as shown in FIG. Figure 2 As shown in D, in the cRGD-PTer-N25 / CPT NPs obtained in Example 1, in order to test the stability of the nanoparticles in physiologically relevant solutions, the nanoparticles were then diluted into water, phosphate buffered saline (PBS) (room temperature) and PBS containing 10 v / v% FBS (37°C) and monitored for 24 hours. The 95% particle size was distributed between 110-120 nm, 115-125 nm and 120-130 nm, respectively. The results are shown in Figure 2C, it showed no significant size change in the three solutions, indicating that it has high colloidal stability at physiological temperature and in the presence of serum proteins. And it is mainly distributed in the range of 160-180 nm. Its morphology was observed using a spherical aberration-corrected transmission electron microscope (Titan G260-300; Frequency Electronics, Inc, UK). The results are shown in Figure 2 As shown in E, CPT and PTeN25 aggregate into the core of the nanoparticles through π-π interaction, and the particle size is 70-90 nm.

[0075] b) Absorption and emission spectrum test. The cRGD-PTer-N25 / CPT NPs obtained in Example 1 were diluted to 10 μM and added to a cuvette to measure its UV-visible absorption spectrum. UV-visible absorption spectrum (Cary8454UV-Vis+Rx2000; Agilent Technologies Inc.UK) was as follows: Figure 2 As shown in A and B, there is characteristic absorption in the range of 500-800nm, with a peak at 584nm; its aggregated emission peak is at 750nm.

[0076] c) Photodynamic performance test. The cRGD-PTer-N25 / CPT NPs obtained in Example 1 were diluted to 10 μM and 1 In vitro detection of cRGD-PTer-N25 / CPT NPs by O2 probe DPBF 1 O2 generation capacity. The results are as follows Figure 2 As shown in F, 407nm is 1 The plasma resonance absorption peak of O2 probe DPBF. 1 After O2 reaction, the absorbance value of its characteristic peak will gradually decrease with the consumption of DPBF, so DPBF can be used to evaluate the PTe-N25-mediated liposome 1 O2 generation capacity. Figure 2 As shown in F, after 808 nm laser irradiation (1.0 W cm -2 )cRGD-PTer-N25 / CPT NPs, the absorbance of DPBF at 407nm showed a continuous and rapid decline. After laser irradiation for 75s (10s each time, 15 times in total), the absorbance of DPBF in cRGD-PTer-N25 / CPTNPs nanosuspension decreased by 50.1%. 1 The above results indicate that PTe-N25 is a good photosensitizer (PS) that can continuously produce a large amount of ROS under NIR irradiation, and ROS will play an important role in subsequent PDT treatment.

[0077] d) Photostability test. The photothermal stability of cRGD-PTer-N25 / CPT NPs solution was evaluated by 808 nm laser irradiation (5 cycles of irradiation / cooling). Figure 3 D), the maximum temperature to which the solution rises remains almost unchanged, indicating that cRGD-PTer-N25 / CPT NPs have good photothermal stability under laser irradiation.

[0078] e) Photothermal performance test. The co-loaded cRGD-PTer-N25 / CPT NPs nanoparticles obtained in Example 3 were diluted to 200uL of suspensions of different concentrations and exposed to laser irradiation (808nm, 1W·cm -2 ) and use a thermocouple to measure the temperature change and record it. Figure 3 As shown in A and 3B, the photothermal conversion efficiency is dependent on the concentration and light intensity, and the maximum temperature can rise to 70°C and 58°C respectively, and the photothermal conversion efficiency PCE = 57.4% ( Figure 3 C), whose photothermal conversion efficiency and photothermal stability are significantly better than the existing FDA-approved ICG for clinical use ( Figure 3 E and 3F).

[0079] Example 3

[0080] Tumor cell uptake of co-loaded CPT and PTeN25 nanomaterials, intracellular ROS production and in vitro antitumor evaluation.

[0081] a) Cellular uptake of co-loaded CPT and PTeN25 nanomaterials. Since the fluorescence intensity of CPT and PTeN25 is not very strong, DiO is used instead of CPT and PTe N25 in this example. Breast cancer 4T1 cells were seeded in a 24-well plate and cultured for 24 hours. The culture medium was replaced with complete culture medium containing co-loaded DiO, DiO@NPs, and DiO@cRGDNPs nanomaterials, respectively. After another 4 hours of incubation, the cells were co-stained with Lyso-Tracker Red in the culture medium for 30 minutes. After washing twice with PBS, the cells were imaged under a microscope. The results are shown in Figure 2. Figure 4 As shown in A, DiO, DiO@NPs, and DiO@cRGD NPs nanoparticles can be effectively internalized by cells within 2 to 4 hours, and flow cytometry also shows the same structure ( Figure 4 B).

[0082] b) Detection of intracellular ROS generation of co-loaded CPT and PTe N25 nanomaterials. A reactive oxygen species detection kit was selected as a ROS indicator. After breast cancer 4T1 cells were cultured in a 24-well plate for 24 h, co-loaded Control, cRGD-PTer-N25 / CPT NPs, and cRGD-PTer-N25 / CPT NPs plus laser groups were added to complete culture medium. After incubation for 4 h, the reactive oxygen species detection kit was added to the culture medium. After another 30 min of incubation, the cells were washed twice with PBS and stimulated with a laser (1 mW cm -2 ) for 5 min. The treated cells were imaged by fluorescence microscopy. Figure 6 As shown in A, the cRGD-PTer-N25 / CPT NPs (abbreviated as PTe N25 in the figure) plus laser nanomaterial group has the highest fluorescence intensity of cells, indicating that ROS is produced in the cells.

[0083] c) In vitro cytotoxicity The cytotoxicity of nanomaterials to breast cancer 4T1 cells was evaluated by MTT assay. 4 ) were inoculated in 96-well plates and cultured at 37°C in 5% CO2 for 24 h. The medium was then replaced with control, free CPT group, CPT NPs group, PTe N25 NPs + laser, PTe N25 / CPT NPs + laser: cRGD-PTerN25 / CPT NPs + laser in complete medium and then incubated for another 6 h. Afterwards, the 96-well plates in the laser irradiation group were exposed to 808 nm laser (1 W cm -2 , 5min) to study the effects of chemotherapy, photodynamic / photothermal therapy or light-enhanced chemotherapy, and then incubate in the dark for another 18h. At the same time, the plates in each group without laser irradiation were incubated in the dark for 24h under the same experimental conditions. Next, a standard MTT assay was performed to assess cell viability. The results are shown in Figure 5 As shown in A, compared with chemotherapy alone (free CPT group and CPTNPS group) and phototherapy (PTer N25 NPs + laser), light-enhanced chemotherapy (PTer N25 / CPT NPs + laser and G8: cRGD-PTer N25 / CPT NPs + laser) can produce significant synergistic therapeutic effects. Live and dead cell staining and cell apoptosis assays also showed the same results ( Figure 5 C and Figure 6 B).

[0084] Example 4

[0085] The nanomaterial cRGD-PTer-N25 / CPT NPs obtained in Example 1 induced HSP70 inhibition in vitro

[0086] The expression level of HSP70 in 4T1 cells treated with different methods was detected by Western blotting ( Figure 7 ) to evaluate the effects of chemotherapy and PTT on HSP70 expression. Briefly, 4T1 cells were plated at 5 × 10 5 The cells were seeded at a density of 100 cells / mL on a 6-well plate for 24 h, and then treated with different conditions (G1: control group;

[0087] G2: laser group; G3: photosensitizer (PTer N25 NPs); G4: CPT; G5: Pter N25 NPs + laser; G6: PterN25 / CPT NPs; G7: cRGD-PTer N25 / CPT NPs; G8: cRGD-PTer N25 / CPT NPs + laser), where the net concentration of CPT substrate in all sample groups was kept at 20.0 mg mL -1 After 6 h, the cells were rinsed with PBS buffer and the culture medium of each sample was replaced with fresh culture medium containing penicillin-streptomycin and cultured for another 12 h. -2 ) treated group, after adding fresh culture medium, near-infrared laser was introduced to irradiate cells for 5 minutes and then cultured for another 12 hours. Afterwards, Western blot analysis was performed with HSP70 antibody according to the conventional protocol. With G5:PTer N25NPs+laser treatment, HSP70 levels increased to 2.4 times compared with saline group G1. This confirms that the increase in PTT temperature can stimulate the expression of HSP70. Compared with saline group G1, the relative level of HSP70 in G2:laser group and G3:photosensitizer (PTer N25 NPs) did not change significantly, indicating that laser and photosensitizer alone had no effect on the expression of HSP70 in cancer cells. However, compared with saline group G1, the relative levels of HSP70 in G4, G6 and G7 groups were significantly reduced to 0.89, 0.84 and 0.87, respectively, indicating that CPT can downregulate the expression of HSP70. In addition, compared with the saline group G1, even under photothermal induction, the expression level of HSP70 in the laser group G8 was significantly downregulated to 0.65, indicating that the active CPT drug was released in tumor cells in response to chemotherapy and downregulated the expression of HSP70, enhancing the sensitivity of tumors to PTe N25-mediated PTT. In general, cRGD and CPT assisted the organic conjugated polymer material PTe N25 to prepare a dual-targeted mediated chemotherapy-photothermal therapy combined treatment system is a promising treatment method for targeted therapy.

[0088] Example 5

[0089] In vivo near-infrared fluorescence imaging evaluation of co-loaded CPT and PTeN25 nanomaterials ( Figure 8 ).

[0090] a) Construction of breast cancer tumor model. Male C57BL / 6 mice (4-5 weeks old) were provided by Xiangya Experimental Animal Center of Central South University and housed in a standard SPF experimental animal room. Breast cancer 4T1 cells (1×10 6 ) was injected subcutaneously into the right back of each mouse to establish a mouse breast cancer tumor subcutaneous model. About 7 days later, when the tumor volume grew to about 100mm 3 When used in vitro, mice can be used for in vivo fluorescence imaging and photothermal imaging experiments.

[0091] b) In vivo near-infrared fluorescence imaging of nanomaterials. Since the fluorescence intensity of camptothecin and organic conjugated polymers is relatively weak, we used cell membrane near-infrared fluorescent probes (DiR) to simulate the above two hydrophobic drugs. In vivo fluorescence accumulation in tumors was performed by intravenous injection of DiR, DiR@NPs, and DiR@cRGDNPs. In vivo fluorescence imaging was performed at predetermined time intervals on the BLT multimodal animal in vivo imaging system AniView100 or the ChemiDocMP imaging system, with an excitation wavelength of 647nm and a fluorescence emission wavelength pass filter of 695nm. Six hours after intravenous injection, the mice were killed according to the time when the maximum fluorescence intensity accumulation was reached in the tumor. Their organs (heart, liver, spleen, lungs, kidneys, intestines) and tumors were harvested and used for ex vivo fluorescence imaging, and the average fluorescence intensity was calculated. The results are shown in Figure 8 As shown, from Figure 8 As can be seen from A and B, the nanomaterials can be enriched in the tumor within 2 hours, lighting up the tumor tissue, and the distribution boundaries of tissues and organs are clear; Figure 8 As can be seen in B, except for the liver, the fluorescence intensity of tumor tissue is significantly higher than that of other internal organs.

[0092] c) In vivo photothermal imaging of nanomaterials. 200 μL of different suspensions were injected intravenously: PBS plus laser, PTeN25 NPs plus laser, PTeN25 / CPT NPs plus laser, and cRGD-PTerN25 / CPT NPs plus laser. Then, an infrared thermal imager was used to record the in vivo photothermal imaging of the animals in each group under the influence of 808 nm laser (1 W cm -2 ) The temperature change of the tumor area after 5 minutes of irradiation was calculated, and the average temperature of the tumor area was calculated. Fig. 9 As shown, the nanomaterial group injected with cRGD-PTerN25 / CPT NPs photosensitizer can achieve local tumor photothermal effect, with the temperature of the tumor area ranging from 53°C to 60°C and the highest temperature of 60°C. This shows that the nanomaterial cRGD-PTerN25 / CPT NPs can be enriched through tumor targeting and produce effective photothermal therapy under laser irradiation.

[0093] Example 6

[0094] In vivo real-time near-infrared fluorescence-guided photosynergistic chemotherapeutic antitumor evaluation of CPT-PTerN25 nanomaterials co-loaded with PTeN25.

[0095] When the tumor volume reaches 100 mm 3 Afterwards, the mice were randomly divided into six groups for different treatments: ①PBS group, intravenous injection of 200μL PBS; ②PBS+laser group, intravenous injection of 200μL PBS, laser irradiation 6h after injection; ③CPT group, intravenous injection of 200μL CPT solution; ④PTer N25 NPs+laser group, laser irradiation 6h after intravenous injection of PTe N25 NPs; ⑤PTer N25 / CPT NPs group, intravenous injection of PTe N25 / CPT NPs; ⑥cRGD-PTer N25 / CPT NPs+laser group, laser irradiation 6h after intravenous injection of cRGD-PTer N25 / CPT NPs. The laser irradiation site and range were determined by near-infrared fluorescence imaging, and the 808nm laser power was 1W·cm -2 , time 10min. The treatment of each group was repeated once a week after the initial treatment. The tumor volume and body weight of the mice were monitored every other day, and all mice were killed 14 days after treatment. The changes in tumor volume and body weight were recorded. Fig.10 , Figure 11-13 As shown, **P>0.05, ***P<0.01, the average tumor volume of the light-enhanced chemotherapy treatment group was significantly lower than that of the other treatment groups (P<0.05), and the weight growth curve of mice was detected, and there was no difference in weight among the groups, indicating that there was no obvious toxicity to the growth of mice after injection of the nanomaterial. This shows that the light-enhanced chemotherapy achieved by the nanomaterial cRGD-PTer N25 / CPT NPs can significantly inhibit the growth of breast cancer.

[0096] At the same time, the obtained nanoparticles were also evaluated for anti-tumor effects under laser irradiation. At the same time, under laser irradiation, the nanoparticles obtained in Comparative Examples 1, 2 and 3 were also evaluated for anti-tumor effects. The results showed that the effect of Comparative Example 1 was worse than that of Example 1, and the effects of Comparative Examples 2 and 3 were worse than those of Comparative Example 1. The chemotherapy drugs and photothermal agents in Comparative Examples 2 and 3 could not play a synergistic role, but could only be simply superimposed.

[0097] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should be protected by the present invention.

Claims

1. An anti-tumor nanoparticle, characterized in that: The nanoparticles include a compound PTe N25, wherein the nanoparticles simultaneously encapsulate camptothecin compounds and the compound PTe N25 with phospholipids; The structural formula of the compound PTe N25 is:

2. The anti-tumor nanoparticle according to claim 1, characterized in that The phospholipid is any one of natural phospholipid, synthetic phospholipid and pegylated synthetic phospholipid or a combination thereof.

3. The anti-tumor nanoparticle according to claim 2, characterized in that: The PEGylated synthetic phospholipids include PEGylated synthetic phospholipids modified with RGD peptide and its analogs.

4. The anti-tumor nanoparticle according to claim 3, characterized in that: The RGD peptide is a linear polypeptide, cyclic peptide or peptidomimetic compound with an arginine-glycine-aspartic acid (RGD) tripeptide sequence as the active center.

5. The anti-tumor nanoparticle according to claim 2, characterized in that: The PEGylated synthetic phospholipids include DSPE-PEG2000-cRGD and DSPE-PEG2000.

6. The anti-tumor nanoparticle according to claim 5, characterized in that: The mass ratio of DSPE-PEG2000:DSPE-PEG2000-cRGD:camptothecin compound:PTer N25 is (2-5):(0.2-0.7):(0.2-0.7):1.

0.

7. The anti-tumor nanoparticle according to any one of claims 1 to 6, characterized in that: The tumor is one or more of breast cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, melanoma, and soft tissue sarcoma.

8. The method for preparing antitumor nanoparticles according to any one of claims 1 to 6, characterized in that: Weigh phospholipids, camptothecin compounds and PTe N25, add solvent to dissolve, and perform ultrasound; then remove the solvent by rotary evaporation, and perform ultrafiltration and centrifugation to obtain anti-tumor nanoparticles.

9. The method for preparing antitumor nanoparticles according to any one of claims 1 to 6, characterized in that: The parameters of the ultrasonic treatment step were as follows: ultrasonic time 15 min, ultrasonic on 3 s, off 3 s, ultrasonic power 150 W.

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