Near-infrared light-regulated conjugated polymer composite nanoparticles and their preparation methods and applications

Abstract

The present invention belongs to the technical field of photothermal gas therapy, and particularly relates to near-infrared light-regulated conjugated polymer composite nanoparticles and a preparation method and application thereof. The present invention develops near-infrared light-regulated conjugated polymer composite nanoparticles CPNPs and CPNPBs. The composite nanoparticle CPNPs is a nanoparticle with near-infrared (NIR) response, and the composite nanoparticle CPNPBs can be used as a near-infrared (NIR)-responsive photothermal nanocarrier and a NO generator in cancer cells. In addition, the above composite nanoparticles all exhibit excellent biocompatibility and thermal effects. More importantly, the combination of the photothermal performance and the NO generation performance of the composite nanoparticle CPNPBs provides a new path for the preparation of hybrid nanomaterials, and the near-infrared light-activated composite nanoparticle CPNPBs can be used to achieve precise photothermal / gas dual-mode therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of photothermal gas therapy, and particularly relates to near-infrared light-regulated conjugated polymer composite nanoparticles, a preparation method thereof, and applications thereof. Background Art

[0002] Cancer is one of the main causes of death globally, seriously threatening human health and well-being. Photothermal therapy (PTT), as a highly promising treatment method, has attracted much attention due to its excellent remote spatio-temporal control accuracy and non-invasive treatment characteristics. It is worth noting that PTT uses light absorbers to convert light energy into heat energy for cell thermal ablation. More importantly, near-infrared light is more conducive to reducing light attenuation in tissues than visible light, which enables therapeutic drugs with high NIR absorbance to achieve better light penetration depth and low toxicity to biological systems. Currently, various types of inorganic nanomaterials have been studied and applied in photothermal therapy, such as rare earth ion-doped nanocrystals, novel metal nanostructures, tungsten oxide nanowires, etc. However, for most inorganic nanomaterials, the controversial long-term toxicity severely limits their potential clinical applications.

[0003] Therefore, organic materials have attracted extensive interest. In particular, semiconductor polymer nanoparticles (SPNs) prepared from semiconductor polymers have broad application prospects in the biomedical field due to their excellent optical properties. It is worth noting that the metal-free nature of SPNs allows them to bypass the toxicity problems caused by metal ions. At the same time, SPNs often have optical advantages, even superior to inorganic semiconductor nanoparticles. In addition, SPNs can effectively convert light energy into mechanical sound waves and heat energy, making them the best candidates for photoacoustic imaging and photothermal therapy. Moreover, compared with carbon nanotubes and gold nanorods, they have stronger light absorption ability and higher photothermal conversion efficiency, resulting in faster temperature rise. Based on these encouraging properties, diagnostic and therapeutic reagents based on semiconductor polymer quantum dots (Pdots) have been used as photothermal nanomodulators for cancer treatment. However, due to the fact that heat shock proteins (HSPs) in cells can inhibit heat stress-induced apoptosis, PTT also has problems of low efficacy and tumor recurrence. Therefore, further overcoming the defects of photothermal therapy is of great significance for tumor treatment.

[0004] At present, gas therapy is considered a very promising tumor treatment strategy due to its high treatment efficacy and biosafety. In the family of gas signaling molecules, nitric oxide (NO) plays extremely important physiological or pathophysiological roles in almost all organ systems. In particular, increasing evidence indicates that NO is a double-edged sword for cancer. Low concentrations of NO can promote tumor growth, but at high concentrations (such as >1 μM), NO can inhibit the growth of solid tumors. At the same time, NO can also enhance the efficacy of chemotherapy by sensitizing multidrug-resistant cancer cells. In addition, NO can not only act as a radiosensitizer but also enhance the efficacy of photodynamic therapy (PDT) by reacting with reactive oxygen species (ROS) to form highly reactive peroxynitrite. Therefore, researchers have developed various NO donors. Among them, N,N'-di-sec-butyl-N,N'-dinitrosophenylenediamine (BNN6) is considered a powerful NO generator for gas therapy. However, BNN6 has the drawback of UV-vis light response, which severely restricts its application and development. To overcome this shortcoming, BNN6 exhibits near-infrared response through π-π stacking with other molecules. Therefore, it is of great necessity and importance to design and prepare a light-controlled NO generator as conjugated polymer nanoparticles for photothermal / gas dual-modal precise therapy. Summary of the Invention

[0005] The present invention develops near-infrared-regulated conjugated polymer composite nanoparticles CPNPs and CPNPBs. The composite nanoparticle CPNPs is a nanoparticle with near-infrared (NIR) response, and the composite nanoparticle CPNPBs can serve as a near-infrared (NIR)-responsive photothermal nanocarrier and NO generator in cancer cells. In addition, the above composite nanoparticles all exhibit excellent biocompatibility and thermal effects. More importantly, the combination of the photothermal performance and NO generation performance of the composite nanoparticle CPNPBs provides a new path for the preparation of hybrid nanomaterials, and the near-infrared light-activated composite nanoparticle CPNPBs can be used to achieve photothermal / gas dual-mode precise therapy.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a near-infrared-regulated conjugated polymer composite nanoparticle CPNPs. The preparation method of CPNPs is as follows: Mix the IN-NDI solution and the F127 solution, add the mixture to deionized water, react under an ice-water bath, remove the organic solvent, filter, and purify to obtain the product.

[0008] Specifically: Prepare IN-NDI solution and F127 solution with THF. The concentration of IN-NDI solution is 1 mg / mL, and the concentration of F127 solution is 10 mg / mL. Mix the above two solutions according to a volume ratio of 1:2, dilute them, and stir vigorously. Then, under ultrasonic treatment, quickly inject the mixed solution into deionized water. The mixture is continuously ultrasonically treated in an ice-water bath for 5 min, and THF is removed with nitrogen. The remaining nanoparticles are obtained after filtration and purification through a 0.22 μm filter membrane.

[0009] Among them, IN-NDI is prepared by the following method:

[0010] Mix the toluene solution of IN and the toluene solution of NDI, add the catalyst Pd(PPh3)4, react at 110 °C for 48 h, and then remove the end groups with (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene and bromobenzene, purify, precipitate, and finally obtain IN-NDI. The synthesis route of the conjugated polymer IN-NDI is as Figure 15 shown in a.

[0011] The present invention provides a near-infrared-regulated conjugated polymer composite nanoparticle CPNPBs. The preparation method of CPNPBs is as follows:

[0012] Mix the IN-NDI solution, BNN6 solution and F127, add the mixture to deionized water, react in an ice-water bath, remove the organic solvent, filter, and wash to obtain the product.

[0013] Specifically: Prepare IN-NDI solution, BNN6 solution and F127 solution with THF. The concentration of IN-NDI solution is 1 mg / mL, the concentration of BNN6 solution is 1 mg / mL, and the concentration of F127 solution is 10 mg / mL. Mix the above three solutions according to a volume ratio of 1:1:2, dilute them, and stir vigorously. Then, under ultrasonic treatment, quickly inject the mixed solution into deionized water. The mixture is continuously ultrasonically treated in an ice-water bath for 5 min, and THF is removed with nitrogen. After the remaining nanoparticles are filtered and purified through a 0.22 μm filter membrane, they are filtered through an ultrafiltration centrifuge tube and washed three times repeatedly to wash away the unencapsulated BNN6, and the upper-layer nanoparticles are collected to obtain the product. The synthesis route of the composite nanoparticle CPNPBs is as Figure 15 shown in b.

[0014] The present invention discovers that the composite nanoparticle CPNPs is a nanoparticle with near-infrared (NIR) response. The composite nanoparticle CPNPBs can be used as a near-infrared (NIR)-responsive photothermal nanocarrier and a NO generator in cancer cells. In addition, the above composite nanoparticles CPNPs and CPNPBs both exhibit excellent biocompatibility and thermal effects, and have insignificant cytotoxicity under dark conditions.

[0015] The composite nanoparticles CPNPs or CPNPBs were injected into the tumor sites of mice. Under laser irradiation, PTT inhibited the tumor growth in mice treated with CPNPs and CPNPBs. In contrast, the tumor growth in mice treated with CPNPBs and laser irradiation showed more significant inhibition within 28 days of monitoring. Therefore, the composite nanoparticles CPNPs or CPNPBs can be used to prepare tumor-suppressing drugs. The specific tumor is a tumor formed by breast cancer cells.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention synthesized a conjugate polymer-based composite nanoparticle CPNPs or CPNPBs with near-infrared stimulus response. CPNPs can be used for photothermal therapy, and CPNPBs can be used for photothermal / NO synergistic therapy. The composite nanoparticle CPNPBs overcame the disadvantages of short diffusion distance and extremely limited lifespan of NO at the same time, realized the precise controllability of NIR on NO release, and the photothermal damage was limited to the irradiated area, greatly reducing the systemic toxicity. It opened up an applicable, efficient and safe idea for designing multiple therapeutic nanodrugs. Description of the Drawings

[0018] Figure 1 1H NMR spectrum of IN-NDI 1

[0019] Figure 2 Gel permeation chromatography (GPC) spectrum of polymer NDI

[0020] Figure 3 UV-Vis-NIR absorption spectrum, linear relationship diagram (insert) between the concentration of IN-NDI dissolved in THF and the absorption at 885 nm

[0021] Figure 4 1H NMR spectrum of BNN6 1

[0022] Figure 5 a) TEM image of CPNPBs; b) DLS distribution image; c) NIR absorption spectra of BNN6, CPNPs and CPNPBs; d) ζ-potentials of CPNPs and CPNPB; e) DLS of different batches of CPNPBs; f) Zeta potential of different batches of CPNPBs

[0023] Figure 6 Graph of the change of Zeta potential of CPNP and CPNPB with storage time at room temperature

[0024] Figure 7 ​​a) UV-Vis-NIR absorption spectrum of BNN6 in absolute ethanol and the linear relationship between the absorption at 248 nm and the concentration (insert). b) UV absorption curve of the ultrafiltrate.

[0025] Figure 8 a) Temperature change curves of 100 μg / mL CPNPs under 808 nm near-infrared irradiation with different power densities. b) Temperature change curves of CPNPBs (IN-NDI concentration is 100 μg / mL, BNN6 is 100 μg / mL) under 808 nm near-infrared irradiation with different power densities. c) Temperature change curves of different concentrations of CPNPs within 10 minutes under 808 nm (0.6 W / cm 2 ) near-infrared irradiation. d) Photothermal images of four different solutions (PBS, BNN6, CPNPs, and CPNPBs) under near-infrared laser (808 nm, 0.6 W / cm 2 ) irradiation for 10 minutes. e) Temperature change curve of CPNPBs under 808 nm (0.6 W / cm 2 ) near-infrared irradiation and five switching cycles. f) Linear relationship between time and -lnθ obtained from the cooling period (applying the linear time data of the cooling period to determine the time constant of the system heat transfer (τs = 160.82178). PCE is 55.6%).

[0026] Figure 9 Temperature change curve of CPNPs under 808 nm (0.6 W / cm 2 ) near-infrared irradiation and five switching cycles.

[0027] Figure 10 Record the temperature continuously for 10 minutes under 808 nm (0.6 W / cm 2 ) laser, and then turn off the laser to record the temperature for 10 minutes.

[0028] Figure 11 a) Dilute 1 M NaNO2 standard solution to different concentrations and mix it with Griess reagent 1 and Griess reagent 2. b) Prepare the NO standard curve by using the UV absorption value at 540 nm.

[0029] Figure 12 a) NO release under 808 nm (0.3 W / cm 2 , 0.6 W / cm 2 , 1 W / cm 2 ) near-infrared laser irradiation of CPNPBs for 10 minutes. b) 808 nm (0.6 W / cm 2) The release of NO from CPNPBs loaded with different concentrations of BNN6 under near-infrared laser irradiation for 10 min. c) 808 nm (0.6 W / cm 2 ) The controlled release curve of NO from CPNPBs NPs under on-off laser irradiation (IN-NDI concentration is 100 μg / mL, BNN6 concentration is 100 μg / mL).

[0030] Figure 13 MCF-7 cells and HeLa cells were treated with CPNPs at different concentrations (0 - 200 μg / mL) for a) biocompatibility evaluation and b) photothermal therapy evaluation (808 nm, 0.6 W / cm 2 )). MCF-7 cells and HeLa cells were treated with CPNPBs at different concentrations for in vitro cell c) biocompatibility evaluation and d) photothermal therapy effect evaluation (808 nm, 0.6 W / cm 2 )). e) Fluorescence images of live / dead cell staining of HeLa cells treated under different conditions. Scale bar = 200 μm.

[0031] Figure 14 After intratumoral injection of PBS, CPNPs, and CPNPBs, a) the corresponding infrared photothermal images of 4T-1 tumor-bearing mice were excited by 808 nm laser (0.6 W / cm 2 , 10 min). b) The curve of tumor volume change in mice within 28 days. c) The change in body weight of mice during treatment. d) Hematoxylin and eosin (H&E) staining images of major organs of normal control group mice and treatment group mice (808 nm, 0.6 W / cm 2 , 10 min) after treatment. Scale bar: 200 μM.

[0032] Figure 15 a) The synthetic route of the conjugated polymer IN-NDI. b) The route of composite nanoparticles CPNPBs synthesized from IN-NDI, BNN6, and F127. Specific Embodiments

[0033] The present invention will be described in more detail below through specific embodiments, so as to facilitate the understanding of the technical solution of the present invention, but it is not used to limit the protection scope of the present invention.

[0034] All reagents used in this embodiment are of analytical grade, and the main experimental reagents required are listed in Table 1.

[0035] Table 1 Main Reagents

[0036]

[0037]

[0038] Example 1

[0039] 1. Synthesis and Characterization of Conjugated Polymer IN-NDI

[0040] According to the classical donor-acceptor (D-A) structure, a new π-conjugated polymer was designed and synthesized using the Stille polymerization reaction with donor monomer IN and acceptor monomer NDI as raw materials. The specific steps are as follows:

[0041] In a 50 mL single-neck flask, IN (IN1443, 0.25 mmol) and NDI (NDI88, 0.25 mmol) were dissolved in 10 mL of toluene under a nitrogen atmosphere. Then, using Pd(PPh3)4 (8.6 mg) as a catalyst, the system was heated to 110 °C and reacted for 48 h. Then, 20 mg of (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene was added and the reaction continued for 5 hours to remove the bromine end groups of the polymer. Then, 0.4 mL of bromobenzene was added and the reaction continued for 6 hours to remove the tin end groups of the polymer. After heating, the crude product was purified in tetrahydrofuran, chloroform, and acetone and precipitated in methanol. Finally, a brownish-black solid product IN-NDI was obtained, which was stored in a small brown glass vial and kept at 4 °C. The successful synthesis of polymer IN-NDI was verified by 1H NMR and UV-visible absorption spectra.

[0042] The results showed that a new type of semiconductor polymer IN-NDI was synthesized in this example, and the chemical structure of the product was verified by 1 1H NMR spectrum ( Figure 1 ). The IN-NDI polymer exhibited an electron donor-acceptor (D-A) alternating main chain, a planar structure, and strong electron deficiency, and thus had broadband optical absorption in the NIR region. The results of gel permeation chromatography (GPC) showed that the number-average molecular weight (Mn) and weight-average molecular weight (Mw) of IN-NDI were 35088 Da and 99343 Da, respectively, and the polydispersity index (PDI) was 2.8 ( Figure 2 ). The linear relationship between the concentration of IN-NDI dissolved in THF and the absorption at 885 nm is as Figure 3 shown.

[0043] 2. Synthesis and Characterization of Nitric Oxide Donor BNN6

[0044] N,N'-di-sec-butyl-N,N'-dinitroso-p-phenylenediamine (BNN6) was synthesized according to the method reported in the literature (Fan J, He N, He Q, Liu Y, Ma Y, Fu X, Liu Y, Huang P, Chen X. A novel self-assembled sandwich nanomedicine for NIR-responsive release of NO. Nanoscale. 2015 Dec 21;7(47):20055-62. doi:10.1039 / c5nr06630a. Epub 2015 Nov 16. PMID:26568270; PMCID:PMC4666708.) as follows: 2.34 mL of BPA was diluted in 18 mL of ethanol and then mixed with NaNO2 solution (20 mL 6 M) under stirring and nitrogen protection. After 30 minutes, hydrochloric acid solution (20 mL 6 M) was added dropwise. After stirring for 4 hours, the beige solid product was collected by centrifugation and dried overnight under freeze vacuum. The successful synthesis of BNN6 was verified by 1H NMR. In this example, the purified BNN6 was characterized by proton nuclear magnetic resonance spectroscopy ( 1 1H NMR), as Figure 4 shown. 1 1H NMR (400 MHz, CDCl3): δ 7.52 (4H), 4.95 - 4.69 (2H), 2.00 - 1.84 (2H), 1.81 - 1.69 (2H), 1.48 (t, J = 7.6 Hz, 6H), 1.08 (td, J = 7.4, 5.3 Hz, 6H).

[0045] 3. Preparation of CPNPs

[0046] Two solutions were prepared with tetrahydrofuran (THF) as the solvent: IN-NDI solution and F127 solution. The concentration of the IN-NDI solution was 1 mg / mL, and the concentration of the F127 solution was 10 mg / mL. The above two solutions were mixed in a volume ratio of 1:2 (3 mL in total) and diluted to 20 mL, and stirred vigorously. Then, under ultrasound, 1 mL of the mixed solution was quickly injected into 10 mL of deionized water (the change in the solvent hydrophobicity caused the hydrophobic part to aggregate to form nanoparticles). The mixture was continuously sonicated in an ice-water bath for 5 min, and THF was removed with nitrogen. The remaining nanoparticles were filtered and purified through a 0.22 μm filter membrane, concentrated to 600 μg / mL, and finally the collected nanoparticles were stored in a refrigerator at 4 °C.

[0047] 4. Preparation of CPNPBs

[0048] Three solutions were prepared separately using tetrahydrofuran (THF) as the solvent: IN-NDI solution, BNN6 solution, and F127 solution. The concentration of the IN-NDI solution was 1 mg / mL, the concentration of the BNN6 solution was 1 mg / mL, and the concentration of the F127 solution was 10 mg / mL. The above three solutions were mixed together in a volume ratio of 1:1:2 (total 4 mL) and diluted to 20 mL, and then stirred vigorously. Then, under ultrasonic conditions, 1 mL of the mixed solution was quickly injected into 10 mL of deionized water. The mixture was continuously ultrasonicated in an ice-water bath for 5 min, and tetrahydrofuran (THF) was removed with nitrogen. The remaining nanoparticles were filtered and purified through a 0.22 μm filter membrane, and then filtered through an ultrafiltration centrifugal tube and washed three times repeatedly to wash away the unencapsulated BNN6. The upper-layer nanoparticles were collected, concentrated to 600 μg / mL, and finally the collected nanoparticles were stored in a refrigerator at 4 °C. The filtrate was recovered, the ultraviolet-visible absorption curve was measured, and the absorption value at 248 nm was taken. By referring to the standard curve of BNN6, the loading rate of BNN6 in CPNPBs was calculated to be 70.15%. Figure 7 a and Figure 7 b.

[0049] The prepared CPNPBs presented a spherical morphology under transmission electron microscopy (TEM) ( Figure 5 a), and the particle size was about 100 nm. Dynamic light scattering (DLS) showed that the hydrodynamic diameter was concentrated at about 103 nm ( Figure 5 b). The absorption spectra of CPNPs, CPNPBs, and BNN6 were measured respectively ( Figure 5 c). It can be seen from the absorption spectra that CPNPBs were successfully synthesized. In addition, the Zeta potential results showed that both CPNPs and CPNPBs had negative potentials of -28 mV and -21 mV ( Figure 5 d), indicating that the nanoparticles could remain stable in aqueous solution. Importantly, within one week, the potential of the nanoparticles remained basically unchanged ( Figure 6 a and Figure 6 b). Zeta potential and DLS indicated that the prepared CPNPBs in different batches had good reproducibility ( Figure 5 e and 5f).

[0050] 5. Photothermal effect and photostability

[0051] The prepared CPNPs and CPNPBs could also show broad absorption in the NIR window and could be used as photothermal reagents for NIR-II photothermal therapy and further utilized in vivo. To evaluate the photothermal performance of CPNPs and CPNPBs, the photothermal effect of CPNPs (CPNPBs) was studied by recording the temperature change under 808 nm near-infrared light irradiation.

[0052] The dependence of the laser irradiation of CPNPs and CPNPBs solutions on the optical power density was studied using different optical power densities (0.1, 0.2, 0.4, 0.6, 0.8, 1 W / cm 2 ). The concentration of the CPNPs solution was 100 μg / mL. When preparing the CPNPBs solution, the concentration of IN-NDI was 100 μg / mL and that of BNN6 was 100 μg / mL.

[0053] In addition, under the near-infrared irradiation of 808 nm (0.6 W / cm 2 ), the concentration dependence was studied using CPNPs solutions with different concentrations (0, 10, 20, 40, 60, 80, 100 μg / mL).

[0054] Under the irradiation of an 808 nm laser, the temperature changes of CPNPs and CPNPBs solutions under near-infrared irradiation with different power densities are shown in Figure 8 a and Figure 8 b. The results show that CPNPs and CPNPBs have a strong dependence on the near-infrared laser power density.

[0055] Figure 8 c plots the temperature change curves of CPNPs solutions with different concentrations (0 - 100 μg / mL) under continuous irradiation with a laser of 808 nm (0.6 W / cm 2 ). The results in c show that for solutions with higher concentrations (such as 80, 100 μg / mL), the temperature can reach up to 59.8 °C after 10 min of irradiation; the maximum temperature of the solution decreases as the solution concentration decreases. Figure 8 c shows that for solutions with higher concentrations (such as 80, 100 μg / mL), the temperature can reach up to 59.8 °C after 10 min of irradiation; the maximum temperature of the solution decreases as the solution concentration decreases.

[0056] Using an infrared thermal image, the temperature changes of four different solutions (PBS 0.1 M, BNN6 100 μg / mL, CPNPs 100 μg / mL, and CPNPBs 100 μg / mL) under the irradiation of an 808 nm (0.6 W / cm 2 ) laser can be visually observed. The maximum temperature of the CPNPBs solution can reach 60 °C ( Figure 8 d), basically meeting the heat dissipation requirements of PTT (>43 °C).

[0057] To evaluate the photothermal stability of CPNPs and CPNPBs solutions, the recyclable temperature changes of 100 μg / mL CPNPs and CPNPBs are shown in Figure 9 and Figure 8 e. CPNPs and CPNPBs have stable photothermal capabilities in five consecutive heating / cooling cycles.

[0058] 6. Photothermal conversion efficiency of CPNPs and CPNPBs

[0059] The photothermal conversion efficiency is an important parameter for evaluating the photothermal conversion ability. To measure the photothermal conversion efficiency, 0.5 mL of an aqueous solution of 100 μg / mL CPNPs (or CPNPBs) was added to an EP tube. Then, the aqueous solution of nanoparticles was placed under 808 nm (0.6 W / cm 2 ) laser irradiation until temperature equilibrium was reached. The laser was turned off, and the solution temperature was cooled to room temperature. The photothermal conversion efficiency (η) was calculated as follows:

[0060]

[0061] where h represents the heat transfer coefficient of B@P@C; S represents the surface area of the container; Tmax and Tmin represent the highest temperature and room temperature during the whole process, respectively; Qdis represents the heat dissipation of water. I is the irradiation laser power (0.6 W / cm 2 ), and A 808 is the absorbance of CPNPs (CPNPBs) at 808 nm. The result of hs was calculated by the following formula.

[0062]

[0063] where τ s is the time constant of the solution heat transfer, which can be obtained from the measured values in Figure 8 (τ sCPNPs = 191.5468, τ sCPNPBs = 160.82178); m is the mass of deionized water dissolving the nanoparticles (500 mg), and c is the heat capacity of water (4.2 J / g). The time constant of the system heat exchange can be obtained from the curve of the cooling time after the laser is turned off and the negative natural logarithm of the temperature change ([[]] Figure 10 and Figure 8 f). After calculation, the photothermal conversion efficiencies of CPNPs and CPNPBs are 55.6% and 46%, respectively.

[0064] 7. Nitric Oxide Standard Curve

[0065] Using a nitric oxide detection kit, a 1 M NaNO2 standard solution was diluted to the following concentrations: 60 μM, 40 μM, 20 μM, 15 μM, 10 μM, 5 μM. 900 μL of Griess reagent I, 900 μL of Griess reagent II, and 900 μL of NaNO2 solution were mixed evenly, and then the ultraviolet absorption curve of the mixed solution was tested. A standard curve of the absorption values of NaNO2 at different concentrations at 540 nm was plotted. Finally, the standard curve equation was obtained as: y = 0.01249x + 0.00708.

[0066] 8. In Vitro NO Detection of CPNPBs

[0067] IN-NDI is an excellent photothermal reagent. In this example, IN-NDI and BNN6 were mixed, and F127 was used to coat the two together by the nano-precipitation method to prepare hydrophilic nanoparticles. Under the irradiation of 808 nm near-infrared laser, a part of IN-NDI generates heat, and the other part is converted into electrons for BNN6 to release NO. The release behavior of NO in CPNPBs was quantitatively studied by the classical Griess method, and the generation of NO controlled by near-infrared light was verified.

[0068] To detect the NO released by CPNPBs, an aqueous solution of CPNPBs with a concentration of 100 μg / mL was placed in a centrifuge tube and irradiated with an 808 nm (0.6 W / cm 2 ) laser. At 10-min intervals, 150 μL of the solution was transferred to a 96-well plate, and then 150 μL of Griess I and Griess II were added. The absorbance at 540 nm was measured with a microplate reader. The release amount of NO was calculated according to the obtained standard curve of the NO concentration absorption value.

[0069] The standard curve of the NO concentration absorption value is as Figure 11 shown in Figure 11 a and

[0070] After IN-NDI and BNN6 combine to form CPNPBs, CPNPBs become very sensitive to near-infrared light ( Figure 12 a–c). The concentration of IN-NDI in CPNPBs was adjusted to 100 μg / mL, and different CPNPBs solutions loaded with different amounts of BNN6 (5, 10, 25, 50, and 100 μg / mL) were prepared. Under the irradiation of an 808 nm (0.6 W / cm 2 ) excitation light for 10 min, the NO release amount increased with the increase in the concentration of BNN6 ( Figure 12 a). When the loading amount of BNN6 was 100 μg mL -1 , the NO release amount could reach 20.9 μg / mL. In addition, the NO release profiles of CPNPBs under 808 nm near-infrared excitation light with different power densities were also studied, and it was found that the response of CPNPBs solution to near-infrared light was power density-dependent ( Figure 12 b).

[0071] The near-infrared light controllability of NO release from CPNPBs was further studied. When the near-infrared light was turned on, NO was rapidly released. Once the near-infrared light was turned off, the release of NO became very slow, accompanied by a small amount of delayed release ( Figure 12c), which indicates that the CPNPBs solution has good near-infrared light controllability for NO release. By controlling the on / off and power of the near-infrared light, the NO concentration can be well controlled as needed, which is of great significance for manipulating the drug concentration within the therapeutic window and reducing the risk of NO poisoning.

[0072] Example 2 Cell Experiments

[0073] 1. Cell Culture and Toxicity Test

[0074] HeLa cells, MCF-7 cells and 4T1 cells were cultured in DMEM cell culture medium containing 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin. All cells were cultured in cell culture flasks in an incubator at 37 °C and 5% carbon dioxide.

[0075] 2. Cytotoxicity Study of CPNPs and CPNPBs

[0076] The MTT method and live / dead cell staining method were used to evaluate the toxicity and photothermal toxicity of the nanoparticles to HeLa cells and MCF-7 cells.

[0077] Toxicity measurement by MTT method: MCF-7 cells and HeLa cells were incubated with different concentrations of CPNPs and CPNPBs respectively, and the cell viability was measured by the MTT method under dark conditions. Specifically, MCF-7 / HeLa cells were seeded in 96-well plates at 7000 cells per well and cultured for 24 h. The next day, the cells were incubated with DMEM cell culture medium containing different concentrations of CPNPs (0, 20, 40, 60, 80, 100, 150, 200 μg / mL) and CPNPBs (0, 20, 40, 60, 80, 100, 150, 200 μg / mL). After 12 h, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated at 37 °C for another 4 h. Then the solution was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the 96-well plate was shaken for 5 min, and the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The control group was untreated cells, and the cell survival rate was calculated. The formula for calculating the cell survival rate (CPR) is: CPR = (average absorbance value of the experimental group - average absorbance value of the zero-adjustment group) / (average absorbance value of the control group - average absorbance value of the zero-adjustment group).

[0078] The results are as Figure 13 shown in Figures 13a and 13c, and the cell viability exceeded 85%. Therefore, CPNPs and CPNPBs have insignificant cytotoxicity and good biocompatibility under dark conditions.

[0079] Photothermal toxicity: The in vitro photothermal toxicity was also evaluated by MTT assay. The method was the same as above, except that after incubating the cells with DMEM basal medium containing different concentrations of CPNPs (0, 20, 40, 60, 80, 100, 150, 200 μg / mL) and CPNPBs (0, 20, 40, 60, 80, 100, 150, 200 μg / mL), the cells were first cultured in an incubator for 4 h, and then irradiated with NIR laser at 808 nm (0.6 W / cm 2 ) for 5 min per well. After irradiation, the cells were cultured for another 12 h, then the solution was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the 96-well plate was shaken for 5 min, and the absorbance at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The control group was untreated cells, and the cell survival rate was calculated. The formula for calculating the cell survival rate (CPR) is: CPR = (mean absorbance value of the experimental group - mean absorbance value of the zero-adjustment group) / (mean absorbance value of the control group - mean absorbance value of the zero-adjustment group).

[0080] The results showed that after irradiating each well of cells with an excitation light at 808 nm (0.6 W / cm 2 ) for 5 min, the cell viability showed an obvious downward trend ( Figure 13 b and 13d). The cell viability of cells incubated with 100 μg / mL CPNPs decreased to 41%, while for the CPNPBs solution loaded with BNN6, since NO that can kill cancer cells can be generated under light illumination, the cell viability of cells incubated with CPNPBs gradually decreased with the increase of the BNN6 loading amount (5 - 100 μg / mL), and the cell viability decreased to 9%. Therefore, it can be concluded that the dual action of photothermal effect and NO can produce a better therapeutic effect.

[0081] Live / dead cell staining: To further detect the cell photothermal toxicity of CPNPBs, the AO / EB double-fluorescence dye method was used to detect the apoptosis of HeLa cells. Specifically, MCF-7 / HeLa cells were seeded into 12-well plates at a density of 200,000 cells per well and cultured for 24 h. The original culture medium was aspirated, and the cells were further incubated with DMEM medium containing different treatment reagents for 12 h: (1) PBS; (2) PBS + NIR; (3) CPNPs; (4) CPNPs + NIR; (5) CPNPBs NPs; (6) CPNPBs + NIR. In the above treatments, the concentration of PBS was 0.1 M, the concentration of all nanoparticles was 100 μg / mL, and NIR was 808 nm 0.6 W / cm 2, The light illumination lasted for 5 min. Discard the original culture medium, and wash each well 3 times with PBS. Add the pre-prepared acridine orange / ethidium bromide (AO / EB) fluorescent dye into the wells, gently shake the well plate to evenly spread the staining solution on the cells. Then observe the cells under a fluorescence microscope and take pictures. Green represents live cells and red represents dead cells.

[0082] It was observed under the fluorescence microscope that under the condition of no light illumination, CPNPBs at all concentrations had no obvious cytotoxicity. Under the condition of light illumination, compared with the control group, the cytotoxicity was significantly enhanced after treatment with CPNPBs ( Figure 13 e), and these results were consistent with those of MTT, indicating that the combined treatment of PTT and NO could effectively kill tumor cells.

[0083] Example 3 Animal Experiment

[0084] 1. Construction of animal model

[0085] Purchase several healthy female mice and raise them in an animal house. Keep the mice in an environment with a temperature of 24 ± 2 °C and a humidity of 60%, and provide circulating water and food. When the nude mice reach 6 weeks of age and their body weight is 20 - 30 g, inject 5×10 7 cells of 4T-1 murine breast cancer subcutaneously into the dorsal side of each mouse. After raising for one week, tumor-bearing mice were obtained. Measure the tumor size of the mice with a caliper every day.

[0086] 2. In vivo photothermal therapy

[0087] When the tumors on the backs of the mice grew to 90 - 120 mm 3 , randomly divide the mice into 6 groups with 5 mice in each group, and perform the following treatments respectively:

[0088] a) Inject 60 μL of 0.1 M PBS into the tumor site;

[0089] b) Inject 60 μL of 0.1 M PBS into the tumor site. After 24 h, irradiate with 808 nm NIR light for 5 min, and the light power density is 0.6 W / cm 2 ; c) Inject 60 μL of CPNPs into the tumor site, with a concentration of 100 μg / mL;

[0090] d) Inject 60 μL of CPNPs into the tumor site, with a concentration of 100 μg / mL. After 24 h, irradiate with 808 nm NIR light for 5 min, and the light power density is 0.6 W / cm 2 ;

[0091] e) Inject 60 μL of CPNPBs into the tumor site, with a concentration of 100 μg / mL;

[0092] f) Inject 60 μL of CPNPBs into the tumor site at a concentration of 100 μg / mL. After 24 h, irradiate with 808 nm NIR light for 5 min at a light power density of 0.6 W / cm 2 .

[0093] Measure the length and width of the tumor every other day and calculate the tumor volume according to the following formula:

[0094] V = L / 2 × W 2 .

[0095] 3. Biosafety study

[0096] Use an infrared thermal camera to record the infrared thermal images of the tumor site of each mouse under 808 nm (0.6 W / cm 2 ) irradiation for 10 min. The temperatures of both CPNPs and CPNPBs can rise to 65 °C ( Figure 14 a), while the PBS group can only rise to 34.3 °C, indicating that CPNPs and CPNPBs have good therapeutic effects in vitro. After the in vivo photothermal effect was verified, the tumor growth after different treatments was monitored for 28 days ( Figure 14 b) to compare the therapeutic effects. In the absence of laser irradiation, the growth of mice treated with CPNPs and CPNPBs was similar to that of the PBS group. Under laser irradiation, PTT inhibited the tumor growth of mice treated with CPNPs and CPNPBs. In contrast, the tumor growth of mice treated with CPNPBs and laser irradiation showed a more significant inhibitory effect within the 28 days of monitoring. In addition, the body weights of the mice were measured every 5 days. The results showed that there was no significant change in the body weights of all mice, indicating that all drugs had no effect on the growth of the mice ( Figure 14 c).

[0097] On the 28th day, the mice were euthanized. After the mice were sacrificed, the tumors and major organs (heart, liver, spleen, lung, kidney) were removed. The collected tumors and major organs were immersed in 10% neutral formalin for fixation, dehydrated until transparent, and then embedded in paraffin. The embedded paraffin blocks were cut into 4-μm sections, and then stained with hematoxylin-eosin (H&E). Finally, photos of the tissue sections were taken with a microscope.

[0098] In the hematoxylin and eosin (H&E) stained images ( Figure 14 d), there were no obvious physiological and morphological damages to the major organs such as the kidneys, spleens, and livers of the mice in each group. Therefore, it can be concluded that while ensuring no obvious damage to the major organs during the treatment, tumor cells can be effectively killed, greatly reducing the systemic toxicity.

[0099] The above-described embodiments are only the preferred embodiments of the present invention and do not limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structure, features, and principles described in the scope of the present invention patent application shall be included within the scope of the present invention's patent application.

Claims

1. A preparation method of near-infrared light-regulated conjugated polymer composite nanoparticles CPNPBs, characterized in that, Mix the IN-NDI solution, BNN6 solution and F127 solution, add the mixture to deionized water, and ultrasonicate for 2 - 5 minutes in an ice-water bath to remove the solvents of the IN-NDI solution, BNN6 solution and F127 solution, then filter and wash to obtain the product; The structural formula of IN-NDI is as follows:

2. The preparation method of a near-infrared light-regulated conjugated polymer composite nanoparticle CPNPBs according to claim 1, characterized in that, The concentration of the IN-NDI solution is 1 mg / mL, the concentration of the BNN6 solution is 1 mg / mL, and the concentration of the F127 solution is 10 mg / mL. When mixing, the IN-NDI solution, BNN6 solution and F127 solution are mixed at a volume ratio of 1∶1∶2.

3. Composite nanoparticles CPNPBs prepared by the method described in claim 1 or 2.

4. Use of the composite nanoparticles CPNPBs described in claim 3 in the preparation of antitumor drugs.

5. The application according to claim 4, wherein The tumor suppressor drug is used under irradiation with 808 nm near-infrared light at a light power density of 0.6 W / cm 2 , and the tumor is a tumor formed by breast cancer cells.

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