Preparation method and application of diagnosis and treatment integrated nanoprobe based on gadolinium-iridium co-doped carbon dots

Abstract

The invention discloses preparation and application of a diagnosis and treatment integrated probe based on gadolinium-iridium co-doped carbon dots, in the preparation process, Gd / Ir-CDs with excellent MRI / CT bimodal imaging ability, I-type photodynamic potential and peroxide dismutase activity and an excellent nano-carrier MPDA with certain photo-thermal conversion ability are synthesized by changing the element doping proportion, and then the Gd / Ir-CDs / MPDA is prepared. Then, the two polymers of the Gd / Ir-CDs and the MPDA are subjected to self-assembly to form a nano-composite, and finally, the diagnosis and treatment integrated nano-probe MPDA-CDs is prepared. The probe is based on Gd / Ir-CDs, the MRI / CT bimodal imaging capacity of the Gd / Ir-CDs is reserved, peroxide dismutase activity and I-type photodynamic therapy under an electron transfer mechanism are achieved through element doping, the photothermal capacity of MPDA is fully combined, and the probe is a diagnosis and treatment integrated probe with PTT / PDT optical combined treatment potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological and medical nanomaterials, and particularly relates to a preparation method and application of a theranostic nanoplatform based on gadolinium-iridium co-doped carbon dots. Background Art

[0002] Currently, the imaging methods commonly used in clinical diagnosis of rheumatoid arthritis include X-ray imaging, ultrasound (US), magnetic resonance imaging (MRI), and computed tomography (CT). In recent years, the application of bimodal imaging probes in the diagnosis of rheumatoid arthritis has been widely studied.

[0003] Carbon dots (CDs) are a type of zero-dimensional (0D) carbon material. As a new material, CDs not only inherit the advantages of traditional semiconductor quantum dots (such as CdS, CdSe, CdTe quantum dots) and small molecule compounds (such as indocyanine green, rhodamine and other fluorescent small molecules), but also have many unique advantages. For example, CDs have good photostability, good biocompatibility, low toxicity, are easy to prepare, and have low cost, and can be used as a fluorescence imaging module for disease diagnosis. At the same time, carbon dots can also be used as therapeutic drugs, mainly in two ways: as a photosensitizer for phototherapy and as a nanozyme for chemodynamic therapy.

[0004] However, how to achieve a theranostic nanoplatform based on carbon dots through element doping for application in bimodal imaging and PTT / PDT precise therapy is a problem that needs to be solved in the current field. Summary of the Invention

[0005] The present invention aims to solve the above technical problems. Before elaborating on the solution of the present invention, the following English abbreviations are explained: Gd: gadolinium; Ir: iridium; MPDA: polydopamine; DMF: N,N-dimethylformamide; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; F127: polyoxyethylene polyoxypropylene ether; TMB: 3,3',5,5'-tetramethylbenzidine.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a preparation method of a theranostic nanoplatform based on gadolinium-iridium co-doped carbon dots, comprising the following steps:

[0008] Preparation of S1, Gd, and Ir co-doped carbon dots Gd / Ir-CDs: Using citric acid and p-phenylenediamine as raw materials for carbon dots, a carbon core is formed by a one-step hydrothermal method, and Gd 3+ / Ir 3+ is chelated and obtained.

[0009] S2. Preparation of MPDA: Prepared by polymerizing dopamine hydrochloride under the action of surfactant F127 and interface modifier TMB.

[0010] S3. Preparation of the integrated diagnosis and treatment probe MPDA-CDs: Prepared by using MPDA as a nanocarrier to load Gd / Ir-CDs after carboxyl activation.

[0011] As a preferred embodiment of the present invention, in the above method for preparing an integrated diagnosis and treatment nanoprobe, step S1 includes:

[0012] S11. Preparation of the carbon dot reaction solution: Citric acid, p-phenylenediamine, gadolinium chloride, and iridium chloride are taken and ultrasonically dissolved in an excessive DMF solvent to obtain it;

[0013] S12. Synthesis of Gd / Ir-CDs: The carbon dot reaction solution prepared in step S11 is subjected to a hydrothermal reaction, and then centrifuged, dialyzed, and dried to obtain it;

[0014] Step S2 includes:

[0015] S21. Preparation of the reaction emulsion: The surfactant F127, the interface modifier TMB, and the solvent ethanol solution are mixed evenly and obtained by ultrasonic treatment;

[0016] S22. Synthesis of MPDA: Under the action of the surfactant F127 and the interface modifier TMB, dopamine hydrochloride and the reaction emulsion prepared in step S21 are fully stirred, and ammonia water is added for reaction to obtain it;

[0017] Step S3 includes:

[0018] S31. Activation of the carboxyl group on the surface of carbon dots: Under the catalysis of EDC and NHS, the carboxyl group on the surface of Gd / Ir-CDs prepared in step S1 is activated to obtain it;

[0019] S32. Synthesis of MPDA-CDs: The Gd / Ir-CDs with the surface carboxyl group activated obtained in step S31 and the MPDA prepared in step S2 are stirred in the dark to obtain it.

[0020] Preferably, in step S11, the molar ratio of citric acid, p-phenylenediamine, gadolinium chloride, iridium chloride to DMF is 1:1:1.2:1:(1-10). Further, the molar ratio of citric acid, p-phenylenediamine, gadolinium chloride, iridium chloride to DMF is 1:1:1.2:1:5.

[0021] Preferably, in step S12, the temperature of the hydrothermal reaction is 160 °C, the time of the hydrothermal reaction is 2 to 4 hours, the centrifugation speed is 10,000 to 13,000 rpm / min, and the MWCO of the dialysis bag selected for dialysis is greater than or equal to 3500. Further, in step S12, the temperature of the hydrothermal reaction is 160 °C, the time of the hydrothermal reaction is 2 hours, the centrifugation speed is 12,000 rpm / min, and the MWCO of the dialysis bag selected for dialysis is 3500.

[0022] Preferably, in step S12, the supernatant after centrifugation is collected, dialyzed in ultrapure water for 1 to 2 days, and freeze-dried to obtain the product. Further, in step S12, the supernatant after centrifugation is collected, dialyzed in ultrapure water for 1 day, and freeze-dried to obtain the product.

[0023] Preferably, in step S21, the molar ratio of F127, TMB, and ethanol is 5:3:2. F127 and TMB are added to ethanol to form a suspension, and a milky white reaction emulsion is formed by ultrasonic treatment.

[0024] Preferably, in step S22, the mass ratio of dopamine hydrochloride to the reaction emulsion is 1:(5 - 10). Dopamine hydrochloride is added to the reaction emulsion and stirred magnetically to obtain solution one. Further, the mass ratio of dopamine hydrochloride to the reaction emulsion is 1:9. Dopamine hydrochloride is added to the reaction emulsion and stirred magnetically to obtain solution one.

[0025] Preferably, in step S22, the molar ratio of dopamine hydrochloride to ammonia water is 1:200. Ammonia water is added dropwise to solution one and stirred for 2 hours.

[0026] Specifically, in step S22, the reaction temperature is 25 °C and the stirring speed is 600 rpm / min.

[0027] Preferably, in step S31, the mass ratio of EDC, NHS, and the Gd / Ir-CDs prepared in step S1 is 10:7:(5 - 10). Further, the mass ratio of EDC, NHS, and the Gd / Ir-CDs prepared in step S1 is 10:7:10.

[0028] Preferably, in step S31, EDC, NHS, and the Gd / Ir-CDs prepared in step S1 are dissolved in a solvent, and stirred and reacted in the dark for 0.5 hour to activate -COOH, obtaining a solution of Gd / Ir-CDs with surface carboxyl groups activated.

[0029] Preferably, in step S32, the mass ratio of MPDA prepared in step S2 to the carboxyl group-activated Gd / Ir-CDs prepared in step S31 is 1:(1 - 5).

[0030] Preferably, in step S32, the MPDA solution is added to the surface carboxyl-activated Gd / Ir-CDs solution, and stirring is continued in the dark for 6 to 8 hours. Further, the MPDA solution is added to the surface carboxyl-activated Gd / Ir-CDs solution, and stirring is continued in the dark for 6 hours to obtain Solution 2.

[0031] Specifically, in step S32, the reaction temperature is 25°C and the stirring speed is 600 rpm / minute.

[0032] Preferably, in step S32, Solution 2 is centrifuged at 13000 rpm for 10 minutes, and the resulting precipitate is dissolved in pure water to obtain the MPDA-CDs solution.

[0033] The present invention also provides an integrated diagnosis and treatment nanosensor prepared by the above preparation method.

[0034] The present invention also provides the application of the above integrated diagnosis and treatment nanosensor in the preparation of a contrast agent.

[0035] The present invention also provides the application of the above integrated diagnosis and treatment nanosensor in the preparation of a therapeutic drug for rheumatoid arthritis.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] The present invention first synthesizes Gd / Ir-CDs with excellent MRI / CT dual-modal imaging ability, type I photodynamic potential and superoxide dismutase activity by changing the element doping ratio, and MPDA, an excellent nanocarrier with certain photothermal conversion ability. Then, these two polymers, Gd / Ir-CDs and MPDA, are self-assembled into a nanocomposite, and finally, a theranostic nanoprobe MPDA-CDs is prepared. Based on Gd / Ir-CDs, this probe not only retains the MRI / CT dual-modal imaging ability of Gd / Ir-CDs, but also realizes type I photodynamic therapy under superoxide dismutase activity and electron transfer mechanism through element doping, and fully combines the photothermal ability of MPDA. It is a theranostic probe with the potential of PTT / PDT optical combined therapy. This probe can achieve complementary imaging of joint soft tissues and hard tissues through two imaging modes of MRI and CT, so as to accurately determine the focus of arthritis and comprehensively evaluate the bone defects and synovial lesions of rheumatoid arthritis. At the same time, by changing the doped elements, Gd / Ir-CDs are endowed with type I photodynamic therapy potential and superoxide dismutase activity, and the type I photodynamic therapy cascaded by superoxide dismutase has a more significant PDT anti-inflammatory effect. And based on the EPR (enhanced permeability and retention) effect at the arthritis site, this probe can stay at the arthritis site, and then realize precise optical therapy under dual-modal imaging localization. After the lesion site is determined by MRI / CT dual-modal imaging, physical localization type PTT / PDT precise treatment of rheumatoid arthritis is efficiently carried out through an external light source, which significantly improves the curative effect while reducing the toxic and side effects, and improves the problem of limited curative effect of a single treatment method. It can be seen that the theranostic nanoprobe of the present invention integrates MRI / CT dual-modal imaging and physical localization type PTT / PDT precise treatment. Description of the Drawings

[0038] Figure 1 It is the synthesis route of the theranostic nanoprobe in Example 1 of the present invention;

[0039] Figure 2 It is the TEM image and DLS of the theranostic nanoprobe in Example 1 of the present invention;

[0040] Figure 3 It is the MRI / CT imaging results in mice in Example 2 of the present invention;

[0041] Figure 4 It is the graph of the change in paw thickness during the treatment of mice in Example 2 of the present invention;

[0042] Figure 5 It is the clinical score for the paw thickness of mice in Example 2 of the present invention. Detailed Embodiments

[0043] The specific embodiments of the present invention will be further described below. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0044] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0045] Example 1 Preparation of a diagnostic and therapeutic integrated nanoprobe

[0046] Its synthesis route is as Figure 1 shown, and the specific preparation method includes the following steps:

[0047] S1. Synthesis of Gd, Ir co-doped carbon dots (Gd / Ir-CDs): Take 192 mg of citric acid, 108 mg of p-phenylenediamine, 37.1 mg of gadolinium chloride and 29.8 mg of iridium chloride in a 50 mL centrifuge tube, add 10 mL of DMF, and ultrasonically dissolve in an ultrasonic cleaner until a black carbon dot reaction solution is formed. Transfer the carbon dot reaction solution to a 50 mL polytetrafluoroethylene inner liner, then put it into a reaction kettle, adjust the oven to 160 °C and put the reaction kettle into the oven after the temperature is stable for 2 hours. After the reaction is completed, take out the reaction kettle, cool it naturally, then ultrasonicate it, and centrifuge it at 12,000 rpm / min for 30 minutes. The obtained supernatant is the Gd / Ir-CDs solution. Dialyze the Gd / Ir-CDs solution with a dialysis bag with MWCO = 3500 in pure water for one day, and finally freeze-dry to obtain Gd / Ir-CDs powder.

[0048] S2. Synthesis of MPDA: Take 0.5 g of F127 and 0.8 mL of TMB, then add 10 mL of 50% ethanol solution, and ultrasonicate until a milky white reaction emulsion is obtained. Take 0.75 g of dopamine hydrochloride into a beaker, then add the reaction emulsion, and stir magnetically until completely dissolved to obtain Solution 1, where the reaction temperature is 25 °C and the stirring speed is 600 rpm / min. Keep stirring, and gradually add 1.875 mL of ammonia water dropwise to Solution 1. After reacting for 2 hours, centrifuge at 13,000 rpm for 10 minutes. The obtained precipitate is washed with ethanol, 50% ethanol solution and pure water respectively (dissolve, ultrasonicate, centrifuge), and finally add 5 mL of pure water to dissolve to obtain the MPDA solution.

[0049] S3. Synthesis of MPDA-CDs: Take 10.00 mg of EDC, 7.00 mg of NHS, and 10 mg of Gd / Ir-CDs prepared in 3.2.3.1. After dissolving with pure water, react for 0.5 hours by magnetic stirring to activate the carboxyl groups on the surface of the carbon dots. Then add 10.00 mg of MPDA prepared in S2 and react for 6 hours to obtain Solution 2. Centrifuge Solution 2 at 13,000 rpm for 10 minutes, and dissolve the obtained precipitate with 5 mL of pure water to obtain the MPDA-CDs solution.

[0050] The above-prepared integrated diagnosis and treatment nanoprobes were observed using a particle size analyzer and transmission electron microscope (TEM). As Figure 2 shown in the TEM image, MPDA-CDs are spherical in aqueous solution, with uniform dispersion. The particle sizes measured by DLS and calculated by TEM are about 328 nm and 288 nm, respectively.

[0051] Example 2 Application of the integrated diagnosis and treatment nanoprobes in the treatment of rheumatoid arthritis under MRI / CT imaging

[0052] (1) Establishment of the arthritis mouse (CIA) model: The collagen-induced arthritis model was constructed by subcutaneous injection of bovine type II collagen and Freund's complete adjuvant (CFA) / Freund's incomplete adjuvant (IFA). First, add 2.5 mL of bovine type II collagen and 2.5 mL of CFA to a centrifuge tube. Under the conditions of ice bath and ultrasound, use a syringe to blow and beat to completely emulsify the solution, that is, a small amount of the emulsion dropped into water can form uniform and stable spherical droplets. The experimental mice were first anesthetized by intraperitoneal injection of 400 μL of 1.25% avertin (0.02 ml·g -1 ), and then 50 μL of the emulsion was subcutaneously injected into the tail for primary immunization. One week later, add 2.5 mL of bovine type II collagen and 2.5 mL of IFA to a centrifuge tube. Under the conditions of ice bath and ultrasound, use a syringe to blow and beat to completely emulsify the solution, that is, a small amount of the emulsion dropped into water can form uniform and stable spherical droplets. The experimental mice were first anesthetized by intraperitoneal injection of 400 μL of 1.25% avertin (0.02 ml·g -1 ), and then 50 μL of the emulsion was subcutaneously injected into the tail for booster immunization. The CIA mice were obtained one week after the booster immunization.

[0053] (2) In vivo imaging: The experimental mice were divided into four groups: healthy blank control group, healthy contrast agent experimental group, arthritis blank control group, and arthritis contrast agent experimental group. One mouse from each of the different experimental groups was taken for in vivo imaging experiments. The experimental mice were first anesthetized by intraperitoneal injection of 400 μL of 1.25% avertin (0.02 ml·g -1) Anesthesia was performed. Then, the mice were fixed in a small animal body coil and T1-enhanced contrast imaging of the coronal plane of the hind paws of the mice was performed on a magnetic resonance imaging instrument. The average signal intensity of the palm of the hind paw was recorded. Then, 100 μL of a contrast agent with a concentration of 0.2 mg·mL -1 was injected through the tail vein. The above scanning process was repeated at 0.5, 1, 1.5, and 2 hours respectively, and the signal intensities of the palm of the hind paw of the mice at different times after injecting the contrast agent were compared.

[0054] After the experiment started, as Figure 3 shown, the imaging pictures of the hind paws of 1.25% avertin-induced arthritic mice showed the most obvious signal enhancement 1 hour after injecting MPDA-CDs. Moreover, the right hind paw with obvious arthritis symptoms had a higher signal intensity than the left hind paw with milder symptoms. This indicated that 1 hour after tail vein injection, the contrast agent was delivered to the arthritis lesion site through blood metabolism. Due to the high retention effect at the arthritis lesion site, MPDA-CDs remained at the arthritis lesion site, and the significantly enriched contrast agent enhanced the signal of the arthritis lesion site, reaching 419% of that before injection. However, 1 hour later, the signal rapidly decreased and basically returned to the normal level 2 hours after injection, proving that MPDA-CDs could be excreted from the body through metabolism and would not remain in the body for a long time to cause a burden on the body. In normal mice and CIA mice injected with PBS, no obvious signal changes were observed, indicating that MPDA-CDs had good specificity. Then, based on the time when MPDA-CDs accumulated at the joint lesion site of the mice, the CT signal intensities of the hind paws of the mice were observed before injecting the contrast agent / PBS and 1 hour after injection. After injecting MPDACD, the pathological features of the joint bone tissue of the mice could be observed more clearly under the same window width and window level. The relative CT value could reach 211% of that before injection. This proved the good MRI / CT dual-modal imaging effect of MPDA-CDs.

[0055] (3) In vivo treatment:

[0056] Taking the primary immunization as Day 0, after the CIA mouse model was constructed on Day 28, the CIA mice were randomly divided into three groups, and 100 μL of PBS and MPDA-CDs were respectively injected through the tail vein for treatment, corresponding to the blank control group, the light-avoiding treatment group, and the laser treatment group (laser wavelength was 808 nm, power was 1 W·cm -2 ). Meanwhile, 5 normal mice without modeling were injected with 100 μL of MPDA-CDs as the normal control group. Every three days, a tail vein injection was performed, and the paw thickness and body weight of the four paws of the mice were recorded to evaluate the arthritis condition and physical health of the mice.

[0057] After the treatment started, as Figure 4 andFigure 5 As shown, the paw thickness and clinical score curves of the mice in the treatment group showed a gradually decreasing trend, while those in the control group gradually increased. Its MRI and CT images also showed that the arthritis symptoms of the mice in the treatment group were significantly alleviated, while severe inflammation and bone erosion occurred in the control group. This indicates that the integrated diagnosis and treatment nanosensor of the present invention can not only actively target the arthritis site and has good MRI and CT imaging contrast functions, but also has a good therapeutic effect on rheumatoid arthritis through PTT / PDT therapy.

[0058] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. A method for preparing a diagnosis and treatment integrated nanoprobe based on gadolinium-iridium co-doped carbon dots, characterized in that: The following steps are involved: Preparation of S1, Gd, and Ir co-doped carbon dots Gd / Ir-CDs: Citric acid and p-phenylenediamine were used as carbon dot raw materials to form carbon cores through a one-step hydrothermal method, and Gd was chelated. 3+ / Ir 3+ and obtained; S2. Preparation of MPDA: dopamine hydrochloride is polymerized under the action of surfactant F127 and interface regulator TMB to obtain MPDA; S3. Preparation of the integrated diagnostic and therapeutic probe MPDA-CDs: It is prepared by using MPDA as a nanocarrier to load carboxyl-activated Gd / Ir-CDs.

2. The preparation method according to claim 1, characterized in that: Step S1 includes: S11, preparation of carbon dot reaction solution: dissolving citric acid, p-phenylenediamine, gadolinium chloride and iridium chloride in excess DMF solvent by ultrasonication; S12, Gd / Ir-CDs synthesis: subjecting the carbon dot reaction solution prepared in step S11 to a hydrothermal reaction, and then subjecting the mixture to centrifugal dialysis and drying to obtain the resultant; Step S2 includes: S21, preparation of reaction emulsion: surfactant F127, interface regulator TMB and solvent ethanol solution are mixed and prepared by ultrasound; S22, MPDA synthesis: dopamine hydrochloride and the reaction emulsion prepared in step S21 are fully stirred under the action of surfactant F127 and interface regulator TMB, and ammonia water is added to react to obtain the product; Step S3 includes: S31, activation of carbon dot surface carboxyl groups: under the catalysis of EDC and NHS, the surface carboxyl groups of Gd / Ir-CDs prepared in step S1 are activated; S32, MPDA-CDs synthesis: Gd / Ir-CDs with surface carboxyl activated obtained in step S31 and MPDA obtained in step S2 are stirred in the dark.

3. The preparation method according to claim 2, characterized in that: In step S11, the molar ratio of citric acid, p-phenylenediamine, gadolinium chloride, iridium chloride and DMF is 1:1:1.2:1:(1-10).

4. The preparation method according to claim 2, characterized in that: In step S21, the molar ratio of F127, TMB and ethanol is 5:3:

2.

5. The preparation method according to claim 2, characterized in that: In step S22, the mass ratio of dopamine hydrochloride to the reaction emulsion is 1:(5-10), and the molar ratio of dopamine hydrochloride to aqueous ammonia is 1:

200.

6. The preparation method according to claim 2, characterized in that: In step S31, the mass ratio of EDC, NHS and Gd / Ir-CDs prepared in step S1 is 10:7:(5-10).

7. The preparation method according to claim 2, characterized in that: In step S32, the mass ratio between the MPDA prepared in step S2 and the carboxyl-activated Gd / Ir-CDs prepared in step S31 is 1:(1-5).

8. An integrated diagnosis and treatment nanoprobe prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the integrated diagnosis and treatment nanoprobe according to claim 8 in the preparation of contrast agents.

10. Use of the integrated diagnosis and treatment nanoprobe according to claim 9 in the treatment of rheumatoid arthritis.

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