Patent Application
20240041787
The present invention relates to the field of nanomaterials, in particular to a photosensitizer molecule and a use thereof in increasing tumor retention time thereof and enhancing the treatment of large-volume tumors.
Photodynamic therapy (PDT), due to its minimal invasiveness and high spatiotemporal precision, results in considerable attention. As a key part of PDT, photosensitizer transfers absorbed energy to surrounding oxygen and generates reactive oxygen species (ROS), and then further reacts with nearby biomacromolecules (such as lipid, protein, and DNA) to kill cancer cells and complete PDT. Therefore, effective enrichment of the photosensitizer at tumor sites is a prerequisite for PDT of cancer patients, and its tumor retention effect directly determines whether the treatment is accurate and effective. However, in the process of in vivo PDT, most organic small-molecule photosensitizers are rapidly cleared from the bloodstream usually within hours or even ten minutes, so that the photosensitizers cannot be effectively enriched and retained at the tumor sites, thereby resulting in low PDT efficiency. Therefore, it is urgent to improve the retention time of photosensitizers at tumor sites, which is of great significance to ensure the accurate and efficient PDT in vivo.
One of the current major strategies to prolong blood circulation and tumor retention time is to nanoengineering the photosensitizers, such as organic polymer nanoparticles (NPs), liposomes or inorganic nanomaterials. Researchers can well control and optimize tumor retention time by changing the physicochemical properties (i.e., size, shape, and charge) of nanosystems. Furthermore, the so-called “passive targeting” effect can further improve the tumor retention time of nanosystems through surface modification with tumor targeting groups (e.g. peptides, antibodies). Although these approaches prolong the retention time of photosensitizers in tumors, these nanosystems are usually cleared from tumors within one to three days, and it is still insufficient to guarantee effective PDT in many cases. Phototherapy still needs to be performed as soon as possible after the injection of photosensitizers, and large doses or multiple injections of photosensitizers are required for the treatment of large-volume tumors or malignant tumors. However, these will increase the risk of treatment during PDT and lead to significant toxic side effects. Therefore, achieving long-term retention time of photosensitizers at tumor sites is highly desirable, but remains one of the key challenges for PDT.
Improving the stability of NPs is a direct way to improve the tumor retention effect, because NPs are usually easy to aggregate or disintegrate in a complex blood environment, which is one of the main reasons for limitations in tumor retention time.
To solve the above technical problems, the present application provides a photosensitizer co-assembly strategy to prolong tumor retention time by greatly improving the stability of nanoparticles (NPs). Retention time of photosensitizer of the present invention at tumor sites has been greatly improved to about 10 days. The photosensitizer synthesizes Bodipy-phenylethynyl anthracene dyad (AN-BDP) by introducing anthracene into BODIPY-meso sites. Due to the strong intermolecular π-π interaction of anthracene, AN-BDP and amphiphilicblock copolymer (distearoyl phosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG2000)) are self-assembled into stable NPs (i.e., AN-BDP NPs). In addition, anthracene can also make AN-BDP excited from a singlet excited state to a triplet excited state and react to O2 to generate 1O2 under the irradiation of light. In case of tail vein injection of AN-BDP NPs, its enrichment and retention time at the tumor sites is up to about 10 days. In a conventional volume-sized mouse tumor model (a tumor initial volume of about 100 mm3), AN-BDP NPs can completely inhibit tumor growth with a single phototherapy injection. In contrast, in a large-volume tumor model (a tumor initial volume of about 350 mm3), only 12% tumor growth inhibition was observed under the same treatment conditions. Since the long tumor retention time, a single injection of AN-BDP NPs can be subjected to 3 times of phototherapy, which significantly improve the treatment effect; while the clinically used photosensitizer Ce6 or Porphyrin NPs encapsulated with the same amphiphilic block copolymer (DSPE-PEG2000) cannot inhibit the growth of large tumors because they are quickly cleared from the tumor sites and only a single phototherapy can be performed under the same conditions. Therefore, improving tumor retention time with stable NPs can be used to treat large-volume tumors multiple times with a single injection, thereby reducing toxic side effects.
The present invention provides a photosensitizer having a structural formula expressed by the general formula I as follows:
wherein in the formula I, R is selected from:
The present invention further provides nanophotosensitizer particles, where the photosensitizer and DSPE-PEG2000 are self-assembled to form nanoparticles.
The present invention further provides a use of the photosensitizer in preparing an antitumor drug, and a use of the nanophotosensitizer particles in preparing an antitumor drug.
Further, in the above technical solutions, the antitumor drug is an antitumor drug for photodynamic therapy.
Further, in the above technical solutions, the antitumor drug is used for multiple photodynamic therapies of a large tumor through a single injection of the photosensitizer or nanophotosensitizer particles.
Further, in the above technical solutions, a retention time of the photosensitizer or the nanophotosensitizer particles at the tumor site is <300 h.
Further, in the above technical solutions, the large tumor is a tumor with a volume of 300-350 mm3.
Further, in the above technical solutions, a frequency of the photodynamic therapy is 1-3 times.
Further, in the above technical solutions, for the large tumor, the treatment effect can be enhanced by one administration and three phototherapies, and the side effects caused by large drug dose and the light dose can be reduced.
The following non-limiting embodiments may enable those skilled in the art to understand the present invention more clearly, but not to limit the invention in any way.
Apoptosis detection kits (Annexin V-FITC) and reactive oxygen species assay kits (DCFH-DA) were purchased from Beyotime Biotechnology. Live/dead cell staining kits (Calcein AM/PI) and Hoechst 33342 were purchased from KeyGEN BioTECH. Commercially available subcellular organelle localization dyes such as nuclear dye Hoechst 33324, lysosomal LysoTracker Green DND-26, mitochondrial MitroTracker Green FM, and Propidium Iodide (PI) were purchased from Thermo Fisher Scientific.
Hela (human cervical cancer cells), MCF-7 (human breast cancer cells) and 4T1 (murine breast cancer cells) were purchased from the Institute of Basic Medical Sciences (IBMS).
Example 1 Molecular Design and Synthesis of an AN-BDP NPs Photosensitizer
(1) Synthesis of an Intermediate 3
3,5-Dimethyl pyrrole aldehyde (100 mg, 0.81 mM) was dissolved in dry CH2Cl2 (15 mL). POCl3 (124 mg, 0.81 mM) was slowly added to the aforesaid solution under an argon atmosphere at 0° C. The reaction solution was stirred at 0° C. for 1 hour, then NEt3 (750 mg, 7.4 mm) was added at room temperature and stirred for 4 hours, then BF3·Et2O (0.93 ml 7.4 mM) was added to react for 2 hours, and then the solvent was evaporated in vacuum, with EtOAc (200 mL) used for extraction. Then the organic layer after extraction was washed with H2O (3×50 mL) and dried with anhydrous Na2SO4. The crude product was purified by silica gel column chromatography (hexane/EtOAc = 5:1) to obtain the intermediate 3 (red crystal) of 99 mg (49%).
(2) Synthesis of an Intermediate 5
Appropriate amounts of Anthraldehyde (2 mmol) and 2,4-dimethylpyrrole (4 mmol) were dissolved in 250 mL of anhydrous CH2Cl2 under N2 atmosphere. One drop of trifluoroacetic acid (TFA) was added and stirred at room temperature overnight. When results of monitoring by thin layer chromatography (TLC) showed complete consumption of the aldehyde, 2 mmol of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was added to the CH2Cl2 solution and further stirred for 15-20 minutes. The reaction mixture was washed with water, dried with magnesium sulfate, and was filtered and evaporated. The crude compound was roughly purified by alumina peroxide column chromatography to obtain a brown-orange solid. Under nitrogen protection, the brown-orange solid and 4 mL of N,N-diisopropylethylamine (DIEA) were dissolved in 150 mL of anhydrous CH2Cl2 (or anhydrous toluene), and the solution was stirred at room temperature for 5 minutes. Then 4 mL of boron trifluoride diethyl etherate (BF3·BFe2) was added to the solution and further stirred for 30 minutes. The reaction solution was washed with water and dried with anhydrous MgSO4, and was filtered and evaporated. The crude compound was purified by silica gel chromatography (CH2Cl2/hexane) to obtain an analytically pure sample, which was recrystallized in MeOH to obtain an orange needle.
(3) Synthesis of a Target Compound
Benzaldehyde (0.31 ml, 2.21 mM), 3a (the intermediate 3 or intermediate 5, 0.58 mM), AcOH (0.10 mL) or catalytic amounts of p-Toluenesulfonic acid, and piperidine (0.10 mL) were dissolved in Toluene (10 mL), and a certain amount of activated 4 e molecular sieves was added, with stirring at 80° C. for 5 hours. After quenching with water, the mixture was cooled to room temperature and extracted with CH2Cl2. The combined organic layers were washed with sodium chloride solution, and was dried with anhydrous Na2SO4 and evaporated. The crude product was purified by TLC (CH2Cl2/n-hexane = 3:1) to obtain a final compound:
AN-BDP: 1H NMR (400 MHz, CDCl3) NMR (400 MHz, CDClmpound:prJ=8.4, 2 H), 7.96 (d, J=8.6, 2H), 7.85 (d, J=16.3, 2H), 7.66 (d, J=7.5, 4H), 7.55 (d, pound:product was purified by TLC xture TOF-HRMS (EI) (m/z): C31H41BF2N2, calculated value: 600.2548; detection value: 600.2543.
13C NMR (101 MHz, CDCl3) δ=152.92, 142.07, 136.63, 136.51, 134.31, 131.34, 130.12, 128.99, 128.82, 128.42, 128.39, 127.61, 127.06, 125.82, 125.27, 119.34, 117.83, 77.33, 77.02, 76.70, 13.60.
Me-BDP: 1H NMR (400 MHz, CDCl3) δ=7.72 (m, 2H), 7.66 .63, 136.51, 134.31, 131.34, 130.12, 128.99, 1J=7.1, 2H), 6.73 (s, 2H), 2.50 (s, 6H), 2.30 (s, 3H). TOF-HRMS (EI) (m/z): [M]+ calcd for C28H25BF2N2: 438.2079; found: 438.2091.
Example 2 Characterization of AN-BDP NPs Photosensitizer Molecules
As shown in
As shown in
Operation steps: 2.5 mL of a DPBF CH2Cl2 solution with an absorbance of about 1.0 and a dichloromethane solution with an AN-BDP absorbance of about 0.3 were prepared and mixed evenly. A 630 nm LED lamp was used to illuminate a cuvette containing the evenly mixed solution, and a corresponding absorption spectrum was measured every 30 s. The 1O2 production level of each sample was evaluated by comparing the changes of absorbance at 415 nm over time. The optical power density of the light source is 5 mW/cm2.
As shown in
It can be seen from
Example 3 Synthesis and Characterization of Nanoparticles (NPs)
(1) Synthesis of NPs
A photosensitizer (AN-BDP or Me-BDP) was dissolved in tetrahydrofuran (THF, 1 mg, 1 mL) to obtain a mixture, then the mixture was added to Milli-Q water containing DSPE-PEG2000 (5 mg, 5 mL) under ultrasonic processing (180 W) and a dispersion liquid was obtained. The obtained dispersion liquid was further kept in ultrasonic processing for 40 minutes to obtain a colloidal dispersion. Thereafter, the colloidal dispersion was dialyzed in the Milli-Q water for 3 days to remove the organic solvent. During the dialysis process, the Milli-Q water was replaced every 6 hours, and finally the dispersion liquid was dialyzed with physiological saline, where the dialysis bag was a regenerated cellulose dialysis bag 3500. The solution in the dialysis bag was collected, that is, the desired NPs.
(2) Characterization of NPs
A certain volume of the NPs solution was added to a sample cell, and the diameter of the NPs was determined by dynamic light scattering (DLS). The morphology of the NPs was measured by transmission electron microscopy (TEM): 2.5 μL of the diluted NPs solution was dropped onto a copper mesh covered with carbon film, and after natural drying, the carbon film was observed with a transmission electron microscope.
AN-BDP was made into NPs by nanoprecipitation. An amphiphilic copolymer (DSPE-PEG2000) was used as the encapsulation matrix. During the assembly process, hydrophobic aromatic planar molecules are randomly accumulated in the core, and hydrophilic PEG chains form a water-soluble shell. The absorption spectra of molecules aggregated in NPs are very different from those in a free state. The size and morphology of the AN-BDP NPs were characterized by using TEM and DLS (as shown in
(3) Stability Testing of the NPs
The prepared NPs were diluted to a certain concentration respectively in phosphate buffer solution (PBS), bovine serum albumin (BSA) and fetal bovine serum (FBS) to observe the changes in absorbance of NPs at different times. Changes in absorbance were used to determine the stability of the NPs.
To verify that strong π-π stacking is beneficial to improving the stability of the NPs, BODIPY with meso-methyl (Me-BDP) was synthesized and Me-BDP NPs were prepared for comparison. Then the stability of AN-BDP NPs and Me-BDP NPs under different conditions in vitro was tested. The changes in normalized absorption intensity ratio were used to quantify the nanostructure retention rate (RNF %). After 48 hours of incubation in pure water, PBS buffer and BSA (45 g/L) at 37° C. and pH 7.4, both AN-BDP NPs and Me-BDP NPs had RNF values>95% (
Example 4 Dynamics Simulation of AN-BDP NPs and Me-BDP NPs
To reveal the possible mechanism of NPs stability, molecular dynamics (MD) simulations of AN-BDP NPs and Me-BDP NPs were performed by using the GROMACS program (
Example 5
The murine breast cancer cells 4T1, Hepg-2 cells (human hepatoma cells) and MCF-7 cells (human breast cancer cells) used in the present invention were cultured in a Dulbecco's Modified Eagle Medium (DMEM) containing a 1% mixed solution of penicillin and streptomycin and 10% fetal bovine serum (FBS) at 37° C. in the environment of 2% O2 and 5% CO2 until the logarithmic phase, and a cell suspension was prepared after trypsinization for subsequent experiments.
(1) Real-Time Fluorescence Imaging of Cellular Uptake of AN-BDP NPs and Me-BDP NPs
100 μL of a 4T1 cell suspension (1×105 cells/mL) was taken and placed in a confocal dish containing 2 mL DMEM medium, and was cultured at 37° C. in an environment of 5% CO2 for 24 h until the logarithmic phase. The cultured cells were washed with a phosphate buffer solution (PBS) 3 times before imaging. 2.0 μM of NPs were incubated with 4T1 cells for different times, and then cell nuclei were stained with Hoechst 33342. Fluorescence images were taken by confocal laser scanning microscopy. The excitation wavelength of NPs is 590 nm, and the emission wavelength is detected in a band range of 600-700 nm. The excitation wavelength of Hoechst 33342 is 405 nm, and the detection band range is 425-475 nm.
The uptake and retention time of a photosensitizer in cells is one of the key factors for the antitumor effect of photodynamics. Only when the photosensitizer is effectively enriched and retained in cells, can the reactive oxygen species (ROS) generated after illumination destroy cancer cells and induce cell apoptosis or death, so as to achieve the purpose of inhibiting tumor growth. Cellular uptake of AN-BDP NPs was investigated by using 4T1 tumor cells (
(2) Cell Viability Test of AN-BDPNPs
Methyl thiazolyl tetrazolium (MTT) method was used to test the cell viability.
Operation steps: MCF-7, HepG-2 and 4T1 cells were inoculated in 96-well plates (1×104 cells per well and incubated in 100 μL of DMEM). Adherent cells were grown to a density of 80% and incubated with different concentrations of AN-BDP NPs for 4 hours. Then a 630 nm OLED lamp (30 mW/cm2) was used for illuminating for 15 minutes. Meanwhile, under the same experimental conditions, dark cytotoxicity of AN-BDP NPs cultured cells without laser irradiation was further studied. After further incubation for 24 hours, a MTT solution (100 wh of 0.5 mg/mL DMEM) was added to each well and the cells were further incubated for 4 hours at 37° C. Then the medium was carefully removed and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. A Bio-Rad microplate reader was used to measure the absorbance at 490 nm and calculate the cell viability:
The test group is the cell culture group treated with different concentrations of AN-BDP NPs; the blank group is the group only with a medium added; the control group is the cell culture group without AN-BDP NPs; OD is the light absorption of a DMSO solution of formazan at 490 nm.
When 12 μM AN-BDP NPs were used under dark conditions, scarcely any cytotoxicity was observed, and the cell viability was above 90%, indicating that AN-BDP NPs had good biocompatibility in the absence of radiation. In contrast, when cells were incubated with the same concentration of AN-BDP NPs under 27 J/cm2 light irradiation (630 nm), tumor cell growth was inhibited by about 70%. The cytotoxicity dependent on the concentration and laser power density was observed (
(3) Imaging of Intracellular Singlet Oxygen Production
Operation steps: 4T1 cells were incubated with 5 μM AN-BDP NPs for 3 hours, then incubated with 5 μM of dichlorodihydrofluorescein diacetate (DCFH-DA) for 0.5 hour, the medium was discarded, the incubated product was washed with PBS for three times and 2 mL of serum-free DMEM was added. In the 1O2 quenched group, 100 μM of NaN3 was added to the cells and incubated for 1 hour before the cells were stained with DCFH-DA. Then, the cells were irradiated with 630 nm light (30 mW/cm2) for 15 minutes (27 J/cm2). Confocal fluorescence imaging was used to observe intracellular 1O2 levels.
As shown in
(4) Subcellular Organelle Colocalization Imaging of AN-BDP NPs
After the 4T1 cells in the confocal dish were grown until the logarithmic phase, 2 μM AN-BDP NPs were added to incubate for 2 h, and then the cells were co-incubated with different commercial dyes for 15 min.
The red fluorescence of AN-BDP NPs was completely superimposed with the green fluorescence of the lysosomal dye LysoTracker Green DND 26 (a commercial lysosomal dye), with a Pearson correlation coefficient of 0.87 (
(5) A Lysosome Destruction Experiment of AN-BDP NPs Under Red Light Irradiation
Acridine orange, also known as 3,6-bis (dimethylamino) acridine zinc chloride hydrochloride (AO), was used as a lysosomal integrity indicator to visually indicate the destruction of lysosomes by the 1O2 generated by excitation of AN-BDP NPs by 630 nm OLED red light.
4T1 cells were inoculated on a 35 mm confocal dish and cultured at 37° C. for 24 hours in a 5% CO2 environment. Then 4T1 cells were exposed to the following different treatments: the control group, untreated; the light group, irradiated with 630 nm red light at a power density of 30 mW/cm2 (27 J/cm2) for 15 minutes; the AN-BDP NPs group, incubated with 10 μM of AN-BDP NPs at 37° C. for 3 hours; the AN-BDP NPs+light group, incubated with 10 μM AN-BDP NPs at 37° C. for 3 hours and irradiated with 630 nm red light at a power density of 30 mW/cm2 (27 J/cm2) for 15 minutes. Before the imaging experiment, all cells were incubated with AO (5 Be) for 0.5 h. Finally, observation by a confocal microscope was performed, and fluorescence images were collected for analysis.
A large amount of red fluorescence (white arrow) was observed in the control group, AN-BDP NPs group and light group, indicating that the lysosomes were intact. However, the red fluorescence of AO of AN-BDP NPs disappeared under 630 nm light irradiation (
(6) An Experiment of Live/Dead Cell Staining Induced by AN-BDP NPs
The 4T1 cells were inoculated in a 35 mm confocal dish and cultured for 24 hours, and after the cells grew until the logarithmic phase, cells in the control group were incubated without any treatment, cells in the light group were irradiated with a 630 nm (30 mW/cm2) LED lamp for 15 minutes, cells in the AN-BDPNPs group were incubated with 12 μM AN-BDP NPs at 37° C. for 4 hours, and cells in the AN-BDP NPs + Light group were incubated with 12 μM AN-BDP NPs at 37° C. for 4 hours, and then were irradiated with a 630 nm (30 mW/cm2) OLED lamp for 15 minutes. The cells were stained with a CalceinAM/PI double staining kit according to the manufacturer's instructions and imaged by a confocal laser scanning microscope (Olympus FV-3000). Calcein: the excitation wavelength is 488 nm, and the emission wavelength for collection is 500-540 nm. PI: the excitation wavelength is 488 nm, and the emission wavelength for collection is 650-690 nm.
Live/dead cell staining experiments were then conducted to confirm the phototherapy effect of AN-BDP NPs under the conditions of 630 nm and 27 J/cm2 light irradiation (
(7) A Validation Experiment of Cell Death Pathway Induced by AN-BDP NPs
The pathway of cell death induced by 1O2 generated by AN-BDP NPs under light irradiation was also investigated. After the 4T1 cells were cultured until the logarithmic phase, 12 μM AN-BDP was added to the cultured cells to incubate for 4 h, and was illuminated with a light source of 630 nm LED (30 mW/cm2) for 15 min. The cells were stained using an Annexin V-FITC/PI Apoptosis Kit and tested by flow cytometry.
Operation steps: 4T1 cells were inoculated in a 35 mm 6-well plate and incubated for 24 hours, and then divided into the following groups: control group with the cells incubated without any treatment, light group with the cells irradiated with a 630 nm (30 mW/cm2) LED lamp for 15 minutes, AN-BDP NPs group with the cells incubated with 12 μM AN-BDP NPs at 37° C. for 4 hours, and AN-BDP NPs+Light group with the cells digested with EDTA-free trypsin after incubation with 12 μM AN-BDP NPs at 37° C. After 4 hours, then centrifuged at 1000 rpm for 5 min to discard the medium. The working solution was prepared and the cells were stained according to the instructions. Finally, states of the cells were tested by flow cytometry.
The control group was similar to the light group and the AN-BDP NPs group, the cell viabilities were above 96%, and no obvious apoptosis signal was detected. In contrast, the percentage of apoptotic cells significantly increased to 35.22% after 630 nm 27 J/cm2 light irradiation of AN-BDP NPs (
Example 6
(1) Xenograft Tumor Modeling (4T1 Breast Cancer)
The mice used in this example were all 6-week-old BALB/c female mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. The relevant experimental operations were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and the regulations of the National Research Committee, and were approved by the Animal Care and Use Committee of Dalian University of Technology. Document number/ethical approval number is 2018-043. A conventional volume tumor model (an initial volume of about 100 mm3) was established. In addition, in order to verify that long retention time is beneficial to multiple treatments of large tumors or malignant tumors, the present invention also established a large volume (an initial volume of about 350 mm3) tumor model.
(2) In Vivo Targeted Biodistribution and Fluorescence Imaging
In order to verify that π-π stacking can improve the stability of NPs so as to prolong the tumor retention time of NPs as well as the tumor targeting and optimal treatment time of NPs, the above tumor-bearing mice were selected, AN-BDP NPs or Me-BDP NPs (100 μL, 200 μM) were tail vein injected into the tumor-bearing mice, and fluorescence imaging was observed at different times after injection (
Normalized fluorescence intensity images of time-dependent tumor regions were plotted according to mouse imaging analysis results (
To quantitatively study the biodistribution of major organs, the mice were killed 3 days later after injection of AN-BDP NPs, and tumors and major organs (heart, liver, spleen, lung, and kidney) were collected for imaging. As shown in
(3) In Vivo Tumor PDT
The fluorescence imaging-guided in vivo solid tumor targeted PDT experiment was conducted mainly to test the tumor suppression effects of the conventional tumor volume model and the large tumor model respectively.
(i) A Conventional Tumor Volume Inhibition Experiment
In view of excellent tumor enrichment and long retention time of AN-BDP NPs, antitumor activity of AN-BDP NPs in mice with conventional size (initially about 100 mm3) tumors was first investigated. The mice were divided into four groups (n=5), including (1) control group with intravenous injection (i.v.) of physiological saline (100 p), (2) light group with irradiation of 200 mW/cm2 635 nm laser for 15 min, (3) AN-BDP NPs group with tail vein injection of AN-BDP NPs (200 vein injec), and (4) AN-BDP NPs+Light group with tail vein injection of 100 μL of 200 μM AN-BDP NPs, and then 48 hours later with 200 mW/cm2 irradiation with 635 nm laser for 15 min. The mice in group (2) treated with physiological saline and laser irradiation (635 nm laser, 200 mW/cm2, 15 min) were used for negative control. Body weights and volumes of the mice were measured every other day.
(ii) A Large-Volume Tumor Inhibition Experiment
The present invention evaluated the therapeutic performance of a large tumor model for AN-BDP NPs-injected mice with an initial tumor volume of about 350 mm3. The mice were divided into six groups (n=5), including (1) control group with intravenous injection (i.v.) of physiological saline (100 μL), (2) Ce6 NPs+Light group with tail vein injection of 100 μL of 200 μM Ce6 NPs and irradiation of 635 nm 200 mW/cm2 laser for 15 min, (3) Porphyrin NPs+light group with tail vein injection of porphyrin (200 hy, , 100 hyrinn) and irradiation of 200 mW/cm2 laser for 15 min, (4) AN-BDP NPs+Light group with tail vein injection of 100 μL of 200 μM LAN-BDP NPs and irradiation of 635 nm 200 mW/cm2 laser for 15 min, (5) AN-BDP NPs+Light 2 group with tail vein injection of 100 μL of 200 μM AN-BDP NPs, and irradiations of 635 nm 200 mW/cm2 laser for 15 min for twice, and (6) AN-BDP NPs+Light 3 group with tail vein injection of 100 μL of 200 μM AN-BDP NPs, and irradiation of 635 nm 200 mW/cm2 laser for 15 min for three times. The fluorescence signal at the tumor site increased in a time-dependent manner, and the fluorescence intensity reached a maximum 8 hours later after injection of Ce6 and Porphyrin. Therefore, groups (2) and (3) received light irradiation (635 nm laser, 200 mW/cm2, 15 min) 8 hours later after tail vein injection. Group (4) received light irradiation (635 nm laser, 200 mW/cm2, 15 min) 24 hours later after tail vein injection of AN-BDPNPs. Group (5) received light irradiation respectively on the 1st and 3rd days after tail vein injection of AN-BDP NPs, and group (6) received light irradiation on the 1st, 3rd and 5th days after tail vein injection of AN-BDP NPs. Body weight and tumor volume were measured every 2 days for 18 days. Major organs (heart, liver, spleen, lung, and kidney) and tumors were collected after treatment, and examination by hematoxylin and eosin (H&E) staining or TUNEL staining and observation by fluorescence microscopy (EVOS XL Core, Life Technologies, USA) were performed for histological analysis.
In the present invention, the antitumor activity of AN-BDP NPs in the mice with tumors of conventional size (initially about 100 mm3) was first tested.
The present invention further evaluated the therapeutic performance of a large tumor model for AN-BDP NPs-injected mice with an initial tumor volume of about 350 mm3 (
(4) In Vivo Biosafety Evaluation
First, the biosafety of AN-BDP NPs was assessed based on blood analysis. The specific steps are as follows: 3 mL of fresh sterile defibrated sheep blood was added to 6 mL of a PBS buffer, centrifuging at 1500 rpm was performed for 8 min, and the supernatant was discarded until the supernatant was clear. Then the obtained erythrocytes were used to prepare a 2% erythrocyte suspension. Different concentrations of AN-BDP NPs were co-incubated with an equal volume of 2% erythrocyte suspension for 4 hours at 37° C. and 180 rpm. After the completion of incubation, complete erythrocytes were removed by centrifugation at 1500 rpm for 8 min, and the supernatant was taken to measure the absorbance at 545 nm with a microplate reader.
The test group is the erythrocyte suspension group treated with different concentrations of AN-BDP NPs; the negative control group is the erythrocyte suspension group incubated with a PBS buffer; the positive control group is the erythrocyte suspension group incubated with deionized water; and OD is the absorbance at 545 nm.
In the present invention, the safety was tested in vivo. Specific steps are as follows: the healthy mice were divided into four groups (n=5), including (1) control group with intravenous injection (i.v.) of physiological saline (100 ph), (2) light group irradiated with 200 mW/cm2 635 nm laser for 15 min, (3) AN-BDP NPs group with tail vein injection of AN-BDP NPs (200 v, 100 v), (4) AN-BDP NPs+Light group with tail vein injection of 100 μL of 200 μM AN-BDP NPs, and then 48 hours later with 200 mW/cm2 irradiation with 635 nm laser for 15 min. The whole blood was taken for analysis. The group with physiological saline was used for negative control.
The in vivo safety of AN-BDP NPs was also evaluated by observing the significant changes in body weights of each group of mice during the treatment period, and the tissue staining of major organs after the treatment.
Good biocompatibility is one of the necessary conditions for biomedical materials. First, blood analysis was made to assess the biosafety of AN-BDP NPs. The material with a hemolysis rate of less than 5% is considered to have good biocompatibility. In the present invention, no obvious hemolysis occurred (
Second, whole blood testing of the mice was also performed for the present invention, and all measured parameters were within a normal range (Table 1), indicating acute inflammation caused by AN-BDP NPs is negligible and the biocompatibility is good.
Notably, the mice in different groups gained weight slowly, but no additional side effects were observed throughout the experiment (
In conclusion, the present invention has demonstrated that strong π-π stacking-stabilized nanophotosensitizers are promising, which can prolong the blood circulation and tumor retention time of nanophotosensitizers during PDT. AN-BDP NPs exhibites excellent stability and may stay at the tumor sites for up to 10 days, which is longer than that of any other existing photosensitizers. In a mouse tumor model for routinely tested tumor volume size (about 100 mm3), single injection and single phototherapy can completely inhibit tumor growth. More importantly, the nanophotosensitizer of the present invention capable of prolonging tumor retention time may be successfully applied to a mouse tumor model of larger tumors (about 100 mm3) for treatment by means of single injection and multiple phototherapies. Our work highlights that rationally designed nanophotosensitizers may be used to achieve multiple PDTs through a single injection, and enhance the PDT effect for the treatment of large-volume tumors or malignant tumors. The present invention provides an available, simple and convenient method to prolong the tumor retention time of the photosensitizer, and further promote the clinical translation of nanomedicine.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111199305.6 | Oct 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/121296 | 9/26/2022 | WO |
