Anti-tumor nanoparticles and their preparation method and application

Abstract

The present invention belongs to the field of biomedical materials and specifically relates to anti-tumor nanoparticles, their preparation methods, and applications. The present invention claims protection for an anti-tumor nanoparticle comprising compound PTe N25, wherein the nanoparticles are simultaneously encapsulated with a camptothecin compound and compound PTe N25 using a phospholipid. The structural formula of compound PTe N25 is: #imgabs0# The present invention utilizes cRGD and camptothecin compounds in conjunction with an organic conjugated polymer material to prepare a dual-targeted chemotherapy-photothermal therapy combined treatment system. This system achieves cRGD-targeted recognition of cancer cells, controlled drug release responsive to spatiotemporal light dual stimulation in the context of a diseased environment, and integrated chemotherapy and photothermal therapy synergistic treatment, significantly reducing drug toxicity and side effects while maintaining anti-tumor efficacy.

Description

Technical Field

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

[0002] Cancer is a serious threat to human health. While chemotherapy is currently one of the main treatment options, its efficacy is limited by toxic side effects, poor targeting, and drug resistance. To improve cancer treatment, researchers are continuously exploring a variety of new treatment approaches. Among them, photothermal therapy, radiotherapy, photodynamic therapy, and gene therapy have garnered widespread attention and research. These treatments utilize light, radiation, and genes to destroy cancer cells, achieving promising results. However, single treatments often fail to completely eliminate tumors. Therefore, researchers are focusing on developing functional combination therapy systems that combine multiple therapies to enhance therapeutic efficacy. These combination therapy systems can target cancer cells in different ways, increasing the targeting and efficacy of treatment while mitigating the side effects of single treatments.

[0003] Photothermal therapy (PTT) is a minimally invasive treatment method that has attracted extensive research and attention. Currently, researchers have successfully developed nanoreagents made of various materials that can exhibit dual-mode functions of PTT and photodynamic therapy (PDT) through laser excitation with specific wavelengths. These materials include metal nanoparticles such as gold, silver, and palladium, semiconductor nanoparticles such as copper, and carbon nanomaterials such as graphene and carbon nanotubes. However, the biometabolism and long-term toxicity of some metal nanoparticles, as well as the biotoxicity of carbon nanomaterials, have limited their application in PTT. In contrast, organic photothermal molecules containing chromophores and capable of absorbing light in the infrared region have attracted much research attention due to their excellent biocompatibility, optical stability, and high photothermal conversion efficiency. However, simple organic small molecules still face several challenges in PTT, such as poor chemical stability, susceptibility to photobleaching, and poor in vivo targeting. These issues prevent the drug from remaining in the tumor site for a sufficient period of time. After intravenous administration, the drug is rapidly metabolized and cleared, failing to achieve effective PTT concentrations. In contrast, organic conjugated polymers have become one of the most promising PTT / PDT agents due to their tunable 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. To improve therapeutic efficacy, combined therapy systems have become a hot topic of research. Therefore, there is an urgent need to develop a chemo-photothermal combined therapy nanosystem to achieve the simultaneous delivery of chemotherapeutic drugs and photothermal agents to tumor sites for combined cancer treatment.

[0004] Chemotherapy is a key component of combination therapy and, alongside surgery, radiotherapy, and immunotherapy, is one of the mainstays of cancer treatment. While these treatments have achieved relatively promising results, they also suffer from numerous drawbacks, such as poor pharmacokinetics, nonspecific drug biodistribution, and low targeting ability. Poor drug solubility and specificity are considered major obstacles to their application in cancer therapy. Therefore, there is an urgent need to overcome these shortcomings and improve tumor therapeutic efficacy. Targeted delivery of toxic chemotherapeutic drugs to cancer cells holds promise for enhancing drug efficacy and reducing side effects. Camptothecins, DNA topoisomerase I inhibitors with potent antitumor activity, are commonly used chemotherapeutic agents. However, their inherent high toxicity, poor water solubility, and structural instability limit their clinical application. To address these clinical needs, researchers have made significant efforts to address this issue. For example, irinotecan and topotecan, derivatives of CPT, have been widely used in the clinical treatment of cancer. However, they still suffer from poor circulation and targeting ability. Therefore, further research and development of new combination therapy strategies are needed to overcome the limitations of chemotherapeutic drugs and achieve precise delivery to tumor cells. This will help improve the effectiveness of treatment and reduce damage to healthy tissue.

[0005] Nowadays, we attach great importance to precision medicine. In the field of cancer treatment, targeted identification of cancer cells plays a vital role. Considering that angiogenesis is one of the main characteristics of tumor development, because 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 high specificity of the β3 integrin receptor makes 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 their affinity for the receptor. This host-guest interaction allows the valves on the targeted nanoparticles to be destroyed under specific pathological environmental conditions (such as hypoxia, weak acidity, 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 its preparation method and application, 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 comprises a compound PTe N25, wherein the nanoparticle is simultaneously encapsulated with a camptothecin compound and the compound PTe N25 by a phospholipid; 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 one or more C1-C6 alkyl groups substituted on the terminal groups of the RGD peptide.

[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 nanoparticle surface, resulting in 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-Asp-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] Phospholipids, camptothecin compounds and PTe N25 were weighed, dissolved in a solvent, and sonicated; the solvent was then removed by rotary evaporation, and ultrafiltration and centrifugation were performed to obtain anti-tumor nanoparticles.

[0026] Preferably, the method for preparing the anti-tumor nanoparticles comprises: weighing a phospholipid, a camptothecin compound, and PTeN25, dissolving them in a solvent, then mixing the PTeN25, phospholipid, and CPT solution and adding them all to 9 ml of ultrapure water, followed by sonication for 10 minutes. The mixture is then placed in a rotary evaporation flask and the solvent is removed by rotary evaporation under reduced pressure. The mixture is then subjected to ultrafiltration and centrifugation (7000 rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain a liposome nanoparticle solution. The product is freeze-dried and subjected to relevant testing and characterization.

[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 has adverse effects 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, pause 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] This invention combines the concepts of spatiotemporal targeting with the concept of cRGD targeted recognition to achieve dual-targeted therapy. This complementary approach significantly improves the efficiency of cancer treatment. This strategy can accurately identify cancer cells and precisely release therapeutic drugs to the target location, thereby enhancing treatment efficacy and minimizing damage to normal tissues.

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

[0038] In the present invention, the organic conjugated polymer PTe N25 is copolymerized with 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 and the aromatic thiophene unit D unit to form PSMA. 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 terpolymers to copolymerize with the aromatic thiophene unit D. By using the A2 unit and the aromatic D thiophene unit for copolymerization, the electronic energy level of PSMA can be easily adjusted. The final 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 complex system prepared by the present invention are mainly manifested in the following aspects:

[0040] 1) The excellent biocompatibility and photothermal conversion capabilities of PTe N25 nanoparticles imbue the entire system with synergistic and efficient photothermal therapy. Furthermore, PTe N25 nanoparticles are easily modified with cRGD, are easily functionalized, and possess high drug-loading capacity. The synergistic effect of cRGD and camptothecin-assisted PTe N25 gives the entire system ideal chemotherapeutic-phototherapeutic capabilities.

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

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

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

[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 allows for flexible functional replacement 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 facilitates post-modification and gives the entire composite drug delivery system good phototherapeutic ability with minimal side effects.

[0047] 3. In this invention, the cRGD cancer cell-targeting molecule and light-controlled molecule simultaneously bind to PTe N25 and CPT nanoparticles, working synergistically to combine dual cancer cell targeting, photothermal therapy, and chemotherapy. Simultaneously, CPT is released for chemotherapy and downregulates HSP70 expression, enhancing tumor sensitivity to PTe N25-mediated PTT. This novel system design provides new insights into the future development of lipid nanoparticles for medical applications and their functional composites. 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any 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 were used to determine 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] Figure 9In vivo photothermal imaging of the nanomaterial cRGD-PTer-N25 / CPT NPs.

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

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

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

[0061] Figure 13 H&E-stained sections of tumors in the nanomaterial-treated group. DETAILED DESCRIPTION

[0062] The technical solutions of the present invention will be further described in detail below by way of specific examples and in conjunction with the accompanying drawings. These examples are intended only to explain the present invention but are by no means intended to limit the present invention. The protection content of the present invention is not limited to the following examples. 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 examples, 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: 1 mg of Pter N25, 2 mg of DSPE-PEG2000, and 0.5 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.2 mg of CPT was dissolved in 1 mL of DMSO. The Pter N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure sodium chloride. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask and the THF solvent was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged at 7000 rpm for 15 minutes in an ultrafiltration centrifuge tube (MW 3500) to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant tests and characterization.

[0067] Comparative Example 1: 3 mg of Pter N25, 3 mg of DSPE-PEG2000, and 0.5 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.5 mg of CPT was dissolved in 1 mL of DMSO. The Pter N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure sodium chloride. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask and the THF solvent was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged (7000 rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant testing and characterization.

[0068] Example 2: 0.6 mg of Pter N25, 4 mg of DSPE-PEG2000, and 0.6 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.6 mg of CPT was dissolved in 1 mL of DMSO. The Pter N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure sodium chloride. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask and the THF solvent was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged (7000 rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant tests and characterization.

[0069] Example 3: 1 mg of Pter N25, 5 mg of DSPE-PEG2000, and 0.7 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.7 mg of CPT was dissolved in 1 mL of DMSO. The Pter N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure sodium chloride. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask and the THF solvent was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged (7000 rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant tests and characterization.

[0070] Comparative Example 2: 1 mg of Pter N25, 3 mg of DSPE-PEG2000, and 0.5 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.5 mg of doxorubicin (DOX) was dissolved in 1 mL of DMSO. The Pter N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure water. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask, and the THF solution was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged (7000 rpm) in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant testing and characterization.

[0071] Comparative Example 3: 1 mg of PTe N25, 3 mg of DSPE-PEG2000, and 0.5 mg of DSPE-PEG2000-cRGD were weighed and dissolved in 1 mL of tetrahydrofuran. 0.5 mg of paclitaxel (PTX) was dissolved in 1 mL of DMSO. The PTe N25, DSPE-PEG2000, and CPT solutions were then mixed and added to 9 mL of ultrapure ethanol. The mixture was sonicated for 10 minutes. The mixture was then placed in a rotary evaporation flask, and the THF solution was removed by rotary evaporation under reduced pressure. The liposome nanoparticle solution was then centrifuged at 7000 rpm in an ultrafiltration centrifuge tube (MW 3500) for 15 minutes to obtain the liposome nanoparticle solution. The product was freeze-dried and subjected to relevant testing and characterization.

[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; Malvern Co., Ltd., Malvern. UK). 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 diluted into water, phosphate buffered saline (PBS) (room temperature) and PBS containing 10v / v% FBS (37°C) and monitored for 24 hours. The 95% particle size distribution was between 110-120nm, 115-125nm and 120-130nm, respectively. The results are shown in Figure 2As shown in C, the morphology of the precipitates was observed using a spherical aberration-corrected transmission electron microscope (Titan G260-300; Frequency Electronics, Inc, UK). Figure 2 As shown in Figure E, CPT and PTeN25 aggregate into the core of nanoparticles through π-π interaction, with a particle size of 70 to 90 nm.

[0075] b) Absorption and emission spectroscopy. The cRGD-PTer-N25 / CPT NPs obtained in Example 1 were diluted to 10 μM and added to a cuvette. The UV-visible absorption spectrum was measured (Cary 8454 UV-Vis+Rx2000; Agilent Technologies Inc. UK). Figure 2 As shown in A and B, there is characteristic absorption in the range of 500-800 nm, with a peak at 584 nm; its aggregated emission peak is at 750 nm.

[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 the O2 probe DPBF is 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 After the addition of cRGD-PTer-N25 / CPT NPs, the absorbance of DPBF at 407 nm showed a continuous and rapid decline. After 75 seconds of laser irradiation (10 seconds each time, a total of 15 times), the absorbance of DPBF in the 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 generate 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 irradiating it with 808 nm laser (5 cycles of illumination / 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 200 μL of suspension at different concentrations and irradiated with laser (808 nm, 1 W·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℃ and 58℃ respectively, with a photothermal conversion efficiency PCE = 57.4% ( Figure 3 C), its photothermal conversion efficiency and photothermal stability are significantly better than the existing FDA-approved ICG ( 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 then replaced with complete medium containing co-loaded DiO, DiO@NPs, and DiO@cRGDNPs nanomaterials. 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 Pter 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 hours, co-loaded control, cRGD-PTer-N25 / CPT NPs, and cRGD-PTer-N25 / CPT NPs plus laser group were added to complete culture medium. After incubation for 4 hours, the reactive oxygen species detection kit was added to the culture medium. After incubation for another 30 minutes, the cells were washed twice with PBS and laser (1mW·cm -2 ) for 5 min. The treated cells were imaged by fluorescence microscopy. Figure 6 As shown in A, the fluorescence intensity of cells in the cRGD-PTer-N25 / CPT NPs (abbreviated as PTe N25 in the figure) plus laser nanomaterial group was the highest, indicating that ROS was generated in the cells.

[0083] c) In vitro cytotoxicity The cytotoxicity of the nanomaterials to breast cancer 4T1 cells was evaluated by MTT assay. 4 ) were seeded in 96-well plates and cultured at 37°C in 5% CO2 for 24 h. The culture medium was then replaced with the control, free CPT group, CPT NPs group, PTe N25 NPs + laser, PTe N25 / CPT NPs + laser: cRGD-PTerN25 / CPT NPs + laser in complete culture medium and 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 of 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 2. 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 levels of HSP70 in 4T1 cells treated with different methods were 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). 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 once 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, cells were irradiated 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 by 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 levels of HSP70 in G2:laser group and G3:photosensitizer (PTer N25 NPs) did not change significantly, indicating that laser and photosensitizer alone have 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. Furthermore, compared with the saline group G1, even under photothermal conditions, the expression level of HSP70 in the laser group G8 was significantly downregulated to 0.65, indicating that the active CPT drug was released into tumor cells in response to chemotherapy and downregulated the expression of HSP70, enhancing the sensitivity of tumors to PTe N25-mediated PTT. In summary, the preparation of a dual-targeted chemotherapy-photothermal therapy system assisted by cRGD and CPT with the organic conjugated polymer material PTe N25 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. After about 7 days, 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. 6 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 2. Figure 8 As shown, from Figure 8 As can be seen from A and B, the nanomaterials can be enriched in the tumor area 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 Figure 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, infrared thermal imaging was used to record the photothermal activity of animals in each group under 808 nm laser (1 W·cm) 6 h after intravenous injection. -2 ) irradiated the tumor area for 5 minutes and calculated the average temperature of the tumor area. Figure 9 As shown, both groups injected with the nanomaterial loaded with cRGD-PTerN25 / CPT NPs photosensitizer achieved localized photothermal effects in the tumor, with temperatures in the tumor region ranging from 53°C to 60°C, with a maximum temperature of 60°C. This indicates that the nanomaterial cRGD-PTerN25 / CPT NPs can be targeted and enriched in tumors and produce effective photothermal therapy under laser irradiation.

[0093] Example 6

[0094] In vivo real-time near-infrared fluorescence-guided photosynergistic chemotherapy anti-tumor evaluation of CPT- and PTeN25-co-loaded nanomaterials.

[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, followed by laser irradiation 6 hours after injection; ③CPT group, intravenous injection of 200μL CPT solution; ④PTer N25 NPs+laser group, intravenous injection of PTe N25 NPs followed by laser irradiation 6 hours later; ⑤PTer N25 / CPT NPs group, intravenous injection of PTe N25 / CPT NPs; ⑥cRGD-PTer N25 / CPT NPs+laser group, intravenous injection of cRGD-PTer N25 / CPT NPs followed by laser irradiation 6 hours later. The laser irradiation site and range were determined by near-infrared fluorescence imaging, and the 808nm laser power was 1W·cm -2 , time 10min. Each group of treatment 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. Figure 10 、 Figure 11-13 As shown in the data (**P>0.05, ***P<0.01), the average tumor volume in the light-enhanced chemotherapy group was significantly lower than that in the other treatment groups (P<0.05). However, the weight growth curves of the mice showed no significant differences between the groups, indicating that the nanomaterial had no significant toxicity to the mice's growth after injection. This indicates that light-enhanced chemotherapy achieved by the nanomaterial cRGD-PTer N25 / CPT NPs can significantly inhibit breast cancer growth.

[0096] At the same time, the anti-tumor effect of the obtained nanoparticles was also evaluated under laser irradiation. At the same time, the anti-tumor effect of the nanoparticles obtained in Comparative Examples 1, 2 and 3 was also evaluated under laser irradiation. 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 did not play a synergistic role, but were 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 compound PTe N25, wherein camptothecin and compound PTe N25 are simultaneously encapsulated by phospholipids; The structural formula of the compound PTe N25 is: The phospholipids include PEGylated synthetic phospholipids; The PEGylated synthetic phospholipids include RGD peptide-modified PEGylated synthetic phospholipids; The RGD peptide is an arginine-glycine-aspartic acid tripeptide.

2. The antitumor nanoparticle according to claim 1, characterized in that The phospholipids also include any one of natural phospholipids and synthetic phospholipids or a combination thereof.

3. The anti-tumor nanoparticle according to claim 1, characterized in that The PEGylated synthetic phospholipids include DSPE-PEG2000-cRGD and DSPE-PEG2000.

4. The anti-tumor nanoparticle according to claim 3, characterized in that The mass ratio of DSPE-PEG2000:DSPE-PEG2000-cRGD:camptothecin:PTer N25 is (2-5):(0.2-0.7):(0.2-0.7):1.

0.

5. The antitumor nanoparticle according to any one of claims 1 to 4, 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.

6. The method for preparing antitumor nanoparticles according to any one of claims 1 to 4, characterized in that: Weigh phospholipids, camptothecin and PTe N25, add solvent to dissolve, and ultrasonicate; then remove the solvent by rotary evaporation, and ultrafiltration and centrifugation to obtain anti-tumor nanoparticles.

7. The preparation method according to claim 6, characterized in that The parameters of the ultrasonic treatment step were as follows: ultrasonic time 15 min, ultrasonic 3 s, pause 3 s, and ultrasonic power 150 W.

Images