Composite nano material as well as preparation method and application thereof
By preparing nanomaterials composed of polydopamine nanoparticles and natural polymers, the problems of lymph node tracing and parathyroid gland protection in existing thyroid surgery have been solved, achieving low-cost, safe, and efficient lymph node tracing and parathyroid gland protection.
Patent Information
- Application Number
- CN202511580600.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-02-03
AI Technical Summary
Existing methods for lymph node tracing and parathyroid negative imaging in thyroid surgery have problems such as high cost, potential cytotoxicity, risk of radionuclide contamination, and insufficient fluorescence penetration, making it difficult to achieve accurate lymph node tracing and parathyroid protection.
A composite nanomaterial was designed, comprising polydopamine nanoparticles as the core and a natural polymer as the coating layer. By controlling the ratio of particle size to coating material, a nanomaterial with uniform and stable particle size was prepared for lymph node tracing and parathyroid negative imaging.
This approach achieves low-cost, high-safety lymph node tracing and parathyroid gland protection, improving lymph node detection rate, reducing the risk of surgical complications, and ensuring maximum protection of parathyroid function.
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Figure CN121445901A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medical new materials, in particular to a composite nanomaterial and a preparation method and application thereof. BACKGROUND
[0002] Thyroid cancer is the most common malignant tumor in the endocrine system and head and neck. The most important treatment for thyroid cancer is surgical resection, and the basic principle is to remove the tumor tissue in one piece and / or to clean the central lymph nodes. Parathyroid glands are small in size and have many anatomical variations, especially lower parathyroid glands, which can be ectopic to the mandibular angle, anterior mediastinum, and even pericardium, and can be embedded in the thymus or thyroid. The damage caused by the parathyroid glands can cause serious short-term and long-term complications. Therefore, accurate identification of parathyroid glands and in-situ reservation of parathyroid glands are still challenging problems in clinical practice.
[0003] The commonly used tracing methods in thyroid surgery include one or a combination of dye method (such as tracing mitoxantrone, nanocarbon), nuclide method (such as 99mTc-labeled sulfur colloid), and fluorescence method (such as indocyanine green). Mitoxantrone, a commonly used dye, is an antitumor drug. It is not only expensive, but also can bind to DNA molecules during cell replication, inhibit nucleic acid synthesis, and cause cell death, which has potential cytotoxicity and mutagenicity. The injection dose of nanocarbon tracer is difficult to control, and it cannot achieve effective tracing. At the same time, it cannot be normally degraded in the human body, and there is a potential safety problem. The nuclide method requires injection 2-30 hours before surgery, and uses a gamma probe to detect radioactivity during surgery. This method not only requires the surgeon to have the qualifications to use radioactive nuclides, but also has the potential risk of nuclide contamination, which causes psychological burden to patients and medical staff. The fluorescence method is similar to the nuclide method, which requires the use of a near-infrared fluorescence imaging instrument to image the lymph nodes during surgery. The fluorescence released by the commonly used fluorescence tracer after excitation has a penetration of only about 9 mm, which makes it difficult to find deep lymph nodes. Improper operation during surgery can also cause fluorescence contamination, making it difficult to identify the position of the lymphatic duct, and ultimately leading to missed detection of lymph nodes. Therefore, the current tracing methods and corresponding tracers all have different degrees of drawbacks, and there is an urgent need for a new medical material that can be easily used, is low in price, and is easy to use for lymph node tracing and parathyroid gland negative imaging during thyroid surgery.
[0004] In a weakly alkaline environment, dopamine can undergo an oxidative self-polymerization reaction using oxygen in the air to generate polydopamine nanoparticles, a process similar to the formation of melanin in the body. Polydopamine exhibits good biocompatibility, stability, and biodegradability. Its particle size is controllable, and it shows lymphatic system tropism, making it a potentially ideal lymph node tracer. However, polydopamine nanoparticles generally have high surface energy, making them prone to aggregation and affecting their stability in vivo. Therefore, surface-modifying materials are needed to encapsulate polydopamine nanoparticles to prevent excessive oxidation and aggregation. Furthermore, the particle size, uniformity, and structural stability of the encapsulated product significantly impact the clinical efficacy of the tracer.
[0005] Based on this, the present invention designs a composite nanomaterial. This composite nanomaterial has stable performance and uniform particle size as a tracer. After being administered through the gland, it can penetrate into the interstitial space and enter the capillary lymphatic vessels and accumulate in the regional lymph nodes, staining the lymph nodes black, so as to simultaneously achieve the purpose of lymph node tracing and negative contrast imaging of the parathyroid gland. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides a composite nanomaterial, its preparation method, and its application.
[0007] In a first aspect, the present invention provides a composite nanomaterial, comprising a core and a coating layer material covering the outside of the core; The core is polydopamine nanoparticles; The coating material is a natural high molecular polymer; The mass ratio between the core and the coating material is 1:1 to 1:20.
[0008] As one possible implementation of the first aspect, the polydopamine nanoparticles have a particle size of 50~400 nm.
[0009] As one possible implementation of the first aspect, the natural polymer is selected from one or more of polyvinylpyrrolidone, dimercaptosuccinic acid, citric acid, chitosan, polyethylene glycol, carboxymethyl dextran, and silicon dioxide.
[0010] As one possible implementation of the first aspect, the particle size of the composite nanomaterial is 100~500nm.
[0011] Furthermore, the particle size of the composite nanomaterial is 200~300nm.
[0012] Secondly, the present invention also provides a method for preparing composite nanomaterials, comprising the following steps: S1. Preparation of polydopamine dispersion: Dopamine hydrochloride powder was dissolved in a Tris-HCl buffer solution with a concentration of 9-11 mM and a pH of 8-9 at a ratio of 1 g: 100-300 mL, and polydopamine nanoparticles were obtained by shaking at room temperature and centrifuging at high speed. S2. Preparation of coating material solution: The coating material was dissolved in physiological saline to obtain a coating material solution with a mass concentration of 5-15 wt%. S3. Preparation of composite nanomaterials: The polydopamine nanoparticles are added to the coating material solution at a mass ratio of 1:1 to 1:20, and ultrasonically dispersed for 20 to 30 minutes to obtain a mixed dispersion. The mixed dispersion is then freeze-dried under vacuum to obtain the composite nanomaterial.
[0013] As one possible implementation of the second aspect, in step S1, the room temperature is 20~37℃; the shaking is performed on a shaker at 60~100r / min for 3~18h; the high-speed centrifugation speed is 10000-12000rmp / min, and the centrifugation time is 10~15min.
[0014] As one possible implementation of the second aspect, the vacuum freeze-drying conditions in step S3 are: operating temperature of -55℃ to -40℃, vacuum degree of 5 to 100 mTorr, and drying time of 24 to 48 h; the ultrasonic dispersion conditions in step S3 are: ultrasonic frequency of 25 kHz, power of 100 W, and temperature of 0 to 4℃.
[0015] Thirdly, the present invention also provides the application of a composite nanomaterial provided in the first aspect above or a composite nanomaterial prepared by the method provided in the second aspect above in a tracer.
[0016] As one possible implementation of the third aspect, the composite nanomaterial is dispersed in physiological saline to obtain a PDA-PVP tracer, wherein the concentration of the composite nanomaterial in the PDA-PVP tracer is 1~10 mg / mL.
[0017] As one possible implementation of the third aspect, the PDA-PVP tracer is used as a positive contrast agent for lymph nodes and / or a negative contrast agent for parathyroid glands.
[0018] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects: First, the composite nanomaterial core and coating material of the present invention have abundant raw material sources, low cost, simple and easy preparation method, good biocompatibility, can be completely metabolized in vivo, few complications, high safety, and are conducive to large-scale mass production and clinical translation.
[0019] Secondly, the composite nanomaterials prepared by this invention have a stable structure and uniform particle size, and can specifically enter the lymphatic system. The traced lymph nodes appear black and negatively visualize the parathyroid glands, achieving precise and efficient removal of regional lymph nodes while maximizing the protection of parathyroid function.
[0020] Third, by controlling the amount of dopamine hydrochloride, the present invention adjusts the particle size of the core polydopamine nanoparticles, further controlling the particle size of the composite nanomaterials, thereby adjusting the retention time of the tracer in lymph nodes and glands, reserving sufficient surgical time window for clinical surgical operations, and improving the lymph node detection rate.
[0021] Fourth, clinical trials have shown that the composite nanomaterials of this invention help reduce complications of permanent hypoparathyroidism caused by surgery, such as numbness in the hands and feet, generalized convulsions, epilepsy, neurasthenia, and diaphragmatic spasm. Attached Figure Description
[0022] Figure 1 A is an appearance diagram of the PDA dispersion prepared in Example 1. Figure 1 B is an image of the PVP solution prepared in Example 1. Figure 1 C is an appearance diagram of the PDA-PVP tracer prepared in Example 1; Figure 2 The results show the effect of oscillation time on the particle size of polydopamine nanoparticles in Experiment Example 1. Figure 3 The results show the effect of PVP solution concentration on the properties of composite nanomaterials in Experiment Example 2. Figure 4 The images shown are D1 and D7 of the PDA and PDA-PVP after standing at room temperature for 1 day and 7 days, respectively, in Experiment Example 3. Figure 5 The images shown are scanning electron microscope (SEM) images of each sample in Experimental Example 4. Figure 5 A is a scanning electron microscope image of the PDA dispersion. Figure 5 B is a scanning electron microscope image of the PVP solution. Figure 5 C is a scanning electron microscope image of the PDA-PVP tracer; Figure 6 The following are the ultraviolet absorption spectra of each sample in Experimental Example 4; among them, Figure 6 A is the ultraviolet absorption spectrum of the PDA sample. Figure 6 B is the UV absorption spectrum of the PVP sample. Figure 6 C is the UV absorption spectrum of the PDA-PVP sample; Figure 7 The particle size distribution diagrams for each sample in Experimental Example 4 are shown below; Figure 7 A is the particle size distribution diagram of the PDA sample.Figure 7 B is the particle size distribution diagram of the PVP sample. Figure 7 C is the particle size distribution diagram of the PDA-PVP sample; Figure 8 Figure 5 shows the experimental results of CCK-8 co-culture of PDA dispersion, PVP solution, and PDA-PVP tracer with rat fibroblasts in Experiment Example 5. Figure 9 The images show H&E staining of the heart, liver, spleen, lungs, and kidneys of rats injected with PDA-PVP tracer and control group (physiological saline) for 28 and 42 days in Experiment Example 6. Figure 10 The graph shows the changes in liver and kidney function and blood routine in rats 42 days after the PDA-PVP tracer was injected into them in Experiment Example 7, specifically including creatinine (CREA), blood urea nitrogen (BUN), and transaminase (ALT, AST). Figure 11 The image shows the tracing effect of the popliteal lymph nodes in SD rats in Experiment Example 8. Figure 11 A is a general comparison diagram of the tracer; Figure 11 B is an HE staining image of the popliteal lymph nodes; Figure 12 The image shows a comparison of the cervical lymph node tracing effects after intrathyroidal injection of PDA-PVP tracer in experimental dogs in Example 9. In the image, A) is the lymph node traced in the PDA-PVP group during surgery; B) is the lymph node traced in the mitoxantrone group during surgery; C) is the lymph node removed from the PDA-PVP group; and D) is the lymph node removed from the mitoxantrone group. Figure 13 The images show a comparison of the contrast effects of PDA-PVP and mitoxantrone tracers on the thyroid and parathyroid glands in experimental dogs during live injection in Example 10. A) shows the parathyroid glands traced in the mitoxantrone group during surgery; the green circle indicates the traced parathyroid glands, and the black circle indicates surgical field contamination. B) shows the parathyroid glands traced in the PDA-PVP group during surgery; the green circle indicates the traced parathyroid glands. C) shows the parathyroid glands removed from the mitoxantrone group. D) shows the parathyroid glands removed from the PDA-PVP group. Detailed Implementation
[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0024] In the embodiments of this application, the terms "exemplary" or "for example" are used to indicate that something is an example, illustration, or description. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design. Specifically, the use of the terms "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.
[0025] The following is a unified explanation of some of the abbreviations used in this application: PDA refers to polydopamine, PVP refers to polyvinylpyrrolidone, and PDA-PVP refers to polydopamine-polyvinylpyrrolidone.
[0026] Unless otherwise specified, all raw materials used in the examples are commercially available.
[0027] In each embodiment, scanning electron microscopy was performed using a Hitachi SU8100 microscope. Microscopy was performed using a Leica DMI3000 inverted phase-contrast microscope. Ultrasonic dispersion was performed using a JY92-IIN ultrasonic transducer (ultrasonic probe) from Ningbo Xinzhi Biotechnology Co., Ltd., with an ultrasonic frequency of 25 kHz and an ultrasonic power of 100 W.
[0028] Example 1: This example describes one method for preparing composite nanomaterials, including the following steps: S1. Preparation of PDA dispersion: 0.1 g of dopamine hydrochloride powder was dissolved in 10 mL of 10 mM Tris-HCl buffer solution with a pH of 8, and the solution was shaken at room temperature for 12 h at 60 r / min and centrifuged at 10000 r / min for 10 min to obtain polydopamine nanoparticles. S2. Preparation of PVP solution: Take 0.5g of coating material (the coating material used in this embodiment is polyvinylpyrrolidone, such as polyvinylpyrrolidone K15 or polyvinylpyrrolidone K30) and dissolve it in physiological saline. Gently mix with a pipette to obtain a coating material solution with a mass concentration of 5wt% (denoted as PVP solution); the appearance of the PVP solution is shown in the figure below. Figure 1 As shown in B; S3. Preparation of PDA-PVP tracer: The polydopamine nanoparticles were added to the coating material solution (PVP solution) at a mass ratio of 1:1. After thorough mixing with a pipette, the mixture was ultrasonically dispersed at 0°C for 20 min at an ultrasonic frequency of 25 kHz and a power of 100 W. Then, the mixture was stirred with a magnetic stirrer for 10 min to obtain a mixed dispersion. The dispersion was then placed in a vacuum freeze dryer and freeze-dried at -55°C and a vacuum degree of 5 mTorr for 48 h to obtain the composite nanomaterial.
[0029] Further redispersing the composite nanomaterial with physiological saline yields PDA-PVP tracers with concentrations of 1–10 mg / ml. The appearance of the PDA-PVP tracer (with a composite nanomaterial concentration of 5 mg / ml) is shown in the image below. Figure 1 As shown in C.
[0030] As a control, the polydopamine nanoparticles obtained in S1 were placed in a vacuum freeze dryer and freeze-dried at -55°C and a vacuum degree of 5 mTorr for 48 h. After UV sterilization for 30 min, they were soaked and washed with 75 wt% ethanol for 1 h, centrifuged at 10000 rpm for 10 min, resuspended in physiological saline, and ultrasonically dispersed at 0°C for 10 min at a frequency of 25 kHz and a power of 100 W to obtain a polydopamine dispersion with a concentration of 10 mg / ml (denoted as PDA dispersion). The appearance of the PDA dispersion is shown in the figure below. Figure 1 As shown in Figure A.
[0031] Example 2: This example describes a second method for preparing composite nanomaterials, including the following steps: S1. Preparation of PDA dispersion: 0.1 g of dopamine hydrochloride powder was dissolved in 20 mL of Tris-HCl buffer solution with a concentration of 9 mM and a pH of 9. The solution was shaken at room temperature for 3 h at 80 r / min and centrifuged at 11000 r / min for 12 min to obtain polydopamine nanoparticles. S2. Preparation of PVP solution: Dissolve 1g of polyvinylpyrrolidone K15 in physiological saline and mix gently with a pipette to obtain a PVP solution with a mass concentration of 10wt%. S3. Preparation of PDA-PVP tracer: The polydopamine nanoparticles were added to the PVP solution at a mass ratio of 1:10 to the coating material. After thorough mixing with a pipette, the mixture was ultrasonically dispersed at 2°C for 25 min at a frequency of 25 kHz and a power of 100 W. Then, the mixture was stirred with a magnetic stirrer for 10 min to obtain a mixed dispersion. The dispersion was then placed in a vacuum freeze dryer and freeze-dried at -50°C and a vacuum degree of 25 mTorr for 30 h to obtain the composite nanomaterial.
[0032] Example 3: This example describes a third method for preparing composite nanomaterials, including the following steps: S1. Preparation of PDA dispersion: 0.1 g of dopamine hydrochloride powder was dissolved in 30 mL of 11 mM Tris-HCl buffer solution with a pH of 9, and the solution was shaken at room temperature for 18 h at 100 r / min and centrifuged at 12000 r / min for 15 min to obtain polydopamine nanoparticles. S2. Preparation of PVP solution: Dissolve 1.5g of polyvinylpyrrolidone K30 in physiological saline and mix gently with a pipette to obtain a PVP solution with a mass concentration of 15wt%. S3. Preparation of PDA-PVP tracer: The polydopamine nanoparticles were added to the PVP solution at a mass ratio of 1:20 to the coating material. After thorough mixing with a pipette, the mixture was ultrasonically dispersed at 4°C for 30 min at a frequency of 25 kHz and a power of 100 W. Then, the mixture was stirred with a magnetic stirrer for 10 min to obtain a mixed dispersion. The dispersion was then placed in a vacuum freeze dryer and freeze-dried at -55°C and a vacuum degree of 100 mTorr for 24 h to obtain the composite nanomaterial.
[0033] Experimental Example 1: This experimental example studies the effect of oscillation time on the particle size of polydopamine nanoparticles.
[0034] 0.1 g of dopamine hydrochloride powder was dissolved in 20 mL of 9 mM Tris-HCl buffer solution (pH 9), and the solution was shaken at 80 rpm at room temperature for 8 h, 12 h, 18 h, 24 h, and 28 h, respectively. The mixture was then centrifuged at 11000 rpm for 12 min to obtain polydopamine nanoparticles. The particle size was determined using a nanoparticle size and potential analyzer. The results are as follows: Figure 2As the oscillation time increased, the diameter of the PDA nanoparticles gradually increased from 168.3 ± 7.64 nm to 685.67 ± 27.47 nm. If the oscillation time was too short, the PDA nanoparticles were too small and easily entered the interendothelial space of blood vessels; conversely, if the oscillation time was too long, the PDA nanoparticles were too large, preventing the tracer from entering the lymphatic endothelial cells. Therefore, to obtain polydopamine nanoparticles of suitable size, the oscillation time needs to be controlled within 3–18 hours.
[0035] Experimental Example 2: This experimental example studies the effect of the concentration of the coating material solution (PVP solution) on the properties of the composite nanomaterial.
[0036] Polydopamine nanoparticles (PDA) were added to PVP (polyvinylpyrrolidone) solutions of different concentrations (3%, 5%, 10%, 15%, 20%). The average particle size of the PDA used in this experiment was 401.6 ± 5.69 nm, and the mass ratio of PVP nanoparticles to PVP was 1:20. After thorough mixing with a pipette, the mixture was ultrasonically dispersed at 4℃ for 30 min at a frequency of 25 kHz and a power of 100 W. The mixture was then stirred with a magnetic stirrer for 10 min to obtain a mixed dispersion. This dispersion was then placed in a vacuum freeze dryer and freeze-dried at -55℃ and a vacuum degree of 100 mTorr for 24 h to obtain the composite nanomaterial. The particle size of the composite nanomaterial was determined using a nanoparticle size and potential analyzer. The results showed ( Figure 3 The concentration of the PVP solution affects the final particle size of the tracer. At too low a concentration (3%), the PVP coating of PDA is incomplete, easily causing aggregation and resulting in larger particle sizes. As the concentration gradually increases, the tracer particle size gradually decreases, reaching its minimum at 10% (402 ± 5.29 nm). With further increases in concentration, due to PVP deposition and multilayer adsorption, the tracer diameter increases; at a concentration of 20%, the tracer particle size is 547.3 ± 6.43 nm, significantly larger than the diameter of capillary lymphatic vessels. Therefore, to obtain composite nanomaterials with suitable particle sizes, the concentration of the PVP solution needs to be controlled between 5 and 15 wt%.
[0037] Experimental Example 3: This experimental example studies the stability of the PDA dispersion and PDA-PVP tracer prepared in Example 1.
[0038] At room temperature, the PDA dispersion and PDA-PVP tracer prepared in Example 1 were placed on a test tube rack and observed for 7 days. Figure 4 As shown, the results indicate that the PDA dispersion began to aggregate and precipitate after standing for 1 day, and completely aggregated and settled at the bottom of the centrifuge tube after standing for 7 days. In contrast, the PDA-PVP tracer did not show any obvious aggregation and sedimentation after standing for 7 days.
[0039] The above results confirm that encapsulating the core with a coating material can effectively maintain the stability of polydopamine nanoparticles in a mixed dispersion. Uniform distribution of polydopamine nanoparticles in the dispersion is a prerequisite for lymph node tracing; polydopamine deposition leads to poor tracer stability, preventing it from passing through the intercellular spaces of lymphatic vessels.
[0040] Experimental Example 4: This experimental example studies the appearance and morphology of the PDA dispersion, PVP solution and PDA-PVP tracer prepared in Example 1.
[0041] (1) Scanning electron microscopy observation: The PDA dispersion, PVP solution and PDA-PVP tracer prepared in Example 1 were characterized. The PDA dispersion, PVP solution and PDA-PVP tracer were ultrasonically dispersed and then dropped onto a silicon wafer, evaporated at room temperature, and then sputtered with gold and observed with a scanning electron microscope.
[0042] Observation results as follows Figure 5 As shown, the PDA nanoparticles in the PDA dispersion have relatively smooth surfaces, are nearly spherical in shape of varying sizes, and exhibit tight intermolecular adhesion, making them difficult to separate (see...). Figure 5 A); PVP material consists of small, uniformly sized, blocky molecules with tight molecular bonds and a relatively smooth surface (see...). Figure 5 B); The PDA-PVP tracer is in the form of nearly spherical particles, with PVP uniformly covering the PDA nanoparticles on the surface. Due to the dispersing effect of PVP, the aggregation of adjacent PDA molecules is reduced, resulting in a more uniform size, with a particle diameter of approximately 300-400 nm (see [link]). Figure 5 C).
[0043] The above results confirm that the composite nanomaterials prepared by the method of the present invention have reduced nanoparticle aggregation and more uniform particle size.
[0044] (2) Measurement of ultraviolet-visible absorption spectrum: The ultraviolet-visible absorption spectra were measured using an ultraviolet-visible absorbance spectrophotometer: The PDA dispersion, PVP solution, and PDA-PVP tracer prepared in Example 1 were diluted with physiological saline to a concentration of 1 mg / mL, and then mixed evenly to obtain PDA samples, PVP samples, and PDA-PVP samples. These samples were then poured into cuvettes and placed in an ultraviolet-visible spectrophotometer. The wavelengths of the samples were measured using the ultraviolet-visible spectrophotometer to determine the maximum ultraviolet absorption peak.
[0045] The measurement results are as follows Figure 6As shown, the maximum ultraviolet absorption wavelength of the PDA sample is 190 nm, the maximum ultraviolet absorption wavelength of the PVP sample is 205 nm, the maximum ultraviolet absorption wavelength of the PDA-PVP sample is 192 nm, and the maximum emission wavelength detected by the ultraviolet-visible spectrometer is 800 nm.
[0046] The above results confirm that PDA and PVP are combined in a non-chemical bond manner, without reacting to generate new substances and changing the original properties of PDA. In other words, the prepared PDA-PVP tracer did not change its original properties.
[0047] (3) Determine particle size: The particle size was determined using a nanoparticle size and potential analyzer: The PDA dispersion, PVP solution and PDA-PVP tracer prepared in Example 1 were diluted with physiological saline to a concentration of 1 mg / mL, mixed thoroughly and then aspirated to obtain PDA sample, PVP sample and PDA-PVP sample, which were then placed into cuvettes and placed in the sample cell of the particle size analyzer to measure the average particle size of the PDA sample, PVP sample and PDA-PVP sample.
[0048] Test results are as follows Figure 7 As shown, the average particle size of the PDA sample is 692.30 ± 2.41 nm, the average particle size of the PVP sample is 12.12 ± 0.15 nm, and the average particle size of the PDA-PVP sample is 376.93 ± 1.92 nm.
[0049] Since the intercellular spaces between capillary lymphatic endothelial cells are approximately 500 nm, much larger than the particle size of the PDA-PVP tracer, the above results confirm that the PDA-PVP tracer prepared in this invention can rapidly penetrate capillary lymphatic vessels and accumulate within lymph nodes, thereby tracing lymph nodes.
[0050] Experimental Example 5: This experimental example studies the cytotoxicity of the PDA dispersion, PVP solution and PDA-PVP tracer prepared in Example 1.
[0051] P3 generation rat fibroblasts were harvested and processed at a concentration of 6 × 10⁻⁶. 4100 µl of each Rat-1 cell was seeded into 96-well plates and cultured for 12 h. Four groups were set up: a blank control group, a PDA dispersion group, a PVP solution group, and a PDA-PVP tracer group, with 5 replicates in each group. 20 µl of each solution was added to a 96-well plate containing adherent Rat-1 cells for co-culture. After 24, 48, and 72 h, the 96-well plates were removed, the culture medium was discarded, and 100 µl of CCK-8 working solution (prepared by mixing CCK-8 detection solution and serum-free culture medium at a 1:10 volume ratio) was added. The 96-well plates were incubated in a cell culture incubator for 2 h, and the absorbance at 450 nm was measured using a microplate reader.
[0052] Test results as follows Figure 8 As shown, rat fibroblasts exhibited good growth under co-culture conditions with the four groups of samples, and the cells remained in a proliferative state after 24, 48, and 72 hours. The cell viability of the PDA group, PVP group, and PDA-PVP group was not significantly reduced compared to the control group.
[0053] The above results confirm that the composite nanomaterials and raw materials prepared in this invention have good biocompatibility.
[0054] Experimental Example 6: This experimental example studies the cytotoxicity of the PDA-PVP tracer prepared in Example 1.
[0055] The chronic toxicity of the PDA-PVP tracer prepared in Example 1 was tested in rats. The testing procedure is as follows: Experimental materials: PDA-PVP tracer prepared in Example 1, physiological saline, SD rats (6-8 weeks old, 150-200g). SD rats were randomly divided into PDA-PVP tracer group (referred to as experimental group) and control (physiological saline) group, with 3 rats in each group.
[0056] Specific experimental procedures: Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution. After complete anesthesia, the rats were fixed on a rat operating table. The fur on the back of the rats was removed, and any remaining fur was cleaned. The skin was disinfected. Rats in the PDA-PVP tracer group were subcutaneously injected with 1 ml of PDA-PVP solution in their backs, while rats in the control group were subcutaneously injected with the same volume of physiological saline in their backs. Aspiration was performed before injection to ensure no blood was found. Pressure was applied to the injection site during needle withdrawal to prevent bleeding and leakage. The rats' general condition, including food intake, water intake, activity, and mental state, as well as local skin reactions on the backs, were observed for 42 days post-injection. Rats were sacrificed on days 28 and 42, and their major organs, including the heart, liver, spleen, lungs, and kidneys, were completely removed for tissue staining and observation. Immediately after removal, the organs were fixed in 5 times their volume of 10% neutral buffered formalin for 24 h, embedded in paraffin, dehydrated using a gradient alcohol process, and sectioned for H&E staining to assess chronic toxicity in vivo.
[0057] During the 42-day observation period, neither group of rats showed any obvious adverse conditions such as redness, swelling, exudation, or ulceration of the skin on their backs, and the skin healing on the backs appeared good to the naked eye. Both the experimental and control groups of rats exhibited good activity and food intake, with no significant differences in their general condition. Rats were sacrificed 28 and 42 days after the injection of the tracer, and their organs (heart, liver, spleen, lungs, and kidneys) were removed for H&E staining.
[0058] Staining results as follows Figure 9 As shown, under a light microscope, the organ sections of the experimental group rats were observed to be intact, with clear structures and no obvious damage. There was no significant difference compared with the control group, and both groups showed obvious normal organ tissue structures.
[0059] The above results confirm that the PDA-PVP tracer prepared in this invention does not have significant chronic toxicity in vivo.
[0060] Experimental Example 7: This experimental example studies the in vivo hematologic toxicity of the PDA-PVP tracer prepared in Example 1.
[0061] Blood samples were collected from rats at the monitoring time points for the PDA-PVP tracer prepared in Example 1. The hematological toxicity and hepatotoxicity of the PDA-PVP tracer were evaluated by detecting key blood biochemical indicators.
[0062] The specific steps are as follows: Blood samples were collected from the tail artery of rats on days 3, 7, 14, 28, 35, and 42 after subcutaneous injection of PDA-PVP tracer and saline. The values of liver and kidney function and complete blood count were measured, including creatinine (CREA), blood urea nitrogen (BUN), and transaminases (ALT, AST) to detect the hematological toxicity of PDA-PVP tracer.
[0063] Test results as follows Figure 10 As shown, the PDA-PVP tracer did not exhibit significant liver and kidney toxicity, and the data from both the experimental and control groups were within the normal range with no significant differences.
[0064] The above results confirm that the PDA-PVP tracer prepared in this invention does not have long-term hematological toxicity.
[0065] Experimental Example 8: This experimental example studies the localization of rat popliteal lymph nodes guided by the PDA-PVP tracer prepared in Example 1.
[0066] The PDA-PVP tracer prepared in Example 1 was tested for its ability to trace rat popliteal lymph nodes. The specific test steps are as follows: ① After anesthetizing the rats in the experimental group with 10% chloral hydrate solution via intraperitoneal injection, they were fixed on the operating table with their faces supine. The fur on both lower limbs was removed and the skin of the lower limbs was disinfected. ② Inject 0.1 ml of PDA-PVP tracer and mitoxantrone into the rat paw pads, respectively. PDA-PVP tracer was injected into the left side and mitoxantrone tracer was injected into the right side. ③ After waiting 5 minutes, dissect the skin and subcutaneous tissue of the lower limb layer by layer. Locate and remove the popliteal lymph nodes in sequence according to the direction of blue-stained and black-stained lymphatic drainage, and mark them. Pay attention to careful operation to avoid cutting the lymphatic vessels. Observe the color development of the popliteal lymphatic vessels and lymph nodes of the rat (the gross comparison diagram is shown in the figure). Figure 11 As shown in A); ④ The extracted lymph nodes were stained with hematoxylin and eosin (HE), and the pathological results of the rat popliteal lymph nodes were observed under a microscope.
[0067] Staining results as follows Figure 11 As shown in Figure B, after injection of the PDA-PVP tracer, black-stained lymphatic vessels and lymph node tissue were observed. HE staining revealed normal lymph node structures such as lymph nodules and germinal centers, with localized PDA-PVP nanoparticle deposition. No obvious leakage was observed. Mitoxantrone injected on the right side also resulted in blue-stained lymph nodes, and good imaging results were obtained bilaterally.
[0068] The above results confirm that the PDA-PVP tracer prepared in this invention can effectively guide lymph node localization.
[0069] Experimental Example 9: This experimental example studies the localization of cervical lymph nodes guided by the PDA-PVP tracer prepared in Example 1.
[0070] The tracking effect of the PDA-PVP tracer prepared in Example 1 on lymph nodes during thyroid surgery was determined. The specific determination process is as follows: Experimental materials: Six experimental dogs (half male and half female, each weighing approximately 10 kg) were selected and randomly divided into two groups (designated as the PDA-PVP tracer group and the mitoxantrone group), with three experimental dogs in each group.
[0071] Specific experimental procedures: Anesthesia was administered via slow injection of 1.0 ml / kg of 3% sodium pentobarbital along the great saphenous vein on the medial side of the hind limb of the experimental dog. After the anesthesia took effect, the dog was fixed in a supine position. The neck was prepared, disinfected with iodine, and draped. A longitudinal incision was made from below the cricoid cartilage to 2 cm above the sternal notch. The skin, subcutaneous tissue, and linea alba were incised to expose the trachea. The thyroid gland was explored and exposed around the thyroid gland below the cricoid cartilage, in the tracheoesophageal groove, and within the neck sheath. The left and right thyroid glands were gently lifted with forceps. 1 ml of PDA-PVP tracer was injected into the PDA-PVP tracer group, and 1 ml of mitoxantrone was injected into the mitoxantrone group. Before injection, the syringe was aspirated, and injection was performed only after no blood was observed. After injection, the needle was withdrawn after pressing the injection site with gauze to prevent leakage. After 5 minutes, the mandibular lymph nodes were explored, and the color development of the mandibular lymph nodes was observed (see [link to relevant documentation]). Figure 12 A, 12B).
[0072] Following the tracer staining instructions, the canine mandibular lymph nodes were removed and stained with hematoxylin and eosin (HE). The pathological results of the lymph nodes were observed under a microscope (see [link to relevant documentation]). Figure 12 C, 12D).
[0073] The experimental results showed that all six mandibular lymph nodes in the PDA-PVP group dogs were detectable, with no difference compared to the mitoxantrone group. Black traces were visible in all six mandibular lymph nodes. However, in the mitoxantrone group, when probing deeper along the traced lymphatic vessels, one side showed lymphatic vessel rupture contaminating the surgical field. Under light microscopy, the lymph node cortex and medulla were clearly distinguishable. Typical lymphoid nodules were observed in the cortex, with a lightly stained germinal center at the center and its top, surrounded by dense small lymphocytes. Under high magnification, black granular material was observed around the germinal center and small lymphocytes. The lymph node tissue structure was normal, and no obvious damage, necrosis, or inflammatory cell infiltration was observed around the black granular deposits. No black granular material deposition was observed in the surrounding blood vessels. Figure 12 ).
[0074] The above results confirm that the PDA-PVP tracer prepared in this invention has a good tracking effect on lymph nodes after intrathyroidal injection, and is less prone to leakage than mitoxantrone, exhibiting superior performance.
[0075] Experimental Example 10: This experimental example studies the negative contrast imaging of parathyroid glands guided by the PDA-PVP tracer prepared in Example 1.
[0076] The effect of the PDA-PVP tracer prepared in Example 1 on negative parathyroid imaging during thyroid surgery was determined. The specific determination process is as follows: Experimental materials: Six experimental dogs (half male and half female, each weighing approximately 10 kg) were selected and randomly divided into two groups (designated as the PDA-PVP tracer group and the mitoxantrone group), with three experimental dogs in each group.
[0077] Specific experimental procedures: Anesthesia was administered via slow injection of 1.0 ml / kg of 3% sodium pentobarbital along the great saphenous vein on the medial side of the hind limb of the experimental dog. After the anesthesia took effect, the dog was fixed in a supine position. The neck was prepared, disinfected with iodine, and draped. A longitudinal incision was made from below the cricoid cartilage to 2 cm above the sternal notch. The skin, subcutaneous tissue, and linea alba were incised to expose the trachea. The thyroid gland was explored and exposed around the thyroid gland below the cricoid cartilage, in the tracheoesophageal groove, and within the neck sheath. The left and right thyroid glands were gently lifted with forceps. 1 ml of PDA-PVP tracer was injected into the PDA-PVP tracer group, and 1 ml of mitoxantrone was injected into the mitoxantrone group. Before injection, the syringe was aspirated, and injection was performed only after no blood was observed. After injection, the needle was withdrawn after applying gauze to the injection site to prevent leakage. After injection, the middle veins of the left and right thyroid glands were cut and ligated. The thyroid gland was pulled inward and anteriorly with hemostatic forceps. The loose tissue on the posterolateral side was bluntly dissected to expose the dorsal side of the thyroid gland. The negative contrast of the parathyroid glands was observed (see [link to relevant documentation]). Figure 13 A and Figure 13 B). Following the instructions for negative contrast imaging with tracer, parathyroid gland tissue from the experimental dogs was removed and stained with hematoxylin and eosin (see [link to relevant documentation]). Figure 13 C and Figure 13 D).
[0078] Depend on Figure 13 As can be seen, after injection of PDA-PVP tracer, the thyroid gland became blackly stained, while a grain-sized tissue on the back remained unchanged in color, consistently appearing pale yellow and unstained, with a significantly different morphology from the surrounding stained thyroid gland. Both PDA-PVP and mitoxantrone tracers can negatively visualize the parathyroid glands, with both showing relatively clear and similar staining effects. The mitoxantrone group showed significant extravasation on one side, contaminating the surgical area, while the PDA-PVP tracer group exhibited clearer boundaries between the thyroid and parathyroid glands, with no contamination or extravasation. HE staining revealed the characteristic chief cells and eosinophils of the parathyroid glands.
[0079] The above imaging results confirm that the negative imaging effect of the PDA-PVP tracer prepared in this invention on the parathyroid gland is not significantly different from that of mitoxantrone. It can achieve the effect of negative imaging of the parathyroid gland by staining the thyroid gland black. Moreover, the PDA-PVP tracer prepared in this invention is less prone to surrounding contamination compared with the mitoxantrone group.
[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.
Claims
1. A composite nanomaterial, characterized in that, Includes the core and the covering material surrounding the core; The core is polydopamine nanoparticles; The coating material is a natural high molecular polymer; The mass ratio between the core and the coating material is 1:1 to 1:
20.
2. The composite nanomaterial as described in claim 1, characterized in that, The polydopamine nanoparticles have a particle size of 50~400nm.
3. The composite nanomaterial as described in claim 1, characterized in that, The natural polymer is selected from one or more of polyvinylpyrrolidone, dimercaptosuccinic acid, citric acid, chitosan, polyethylene glycol, carboxymethyl dextran, and silicon dioxide.
4. The composite nanomaterial as described in claim 1, characterized in that, The particle size of the composite nanomaterial is 100~500nm.
5. The method for preparing a composite nanomaterial as described in claim 1, characterized in that, Includes the following steps: S1. Preparation of polydopamine dispersion: Dopamine hydrochloride powder was dissolved in a Tris-HCl buffer solution with a concentration of 9-11 mM and a pH of 8-9 at a ratio of 1 g: 100-300 mL, and polydopamine nanoparticles were obtained by shaking at room temperature and centrifuging at high speed. S2. Preparation of coating material solution: The coating material was dissolved in physiological saline to obtain a coating material solution with a mass concentration of 5-15 wt%. S3. Preparation of composite nanomaterials: The polydopamine nanoparticles are added to the coating material solution at a mass ratio of 1:1 to 1:20, and ultrasonically dispersed for 20 to 30 minutes to obtain a mixed dispersion. The mixed dispersion is then freeze-dried under vacuum to obtain the composite nanomaterial.
6. The method for preparing a composite nanomaterial as described in claim 5, characterized in that, In step S1, the room temperature is 20~37℃; the shaking is performed on a shaker at 60~100r / min for 3~18h; the high-speed centrifugation speed is 10000-12000rmp / min, and the centrifugation time is 10~15min.
7. The method for preparing a composite nanomaterial as described in claim 5, characterized in that, The vacuum freeze-drying conditions described in step S3 are: operating temperature of -55℃ to -40℃, vacuum degree of 5 to 100 mTorr, and drying time of 24 to 48 h; the ultrasonic dispersion conditions described in step S3 are: ultrasonic frequency of 25 kHz, power of 100 W, and temperature of 0 to 4℃.
8. The application of a composite nanomaterial as described in any one of claims 1 to 4 or a composite nanomaterial prepared by the method described in any one of claims 5 to 7 in tracers.
9. The application as described in claim 8, characterized in that, The composite nanomaterials were dispersed in physiological saline to obtain a PDA-PVP tracer, wherein the concentration of the composite nanomaterials in the PDA-PVP tracer was 1~10 mg / mL.
10. The application as described in claim 9, characterized in that, The PDA-PVP tracer is used as a positive contrast agent for lymph nodes and / or a negative contrast agent for parathyroid glands.