CN113559084B - Drug-loaded ultra-small ferroferric oxide nanocluster based on micro-fluidic chip and preparation method and application thereof - Google Patents

Drug-loaded ultra-small ferroferric oxide nanocluster based on micro-fluidic chip and preparation method and application thereof Download PDF

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CN113559084B
CN113559084B CN202110779874.1A CN202110779874A CN113559084B CN 113559084 B CN113559084 B CN 113559084B CN 202110779874 A CN202110779874 A CN 202110779874A CN 113559084 B CN113559084 B CN 113559084B
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CN113559084A (en
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杨瑞
史向阳
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Donghua University
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Abstract

The invention relates to a drug-loading ultra-small ferroferric oxide nanocluster based on a microfluidic chip and a preparation method and application thereof. The method comprises the following steps: preparing ultra-small ferroferric oxide nanoparticles, preparing ultra-small ferroferric oxide nanoclusters, preparing 4T1 cell membrane vesicle suspension, preparing a microfluidic chip, and preparing the drug-loaded ultra-small ferroferric oxide nanoclusters coated by cell membranes. The method takes a microfluidic chip as a reactor to prepare the cell membrane coated drug-loaded ultra-small ferroferric oxide nanocluster, has good biological safety, specifically targets a tumor area, selectively delivers the drug and realizes T 2 /T 1 The magnetic resonance imaging with the dual-mode conversion realizes the photothermal therapy, the chemical dynamic therapy and the chemotherapy three-mode combined therapy of the tumor, and has potential application prospect in the aspect of cancer diagnosis and treatment.

Description

Drug-loading ultra-small ferroferric oxide nanocluster based on micro-fluidic chip and preparation method and application thereof
Technical Field
The invention belongs to the field of nano medical diagnosis and treatment materials and preparation and application thereof, and particularly relates to a drug-loaded ultra-small ferroferric oxide nanocluster based on a microfluidic chip and a preparation method and application thereof.
Background
Recent data published by the world health organization show that cancer remains the major fatal disease facing all humans, with both morbidity and mortality rates on the rise. However, early diagnosis and treatment of cancer can greatly improve the cure rate. Surgical resection, radiotherapy and chemotherapy are the main treatment means of cancer at present, and have certain problems. Patients are at risk of recurrence if the surgical resection is incomplete and require long-term clinical observation after surgery; radiotherapy and chemotherapy are associated with significant side effects due to lack of targeting. Thanks to the rapid development of nanotechnology, various types of nanomaterials have been developed for the research of tumor diagnosis and treatment integration. The multifunctional nano material integrating multiple imaging and treatment means is expected to be used for accurate detection and treatment of cancers, and provides a chance for early diagnosis and treatment of tumors.
Ultra-small ferroferric oxide (Fe) 3 O 4 ) The nano-particles are used as an FDA approved nano-material and have good biocompatibility and T 1 Magnetic Resonance Imaging (MRI) features. Interestingly, when the ultra-small ferroferric oxide nanoparticles are agglomerated to further form the nanoclusters with larger sizes, the ultrafine ferroferric oxide nanoparticles are converted into the ultrafine ferroferric oxide nanoparticles with T 2 Contrast agents with magnetic resonance imaging properties, whereby a dynamic T-conversion from ultra-small ferroferric oxide nanoclusters to ultra-small ferroferric oxide nanoparticles can be achieved 2 /T 1 Bimodal MRI (Liang Jia, et al, Nano Today,2021,36, 101022). Because the Glutathione (GSH) content of the tumor microenvironment is higher, the ultra-small ferroferric oxide nano particles can be connected into clusters by utilizing disulfide bonds responded by the GSH. The disulfide bond is broken in a high-concentration GSH tumor microenvironment, and clusters are re-dispersed into single ultra-small ferroferric oxide nano particles, so that GSH in tumor cells is consumed, the tumor cells are sensitive to active oxygen (ROS), and the specificity T of tumor parts is realized 2 /T 1 Bimodal magnetic resonance imaging and drug delivery. Meanwhile, in a slightly acidic tumor microenvironment, the ultra-small ferroferric oxide nanoparticles can release iron ions to react with H in the tumor microenvironment 2 O 2 In effect, Reactive Oxygen Species (ROS) are generated by Fenton reaction (Fenton), resulting in the implementation of chemokinetic therapy (CDT) (Keyi Luo, et al. ACS appl. mater. interfaces,2020,12, 22650-.
The enhanced penetration and retention Effect (EPR) of the nanoparticles in tumor tissues can realize the enrichment of the nanomaterials at tumor sites. However, current nano-delivery systems for in vivo applications still face problems with immune clearance, protein adhesion and lack of targeting. Research shows that immune escape protein and homologous targeting protein exist on the surface of cancer cell membrane, which can promote immune escape (Mingjun Xuan, et al. Natl. Sci. Rev.,2019,6, 551-. In order to further improve the targeting property and delivery efficiency of the medicine and reduce the toxicity to normal tissues and cells, the specific cancer cell membrane is coated on the surface of the nano-carrier, thereby realizing the precise imaging and homologous targeted therapy of the tumor part and having wide prospect in the field of personalized therapy of cancer.
In recent years, nanomaterials for cancer diagnosis and treatment have been the hot point of research, but the clinical transformation of nanomaterials is hindered. One of the main reasons is that reproducible synthesis of nanomaterials with the same properties and sufficient amounts still presents difficulties. Microfluidics as a highly crossed science and technology enables precise control of the reaction sequence in terms of material preparation and the generation of nanomaterials with defined dimensions and morphology (Xin Zhao, et al.small,2019,16, 1901943). Because the reaction environment in the microfluidic chip is uniform, the production efficiency and monodispersity of the nano material prepared by the microfluid are far higher than those of the traditional method. In addition, the structure and the function of the prepared nano material can be flexibly controlled by designing and customizing the micro-channel, introducing a specific physicochemical process and combining a functional agent. Therefore, the microfluid technology provides a good platform for synthesizing high-quality multifunctional nano materials, and is expected to promote the development and clinical transformation of nano medicine.
The retrieval of domestic and foreign documents and patent results shows that: cell membrane coated cisplatin-loaded GSH (glutathione) response type ultra-small ferroferric oxide nanocluster prepared based on microfluidic chip and used for dynamic T of tumor 2 /T 1 A bimodal MRI-guided photothermal therapy/chemokinetic therapy/chemotherapy triple-modal combination therapy method is not reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a drug-loaded ultra-small ferroferric oxide nanocluster based on a microfluidic chip, and a preparation method and application thereof, so as to overcome the defects that the preparation of the traditional wet chemical method in the prior art is not easy to control, the product appearance is irregular, the repeatability is poor, and false positive is easy to appear in single-mode imaging of tumors.
The invention provides a drug-loaded ultra-small ferroferric oxide nanocluster based on a micro-fluidic chip, which is characterized in that activated ultra-small ferroferric oxide nanoparticles are reacted with cystamine dihydrochloride to obtain the ultra-small ferroferric oxide nanocluster, the ultra-small ferroferric oxide nanocluster is mixed with dopamine hydrochloride solution and subjected to ultrasonic treatment, then the mixture is injected into a first sample inlet of the micro-fluidic chip, 4T1 cell membrane vesicle suspension is injected into a second sample inlet of the micro-fluidic chip, Tris buffer solution is injected into a third sample inlet of the micro-fluidic chip, cis-platinum water solution is injected into a fourth sample inlet of the micro-fluidic chip, and the generated drug-loaded ultra-small ferroferric oxide nanocluster coated by a cell membrane is collected through a sample outlet.
The invention also provides a preparation method of the drug-loaded ultra-small ferroferric oxide nanocluster based on the microfluidic chip, which comprises the following steps:
(1) dissolving ferric salt in a solvent, performing ultrasonic treatment, adding sodium citrate, stirring, adding anhydrous sodium acetate, continuously stirring, performing solvothermal reaction, cooling, centrifuging, and drying to obtain the Fe nanoparticles of ultra-small ferroferric oxide 3 O 4 NPs；
(2) The ultra-small ferroferric oxide nano-particles Fe in the step (1) are treated 3 O 4 NPs are dispersed in ultrapure water, activated by EDC and NHS, then dripped into cystamine dihydrochloride solution for reaction, and dialyzed to obtain the ultra-small ferroferric oxide nanocluster Fe 3 O 4 NCs solution;
(3) centrifuging 4T1 cells, adding hypotonic cell lysate into the obtained 4T1 cell sediment, repeatedly freezing and thawing and crushing, extracting 4T1 cell membranes by gradient centrifugation, suspending in PBS solution to obtain 4T1 cell membrane suspension, and extruding to obtain 4T1 cell membrane vesicle suspension;
(4) designing an S-shaped microfluidic channel structure comprising five sample inlets and one sample outlet and gradually expanding, printing a mask, photoetching and preparing a mold on a silicon wafer, reversing the mold, taking a slide glass as a substrate of the obtained microfluidic channel cover sheet, and carrying out plasma bonding to obtain a microfluidic chip;
(5) the ultra-small ferroferric oxide nanocluster Fe in the step (2) 3 O 4 Mixing the NCs solution with the dopamine hydrochloride solution, performing ultrasonic treatment, injecting the mixture into a first sample inlet of the microfluidic chip in the step (4), injecting the 4T1 cell membrane vesicle suspension in the step (3) into a second sample inlet of the microfluidic chip in the step (4), injecting a Tris buffer solution into a third sample inlet of the microfluidic chip in the step (4), injecting a cis-platinum aqueous solution into a fourth sample inlet of the microfluidic chip in the step (4), and collecting the generated cell membrane coated drug-loaded ultra-small ferroferric oxide nanocluster FDPC through a sample outlet.
Preferably, in the above method, in the step (1), the ferric salt is ferric chloride; the solvent is diethylene glycol.
Preferably, in the method, the ratio of the iron salt, the solvent, the sodium citrate and the anhydrous sodium acetate in the step (1) is 0.60-0.66 g, 36-42 mL, 0.46-0.48 g and 1.3-1.4 g.
Preferably, in the method, the ultrasonic treatment in the step (1) is carried out for 20-40 min until the iron salt is completely dissolved.
Preferably, in the above method, the stirring in step (1) is: stirring for 1-3 h at 75-85 ℃ in an air atmosphere.
Preferably, in the method, the solvothermal reaction temperature in the step (1) is 180-220 ℃, and the solvothermal reaction time is 3-5 hours.
Preferably, in the above method, the ultra-small ferroferric oxide nanoparticles Fe in the step (2) 3 O 4 The mass ratio of NPs to EDC to NHS to cystamine dihydrochloride is 40-60: 196 to 216: 114 to 134: 88.5 to 108.5.
Preferably, in the above method, the reaction temperature in the step (2) is room temperature, and the reaction time is 2-4 days
Preferably, in the above method, the EDC and NHS activation in the step (2) is: firstly adding EDC, stirring and reacting for 0.5-1.5 h, then adding NHS, and stirring and reacting for 2-4 h.
Preferably, in the above method, the ratio of 4T1 cells to hypotonic cell lysate in step (3) is 10 7 2-4 mL.
Preferably, in the above method, the volume fraction of PMSF in the hypotonic cell lysate in step (3) is 10%.
Preferably, in the above method, the process parameters of the repeated freeze-thaw disruption in the step (3) are as follows: and (3) quickly freezing at the temperature of minus 10 to minus 30 ℃, quickly thawing at the temperature of 30 to 50 ℃, and repeating for 2 to 4 times.
Preferably, in the above method, the gradient centrifugation in step (3) is: centrifuging at the centrifugal temperature of 4 ℃ for 5-15 min by a centrifugal force of 600-800 g, removing the precipitate, leaving the supernatant, centrifuging at the rotating speed of 12000-14000 rpm for 20-40 min, removing the supernatant, and leaving the precipitate.
Preferably, in the above method, the extruding in the step (3) is: the 4T1 cell membrane suspension was extruded 8-14 times using an Avanti micro extruder.
Preferably, in the above method, the protein concentration of the 4T1 cell membrane vesicle suspension in the step (3) is 1.8-2.2 mg/mL.
Preferably, in the method, the temperature of the PDMS inverse mold in the step (4) is 75-85 ℃.
Preferably, in the above method, the process parameters of plasma bonding in step (4) are: the vacuum degree is 18-22 Pa, and the bonding treatment time in the air is 70-90 s.
Preferably, in the above method, in step (4), all the microfluidic channels have a height of 50 μm, the widths of the microfluidic channels of the first sample inlet, the second sample inlet and the third sample inlet are 100 μm, the widths of the rest of the microfluidic channels are 300 μm, and the total length from the inlet to the outlet of the channel is 37.8 mm.
Preferably, in the above method, the ultra-small ferroferric oxide nanoclusters Fe in the step (5) 3 O 4 The mass ratio of NCs to dopamine hydrochloride to 4T1 cell membrane vesicles to cisplatin is 6-8: 2-3: 0.5-1.5: 9-11.
Preferably, in the above method, the ultra-small ferroferric oxide nanocluster Fe in the step (5) 3 O 4 The concentration of the NCs solution is 1-2 mg/mL.
Preferably, in the method, the concentration of the dopamine hydrochloride solution in the step (5) is 10-20 mg/mL.
Preferably, in the above method, the concentration of the cisplatin aqueous solution in the step (5) is 0.6-2.4 mg/mL.
Preferably, in the above method, the concentration of Tris buffer in the step (5) is 10mM, and the pH is 8.5.
Preferably, in the method, the flow rates of the first sample inlet, the second sample inlet, the third sample inlet and the fourth sample inlet in the step (5) are respectively 4-6 mL/h, 0.2-1 mL/h, 20-35 mL/h and 8-9.5 mL/h.
Preferably, in the above method, the injecting in step (5) is performed by using a micro-syringe pump.
The invention also provides T responsive to GSH preparation of the drug-loaded ultra-small ferroferric oxide nanocluster 2 /T 1 Application in bimodal nuclear magnetic resonance imaging contrast and multimodal tumor cell proliferation inhibiting medicines.
The invention synthesizes ultra-small ferroferric oxide nano particles (Fe) with stable surface citric acid by a solvothermal method 3 O 4 NPs) activating carboxyl, then taking cystamine (Cys) as a connecting agent to obtain GSH-responsive ultra-small ferroferric oxide nanoclusters, then taking the ultra-small ferroferric oxide nanoclusters, dopamine hydrochloride, 4T1 cell membrane vesicles, Tris buffer solution and cisplatin as raw materials, and performing one-step reaction by a microfluidic chip technology through controlling flow rate ratio to obtain the cell membrane-coated drug-loaded ultra-small ferroferric oxide nanoclusters (FDPC). The cell membrane can enhance the biocompatibility and targeting property of the nano material. Coated on Fe 3 O 4 Polydopamine (PDA) on the surface of NCs has good photo-thermal property, and phenolic hydroxyl on the surface of PDA can be coordinated with cisplatin as a chemotherapeutic drug. Fe 3 O 4 Disulfide bonds in NCs are destroyed by GSH in the tumor microenvironment and redistributed to Fe 3 O 4 NPs, T for in vivo tumor models 2 /T 1 Bimodal MRI. At the same time, Fe 3 O 4 NPs can release iron ions in acidic tumor microenvironment for chemodynamic therapy (CDT) of tumors, thereby realizing T 2 /T 1 Bi-modal MRI guided photothermal/chemodynamic/chemotherapyTrimodal tumor targeted therapy.
The physical and chemical properties of the prepared cell membrane-wrapped drug-loaded ultra-small ferroferric oxide nanocluster (FDPC) are characterized by means of ultraviolet visible absorption spectroscopy (UV-Vis), Zeta potential and dynamic light scattering analysis (DLS), infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), a Transmission Electron Microscope (TEM), inductively coupled plasma emission spectroscopy (ICP-OES) and the like. Determination of T of cell membrane-wrapped drug-loaded ultra-small ferroferric oxide nanoclusters (FDPCs) by means of nuclear magnetic resonance imaging analyzer 2 /T 1 Bimodal imaging performance. Then, the cytotoxicity of the membrane-wrapped drug-loaded ultra-small ferroferric oxide nanocluster (FDPC) is evaluated by a CCK-8 method, the chemical kinetic treatment effect of the membrane-wrapped drug-loaded ultra-small ferroferric oxide nanocluster (FDPC) is verified by a laser Confocal microscope (Confocal), the apoptosis influence of the membrane-wrapped drug-loaded ultra-small ferroferric oxide nanocluster (FDPC) on cancer cells is detected by a flow cytometer, and the photo-thermal heating condition of the tumor position of a tumor-bearing nude mouse is observed by in-vivo thermal imaging. Finally, a nude mouse subcutaneous tumor model of 4T1 is constructed, and the T of FDPC is examined 2 /T 1 Bimodal magnetic resonance imaging and photothermal therapy/chemokinetic therapy/chemotherapy triple-modal combined treatment effect. The specific test results are as follows:
zeta potential and hydrodynamic diameter test results
Referring to the specification and attached table 1, respectively, the ultra-small ferroferric oxide nano particles (Fe) 3 O 4 NPs), ultra-small ferriferrous oxide nanocluster (Fe) 3 O 4 NCs), drug-loaded ultra-small ferroferric oxide nanoclusters (FDPs), and hydrodynamic diameter and potential change of the drug-loaded ultra-small ferroferric oxide nanoclusters (FDPCs) coated with cell membranes. As shown in the attached Table 1, Fe 3 O 4 The potential of NPs was-32.1 mV, and the hydrated particle size was 154.4 nm. Coupling of Fe with cystamine 3 O 4 After NPs, the potential changed to-21.3 mV and the hydrated particle size increased to 235.0nm, demonstrating Fe 3 O 4 Successful synthesis of NCs. When Fe 3 O 4 After NCs encapsulated polydopamine and the chemotherapeutic drug cisplatin loaded, the potential further increased to-2.8 mV and the hydrated particle size changed to 222.3nm, which is Fe due to polydopamine encapsulation 3 O 4 The NCs hydrated particle size decreased, indicating successful encapsulation of dopamine and successful loading of cisplatin. After coating the cell membrane, the potential was changed to-16.3 mV and the hydrated particle size was changed to 233.6nm, indicating successful coating of the cell membrane on the FDPC surface. As shown in FIG. 2, the hydrodynamic diameter of FDPC in water, PBS solution and DMEM medium can be constant for a long time, demonstrating that FDPC has good colloidal stability.
2. Ultraviolet (UV-Vis) test results
Referring to the attached figure 3 in the specification, A is Fe respectively 3 O 4 NPs、Fe 3 O 4 The ultraviolet-visible absorption spectrograms of NCs, FDP and FDPC are used for observing the characteristic absorption peak of polydopamine in the near infrared region of 600-800nm, thereby proving the successful package of polydopamine. As can be seen from the figure, the UV absorption of the 280nm protein increased after the cell membrane was encapsulated, indicating successful encapsulation of the cell membrane.
3. Infrared (FTIR) test results
Referring to B in the attached figure 3 of the specification, which is Fe respectively 3 O 4 NPs、Fe 3 O 4 Infrared spectra of NCs, FDP, FDPC, 591cm as shown in B of figure 3 -1 The characteristic absorption peak nearby is attributed to Fe-O of the ultra-small ferroferric oxide nano-particles, 557cm -1 The nearby characteristic absorption peak is attributed to S-S of cystamine, proving that Fe 3 O 4 Successful synthesis of NCs. 2923 and 2850cm -1 Nearby enhanced characteristic absorption peaks are C-H stretching and bending vibration characteristic peaks of cystamine, polydopamine and membrane protein, 1465cm -1 The nearby characteristic peak is attributed to-C ═ C-stretching vibration of polydopamine and membrane proteins, further demonstrating successful encapsulation of polydopamine and cell membranes.
X-ray photoelectron Spectroscopy (XPS) test results
Referring to C and D in the attached figure 3 of the specification, C in the attached figure 3 is Fe respectively 3 O 4 NPs、Fe 3 O 4 X-ray photoelectron spectroscopy of NCs, FDP, FDPC. Wherein, in Fe 3 O 4 NPs、Fe 3 O 4 XP of NCs, FDP, FDPCFe element is observed in the S map, and the strength of Fe binding energy is in a descending trend, which is due to Fe 3 O 4 The NPs surface is sequentially modified by cystamine, wrapped by polydopamine and coated by cell membranes. In Fe 3 O 4 No N element was observed in XPS spectra of NPs, but in Fe 3 O 4 N element is observed in XPS spectra of NCs, FDP and FDPC, and the strength of N binding energy is in an increasing trend, further showing that Fe 3 O 4 The NPs surface is sequentially modified by cystamine, wrapped by polydopamine and coated by cell membranes. Pt element was observed in XPS spectra of FDP and FDPC, demonstrating successful loading of Pt. In FIG. 3, D is the Fe 2p high resolution XPS spectrum of FDPC, 710.52, 712.84, 724.08, 726.65, 715.17, 719.14, 729.49 and 733.21eV correspond to Fe respectively 2+ 2p 3/2 、Fe 3+ 2p 3/2 、Fe 2+ 2p 1/2 、Fe 3+ 2p 1/2 、Fe 2+ 2p 3/2 satellite、Fe 3+ 2p 3/2 satellite、Fe 2+ 2p 1/2 satelite and Fe 3+ 2p 1/2 satelite, which demonstrates Fe in FDPC 3 O 4 Is present.
Results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
See figure 4 for the SDS-PAGE results of 4T1 Cell Membranes (CM), FDPC and FDP. Protein content in the cell membrane suspension and FDPC is determined by using a BCA protein quantitative kit, and then PBS is used for regulating the protein content to be 1 mg/mL. Add 5. mu.L of protein Marker to the first protein lane, and then add 20. mu.L of cell membrane suspension (CM), FDPC and FDP (200. mu.g in 1mL PBS) to the lanes sequentially, set the gel-running current at 100A for 30 min. As shown in fig. 4, the FDP group did not run out of lanes with protein streaks, whereas the CM and FDPC groups ran out of similar protein streaks, demonstrating successful coating of the cell membrane on the FDP surface.
TEM test results
See specification A, B, C in fig. 5 for TEM images of FDPC at different magnifications, respectively. As can be seen from A in FIG. 5, FDPC has a very uniform size distribution with an average particle size of 205.5nm and good dispersibility. It can be clearly observed from fig. 5B and C that a cell membrane with a thickness of 9.8nm exists at the periphery of FDPC, the particle size of the polydopamine-coated ultra-small ferroferric oxide nanocluster residing in the core is 17.6nm, and the particle size of the inner-coated ultra-small ferroferric oxide nanocluster is 3.0 nm. Taken together, the successful synthesis of FDPC is demonstrated.
7. Material T 1 And T 2 Relaxation Performance test results
See description FIG. 6, wherein A and B are respectively FDPC T in the presence and absence of GSH (10mM) 1 And T 2 Graph of change in relaxation behavior. As shown in FIGS. 6A and B, r of FDPC without GSH 2 Is 20.06mM -1 s -1 ，r 1 Is 1.05mM -1 s -1 At this time, r 2 /r 1 Greater than 5 indicates that FDPC has a good T in the absence of GSH 2 Imaging performance; and r of FDPC in the presence of GSH 2 Reduced to 3.73mM -1 s -1 ，r 1 Increased to 2.22mM -1 s -1 At this time, r 2 /r 1 Less than 5 indicates that FDPC has good T in the presence of GSH 1 And (4) imaging performance. From the above, it can be demonstrated that FDPC has good T with GSH as the response mechanism 2 /T 1 The bimodal MRI performance can be used as good T in MRI molecular imaging diagnosis 2 /T 1 A bimodal contrast agent.
8. In vitro photothermal performance test results
Referring to A in FIG. 7 of the specification, temperature rise of FDPC at different concentrations (25, 50, 100. mu.g/mL) under 808nm laser irradiation was examined. At a certain power density (1W/cm) 2 ) The warming effect of FDPC increases gradually with increasing concentration, with a concentration of 100. mu.g/mL, the temperature of FDPC increases by 20.6 ℃ and the water increases by only 0.9 ℃. Referring to B of the specification, FIG. 7, the photo-thermal stability of FDPC (100. mu.g/mL) was tested by five cycles of temperature increase and decrease, using 808nm laser irradiation for 5min to complete a temperature increase process, and then turning off the laser to cool to the initial temperature to complete a temperature decrease process. It can be seen from the figure that the temperature change between five cycles is not obviousThe difference shows that the prepared FDPC has good photo-thermal stability. According to the temperature-rising cooling curve shown in the specification and shown in the figure 7C, the relation between the cooling time D shown in the figure 7 and the opposite number of the natural logarithm of the driving temperature is obtained, the heat transfer coefficient of the FDPC is 171s through linear fitting, and the photothermal conversion efficiency is calculated to be 46.2% according to the formula.
9. Drug release test results
Referring to fig. 8, a and B in the specification are drug release curves of FDPC under different conditions (pH 7.4, pH 6.5, pH 7.4+ GSH, pH 6.5+ GSH), respectively, Fe and Pt released amounts at different time points are detected by ICP-OES, and cumulative release rates are calculated according to initial Fe and Pt contents. As can be seen from a in fig. 8, the cumulative amount of Fe released at pH 7.4 is only 5.8%, whereas the cumulative amount of Fe released in the presence of GSH (10mM) can be increased to 6.3% due to the fact that GSH accelerates the dissociation of the ultra-small magnetite clusters into individual magnetite particles. At pH 6.5, the cumulative amount of Fe released increased to 11.9%, since Fe accelerated under weakly acidic conditions 3 O 4 And the release of medium Fe is realized, and the cumulative release amount of Fe is increased to 13.0% under the condition of GSH based on the dissociation effect of GSH on the ultra-small ferroferric oxide cluster. As can be seen from B in fig. 8, the cumulative amount of released Pt was only 11.5% at pH 7.4, and increased to 27.6% at pH 6.5, due to hydrogen ions (H) under acidic conditions + ) Will interact with the lone electron pair of oxygen atoms in the hydroxyl group of catechol on the surface of polydopamine to result in [ Pt (H) 2 O) 2 (NH 2 ) 2 ] 2+ And (4) separating to realize acid response release of Pt. And the cumulative release of Pt increased slightly to 29.3% in the presence of GSH, which benefits from GSH changing or even destroying the polymeric state of polydopamine.
10. Results of cytotoxicity test
See the description and figure 9 for the CCK-8 cell viability experiment of 4T1 cells under different treatment modes. From the CCK-8 results, it can be seen that at the same Pt concentration, FDPC exhibits stronger cytotoxicity than free Pt and FDP due to the targeting effect of the cell membrane, which can enhance the uptake of nanomaterials by cells. Meanwhile, at the same Pt concentration, both the FDP + NIR and FDPC + NIR experimental groups showed stronger cytotoxicity than the group without 808nm laser irradiation, which is due to the good photothermal properties of polydopamine. In summary, the photothermal/chemodynamic/chemotherapy-integrated FDPC + NIR group had the strongest killing effect on 4T1 cells.
11. Results of intracellular GSH content assay
See description figure 10, in which a is the result of the measurement of intracellular GSH content. PBS was used as a blank control group, PBS + NIR and FDP, FDP + NIR, FDPC and FDPC + NIR with the same iron concentration were used as experimental groups, and after co-incubation with 4T1 cells in an incubator for 4h, fresh medium was changed, wherein the PBS + NIR, FDP + NIR and FDPC + NIR groups were irradiated with laser light for 5min (808nm, 1.0W/cm) 2 ) And continuously culturing for 24h, washing the cells with PBS three times after the culture is finished, digesting the cells with trypsin, centrifuging the cells at 1000rpm for 5min, then resuspending the cells with 500 mu L of PBS solution, centrifuging the cells again, collecting cell precipitates, and detecting the level of the GSH in the cells by using a GSH and GSSG detection kit. Experimental results as shown in fig. 10, a different reduction in both FDP and FDPC treated cellular GSH levels occurred for the same Fe concentration compared to the PBS group, with lower levels of FDPC treated cellular GSH due to targeting of the FDPC peripheral cell membrane. The GSH levels within the cells of both the FDP + NIR and FDPC + NIR groups were slightly reduced under laser irradiation compared to the laser-free groups (FDP and FDPC), since hyperthermia was able to promote the production of ROS, further depleting GSH.
12. Intracellular ROS content test results
See description, accompanying drawings 10, wherein B and C are the detection of intracellular ROS content by flow cytometry and laser confocal microscopy, respectively. PBS was used as a blank control group, PBS + NIR and FDP, FDP + NIR, FDPC and FDPC + NIR with the same iron concentration were used as experimental groups, and after co-incubation with 4T1 cells in an incubator for 4h, fresh medium was changed, wherein the PBS + NIR, FDP + NIR and FDPC + NIR groups were irradiated with laser light for 5min (808nm, 1.0W/cm) 2 ) Culturing for 24h, washing with PBS for three times after culturing, and adding 2 μ L of ROS probe into each well under dark conditionAnd (3) incubating the needle and 2000 mu L of DMEM medium in an incubator for 20 minutes, washing the needle and the DMEM medium with PBS for three times after the incubation is finished, digesting the cells with pancreatin, collecting the cells after 1000-5 min, resuspending the cells with PBS, and detecting the content of ROS in the cells by a flow cytometer. As shown in fig. 10B, FDP and FDPC treated cells with the same Fe concentration both increased their intracellular ROS levels to different degrees compared to the PBS group, which was 1.9 times higher due to the homologous targeting of FDPC peripheral cell membranes. The higher ROS content in the cells of the FDP + NIR and FDPC + NIR groups under laser irradiation compared to the laser irradiation free groups (FDP and FDPC), also thanks to the fact that hyperthermia has a promoting effect on ROS production.
PBS was used as a blank control group, PBS + NIR and FDP, FDP + NIR, FDPC and FDPC + NIR at the same iron concentration were used as experimental groups, and after incubation with 4T1 cells in an incubator for 4h, the fresh medium was changed, wherein the PBS + NIR, FDP + NIR and FDPC + NIR groups were irradiated with laser for 5min (808nm, 1.0W/cm) 2 ) The culture was continued for 24 hours, and after the completion of the culture, the cells were washed three times with PBS. Under the condition of keeping out of the sun, adding 1 mu L ROS probe and 1000 mu L DMEM culture medium into each well, incubating for 20min in an incubator, washing for three times by PBS after incubation is finished, then fixing for 15min by 2.5% glutaraldehyde, staining for 5min by DAPI after fixing, and observing the green fluorescence signal of the cells under an oil mirror. As shown in fig. 10, C, different intensities of green fluorescence signals were observed for both FDP and FDPC treated cells at the same Fe concentration compared to the PBS group, with stronger green fluorescence signals in FDPC treated cells due to targeting properties of the FDPC coated cell membrane. The green fluorescence signal in the cells of the FDP + NIR and FDPC + NIR groups was stronger under laser irradiation than in the laser-free groups (FDP and FDPC), which also benefits from the thermal treatment promoting ROS generation.
13. Results of intracellular LPO content test
See description accompanying fig. 11 for the results of intracellular LPO content testing. PBS is used as a blank control group, PBS + NIR and FDP, FDP + NIR, FDPC and FDPC + NIR with the same iron concentration are used as an experimental group, after the cells are co-incubated with 4T1 in a culture box for 4h, the fresh culture medium is replaced, wherein the PBS + NIR, FDP + NIR and FDPC + NIR groups are subjected to laser irradiation for 5min(808nm，1.0W/cm 2 ) The culture was continued for 24 hours, and after the completion of the culture, the cells were washed three times with PBS. Under the condition of keeping out of the sun, 1 mu L of LPO probe and 500 mu L of DMEM medium are added into each well, the mixture is incubated in an incubator for 20min, washed three times by PBS after the incubation is finished, then fixed for 15min by 2.5% glutaraldehyde, stained for 5min by DAPI after the fixation, and then the red and green fluorescence signals of the cells are observed under an oil mirror. As shown in FIG. 11, when the probe shows red fluorescence, it indicates that the probe is not oxidized by LPO; when the probe shows green fluorescence, it indicates that the probe is oxidized by LPO in the cell. FDP and FDPC treated cells showed significantly increased green fluorescence signal, significantly decreased red fluorescence signal, and stronger green fluorescence signal due to the targeting of FDPC peripheral cell membrane compared to the PBS group. Compared with the laser irradiation free group (FDP and FDPC), under laser irradiation, the green fluorescence signals in cells of the FDP + NIR and FDPC + NIR groups were stronger and the red fluorescence signals were weaker, since the photothermal treatment promoted the generation of ROS, thereby further increasing the intracellular LPO level.
14. Apoptosis test results
See description figure 12 for apoptosis test results. PBS was used as a blank control group, PBS + NIR and FDP, FDP + NIR, FDPC and FDPC + NIR with the same iron concentration were used as experimental groups, and after co-incubation with 4T1 cells in an incubator for 4h, fresh medium was changed, wherein the PBS + NIR, FDP + NIR and FDPC + NIR groups were irradiated with laser light for 5min (808nm, 1.0W/cm) 2 ) And continuously culturing for 2h, washing the cells with PBS for three times after the culture is finished, digesting the cells with pancreatin, collecting the cells after 1000-5 min, resuspending the cells with PBS, staining the cells for 15min by an Annexin V-FITC/PI apoptosis detection reagent under the condition of keeping away from light, and detecting the apoptosis condition of the cells by a flow cytometer. As shown in fig. 11, the apoptosis rates of the FDP and FDPC groups were significantly increased compared to the PBS group, and in addition, the FDPC group showed a higher apoptosis rate, 4 times higher than that of the FDP group, due to the targeting of the FDPC peripheral cell membrane. The FDP + NIR and FDPC + NIR groups showed higher apoptosis and necrosis rates under laser irradiation compared to the laser irradiation-free group (FDP and FDPC), since photothermal treatment further promoted apoptosis and necrosis of cells, indicating photothermal treatment ∑ erThe combined treatment of the three modes of chemical dynamic therapy and chemotherapy has the best anti-tumor effect.
15. In vivo tumor thermography test results
See description figure 13 for in vivo thermographic results of tumor bearing mice. PBS was used as a blank control group, and the blank control group was irradiated with 808nm laser for 5min (1W/cm) 2 ) The temperature at the tumor site of the mice increased only 1.9 ℃, mainly due to the small amount of heat generated by the absorption of laser radiation by the organism itself. Compared to the PBS group, the temperatures of the tumor sites of the mice in the FDC and FDPC groups increased by 14.2 and 14.7 ℃, respectively, due to the excellent photothermal conversion efficiency of polydopamine, while no significant difference was exhibited between the FDC and FDPC groups.
16. In-vivo magnetic resonance bimodal imaging test results
See description figure 14 for in vivo magnetic resonance bimodal imaging of tumor-bearing mice. As shown in A in FIG. 14, due to Fe 3 O 4 Lack of targeting and responsiveness of NPs, Fe 3 O 4 Significant T was observed throughout the mice in the groups 2 And T 1 MR signal and exhibits no dynamic T at different time points (0, 15, 30, 45, 75min) 2 /T 1 Characterization of bimodal MRI. Fe obtained by modification of cystamine 3 O 4 Tumor internal T of tumor-bearing mice 15min after tail vein injection of NCs 2 The MR signal strength reaches a minimum and, over time, T 2 The MR signal intensity gradually rises (as in B of fig. 14); after 30min of injection, the tumor site became clearly bright, T 1 The MR signal peaked (C in FIG. 14), indicating that the high GSH concentration at the tumor site resulted in cluster-structured Fe 3 O 4 Dissociation of NCs to achieve dynamic T at the tumor site 2 /T 1 Bimodal MRI, but due to lack of targeting, still allows significant magnetic resonance signals to be observed throughout the mouse. Due to the homologous targeting characteristic of the FDPC coated cell membrane, after 15min of tail vein injection, the internal T of the tumor-bearing mouse 2 The MR signal reaches a minimum and is clearly more than Fe at the same time point 3 O 4 T in tumors of mice in NCs group 2 Good MRI effectOver time, T 2 The MR signal gradually rises (as in fig. 14B); after 30min of injection, it was observed that the tumor site became significantly bright, T 1 The MR signal reaches the peak value and is obviously higher than that of Fe at the same time point 3 O 4 Tumor T of mice in NCs group 1 MR signals (C in FIG. 14) further demonstrate that high concentration of GSH at the tumor site can cause disulfide bond cleavage, promoting cluster-structured FDPC degradation, and realizing dynamic T at the tumor site 2 /T 1 Bimodal precision MRI.
17. Evaluation of in vivo therapeutic Effect
Referring to the specification, in the attached figure 15, A is a schematic view of the combined treatment process, and the combined treatment process is mainly divided into 6 groups: PBS group, Pt group, FDC group, FDPC group, FDC + NIR group, and FDPC + NIR group. Constructing 4T1 subcutaneous tumor model in nude mice until the model grows to 200mm 3 Treatment was started on the left and right, with 4 groups receiving 100 μ L of PBS, Pt, FDC and FDPC, respectively, tail vein injections (Fe concentration 1mg/mL, corresponding to a Pt concentration of 208 μ g/mL); the remaining 2 groups were subjected to tail vein injection of 100. mu.L of FDC and FDPC (Fe concentration 1mg/mL), respectively, and after 15min, irradiated with 808nm near-infrared laser for 5min (1.0W/cm) 2 ) The treatment is performed once every 4 days, and the treatment is performed 3 times in a treatment course. All mice were euthanized by day 14 after the end of treatment, and tumor tissues were taken for H treatment, respectively&E and TUNEL staining. As shown in B and D in fig. 15 of the drawings, the tumor volume of the PBS control group mouse is increased most, and the tumor volume of the cell membrane-coated drug-loaded ultra-small ferroferric oxide nanocluster combined photothermal therapy FDPC + NIR group is decreased most, thereby showing the best therapeutic effect. As shown in fig. 15, C, the body weight of Pt group mice decreased significantly throughout the treatment, indicating that Pt had severe side effects, while the body weight of other group mice remained substantially stable with no significant changes, demonstrating that FDPC had good biocompatibility. Referring to FIG. 15 of the specification, E, through H&E and TUNEL staining examines the necrosis and apoptosis of the tumors of mice in each treatment group, and the necrosis and apoptosis of the FDPC + NIR group are most obvious from the graph, thereby proving that the chemotherapy/chemodynamic treatment/photothermal treatment triple-modal combined treatment has the best effect.
Advantageous effects
(1) The method has the advantages of easy realization of reaction conditions, strong controllability, easy operation, environment-friendly raw materials, low cost and good development prospect.
(2) The cell membrane-coated cisplatin-loaded ultra-small ferroferric oxide nanocluster (FDPC) prepared by the invention has good GSH and pH dual-response performance. Ferroferric oxide (Fe) with cluster structure in tumor microenvironment (high-concentration GSH) 3 O 4 NCs) will disperse into individual ferroferric oxides (Fe) 3 O 4 ) Nanoparticles, corresponding T 2 MRI imaging to T 1 MRI imaging, T realization of material 2 /T 1 Bimodal MRI. In the tumor microenvironment (weakly acidic, pH about 6.5), hydrogen ion (H) + ) Will interact with the lone electron pair of oxygen atoms in the hydroxyl group of catechol on the surface of polydopamine to result in [ Pt (H) 2 O) 2 (NH 2 ) 2 ] 2+ The separation is realized, the acid response release of Pt is realized, and the toxic and side effects on normal cells are reduced.
(3) The cell membrane-coated cisplatin-loaded ultra-small ferroferric oxide nanocluster (FDPC) prepared by the invention has good biocompatibility and higher photothermal conversion efficiency. By means of the homologous targeting effect of the cell membrane, the specific accumulation of the antitumor drug in the tumor is increased, the tumor diagnosis and treatment effect is enhanced, meanwhile, the toxic and side effects of Pt are reduced, and the application prospect is provided for constructing a safe and efficient drug carrier and imaging-guided tumor combined treatment.
(4) After the cell membrane-coated cisplatin-loaded ultra-small ferroferric oxide nanocluster (FDPC) prepared by the invention enters a mouse body through tail vein injection, the T of GSH response in the tumor can be realized 2 /T 1 The double-mode MRI conversion can also realize the chemotherapy/chemical dynamic therapy/photothermal therapy triple-mode combined treatment of the tumor, further enhances the anti-tumor effect and has potential clinical application value.
Drawings
FIG. 1 is a schematic diagram of the synthesis and application of FDPC prepared by the present invention, wherein 1 is a first sample inlet, 2 is a second sample inlet, 3 is a third sample inlet, 4 is a fourth sample inlet, and 5 is a sample outlet;
FIG. 2 is a graph of hydrodynamic diameter versus time for FDPC prepared according to the present invention in ultrapure water, PBS solution (pH 7.4) and DMEM medium, respectively;
FIG. 3 shows Fe prepared by the present invention 3 O 4 NPs、Fe 3 O 4 UV spectrograms (A), IR spectrograms (B), XPS spectrograms (C) of NCs, FDP and FDPC and Fe 2p high resolution XPS spectrograms (D) of FDPC;
FIG. 4 is a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the cell membrane suspension (CM), FDPC and FDP prepared in the present invention;
FIG. 5 is a TEM image of FDPC prepared according to the present invention, with the scales (Scale bar) at 200nm (A), 50nm (B), 20nm (C);
FIG. 6 is a T of FDPC prepared according to the invention in the presence or absence of GSH 2 Relaxation Rate (r) 2 ) (A) and T 1 Relaxation Rate (r) 1 )(B)；
FIG. 7 is an in vitro photothermal performance analysis of FDPC prepared in accordance with the present invention: a certain power (1W/cm) 2 ) The method comprises the following steps of (1) obtaining a temperature-rise curve (A) of FDPC (fully drawn yarn) under different concentrations when the FDPC is irradiated for 5min under 808nm laser, continuously obtaining photothermal stability (B) of five cycles of temperature rise/temperature reduction, obtaining a temperature-time relation graph (C) in a laser irradiation stage and a laser removal cooling stage, and obtaining a linear fitting cooling time-driving temperature natural logarithm opposite relation (D);
FIG. 8 is a graph showing the Fe release profile (A) of FDPC prepared according to the present invention under various conditions and the Pt release profile (B) of FDPC under various conditions;
FIG. 9 is a graph of cell viability of free Pt, FDP, FDPC and FDP and FDPC after 24h co-incubation with 4T1 cells in the presence of laser irradiation (5 min);
FIG. 10 is a graph of the effect of PBS, FDP and FDPC on the levels of GSH and ROS in 4T1 cells in the presence and absence of laser irradiation: effect of intracellular GSH content of 4T1 (a), quantitative analysis of intracellular ROS content of 4T1 (B), qualitative analysis of intracellular ROS content of 4T1 (C), Scale bar (Scale bar) 50 μm;
FIG. 11 shows the effect of PBS, FDP and FDPC on the intracellular LPO content of 4T1 in the presence or absence of laser irradiation, with a 50 μm Scale (Scale bar);
FIG. 12 is a graph of the effect of PBS, FDP and FDPC on apoptosis of 4T1 cells in the presence and absence of laser irradiation;
FIG. 13 is a thermal image of tumors in a tumor-bearing mouse with PBS, FDC and FDPC and their corresponding temperature-increasing curves;
FIG. 14 shows Fe prepared by the present invention 3 O 4 NPs、Fe 3 O 4 T of tumor-bearing mice at different time points before and after tail vein injection of NCs and FDPC 2 /T 1 Bimodal MRI map (A), T of the corresponding tumor site 2 MR signal-to-noise ratio variation plots (B) and T 1 MR signal-to-noise ratio variation graph (C);
FIG. 15 is a schematic view showing the treatment process of tumor-bearing mice (A), the relative tumor volume change of each group of mice (B), the body weight change of each group of mice (C), the tumor image of each group of mice obtained after the treatment is finished (D), and the tumor tissue H & E and TUNEL staining results of each group of mice at 14 days after the treatment is finished (E), respectively; scale (Scale bar) 50 μm.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Unless otherwise specified, all chemical reagents were commercially available and used without further purification. Polydimethylsiloxane (PDMS) was purchased from Dow Corning, USA. Iron (III) chloride was purchased from Adamas reagent limited, shanghai, china. Sodium citrate, anhydrous sodium acetate and dopamine hydrochloride were purchased from national drug-controlled chemical agents, ltd (shanghai, china). Cystamine dihydrochloride is available from sigma aldrich trade, inc (shanghai, china). Cisplatin was purchased from Beijing Huafeng technologies, Inc. (Beijing, China). EDC and NHS were purchased from carbofuran technologies ltd (shanghai, china). SDS sample buffer and SDS-polyacrylamide gels were purchased from Tanon Science & Technology co., Ltd. (shanghai, china). DMEM medium, fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (HyClone, Thermo Scientific, Logan, UT), and trypsin 0.25% solution (HyClone) were purchased from jino biomedical technologies, inc (hangzhou, china). C11 BODIPY 581/591 was purchased from shanghai macrolobal biotechnology limited, shanghai, china. Cell Counting Kit-8(CCK-8) and Annexin V-FITC/PI apoptosis detection kits were purchased from 7Sea Biotech Co., Ltd. (Shanghai, China). The GSH and GSSH detection kit and the ROS detection kit are purchased from Shanghai Binminba biology company (Shanghai, China). Avanti extruders are available from Avanti Polar Lipids, Inc. 4T1 cells (murine breast cancer cell line) were obtained from the institute of biochemistry and cell biology, national academy of sciences. Nude mice, 4-5 weeks old, were purchased from shanghai slyke laboratory animal center (shanghai, china). The water used in all experiments with a resistivity higher than 18.2 M.OMEGA.cm was purified by a laboratory water purification system (Cascada I, PALL, Beijing, China). Regenerated cellulose dialysis membranes with a molecular weight cut-off (MWCO) of 10000 were purchased from Fisher (pittsburgh, pa).
Example 1
(1) Designing a micro-fluidic channel structure by using Auto CAD software, wherein the micro-fluidic channel is designed by adopting a gradually enlarged S-shaped channel and comprises five inlets, one outlet and a gradually enlarged S-shaped micro-fluidic channel, the height of the channel is 50 mu m, the width of the micro-fluidic channel of the sample inlets 1, 2 and 3 is 100 mu m, the width of the rest micro-fluidic channels is 300 mu m, and the total length from the inlet to the outlet of the channel is 37.8mm (shown in figure 1); printing a mask plate on the designed chip by using a high-resolution printer, preparing a micro-fluidic chip mould on a silicon chip by using a photoetching technology, finally performing mould inversion by using the prepared chip mould, uniformly mixing Polydimethylsiloxane (PDMS) and a curing agent according to the mass ratio of 10:1, discharging bubbles in vacuum, pouring into the mould, and then performing vacuum drying for 2 hours at 80 ℃ to obtain a corresponding PDMS micro-fluidic channel cover plate; and (3) processing for 80s in an air atmosphere by setting the vacuum degree to be 20Pa through a plasma bonding technology, and bonding the glass slide serving as a substrate with the PDMS microfluidic channel cover plate to obtain the microfluidic chip.
(2) 0.6484g of anhydrous ferric chloride solution is takenDissolving in 40mL of diethylene glycol, dissolving 0.471g of sodium citrate (Na3Cit) in the solution, stirring for 2 hours at 80 ℃ in an air atmosphere, cooling to 55 ℃ after the sodium citrate is completely dissolved, adding 1.312g of anhydrous sodium acetate into the solution, continuously stirring until the sodium acetate powder is completely dissolved, transferring the solution into a 50mL high-pressure reaction kettle, and reacting for 4 hours at 200 ℃; naturally cooling to room temperature after the reaction is finished, transferring the product into a 50mL centrifuge tube, centrifuging for 30min at the rotation speed of 13000rpm, discarding supernatant, redissolving the precipitate with absolute ethyl alcohol, centrifuging for 30min at the rotation speed of 13000rpm, repeating the operation for 3 times, and drying the precipitate in a vacuum drying oven at 60 ℃ to obtain the ultra-small ferroferric oxide nanoparticles (Fe) with stable citric acid on the surface 3 O 4 NPs)。
(3) Taking 50mg of Fe 3 O 4 NPs were dissolved in 15mL of ultrapure water, and stirring was continued, EDC (206.5mg, 1mL of ultrapure water) was added dropwise to the solution, and after 30min, NHS (124.02mg, 1mL of ultrapure water) was added dropwise to the solution to activate Fe 3 O 4 Carboxyl groups on NPs 3 h. Subsequently, cystamine dihydrochloride aqueous solution (98.5mg, 1mL of ultrapure water) was dropped into the above solution, reacted at room temperature for 72 hours, dialyzed for 1 day with a fiber membrane dialysis bag having a cut-off molecular weight of 10000 in PBS buffer (10mM, pH 7.4), and dialyzed for 2 days in ultrapure water to obtain Fe 3 O 4 NCs solution.
(4) Taking 4T1 cells 10 in logarithmic growth phase 7 And centrifuging (1000rpm,5min) to obtain cell sediment, washing with sterile PBS solution once, adding 3mL of hypotonic cell lysis buffer solution (containing 10% PMSF by volume fraction) into the sediment, performing ice bath for 15min, and repeatedly freezing and thawing (quick freezing at (-20 ℃ and quick thawing at 40 ℃) for 3 times by a freezing and thawing method. Setting the centrifugal temperature at 4 ℃, centrifuging for 15min at the centrifugal force of 700g, and removing precipitates; centrifuging the supernatant at 13500rpm for 30min, keeping precipitate, and suspending the precipitate in 500 μ L PBS solution to obtain 4T1 cell membrane suspension; the cell membrane vesicle suspension was extruded 11 times using an Avanti micro-extruder to give a cell membrane vesicle suspension.
(5) Ultra-small ferroferric oxide nanoclusters (Fe) 3 O 4 NCs) solution (1.39mg/mL, 5mL) with dopamine hydrochlorideMixing the solutions (16mg/mL, 165 mu L), performing ultrasonic treatment for 3min to uniformly mix the solutions, and injecting the mixture into the first sample inlet 1 by a micro-injection pump at the flow rate of 5 mL/h; tris buffer (pH 8.5, 10mM, 0.5mL) was injected into the second injection port 2 by a micro syringe pump at a flow rate of 0.5 mL/h; tris buffer (pH 8.5, 10mM, 56mL) is injected into the third sample injection port 3 by a micro-injection pump at the flow rate of 28 mL/h; and (3) injecting a cisplatin aqueous solution (1.2mg/mL, 8.7mL) into a fourth sample inlet 4 by a micro-injection pump at a flow rate of 8.7mL/h, and collecting through a sample outlet 5 to obtain the drug-loaded ultra-small ferroferric oxide nanocluster (FDP).
(6) Ultra-small ferroferric oxide nanoclusters (Fe) 3 O 4 NCs) solution (1.39mg/mL, 5mL) and dopamine hydrochloride solution (16mg/mL, 165 mu L) are mixed, ultrasonically mixed for 3min, and injected into the first sample inlet 1 by a micro-injection pump at the flow rate of 5 mL/h; injecting a cell membrane vesicle solution (2.0mg/mL, 0.5mL) into the second sample inlet 2 by a micro-injection pump at a flow rate of 0.5 mL/h; and injecting Tris buffer solution (pH 8.5, 10mM and 56mL) into the third injection port 3 by a micro-injection pump at the flow rate of 28mL/h, injecting Tris buffer solution (pH 8.5, 10mM and 8.7mL) into the fourth injection port 4 by a micro-injection pump at the flow rate of 8.7mL/h, and collecting the Tris buffer solution through the sample outlet 5 to obtain the cell membrane coated ultra-small ferroferric oxide nanoclusters (FDC).
(7) Ultra-small ferroferric oxide nanoclusters (Fe) 3 O 4 NCs) solution (1.39mg/mL, 5mL) and dopamine hydrochloride solution (16mg/mL, 165 mu L) are mixed, ultrasonically mixed for 3min, and injected into the first sample inlet 1 by a micro-injection pump at the flow rate of 5 mL/h; injecting a cell membrane vesicle solution (2.0mg/mL, 0.5mL) into the second sample inlet 2 by a micro-injection pump at a flow rate of 0.5 mL/h; tris buffer (pH 8.5, 10mM, 56mL) was injected into the third injection port 3 by a micro syringe pump at a flow rate of 28 mL/h; cisplatin aqueous solution (1.2mg/mL, 8.7mL) is injected into the fourth sample inlet 4 by a micro-injection pump, and the flow rate is 8.7 mL/h; and collecting through a sample outlet 5 to obtain the cell membrane coated drug-loaded ultra-small ferroferric oxide nanocluster (FDPC).
Example 2
For Fe prepared in example 1 3 O 4 NPs、Fe 3 O 4 NCs, FDP and FDPC. Fe 3 O 4 NPs、Fe 3 O 4 The hydrodynamic diameters and zeta surface potentials of NCs, FDP and FDPC are shown in Table 1, due to Fe 3 O 4 Surface modification of NPs with carboxyl groups, Fe 3 O 4 The potential of NPs was-32.1 mV, and the hydrated particle size was 154.4 nm. When cystamine is coupled with Fe 3 O 4 NPs form Fe 3 O 4 At NCs, the potential was changed to-21.3 mV and the hydrated particle size increased to 235.0nm due to the amino groups at both ends of cystamine and Fe 3 O 4 The carboxyl on the surface of the NPs generates amide reaction to obtain Fe with cluster structure 3 O 4 NCs, causing the hydrated particle size to increase and the potential to rise. When Fe 3 O 4 After NCs wrap polydopamine and the chemotherapy drug cisplatin is carried, the potential of FDP is further increased to-2.8 mV, and the hydrated particle size is changed to 222.3nm, which is because the wrapping effect of the polydopamine causes Fe 3 O 4 The NCs hydrated particle size shrinks and phenolic hydroxyl groups on the surface of polydopamine are further coordinated with Pt, demonstrating successful encapsulation of polydopamine and successful loading of cisplatin. After coating the cell membrane, the potential of FDPC was again lowered to-16.3 mV and the hydrated particle size was changed to 233.6nm, indicating successful coating of the cell membrane on the surface of FDPC.
TABLE 1
Sample Zeta potential(mV) Hydrodynamic(nm) PDI
Fe 3 O 4 NPs -32.1±1.710 154.4±18.455 0.448±0.099
Fe 3 O 4 NCs -21.3±0.350 235.0±2.730 0.189±0.063
FDP -2.8±0.615 222.3±28.290 0.224±0.014
FDPC -16.3±0.191 233.6±16.020 0.401±0.072
As shown in A in FIG. 3, Fe 3 O 4 After the poly-dopamine is wrapped by NCs, the characteristic absorption peak of the poly-dopamine is observed in the near infrared light region of 600-800nm, and the successful wrapping of the poly-dopamine is proved. As can be seen from the figure, the UV absorption of the 280nm protein increased after the cell membrane was encapsulated, indicating successful encapsulation of the cell membrane. As shown in FIG. 3B, 591cm -1 The characteristic absorption peak nearby is attributed to Fe-O of the ultra-small ferroferric oxide nano-particles, 557cm -1 The nearby characteristic absorption peak is attributed to S-S of cystamine, proving that Fe 3 O 4 Successful synthesis of NCs. 2923 and 2850cm -1 Nearby enhanced characteristic absorption peaks are C-H stretching and bending vibration characteristic absorption peaks of cystamine, polydopamine and membrane protein, 1465cm -1 The nearby characteristic peak is attributed to-C ═ C-stretching vibration of polydopamine and membrane proteins, demonstrating successful encapsulation of polydopamine and cell membranes.
As shown by C in FIG. 3, in Fe 3 O 4 NPs、Fe 3 O 4 Fe element was observed in XPS spectra of NCs, FDP and FDPC, and the strength of Fe binding energy was in a decreasing trend, because of Fe 3 O 4 The surface of NPs is in turn modified with cystamine, wrapped with polydopamine, and cell membrane-coated, resulting in a reduction in the Fe content of the surface. In Fe 3 O 4 No N element was observed in XPS spectra of NPs, but in Fe 3 O 4 N elements are observed in XPS spectra of NCs, FDP and FDPC, and the strength of N binding energy is in an increasing trend, further proving that Fe 3 O 4 The NPs surface is sequentially modified by cystamine, wrapped by polydopamine and coated by cell membranes. Pt element was observed in XPS spectra of FDP and FDPC, demonstrating successful loading of Pt. In FIG. 3, D is the Fe 2p high resolution XPS spectrum of FDPC, 710.52, 712.84, 724.08, 726.65, 715.17, 719.14, 729.49 and 733.21eV correspond to Fe respectively 2+ 2p 3/2 、Fe 3+ 2p 3/2 、Fe 2+ 2p 1/2 、Fe 3+ 2p 1/2 、Fe 2+ 2p 3/2 satellite、Fe 3+ 2p 3/2 satellite、Fe 2+ 2p 1/2 satelite and Fe 3+ 2p 1/2 satelite, which demonstrates Fe in FDPC 3 O 4 Is present.
FIG. 4 shows the results of SDS-PAGE of CM, FDPC and FDP. The protein content in the cell membrane suspension and the FDPC is determined by using a BCA protein quantitative kit, and the protein content is adjusted to be 1mg/mL by using PBS. Add 5. mu.L of protein Marker to the first protein lane, and then add 20. mu.L of cell membrane suspension (CM), FDPC and FDP (200. mu.g in 1mL PBS) to the lanes sequentially, set the gel-running current at 100A for 30 min. As shown in fig. 4, the FDP group did not run out of the lane with protein streaks, whereas the CM and FDPC groups ran out of similar protein streaks, demonstrating successful coating of the cell membrane on the FDP surface.
In FIG. 5, A is a TEM image of FDPC, which is very uniform in the size distribution, has an average particle diameter of 205.5nm, and is excellent in dispersibility. From fig. 5B and 5C, it is evident that a cell membrane with a thickness of 9.8nm exists at the periphery of FDPC, the core of the cell membrane is an ultra-small ferroferric oxide nanocluster wrapped by polydopamine, and the particle size is 17.6 nm. As shown in FIG. 5C, ultra-small ferroferric oxide nanoparticles with a particle size of 3.0nm were observed inside polydopamine, which, taken together, demonstrated the successful synthesis of FDPC.
Example 3
The content of Fe element in FDPC was determined by ICP-OES test method. In order to test the GSH response performance of FDPC, FDPC is respectively prepared into GSH-containing aqueous solutions with the concentration gradient of 1mL (the concentration of Fe element is respectively 0.1, 0.2, 0.4, 0.8 and 1.6 mM). Separately determining T of FDPC in the Presence and absence of GSH 2 And T 1 The relaxation time, the inverse of the relaxation time and the Fe concentration are linearly fitted, and the slope is the relaxation rate (shown in FIG. 6) (T) 1 Relaxation rate of imaging by r 1 Denotes, T 2 Relaxation rate of imaging with r 2 Representation). Generally, r 2 /r 1 Less than 5 indicates that the material has a good T 1 Imaging performance; if r is 2 /r 1 Greater than 5 indicates that the material has a good T 2 And (4) imaging performance. As shown in FIG. 6, r of FDPC in the absence of GSH 2 Is 20.06mM -1 s -1 And r is 1 Is 1.05mM -1 s -1 At this time r 2 /r 1 Greater than 5, indicates that FDPC has a good T in the absence of GSH 2 And (4) imaging performance. R of FDPC in the Presence of GSH 2 Reduced to 3.73mM -1 s -1 And r is 1 Increased to 2.22mM -1 s -1 At this time r 2 /r 1 Less than 5, indicating that FDPC has a good T in the presence of GSH 1 Imaging performance, due to cleavage of the disulfide bond of cystamine by GSH, disrupted the cluster structure of FDPC. Taken together, the above results further demonstrate that FDPC has good T with GSH as the response mechanism 2 /T 1 The bimodal MRI performance can be used as good T in MRI molecular imaging diagnosis 2 /T 1 A bimodal contrast agent.
The in vitro photothermal performance test results of FDPC are shown in FIG. 7, wherein A in FIG. 7 is the temperature rise of FDPC with 808nm laser irradiation under different Fe concentrations (25, 50, 100. mu.g/mL). At a certain power density (1W/cm) 2 ) The warming effect of FDPC increases gradually with increasing concentration, with a concentration of 100. mu.g/mL, the temperature of FDPC increases by 20.6 ℃ and the water increases by only 0.9 ℃. The photo-thermal stability of FDPC (100 μ g/mL) was tested by five cycles of temperature increase and decrease, wherein a temperature increase process was completed by irradiating with 808nm laser for 5min, and then closing the laser to cool to the initial temperature to complete a temperature decrease process, and as a result, as shown in fig. 7B, no significant difference was found between five cycles, indicating that the prepared FDPC had good photo-thermal stability. Further, from the temperature-increasing cooling curve in C in fig. 7, the relationship between the cooling time D in fig. 7 and the inverse number of the natural logarithm of the driving temperature was obtained, and the heat transfer coefficient of FDPC was found to be 171s by linear fitting, and the photothermal conversion efficiency thereof was calculated to be 46.2% by the formula.
Four kinds of PBS buffers each having a pH of 7.4, 6.5, GSH-containing pH 7.4, and GSH-containing pH 6.5 were prepared, FDPC was prepared to be dissolved in 1mL of the four PBS buffer solutions described above to prepare a solution having an Fe concentration of 500 μ g/mL, and then placed in a dialysis bag, and the dialysis bag was placed in a container containing 19mL of the four different buffer solutions described above and shaken in a constant temperature shaker at 37 ℃. And (3) sucking 1mL of dialysis bag external liquid at different time points, supplementing a corresponding PBS buffer solution into the container, and determining the contents of Fe and Pt by an ICP-OES test method after the 1mL of dialysis bag external liquid is digested by aqua regia. And after the slow release is finished, drawing a drug release curve of the FDPC under different conditions. As shown in a of fig. 8, the cumulative amount of released Fe is only 5.8% at pH 7.4, whereas the cumulative amount of released Fe can be increased to 6.3% when GSH (10mM) is present, since GSH accelerates the dissociation of clusters. At pH 6.5, the cumulative amount of Fe released increased to 11.9%, since Fe accelerated under weakly acidic conditions 3 O 4 Release of medium Fe; and the cumulative amount of Fe released in the presence of GSH increased to 13.0% based on the dissociation of clusters by GSH. As can be seen from B in fig. 8, the cumulative release amount of Pt was only 11.5% at pH 7.4, and increased to 27.6% at pH 6.5, due to hydrogen ions (H) under acidic conditions + ) Will be isolated with oxygen atom in catechol hydroxyl group on polydopamine surfaceThe pairs interact to result in [ Pt (H) 2 O) 2 (NH 2 ) 2 ] 2+ The Pt is separated, and the acid response release of the Pt is realized; on this basis, the cumulative release of Pt increased slightly to 29.3% due to the presence of GSH, which benefits from GSH altering and even destroying the polymeric state of polydopamine.
Example 4
The cytotoxicity of free Pt, FDP, FDPC, and FDP and FDPC under the laser participation was evaluated using 4T1 cells as a cell model. 4T1 cells were plated at 1X 10 4 The density of each cell per well was seeded in 5 96-well plates in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. The medium from 3 of the 96-well plates was then changed to medium containing free Pt, FDP, FDPC (all Pt concentrations were set to 0. mu.g/mL, 1.3. mu.g/mL, 2.6. mu.g/mL, 5.2. mu.g/mL, 10.4. mu.g/mL, 20.8. mu.g/mL) with cells in 5% CO 2 CO-culturing at 37 deg.C for 4 hr, then changing the culture medium to new DMEM medium, and continuing to culture in 5% CO 2 And co-culturing at 37 ℃ for 24 h. The media of the remaining 2 96-well plate cell culture plates were changed to media containing FDP, FDPC (Pt concentrations were all set to 0. mu.g/mL, 1.3. mu.g/mL, 2.6. mu.g/mL, 5.2. mu.g/mL, 10.4. mu.g/mL, 20.8. mu.g/mL) and cells at 5% CO 2 CO-culturing at 37 deg.C for 4 hr, changing the culture medium to new DMEM, irradiating with 808nm laser for 5min, and continuously adding 5% CO 2 And co-culturing at 37 ℃ for 24 h. For 5 pieces of 96-well plates, the cell culture plates were washed three times with PBS, followed by addition of 10% (v/v) CCK-8 (10. mu.L) in DMEM medium (100. mu.L/well) and further incubation in the incubator for 4 hours. And finally, testing the absorbance of each hole at the position with the wavelength of 450nm by using an enzyme-labeling instrument, taking the cells treated by the DMEM medium as a blank control, and marking the cell activity as 100%. CCK-8 results are shown in fig. 9, where FDPC exhibits greater cytotoxicity than free Pt and FDP at the same Pt concentration due to the targeting effect of the cell membrane, which enhances cellular uptake of nanomaterials. Meanwhile, at the same Pt concentration, the FDP + NIR and FDPC + NIR experimental groups all showed stronger cytotoxicity than the group without 808nm laser irradiation (FDP and FDPC groups), thanks to the good photothermal properties of polydopamine.In conclusion, the FDPC + NIR group integrated with photothermal/chemodynamic/chemotherapy had the strongest killing effect on 4T1 cells.
Example 5
The effect of FDP, FDPC and the involvement of laser on intracellular GSH content was evaluated using 4T1 cells as a cell model. 4T1 cells were plated at 2X 10 5 The density of each cell per well was seeded in 3 6-well plates in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. Then, the old medium was removed and replaced with new DMEM medium, the medium containing FDP and FDPC (Fe concentration was 50. mu.g/mL) and the cells were incubated at 5% CO 2 Co-culturing at 37 deg.C for 4 hr, and then changing the culture medium to new DMEM medium, wherein three wells of each 6-well plate are treated with 808nm laser (1.0W/cm) 2 ) Irradiating for 5min under 5% CO 2 And co-culturing at 37 ℃ for 24 h. Washed three times with PBS, followed by trypsinization of the cells, centrifuged at 1000rpm for 5min, resuspended in 500 μ L PBS solution, centrifuged again to collect cell pellets, and intracellular GSH levels were measured using the GSH and GSSG assay kit (available from bi yun day biotechnology). As shown in fig. 10, the levels of both FDP and FDPC treated cellular GSH were reduced to different degrees for the same Fe concentration compared to the PBS group, with lower levels of FDPC treated cellular GSH due to targeting of the FDPC peripheral cell membrane. The GSH levels in the cells of both the FDP + NIR and FDPC + NIR groups were slightly reduced under laser irradiation compared to the laser-free groups (FDP and FDPC), due to the promoting effect of hyperthermia on ROS production, further depleting GSH, indicating that depletion of intracellular GSH by FDPC under laser irradiation was most pronounced.
The effects of FDP, FDPC and FDP and FDPC under the participation of laser on the ROS content in the cells are quantitatively evaluated by taking 4T1 cells as a cell model. 4T1 cells were plated at 2X 10 5 The density of each cell per well was seeded in 3 6-well plates in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. Then, the old medium was removed and replaced with new DMEM medium, the medium containing FDP and FDPC (Fe concentration was 50. mu.g/mL) and the cells were incubated at 5% CO 2 Co-culturing at 37 deg.C for 4 hr, and changing the culture medium to new DMEM medium with 6-well platesThree holes in the sample (2) are lased at 808nm (1.0W/cm) 2 ) Irradiating for 5min under 5% CO 2 And co-culturing at 37 ℃ for 24 h. Washing with PBS for three times, adding 2 mu L of DCFH-DA and 2000 mu L of DMEM culture medium into each well under the condition of keeping out of the sun, incubating for 20min in an incubator, washing with PBS for three times after incubation is finished, digesting the cells with pancreatin, collecting the cells at 1000rpm and 5min, resuspending the cells with PBS, and detecting the content of ROS in the cells by a flow cytometer. As shown in fig. 10B, FDP and FDPC treated cells with the same Fe concentration both increased their ROS content to different extents compared to the PBS group, which was 1.9 times higher than the PBS group due to the homologous targeting of the FDPC peripheral cell membrane, whereas the ROS content of the PBS + NIR group did not change significantly, indicating that near-infrared laser irradiation alone did not cause an increase in intracellular ROS. The higher ROS content in cells of the FDP + NIR and FDPC + NIR groups under laser irradiation, as compared to the laser irradiation free groups (FDP and FDPC), is facilitated by the hyperthermia.
The effects of FDP, FDPC and FDP and FDPC in the presence of laser on intracellular ROS content were further qualitatively evaluated using 4T1 cells as a cell model. 4T1 cells were plated at 1X 10 4 The density of each cell per well was seeded in 6 confocal dishes and placed in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. Then, the old medium was removed and replaced with new DMEM medium, the medium containing FDP and FDPC (Fe concentration was 50. mu.g/mL) and the cells were incubated at 5% CO 2 Co-cultivation at 37 ℃ for 4h, then the medium was changed to fresh DMEM medium, with 3 confocal dishes (DMEM medium, FDP, FDPC) continuing at 5% CO 2 Co-culture was carried out at 37 ℃ for 24h, and the remaining 3 confocal dishes (DMEM medium, FDP, FDPC) were irradiated with a 808nm laser (1.0W/cm) 2 ) Irradiating for 5min under 5% CO 2 The cells were co-cultured at 37 ℃ for 24 hours, and then washed three times with PBS after the culture. Under the condition of keeping out of the light, 1 mu L of ROS probe and 1000 mu L of LDMEM culture medium are added into each well, the mixture is incubated in an incubator for 20min, washed three times by PBS after the incubation is finished, then fixed by 2.5% glutaraldehyde for 15min, stained by DAPI for 5min after the fixation, and then the green fluorescence signal of the cells is observed under an oil mirror. As shown at C in FIG. 10, phaseCompared with the PBS group, the green fluorescence signals with different intensities are observed in the FDP and FDPC treated cells with the same Fe concentration, the green fluorescence signals in the FDPC treated cells are stronger due to the targeting property of the FDPC-coated cell membranes, and the PBS + NIR group does not show obvious ROS signals, which indicates that independent near infrared laser irradiation does not cause the increase of ROS in the cells. Compared with the laser irradiation free group (FDP and FDPC), under the laser irradiation, the green fluorescence signals in the cells of the FDP + NIR and FDPC + NIR groups are stronger, and the promotion effect of the thermotherapy on the generation of ROS is also benefited, so that the promotion of the generation of the ROS in the cells by the FDPC under the laser irradiation is proved to be the most obvious.
Example 6
The effects of FDP, FDPC and FDP and FDPC in the presence of laser on the intracellular LPO content were evaluated using 4T1 cells as a cell model. 4T1 cells were plated at 1X 10 4 The density of each cell per well was seeded in 6 confocal dishes and placed in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. Then, the old medium was removed and replaced with new DMEM medium, the medium containing FDP and FDPC (Fe concentration was 50. mu.g/mL) and the cells were incubated at 5% CO 2 Co-culture at 37 ℃ for 4h, then change the medium to new DMEM medium, in which 3 confocal dishes (DMEM medium, FDP, FDPC) were continued at 5% CO 2 Co-culture was carried out at 37 ℃ for 24h, and the remaining 3 confocal dishes (DMEM medium, FDP, FDPC) were irradiated with a 808nm laser (1.0W/cm) 2 ) Irradiating for 5min under 5% CO 2 The cells were co-cultured at 37 ℃ for 24 hours, and then washed three times with PBS after the culture. Adding 1 mu L C11 BODIPY probe and 500 mu L DMEM medium into each well under the condition of keeping out of the light, incubating for 20min in an incubator, washing for three times by PBS after incubation is finished, then fixing for 15min by 2.5% glutaraldehyde, staining for 5min by DAPI after fixing, and observing red and green fluorescence signals of the cells under an oil mirror. As shown in FIG. 11, when the probe showed red fluorescence, it indicates that the probe was not Oxidized by LPO (Non-Oxidized C11 BODIPY); when the probe showed green fluorescence, it was indicated that the probe was Oxidized by LPO in the cell (Oxidized C11 BODIPY). FDP and FDPC treated cells showed significantly increased green fluorescence signal and significantly decreased red fluorescence signal compared to the PBS group, in addition to target of FDPC peripheral cell membraneIn the positive effect, the green fluorescence signal was stronger, whereas the PBS + NIR group showed no significant increase in LPO levels, indicating that near-infrared laser irradiation alone did not cause an increase in intracellular LPO. Compared with the laser irradiation free group (FDP and FDPC), the green fluorescence signals and the red fluorescence signals in the cells of the FDP + NIR and FDPC + NIR groups were stronger and weaker under laser irradiation, since the photothermal treatment promoted the generation of ROS, thus exacerbating the increase in the intracellular LPO level, demonstrating that FDPC leads to the highest intracellular LPO level under near-infrared laser irradiation.
Example 7
The cell model of 4T1 cells was used to evaluate the effects of FDP, FDPC and FDP and FDPC on apoptosis in the presence of laser. 4T1 cells were plated at 2X 10 5 The density of each cell per well was seeded in 3 6-well plates in 5% CO 2 Incubation was carried out at 37 ℃ for 24 h. Then, the old medium was removed and replaced with new DMEM medium, the medium containing FDP and FDPC (Fe concentration was 50. mu.g/mL) and the cells were incubated at 5% CO 2 Co-culturing at 37 deg.C for 4 hr, and then changing the culture medium to new DMEM medium, wherein three wells of each 6-well plate are treated with 808nm laser (1.0W/cm) 2 ) Irradiating for 5min under 5% CO 2 And co-culturing at 37 ℃ for 2 h. Washing with PBS for three times, digesting the cells with pancreatin, collecting the cells after 1000-5 min of rotation, resuspending the cells with PBS, adding 5 mul Annexin V-FITC into each tube under the condition of keeping out of the sun, reacting for 10min at room temperature, adding 10 mul PI staining solution, reacting for 5min at room temperature, and detecting the apoptosis condition by a flow cytometer. As shown in fig. 12, the apoptosis rates of the FDP and FDPC groups were significantly increased compared to the PBS group, and in addition, the FDPC group showed a higher apoptosis rate due to the targeting of the peripheral cell membrane of FDPC, which was 4 times higher than that of the FDP group, while the PBS + NIR group did not show a significant difference, indicating that the near infrared laser irradiation alone did not cause apoptosis. Compared with the laser irradiation free group (FDP and FDPC), the FDP + NIR and FDPC + NIR groups showed higher apoptosis rate and cell necrosis rate under laser irradiation, since the photothermal therapy further promotes the apoptosis and necrosis of the cells, indicating that the antitumor effect of the photothermal therapy/chemodynamic therapy/chemotherapy triple-mode combined therapy is the best.
Example 8
In vivo thermography experiments: 4T1 subcutaneous tumor model was constructed in female nude mice at 4-5 weeks, 100. mu.L LPBS and FDC and FDPC dissolved in 100. mu.L PBS (Fe concentration 1mg/mL) were injected into caudal vein, and 808nm near infrared laser (1.0W/cm) was applied after 15min 2 ) The tumor sites of each group of tumor-bearing mice were irradiated for 5min, and the temperature rise at the tumor sites was recorded by an infrared camera, as shown in FIG. 13. PBS as a blank control group, after 808nm near-infrared laser irradiation for 5min, the temperature of the tumor part of the mouse only rises by 1.9 ℃, which is mainly caused by a small amount of heat generated by the absorption of laser radiation by the organism. Compared to the PBS group, the temperatures of the tumor sites of the mice in the FDC and FDPC groups increased by 14.2 and 14.7 ℃, respectively, due to the excellent photothermal conversion efficiency of polydopamine, and no significant difference was exhibited between the FDC and FDPC groups.
Example 9
4T1 subcutaneous tumor model was constructed in female nude mice at 4-5 weeks, and 100. mu.L Fe was injected into tail vein 3 O 4 NPs、Fe 3 O 4 NCs and FDPC in PBS buffer (Fe concentration 1mg/mL), tumor sites T of mice with tumor at different time points (0, 15, 30, 45, 75min) after injection of the material were scanned by NMR 2 And T 1 MRI, evaluation of T thereof 2 /T 1 Bimodal MR contrast effect (as in fig. 14). As shown in A in FIG. 14, due to Fe 3 O 4 Lack of targeting and responsiveness of NPs, Fe 3 O 4 Significant T was observed throughout the mice in the groups 2 And T 1 MR signal and exhibits no dynamic T at different time points (0, 15, 30, 45, 75min) 2 /T 1 Characterization of bimodal MRI. Fe obtained by modification of cystamine 3 O 4 Tumor internal T of tumor-bearing mice 15min after tail vein injection of NCs 2 The MR signal reaches a minimum and, over time, T 2 The MR signal intensity gradually rises (as in B of fig. 14); after 30min of injection, the tumor site became clearly bright (as in A in FIG. 14), T 1 The MR signal peaked (C in FIG. 14), indicating that the high concentration of GSH environment at the tumor site causes cluster-junctionsStructural Fe 3 O 4 Degradation of NCs to achieve dynamic T at tumor sites 2 /T 1 Bimodal MRI, but due to lack of targeting, still allows significant magnetic resonance signals to be observed throughout the mouse. Due to the homologous targeting characteristic of FDPC (fully drawn protein) wrapped cell membrane, after 15min of tail vein injection, the internal T of tumor of the tumor-bearing mouse 2 The MR signal reaches a minimum and is clearly more Fe than at the same point in time 3 O 4 T in tumors of mice in NCs group 2 MRI works well (A in FIG. 14), with T over time 2 The MR signal intensity gradually increases (as in B of fig. 14); after 30min of injection, it was observed that the tumor site became significantly bright, T 1 The MR signal reaches the peak value and is obviously higher than that of Fe at the same time point 3 O 4 T in tumors of mice in NCs group 1 MR signals (C in FIG. 14) further demonstrate that high concentration of GSH at the tumor site can cause disulfide bond cleavage, promoting cluster-structured FDPC degradation, and realizing dynamic T at the tumor site 2 /T 1 Bimodal precision MRI.
Example 10
Refer to the description and FIG. 15B for a schematic representation of the combined treatment process. Constructing 4T1 subcutaneous tumor model in nude mice until tumor volume reaches 200mm 3 On the left and right, tumor-bearing nude mice were randomly divided into 6 groups (PBS, Pt, FDC, FDPC, FDC + NIR, FDPC + NIR) of 5 mice each. 4 groups of the four drugs were injected into tail vein with 100. mu.L PBS, Pt, FDC and FDPC (Fe concentration 1mg/mL, corresponding Pt concentration 208. mu.g/mL); the remaining 2 groups were subjected to tail vein injection of 100. mu.L of FDC and FDPC (Fe concentration of 1mg/mL), respectively, and after 15min, the tumor site was irradiated with 808nm near-infrared laser for 5min (1.0W/cm) 2 ) Every 4 days, 3 treatment courses were given, as shown in a in fig. 15. The tumor volume and the weight of the mouse were measured every 2 days, and the tumor volume and the relative tumor volume were calculated by the following formulas (1) and (2), respectively.
Tumor volume (V) ═ a × b 2 /2 (1)
a and b represent the maximum and minimum of the tumor diameter, respectively.
Relative tumor volume ═ V/V 0 (2)
V and V 0 The tumor volume after administration and the tumor volume before administration are represented, respectively.
All mice were euthanized by day 14 after the end of 3 treatments, and tumor tissues were then taken for H & E and TENEL staining, respectively. As shown in B and D in fig. 15, the tumor volume of the PBS control group mouse increased most, and the tumor volume of the cell membrane-wrapped drug-loaded ultra-small ferroferric oxide nanocluster combined photothermal therapy FDPC + NIR group decreased most, showing the best therapeutic effect. As shown in fig. 15C, the body weight of the Pt group mice decreased significantly throughout the treatment, indicating that Pt had severe toxic side effects, while the body weight of the other group mice remained essentially stable with no significant change, demonstrating that the other group materials all had good biocompatibility. As shown in E in FIG. 15, H & E and TUNEL staining was used to examine the necrosis and apoptosis of the tumors in the mice of each treatment group, and the necrosis and apoptosis effects of the tumor tissues of the FDPC + NIR group are most obvious, thus proving that the effect of the photothermal therapy/chemodynamic therapy/chemotherapy combination is the best.

Claims (6)

1. A preparation method of a drug-loaded ultra-small ferroferric oxide nanocluster based on a microfluidic chip comprises the following steps:
(1) dissolving ferric salt in a solvent, performing ultrasonic treatment, adding sodium citrate, stirring, adding anhydrous sodium acetate, continuously stirring, performing solvothermal reaction, cooling, centrifuging, and drying to obtain the Fe nanoparticles of ultra-small ferroferric oxide 3 O 4 NPs；
(2) The ultra-small ferroferric oxide nano-particles Fe in the step (1) are treated 3 O 4 NPs are dispersed in ultrapure water, activated by EDC and NHS, then dripped into cystamine dihydrochloride solution for reaction, and dialyzed to obtain the ultra-small ferroferric oxide nanocluster Fe 3 O 4 NCs solution;
(3) centrifuging 4T1 cells, adding hypotonic cell lysate into the obtained 4T1 cell sediment, repeatedly freezing and thawing and crushing, extracting 4T1 cell membranes by gradient centrifugation, suspending in PBS solution to obtain 4T1 cell membrane suspension, and extruding to obtain 4T1 cell membrane vesicle suspension;
(4) designing an S-shaped microfluidic channel structure comprising five sample inlets and one sample outlet and gradually expanding, printing a mask, photoetching a silicon wafer to prepare a mold, performing reverse molding, and performing plasma bonding on the obtained microfluidic channel cover plate by using a glass slide as a substrate to obtain a microfluidic chip, wherein the temperature of the reverse molding is 75-85 ℃; the technological parameters of plasma bonding are as follows: the vacuum degree is 18-22 Pa, and the bonding treatment time in the air is 70-90 s;
(5) the ultra-small ferroferric oxide nanocluster Fe in the step (2) 3 O 4 Mixing NCs solution and dopamine hydrochloride solution, performing ultrasonic treatment, injecting the mixture into a first sample inlet of the microfluidic chip in the step (4), injecting 4T1 cell membrane vesicle suspension in the step (3) into a second sample inlet of the microfluidic chip in the step (4), injecting Tris buffer solution into a third sample inlet of the microfluidic chip in the step (4), injecting cis-platinum aqueous solution into a fourth sample inlet of the microfluidic chip in the step (4), and collecting the generated cell membrane coated drug-loaded ultra-small ferroferric oxide nanocluster FDPC through a sample outlet, wherein the ultra-small ferroferric oxide nanocluster Fe B is subjected to ion treatment 3 O 4 The mass ratio of NCs to dopamine hydrochloride to 4T1 cell membrane vesicles to cisplatin is 6-8: 2-3: 0.5-1.5: 9-11; the flow rates of the first sample inlet, the second sample inlet, the third sample inlet and the fourth sample inlet are respectively 4-6 mL/h, 0.2-1 mL/h, 20-35 mL/h and 8-9.5 mL/h.
2. The method according to claim 1, wherein the iron salt in the step (1) is ferric chloride; the solvent is diethylene glycol; the ratio of the ferric salt, the solvent, the sodium citrate and the anhydrous sodium acetate is 0.60-0.66 g, 36-42 mL, 0.46-0.48 g and 1.3-1.4 g.
3. The method according to claim 1, wherein the stirring in the step (1) is: stirring for 1-3 h at 75-85 ℃ in an air atmosphere; the solvothermal reaction temperature is 180-220 ℃, and the solvothermal reaction time is 3-5 h.
4. The preparation method according to claim 1, wherein the ultra-small ferroferric oxide nanoparticles Fe in the step (2) 3 O 4 The mass ratio of NPs to EDC to NHS to cystamine dihydrochloride is 40-60: 196 to 216: 114 to 134: 88.5 to 108.5; the reaction temperature is room temperature, and the reaction time is 2-4 days.
5. The method of claim 1, wherein the EDC and NHS activation in step (2) is: firstly adding EDC, stirring and reacting for 0.5-1.5 h, then adding NHS, and stirring and reacting for 2-4 h.
6. The method according to claim 1, wherein the ratio of 4T1 cells to hypotonic cell lysate in step (3) is 10 7 2-4 mL; the technological parameters of repeated freeze thawing and crushing are as follows: quickly freezing at minus 10 to minus 30 ℃, quickly thawing at 30 to 50 ℃, and repeating for 2 to 4 times; the extrusion is as follows: extruding the 4T1 cell membrane suspension by using an Avanti miniature extruder for 8-14 times; the protein concentration of the 4T1 cell membrane vesicle suspension is 1.8-2.2 mg/mL.
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