Near-infrared second-region small organic molecule photosensitizer nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of near-infrared second-region small organic molecule photosensitizer nanoparticles (L8-4F NPs) with high photothermal conversion efficiency and an imaging function and application of the near-infrared second-region small organic molecule photosensitizer nanoparticles (L8-4F NPs) in efficient photothermal therapy of 4T1 tumors and multi-mode imaging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a near-infrared II organic small molecule photosensitizer nanoparticle (L8-4F NPs) with high photothermal conversion efficiency and imaging function, its preparation method, and its application in efficient anti-4T1 tumor photothermal therapy and multimodal bioimaging (fluorescence imaging / photoacoustic imaging). Background Technology

[0002] Photosensitizers are key materials in photothermal and photodynamic therapy for tumors. Currently, many photosensitizers have shown some efficacy in tumor treatment under optical imaging guidance. However, traditional photosensitizers such as BODIPY and tetraphenylethylene have simple structures and suffer from problems such as short absorption wavelengths, limited imaging modes, and insufficient therapeutic effects.

[0003] Organic fused-ring receptor small molecules, with their unique ADADA structural characteristics and large conjugated planes, exhibit significant near-infrared absorption and high molar absorptivity, making them widely applicable in various fields such as organic solar cells, perovskites, and OLEDs. This invention is the first to apply L8-4F to multifunctional photothermal therapy, and successfully prepared organic photosensitizers (L8-4F NPs) using nano-co-precipitation technology. This innovative approach aims to construct a multifunctional nanoplatform capable of achieving highly efficient phototherapy for 4T1 breast cancer guided by near-infrared fluorescence imaging and photoacoustic imaging. Summary of the Invention

[0004] The primary objective of this invention is to provide a highly efficient near-infrared II organic small molecule photosensitizer nanoparticle (L8-4F NPs) for multi-mode imaging.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned nanoparticles.

[0006] Another object of the present invention is to provide the application of the above-mentioned nanoparticles in efficient photothermal therapy against 4T1 tumors and multimodal bioimaging.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The present invention provides an organic small molecule compound, named L8-4F. This compound is the core active ingredient constituting subsequent nanoparticles, and it has strong absorption in the wavelength range of 500-800 nm, possessing the potential to become a photothermal agent and imaging agent.

[0008] This invention provides lipid nanoparticles prepared from the aforementioned small organic molecule compound L8-4F and a surfactant. The surfactant is preferably Pluronic F127. The nanoparticles are named L8-4F NPs, wherein the mass ratio of L8-4F to F127 is 1:1 to 1:50; for example, 1:5, 1:10, and 1:20.

[0009] The L8-4F-NPs provided by this invention possess the following physicochemical properties: 1) Particle size: Measured by dynamic light scattering (DLS), their hydrated particle size ranges from 60 nm to 80 nm. This size range is beneficial for enrichment in tumor tissues through enhanced permeation and retention (EPR) effects; 2) Zeta potential: Their Zeta potential is approximately -35 mV. The higher negative charge contributes to the dispersion stability of the nanoparticles in aqueous solution and influences their interaction with cells.

[0010] Optical properties: 1) Absorption spectrum: as shown in the appendix Figure 1 and 2 As shown, the absorption spectrum of L8-4F NPs exhibits a redshift compared to free L8-4F molecules, which is more favorable for photothermal therapy within the NIR-II window. 2) Emission spectrum: Its fluorescence emission peak is broad, covering the NIR-II region from 750 nm to 1150 nm, making it suitable for deep tissue fluorescence imaging.

[0011] Stability: The nanoparticles can be stably stored at room temperature and pressure for at least 7 days without significant changes in particle size distribution, indicating that they have good physical stability.

[0012] This invention also provides a method for preparing nanoparticles, characterized by comprising the following steps: dissolving compound L8-4F and surfactant F127 in an organic solvent and mixing them evenly; injecting the mixed solution into water, subjecting it to ultrasonic treatment, and then stirring to remove the organic solvent; purifying the resulting solution by centrifugation and freeze-drying to obtain the nanoparticles. The organic solvent is tetrahydrofuran; the ultrasonic treatment uses a probe-type ultrasonic generator.

[0013] The method for preparing nanoparticles of the present invention is further preferably as follows:

[0014] (1) Dissolution: Dissolve 1 mg of compound L8-4F and 10 mg of surfactant F127 in 1 mL of organic solvent tetrahydrofuran (THF).

[0015] (2) Mixing and initial dispersion: After ultrasonication, the mixture was injected into 18 mL of pure water and ultrasonicated for 10 min to obtain the initial product.

[0016] (3) Purification: The initially formed nanoparticle solution was magnetically stirred overnight to completely remove the organic solvent THF.

[0017] (4) Concentration and preservation: The solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff: 10 kDa), centrifuged at 4000 rpm for 20 minutes, the concentrate was collected, and then freeze-dried to obtain solid L8-4F NPs powder.

[0018] The method for preparing nanoparticles of the present invention is further preferably as follows:

[0019] L8-4F and F127 were mixed at a mass ratio of 1:10 and then sonicated to prepare ADADA-type organic photosensitizers L8-4FNPs. The prepared L8-4F NPs had a particle size of 60–80 nm and a Zeta potential of -35–38 mV. They exhibited an absorption wavelength range of 600–900 nm and an emission wavelength of 750–1150 nm, providing a wide imaging range and excellent photothermal conversion efficiency of up to 58%. They also possessed good water solubility, facilitating intravenous injection. At low concentrations, irradiation of their aqueous solution with an 808 nm near-infrared laser could raise the local temperature by up to approximately 55 °C, meeting the temperature difference requirements for tumor killing. Compared to ICG, which has poor photostability, these nanoparticles exhibited good photostability; their photothermal conversion ability and absorption spectrum remained unchanged after repeated light irradiation (the heating effect of ICG aqueous solution decreased significantly with increasing laser irradiation cycles). In vitro cell experiments verified their biocompatibility and excellent cancer cell killing effect. Furthermore, the fluorescence and photoacoustic imaging images of the in vitro aqueous solution, as well as the concentration-signal curves of fluorescence and photoacoustic imaging, show that the imaging capability increases with increasing concentration, indicating that it has the ability to guide in vivo anti-tumor applications through fluorescence and photoacoustic imaging.

[0020] The present invention also provides the application of the nanoparticles in highly efficient anti-4T1 photothermal therapy; the highly efficient photosensitizer is used to treat tumors, preferably 4T1 breast cancer.

[0021] The present invention also provides the application of the nanoparticles in multimodal bioimaging; the imaging agent is a fluorescent imaging agent or a photoacoustic imaging agent.

[0022] In this invention, L8-4F is a small organic molecule with high photothermal conversion efficiency, a mature synthetic route, and relatively low cost. It has been developed for the first time for application in multifunctional photothermal therapy. Its parent core is an electron-deficient benzothiadiazole unit, with both ends capped by 5,6-difluoro-3-(dicyanomethylene)indole via Knoevenagel condensation. It has the following structural formula:

[0023]

[0024] In this invention, the structural formula of the Prünnicke F127 is shown in the figure below:

[0025]

[0026] Compared with the prior art, the present invention has the following significant advantages:

[0027] 1) Highly Efficient Photothermal Therapy: L8-4F was applied to photothermal therapy for the first time. Nanoparticles prepared from L8-4F and Pluronic F127 exhibited a photothermal conversion efficiency of up to 58%, far exceeding the clinical control ICG (15%), and could effectively kill tumor cells. It showed significant killing effects on tumors in 4T1 tumor-bearing mice, and within 15 days of treatment, most or even completely ablated the tumors.

[0028] 2) Integrated fluorescence and photoacoustic tumor imaging; It combines NIR-II fluorescence imaging and photoacoustic imaging functions, enabling lesion localization before treatment, real-time guidance during treatment, and efficacy evaluation after treatment, truly achieving integrated diagnosis and treatment.

[0029] 3) High biocompatibility: It has extremely low toxicity to normal cells, and the cell survival rate under dark toxicity exceeds 90%, showing good biocompatibility; compared with the control group, the weight of the 4T1 tumor-bearing mice after treatment was not significantly different, and there was no obvious inflammation in the organs. Blood routine tests showed that photosensitizer treatment guided the recovery of inflammation.

[0030] 4) Excellent stability: Nanoparticles have stable physicochemical properties, are easy to store and transport; they have good photostability and can be used for repeated treatments. Attached Figure Description

[0031] Figure 1 The optimized process parameters for the feed ratio in the preparation of the near-infrared II organic small molecule photosensitizer L8-4F NPs in Example 1 are as follows.

[0032] Figure 2 The images show the UV-Vis absorption spectrum of the near-infrared II organic small molecule L8-4F in tetrahydrofuran solution and the UV-Vis absorption spectrum and fluorescence emission spectrum of the near-infrared II organic photosensitizer L8-4F NPs in aqueous solution, as well as the near-infrared II organic photosensitizer L8-4F NPs in aqueous solution.

[0033] Figure 3 The temperature rise curves of aqueous solutions of L8-4F NPs, a near-infrared II organic small molecule photosensitizer of different concentrations in Example 3, under the action of an 808nm laser.

[0034] Figure 4 The temperature rise and fall curves of the aqueous solution of the near-infrared II organic small molecule photosensitizer L8-4F NPs in Example 3 under the action of an 808nm laser.

[0035] Figure 5 The photothermal conversion efficiency curve of the near-infrared II organic small molecule photosensitizer L8-4F NPs in Example 3 is shown.

[0036] Figure 6 Bar chart showing cell viability after co-culturing near-infrared II organic small molecule photosensitizer L8-4F NPs with mouse breast cancer (4T1) cells in Example 4:

[0037] Figure 7 Bar chart showing cell viability after co-culturing different concentrations of the near-infrared II organic small molecule photosensitizer L8-4F NPs with mouse hippocampal neurons (HT22) in Example 5:

[0038] Figure 8 Fluorescence imaging images of aqueous solutions of L8-4F NPs, a near-infrared II organic small molecule photosensitizer, at different concentrations in Example 6.

[0039] Figure 9 This is a fluorescence imaging image of 4T1 tumor-bearing mice after tail vein injection of photosensitizer L8-4F NPs in Example 6.

[0040] Figure 10 Photoacoustic images of aqueous solutions of L8-4F NPs, a near-infrared II organic small molecule photosensitizer, at different concentrations in Example 7.

[0041] Figure 11 This is a 3D photoacoustic image of 4T1 tumor-bearing mice after intravenous injection of the photosensitizer L8-4F NPs in the tail vein, as shown in Example 7.

[0042] Figure 12 This is a real-time change graph of tumor temperature during photothermal therapy after tail vein injection of the near-infrared II organic small molecule photosensitizer L8-4F NPs into 4T1 tumor-bearing mice in Example 8. The PBS+L group served as a control.

[0043] Figure 13 The change in tumor volume during photothermal therapy after injecting photosensitizer L8-4F NPs into the tail vein of 4T1 tumor-bearing mice in Example 8.

[0044] Figure 14 The change in body weight of mice during photothermal therapy following the tail vein injection of photosensitizer L8-4F NPs into 4T1 tumor-bearing mice in Example 8.

[0045] Figure 15 The image shows the dissected tumor after photothermal treatment of 4T1 tumor-bearing mice following tail vein injection of photosensitizer L8-4F NPs in Example 8.

[0046] Figure 16 The image shows the H&E staining of anatomical organs after photothermal treatment following the tail vein injection of photosensitizer L8-4F NPs into 4T1 tumor-bearing mice in Example 8. Detailed Implementation

[0047] The following experiments and examples are used to further illustrate, but are not limited to, the present invention.

[0048] In this invention, L8-4F was purchased from Jiaxing Hepu Optoelectronics Technology Co., Ltd.;

[0049] The polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer poloxamer 407 / Planick F127 (F127) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0050] Example 1

[0051] Preparation method and optimization of lipid nanoparticles L8-4F NPs

[0052] Organic small molecules L8-4F and F127 were dissolved in 1 mL of THF at concentrations of 1:5, 1:10, and 1:20, respectively. After mixing and sonicating, 18 mL of pure water was added, and the mixture was sonicated for 10 min to obtain a preliminary product. The product was stirred overnight to remove THF, then transferred to an ultrafiltration centrifuge tube (10 kDa) and centrifuged for 20 min at 4000 rpm. The product was then freeze-dried and stored for later use to obtain lipid nanoparticles L8-4F NPs. The optimal feed ratio (1:10) resulted in the smallest hydrated particle size (66.02 nm) of the nanoparticles. Figure 1 .

[0053] The nanoparticles have a particle size of 60–80 nm and a zeta potential of approximately -35 mV. The nanoparticles are stable when stored under refrigeration for more than 7 days without significant changes in particle size.

[0054] Example 2

[0055] Optical behavior of organic small molecule L8-4F and organic photosensitizer L8-4F NPs

[0056] L8-4F and L8-4F NPs solutions were prepared separately using THF and pure water, with a volume of 2 mL and a concentration of 100 μg / mL, respectively. The UV absorption and fluorescence emission spectra were then measured. Figure 2 As shown, L8-4F exhibits a wide absorption range, with strong absorption in the 500–800 nm range. The prepared L8-4F NPs show a redshift of nearly 100 nm, which is more beneficial for photothermal therapy in the near-infrared II region. Furthermore, analysis of its fluorescence spectral behavior reveals a broad emission peak in the 750–1150 nm range. Therefore, this demonstrates that L8-4F NPs possess the fundamental properties for near-infrared II imaging and photothermal therapy.

[0057] Example 3

[0058] Photothermal performance experiment of organic photosensitizer L8-4F NPs

[0059] Aqueous solutions of photosensitizer L8-4F NPs with concentrations of 0.07, 0.28, 0.7, 1.4, and 2.8 μM were prepared and tested at 808 nm and 1 W / cm². 2Under laser irradiation, the temperature change of the aqueous solution was monitored in real time over 10 minutes using a near-infrared thermal imaging camera, and a temperature-time curve was plotted. The results are shown below. Figure 3 As shown, under light irradiation, the system temperature rises rapidly, with the temperature change of the 2.8 μM L8-4FNPs aqueous solution reaching as high as 55 °C, exhibiting excellent photothermal conversion effect.

[0060] Subsequently, the sample was continuously irradiated with a 2.8 μM L8-4F NPs aqueous solution four times, and the changes in temperature rise were observed. Figure 4 As shown, using indocyanine green (ICG, a near-infrared visible fluorescent dye approved by the US Food and Drug Administration in 1956) aqueous solution as a reference, it was found that under the same conditions, the maximum temperature of the ICG aqueous solution decreased with increasing laser irradiation times, while the temperature of the L8-4F NPs aqueous solution remained almost unchanged. This indicates that L8-4F NPs has good photostability, ensuring the stability of subsequent in vitro and in vivo experiments. Based on the temperature rise and fall curves and the absorbance at corresponding concentrations, the photothermal conversion efficiency of L8-4F NPs was calculated to be approximately 58%, compared to only 15% for ICG in aqueous solution, demonstrating a significant improvement in photothermal conversion efficiency. Figure 5 As shown.

[0061] Example 4

[0062] Investigation of the toxicity of lipid nanoparticles L8-4F NPs to cancer cells

[0063] The cytotoxicity of L8-4F NPs to cancer cells was investigated using murine breast cancer cells 4T1 as an example, focusing on phototoxicity and dark toxicity. 4T1 cells were cultured at 10 μL per well. 5 Cells were seeded at a density of 200 μL in 96-well plates; after 24 hours of culture, 100 μL of fresh medium containing L8-4F NPs was added to each well (concentrations of 0, 0.35, 0.70, 1.40, 2.10, 2.80, and 3.50 μM for each group). After 12 hours of culture, the light group received 1 W / cm² light for 10 min. 2 The cells were irradiated with an 808 nm laser and cultured for 12 h. The old culture medium was then discarded, and basal medium solution containing 10% CCK-8 was added to each well. After incubation for 0.5 hours, the absorbance at 450 nm was immediately recorded using a microplate reader to evaluate the cytotoxicity of L8-4F NPs. The results showed that when the concentration of L8-4F NPs reached 3.50 μM, the survival rate of 4T1 cells remained above 80%. Figure 6This indicates that L8-4F NPs have low dark cytotoxicity, while under laser irradiation, when the concentration of L8-4F NPs is only 0.35 μM, it exhibits significant phototoxicity to 4T1 cells.

[0064] Example 5

[0065] Investigation of the cytotoxicity of lipid nanoparticles L8-4F NPs to normal cells

[0066] The normal cytotoxicity of lipid nanoparticles L8-4F NPs was demonstrated using human neuronal immortalized cells HT22 as an example. HT22 cells were cultured at 10 nanoparticles per well. 5 Cells were seeded at a density of 200 μL in 96-well plates. After 24 hours of culture, 200 μL of fresh medium containing L8-4F NPs was added to each well (concentrations of 0, 0.35, 0.70, 1.40, 2.10, 2.80, and 3.50 μM for each group). After another 24 hours of culture, the fresh medium was discarded, and basal medium solution containing 10% CCK-8 was added to each well. After 0.5 hours of incubation, the absorbance at 450 nm was immediately recorded using a microplate reader to evaluate the normal cell cytotoxicity of L8-4F NPs. The results showed that when the concentration of L8-4F NPs reached 3.5 μM, the cell viability remained above 90%. Figure 7 This indicates that L8-4F NPs have high cell safety.

[0067] Example 6

[0068] In vitro and in vivo fluorescence imaging capabilities of lipid nanoparticles L8-4F NPs

[0069] The fluorescence imaging capability of lipid nanoparticles L8-4F NPs was investigated using photosensitizer solutions of different concentrations. An initial concentration of 1 mg / mL was prepared and then serially diluted (2, 4, 8, 16, 32 times). A PBS control group was also included. Imaging was performed using a Biospace Lab in vivo imaging system. Linearity was fitted based on the semi-quantitative data of concentration and fluorescence signal, as shown in the figure. Figure 8 As shown, we can see that the intensity of fluorescence imaging increases with increasing concentration, which indicates that L8-4F NPs have good fluorescence imaging capabilities.

[0070] Based on this, PBS solution containing L8-4F nanoparticles (100 μL, 2.5 mg / mL) was intravenously injected into 4T1 tumor-bearing mice on the back. Fluorescence images were then collected at 1, 2, 4, 8, 12, 24, and 48 hours post-injection (excitation wavelength 808 nm, using a 900 nm low-pass filter). The mean fluorescence intensity of the obtained mouse tumors and tissues was quantitatively analyzed, such as... Figure 9The results showed that the fluorescence intensity at the tumor site gradually increased over time, reaching a peak 8 hours after injection, indicating that 8 hours after administration is the optimal time for phototherapy.

[0071] Example 7

[0072] In vivo and in vitro photoacoustic imaging capabilities of lipid nanoparticles L8-4F NPs

[0073] The photoacoustic imaging capability of lipid nanoparticles L8-4F NPs was investigated using PBS solutions of different concentrations. The methodological steps were largely consistent with fluorescence imaging, except that imaging was performed using an Endra inc NEXUS 128 in vivo imaging system. Linear relationships were fitted based on the concentration and semi-quantitative photoacoustic signal data, such as... Figure 10 As shown, similar to fluorescence imaging, the intensity of photoacoustic imaging increases with increasing concentration, indicating that L8-4F NPs have good photoacoustic imaging capabilities.

[0074] Based on this, PBS solution containing L8-4F NP (2.5 mg / mL, 100 μL) was intravenously injected into tumor-bearing mice, and 3D photoacoustic imaging was performed using an 808 nm laser on an Endra inc NEXUS 128 photoacoustic imaging system at different time intervals (0, 1, 8, 12, 24, 48, and 72 hours after injection). The results showed that the tumor signal rapidly increased within 0–6 hours after administration, reaching a peak at 8 hours. Figure 11 Furthermore, the strong photoacoustic signals clearly present three-dimensional images, fully demonstrating its excellent in vivo photoacoustic imaging capabilities, providing an important basis for guiding tumor surgical resection and developing precise personalized treatment plans.

[0075] Example 8

[0076] In vivo antitumor effect study of lipid nanoparticles L8-4F NPs

[0077] The in vivo antitumor effect of lipid nanoparticles L8-4F NPs was evaluated in 4T1 tumor-bearing mice. Tumors with a volume of 100±30 mm were included in the study. 3 Female Balb / c mice were randomly divided into four groups (n=6): PBS group, PBS+L group, L8-4F NPs group, and L8-4F NPs+L group. Each group was intravenously injected with 100 μL of PBS or NPs (2.5 mg / mL). Eight hours later, they were subjected to 808 nm laser (0.6 W / cm²) treatment. 2 The tumor site was irradiated for 10 minutes. Real-time temperature changes at the tumor site were monitored and recorded using a Fortic infrared thermal imaging camera in both the PBS+L and NPs+L groups (e.g., ...). Figure 12Tumor volume and body weight of mice were recorded every two days (the day of the first administration was recorded as day 0), see [link to relevant documentation]. Figure 13 14. On day 15, the experimental mice were euthanized, the tumors were removed, and their size and shape were recorded. Figure 15 Organs such as the heart, liver, spleen, lungs, and kidneys were subjected to H&E staining to observe tissue morphology, such as... Figure 16 Based on the above data, it was found that the temperature of the irradiated tumors in the NPs+L group increased significantly from 34℃ to 62℃. Generally, an anti-cancer effect can be achieved when the temperature rises above 42℃. In contrast, the PBS+L group only showed a temperature increase of about 6℃, with negligible impact. When monitoring changes in tumor volume in mice, the NPs+L group showed a decreasing trend, and the tumors almost disappeared after the fourth treatment, while the PBS and PBS+L groups continued to grow, strongly demonstrating that L8-4FNPs can effectively kill cancer cells under low-power light irradiation. In addition, no significant decrease in mouse weight was observed during treatment. H&E staining was performed on the heart, liver, spleen, lungs, and kidneys of mice treated with different formulations. The staining results of each organ were similar, and no significant damage was observed. These results indicate that L8-4F NPs have good biosafety and biocompatibility.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A near-infrared two-region organic small-molecule photosensitizer nanoparticle, characterized in that, The nanoparticles comprise compound L8-4F and a surfactant, have a particle size of 60-80 nm, and a Zeta potential of about -35 mV.

2. The nanoparticle of claim 1, wherein, The surfactant is pluronic F127.

3. The nanoparticle according to claim 1 or 2, characterized in that, The nanoparticles have a photo-thermal conversion efficiency of 50-65%, preferably 58%, under 808 nm laser irradiation.

4. A method of preparing the nanoparticle according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: dissolving compound L8-4F and a surfactant in an organic solvent and mixing uniformly; injecting the mixed solution into water, removing the organic solvent by stirring after ultrasonic treatment; and centrifuging the obtained solution, followed by lyophilization to obtain the nanoparticles.

5. The method of claim 4, wherein, The organic solvent is tetrahydrofuran.

6. The method of claim 4, wherein, The ultrasonic treatment uses a probe-type ultrasonic generator.

7. Use of the nanoparticles according to any one of claims 1-3 in the preparation of a high-efficiency anti-4T1 tumor photosensitizer.

8. Use according to claim 7, characterized in that, The photosensitizer is used for photothermal treatment of tumors, preferably the tumor is 4T1 breast cancer, the laser wavelength is 808nm, the power is 0.6-1.0W / cm 2 , and the irradiation time is 5-10min.

9. Use of the nanoparticle according to any one of claims 1 to 3 in multimodal bioimaging, characterized in that, The laser wavelength for bio-imaging is 808 nm, and a 900 nm filter is selected.

10. Use according to claim 10, characterized in that, The multi-modal bio-imaging agent is a fluorescence imaging agent or a photoacoustic imaging agent.

11. A photosensitizer nanoparticle, characterized in that, A pharmaceutical composition comprising the nanoparticles according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

Images