CN117797273B - Preparation method of mesoporous polydopamine nanometer diagnosis and treatment agent for MRI (magnetic resonance imaging) visual cancer treatment - Google Patents
Preparation method of mesoporous polydopamine nanometer diagnosis and treatment agent for MRI (magnetic resonance imaging) visual cancer treatment Download PDFInfo
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Abstract
本发明属于磁共振成像技术领域，具体涉及一种用于MRI可视化癌症治疗的介孔聚多巴胺纳米诊疗剂的制备方法。本发明以钆离子(Gd3+)和精氨酸(Arg)掺杂的介孔聚多巴胺(MPDA)纳米粒子为载体，负载抗炎药美洛昔康(MX)制备得到纳米制剂MX@Arg‑Gd‑MPDA(MAGM)，用于T1‑T2双模MRI引导的癌症光热和抗炎治疗，提高癌症治疗效率，降低复发风险。同时，本发明制备的纳米制剂具备良好的生物相容性、肿瘤微环境响应性和药物缓释能力，可有效规避常规药物副作用大、靶向性差、体内循环时间短等问题，为MRI引导癌症的治疗提供了更有力支撑，具有重要的潜在应用价值。
The present invention belongs to the field of magnetic resonance imaging technology, and specifically relates to a method for preparing a mesoporous polydopamine nano-diagnostic agent for MRI-visualized cancer treatment. The present invention uses mesoporous polydopamine (MPDA) nanoparticles doped with gadolinium ions (Gd 3+ ) and arginine (Arg) as carriers, and loads anti-inflammatory drug meloxicam (MX) to prepare nano-preparation MX@Arg‑Gd‑MPDA (MAGM), which is used for T1‑T2 dual-mode MRI-guided cancer photothermal and anti-inflammatory treatment, improves cancer treatment efficiency, and reduces the risk of recurrence. At the same time, the nano-preparation prepared by the present invention has good biocompatibility, tumor microenvironment responsiveness, and drug sustained release ability, which can effectively avoid the problems of large side effects, poor targeting, and short in vivo circulation time of conventional drugs, and provides more powerful support for the treatment of MRI-guided cancer, and has important potential application value.
Description
Technical Field
The invention belongs to the technical field of magnetic resonance imaging, and particularly relates to a preparation method of a mesoporous polydopamine nanometer diagnosis and treatment agent for MRI (magnetic resonance imaging) visual cancer treatment.
Background
Magnetic Resonance Imaging (MRI) is widely used because of its advantages of strong soft tissue contrast and high spatial resolution, etc., which are exhibited in clinical disease diagnosis. The basic principle is that a patient is placed in a special magnetic field, and resonance of hydrogen nuclei in the body is excited by applying radio frequency pulses. After the radio frequency pulse stops, the hydrogen atomic nucleus sends out signal at specific frequency and releases energy, and the signal is processed by the electronic computer to form magnetic resonance image. When human tissue is diseased, the transverse and longitudinal relaxation times of hydrogen nuclei change, resulting in changes in MRI images, so that the diseased location or damage condition can be deduced. In the diagnosis of some diseases, such as cancer, it is often desirable to use contrast agents to shorten the relaxation time of surrounding water protons, thereby enhancing tissue contrast and improving MRI sensitivity and accuracy. MRI contrast agents generally fall into two categories. One is a T1 contrast agent that shortens the longitudinal relaxation time of protons, mainly comprising Gd-DTPA, mn-DPDP, etc. commonly used clinically, which exhibit brighter signals in T1 weighted imaging. The other is a T2 contrast agent that shortens the proton transverse relaxation time, mainly comprising superparamagnetic iron oxide (SPIOs) or the like, which exhibit darker signals in T2-weighted imaging. However, these currently used contrast agents have problems of high toxicity, low sensitivity, and the like, and are difficult to meet clinical demands. Therefore, there is an urgent need to develop new safer and more sensitive contrast agents.
In recent years, constructing a nano-drug delivery system with an MRI contrast function becomes a research hot spot, which has a wide application prospect in monitoring treatment progress and improving treatment accuracy. For example, manganese carbonate@polydopamine (MnCO 3 @pda) core-shell nanocomposites are used for MRI-guided photothermal therapy (PTT) treatment, whereas chitosan-derived glycolipid nanoparticles are used for MRI-guided photodynamic therapy (PDT) treatment. These nanomedicine delivery systems exhibit more sensitive contrast effects than commercial contrast agents and achieve desirable tumor suppression under MRI guidance. In addition, the continuous development of nanotechnology provides new possibilities for developing T1-T2 dual-mode MRI contrast agents, and the dual-mode contrast agents can meet the requirements of different tissues on T1/T2 weighted imaging, so that the accuracy of disease diagnosis is further improved. Currently, two strategies are mainly adopted for developing a T1-T2 dual-mode contrast agent: one strategy is to combine a T1 contrast agent with a T2 contrast agent to build a new composite contrast agent. During imaging, the two images respectively exert respective contrast capability, and are integrally represented as T1-T2 dual-mode contrast. For example, gadolinium-labeled magnetite nanoparticles, core/shell structured ferroferric oxide/silica/gadolinium carbonate nanoparticles, and the like are utilized. Another strategy is to achieve the effect of T1-T2 dual mode radiography by changing the spatial conformation around the metal ion so that it affects both the proton resonances in the lateral and longitudinal directions, thereby shortening the relaxation time. For example, a novel composite nanosystem (Gd-PDA-ce6@gd-MOF, GPCG) based on gadolinium doped polydopamine nano-ions and a metal organic framework structure. GPCG has the ability to perform T1-T2 dual mode MRI contrast, benefiting from Metal Organic Framework (MOF) structures.
Photothermal therapy (PTT) is an emerging treatment that is widely studied to achieve tumor ablation by converting light energy into heat energy. It has the advantages of small trauma, low toxicity, high efficiency and the like, and is considered as a promising therapeutic strategy. However, studies have shown that release of intracellular components into the extracellular environment after PTT induces cancer cell necrosis will trigger an inflammatory response. Inflammatory factors generated by this process may activate pro-survival genes in residual cancer cells, increasing the risk of cancer recurrence. Therefore, it becomes necessary to explore anti-inflammatory and photothermal combined therapeutic strategies to alleviate the inflammatory response caused by photothermal therapy. The development of nanomaterials provides a viable strategy for achieving combination therapy, and combining a photothermal conversion agent with an anti-inflammatory agent to construct a multifunctional nanomaterials becomes an effective way to solve the above problems.
Polydopamine (polydopamine, PDA) has excellent biocompatibility, metal chelation, free radical removal and photothermal conversion capabilities as an artificial melanin material. The nanometer material is formed by self-polymerization of dopamine in alkaline environment, and the nanometer material prepared based on PDA has been widely applied in the fields of biological imaging, drug delivery and the like. In particular its ability to chelate metals and good compatibility in organisms. By combining the PDA with other functional components, integration of a variety of therapeutic and imaging functions can be achieved, providing a more comprehensive and effective solution for cancer treatment and diagnosis. Therefore, the development based on PDA not only shows wide application prospect in the biomedical field, but also has important potential value in the aspect of constructing the T1-T2 dual-mode MRI contrast agent with the effects of photo-thermal and anti-inflammatory cancer treatment.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a method for generating nano particles by doping gadolinium and arginine into polydopamine and loading drug Meloxicam (MX), and the nano particles not only have the capacity of T1-T2 dual-mode imaging, but also have enhanced photo-thermal performance and oxidation resistance by doping gadolinium ions and arginine.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
The invention provides a preparation method of a mesoporous polydopamine nanometer diagnosis and treatment agent, which comprises the following steps:
S1, dissolving dopamine hydrochloride and gadolinium trichloride hexahydrate in water, and stirring in a dark place to prepare a dopamine-gadolinium chelate solution;
s2, adding the surfactant F127 and the dopamine-gadolinium chelate solution obtained in the step S1 into an ethanol-water mixed solution, and stirring the mixed solution in a dark place until the mixed solution is uniformly mixed;
s3, adding 1,3, 5-Trimethylbenzene (TMB) solution while shaking in water bath ultrasonic, and continuously dispersing to be milky;
s4, adding arginine (Arg) solution under stirring, and stirring and uniformly mixing in a dark place;
S5, centrifuging, re-suspending and washing the solution stirred in the step S4 to obtain Arg-Gd-MPDA (AGM) nano particles;
S6, dispersing Arg-Gd-MPDA (AGM) nano particles in the step S4 in water, mixing and stirring the Arg-Gd-MPDA (AGM) nano particles with a DMSO solution of Meloxicam (MX), collecting precipitate, and washing to remove DMSO to obtain the MX@Arg-Gd-MPDA (MAGM) nano diagnosis and treatment agent.
According to the invention, gd 3+ and Arg doped MPDA nano particles are used as carriers, and an anti-inflammatory agent MX is loaded to prepare the MAGM nano preparation, so that photo-thermal and anti-inflammatory treatment of the cancer under the guidance of T1-T2 double-mode MRI is realized, the treatment efficiency of the cancer is improved, and the recurrence risk is reduced. Firstly, the mesoporous nano structure greatly improves the encapsulation and delivery efficiency of the medicine, and has important significance for prolonged retention and slow release of the medicine. And secondly, the doping of arginine enhances the free radical scavenging and photo-thermal conversion performance of the polydopamine material, thereby being beneficial to improving the disease treatment efficiency. Again, the gm chelated Gd 3+ has the potential to be a T1-T2 dual mode MRI contrast agent under mesoporous spatial structure, providing a more powerful support for MRI-guided cancer treatment.
In a preferred embodiment of the present invention, in step S1, the mass ratio of the dopamine hydrochloride to the gadolinium chloride hexahydrate is 20 to 25:1.
As a preferred embodiment of the present invention, in the step S1, the parameter of the light-shielding stirring is 150-200 rpm, 22-26 hours.
As a preferred embodiment of the invention, in the step S1, the mass volume ratio of the gadolinium trichloride hexahydrate to the water is 1mg to 1.5mL.
As a preferred embodiment of the present invention, in the step S2, the mass-volume ratio of F127 to the dopamine-gadolinium chelate solution is 100-150 mg/1 mL.
As a preferred embodiment of the present invention, in the step S2, the parameter of the light-shielding stirring is 150 to 200rpm for 15 to 25 minutes.
In a preferred embodiment of the present invention, in step S2, the volume ratio of the ethanol-water mixed solution to the dopamine-gadolinium chelate solution is 40:1-1.2.
In step S3, as a preferred embodiment of the present invention, the parameters of the ultrasound are: 4kHz, 2-5 min.
As a preferred embodiment of the present invention, in the step S4, the concentration of the arginine solution is 17-19mg/mL, and the parameter of light-shielding stirring is 150-200 rpm and 6-8 h.
In step S5, the solvent to be washed is ethanol or ultrapure water, as a preferred embodiment of the present invention.
In step S6, as a preferred embodiment of the present invention, the volume ratio of the aqueous Arg-Gd-MPDA (AGM) solution to the DMSO solution of Meloxicam (MX) is 1:1.
The second aspect of the invention provides a mesoporous polydopamine nanometer diagnosis and treatment agent prepared by the preparation method of the first aspect.
The third aspect of the invention provides an application of the mesoporous polydopamine nano diagnosis and treatment agent in preparation of contrast agents.
The third aspect of the invention provides an application of the mesoporous polydopamine nanometer diagnosis and treatment agent in preparing an anti-tumor drug.
As a preferred embodiment of the present invention, the antitumor drug is an antitumor drug.
Compared with the prior art, the invention has the beneficial effects that:
The invention discloses a preparation method of a mesoporous polydopamine nanometer diagnosis and treatment agent, which takes gadolinium ions (Gd 3+) and arginine (Arg) doped Mesoporous Polydopamine (MPDA) nanometer particles as carriers and loads an anti-inflammatory drug Meloxicam (MX) to prepare a nanometer preparation MX@Arg-Gd-MPDA (MAGM) for T1-T2 dual-mode MRI guided photo-thermal and anti-inflammatory treatment of cancers. In general, the present invention has the following advantages:
(1) Aiming at the problems of low photo-thermal performance and limited oxidation resistance of polydopamine nano particles, arginine is doped in mesoporous polydopamine, so that a donor-acceptor microstructure is built in the mesoporous polydopamine, the energy band gap is reduced, and the electron transfer process is promoted, thereby enhancing the near infrared light absorption and photo-thermal conversion performance of polydopamine; meanwhile, effective inter-unit pi conjugation in the polydopamine is destroyed, so that weak aggregates are formed, the concentration of internal free radicals is further improved, and the free radical scavenging effect is enhanced.
(2) Aiming at the problem of influence of MRI artifact on a diagnosis result in a single mode in magnetic resonance imaging, the invention realizes T1-T2 bimodal magnetic resonance imaging by doping Gd 3+ in mesoporous polydopamine, so that the MRI imaging result is more accurate and has statistical significance.
(3) The nano preparation prepared by the invention has good biocompatibility, tumor microenvironment responsiveness and drug slow release capability, and can effectively avoid the problems of large side effect, poor targeting property, short in vivo circulation time and the like of the conventional drugs.
Drawings
Fig. 1 is a graph showing a change of a mixed solution in a preparation process of the MAGM nanoparticle (1 is a mixed solution of dopamine-gadolinium chelate and F127 dispersed in ethanol and water, 2 is a solution after TMB is added, 3 is a solution after arginine is added, and 4 is a MAGM dispersion);
FIG. 2 is a transmission electron microscope image (A) and a particle size distribution diagram (B) of MAGM nanoparticles;
FIG. 3 is an EDS energy spectrum of MAGM nanoparticles;
FIG. 4 shows (A) the temperature rise of different samples (618 nm,1W/cm 2, 100. Mu.g/mL); the temperature rise of MAGM under the conditions of (B) different concentrations (618 nm,1W/cm 2) and (C) different powers (618 nm,100 mug/mL); (D) The photothermal conversion efficiency of MAGM (806 nm,1W/cm 2, 100. Mu.g/mL);
FIG. 5 is a temperature change curve of MAGM (806 nm,1W/cm 2, 100. Mu.g/mL);
FIG. 6 shows (A) DPPH radical scavenging by GM, AGM and MAGM (20. Mu.g/mL) in 20 minutes; (B) Ultraviolet visible absorption spectra of solutions after GM, AGM and MAGM (100. Mu.g/mL) treatment;
FIG. 7 is (A) T1 and (B) T2 weighted magnetic resonance imaging of AGM and Gd-DTPA; longitudinal (C) and transverse (D) relaxations (field strength=3t) of AGM and Gd-DTPA;
FIG. 8 is (A) T1 and (B) T2 weighted magnetic resonance imaging of AGM and Gd-DTPA; longitudinal (C) and transverse (D) relaxations (field strength=7t) of AGM and Gd-DTPA;
FIG. 9 shows the viability of 4T1 cells after treatment under different conditions (NIR: 806 nm,1W/cm 2, 3 min); at the same concentration MX, AGM, MAGM, AGM +NIR, MAGM+NIR are indicated sequentially from left to right.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
Example 1 preparation method of mesoporous polydopamine nanometer diagnosis and treatment agent (MX@Arg-Gd-MPDA)
The Arg-Gd-MPDA (AGM) nano-carrier is prepared by chelating gadolinium ions in mesoporous polydopamine and doping arginine, and then an anti-inflammatory drug Meloxicam (MX) is loaded to prepare the MX@Arg-Gd-MPDA (MAGM) nano-diagnosis and treatment agent.
The specific method comprises the following steps:
1. the preparation method of the gadolinium and arginine doped mesoporous polydopamine Arg-Gd-MPDA (AGM) comprises the following steps:
(1) Dissolving dopamine hydrochloride and gadolinium trichloride hexahydrate in ultrapure water, and stirring in a dark place (180 rpm, 24 hours) to obtain a dopamine-gadolinium chelate solution; the mass ratio of the dopamine hydrochloride to the gadolinium chloride hexahydrate is 23:1, a step of; the mass volume ratio of gadolinium trichloride hexahydrate to ultrapure water is 1 mg:1-1.3 mL;
(2) Adding the surfactant F127 and the dopamine-gadolinium chelate solution in the step (1) into a mixed solution of absolute ethyl alcohol and ultrapure water (volume ratio is 1:1), and stirring in a dark place until the mixture is uniformly mixed (stirring parameters are 180rpm and 20 min); the mass volume ratio of F127 to the dopamine-gadolinium chelate solution is 120 mg/1 mL; the volume ratio of the absolute ethyl alcohol-ultrapure water mixed solution to the dopamine-gadolinium chelate solution is 40:1.1;
(3) Adding 1,3, 5-Trimethylbenzene (TMB) solution while shaking in water bath ultrasonic, and continuously dispersing to be milky (4 kHz, 3 min) by ultrasonic, wherein the volume ratio of TMB to the mixed solution in the step (2) is 41:1, a step of;
(4) Dropwise adding an arginine (Arg) aqueous solution (18 mg/mL) under magnetic stirring, and uniformly stirring and mixing (180 rpm, 7 h) in a dark place, wherein the volume ratio of the arginine aqueous solution to the mixed solution in the step (3) is 12.6:1, a step of;
(5) And finally, subpackaging the obtained solution into a centrifuge tube, centrifuging (13000 rpm,10 min), re-suspending the obtained precipitate in absolute ethyl alcohol, washing with water bath ultrasonic waves (4 kHz, 28 min), and repeatedly washing for 2 times to obtain the product, namely the AGM (Arg-Gd-MPDA) nano-particles. Meanwhile, in step (4), the Arg solution was replaced with a Tris (Tris) solution (20 mg/mL), and Gd-MPDA (GM) was obtained for the subsequent experimental control by the same procedure as that of the preparation and centrifugal washing.
2. The preparation method of the nanoparticle MX@Arg-Gd-MPDA (MAGM) loaded with the anti-inflammatory drug Meloxicam (MX) comprises the following steps:
(6) Dispersing the product AGM obtained in the step (5) in pure water (1 mg/mL), mixing with a DMSO solution of MX (1 mg/mL) (the volume ratio of the two solutions is 1:1), stirring and uniformly mixing for 24 hours, separating the reacted solution into centrifuge tubes, centrifuging (13000 rpm,10 min) to collect the precipitate, washing with absolute ethyl alcohol and ultrapure water to remove the DMSO, and then re-suspending and collecting with ultrapure water to obtain the MAGM nanoparticles.
Example 2 characterization of mesoporous polydopamine nanodiagnosis and treat (MAGM)
(1) Color change of mixed solution in MAGM nanoparticle preparation process
As shown in fig. 1, the dopamine-gadolinium chelate and F127 are dispersed in a mixed solution of ethanol and water to form a white transparent solution; after TMB is gradually added under the ultrasonic of water bath, the solution gradually turns into milky white; after the arginine solution is added, the dopamine polymerization reaction liquid turns grey due to the alkaline environment provided by the arginine solution; after 8 hours of reaction, black nanoparticle dispersion liquid is formed, and the product AGM nanoparticles can be obtained after centrifugal cleaning; after mixing and stirring the product AGM with MX, the mixture was centrifugally washed and the MAGM dispersion, which gave a black solution, was collected.
(2) MAGM morphology and particle size characterization
And (3) dripping 10 mu L of diluted MAGM dispersion (50 mu g/mL) on the front surface of a copper mesh of a carbon support film, naturally drying in the air at room temperature in a dryer, and observing the microscopic morphology (morphology, particle size and dispersion condition) of the MAGM under the condition of 200KV voltage by using a Transmission Electron Microscope (TEM). As shown in FIG. 2A, the prepared MAGM has a narrow particle size distribution range, is spherical, has uniform particle size, is about 200nm, and has a uniformly distributed mesoporous structure. Meanwhile, the prepared MAGM nano particles are resuspended in ultrapure water, diluted to a certain concentration (50 mug/mL) and then are uniformly subjected to ultrasonic treatment, and a Dynamic Light Scattering (DLS) method is used for detecting the hydration particle size, the polydispersity coefficient and the Zeta potential of the nano particle sample. As shown in Table 1 and FIG. 2B, MAGM nanoparticles were excellent in dispersibility and had a hydrated particle diameter of about 247 nm. The reason why the transmission electron microscope and the Dynamic Light Scattering (DLS) measurement result are different is that the latter obtains a particle size of the nanoparticle in a hydrated state, and the solvent effect causes the nanoparticle to display a larger hydrodynamic volume.
(3) EDS energy spectrum analysis and ICP-AES characterization
Diluting MAGM nano particles (50 mug/mL) and fully performing ultrasonic dispersion, dripping 10 mug of solution on a copper mesh, drying, and determining the distribution of four elements of carbon (C), oxygen (O), nitrogen (N) and Gd in the MAGM by X-ray energy spectrum analysis (EDS) under a 200kV transmission electron microscope. Meanwhile, 1mL of MAGM solution (1 mg/mL) is taken, a proper amount of concentrated nitric acid (2-4 mL) is added, the solution is heated for more than 48h in an oil bath until the solution is colorless, the solution is diluted by ultrapure water and is fixed to 10mL (acid content is less than 5% by weight percent), and the content of Gd element in MAGM nano particles is measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). As shown in FIG. 3, C, N, O, gd elements are uniformly distributed in the nanoparticle, and the content of Gd element is 3.06% (wt%) by ICP-AES determination. Successful chelation of Gd provides a basis for use of MAGM for MRI contrast. Further, the Arg doping amount in MAGM was calculated to be 3.22% (wt%) by quantitative analysis of N element.
(4) In vitro photo-thermal performance of MAGM using 808nm near infrared laser
The photo-thermal conversion efficiency and photo-thermal stability of MAGM were investigated. 1) Comparing the photo-thermal conversion performance of different samples: 2mL of ultrapure water, GM, AGM and MAGM (100. Mu.g/mL) samples were subjected to near infrared laser irradiation at 1W/cm 2, and the real-time temperature of the solution was recorded over 10 min; 2) The effect of concentration on temperature rise was investigated: 2mL of MAGM samples with different concentrations (50, 100 and 150 mug/mL) were subjected to near infrared laser irradiation of 1W/cm 2, and the real-time temperature of the solution within 10min was recorded; 3) The effect of laser power on temperature rise was explored: 2mL of MAGM (100 mug/mL) samples were subjected to near infrared laser irradiation with different powers (0.5, 1, 1.5W/cm 2), and the real-time temperature of the solution within 10min was recorded; 4) Photo-thermal conversion efficiency study: carrying out 1W/cm 2 near infrared laser irradiation on a 2mL MAGM (100 mug/mL) sample until the temperature is stable (the temperature is not changed within 2 min), then naturally cooling, recording the real-time temperature in the whole process of heating and cooling, and calculating the photo-thermal conversion efficiency of the MAGM; 5) Photo-thermal stability study: 2mL of 100 mug/mL MAGM sample is subjected to near infrared laser irradiation of 1W/cm 2 for 15min, then the irradiation is stopped, the solution temperature is waited to be reduced to the initial temperature, and the MAGM sample is taken as a cycle, four times of cycles are performed on the MAGM, the real-time temperature of the whole process is recorded, and the photo-thermal stability of the MAGM is studied.
As shown in fig. 4A, the temperature of the Arg-doped AGM group is raised by 34 ℃ higher than that of the Arg-undoped GM group (22 ℃) to prove that the Arg doping further improves the photo-thermal conversion performance of polydopamine, and the photo-thermal conversion efficiency can reach 56.5% (fig. 4D), because the Arg possibly breaks down the compact microstructure of polydopamine, builds a donor-acceptor structure in the Arg, promotes electron transfer, and reduces non-thermal radiation transition. Then, the influence factors of the MAGM heating effect are studied by changing the concentration and the laser power, and the result is that the MAGM heating effect is enhanced along with the increase of the concentration and the power, as shown in fig. 4B and 4C, so that the sample concentration, the irradiation power and the time can be adjusted according to different temperature requirements in practical application, and the optimal effect is achieved. In addition, the photo-thermal stability of the MAGM is evaluated through the cyclic heating and cooling process (figure 5), and after 4 times of circulation, the highest temperature is only reduced by 1.6 ℃, so that the photo-thermal conversion capability of the MAGM is not obviously affected by cyclic irradiation, the photo-thermal stability is achieved, and the method has a wide application prospect in PTT.
(5) In vitro free radical scavenging Performance study
Evaluation of the free radical scavenging properties of GM, AGM and MAGM by DPPH and salicylic acid method (evaluation of DPPH method with reference to DPPH method "Peralta E,Roa G,Hernandez-Servin J A,et al.Hydroxyl Radicals quantification by UV spectrophotometry[J].Electrochimica Acta,2014,129:137-141.").. Evaluation of salicylic acid method with reference to "Adolfsson K H,Huang P,Golda-Cepa M,et al.Scavenging of DPPH by Persistent Free Radicals in Carbonized Particles[J].Advanced Sustainable Systems,2023,7(3):2200425."; is shown in fig. 6), all three have DPPH and OH scavenging ability, the free radical of GM group is lower, AGM after arginine doping shows more excellent scavenging ability, because the doped arginine breaks down the internally dense microstructure of polydopamine, forming weak polymers with stronger free radical capturing ability, MAGM shows similar free radical scavenging rate as AGM, indicating that the loading of MX does not affect the capturing of free radicals by nanoparticles.
(6) In vitro magnetic resonance imaging capability
The T1 and T2 weighted magnetic resonance imaging capabilities of AGM and Gd-DTPA (Magnevist) contrast agents were compared using a clinically used 3T magnetic resonance imager. In T1-weighted MRI (FIG. 7), AGM showed superior contrast capability to Gd-DTPA, with r1 values (74.86 mM -1·s-1) much higher than Gd-DTPA (4.99 mM-1 s -1) because Gd3+ chelated mesoporous nanoparticles significantly affect water molecule relaxation. This effect on water molecule relaxation gives AGM a high r2 (70.66 mM -1·s-1) at the same time, which also shows excellent effect in T2 weighted imaging (fig. 7), whereas Gd-DTPA has r2 value of only 4.82mM -1·s-1 and cannot be used for T2 imaging. Therefore, the AGM chelated with Gd 3+ has higher T1 sensitivity than Gd-DTPA, and simultaneously has the T1-T2 dual-mode contrast capability, thereby having important significance for improving the accuracy of disease diagnosis and treatment. Meanwhile, in order to further research the application prospect of the AGM serving as the T1-T2 dual-mode MRI contrast agent in ultra-high field intensity magnetic resonance imaging, the contrast performance of the AGM under the field intensity of 7T is also evaluated. In T1 weighted imaging, AGM showed better contrast effect than Gd-DTPA (fig. 8). The r1 value of AGM was reduced to 9.76mM -1·s-1, but still higher than Gd-DTPA (4.63 mM -1·s-1) compared to the experimental results at 3T field strength. The reason for the decrease of the relaxation rate is that the interaction of AGM and water molecules changes along with the increase of the field intensity, so that the longitudinal relaxation process of the water molecules is limited, and the Gd-DTPA as a small molecule is less influenced by the field intensity, so that the relaxation rate is not obviously decreased. In addition, AGM also shows T2 contrast capability at 7T field strength (FIG. 8), and has a higher r2 (121.77 mM -1·s-1), which can be considered an excellent T2 contrast agent at ultra-high field strength. In conclusion, the AGM also has the T1-T2 dual-mode MRI contrast capability with higher sensitivity under the field intensity of 7T, and further widens the application prospect in the field of magnetic resonance imaging.
(7) Cytotoxicity study
The photothermal anticancer effect of MAGM was studied from the level of mouse breast cancer cells (4T 1). Culture of 4T1 cells (5000 cells/well, 24 h) was performed using 96-well plates, and the effect of MX, AGM and MAGM on 4T1 cell viability with or without laser irradiation was examined by CCK8 experiments. As shown in figure 9, the three components have no obvious toxicity to cells, and the good biocompatibility of the nano particles is reflected. Laser light (806 nm,1W/cm 2, 3 min) was applied to the AGM and MAGM groups to evaluate the effect of photothermal treatment, and the cell viability of both groups showed a similar trend of decreasing to about 50% at a concentration of 100. Mu.g/mL, and only about 15% at a continued increase to 200. Mu.g/mL. From this, it was demonstrated that photothermal treatment with AGM and MAGM has a significant killing effect on 4T1 cells.
In summary, the Arg-Gd-MPDA (AGM) nano-carrier is prepared by utilizing gadolinium ions and arginine doped mesoporous polydopamine, and then the anti-inflammatory drug Meloxicam (MX) is loaded to prepare the MX@Arg-Gd-MPDA (MAGM) nano diagnosis and treatment agent, so that the nano diagnosis and treatment agent not only has the T1-T2 dual-mode imaging capability, but also has enhanced photo-thermal performance and oxidation resistance, provides more powerful support for the treatment of MRI guided cancers, and has important potential application value.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
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