CN113440626A

CN113440626A - Targeting nano magnetic resonance contrast agent for articular cartilage damage and preparation and application thereof

Info

Publication number: CN113440626A
Application number: CN202011105510.7A
Authority: CN
Inventors: 鹿蓉; 陈爽; 陶虹月; 张宇阳; 陆遥; 周璐; 李慧逖
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2020-03-30
Filing date: 2020-10-15
Publication date: 2021-09-28

Abstract

The invention belongs to the technical field of molecular imaging, and relates to a targeted nano magnetic resonance contrast agent for articular cartilage damage, and preparation and application thereof. The contrast agent HAs a structure of HA-DTPA-Gd, hyaluronic acid HA is linked with gadolinium ions Gd (III) through diethylenetriaminepentaacetic acid DTPA, and the molecular structural formula of the contrast agent is shown as a formula (I). The contrast agent can be injected through a joint cavity, is nano-scale in size, is combined with extracellular matrix of a cartilage defect area based on the surface effect of nano-particles, is more easily and specifically aggregated in the cartilage damage area, and has MRIT1 weighted delay increaseThe strengthening degree of the strong posterior focal zone is obvious compared with the strengthening degree of a clinical gadolinium-containing contrast agent, so that the cartilage damage focus can be accurately and visually displayed, the accuracy and the safety of treatment can be clinically improved, the curative effect of a knee joint cartilage damage treatment mode can be evaluated, and the prognosis of a patient can be evaluated.

Description

Targeting nano magnetic resonance contrast agent for articular cartilage damage and preparation and application thereof

Technical Field

The invention belongs to the technical field of molecular imaging, relates to a targeted nano magnetic resonance contrast agent for articular cartilage damage and preparation and application thereof, and particularly relates to a mucopolysaccharide modified articular cartilage damage specific nano magnetic resonance contrast agent based on Hyaluronic Acid (HA), and a preparation method and application thereof.

Background

Articular cartilage damage is a common and frequently encountered disease in clinic and affects a wide range of people. The cartilage covers the surface of the joint and plays the roles of reducing friction generated by joint movement, absorbing mechanical shock and distributing joint load; cartilage is composed of dispersed chondrocytes and extracellular matrix, and lacks blood supply. The extracellular matrix mainly comprises collagen (15-20%), proteoglycan (3-10%) and moisture (65-80%). The factors causing cartilage damage are various, acute and chronic injuries, obesity, aging, heredity, immunity and the like are all factors causing cartilage cell damage, and the damaged cartilage cells release neutral protease, collagenase and the like to degrade proteoglycan and collagen fiber networks in a cartilage matrix, so that cartilage structure damage, edema and viscoelasticity are reduced, the biomechanical function of cartilage is damaged, and cartilage damage and degeneration are caused. Cartilage damage easily causes joint pain, edema and mobility reduction, joint cartilage in vivo cannot be regenerated, self-repair capacity is extremely limited, once damage occurs, self-healing is difficult, irreversible damage is caused if diagnosis and treatment are not carried out in time, secondary osteoarthritis is gradually developed, joint function loss is finally caused, and life quality of patients is seriously influenced. Therefore, timely diagnosis of articular cartilage damage is the key to clinical early intervention.

Currently, arthroscopy is an important means for diagnosing articular cartilage damage, but it is an invasive examination, has high technical requirements for operators, may cause postoperative complications, and cannot evaluate cartilage thickness, cartilage surrounding structures, and the like. MRI has the advantages of multi-azimuth, multi-sequence, multi-parameter imaging, high tissue resolution and the like, becomes the only non-invasive examination method which can effectively display the morphological change of articular cartilage at present, and can display the surface, internal characteristics, surrounding tissues and the like of the cartilage. However, early cartilage damage or degeneration occurs before the morphology has not changed, with loss of proteoglycans within the cartilage matrix and disruption of the collagen fiber network. The conventional MRI sequence can only show the change of the morphological structure such as thickness and integrity of cartilage, but can not show the change of the structure of proteoglycan and collagen in the cartilage, so that the degeneration of cartilage can not be detected early. With the high occurrence of sports injury and obesity and the trend of aging of the population in China, the clinical requirements on the diagnosis and treatment evaluation of articular cartilage injury are increasingly improved, and MRI is expected to sensitively discover the morphological change of cartilage and even the change of biochemical components of cartilage matrix in the early stage of cartilage injury and degeneration, quantitatively and qualitatively evaluate the cartilage repair condition in the follow-up process after treatment and achieve the same effect as arthroscopy and even pathological biopsy; therefore, cartilage imaging is an important research field for image medicine and sports medicine, and provides important basis for clinical decision and evaluation of various surgical or non-surgical curative effects.

With the development of MRI, many new sequences that can display cartilage with high resolution have appeared, such as sequences that can display cartilage morphology with high resolution (3D-FSPGR, 3D-DESS, 3D-FLASH, 3D-SSFP, etc.), and functional sequences that can reflect biochemical composition of cartilage matrix (T2-mapping, dgermric, T1-mapping, DWI, T1 ρ mapping, UTE, etc.), which have improved diagnostic ability for early cartilage injury degeneration and monitoring ability after treatment to some extent, but in principle only indirectly reflect proteoglycan (e.g., dgermric, T1 ρ mapping) and collagen fiber component (e.g., T2-mapping, UTE) in cartilage matrix, are susceptible to joint fluid, water in cartilage matrix, etc., lack specificity, and are inevitably affected by magic angle effect, lack accuracy. At present, in the field of cartilage MRI imaging, the research of cartilage specific imaging, namely cartilage molecular imaging probes, is blank, a targeted contrast agent capable of being specifically combined with the internal structure of cartilage is also lacked in the imaging of the cartilage, and the imaging of the cartilage can be sensitive and accurate. The MRI probes are mainly divided into two types, one is a paramagnetic probe containing Gd and can strengthen the signal of T1WI, and the other is a probe containing superparamagnetic iron oxide and has the function of negative contrast in T2 WI. The ideal magnetic resonance molecular imaging probe has the following characteristics that 1, the marked molecules need to reach certain chemical purity; 2. the binding of the labeled molecule to the target should be highly specific; 3. the molecular weight is small, and the cell membrane is easy to pass through; 4. the compound is kept stable during imaging in order to obtain a sharp image.

In recent years, the development and application research trend of magnetic resonance contrast agents is to chemically modify the basic skeleton of diethylenetriamine Pentaacetic Acid (DTPA) to improve the selectivity and adaptability of the contrast agent to specific organs and pathological changes. Research reports that based on the existence of a large number of asialoglycoprotein receptors on the surfaces of liver parenchymal cells of mammals, the asialoglycoprotein receptors can selectively identify and combine glycoproteins containing D-galactosyl, and the liver targeting contrast agent can be synthesized by introducing D-galactose into DTPA and DOTA; the folic acid and DTPA are combined to synthesize the folic acid group-containing contrast agent by utilizing the characteristic that tumors can selectively take folic acid, and the contrast agent has better targeting property on the tumors; the vitamin B6 derivatives are used for modifying DTPA to synthesize a series of liver and gall targeted contrast agents; reacting a series of hydrophobic amino acids and short peptides with DTPA, wherein the Gd (III) complex of the obtained ligand has T1 relaxivity equivalent to Gd-DTPA, and the like. Based on the above research, the present inventors initially synthesized Gd (DTPA-HA) and contrast agent by using hyaluronic acid and glucosamine as affinity targets, using DTPA as a parent metal chelator molecule, coupling via covalent bonds (ester bond or amide bond), and introducing Gd ions (a specific synthetic route is shown in fig. 1).

At present, gadolinium (Gd (III)) containing compounds and chelating agents are used as main raw materials to prepare a small molecular gadolinium chelate through a chelation reaction. The patent with publication number CN109867635A discloses a T1 type micelle magnetic resonance imaging contrast agent and a preparation method thereof, the micelle magnetic resonance imaging contrast agent is formed by self-assembling a solution based on an amphiphilic micromolecule gadolinium-containing complex into micelles, and has the advantages of high relaxation rate, excellent water solubility and good biocompatibility. The MRI contrast agent disclosed by the invention takes amino modified Gd-HP-DO3A as a basic framework and is linked with alkyl chains with different lengths through amido bonds, so that a hydrophobic long-chain modified amphiphilic micromolecule contrast agent is constructed, and the contrast agent can be self-assembled into micelles in an aqueous solution to realize the abrupt increase of the relaxation rate; meanwhile, the preparation method of the T1 type micelle magnetic resonance imaging contrast agent provided by the invention is short in route and easy to operate.

The contrast agent containing metal ions is absorbed or coated in micron-sized or nano-sized particles by researchers, and the application effect of the contrast agent in a specific field is enhanced by utilizing special parameters and performances of the particles such as particle size, surface charge, adsorbability and the like. Patent publication No. CN110755618A discloses a hyaluronic acid-copper (II) composite nanoparticle and a preparation method thereof, wherein the copper (II) composite nanoparticle is prepared by taking hyaluronic acid and copper (II) salt modified by diethylenetriamine bridging as raw materials. The preparation method disclosed by the invention is simple and convenient to operate, the raw materials are cheap and easy to obtain, and the copper (II) composite nanoparticles modified by the hyaluronic acid have targeting property and good biocompatibility on cancer cells and have lower toxic and side effects. In addition, the diethylenetriamine-modified hyaluronic acid-copper (II) composite nanoparticles have good absorption in a near infrared region, and have the possibility of photo-thermal conversion. The photothermal property research proves that the photo-thermal composite material has good photo-thermal properties. The hyaluronic acid-copper (II) composite nanoparticle photothermal therapy has wide application prospect in the field of cancer therapy. The patent with publication number CN110787294A discloses a preparation method of hyaluronic acid-melanin nanoparticles, which combines aminated hyaluronic acid with melanin nanoparticles through Schiff base reaction/Michael addition reaction to obtain hyaluronic acid-melanin nanoparticles, and improves the cellular uptake efficiency of natural melanin nanoparticles while enhancing the biocompatibility of natural melanin nanoparticles, so that the natural melanin nanoparticles have the target recognition property for cancer cells; the prepared hyaluronic acid-melanin nano particles have excellent biocompatibility, no toxicity and better dispersibility and stability than those before modification, and the surface of hyaluronic acid is rich in carboxyl and hydroxyl and can be connected with various anti-cancer drugs through modification, so that an available carrier is provided for anti-cancer treatment. The patent with the publication number of CN101862461A discloses a gadolinium-containing macromolecular contrast agent HA-DTPA-Gd for specific imaging of lymphatic system and a preparation method thereof. The molecular structure of the contrast agent is as follows: the macromolecular MRI contrast agent prepared by the invention has large molecular weight, and after interstitial administration, the macromolecular MRI contrast agent is mainly absorbed by lymphatic capillaries and can not enter capillary vessels; meanwhile, the main chain of the contrast agent is hyaluronic acid molecules, so that the contrast agent is easily absorbed by a lymphatic endothelial hyaluronic acid receptor LYVE-1 in a lymphatic capillary to enter a lymphatic system, lymphatic active targeting is realized, interference of the lymphatic vessel in lymphatic system nuclear magnetic resonance examination is avoided, and the sensitivity and specificity of lymphatic qualitative diagnosis are improved. The patent with publication number CN107802888A discloses a preparation method of a nanofiber scaffold for promoting cartilage regeneration, which has good biocompatibility, can protect the bioactivity of growth factors, prolong the release time of the growth factors, and is beneficial to promoting cartilage repair and regeneration.

The patent describes that the targeted nano contrast agent is mostly focused on tumor imaging and biological scaffolds, the patent of the cartilage imaging nano contrast agent is less, and the patent with the publication number of CN109620974A discloses an osteoarthritis cartilage targeted gadolinium-based magnetic resonance imaging contrast agent and a preparation method thereof, wherein the contrast agent comprises Gd₂CO₃Nanoparticles of Gd in₂CO₃Watch with nanoparticlesThe bread is wrapped with a dopamine shell, the surface of the dopamine shell is loaded with cartilage targeting polypeptide through end group modified polyethylene glycol, and the amino acid sequence of the cartilage targeting polypeptide is DWRVIIPPRPSA; the osteoarthritis cartilage targeted gadolinium-based magnetic resonance imaging contrast agent realizes osteoarthritis cartilage specific targeting and MRI imaging. The contrast agent is easy to diffuse into joint cavities and mix with effusion of the surrounding joint cavities freely, clinical Gd agent has low relaxation rate and poor cartilage permeability, not only interferes the imaging effect of the defect area of the joint cartilage, but also has less obvious delayed strengthening degree.

Hyaluronic Acid (HA) is a mucopolysaccharide formed by polymerization of disaccharide unit composed of gluconic Acid and acetyl hexosamine, and HAs a molecular formula of (C)₁₄H_2lO_llN) N, molecular weight Mw N × 378+ 18. High molecular weight hyaluronic acid is the major component of joint synovial fluid, one of the components of the cartilage matrix. Clinically, the hyaluronic acid and the preparation thereof injected into joints are widely applied to the treatment of osteoarthritis, and have the characteristics of sterility, no toxicity, no antigenicity, no chemotaxis, no foreign body reaction and the like. A great deal of research at home and abroad proves that the research on the mechanism of osteoarthritis has the following well-known characteristics: HA can increase the content of hyaluronic acid in joint cavities, covers the surfaces of articular cartilage and synovium, can repair damaged physiological barriers, prevents cartilage matrix from further losing and protects articular cartilage; 2. hyaluronic acid can enter a damaged cartilage layer to be combined with proteoglycan in a non-covalent manner to form local molecules specific to the articular cartilage matrix, namely, the polymin is filled in the damaged collagen mesh, which is helpful for anchoring proteoglycan in the matrix, preventing the matrix from being further lost and repairing cartilage; 3. enhancing the lubricating effect of joint synovial fluid and improving the motion function of joints; 4. limit the diffusion of inflammatory mediators and play a role in chemoprotection of articular cartilage; 5. promoting synovial cells to synthesize self hyaluronic acid; 6. the compound has a stabilizing effect on nociceptors, and inhibits the excitability of the synovial membrane and the nociceptors under the synovial membrane; wherein, the 2 nd point: the hyaluronic acid can be non-covalently combined with proteoglycan in the damaged cartilage layer to form polymer filled in the damaged collagen network, and the inventor of the invention imagines to utilize hyaluronic acidThe acid polysaccharide molecule is used as an important basis for preparing a cartilage magnetic resonance molecular imaging probe by using a carrier.

According to the background, when the nano magnetic resonance contrast agent is used for cartilage contrast, the nano magnetic resonance contrast agent can quickly enter a joint cavity after being injected into the joint cavity and drain to a cartilage defect area, the preparation process is simple, safe and effective, the inventor intends to use mucopolysaccharide compound hyaluronic Acid as a targeting functional molecule, and gadolinium diethylenetriamine Pentaacetic Acid (Gd (III) and DTPA-Gd) as a carrier, so that the relaxation rate and the delayed enhanced signal-to-noise ratio change degree of the contrast agent are improved, and the novel nano magnetic resonance contrast agent for the joint cartilage targeting is constructed by utilizing the high permeability of nano particles and the affinity of hyaluronic Acid.

Disclosure of Invention

The invention overcomes the defects in the prior art, provides a targeted nano magnetic resonance contrast agent for an articular cartilage damaged area, and preparation and application thereof, and particularly relates to a hyaluronic acid modified nano targeted contrast agent.

According to the invention, biological macromolecule Hyaluronic Acid (HA) and gadolinium (Gd (III)) ions are connected by the gadolinium-containing contrast agent to form a HA and Gd-DTPA binary conjugate, the nano target contrast agent is prepared, the distribution of cartilage damage areas can be visually displayed by the specific combination of joint cavities and articular cartilage, and the detection rate and accuracy of the cartilage damage areas are improved.

The invention also aims to provide a preparation method of the nano-scale gadolinium-containing contrast agent.

In order to achieve the purpose, the invention adopts the following steps: hyaluronic Acid (HA) and gadolinium (Gd (III)) are connected through ions to form a HA-Gd-DTPA binary conjugate, nanoparticles are formed after freeze-drying, the nanomaterials penetrate into an extracellular matrix of cartilage through the affinity of articular cavity cartilage based on surface effect, and the nanomaterials are enriched in cartilage damaged areas due to the disorder of fibrous skeletons of the cartilage damaged areas.

In the invention, the structure of the contrast agent is HA-DTPA-Gd, Hyaluronic Acid (HA) is connected with gadolinium ions (Gd (III)) through diethylenetriaminepentaacetic acid (DTPA), and the molecular structure of the contrast agent is shown as the formula (I):

preferably, the contrast agent is gadolinium-containing specific nano magnetic resonance contrast agent HA-DTPA-Gd NPs.

Preferably, the diameter of the HA-DTPA-Gd NPs is 50-100 nm; the HA-DTPA-Gd NPs are high in safety, biodegradable, capable of mainly improving the strengthening time of tissue T1, capable of being connected with various ligands to form a targeted contrast agent and high in sensitivity and specificity.

More preferably, the particle size of the HA-DTPA-Gd NPs is 50-100 nm.

Preferably, the immunofluorescent label comprises 5-carboxyfluorescein (5-FAM SE).

Preferably, the molecular weight of the hyaluronic acid is 990,000 Da.

The preparation method of the contrast agent comprises the following steps:

a. dissolving 200mg hyaluronic acid HA (0.5mM) with molecular weight of 990,000Da in 50mL test tube, adding 20mL double distilled water, stirring at room temperature for 1 hr until it is clear and transparent, adding 1% (2-3mg)5-FAM SE, and stirring for 1-2 hr; adding 90mg of diethylenetriaminepentaacetic dianhydride (0.25mM) to 600. mu.L of dimethyl sulfoxide to dissolve, and stirring at room temperature for 2-3 h; adding 105mg of gadolinium chloride hexahydrate (0.25mM), stirring for 0.5h, adding the reaction solution into a dialysis bag with the molecular weight cutoff of 30000, dialyzing for 24h-48h in double distilled water, and finally preparing 14.26mmol/L solution A by taking care of using tinfoil paper to protect from light: wherein the respective mass ratios of the diethylene triamine pentaacetic dianhydride, the hyaluronic acid, the gadolinium chloride hexahydrate and the 5-carboxyfluorescein are as follows: 1: 1: 0.8: 0.8;

b. carrying out freeze drying treatment on the solution A to generate nano-particle HA-DTPA-Gd NPs, and obtaining a product, namely the nano-scale hyaluronic acid modified gadolinium-containing Gd (III) contrast agent;

the buffer solution has the buffering capacity of 4.0-6.0 of pH value;

the solvent is aprotic polar solution;

the aprotic polar solution is: dimethyl sulfoxide;

the gadolinium-containing Gd (III) -ion compound is a gadolinium-containing compound capable of dissociating Gd (III) -ions in an aqueous solution and a hydrate thereof;

the above gadolinium-containing gd (iii) ion compound: gadolinium chloride and hydrates thereof.

Compared with the prior art, the invention has the beneficial effects that:

(1) better penetration into the extracellular matrix of chondrocytes and into chondrocyte crypts, based on the surface effect of nanomaterials;

(2) the contrast agent is specifically combined with hyaline cartilage, the combination amount is indirectly reflected through 5-FAM SE and DAPI, and ICP-MS is used for detecting the synthesis rate, so that the distribution of cartilage damage areas can be prompted earlier, the clinical improvement of the accuracy and safety of treatment can be facilitated, the curative effect can be evaluated, and the prognosis can be evaluated;

(3) the hyaluronic acid contrast agent has medicine components for improving the healing degree of cartilage, and is hopeful to provide help for timely intervention through targeted medicine delivery, so that the integration of development and treatment is realized.

The size of the nano MRI contrast agent prepared by the invention is nano-scale, so that the nano-MRI contrast agent can better permeate into hyaline cartilage based on the nano surface effect after interstitial administration; meanwhile, the main chain of the contrast agent is hyaluronic acid molecules, so that the affinity with cartilage extracellular matrix is high, the active targeting of a cartilage defect area is realized, the interference of joint cavity effusion in the magnetic resonance imaging examination of cartilage damage is avoided, the efficiency and safety of delayed enhancement of the cartilage damage area are improved, and the sensitivity and specificity are improved; in animal in vivo simulation experiments, the relaxation rate of the contrast agent is higher than that of a conventional gadolinium contrast agent clinically used, and the high efficiency of the imaging effect is proved. Meanwhile, the synthesis rate of the MRI contrast agent prepared by the method is high, and is up to 57.7% through ICP test, so that the MRI signal intensity of the contrast agent is effectively ensured.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of the principle of action of targeted nanosonic contrast agents;

FIG. 2 is a representation of a targeted nanosonic contrast agent; wherein, a is a compound synthesis picture, and b and c are electron micrographs; d is the particle size distribution;

FIG. 3 is a magnetic sensitivity profile of a targeted nanoscopic contrast agent; wherein a is relaxation rate imaging of a Gd-HA NPs group, an HA-DTPA-Gd group and a Gd-DTPA group; b is a relaxation rate linear regression graph of Gd-HA NPs groups, HA-DTPA-Gd groups and Gd-DTPA groups with different concentrations;

FIG. 4 shows delta SNR T1 enhancement levels before, 1h, 2h, and 3h after injection for three MRI imaging joint cavities; wherein a and b are 3D T1WI MR images and pseudo-color images of Gd-HA NPs group, HA-DTPA-Gd group and Gd-DTPA group before injection, 1h, 2h and 3h after enhancement; c-d is the SNR change degree of the cartilage defect area of 1h, 2h and 3h before and after the injection of the Gd-HA NPs group, the HA-DTPA-Gd group and the Gd-DTPA group; e-f is the SNR change degree of the normal cartilage area of 1h, 2h and 3h before and after the injection of the Gd-HA NPs group, the HA-DTPA-Gd group and the Gd-DTPA group.

FIG. 5 shows tissue affinity and permeability of Gd-HA NPs and cartilage. Wherein a is a fluorescence staining chart of joint cavity injection of a 5-carboxyfluorescein labeled Gd-DTPA-HA group and a Gd-HA NPs group for 2 h; b is the average fluorescence intensity between the two groups, and the distribution of Gd-HA NPs in cartilage is obviously higher than that of Gd-DTPA-HA group; c-E are H & E, safranin fast green and PAS glycogen stained tissue sections, respectively.

FIG. 6 is a schematic view; the Gd-HA NPs group was metabolized and toxic in animals. Wherein a and b are body temperature and body weight changes of 0h-7 d; c. d is the blood routine and blood biochemical detection results between 1d, 3d and 7d and a normal control group after Gd-HA NPs are injected; e. f is Gd (III) ion concentration metabolism condition of heart, liver, lung, kidney, blood, urine and feces between 1d, 3d and 7d after Gd-HA NPs injection and a normal control group.

FIG. 7 is a schematic diagram of establishing an in vitro cell model and analyzing the influence of the targeted nano-magnetic resonance contrast agent on the survival rate of each cell line, namely an MTT cell activity detection diagram; in vitro experiments the degree of cell activity of the 4 cell lines was determined at different Gd (III) concentrations, where a-d were L929, HUVEC, MC3T3 and ATDC5 cell lines, respectively.

FIG. 8 is a toxicity study of various major organs in vivo with targeted nanomagnetic resonance contrast agents; and (3) slicing heart, liver, spleen, lung and kidney tissues of 1d, 3d and 7d of mice injected with Gd-HA NPs and a normal control group.

Detailed Description

The present invention is further illustrated by the following examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that, for a person skilled in the art, several modifications and improvements can be made without departing from the inventive concept, which falls within the scope of the invention.

Example 1: preparation and physicochemical characterization of targeted nano magnetic resonance contrast agent

a. Dissolving 200mg hyaluronic acid HA (0.5mM) with molecular weight of 990,000Da in a 50mL test tube, adding 20mL double distilled water, stirring at room temperature for 1h until the solution is clear and transparent, adding 1% (2-3mg)5 carboxyl fluorescein (5-FAM SE), and stirring for 1-2 h; adding 90mg of diethylenetriaminepentaacetic dianhydride (0.25mM) to 600. mu.L of dimethyl sulfoxide to dissolve, and stirring at room temperature for 2-3 h; adding 105mg of gadolinium chloride hexahydrate (0.25mM), stirring for 0.5h, adding the reaction solution into a dialysis bag with the molecular weight cutoff of 30000, dialyzing for 24h-48h in double distilled water, and finally preparing 14.26mmol/L solution A by taking care of using tinfoil paper to protect from light: wherein the respective mass ratios of the diethylene triamine pentaacetic dianhydride, the hyaluronic acid, the gadolinium chloride hexahydrate and the 5-carboxyfluorescein are as follows: 1: 1: 0.8: 0.8;

b. carrying out freeze drying treatment on the solution to generate nano-particle Gd-HA NPs, and obtaining a product, namely the nano-hyaluronic acid modified gadolinium-containing Gd (III) contrast agent;

dissolving a nano contrast agent in absolute ethyl alcohol, and carrying out ultrasonic treatment for 15-20min at 12000rpm to obtain water-dispersible nanoparticles;

characterization of targeted nanomagnetic resonance contrast agents: the particle morphology was observed by transmission electron microscopy, and the binding product was analyzed by Fourier transform infrared spectroscopy, the result being shown in FIG. 2, in which Gd-HA NPs were regularly rounded in shape and were in the form of dispersed single-globules of snowFlower-like, with an average particle size of 50 nm. The characteristic peaks of HA obtained by Fourier transform infrared spectroscopy analysis are respectively located at 1613 cm and 1567cm^-1The absorption peak of Gd-DTPA is 1633cm^-1In the vicinity, Gd-DTPA-HA exhibited characteristic peaks for HA and Gd-DTPA. A peak of Gd-DTPA; the C-N and N-H shock intensities increased and the peak of Gd-DTPA-HA shifted to 1591 and 1024cm^-1。

Example 2: in vitro relaxation rate research of targeted nano magnetic resonance contrast agent

Taking a proper amount of nano magnetic resonance contrast agent solution samples, preparing HA-DTPA-Gd NPs solutions with different concentrations by using double distilled water, and respectively placing Gd (III) with the concentration of 0.0005-0.06 mM in 2ml centrifuge tubes. And then DTPA-Gd with corresponding gadolinium concentration is respectively configured for comparison. The tubes were placed in sequence on a plastic tube rack and then placed in a box filled with water. The negative enhancement effect was evaluated by performing T1 Map sequence scan using clinical 3.0T MR System (Discovery MR750, GE Medical System, USA) and animal coil kit. The T1 Map was post-processed using GE Function Tool 4.6 specific software.

Magnetic susceptibility as shown in fig. 3a, T1 gradually increased with the gradually increasing gadolinium ion concentration compared to the control. Through measurement and calculation, the T1 fish eating time and the concentration of the contrast agent have a good linear relation, and the slope of a corresponding linear curve gradually increases along with the increase of the concentration of the contrast agent. Different Gd (III) concentrations and responses in the two groups of solutions were plotted as scattergrams 1/T1. As shown in FIGS. 3b-d, the Gd-HA NPs linear regression equation is: y-12.51 x +0.4725 (R2-0.9850, P)<0.05) having a T1 relaxation rate of 12.51mM^-1·s^-1(ii) a The linear regression equation of Gd-DTPA-HA is as follows: y 8.37x +0.4857(R2 0.9932, P)<0.05) having a T1 relaxation rate of 8.37mM^-1·s^-1(ii) a The linear regression equation of Gd-DTPA is as follows: y 3.72x +0.5578(R2 0.8953, P)<0.05) having a T1 relaxation rate of 3.72mM^-1·s^-1。

Example 3: and (3) researching the signal-to-noise ratio change in vivo of the targeted nano magnetic resonance contrast agent.

Gd-HA-NPs, Gd-DTPA-HA and Gd-DTPA are respectively injected into a rabbit knee joint cartilage injury model. After injection, the knee joint was imaged with a siemens Verio 3T magnetic resonance imaging system equipped with a rabbit knee coil.

As shown in fig. 4a and b, neither the articular cartilage (red dotted line) nor the damaged area (blue dotted line) was clearly visible before the two contrast agent injections. After 1h of intra-articular injection of Gd-DTPA, the signal intensity of the whole joint cavity is increased, but the lesion image is only slightly enhanced, and the cartilaginous contour is still unclear. The signal intensity of cartilage and injury sites remained very close 2h and 3h after injection. 1h after injection, increased signal intensity was observed in cartilage and injured sites in the Gd-DTPA-HA group and Gd-HA-NP group. The MRI signals of cartilage and lesions were significantly enhanced 2h after injection. The MRI signal intensity at 3h post-injection decreased slightly, indicating that the contrast agent began to metabolize.

SNR values were determined from T1-weighted MRI images of cartilage and injury sites. The Gd-HA-NP group showed significantly higher Δ SNR magnitude at the lesion site compared to the Gd-DTPA-HA and Gd-DTPA groups (fig. 4c, d). The Δ SNR values measured by the Gd-DTPA group fluctuate around 1.3, indicating that the signals generated by these lesions are less affected by the clinical application of Gd-DTPA. After 2h of injection of Gd-HA NPs, the delta SNR of the focus is obviously increased by 2.3 times, and after 3h of injection, the delta SNR is still obviously higher than those of a Gd-DTPA-HA group and a Gd-DTPA group. In addition, NPs also enhanced the cartilage images obtained, whereas the Δ SNR values of the Gd-HA-NP group were very close to the Gd-DTPA-HA group (FIG. 4e, f). Thus, the synthetic Gd-HA-NPs are more sensitive to cartilage damage than the other two contrast agents, and can enhance MRI detection of cartilage damage.

Example 4: pathological histology and fluorescein distribution research of targeted nano magnetic resonance contrast agent cartilage section

2h after injection of 5-carboxyfluorescein labeled Gd-DTPA-HA, green fluorescence was observed only on the cartilage surface (FIG. 5 a); in contrast, in the group of 5-FAM-labeled Gd-HA-NPs, strong fluorescence was detected throughout the thickness of the cartilage. Notably, fluorescence was observed even at the cartilage pits for the group of Gd-HA NPs, suggesting that Gd-HA-NPs may penetrate cartilage depth to improve MR imaging quality. In addition, cartilage was stained with H & E, Safranin O-fast and PAS after injection. The results of H & E and Safranin O-fast staining compared to healthy controls showed that neither the Gd-DPTA-HA nor Gd-HA NPs group had damaged or inflammatory response after exposure to contrast agent (fig. 5c, 5 d); meanwhile, cartilage turned purple-red after PAS staining due to the presence of oligosaccharides of glycoproteins or other structural proteins, indicating that Gd-HA NPs did not have any adverse effect on cartilage structure or composition (fig. 5 e); the above results indicate that the synthesized NPs can be used as an effective MRI contrast agent for the detection of cartilage damage in vivo.

Example 5: in vitro cell line toxicity research of nano magnetic resonance contrast agent

MTT cell activity assay: cell lines L929, HUVEC, MC3T3 and ATDC5 were taken at 1X 10 in log phase growth³The density of each well was inoculated into a 96-well plate, 200. mu.l of the culture medium per well was incubated at 37 ℃ with 5% CO2 and humidity>Culturing for 24h in a 95% incubator, discarding the culture solution when the cells are nearly fused, adding Gd-HA NPs with different concentrations, washing twice with PBS, adding 200 mu L of MTT solution (0.05%) into each hole, continuously incubating for 4h at 37 ℃, terminating the culture, carefully removing the culture solution in the holes, adding 150 mu L of DMSO into each hole, and shaking for 10min to fully dissolve the formazan. Color comparison: the 490nm wavelength is selected, and the absorbance of each well is measured on an enzyme linked immunosorbent assay.

The MTT cell activity test result shows that the nano magnetic resonance contrast agent with different concentrations (0,0.01,0.02,0.05,0.1,0.2 and 0.4mM) has no obvious inhibition effect on the proliferation of four groups of cell lines L929, HUVEC, MC3T3 and ATDC5, and has low cytotoxicity, as shown in figure 7.

Example 6: in vivo toxicity study of nano magnetic resonance contrast agent

Injecting a nano contrast agent into joint cavities of 4 groups of mice of a normal control group, a 1-day group, a 3-day group and a 7-day group, and carrying out routine monitoring on the weight, the body temperature, the blood urine feces, the blood biochemistry and the blood of the four groups of mice; carrying out aqua regia dissolution on heart, liver, spleen, lung and kidney tissues, and respectively carrying out inductively coupled plasma-mass spectrometry to determine the content of Gd (III).

The in vivo experiment result shows that the NPs contrast agent has no toxic or side effect on the growth of mice, the blood system and the functions of main organs; is firstly metabolized by the kidney and the liver and then is discharged out of the body along with urine, and has good biological safety.

Example 7: section of each important organ tissue in vivo

Respectively establishing 4 groups of normal control group, 1 day group, 3 day group and 7 day group mice, injecting nano contrast agent into joint cavities, and respectively carrying out H & E staining on heart, liver, spleen, lung and kidney of the four groups of mice.

H & E stained sections the results are shown in figure 8, and the mouse heart, liver, spleen, lung and kidney microstructures are not significantly cytotoxic and structurally compromised.

While particular embodiments of the present invention have been described, it will be understood that the invention is not limited to the particular embodiments described, but is capable of various changes and modifications within the scope of the appended claims by those skilled in the art without departing from the spirit of the invention.

Claims

1. A targeted nano magnetic resonance contrast agent for articular cartilage injury is characterized in that the structure of the contrast agent is HA-DTPA-Gd, hyaluronic acid HA is linked with gadolinium ions Gd (III) through DTPA, and the molecular structural formula of the contrast agent is shown as the formula (I):

2. the preparation method of the targeted nano magnetic resonance contrast agent for articular cartilage damage of claim 1 is characterized by comprising the following steps:

b. and (3) carrying out freeze-drying treatment on the solution A to generate nano-particle HA-DTPA-Gd NPs, thus obtaining the product, namely the nano-hyaluronic acid modified gadolinium-containing Gd (III) contrast agent.

3. The method according to claim 2, wherein the buffer solution is a buffer solution having a buffering capacity of 4.0 to 6.0 pH.

4. The method of claim 2, wherein the HA-modified gadolinium-containing contrast agent HAs a diameter of about 50nm to 100 nm; the immunofluorescent label comprises 5-carboxyfluorescein.

5. The method according to claim 2, wherein the chelating agent is diethylenetriaminepentaacetic dianhydride.

6. The method according to claim 2, wherein the solvent is an aprotic polar solvent.

7. The method according to claim 6, wherein the aprotic polar solvent is: dimethyl sulfoxide (DMSO).

8. The method according to claim 2, wherein the gadolinium-containing Gd (III) -ion compound is a gadolinium-containing compound capable of dissociating Gd (III) -ion in an aqueous solution, and a hydrate thereof.

9. The method according to claim 8, wherein the gadolinium-containing Gd (III) ionic compound is: gadolinium chloride and hydrates thereof.

10. The preparation method according to claim 8, wherein the nano-particle size of the hyaluronic acid modified gadolinium-containing compound contrast agent is 50-100 nm.