Superficial tumor targeted multi-mode diagnosis and treatment nanoprobe, preparation method and application
By designing a superficial tumor-targeted multimodal nanoprobe containing PLGA shell, photosensitizer, magnetic resonance imaging agent and targeting molecules, the problems of early diagnosis and treatment of superficial tumors such as breast cancer are solved, and high-precision diagnosis and effective therapeutic effects are achieved.
Patent Information
- Application Number
- CN202510051434.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art has the challenges of difficulty in the early diagnosis and treatment of superficial tumors such as breast cancer, which are difficult to diagnose accurately, have great side reactions, and lack of efficient treatment.
A superficial tumor-targeted multimodal diagnosis and treatment nanoprobe was designed, and photosensitizers were encapsulated with PLGA shells, and copper diethyldithiocarbamate was embedded on the shells and chitosan-targeted molecular coatings were achieved to achieve photoacoustic-fluorescence-magnetic resonance trimodal imaging and chemotherapy effects.
It improves the bioavailability and selectivity of drugs, reduces side effects, achieves high-precision diagnosis and effective treatment effects of early tumors, and has the ability to integrate diagnosis and treatment.
Smart Images
Figure CN119818672A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of nanoprobes, and in particular to a superficial tumor targeted multimodal diagnosis and treatment nanoprobe, a preparation method and an application thereof. Background Art
[0002] Breast cancer is a type of superficial tumor. Breast cancer has become one of the most common cancers in the world. Although the clinical diagnosis and treatment techniques for breast cancer are becoming increasingly sophisticated, early-stage lesions still face arduous challenges in terms of accurate diagnosis, reduction of side effects, and efficient treatment.
[0003] Nanomedicine is a new field of medicine that uses nanomaterials to diagnose and treat diseases at the molecular level. Nanomedicine technology can achieve a variety of biological functions by designing different sizes and shapes for nanomaterials, changing the chemical composition and surface characteristics of nanomaterials, and regulating the optical, electronic, magnetic and other properties of nanomaterials. By combining targeting molecules with nanomaterials, nanomaterials can be given specific recognition properties, so that targeted nanoprobes can accurately deliver imaging agents and drugs to the lesion site, significantly improving diagnostic specificity and enhancing therapeutic effects.
[0004] Although nanodrug delivery systems currently have the advantages of low invasiveness, high drug utilization efficiency and few side effects, single nanomedicine treatment modes, such as chemotherapy, photothermal therapy, photodynamic therapy, sonodynamic therapy or immunotherapy, cannot be carried out at the same time and often fail to achieve effective treatment results.
[0005] Multimodal imaging technology provides rich imaging information and, to a certain extent, makes up for the shortcomings of each imaging method and evaluates tumor conditions from multiple angles. Combining nano-targeting technology with multimodal imaging technology can detect tumor lesions earlier, improve the diagnostic efficiency of imaging, diagnose diseases more accurately, and start treatment in a timely manner.
[0006] Based on the above nanomedicine technology, multimodal imaging technology and the shortcomings of existing nanodrug delivery systems, the inventor used this as motivation for research and development, and designed a targeted multimodal diagnostic and therapeutic nanoprobe for superficial tumors (such as breast cancer), as well as a preparation method and application, and therefore applied for a patent for protection. Summary of the invention
[0007] The present invention aims to provide a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe, a preparation method and an application thereof, for the diagnosis and treatment of superficial tumors (such as breast cancer) to improve the diagnostic and therapeutic effects.
[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a superficial tumor targeted multimodal diagnostic and therapeutic nanoprobe, comprising a portion of photosensitizer, the outer side of which is wrapped with a PLGA shell, the PLGA shell is inlaid with copper diethyldithiocarbamate, the outer side of the PLGA shell is wrapped with a chitosan-targeting molecule coating, and the chitosan-targeting molecule coating contains another portion of photosensitizer.
[0009] The principle and advantage of the scheme of the present application are: the nanoprobe in the present application has a chitosan-targeting molecule coating, the chitosan-targeting molecule coating includes chitosan and targeting molecules, chitosan is located on the side of the chitosan-targeting molecule coating close to the PLGA shell, and the targeting molecule is located on the side of the chitosan-targeting molecule coating away from the PLGA shell, chitosan is adhesive and positively charged, and chitosan adsorbs the targeting molecule on the surface of the nanoprobe by adsorption of positive and negative charges, so that the targeting molecule is more stably located on the surface of the nanoprobe. For the targeting molecule, the targeting molecule itself can specifically recognize superficial tumor cells with high expression of CD44 and has high biocompatibility and non-immunogenicity, thereby giving the nanomaterial tumor targeting and relatively prolonging the in vivo circulation time of the nanomaterial.
[0010] At the same time, the nanoprobe contains copper diethyldithiocarbamate, which is itself a chemotherapy drug, thus being able to treat superficial tumor cells.
[0011] In addition, copper diethyldithiocarbamate not only has a chemotherapeutic effect, but can also give the nanoprobe magnetic resonance imaging and photoacoustic imaging capabilities; and the photosensitizer in the nanoprobe in the present application gives the nanoprobe photoacoustic imaging and fluorescence imaging capabilities, so that the entire nanoprobe can achieve photoacoustic-fluorescence-magnetic resonance triple-modal imaging, thereby having the ability of integrated diagnosis and treatment.
[0012] The nanoprobe of the present application is an integrated nanoprobe for effective diagnosis and treatment of superficial tumors, which has biocompatibility, photoacoustic-magnetic resonance-fluorescence tri-modal imaging characteristics, tumor targeting characteristics, photothermal warming characteristics, and chemotherapy killing characteristics.
[0013] Compared with the prior art, the nanoprobe of the present application has the following beneficial effects: 1. Compared with traditional chemotherapy, nanoprobes based on nanomedicine technology improve the bioavailability and selectivity of drugs and reduce side effects.
[0014] 2. In terms of early tumor screening, multimodal imaging provides rich imaging information, and nanoprobes with targeted functions specifically accumulate at the tumor site, which can detect more hidden cancer lesions.
[0015] 3. It has the function of precision medicine. The probe has the ability of photothermal heating and effective chemotherapy drug release, and can achieve effective cancer cell killing through targeted aggregation.
[0016] 4. Cell pyroptosis is a form of programmed cell death, and the photosensitizer in the nanoprobe in this application has a photothermal effect, while chemotherapy plays a certain auxiliary role. The combined effect of photothermal and chemotherapy leads to the pyroptosis of tumor cells.
[0017] In addition, it is necessary to explain that at the beginning of the study, the inventor's original intention was to encapsulate the photosensitizer inside the nanoprobe to achieve the purpose of the nanoprobe having the ability of photoacoustic imaging and fluorescence imaging, and to adsorb the targeting molecule by arranging chitosan on the nanoprobe so that the nanoprobe has the ability of targeting. However, due to the use of the preparation method in this application, the inventor did not expect that during the preparation process, chitosan not only electrostatically adsorbs the targeting molecule, but also electrostatically adsorbs the photosensitizer that is not encapsulated inside the nanoprobe, so that the chitosan-targeting molecule coating also has a part of the photosensitizer. After the photosensitizer is adsorbed on the chitosan-targeting molecule coating, the photosensitizer on the nanoprobe has two parts, one part is located inside the nanoprobe, and the other part is located on the chitosan-targeting molecule coating of the nanoprobe, so that the encapsulation rate and loading rate of the photosensitizer are significantly improved compared to only being located inside the nanoprobe, thereby making the nanoprobe photoacoustic imaging and fluorescence imaging capabilities, and the ability to cause cell pyroptosis more significant.
[0018] Preferably, as an improvement, the encapsulation efficiency of the photosensitizer is 50-60%. Thus, the encapsulation efficiency of the nanoprobe without the chitosan-targeting molecule coating (the photosensitizer is only located inside the nanoprobe) is about 9%, and the encapsulation efficiency is increased by 5-6 times, and the encapsulation efficiency of the photosensitizer in the present application is significantly improved.
[0019] Preferably, as an improvement, the loading rate of the photosensitizer is 6-8%. Thus, for the nanoprobe without chitosan-targeting molecule coating (the photosensitizer is only located inside the nanoprobe), the loading rate of the photosensitizer is about 1%, and the loading rate is increased by 6-8 times, and the loading rate of the photosensitizer in the present application is significantly increased.
[0020] Preferably, as an improvement, the photosensitizer is indocyanine green or IR780 photosensitizer.
[0021] Preferably, as an improvement, the targeting molecule is hyaluronic acid, and the chitosan-targeting molecule coating is a chitosan-hyaluronic acid coating.
[0022] To achieve the above object, the present invention also adopts the following technical solution: a method for preparing a superficial tumor targeted multimodal diagnosis and treatment nanoprobe, comprising the following steps: S1. Preparing a primary emulsion: mixing PLGA, copper diethyldithiocarbamate and dichloromethane, and dissolving the mixture using an ultrasonic oscillator, then adding an aqueous solution of ICG to the mixture and emulsifying the mixture to obtain a primary emulsion; S2, preparing a double emulsion; adding a polyvinyl alcohol solution and a chitosan solution to the primary emulsion, and emulsifying to obtain a double emulsion; S3, then adding isopropanol and magnetic beads to the double emulsion, and stirring magnetically to evaporate the dichloromethane to obtain IC@PC NPs; S4, washing the IC@PC NPs by high-speed centrifugation for multiple times, resuspending them in pure water, adding hyaluronic acid, and mixing them to obtain IC@PCH NPs; S5. Finally, the IC@PCH NPs were washed, resuspended in pure water, and stored at 4 °C in the dark.
[0023] Thus, the preparation method of the present application realizes the preparation of the superficial tumor targeted multimodal diagnosis and treatment nanoprobe in the present application. In addition, in S2, the negatively charged ICG that is not encapsulated in the nanoprobe is attracted by the positively charged chitosan and loaded on the surface of the nanoprobe, which significantly improves the encapsulation rate and loading rate of the indocyanine green photosensitizer on the nanoprobe.
[0024] Preferably, as an improvement, in S2, the concentration of the polyvinyl alcohol solution is 5%, the concentration of the chitosan solution is 2%; and the volume ratio of the chitosan solution to the polyvinyl alcohol solution is 1:1.
[0025] In this preparation method, the ratio of polyvinyl alcohol solution and chitosan solution is particularly important. When polyvinyl alcohol is more and chitosan is less, the prepared IC@PC NPs do not have a positive charge, so they cannot adsorb hyaluronic acid or indocyanine green. When polyvinyl alcohol is less and chitosan is more, the surface tension will increase and the nanoprobe will not be able to form a ball. After adjusting the dosage many times, the inventor found that when the polyvinyl alcohol solution and the chitosan solution are in the above ratio, IC@PCNPs with a positive charge on the surface can be obtained, so that hyaluronic acid can be adsorbed and indocyanine green can be adsorbed, and the nanoprobe in this application can be obtained, and the indocyanine green encapsulation rate and loading rate have the unexpected effect of significantly improving.
[0026] Preferably, as an improvement, the chitosan solution is chitosan dissolved in 3% acetic acid.
[0027] Preferably, as an improvement, the high-speed centrifugation speed is 9000-11000 rpm / min.
[0028] In order to achieve the above-mentioned purpose, the present invention also adopts the following technical solution: application of a superficial tumor targeted multimodal diagnostic and therapeutic nanoprobe in an imaging agent.
[0029] Since the nanoprobe in the present application has the triple-modal imaging effects of photoacoustic-fluorescence-magnetic resonance, it can be used as an imaging agent.
[0030] In order to achieve the above-mentioned purpose, the present invention also adopts the following technical solution: application of a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe in drugs for treating superficial tumors.
[0031] Using the nanoprobes in the present application for drugs to treat superficial tumors can significantly improve the therapeutic effect of superficial tumors, and the therapeutic drugs have the advantages of high diagnostic accuracy and few side effects.
[0032] Preferably, as a refinement, the superficial tumor is breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 The preparation process and internal structure of the IC@PCH nanoprobe of the present invention are shown.
[0034] Figure 2 Shown are (A) scanning electron microscopy images of IC@PCH NPs and (B) transmission electron microscopy images of IC@P NPs and (C) IC@PCH NPs.
[0035] Figure 3 Schematic diagram showing the (A) particle size and potential, (B) UV spectrum, (C) IR spectrum, and (D) X-ray diffractometer of the IC@PCH nanoprobe.
[0036] Figure 4 Photos of four nanoparticle solutions.
[0037] Figure 5 is the standard curve of ICG.
[0038] Figure 6 Shown are the photothermal heating curves of (A) IC@PCH NPs and (B) IC@P NPs, and (C) the temperature curves of IC@PCH NPs under four heating and cooling cycles.
[0039] Figure 7 Schematic diagram of in vitro (A) photoacoustic / (B) magnetic resonance / (C) fluorescence imaging.
[0040] Figure 8 Schematic diagram showing the phagocytosis experiment.
[0041] Fig. 9 Schematic diagram showing the in vitro safety and killing experiments.
[0042] Fig.10 Shown are in vivo (A) photoacoustic / (B) magnetic resonance / (C) fluorescence imaging.
[0043] Fig.11 Schematic diagram showing the photothermal conversion capability in vivo.
[0044] Fig.12 Schematic diagram showing in vivo treatment and safety.
[0045] Fig.13 This is a photo of 4T1 cells under an optical microscope after IC@PCH NPs+laser treatment.
[0046] Fig.14 Figure 3 shows (A) heat map of differentially expressed genes between the control group and IC@PCH NPs+laser group; (B) representative KEGG pathways of significantly differentially expressed genes between the control group and IC@PCH NPs+laser group.
[0047] Fig.15 Shown are (A) schematic diagram of the mechanism of photothermal chemotherapy-induced pyroptosis; (B) expression levels of NLRP3, Caspase-1, cleaved Caspase-1, GSDMD, and GSDMD-N in 4T1 cells; (C) quantitative analysis of (B); n=3.
[0048] Fig.16 (A) When the dosage of PVA and CS is appropriate, the digital photo (left) and scanning electron microscope image (right) of IC@PCH NPs solution; (B) When the dosage of PVA is less and CS is more, the digital photo (left) and upper and lower layer optical microscope images (right) of IC@PCH NPs solution; (C) When the dosage of PVA is more and CS is less, the Zeta potential of IC@PC NPs and IC@PCH NPs. DETAILED DESCRIPTION
[0049] The following is further described in detail through specific implementation methods: This embodiment discloses a superficial tumor (superficial tumors such as breast cancer, but not limited to breast cancer, such as prostate cancer, pharyngeal cancer cells) targeted multimodal diagnosis and treatment nanoprobe, including a portion of photosensitizer, the outside of which is wrapped with a PLGA shell, the PLGA shell is spherical, the PLGA shell is inlaid with copper diethyldithiocarbamate, the outside of the PLGA shell is wrapped with a chitosan-targeting molecule coating, and the chitosan-targeting molecule coating contains another portion of photosensitizer. The chitosan-targeting molecule coating in this embodiment includes chitosan and a targeting molecule, and the chitosan is closer to the PLGA shell than the targeting molecule.
[0050] The photosensitizer in this embodiment is specifically indocyanine green for illustration. Of course, in other embodiments, it can also be other photosensitizers that can play a role in photothermal conversion, photoacoustic imaging, and fluorescence imaging, such as IR780 and other photosensitizers. In this embodiment, the targeting molecule is specifically hyaluronic acid for illustration, and the chitosan-targeting molecule coating is specifically chitosan-hyaluronic acid coating. Of course, in other embodiments, other targeting molecules that can target tumors can also be used, such as folic acid that can target folate receptors and mannose that can target mannose receptors.
[0051] Combination Figure 1 As shown, this embodiment also discloses a method for preparing a superficial tumor targeted multimodal diagnosis and treatment nanoprobe, comprising the following steps: S1. Preparing primary emulsion: 50 mg of PLGA, 5 mg of copper diethyldithiocarbamate (CuET) and 2 mL of dichloromethane were added to a 50 mL centrifuge tube, mixed, and treated with an ultrasonic oscillator for 30 s to dissolve the mixture. Subsequently, 200 μL of an aqueous solution of indocyanine green (ICG) (15 mg / mL) was added to the mixture, and an ultrasonic sonicator (VCX130; Sonics and Materials) was used to emulsify the mixture at a power of 100 W for 3 min to obtain a primary emulsion; S2, preparing a double emulsion; adding 4 mL of 5% polyvinyl alcohol (PVA) solution and 4 mL of 2% chitosan (CS) solution (the chitosan solution in this embodiment is dissolved in 3% acetic acid) to the primary emulsion, and emulsifying for 2 minutes at a power of 50 W using an ultrasonic sonicator to obtain a double emulsion; S3, then the emulsion was transferred to a 50 ml beaker, and 10 ml of isopropanol and magnetic beads were added, and magnetic stirring was performed for 3 h to evaporate the dichloromethane to obtain formed IC@PC NPs; S4, washing IC@PC NPs by high-speed centrifugation (centrifugal speed is 9000-11000 rpm / min, preferably 10000 rpm / min, 10 min in this embodiment) for 3 times, resuspending in pure water, adding 10 mg hyaluronic acid (HA), placing on a shaker and mixing thoroughly for 1 h, to obtain IC@PCH NPs; S5. Finally, the IC@PCH NPs were washed, resuspended in pure water, and stored in a refrigerator at 4 °C away from light.
[0052] In addition, this embodiment also discloses a method for preparing IC@P NPs, comprising the following steps: S1. Preparing primary emulsion: 50 mg of PLGA, 5 mg of copper diethyldithiocarbamate and 2 mL of dichloromethane were added to a 50 mL centrifuge tube, and the mixture was treated with an ultrasonic oscillator for 30 s to dissolve the mixture. Subsequently, 200 μL of an aqueous solution of indocyanine green (ICG) (15 mg / mL) was added to the mixture, and an ultrasonic sonicator (VCX130; Sonics and Materials) was used to emulsify the mixture at a power of 100 W for 3 min to obtain a primary emulsion; S2, preparing a double emulsion; adding 8 mL of 5% polyvinyl alcohol (PVA) solution to the primary emulsion, and emulsifying for 2 minutes at a power of 50 W to obtain a double emulsion; S3, then the emulsion was transferred to a 50 ml beaker, and 10 ml of isopropanol and magnetic beads were added, and magnetic stirring was performed for 3 h to evaporate the dichloromethane to obtain formed IC@P NPs; S4. Wash the IC@P NPs three times by high-speed centrifugation (centrifugal speed 10000 rpm / min, 10 min), resuspend in pure water, and store in a refrigerator at 4 degrees Celsius away from light.
[0053] In this embodiment, for IC@PCH NPs, IC@PC NPs and IC@P NPs, I represents ICG, C before @ represents CuET, P represents PLGA, C after @ represents CS, and H represents HA.
[0054] Experimental Section A series of experiments are conducted below to demonstrate the relevant performances, advantages and effects of the IC@PCH NPs in this application.
[0055] 1. Characterization of physical and chemical properties Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to detect the morphological characteristics of IC@P and IC@PCH nanoprobes obtained by the above method. Figure 2 As shown in A, the SEM image shows that IC@PCH NPs show a spherical shape with a smooth surface and uniform particle size. Figure 2 B) Compared with IC@PCH NPs ( Figure 2 C) The halo on the surface is thicker, proving that HA / CS modification is successful.
[0056] Particle size potential instrument results ( Figure 3A) shows that IC@P NPs are about 219.8 nm, and after loading CS and HA, the size increases to 248.4 nm and 259.2 nm, which is consistent with the TEM results. In aqueous solution at pH = 7.4, the ζ charge of IC@P NPs is -17.67 mV, which increases to 21.57 mV after loading CS and reverses to -16.17 mV after loading HA, indicating that HA is loaded by the method of attraction between positive and negative charges.
[0057] The drug loading performance of IC@PCH NPs was analyzed using UV-visible spectrophotometer. The results showed that IC@PCH NPs had obvious absorption peaks at 263nm, 545nm and 699nm, which was the result of successful loading of ICG and CuET ( Figure 3 B). In addition, combined Figure 3 As shown in Figure C, the Fourier transform infrared spectrum of IC@PCH NPs shows a stretching vibration peak of the C=S double bond at 1020-1200cm-1, which is the characteristic absorption peak of CuET, confirming the existence of CuET, while the absorption peak at 1747cm-1 is attributed to the asymmetric stretching vibration absorption peak of the unsaturated group C=O double bond, which comes from PLGA, thus proving the existence of PLGA. Figure 3 As shown in (D), no obvious differences were found in the powder X-ray diffraction patterns of IC@PCH NPs and CuET, which indicated that the loading process did not change the crystal structure of CuET.
[0058] Combination Figure 4 As shown, the inventors used I@P NPs (containing ICG and PLGA), C@P NPs (containing CuET and PLGA), IC@PCH NPs and IC@P NPs for comparison, showing that I@P NPs were green, C@P NPs were brown, and IC@PCH NPs and IC@P NPs were between these two colors, indicating that both IC@PCH and IC@P were successfully loaded with ICG and CuET. Compared with IC@P NPs, IC@PCH NPs were darker green, indicating that the ICG loading amount of IC@PCH NPs was greater than that of IC@P NPs, which could explain that the negatively charged ICG that was not encapsulated in the nanoparticles was attracted by the positively charged IC@PC NPs and loaded on the surface of the nanoparticles.
[0059] The inventors tested the ICG encapsulation efficiency and drug loading of the nanoprobes by preparing ICG standard solutions of different concentrations, measuring the ultraviolet absorbance of the solutions of each concentration at a wavelength of 780 nm using an ultraviolet spectrophotometer, and drawing an ICG 780 nm ultraviolet standard curve (e.g. Figure 5 as shown).
[0060] Because ICG is a water-soluble reagent, the concentration of ICG not encapsulated into nanoparticles in the supernatant was collected and measured, and then the concentration of ICG was calculated according to the Figure 5 The mass of unencapsulated ICG is estimated from the standard curve in. The specific calculation formulas for ICG encapsulation efficiency and drug loading are as follows: EEICG = [(mass of ICG added - mass of ICG in supernatant) / mass of ICG added] × 100% LCICG = [(mass of ICG added - mass of ICG in supernatant) / total mass of nanoparticles] × 100% After calculation, it was found that the encapsulation rate of ICG in IC@P NPs was about 9.13%, and the loading rate was about 1.00%; the encapsulation rate of ICG in IC@PCH NPs was about 52.40%, and the loading rate was about 7.33%. Compared with IC@P NPs, the ICG loading rate of IC@PCH NPs increased from 1.00% to 7.33%, and the encapsulation rate increased from 9.13% to 52.40%. The encapsulation rate and loading rate increased significantly, indicating that due to the setting of the chitosan-hyaluronic acid coating, the negatively charged ICG that was not encapsulated in the nanoparticles was attracted by the positively charged IC@PC NPs and loaded on the surface of the nanoparticles.
[0061] 2. Magnetothermal Heating Characteristics and Magnetothermal Stability Evaluation of IC@PCH NPs The IC@PCH NPs in this application were diluted with double distilled water to different concentrations and tested under 808 nm laser irradiation (1.5 W / cm2).
[0062] like Figure 6 As shown in A, during laser irradiation, the temperature of PBS and different concentrations of IC@PCH NPs (0.5, 1, 2, and 4 mg / mL) increased in a concentration-dependent manner, and the warming effect was obvious.
[0063] At the same time, the photothermal capabilities of IC@PCH NPs and IC@P NPs were compared. Figure 6 As shown in B, the temperature of PBS and different concentrations of IC@P NPs (2, 4, 6, 8 mg / mL) increased in a concentration-dependent manner during 808 nm laser irradiation (1.5 W / cm2). Compared with IC@PCH NPs, IC@P NPs need to reach a higher concentration to produce a similar temperature increase effect, so IC@P NPs have a poorer temperature increase ability. Therefore, compared with IC@P NPs, IC@PCH NPs have higher ICG encapsulation and loading rates, so they have higher therapeutic efficiency and safety.
[0064] like Figure 6As shown in Figure C, the IC@PCH NPs solution was exposed to 1.5 W / cm2 laser four times. The maximum temperature of the red curve in the figure did not decrease significantly, indicating that IC@PCH NPs have good photothermal heating properties and magnetothermal stability and can be used for subsequent treatment.
[0065] 3. In vitro PA / MR / FI imaging capabilities of IC@PCH NPs The inventors verified the in vitro PA / MR / FI imaging capabilities of IC@PCH NPs. Figure 7 As shown in Figure A, the inventors found that different concentrations of IC@PCH NPs produced different intensities of photoacoustic signals in in vitro photoacoustic (PA) imaging, and they were linearly correlated. Figure 7 As shown in B, in magnetic resonance imaging, the intensity of magnetic resonance (MR) signals increases with the increase of Cu2+ concentration, and the signal on the image changes from dark to bright. Figure 7 As shown in Figure C, in fluorescence imaging, within a certain range, the concentration of IC@PCH NPs is proportional to the fluorescence signal intensity. The above results show that IC@PCH NPs have PA / MR / FI multimodal imaging capabilities.
[0066] IV. In vitro targeting of IC@PCH NPs MCF-10A cells (normal breast cells) were co-incubated with IC@PCH-DiI NPs (IC@PCH NPs labeled with DiI), and 4T1 cells (breast cancer cells) were co-incubated with IC@P-DiI NPs (IC@P NPs labeled with DiI) and IC@PCH-DiI NPs, respectively. The concentration of the nanoparticles was 1 mg / mL and the volume was 200 μL. When incubated for 1, 2, and 3 hours, the aggregation of the nanoparticles (red) around the cell nucleus (blue) was observed under a laser confocal microscope. Because the hyaluronic acid-coated nanoprobes are more likely to bind to 4T1 cells overexpressing the CD44 receptor through ligand-receptor interactions, the binding Figure 8 As shown, extensive red fluorescence was observed in the cytoplasm of the 4T1+IC@PCH NPs group, IC@P NPs without hyaluronic acid encapsulation gathered less around 4T1 cells, and very little red fluorescence was observed in MCF-10A cells with low expression of CD44 receptors. These results demonstrate that IC@PCH nanoparticles can target cancer cells with overexpression of CD44 receptors, improving the accuracy of cancer diagnosis and treatment. Since superficial tumors usually have cancer cells with overexpression of CD44 receptors, the nanoprobes in this application can be used not only for breast cancer, but also for other superficial tumors.
[0067] 5. Antitumor effect of IC@PCH NPs combined with photothermal-chemotherapy in vitro like Fig. 9As shown in A, the biosafety of IC@PCH NPs on human umbilical vein endothelial cells (HUVEC), normal breast cells (MCF-10A) and breast cancer cells (4T1) was evaluated by CCK8 method. Different concentrations of nanoprobes were added to each cell group and incubated for 24 hours, and then reacted with CCK8. Finally, the absorbance was measured at a wavelength of 450 nm to calculate the cell survival rate. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD experimental group OD zero well) / (OD negative control group OD zero well) × 100%. After statistical analysis, P>0.05, the difference was not statistically significant, so it was considered that IC@PCH NPs had no cytotoxicity to MCF-10A cells and HUVEC cells, indicating that IC@PCH NPs made of safe and FDA-approved materials are biocompatible.
[0068] like Fig. 9 As shown in B, 4T1 cells were divided into 7 groups, including control group, laser group only, Free CuET group (chemotherapy only), C@PCH NPs (chemotherapy + targeting), I@PCH NPs+laser (photothermal + targeting), IC@P NPs+laser (photothermal + chemotherapy), IC@PCH NPs+laser (photothermal + chemotherapy + targeting), and were treated accordingly, and the cell survival rate was calculated using the CCK8 method. Compared with other groups, the cell survival rate of IC@PCH NPs+laser (photothermal + chemotherapy + targeting) was the lowest, and after statistical analysis, P<0.01, the difference was statistically significant. As can be seen from the figure, repeating the experiment with different concentrations of NPs produced similar results, and the efficacy increased with increasing concentration.
[0069] This shows that IC@PCH NPs have a combined photothermal-chemotherapy effect and have a better therapeutic effect on tumors.
[0070] VI. In vivo PA / MR / FI imaging of IC@PCH NPs like Fig.10 As shown, cancer cells were implanted subcutaneously on the right back of the experimental animals to establish a mouse tumor model. When the tumor volume reached 100-150 mm3, IC@P NPs and IC@PCH NPs solutions (both 5 mg / mL, 200 μL) were injected into the mice through the tail vein. Photoacoustic imaging and MR were used for scanning imaging before and 1h, 2h, 3h, 6h, and 12h after injection, and small animal in vivo photoacoustic imaging was used for scanning imaging before and 0.5h, 1h, 2h, 3h, and 6h after injection. The PA / MR / FI images of the two groups of mice were compared and analyzed.
[0071] like Fig.10As shown in A, the PA signal intensity at the tumor site gradually increased after the injection of the nanoprobe and reached the maximum value at 2 hours, indicating that a large number of IC@P NPs and IC@PCH NPs accumulated at the tumor site. At the same time, the signal intensity of the IC@PCH NPs group was stronger than that of the IC@P NPs group at each time point, so IC@PCH NPs had better tumor targeting and imaging capabilities. Fig.10 As shown in B, the targeting aggregation effect was strongest 2 hours after IC@PCH NPs were injected into mice, and some NPs still remained after 12 hours. Fig.10 As shown in Figure C, the fluorescence signal peaked at 0.5 h after IC@PCH NPs accumulated in tumor tissue, and the fluorescence signal of the IC@PCH NPs group was stronger than that of the IC@P NPs group. It can be seen that the IC@PCH probe combines the advantages of the three imaging modes of PA / MR / FI and has a good prospect for in vivo imaging diagnosis.
[0072] VII. Photothermal conversion capability of IC@PCH NPs in vivo When the tumor volume grew to about 0.5 cm3, the 4T1 breast cancer tumor-bearing mice were randomly divided into 4 groups: PBS + Laser group, IC@P + Laser group, I@PCH + Laser group, and IC@PCH + Laser group. Two hours after the corresponding drugs (5 mg / mL, 200 μL) were injected intravenously into each group of mice, the tumor area was irradiated with 1.5 W / cm2 laser for 6 minutes, and the temperature of the tumor area was monitored by a thermal imaging camera ( Fig.11 The results showed that the temperature of the laser-only group slightly increased to 38.5°C, which was lower than the hyperthermia threshold of 42°C, indicating that 808 nm near-infrared laser was almost harmless to normal tissues and suitable for clinical applications. The temperature of the IC@PCH+ Laser group quickly increased to 53.4°C, indicating that IC@PCH NPs had excellent in vivo thermal efficiency and could effectively kill tumor tissues.
[0073] 8. In vivo therapeutic efficacy and safety of IC@PCH NPs Twenty tumor-bearing mice were randomly divided into five groups: Control group, Laser Only group, C@PCH NPs group, I@PCHNPs + Laser group, and IC@PCH NPs + Laser group. Two hours after tail vein injection (all 5 mg / mL, 200 μL), the tumor was irradiated with 808 nm laser (1.5 W / cm2) for 6 min. The whole course of treatment was only treated once. During the whole treatment cycle, the tumor volume was measured every 2 days ( Fig.12A), it was observed that the tumor growth in the IC@PCH NPs+Laser group was significantly inhibited, and the difference was statistically significant (P<0.05). The tumor inhibition effect of the I@PCH NPs+Laser group and the C@PCH NPs group was relatively mild. As a control, the Control group and the Laser Only group showed a significant increase in tumor volume. The tumor growth inhibition rate was statistically analyzed and found to be P>0.05, indicating that there was no statistical difference, indicating that there was no therapeutic effect. In addition, combined with Fig.12 The above results were further confirmed by the size of harvested tumors from each treatment group, shown in B.
[0074] Healthy BALB / c mice were intravenously injected with IC@PCH NPs (8 mg / mL, 200 μL) on days 5, 10, 15, and 20, respectively, and the control group was given normal saline, and the main organs of the mice were collected for analysis. Fig.12 As shown in (C), hematoxylin-eosin (H&E) staining of major organs showed almost no histopathological changes, indicating that IC@PCH NPs had good biocompatibility and biosafety.
[0075] IX. Verification of the effect of nanoprobes on cell pyroptosis Combination Fig.13 As shown, Fig.13 This is a photo of 4T1 cells under an optical microscope after IC@PCH NPs + laser treatment (the specific treatment method is: 4T1 cells were inoculated in a culture dish and incubated for 24 hours, and then IC@PCH NPs (1 mg / mL, 200 μL) were added and incubated for 4 hours. Subsequently, the cells were washed 3 times with PBS to remove the unphagocytosed nanoparticles. Finally, 808 nm laser irradiation (1.5 W / cm2, 6 minutes) was used. After IC@PCH NPs + laser treatment, the tumor cells were swollen and enlarged, and there were many bubble-like protrusions.
[0076] In order to study the mechanism of action of IC@PCH NPs-mediated photothermal chemotherapy, RNA-seq sequencing and bioinformatics analysis were performed on 4T1 cells in the control group and IC@PCH NPs + laser group. Fig.14 As shown in A, blue indicates down-regulated genes, and red indicates up-regulated genes. In order to better understand the main metabolic and signaling pathways of differentially expressed genes (DEGs) in the IC@PCH NPs+laser group, the inventors performed KEGG pathway enrichment analysis. The results showed that combined with Fig.14 As shown in (B), insulin resistance, allogeneic transplant rejection, cell death-related pathways, and the NLRP3 signaling pathway (the NLRP3 signaling pathway is closely related to a new mode of death, namely, pyroptosis) were mainly involved in the therapeutic process of IC@PCH NPs.
[0077] Combination Fig.15 As shown in A, pyroptosis is a type of inflammatory programmed cell death triggered by gasdermin-mediated pore formation. After pattern recognition receptors (such as TLRS and NLRs) recognize intracellular and extracellular signals, they bind to adapters and procaspase-1 to form inflammasomes, thereby activating caspase-1. Activated caspase-1 can cause the release of proinflammatory factors such as interleukin (IL)-1β and IL-18, and on the other hand, it can cleave gasdermin D (GSDMD) into GSDMD-N, which then non-selectively penetrates the cell membrane, ultimately leading to cell swelling, lysis, and death.
[0078] Optical microscopy and RNA-seq sequencing results showed that IC@PCH NPs mediated 4T1 cell death through the pyroptosis pathway. Fig.15 As shown in B, the inventors used Western blotting to evaluate the expression levels of NLRP3, Caspase-1, cleaved Caspase-1, GSDMD and GSDMD-N proteins in 4T1 cells after different treatments (grouping: control group, laser only group, C@PCH NPs group, I@PCH NPs+laser group, IC@PCH NPs+laser group, the concentration of each group was 1 mg / mL, and the laser was irradiated with 808 nm laser (1.5 W / cm2, 6 minutes)). The results of Western blotting were quantitatively analyzed and combined with Fig.15 As shown in Figure C, the levels of key proteins of pyroptosis in the I@PCH NPs+laser group and the IC@PCH NP+laser group were significantly increased, which is related to the dominant role of photothermal effect in the treatment. At the same time, the expression level of key proteins of pyroptosis in the IC@PCH NP+laser group was significantly higher than that in the I@PCH NPs+laser group, which demonstrated the effective synergistic effect of chemotherapeutic drugs. In summary, it is concluded that IC@PCH NPs can induce tumor cell pyroptosis by initiating the Caspase-1-dependent classical pyroptosis pathway, thereby inhibiting tumor cell proliferation.
[0079] 10. Verification of PVA and CS dosage ratio like Fig.16 A, when the PVA and CS dosages in the above preparation method are used, the digital photo shows that the IC@PCH NPs solution is uniform, and the nanoparticles under the scanning electron microscope are uniform in size and spherical in morphology.
[0080] like Fig.16B, when PVA is less and CS is more (specifically PVA is 3mL and CS is 5mL), the digital photo shows that the IC@PCH NPs solution is layered, and the optical microscope shows that the lower layer is irregular aggregates, and the upper layer is flocculent and a small amount of nanoparticles. This is because the surface tension increases and the nanoparticles cannot form balls.
[0081] like Fig.16 C. When there is more PVA and less CS (specifically, PVA is 5 mL and CS is 3 mL), IC@PC NPs are electrically neutral and do not have a positive charge. They cannot adsorb hyaluronic acid and cannot reduce the charge of IC@PCH NPs.
[0082] Therefore, the dosage ratio of PVA and CS in this application plays an important role in the successful preparation of nanoprobes. If the dosage ratio of PVA and CS is too high or too low, either spherical nanoprobes cannot be prepared, or nanoparticles with electrostatic adsorption function cannot be prepared.
[0083] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe, characterized in that: The invention comprises a part of photosensitizer, the outer side of which is wrapped with a PLGA shell, the PLGA shell is inlaid with copper diethyldithiocarbamate, the outer side of the PLGA shell is wrapped with a chitosan-targeting molecule coating, and the chitosan-targeting molecule coating contains another part of photosensitizer.
2. A superficial tumor targeting multimodal diagnosis and treatment nanoprobe according to claim 1, characterized in that: The encapsulation efficiency of the photosensitizer was 50-60%.
3. The superficial tumor targeting multimodal diagnosis and treatment nanoprobe according to claim 1, characterized in that: The loading rate of photosensitizer was 6-8%.
4. A method for preparing a superficial tumor targeting multimodal diagnosis and treatment nanoprobe according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Preparing a primary emulsion: mixing PLGA, copper diethyldithiocarbamate and dichloromethane, and dissolving the mixture using an ultrasonic oscillator, then adding an aqueous solution of ICG to the mixture and emulsifying the mixture to obtain a primary emulsion; S2, preparing a double emulsion; adding a polyvinyl alcohol solution and a chitosan solution to the primary emulsion, and emulsifying to obtain a double emulsion; S3, then adding isopropanol and magnetic beads to the double emulsion, and stirring magnetically to evaporate the dichloromethane to obtain IC@PCNPs; S4, washing the IC@PC NPs by high-speed centrifugation for multiple times, resuspending them in pure water, adding hyaluronic acid, and mixing them to obtain IC@PCH NPs; S5. Finally, the IC@PCH NPs were washed, resuspended in pure water, and stored at 4 °C in the dark.
5. The method for preparing a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe according to claim 4, characterized in that: In S2, the concentration of the polyvinyl alcohol solution is 5%, the concentration of the chitosan solution is 2%, and the volume ratio of the chitosan solution to the polyvinyl alcohol solution is 1:
1.
6. The method for preparing a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe according to claim 5, characterized in that: The chitosan solution is chitosan dissolved in 3% acetic acid.
7. The superficial tumor targeted multimodal diagnosis and treatment nanoprobe, preparation method and application according to claim 4, characterized in that: The high-speed centrifugation speed is 9000-11000 rpm / min.
8. Application of a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe as described in any one of claims 1-3 as an imaging agent.
9. Use of a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe as described in any one of claims 1-3 in drugs for treating superficial tumors.
10. The use of a superficial tumor-targeted multimodal diagnostic and therapeutic nanoprobe according to claim 9, characterized in that: The superficial tumor is breast cancer.