CN112603999A - Tumor microenvironment response type nanoparticles based on bionic engineering and preparation method and application thereof - Google Patents
Tumor microenvironment response type nanoparticles based on bionic engineering and preparation method and application thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the technical field of biomedicine, and particularly relates to a tumor microenvironment response type nanoparticle based on bionic engineering and a preparation method and application thereof. The nanoparticle comprises a chelate shell layer formed by tannic acid and ferric ions, a cancer cell membrane is also adsorbed outside the chelate shell layer, and a photosensitizer is loaded in the chelate shell layer. The nanoparticle improves the stability of a tumor microenvironment through cancer cell membrane modification, and the nanoparticle also has tumor targeting, so that the imaging and tumor treatment effects of the nanoparticle are improved. The nano-particle can be used as a T1 weighted nuclear magnetic resonance, photoacoustic imaging and photothermal imaging contrast agent, a photodynamic therapy medicament and a photothermal therapy medicament, and is widely applied to cancer treatment and diagnosis.
Description
Technical Field
The invention relates to the technical field of biomedicine, and particularly relates to a tumor microenvironment response type nanoparticle based on bionic engineering and a preparation method and application thereof.
Background
Photothermal therapy (PTT) is a promising therapeutic alternative to traditional radiotherapy and chemotherapy, and is also considered a tumor-specific therapeutic modality with high spatiotemporal selectivity and low toxicity. Photothermal therapy specifically refers to a photothermal conductive agent that converts light energy into heat energy and then raises the temperature at the tumor site to induce cancer cell death.
The material formed by chelating metal ions and tannic acid is a novel organic-inorganic hybrid material and has the advantages of high photo-thermal conversion efficiency, strong photo-thermal stability and good biocompatibility. Among them, nanoparticles prepared from chelates formed by iron ions and tannic acid have good photo-thermal properties in the Near Infrared (NIR) region, and show great potential in the preparation of nano-drugs. However, the exogenous or endogenous acidic tumor microenvironment (including endosomes having a pH of 5.5 to 6.0, lysosomes having a pH of 4.5 to 5.0, etc.) promotes the decomposition of the chelate complex formed by iron ions and tannic acid, which shows a pH-dependent decomposition behavior after reaching the tumor site, so that the chelate complex cannot stably exist at the tumor site, and its photothermal efficacy cannot be sufficiently exerted. In addition, the non-targeting property of the chelate formed by iron ions and tannic acid is an obstacle which must be overcome by an ideal antitumor drug.
Disclosure of Invention
The invention aims to provide a tumor microenvironment response type nanoparticle based on bionic engineering, the stability of the tumor microenvironment of the nanoparticle is improved through the modification of a cancer cell membrane, and the nanoparticle also has tumor targeting, so that the imaging and tumor treatment effects of the nanoparticle are improved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a tumor microenvironment response type nanoparticle based on bionic engineering comprises a chelate shell formed by tannic acid and ferric ions, and a cancer cell membrane is adsorbed outside the chelate shell.
By adopting the technical scheme, the technical principle is as follows: the chelate formed by the tannic acid and the ferric ion has good photothermal performance in a Near Infrared (NIR) region, and can be used as a contrast agent and a photothermal treatment drug to be applied to clinical practice. However, the chelate complex formed by tannic acid and ferric ion shows a pH-dependent decomposition behavior after reaching the tumor site, which is disadvantageous for its imaging effect and photothermal therapeutic effect. Meanwhile, a chelate shell formed by tannic acid and ferric ions is attached to a cancer cell membrane, so that tumor targeting is realized and the chelate shell is stabilized.
Attaching a specific cancer cell membrane to the outside of the nanoparticle is a means for targeting the nanoparticle to a specific tumor site in the prior art. The cancer cell membrane is attached to the outside of a chelate shell formed by tannic acid and ferric ions, so that the targeting function of the nanoparticle is realized. After the chelate particle with the cancer cell membrane attached to the surface is prepared, the nanoparticle is found to no longer present a pH-dependent decomposition phenomenon, so that the stability of the chelate formed by the tannic acid and the ferric ion is increased, and the imaging effect and the photothermal treatment effect of the nanoparticle can be better realized.
The invention has the beneficial effects that:
(1) the metal ion tannin material is a novel organic-inorganic hybrid material, which integrates ferric ions and a plurality of treatment mechanisms into a single nano platform. The nano particles in the technical scheme have the properties of photothermal imaging (PTI) and photoacoustic imaging (PAI), and can be used as a contrast agent to be applied to clinical practice.
(2) The nanoparticles in the technical scheme have the effect of photothermal therapy (PTT). After chelation of ferric ions and tannic acid, the compound has high photo-thermal conversion efficiency, and can generate thermal effect under the irradiation of 600-900nm laser, thereby realizing the photo-thermal treatment of tumors.
(3) The nano particles in the technical scheme contain ferric ions and have the function of enhancing nuclear magnetic resonance T1 weighted magnetic resonance imaging.
(4) The outer layer of the nano particle is attached with a cancer cell membrane, and a targeting function can be obtained without modifying a functional group. The cancer cell membrane also has the function of protecting the metal ion tannic acid from the erosion of the tumor microenvironment. And the cancer cell membrane has wide acquisition ways and can be easily extracted from almost all types of cancer cells.
Further, a photosensitizer is loaded in the chelate shell layer.
With the above technical solution, the use of a photosensitizer may initiate photodynamic therapy (PDT). Wherein the photosensitizer is a substance which can absorb radiation energy and generate photochemical change through excitation to generate a reactive intermediate (free radical or cation) with the polymerization initiating capability.
Further, the ferric ion is provided by ferric chloride.
By adopting the technical scheme, the ferric trichloride is a common chemical reagent, is easy to obtain and has low cost.
Further, the photosensitizer is protoporphyrin.
By adopting the technical scheme, the chelate consisting of ferric ions and tannic acid and PpIX have the effect of synergistically enhancing the apoptosis of cancer cells under the condition of simultaneously receiving 660nm laser radiation (photodynamic therapy) and 808nm laser radiation (photothermal therapy).
Further, the cancer cell membrane is from a breast cancer cell.
By adopting the technical scheme, the breast cancer is one of high-incidence cancers, and the detection and treatment effects on related tumors can be realized by adopting the breast cancer cell membrane to prepare the nano particles.
Further, a preparation method of the tumor microenvironment responsive nanoparticle based on the bionic engineering, which comprises the following steps:
step (1): dispersing a methanol solution of protoporphyrin in water; then under the ultrasonic action, sequentially adding a tannic acid solution and a ferric trichloride solution to obtain PFTNPs;
step (2): dissolving cancer cell membranes in water, adding PFTNPs in the step (1) to obtain a mixture, and performing ultrasonic dispersion treatment on the mixture to obtain MPFTNPs.
By adopting the technical scheme, the tannin and the ferric trichloride can form a chelate shell outside protoporphyrin, and then cancer cell membranes are attached to the outer layer of the chelate shell.
Further, in step (2), the cancer cell membrane is derived from MDA-MB-231 human breast cancer cells.
By adopting the technical scheme, the MDA-MB-231 human breast cancer cell is a common standard cell strain and is easy to obtain.
Further, the cancer cell membrane is prepared by the following method: and (3) carrying out freeze thawing treatment on the MDA-MB-231 human breast cancer cells for multiple times, and then centrifuging to obtain cancer cell membranes.
By adopting the technical scheme, the cells are cracked through repeated freeze thawing, and the required cell membranes are separated.
Further, an application of the tumor microenvironment responsive nanoparticle based on the bionic engineering in the preparation of the contrast agent.
By adopting the technical scheme, the ferric ions and the tannin chelate have the properties of photothermal imaging and photoacoustic imaging, and the ferric ions have the function of enhancing nuclear magnetic resonance T1 weighted magnetic resonance imaging.
Further, the application of the tumor microenvironment responsive nanoparticles based on the bionic engineering in preparing photodynamic therapy medicines or photothermal therapy medicines.
By adopting the technical scheme, the protoporphyrin can start photodynamic therapy, and has high photo-thermal conversion efficiency after chelation of ferric ions and tannic acid, and has the effect of photo-thermal treatment.
Drawings
FIG. 1 is a scanning electron micrograph of MPFTNPs in example 2 of the present invention;
FIG. 2 is a transmission electron microscope of MPFTNPs in example 2 of the present invention;
FIG. 3 is a photograph of MPFTNPs confocal laser microscopy in example 2 of the present invention;
FIG. 4 is a graph showing the particle size distribution of PFTNPs and MPFTNPs in examples 1 and 2 of the present invention;
FIG. 5 is a graph showing the potential distribution of PFTNPs and MPFTNPs in examples 1 and 2 of the present invention;
FIG. 6 shows the experimental results of confocal laser microscopy according to Experimental example 1 of the present invention;
FIG. 7 shows the results of the flow cytometry experiment of Experimental example 1 of the present invention;
FIG. 8 shows the results of the confocal laser test in Experimental example 2;
FIG. 9 shows photothermal effect curves (different particle concentrations) of Experimental example 3 of the present invention;
FIG. 10 is a photothermal effect curve (different irradiation intensities) of Experimental example 3 of the present invention;
FIG. 11 shows photothermal effect curves (different compositions) of Experimental example 3 of the present invention;
FIG. 12 is a graph showing the photo-thermal stability of MPFTNPs in Experimental example 3 of the present invention;
FIG. 13 is a photothermal curve of PFTNPs of Experimental example 4 of the present invention under different pH conditions;
FIG. 14 is a photothermal curve of MPFTNPs of Experimental example 4 of the present invention under different pH conditions;
FIG. 15 is a graph showing photothermal conversion efficiency of MPFTNPs of Experimental example 5 of the present invention;
FIG. 16 is the result of the in vitro MDA-MB-231 cytotoxicity assay (CCK-8) for MPFTNPs of Experimental example 6 of the present invention;
FIG. 17 shows the results of the in vitro MDA-MB-231 cytotoxicity assay (flow cytometry) for MPFTNPs of Experimental example 6 of the present invention;
FIG. 18 shows the results of in vitro cytotoxicity assays for MPFTNPs of Experimental example 7 of the present invention;
FIG. 19 shows the results of in vitro cytotoxicity assays of MPFTNPs of Experimental example 8 of the present invention;
FIG. 20 shows in vitro MRI images and quantitative analysis results of Experimental example 9 of the present invention;
FIG. 21 shows the result of in vivo MRI imaging in Experimental example 9 of the present invention;
FIG. 22 is a histogram of in vitro PAI signal intensity for Experimental example 10 of the present invention;
FIG. 23 is an in vivo PAI image of Experimental example 10 of the present invention;
FIG. 24 is an in vivo PTI image of Experimental example 11 of the present invention;
FIG. 25 is a graph showing tumor growth curves of different treatment groups in Experimental example 12 of the present invention;
FIG. 26 is a graph showing the body weight changes of experimental mice under different treatment conditions in Experimental example 12 of the present invention;
FIG. 27 is a graph showing the tumor inhibition rate under different treatment conditions in Experimental example 12 of the present invention;
FIG. 28 is a graph showing H & E staining of different tissues under different treatment conditions in Experimental example 12 of the present invention;
FIG. 29 is a SDS-PAGE analysis result chart of Experimental example 13 of the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1: preparation of PFTNPs
PpIX (protoporphyrin, 10mg) was first dissolved in 2mL of methanol to obtain a solution of PpIX in methanol. 200. mu.l of PpIX in methanol was added rapidly to deionized water (5-30 mL, 20mL used in this example), and 40. mu.l of TA solution (40mg/mL) and 40. mu.l of FeCl were added sequentially under continuous sonication (sonication power 50-100W, 100W used in this example)3.6H2O (10mg/mL) to obtain nanoparticles dispersed in the aqueous phase. Next, the nanoparticles were washed with deionized water and centrifuged at 10000rpm for 10min, and then portions of the nanoparticles were taken and neutralized with NaOH to obtain PFTNPs (i.e., PpIX @ FeIII/TA).
Example 2: preparation of MPFTNPs
Synthesis of PFTNPs
See example 1 for the preparation.
2. Cancer cell culture
MDA-MB-231 human breast cancer cells containing 5% CO at 37 deg.C2The culture medium was high-glucose DMEM medium supplemented with 10% FBS (v/v) and 1% penicillin streptomycin (v/v).
3. Extraction of cancer cell membranes
MDA-MB-231 cell membranes were extracted using a membrane protein extraction kit according to the instructions provided by the Biooitine Biotech company. Briefly, 1X 10 of the collected8The cells were resuspended in 3ml of membrane protein extract and 1mM PMSF was added. The cells were gently and fully suspended and then cultured in an ice bath for 15 min. The MDA-MB-231 cell suspension was frozen at-80 ℃ and then thawed three times. The obtained suspension was centrifuged at 700g for 10min, and the supernatant was centrifuged at 4 deg.C (14000rpm, 30min) to precipitate cell membrane debris, thereby obtaining MDA-MB-231 cell membranes. Finally, the precipitate was collected and lyophilized for further use. All cell membrane extraction processes were performed in an ice bath.
Preparation and characterization of MPFTNPs
Dissolving MDA-MB-231 cell membranes in deionized water, and carrying out ultrasonic treatment for 2.5min (ultrasonic intensity is 50W), wherein the concentration of the MDA-MB-231 cell membranes is 1mg/mL, so as to obtain a membrane solution. Then, 200mL of PFTNPs (prepared in step 1 of this example, 2mg/mL) were added to the membrane solution, and the mixture was sonicated for 2.5min (sonication intensity 50W) to obtain nanoparticles dispersed in the aqueous phase. And centrifuging at 4 deg.C (8000rpm, 10min), collecting the nanoparticle part, washing the nanoparticles with deionized water twice, and removing the excessive cell membranes to obtain cancer cell membrane modified PFTNPs (MPFTNPs). The morphology of the prepared MPFTNPs was observed with a transmission electron microscope (TEM, Hitachi H-7600, Japan) and a scanning electron microscope (SEM, Hitachi S3000N, Japan) (FIGS. 1 and 2). The MPFTNPs confocal laser microscopy picture is shown in FIG. 3. The particle size distribution and zeta potential of MPFTNPs were measured using a dynamic laser scattering particle sizer (ZEN3600, Malvern instruments, UK) (FIGS. 4 and 5, average particle size of PFTNPs 247.4nm, average particle size of MPFTNPs 295.3 nm; average potential of PFTNPs 32.3mV, average potential of MPFTNPs-15.4 mV).
The following is the experimental part of this patent, all data are expressed as mean Standard Deviation (SD), and the significance of differences between groups (p <0.05, p <0.01, p <0.001) was assessed by one-way anova and student's t-test.
Experimental example 1: cellular uptake performance of MPFTNPs
MDA-MB-231 cells, HepG2 cells and PANC-1 cells were seeded overnight in glass confocal dishes. MPFTNP solution at a concentration of 40. mu.g/mL was added, the cells were incubated for 1h, then the cancer cells were washed three times with fresh DMEM medium, stained with DAPI for 10min, and the cellular uptake performance of MPFTNPs was observed under CLSM (LSM710, Carl-Zeiss, Germany). The experimental result is shown in FIG. 6, which proves that MPFTNP has stronger targeting property on MDA-MB-231 cells.
And the cell uptake behavior was measured by flow cytometry (BD-FACSVantage SE, USA). Similar to the CLSM assay method, MDA-MB-231 cells, PANC-1 cells, and HepG2 cells were seeded in 6-well plates and incubated with MPFTNPs at a dose of 40 mg/mL. After 1h of incubation, cells were washed with DMEM medium and analyzed by flow cytometry. The experimental result is shown in FIG. 7, which proves that MPFTNP has stronger targeting property on MDA-MB-231 cells.
Experimental example 2: detection of reactive oxygen species levels
Intracellular ROS levels were first determined by CLSM (LSM710, Carl-Zeiss, Germany) using DCFH-DA. ROS-producing ability was compared in 6 groups (untreated control group, only 660nm laser group, PFTNPs +660nm laser group, MPFTNPs group, and MPFTNP +660nm laser group). MDA-MB-231 cells were seeded into 35mm confocal culture dishes (2X 10 cells per well)5Individual cells) for 8h, co-cultured with different nanopreparations overnight, then co-cultured with DCFH-DA at a concentration of 10. mu.M for 20min, and the experimental group to be irradiated was treated with a 660nm laser (100 mW. cm) after co-culture with DCFH-DA-2) After 5min of irradiation and 1h of incubation, cells were washed 3 times with fresh PBS and examined with CLSM. The experimental results are shown in FIG. 8, MPFTNPs +660nm laser set, PFTNPs +660nm laser set, MPFTNPs set, PFTNThe active oxygen generation amount of the Ps group, the laser group with only 660nm and the control group is in a descending trend, which shows that the targeting of PFTNPs is increased by the cancer cell membrane, the capability of promoting the increase of the intracellular ROS level by the nano particles is enhanced, and the ROS level increase can effectively kill cancer cells.
Experimental example 3: in vitro photothermal Properties of MPFTNPs
At a power density of 2.0 W.cm-2808nm laser irradiation of MPFTNPs at different concentrations (400, 200, 100, 50 and 25. mu.g/mL) in 96-well plates for 10min, and real-time temperature recording with an infrared camera (Fotric 226, Shanghai, China). The experimental results are shown in fig. 9, and the photothermal effect of MPFTNPs increases with increasing particle concentration.
MPFTNPs at a concentration of 400. mu.g/mL were irradiated at different intensities (2.0, 1.5, 1.0 and 0.5W-cm)-2) Irradiating for 10 min. Experimental results as shown in fig. 10, the photothermal effect of MPFTNPs increases with increasing irradiation intensity.
Further evaluation of the active ingredients of MPFTNPs: FeIIITA (400. mu.g/mL, nanoparticle of ferric iron and tannic acid), cancer cell membrane (1mg/mL), PpIX solution (400. mu.g/mL), TA solution (10mg/mL) and FeCl3The solution (10mg/mL) was added to a 96-well plate and the plate was irradiated with a laser (2.0W. cm) at 808nm-2) The thermal profile of the mixture was measured after 10min of irradiation. Experimental results as shown in fig. 11, the main source of photothermal effect of MPFTNPs is FeIIITA, and the presence of cancer cell membrane enhances the photothermal effect of FeIIITA.
To examine the photostability of MPFTNPs, MPFTNPs solutions were exposed to 808nm laser light (2.0W cm)-2) Under irradiation until the temperature of the MPFTNPs rises to 60 ℃. Subsequently, the MPFTNPs suspension was cooled to room temperature by turning off the 808nm laser. The laser switching program was repeated for 5 cycles, and the temperature change was monitored by an infrared camera to calculate the photothermal conversion efficiency. The experimental result is shown in fig. 12, the photo-thermal stability of the particles can be enhanced by combining the cancer cell membrane and PFTNPs, the MPFTNPs have better in-vitro photo-stability, and the phenomenon of photo-thermal effect reduction caused by acid hydrolysis and complexation of ferric iron and tannin can be overcome to a certain extent.
Experimental example 4: photothermal performance of PFTNPs and MPFTNPs in different pH environments
To study the photothermal properties of PFTNPs and MPFTNPs in different pH environments, 808nm laser was used at 2.0W cm-2The dispersions of nanoparticles (PFTNPs and MPFTNPs) at different pH values (pH 4.5, 5.5 and 7.0) were irradiated, the suspensions of PFTNPs and MPFTNPs were monitored in real time for 10min for temperature change, and the corresponding thermographic images were recorded. As shown in fig. 13 and 14, the stability of the MPFTNPs in a low pH environment is enhanced by the cancer cell membrane, and the MPFTNPs have a more stable and effective photothermal effect.
Experimental example 5: photothermal conversion efficiency of MPFTNPs
Add 200. mu.l MPFTNPs suspension to 96-well plate and use 808nm laser at 2.0W cm-2Is irradiated. The laser is then turned off after the temperature reaches steady state. The heating and cooling process was recorded using a thermal infrared imager as shown in fig. 15.η (photothermal conversion efficiency) of MPFTNPs was calculated according to the following formula:
in equation (1), ε represents the conversion efficiency value, TMax is the minimum equilibrium temperature, QS is the light absorption of distilled water, I is the laser energy (MW) of a 808nm laser, and the absorbance value of the MPFTNPs suspension is related to A λ by UV-Vis spectroscopy at 808 nm. The hS value is calculated according to equation (2).
In the formula (2), m represents the mass of water (200mg), and C represents the specific heat capacity (4.2J/(g. k)). The time constant (τ s) was calculated from the linear time-dependent data collected during cooling and was determined to be 213.3 s. From equation (2), hS is calculated to be 3.938X 10-3W/K。
Finally, η for MPFTNPs is calculated according to equation (1). Tmax was 36.5 ℃, Qs was 1.68mW, and I was 0.64 mW. The A lambda of the sample is 0.49 measured by 808nm ultraviolet-visible spectrum. The η of MPFTNPs under 808nm laser irradiation was calculated to be 33.2%.
Experimental example 6: study on PDT/PTT in vitro induced MDA-MB-231 cytotoxicity
To evaluate MPFTNPs, the following experimental groups were set up: control group, laser group (660nm +808nm), MPFTNPs group, MPFTNPs +660nm laser (100 mW. cm)-25min) group (as PDT group), MPFTNPs +808nm laser (2W. cm)-210min) group (as PTT group) and MPFTNPs +660nm laser (100 mW. cm)-25min) +808nm laser (2W cm)-210min) group (as PDT/PTT group). MDA-MB-231 cells were seeded in 96-well plates (10 per culture dish)4Individual cells) was cultured overnight. MDA-MB-231 cells treated with 100. mu.l MPFTNPs (100. mu.g/mL) were incubated in DMEM for an additional 4h, followed by various treatments as described above. Finally, after a total volume of 2h with 10. mu.l CCK-8 solution, cell viability was assessed with a microplate reader at an absorbance of 450 nm. Experimental results as shown in fig. 16, MPFTNPs have photodynamic and photothermal effects, and can achieve the effect of inhibiting cancer cell proliferation in vitro by PDT and/or PTT. Wherein PDT and PTT are used in combination to synergistically increase the therapeutic effect of MPFTNPs.
MDA-MB-231 cells were seeded in 6-well plates (2X 10 cells per well)5Individual cells) overnight, co-cultured with different nanopreparations (100. mu.g/mL) for 4h, then irradiated with a 660nm laser (100 mW. cm)-25min) and/or 808nm laser (2W cm)-210min) cells were further irradiated at room temperature and then detected after staining the cells with a flow cytometer. The experimental component groups are as follows: control group, laser group (660nm +808nm), MPFTNPs group, PFTNPs group, MPFTNPs +660nm laser (100 mW. cm)-25min) group (as PDT group), MPFTNPs +808nm laser (2W. cm)-210min) group (as PTT group) and MPFTNPs +660nm laser (100 mW. cm)-25min) +808nm laser (2W cm)-210min) group (as PDT/PTT group). As shown in FIG. 17, the results of the experiments demonstrated that the PDT/PTT group had the strongest apoptosis-promoting effect, and the PDT group had the second highest apoptosis-promoting effect, which was much different from those of the PDT/PTT group and the PTT group, while the MPFTNPs group and PFTNPs group had the smaller difference in apoptosis-promoting effect. 660nm laser (100mW cm)-25min) can excite the nano particle to generate photodynamic therapy effect (PpIX responds to 660nm laser to realize photodynamic therapy), 808nm laser (2W cm)-210min) can beActivating the photothermal therapy effect of the nano particles (the chelate formed by ferric ions and tannic acid responds to 808nm laser to realize photothermal therapy). The apoptosis rate of PDT group is 19.12%, the apoptosis rate of PTT group is 33.31%, and 660nm laser radiation and 808nm laser radiation (PDT/PTT group) are simultaneously carried out on cancer cells, the apoptosis rate reaches 57.1%, which is larger than the sum of the apoptosis rates of PDT group and PTT group, thus indicating that the chelate composed of ferric ion and tannic acid and PpIX have the effect of synergistically enhancing the cancer cell apoptosis under the condition of simultaneously receiving 660nm laser radiation and 808nm laser radiation.
Experimental example 7: in vitro cytotoxicity assay
MDA-MB-231 cells were seeded in 96-well plates (10 per well)4Individual cells) was cultured overnight. The 96-well plates were then loaded with different concentrations of PFTNPs and MPFTNPs (ranging from 6.25 to 400. mu.g/ML) and incubated for an additional 24 h. After incubation with 10. mu.L of CCK-8 solution for 2h, the cytotoxicity of PFTNPs and MPFTNPs was analyzed. As shown in fig. 18, PFTNPs at high concentration have strong cytotoxicity without laser treatment, while MPFTNPs have low cytotoxicity.
Experimental example 8: cell staining experiment
MDA-MB-231 cells were seeded into 35mm confocal culture dishes (2X 10 cells per dish)5Individual cells) overnight, co-cultured with different nanoparticies for 4h, then in an ice bath with a 660nm laser (100mW · cm)-25min) and/or 808nm laser (2W cm)-210min) cells were further irradiated at room temperature. Thereafter, the cells were incubated with calcein (2. mu.M) and PI (4. mu.M) at 37 ℃ with 5% CO2Co-culturing for 30min in humidified air. The experiment was set up with the following experimental groups: control group, laser group (660nm +808nm), MPFTNPs group, MPFTNPs +660nm laser (100 mW. cm)-25min) group (as PDT group), MPFTNPs +808nm laser (2W. cm)-210min) group (as PTT group) and MPFTNPs +660nm laser (100 mW. cm)-25min) +808nm laser (2W cm)-210min) group (as PDT/PTT group). Cells were washed three times with PBS and visualized with CLSM. Experimental results As shown in FIG. 19 the experimental results were consistent with those of Experimental example 6, and the PDT/PTT group hadHigh tumor cell killing effect.
In the following experimental examples, animal and tumor models were constructed as follows: female Balb/c nude mice (6 weeks old) were purchased from Beijing Fukang Biotech, Inc., China. Mice were injected subcutaneously on the right back with MDA-MB-231 cells (2X 10) dispersed in 100. mu.l PBS6) An MDA-MB-231 tumor-bearing model was prepared. When the tumor size reaches about 100mm2In vivo treatment experiments were performed using tumor-bearing mice.
Experimental example 9: in vitro and in vivo MRI performance
In vitro MRI experiments: t1 weighted MRI signals were measured in vitro for different concentrations of MPFTNPs. Then, MPFTNP solutions were prepared and diluted to the indicated concentrations (Fe concentrations: 1.6, 1.2, 0.8, 0.6, 0.4 and 0.3mM) to test the MRI performance of Eppendorf tubes. A 3.0T gradient echo sequence of a magnetic resonance imaging system (siemens medical system) is used to obtain a dilution concentration image and obtain a corresponding T1 relaxation time. The results of the experiment are shown in FIG. 20, and the MRI signal increases with increasing iron ion concentration.
In vivo MRI experiments: after tail vein injection of MPFTNPs (dose: 20mg/kg), MRI signals were collected for tumor and non-tumor tissue areas at predetermined time points (0, 2, 6 and 24 h). MRI parameter setting: gradient echo sequence (repetition Time (TR)/echo Time (TE))790/15ms, layer thickness 3.00mm, substrate size 320X 320. The experimental results are shown in figure 21, with increasing treatment time, the in vivo MRI signal increased significantly.
Experimental example 10: in vitro and in vivo PAI Performance
To investigate the potential of MPFTNPs as PAI contrast agents, PAI images at diluted concentrations were measured with a Vevo-LAZR photoacoustic imaging system (Visual sonic inc., toronto, canada). MPFTNPs and PFTNPs were tested at different pH values (pH 7.0, 5.5 and 4.5) and at fixed concentrations (450 and 600 μ g/mL). In the Vevo-LAZR photoacoustic imaging system, the signal intensity of a region of interest (ROI) was quantitatively analyzed using software. The experimental results are shown in fig. 22, and it is known from the experimental results that the stability of MPFTNPs in acidic environment is better than that of PFTNPs, indicating that the stability of MPFTNPs in cancer microenvironment is better.
PAI activity was assessed in MDA-MB-231 tumor-bearing nude mice. Mice were anesthetized with sodium pentobarbital, and then a solution of MPFTNPs (dose: 10mg/kg) or a solution of PFTNPs (dose: 10mg/kg) was injected intravenously. PAI images of the tumor are then acquired at predetermined time intervals (0, 2, 4, and 24h) and the corresponding PAI signals are measured. PAI parameters were set as follows: PAI gain: 42db of; depth of focus: 12 mm. Experimental results as shown in fig. 23, the in vivo photoacoustic signals of MPFTNPs were superior to PFTNPs and the imaging was enhanced with increasing processing time.
Experimental example 11: in vivo PTI Properties
In vivo performance of PTI was assessed in MDA-MB-231 tumor-bearing nude mice. Mice were anesthetized with sodium pentobarbital, followed by intravenous injection of MPFTNPs solution (dose: 20mg/kg) or PFTNPs solution (dose: 20 mg/kg). Under 808nm laser irradiation (2W cm)-25min), PTI images of the tumors were obtained 24h after injection. The experimental results are shown in fig. 24, and the photothermal imaging effect of the MPFTNPs is stronger than that of the PFTNPs.
Experimental example 12: in vivo PDT/PTT co-therapy
The tumor volume reaches 60mm3Thereafter, nude mice bearing MDA-MB-231 tumors were randomized into 6 groups (6 per group): (i) control group (treated with 5% glucose solution), (ii) laser only group (660nm laser, 100 mW. cm)-25 min; 808nm laser, 2W cm-25min), (iii) simple MPFTNPs group (20mg/kg), (iv) MPFTNPs +660nm laser (100 mW. cm)-25min) as PDT group, (v) MPFTNPs +808nm laser (2W. cm)-25min) as PTT group, (vi) MPFTNPs +660nm laser (100 mW. cm)-25min) +808nm laser (2W cm)-25min) as PDT/PTT group. The second, fourth and sixth groups use 660nm laser (100m W cm)-2) The tumor was irradiated for 5min (20 s after irradiation cooled to 30 ℃). Tumor volume and weight were recorded every other day for 2 weeks of observation period for each mouse. Tumor volumes were calculated as follows: length x width2/2. Changes in tumor volume are indicated by V/V0 (V0 was set to the initial tumor volume in the experiment). As shown in FIGS. 25, 26 and 27, the results of the experiments revealed that the PDT/PTT combination treatment had a strong tumor-inhibiting effect.
Each group was sacrificed 1 mouse 24H after treatment, and tumors and major organs (heart, liver, spleen, lung, kidney, brain) were preserved in 4% paraformaldehyde and H & E stained. Finally, tumor tissue was subjected to TUNEL and PCNA staining to evaluate tumor proliferation. The experimental results are shown in FIG. 28, and it is clear from the experimental results that the PDT/PTT synergistic treatment has a strong tumor suppression effect.
Experimental example 13: membrane protein analysis
The membrane protein of cell membrane (MDA-MB-231 human breast cancer cells) and MPFTNPs were analyzed by SDS-PAGE, and the results are shown in FIG. 29 (cell membrane lane for A and MPFTNPs lane for B), which proves that the MPFTNPs retain the membrane protein on the cell membrane surface.
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications 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 practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
1. A tumor microenvironment response type nanoparticle based on bionic engineering is characterized by comprising a chelate shell formed by tannic acid and ferric ions, and a cancer cell membrane is also adsorbed outside the chelate shell.
2. The tumor microenvironment responsive nanoparticle based on biomimetic engineering according to claim 1, wherein a photosensitizer is loaded in the chelate shell.
3. The tumor microenvironment responsive nanoparticle based on biomimetic engineering according to claim 2, wherein the ferric ions are provided by ferric chloride.
4. The biomimetic engineering based tumor microenvironment responsive nanoparticle according to claim 3, wherein the photosensitizer is protoporphyrin.
5. The biomimetic engineering based tumor microenvironment-responsive nanoparticle according to claim 4, wherein the cancer cell membrane is derived from breast cancer cells.
6. The preparation method of the tumor microenvironment responsive nanoparticle based on the biomimetic engineering according to claim 5, comprising the following steps:
step (1): dispersing a methanol solution of protoporphyrin in water; then under the ultrasonic action, sequentially adding a tannic acid solution and a ferric trichloride solution to obtain PFTNPs;
step (2): dissolving cancer cell membranes in water, adding PFTNPs in the step (1) to obtain a mixture, and performing ultrasonic dispersion treatment on the mixture to obtain MPFTNPs.
7. The method of claim 6, wherein in step (2), the cancer cell membrane is derived from MDA-MB-231 human breast cancer cells.
8. The method of claim 7, wherein the cancer cell membrane is prepared by: and (3) carrying out freeze thawing treatment on the MDA-MB-231 human breast cancer cells for multiple times, and then centrifuging to obtain cancer cell membranes.
9. Use of the tumor microenvironment responsive nanoparticle based on biomimetic engineering according to any of claims 1-5 in the preparation of a contrast agent.
10. The application of the tumor microenvironment responsive nanoparticle based on biomimetic engineering in preparation of photodynamic therapy drugs or photothermal therapy drugs.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113181211A (en) * | 2021-05-12 | 2021-07-30 | 南京大学深圳研究院 | Fe2O3@ TA-Pt nano composite material and preparation method and application thereof |
| CN113577306A (en) * | 2021-07-13 | 2021-11-02 | 中国科学院长春应用化学研究所 | Preparation of double-targeting pH stimulus-responsive nano particles and application of nano particles in tumor diagnosis and treatment |
| CN115192544A (en) * | 2022-07-12 | 2022-10-18 | 北京大学 | Iron chelate nanoparticle for inducing iron death and preparation and application thereof |
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2020
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Non-Patent Citations (1)
| Title |
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| BIN QIAO等: ""Artificial Nanotargeted Cells with Stable Photothermal Performance for Multimodal Imaging-Guided Tumor-Specific Therapy"", 《ACS NANO》, vol. 14, 28 September 2020 (2020-09-28), pages 1 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113181211A (en) * | 2021-05-12 | 2021-07-30 | 南京大学深圳研究院 | Fe2O3@ TA-Pt nano composite material and preparation method and application thereof |
| CN113577306A (en) * | 2021-07-13 | 2021-11-02 | 中国科学院长春应用化学研究所 | Preparation of double-targeting pH stimulus-responsive nano particles and application of nano particles in tumor diagnosis and treatment |
| CN113577306B (en) * | 2021-07-13 | 2023-02-28 | 中国科学院长春应用化学研究所 | Preparation of double-targeting pH stimulus-responsive nano particles and application of nano particles in tumor diagnosis and treatment |
| CN115192544A (en) * | 2022-07-12 | 2022-10-18 | 北京大学 | Iron chelate nanoparticle for inducing iron death and preparation and application thereof |
| CN115192544B (en) * | 2022-07-12 | 2024-01-02 | 北京大学 | Iron chelate nanoparticle for inducing iron death and preparation and application thereof |
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