CDs-SA-PTX nano-composite as well as preparation method and application thereof
By loading PTX into the modified CDs-SA nanocomplex, the problems of poor water solubility and insufficient targeting of PTX are solved, and the effect of improving efficacy and reducing side effects in breast cancer treatment is achieved.
Patent Information
- Application Number
- CN202510191056.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-20
- Publication Date
- 2025-05-16
AI Technical Summary
The existing chemotherapeutic drug paclitaxel (PTX) is poor in water solubility when treating breast cancer, causing systemic toxicity, and prone to drug resistance and reducing efficacy.
CDs-SA-PTX nanocomplex was prepared by modifying sialic acid (SA) on the surface of carbon dots (CDs), forming CDs-SA, and loading paclitaxel (PTX) on CDs-SA, targeted transmission and controlled release of drugs were achieved.
It improves the accumulation and release efficiency of PTX in the tumor site, enhances the selectivity of cancer cells, improves the efficacy of chemotherapy, and reduces the toxic effect on normal tissues.
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Figure CN120000786A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a nanocomposite, in particular to a CDs-SA-PTX nanocomposite and a preparation method and application thereof. Background Art
[0002] Breast cancer is one of the most common malignant tumors in women, accounting for 7-10% of all malignant tumors in the body, and has become a major cause of threat to women's life and health. In the clinical treatment of breast cancer, chemotherapy is still an important means, but due to its dose limitation, it is easy to cause damage to healthy tissues, and patients may develop drug resistance, resulting in unsatisfactory chemotherapy efficacy.
[0003] Carbon dots (CDs) have broad application prospects in bioimaging and cancer photothermal therapy due to their excellent fluorescence properties, biocompatibility, stability and ease of functional modification. Sialic acid (SA) is a naturally occurring carbohydrate that can actively target selectins in the plasma membrane of cancer cells. Sialic acid has become a player in the field of drug delivery due to its excellent biocompatibility, low toxicity and abundant drug-carrying functional groups.
[0004] Paclitaxel (PTX) is a chemotherapy drug with good efficacy in treating breast cancer. However, due to its poor water solubility and the fact that it will inevitably diffuse into certain normal tissues, PTX is prone to cause systemic toxicity. Therefore, it is necessary to develop an intelligent platform to achieve targeted delivery and controlled release of anticancer drugs in tumors to improve the therapeutic effect and reduce side effects. Summary of the invention
[0005] The purpose of the present invention is to provide a CDs-SA-PTX nanocomposite and a preparation method and application thereof, so as to achieve targeted delivery and controlled release of the anticancer drug paclitaxel (PTX) in tumors.
[0006] To achieve the above object, according to one aspect of the present invention, a CDs-SA-PTX nanocomplex is provided, which includes paclitaxel (PTX) and CDs-SA, wherein the CDs-SA is prepared by modifying sialic acid (SA) on the surface of carbon dots (CDs) through an amide reaction; and paclitaxel (PTX) is loaded on the CDs-SA.
[0007] Furthermore, the mass ratio of paclitaxel (PTX) to CDs-SA is 1:1.
[0008] According to another aspect of the present invention, provided is a method for preparing the above-mentioned CDs-SA-PTX nanocomposite, comprising the following steps: Step 1, preparing CDs-SA; Sialic acid (SA) was dissolved in PBS solution to obtain SA solution, NHS and EDC were added to the SA solution, and the solution was stirred at room temperature to obtain an activated SA solution; the dissolved CDs were mixed with the activated SA solution, and the solution was stirred at room temperature to obtain a solution, and then dialyzed to obtain CDs-SA; Step 2, preparing CDs-SA-PTX; The CDs-SA obtained in step 1 is added to the ethanol solution of paclitaxel (PTX), and the mixture is stirred to obtain a mixed solution. The mixed solution is centrifuged, washed, and dried to obtain a CDs-SA-PTX solid.
[0009] Furthermore, in step 1, 20 mg of sialic acid (SA) was weighed and dissolved in 10 mL of PBS solution with a pH of 7.4, 18 mg of NHS and 18 mg of EDC were added to the SA solution, and the mixture was stirred at room temperature for 15 min to obtain an activated SA solution; 20 mg of freeze-dried CDs solid was dissolved in 10 mL of deionized water, and after complete dissolution, the mixture was mixed with the activated SA solution, stirred at room temperature for 12 h, and the obtained solution was dialyzed to obtain CDs-SA.
[0010] Furthermore, the CDs described in step 1 are obtained by dissolving 1,2,4-triaminobenzene dihydrochloride in deionized water and then adding triethylenetetramine to carry out a hydrothermal reaction.
[0011] Furthermore, in step 1, 196 mg of 1,2,4-triaminobenzene dihydrochloride was weighed and dissolved in deionized water, and ultrasonically dissolved, 1.2 mL of triethylenetetramine was added, and the obtained solution was transferred to a polytetrafluoroethylene-lined autoclave, and the autoclave was placed in an oven and the temperature was set to 90 ° C. The reaction was carried out for 12 h, and the reaction was naturally cooled to room temperature to obtain a dark brown liquid; the dark brown liquid was centrifuged and the supernatant was collected, and then filtered using a 0.22 μm microporous filter membrane, and finally dialyzed to obtain a brown solution, and the obtained solution was freeze-dried to obtain CDs.
[0012] Furthermore, in step 2, PTX is dissolved in ethanol to prepare PTX solutions of different concentrations, and 10 mg of CDs-SA is added to the PTX solutions of different concentrations and stirred overnight. The mixed solution is centrifuged to collect the precipitate, which is washed with ethanol 3-4 times and freeze-dried to obtain CDs-SA-PTX solid.
[0013] According to another aspect of the present invention, provided is the use of the above-mentioned CDs-SA-PTX nanocomplex in the preparation of a drug for the combined photothermal / chemotherapy treatment of breast cancer.
[0014] The present invention modifies the surface functional groups of CDs to solve the disadvantage of poor water solubility of PTX. The use of SA can enhance the selectivity of PTX for cancer cells and increase the accumulation of PTX at the tumor site. The nanocomposite synthesized by the present invention can improve the targeted uptake of hydrophobic drugs, help improve the bioavailability of oral drugs, and provide new possibilities for improving the efficacy of chemotherapy and reducing the side effects of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In Figure 1 , (a), (b) and (c) are TEM images of CDs, CDs-SA and CDs-SA-PTX, respectively; (d), (e) and (f) are particle size distribution diagrams of CDs, CDs-SA and CDs-SA-PTX, and the inset of (a) is the HRTEM image of CDs; Figure 2 shows the XRD patterns of CDs, CDs-SA and CDs-SA-PTX; Figure 3 shows the XPS general spectra of CDs, CDs-SA and CDs-SA-PTX (a), and the detailed spectra of C1s (b), N1s (c), and O1s (d) of CDs-SA-PTX. FIG4 is a UV-visible absorption spectrum of CDs, SA, CDs-SA, PTX and CDs-SA-PTX; FIG5 is a fluorescence spectra of CDs, SA, CDs-SA, PTX and CDs-SA-PTX; FIG6 is the FT-IR spectra of CDs, SA, CDs-SA, PTX and CDs-SA-PTX; FIG7 is a Zeta potential diagram of CDs, SA, CDs-SA, PTX and CDs-SA-PTX; Figure 8 shows the drug loading of CDs-SA-PTX at different drug loading ratios; Figure 9 shows the release rate of PTX in CDs-SA-PTX at pH 7.4 and 5.5; Fig.10 The temperature change curves of CDs-SA-PTX with different concentrations under 660 nm (1.5 W) laser irradiation (a) and the temperature change curves of CDs-SA-PTX with different power laser irradiation (b); Fig.11 The temperature variation of H2O, CDs, CDs-SA and CDs-SA-PTX under 660 nm (1.5 W) laser irradiation over time; Fig.12The infrared images of H2O, CDs, CDs-SA and CDs-SA-PTX changing with time under 660 nm (1.5 W) laser irradiation; Fig.13 is the thermal cycle curve of CDs-SA-PTX under 660 nm laser irradiation; Fig.14 The temperature rise and fall curves of CDs-SA-PTX irradiated by 660 nm laser (1.5 W) (a); the linear relationship between time and -ln (θ) (b); Fig.15 The effect of CDs-SA-PTX on the viability of MCF-7 cells and HUVEC cells; Fig.16 The effects of different treatment groups on the survival rate of MCF-7 cells (n=6, *** indicates p<0.001); Fig.17 The uptake of PBS, CDs, CDs-SA and CDs-SA-PTX by MCF-7 cells; Fig.18 This is the laser confocal microscope result after MCF-7 was treated with PBS, DNase and RNase; Fig.19 The killing effects of PBS group, NIR group, CDs-SA group, CDs-SA + NIR group, PTX group, CDs-SA-PTX group and CDs-SA-PTX + NIR group on MCF-7 cells; Fig. 20 Apoptosis of MCF-7 cells after treatment with PBS, NIR, CDs-SA, CDs-SA + NIR, PTX, CDs-SA-PTX and CDs-SA-PTX + NIR (a), apoptosis rate of each group (b), (*** represents p < 0.001); Fig.21 In the figure, (a) is the distribution map of CDs-SA-PTX in mice, and the fluorescence images were collected at 0, 3, 6, 9, 12, and 24 h after injection. (b) is the in vitro fluorescence image of the main organs and tumors of mice 9 h after CDs-SA-PTX injection. Fig. 22 Photothermal imaging of mice injected with Saline, CDs, CDs-SA and CDs-SA-PTX into the tail vein and then irradiated with 660 nm (1.5W) laser for 10 min; Fig.23Images of mice in different treatment groups after two weeks of treatment (a), tumor images (b), weight changes of mice in each group during treatment (c), tumor weight changes (d), and relative tumor volume changes (e), (*** represents p<0.001); Fig.24 Are the blood routine indicators of mice in each group; Fig.25 Blood biochemical indexes of mice in each group; Fig.26 HE staining images of the main organs (heart, liver, spleen, lung, kidney) and tumor tissues of mice in each group; Fig. 27 Schematic diagram of the preparation and application of CDs-SA-PTX nanocomposites. DETAILED DESCRIPTION
[0016] In the following embodiments, a new type of nucleolus-targeted orange light CDs was prepared by hydrothermal method, and SA was modified on the surface of CDs by amide reaction to make it specific and selective for tumor tissue. On this basis, dual-targeted nanoparticles (CDs-SA-PTX) were designed to achieve combined chemotherapy and photothermal therapy. The morphology, elemental composition, surface functional groups, and optical properties of CDs-SA-PTX were tested by basic characterization methods, and the drug release rate of CDs-SA-PTX was determined at pH 5.5 and 7.4. The dual-targeting performance and fluorescence imaging ability of CDs-SA-PTX for tumor cells and cell nucleoli were evaluated by cell uptake experiments. The ability of CDs-SA-PTX to inhibit cancer cell growth by chemotherapy and photothermal combination was evaluated by live-dead cell staining and cell apoptosis experiments. The therapeutic effect of CDs-SA-PTX on tumor tissue was evaluated by animal in vivo experiments, and the blood routine and biochemical indicators of mice were tested, and combined with tissue HE staining observation, it was proved that CDs-SA-PTX has good biosafety. The CDs-SA-PTX nanocomplex achieves combined photothermal and chemotherapy treatment of tumors, providing a theoretical basis for further research on breast cancer treatment.
[0017] 1. Preparation of CDs-SA-PTX Nanocomposites 1.1 Preparation of CDs Orange CDs were synthesized by a one-step hydrothermal method. 196 mg of 1,2,4-triaminobenzene dihydrochloride was weighed and dissolved in deionized water (10 mL). Ultrasonication was performed for 10 min to completely dissolve it. 1.2 mL of triethylenetetramine was added and the solution was transferred to a polytetrafluoroethylene-lined autoclave. The autoclave was placed in an oven and the temperature was set to 90 °C. The reaction was carried out for 12 h and the reaction was naturally cooled to room temperature to obtain a dark brown liquid. The dark brown liquid was centrifuged for 10 min (15000 rpm) to collect the supernatant, then filtered using a microporous filter membrane (0.22 μm), and finally dialyzed for 12 h (MWCO, 500 Da) to obtain a brown solution. The obtained solution was freeze-dried for later use.
[0018] 1.2 Preparation of CDs-SA Weigh 20 mg of sialic acid (SA) and dissolve it in 10 mL of PBS solution (pH 7.4). Add 18 mg of NHS and 18 mg of EDC to the SA solution and stir at room temperature for 15 min to obtain activated SA. Dissolve 20 mg of freeze-dried CDs solid in 10 mL of deionized water, mix with the activated SA solution after complete dissolution, and stir at room temperature for 12 h. Dialyze the above solution (MWCO, 1000Da) for 6 h to obtain CDs-SA, which is freeze-dried and set aside.
[0019] 1.3 Preparation of CDs-SA-PTX PTX was dissolved in ethanol to prepare PTX solutions of different concentrations, and 10 mg CDs-SA was added to the PTX solutions of different concentrations and stirred overnight. The mixed solution was centrifuged for 10 min (10,000 rpm) to collect the precipitate, which was washed with ethanol 3-4 times and freeze-dried to obtain CDs-SA-PTX solid.
[0020] 1.4 Characterization of materials The surface morphologies of CDs, CDs-SA and CDs-SA-PTX were observed by TEM. Figure 1 (a) It can be seen that CDs present a uniformly distributed spherical structure. The HR-TEM image of CDs ( Figure 1 (a) Inset) shows well-resolved lattice fringes with a spacing of 0.34 nm, which is attributed to the (002) lattice plane of graphite, indicating that CDs have a graphite-like structure. [ .
[0021] Figure 1 (b) and Figure 1 (c) TEM images of CDs-SA and CDs-SA-PTX, respectively. It can be seen that the CDs and CDs-SA-PTX nanoparticles are evenly distributed and have a spherical structure. Figure 1 (d), 1(e), and 1(f) show that the average size of CDs is 1.8 nm, while the average size of CDs-SA is 11.0 nm, and the particle size is significantly increased, proving the successful connection of SA. Compared with CDs-SA, the average size of CDs-SA-PTX nanoparticles (15.1 nm) is further enlarged, indicating that PTX is successfully loaded.
[0022] XRD patterns of CDs, CDs-SA and CDs-SA-PTX ( Figure 2 ) shows that CDs has a diffraction peak near 2θ=18.8°, indicating the presence of amorphous carbon particles in CDs. CDs-SA has a broad diffraction peak at 2θ=21.1° similar to the graphite lattice spacing, indicating amorphous properties. The broad diffraction peak of CDs-SA-PTX at 2θ=24.6° is caused by disordered carbon atoms.
[0023] XPS was used to characterize the elemental compositions of CDs, CDs-SA and CDs-SA-PTX. Figure 3 (a) It can be seen that CDs, CDs-SA and CDs-SA-PTX only contain C, N and O elements. The changes in the content of each element in CDs, CDs-SA and CDs-SA-PTX are shown in Table 1. The fine spectrum of C1s of CDs-SA-PTX is shown in Figure 3 As shown in (b), the peaks at 284.6 eV, 286.1 eV, and 288.33 eV correspond to the binding energies of CC, CN, and C=O bonds, respectively. Figure 3 (c)), peaks at 399.1 eV, 400.5 eV and 401.7 eV can be found, which are caused by C=NC, N-(C)3 and CNH bonds, respectively. Figure 3 (d) shows the fine map of O1s, with the peaks at 531.9 eV, 533.6 eV, and 535.3 eV originating from the binding energies of CO, C=O, and O=CO.
[0024] Table 1 Contents of elements in CDs, CDs-SA and CDs-SA-PTX name C1s (%) N1s (%) O1s (%) CDs 53.78 12.64 10.53 CDs-SA 38.66 35.52 25.82 CDs-SA-PTX 61.8 14.87 23.33 Figure 4 The UV-visible absorption spectra of CDs, CDs-SA and CDs-SA-PTX are shown in Figure 2. CDs has an absorption peak at 440 nm, and the absorption peaks of CDs-SA and CDs-SA-PTX at 440 nm are weakened but the peak positions remain unchanged. The fluorescence spectra of CDs, CDs-SA and CDs-SA-PTX are shown in Figure 2. Figure 5As shown in the figure, at the maximum excitation wavelength of 440 nm, CDs exhibited its maximum emission wavelength, i.e., 590 nm. After modification with SA, the fluorescence intensity of CDs-SA decreased, and after PTX loading, the fluorescence intensity decreased further. Both the UV spectrum and fluorescence spectrum results indicate the successful modification of SA and the successful loading of PTX.
[0025] The FT-IR spectra of CDs, CDs-SA and CDs-SA-PTX are shown in Figure 6 As shown, CDs at 3434 cm -1 、1636 cm -1 、1567 cm -1 、1497 cm -1 and 1328 cm -1 There are characteristic absorption peaks at 3434 cm -1 The characteristic peak at 1636 cm is attributed to the stretching vibration of OH. -1 and 1567 cm -1 The characteristic peaks at 1497 cm are caused by the stretching vibrations of the CH bond, C=O bond, and NH bond. -1 and 1328 cm -1 The characteristic peaks appearing at 1246 cm-1 are related to the C=C bond and CN stretching vibration. -1 and 1031 cm -1 There is a characteristic absorption peak at 1246 cm -1 and 1031 cm -1 The characteristic absorption peak at 1246 cm -1 The characteristic band at 1031 cm is due to the stretching vibration of the OH bond. −1 The characteristic peak of CDs-SA-PTX belongs to C−O stretching vibration. -1 There is a characteristic peak at , which is attributed to the stretching vibration of C=O in PTX. From the potential diagrams of CDs, CDs-SA and CDs-SA-PTX ( Figure 7 ), the potential of CDs is 7.7 mV, the potential of CDs-SA is 13.1 mV, the potential of PTX is -4.3 mV, and the potential of CDs-SA-PTX is 6.7 mV, indicating that PTX can be adsorbed on the surface of CDs-SA by electrostatic adsorption.
[0026] 2. Determination of drug loading Solutions with a PTX mass to CDs-SA mass ratio of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, and 1.75:1 were prepared for drug loading experiments. 5 mg of CDs-SA was added to each concentration of PTX solution (5 mL) and mixed and stirred overnight. After the mixed solution was centrifuged and the precipitate was washed with ethanol 3-4 times (10 min, 10000 rpm), the supernatant was collected and its absorbance value A at 230 nm was measured. According to the drawn PTX standard curve, the concentration of PTX in the supernatant was calculated. The calculation formula of drug loading (DLC) is as follows: Drug loading capacity (DLC) = (mass of loaded drug / total mass of nanocomplex) × 100%.
[0027] Depend on Figure 8 It can be seen that the mass of CDs-SA remains unchanged, and the drug loading gradually increases with the increase of PTX mass. When the mass of PTX and CDs-SA is 1:1, the drug loading reaches the maximum. As the mass of PTX continues to increase, the drug loading gradually decreases. Therefore, the drug loading ratio of 1:1 is selected as the optimal ratio for subsequent experiments.
[0028] 3. Release of drug PTX The pH responsiveness of CDs-SA-PTX was studied. The release rates of PTX at pH 7.4 and 5.5 were shown in Figure 2. Fig. 9 As shown in the figure, within 36 hours, the release rate of PTX in PBS at pH 7.4 was only 24.2%, but the release rate in pH 5.5 was 65.7%. Compared with pH 5.5, PTX had no obvious release behavior at pH 7.4, indicating that the release of PTX was affected by pH. The reason for this phenomenon may be that under acidic conditions, the unmodified amino groups of CDs-SA were protonated, and the electrostatic attraction to PTX decreased, so the drug binding affinity was weakened, which promoted drug release.
[0029] 4. In vitro photothermal performance measurement of CDs-SA-PTX nanocomplex The photothermal effect of CDs-SA-PTX nanocomposites was measured using a 660 nm laser and a thermocouple microprobe (accuracy 0.1°C).
[0030] Fig.10(a) shows the photothermal performance of CDs-SA-PTX with different concentrations under laser irradiation (660 nm, 1.5 W). The temperature of CDs-SA-PTX with different concentrations rises to 34.6°C, 38.7°C and 45.7°C, respectively. The temperature of CDs-SA-PTX solution increases with the concentration, indicating that CDs-SA-PTX is concentration-dependent. Fig.10 (b) It can be seen that the temperature of CDs-SA-PTX solution increases with the increase of 660 nm laser power. Fig.11 The photothermal effects of H2O, CDs, CDs-SA and CDs-SA-PTX were studied. H2O was used as the control group, and its temperature rose to 29.8℃ within 10 min, while the final temperature of the CDs-SA-PTX group solution was 45.7℃, which could induce tumor cell death, indicating that CDs-SA-PTX has a good photothermal effect.
[0031] Fig.12 Figure 2 is the photothermal imaging of H2O, CDs, CDs-SA and CDs-SA-PTX. The temperature rise of CDs, CDs-SA and CDs-SA-PTX under laser irradiation is basically the same, indicating that the CDs-SA-PTX nanocomposite has excellent photothermal imaging ability.
[0032] Fig.13 The photothermal stability of CDs-SA-PTX was investigated. The results showed that after 5 heating and cooling cycle experiments, the maximum temperature of the CDs-SA-PTX solution remained unchanged, proving its good photothermal stability.
[0033] From the picture Fig.14 (a) and Figure 2-14 (b), we can get the system time constant (τ s ) is 278. The calculated photothermal conversion efficiency of CDs-SA-PTX nanocomposite is 43.8%, indicating that it has strong light absorption ability and good photothermal performance.
[0034] 5. Cell experiments of CDs-SA-PTX nanocomplexes 5.1 Cytotoxicity assay The biocompatibility of CDs-SA-PTX was evaluated by MTT assay. Fig.15 The dark toxicity of CDs-SA to MCF-7 cells and HUVEC cells was shown. After incubation with 300 μg / mL CDs-SA, the cell survival rate of MCF-7 cells and HUVEC cells was still above 85%, indicating that CDs-SA had no toxic effect on MCF-7 cells and HUVEC cells and exhibited good biocompatibility.
[0035] In order to evaluate the therapeutic effects of different materials, the MTT method was used to determine the survival rate of MCF-7 cells. Fig.16 It can be seen that when MCF-7 cells were co-incubated with PTX or CDs-SA-PTX alone, the cell viability was 33.9% and 30.1%, respectively, while when CDs-SA-PTX was incubated with 660 nm laser irradiation, the cell viability dropped to 13.7%, indicating that the strategy of combined chemotherapy and photothermal therapy has an excellent killing effect on MCF-7 cells. 5.2 Cell uptake experiment Laser confocal microscopy was used to observe the uptake of PBS, CDs, CDs-SA, and CDs-SA-PTX by MCF-7 cells. Fig.17 As shown, DAPI is used for nucleus localization, showing blue fluorescence, and CDs shows orange fluorescence. At an emission wavelength of 590 nm, the CDs orange fluorescence signal mainly appears in the nucleus and nucleolus, and the fluorescence intensity in the nucleolus is higher than that in the nucleus, indicating that CDs has nucleolar targeting ability. In the CDs-SA-PTX group, it can be observed that the fluorescence distribution is uniform, and the fluorescence intensity in the nucleolus is significantly higher than that in the cytoplasm and nucleus. This result shows that CDs-SA-PTX still has excellent nucleolar targeting ability and that cells have good uptake ability.
[0036] 5.3 Nuclease digestion experiment of CDs The specific targeting ability of CDs to the nucleolus was investigated by nucleic acid enzymatic hydrolysis experiments. MCF-7 cells were treated with PBS, RNase, and DNase. Fig.18 It can be seen that the fluorescence intensity of the nucleolus after DNase treatment is still stronger than that of the cell nucleus. After RNase treatment, the fluorescence is evenly distributed in the cell nucleus, and the fluorescence intensity of the nucleolus is not prominent, indicating that CDs can specifically bind to RNA after entering the nucleolus.
[0037] 5.4 Live and dead cell staining experiment The therapeutic effect of CDs-SA-PTX nanocomplex on MCF-7 cells was evaluated by Calcein-AM / PI live cell / dead cell double staining method. MCF-7 cells were co-stained with Calcein-AM (for marking live cells) and PI (for marking dead cells). The results are shown in Fig.19, green fluorescence indicates live cells, and red fluorescence indicates dead cells. The PBS group, NIR group, and CDs-SA group all showed only green fluorescence signals, indicating that no cell death occurred. Free PTX and CDs-SA-PTX without laser irradiation showed obvious red and green fluorescence signals, indicating partial cell death. Only red fluorescence was observed in the CDs-SA-PTX+NIR group, indicating that chemotherapy and photothermal effects caused complete cell death. The results showed that CDs-SA-PTX has the advantage of combined therapy in vitro.
[0038] 5.5 Cell apoptosis assay Flow cytometry was used to analyze cell apoptosis. Fig. 20 The results showed that there was almost no cell apoptosis in the PBS group, NIR group and CDs-SA group, while after treatment with CDs-SA-PTX+NIR, the apoptosis rate of MCF-7 cells was significantly higher than that in the CDs-SA+NIR group, and even higher than that in the free PTX and CDs-SA-PTX groups, indicating that the strategy of combined chemotherapy and photothermal therapy showed a strong ability to kill cancer cells.
[0039] 6. Animal model experiments of CDs-SA-PTX nanocomplexes Six-week-old Balb / c female mice were selected for model mouse establishment. The right armpit of the mice was first depilated, and then 4T1 cells were inoculated into the right armpit of the mice. The health status and tumor size of the mice were observed every day. When the tumor volume grew to 100 mm 3 In vivo experiments were performed in mice.
[0040] 6.1 Fluorescence imaging of mice The biodistribution behavior of CDs-SA-PTX in vivo was evaluated. Fig.21 (a) It can be seen that 6 hours after tail vein injection, fluorescence accumulation began to appear in the tumor site. At 9 hours, the fluorescence of the tumor site reached the maximum. However, as time went by, the fluorescence of the mouse tumor site gradually weakened, while the fluorescence of the liver site began to increase, indicating that CDs-SA-PTX began to be metabolized from the liver. At 9 hours, the main organs and tumor tissues of the mice were dissected for fluorescence imaging ( Fig.21 (b) The results showed that the fluorescence signal was mainly concentrated in the tumor and liver. The above results prove that CDs-SA-PTX has low biological toxicity and strong tumor targeting ability.
[0041] 6.2 Photothermal imaging of mice Fig. 22This is a photothermal image of mice recorded by a near-infrared imager. Within 10 minutes of laser irradiation, the temperature of the tumor site in the Saline group did not increase significantly. Compared with the CDs group, the CDs-SA group can be more effectively enriched at the tumor site, indicating that the modification of SA makes CDs-SA tumor-targeted. At the same time, the color of the tumor site in the CDs-SA-PTX group turned red, and the imaging effect was good. The above results confirm that CDs-SA-PTX can be thermally ablated in vivo and has good photothermal imaging effects.
[0042] 6.3 In vivo tumor treatment effects Fig.23 (a) and Fig.23 (b) Photos of mice in each group after treatment and tumors. The tumor sizes of the Saline group, NIR group, and CDs-SA group were almost the same, indicating that NIR and CDs-SA had no therapeutic effect on mouse tumors. The tumor sizes of the CDs-SA+NIR, PTX, and CDs-SA-PTX groups became smaller, and the tumors of the CDs-SA-PTX+NIR group almost completely disappeared, indicating that the combined chemotherapy and photothermal therapy was effective. The average weight of mice after 14 days of treatment is shown in Figure 2. Fig.23 As shown in (c), the body weight of mice in each group increased slightly within 15 days, demonstrating that the CDs-SA-PTX nanocomplex had no systemic toxicity. Fig.23 (d) is the average weight of tumors in each group after 14 days of treatment. Fig.23 (e) is the relative tumor volume of mice. The tumor volumes of the Saline group, NIR group, and CDs-SA group were 8.3 times, 8.1 times, and 8.4 times of the initial volume, respectively, indicating that single laser irradiation and CDs-SA incubation did not inhibit tumor growth. The tumor volumes of the CDs-SA+NIR group, PTX group, and CDs-SA-PTX group were 4.3 times, 2.4 times, and 2.2 times of the initial volume, respectively, indicating that single photothermal therapy or single chemotherapy had a certain inhibitory effect on tumors. The tumor in the CDs-SA-PTX+NIR group shrank to 0.6 times of the initial volume after 14 days, confirming that the CDs-SA-PTX+NIR group had an excellent combined chemotherapy and photothermal inhibition of tumor growth.
[0043] 6.4 Biosafety Assessment The biocompatibility of CDs-SA-PTX nanocomplexes was evaluated by blood routine examination, biochemical indices and HE staining. The analysis results of blood routine examination (WBC, RBC, PLT, MCH, MCV, MCHC, HCT, HGB) and blood biochemical indices (ALT, AST, ALB, TP, BUN, CRE) of mice in each group are shown in Figure 2. Fig.24 and Fig.25As shown, there were no significant differences in the parameters among the groups, indicating that CDs-SA-PTX had no side effects on the blood, liver, and kidneys.
[0044] Fig.26 HE staining results of the heart, liver, spleen, lung, kidney and tumor tissues of mice. Compared with the Saline group, the shape, size and degree of necrosis of tumor cells in the NIR group and CDs-SA group did not change significantly. The morphology of tumor cells in the CDs-SA+NIR group, PTX group and CDs-SA-PTX group changed to a certain extent, and the CDs-SA-PTX+NIR group showed the most significant cell lysis and loss of cell morphology. The cell structure and morphology of the main organs (heart, liver, spleen, lung, kidney) of each group were normal. This result shows that nanocomposites can damage tumor cells without obvious toxicity to the main organs.
[0045] In summary, the CDs-SA-PTX obtained by the above implementation method exhibits good photothermal conversion efficiency and nucleolar targeting. In vitro cytotoxicity tests showed that compared with free PTX, CDs-SA-PTX after 660 nm laser irradiation had a higher killing efficiency on MCF-7 cells. The results of mouse fluorescence and photothermal imaging showed that CDs-SA-PTX can be efficiently enriched in tumor tissues. The results of mouse blood routine, biochemistry and HE staining proved that CDs-SA-PTX has excellent biosafety.
Claims
1. A CDs-SA-PTX nanocomposite, characterized in that: The invention comprises paclitaxel (PTX) and CDs-SA, wherein the CDs-SA is prepared by modifying sialic acid (SA) on the surface of carbon dots (CDs) through an amide reaction; and paclitaxel (PTX) is loaded on the CDs-SA.
2. The CDs-SA-PTX nanocomposite according to claim 1, characterized in that: The mass ratio of paclitaxel (PTX) to CDs-SA is 1:
1.
3. The method for preparing the CDs-SA-PTX nanocomposite according to claim 1 or 2, characterized in that: The following steps are involved: Step 1, preparing CDs-SA; Sialic acid (SA) was dissolved in PBS solution to obtain SA solution, NHS and EDC were added to the SA solution, and the solution was stirred at room temperature to obtain an activated SA solution; the dissolved CDs were mixed with the activated SA solution, and the solution was stirred at room temperature to obtain a solution, and then dialyzed to obtain CDs-SA; Step 2, preparing CDs-SA-PTX; The CDs-SA obtained in step 1 is added to the ethanol solution of paclitaxel (PTX), and the mixture is stirred to obtain a mixed solution. The mixed solution is centrifuged, washed, and dried to obtain a CDs-SA-PTX solid.
4. The method for preparing the CDs-SA-PTX nanocomposite according to claim 3, characterized in that: In step 1, 20 mg of sialic acid (SA) was weighed and dissolved in 10 mL of PBS solution with a pH of 7.
4. 18 mg of NHS and 18 mg of EDC were added to the SA solution and stirred at room temperature for 15 min to obtain an activated SA solution. 20 mg of freeze-dried CDs solid was dissolved in 10 mL of deionized water. After complete dissolution, it was mixed with the activated SA solution and stirred at room temperature for 12 h. The obtained solution was dialyzed to obtain CDs-SA.
5. The method for preparing the CDs-SA-PTX nanocomposite according to claim 3 or 4, characterized in that: The CDs described in step 1 are obtained by dissolving 1,2,4-triaminobenzene dihydrochloride in deionized water and then adding triethylenetetramine to carry out hydrothermal reaction.
6. The method for preparing the CDs-SA-PTX nanocomposite according to claim 5, characterized in that: In step 1, 196 mg of 1,2,4-triaminobenzene dihydrochloride was weighed and dissolved in deionized water, and ultrasonication was performed to completely dissolve it. 1.2 mL of triethylenetetramine was added, and the obtained solution was transferred to a polytetrafluoroethylene-lined autoclave. The autoclave was placed in an oven and the temperature was set to 90 ° C. The reaction was carried out for 12 h, and the reaction was naturally cooled to room temperature to obtain a dark brown liquid. The dark brown liquid was centrifuged and the supernatant was collected, and then filtered using a 0.22 μm microporous filter membrane, and finally dialyzed to obtain a brown solution. The obtained solution was freeze-dried to obtain CDs.
7. The method for preparing the CDs-SA-PTX nanocomposite according to claim 3 or 6, characterized in that: In step 2, PTX is dissolved in ethanol to prepare PTX solutions of different concentrations, and 10 mg of CDs-SA is added to the PTX solutions of different concentrations and stirred overnight. The mixed solution is centrifuged to collect the precipitate, which is washed with ethanol 3-4 times and freeze-dried to obtain CDs-SA-PTX solid.
8. Use of the CDs-SA-PTX nanocomposite according to claim 1 or 2 in the preparation of a drug for combined photothermal / chemotherapy treatment of breast cancer.