Fluorescence-labeled bionic uricase-loaded hollow Prussian blue nanoparticles as well as preparation method and application thereof

Abstract

The invention provides a fluorescence-labeled bionic uricase-loaded hollow Prussian blue nanoparticle and a preparation method and application thereof.The fluorescence-labeled bionic uricase-loaded hollow Prussian blue nanoparticle has a fluorescence label, takes hollow Prussian blue as an inner core and takes a neutrophil-like cell membrane as a shell, and the hollow Prussian blue core and the neutrophil-like cell membrane are combined to form the fluorescence-labeled bionic uricase-loaded hollow Prussian blue nanoparticle. Uricase is loaded in the hollow Prussian blue; the preset emission wavelength of the near-infrared laser equipment is 808nm, the laser power density is 1W / cm < 2 >, and the action time is 5min; when the fluorescent mark shows that the nanoparticles reach the preset action part, the near-infrared laser equipment is started. The fluorescently-labeled bionic uricase-loaded hollow Prussian blue nanoparticles have the effects of clearing urate crystals and reducing the active oxygen level, and meanwhile, Prussian blue has good photo-thermal conversion efficiency and can be combined with photo-thermal therapy to remarkably relieve acute gout and reduce the degree of swelling of the sole of a mouse.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles and a preparation method and application thereof. Background Art

[0002] Gout is an inflammatory disease caused by excessive uric acid production or reduced excretion due to purine metabolism disorders in the body. Urate crystals are deposited in the joints, causing joint inflammation and destruction. Repeated attacks of acute gout can lead to multiple complications such as renal insufficiency, coronary heart disease, and metabolic syndrome. Therefore, for the treatment of gout, the authoritative treatment guidelines recommend that in addition to lowering uric acid treatment, anti-inflammatory treatment should also be used to prevent the onset of gout, and the duration of anti-inflammatory treatment should be no less than 6 months. Current research results show that macrophages residing in the joint cavity are the initiating cells of inflammation. Receptors such as CD14 and TLR4 expressed on the macrophage membrane can bind to MSU crystals and mediate their phagocytosis of MSU crystals. Then, under the stimulation of external factors such as LPS, fatty acids, and ATP, the assembly of NLRP3 inflammasomes in macrophages is finally triggered, releasing IL-1β, further causing the infiltration of neutrophils and the release of IL-6 and TNF-α, leading to abnormal symptoms such as pain and swelling in the joints. In response to the above pathogenesis, the only biological agents currently developed are canakinumab and anakinra, which exert anti-inflammatory effects by antagonizing IL-1β. Although both have strong anti-inflammatory effects, they also have large side effects and are only suitable for patients with refractory gout with heart and kidney dysfunction. Other commonly used anti-gout drugs in clinical practice, such as non-steroidal anti-inflammatory drugs, colchicine, and glucocorticoids, all have large side effects and are suitable for a limited population. Therefore, there is an urgent need to develop safer and more effective anti-gout treatment drugs in clinical practice.

[0003] Uricase deficiency is one of the key factors in the occurrence of gout, and its absence leads to the obstruction of uric acid metabolism in the body. As the end product of purine metabolism, uric acid should be degraded into water-soluble allantoin under the action of uricase, so as to maintain its steady-state concentration in the body. However, uricase deficiency hinders this process, and uric acid continues to accumulate in tissues, forming urate crystals, which stimulate joints and surrounding tissues, leading to the occurrence and development of gout. Therefore, treatment strategies for uricase deficiency are particularly important. Supplementing uricase or taking other methods to promote the metabolism and excretion of uric acid can help control the development of gout and relieve gout symptoms. However, as an exogenous enzyme, uricase will inevitably induce immunogenic reactions when used in the human body. In addition, uricase-mediated uric acid dissolution will produce hydrogen peroxide (H 2 O 2 ) as a byproduct, especially in the slow metabolism and closed internal environment of the joint cavity, H 2 O 2The accumulation of H2O2 is more prominent, which brings additional challenges to the treatment of gout. To solve this problem, the present invention provides an innovative treatment method, which is to use hollow Prussian blue nanoparticles to load uricase, wrap the neutrophil-like membrane on the outer layer, and perform fluorescent labeling. The principle of this method is that the loaded uricase can effectively degrade monosodium urate crystals through biocatalytic reactions, and the hollow Prussian blue can serve as a carrier of uricase and eliminate H2O2 generated by the degradation of urate crystals catalyzed by uricase. 2 O 2 , and also reduces the reactive oxygen species produced in macrophages during gout attacks, thereby successfully blocking the inflammatory amplification during the development of gout. At the same time, the wrapping of neutrophil cell membranes is conducive to the targeted delivery of drugs to the site of gout inflammation, achieving effective treatment of gout. The catalytic formula of the uricase catalytic cascade is as follows:

[0004] Summary of the invention

[0005] The present invention provides a fluorescently labeled biomimetic uricase-loaded hollow Prussian blue nanoparticle, a preparation method and an application thereof. The biomimetic uricase-loaded hollow Prussian blue nanoparticle has a fluorescent label, uses a hollow Prussian blue as a core, uses a neutrophil-like cell membrane as an outer shell, and uricase is loaded in the hollow Prussian blue. The drug can be well targeted and delivered to the gout inflammation site, thereby achieving effective treatment of gout.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: an application of a fluorescently labeled biomimetic hollow Prussian blue nanoparticle loaded with uricase and a near-infrared laser device in the preparation of a drug delivery system for treating acute gout, wherein the biomimetic hollow Prussian blue nanoparticle loaded with uricase has a fluorescent label, a hollow Prussian blue as a core, a neutrophil-like membrane as a shell, and uricase is loaded in the hollow Prussian blue; the near-infrared laser device has a preset emission wavelength of 808nm and a laser power density of 1W / cm 2 , the action time is 5 minutes; when the fluorescent marker shows that the nanoparticles have reached the predetermined action site, the near-infrared laser device is started.

[0007] Preferably, the preparation method of the fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles is as follows:

[0008] S1: Preparation of hollow Prussian blue HMPB by synthesis and acid etching;

[0009] S2: first, HMPB is uniformly dispersed in PBS to obtain an HMPB dispersion, and then a 1 mg / mL uricase solution dissolved in PBS is added dropwise to the HMPB dispersion, stirred and centrifuged to obtain uricase-loaded hollow Prussian blue nanoparticles UPB;

[0010] S3: HL-60 cells were induced to form neutrophil-like cells by dimethyl sulfoxide (DMSO), and then the neutrophil-like cell membrane was separated and extracted by extrusion method;

[0011] S4: The neutrophil-like membrane obtained in S3 was wrapped in the outer layer of UPB by extrusion method to obtain the biomimetic hollow Prussian blue nanoparticles UPB@NM loaded with uricase;

[0012] S5: Mixing DiR fluorescence with a concentration of 1 mg / mL with the UPB@NM obtained in S4, wherein the concentration of UPB@NM is 1 mg / mL, to obtain the fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles DiR-UPB@NM.

[0013] Preferably, the specific process of S1 is:

[0014] S11: K 3 [Fe(CN) 6 ] and PVP K30 were dissolved in 0.01M HCl solution, and a mixed solution was obtained after magnetic stirring for 30 min. The mixed solution was placed in a reaction kettle, reacted at 70°C-90°C for 20 h, cooled to room temperature, centrifuged and the precipitate was collected, and the precipitate was washed with deionized water and ethanol for 3 times respectively to obtain solid Prussian blue, which was then dried at room temperature;

[0015] S12: The solid Prussian blue and PVP K30 obtained in S11 were dissolved in a 1 M HCl solution, stirred for 2 h to obtain a blue mixed solution, placed in a reactor, reacted at 130°C-150°C for 4 h, cooled to room temperature, centrifuged and collected the precipitate, washed the precipitate three times with deionized water and ethanol respectively, to obtain a hollow Prussian blue HMPB.

[0016] Preferably, in S11, K 3 [Fe(CN) 6 ], the mass volume ratio of PVP K30 and HCl solution is 132 mg:3 g:40 mL.

[0017] Preferably, the mass volume ratio of solid Prussian blue, PVP K30 and HCl solution in S12 is 1 mg:5 g:1 mL.

[0018] Preferably, the centrifugation conditions in S11 and S12 are both 10000 rpm-12000 rpm for 25 min-35 min.

[0019] Preferably, the specific process of S3 is:

[0020] HL-60 cells were cultured in RPMI-1640 medium, and the inducer DMSO was added to the medium. The concentration of DMSO was 1.25%. After 5 days of culture, the cells were collected and cultured at 2.0×10 7 The cells were suspended at a density of 100 cells / mL in TM buffer, which has a pH of 7.4 and contains 0.01 M Tris and 0.001 M MgCl 2 , placed at 4°C for 30 minutes, after 5 freeze-thaw cycles in liquid nitrogen, the cells were squeezed 25-35 times by a micro-squeezer to destroy the cells to obtain a mixture, and the mixture was centrifuged at 10000×g and 4°C for 15min-25min to precipitate the intracellular substances, the supernatant was collected, and centrifuged at 100000×g and 4°C for 45min-60min. The final precipitate was collected as the purified neutrophil-like membrane and stored at -80°C for future use.

[0021] Preferably, the specific process of S4 is:

[0022] 1 mL UPB and 1 × 10 8 The cell membranes extracted from HL-60 cells were mixed and squeezed back and forth through a 400nm polycarbonate membrane using a 1mL micro-extruder for 12-25 times. The collected preparation was the biomimetic uricase-loaded hollow Prussian blue nanoparticles UPB@NM.

[0023] Compared with the prior art, the at least one technical solution adopted in the embodiments of this specification can achieve the following beneficial effects:

[0024] First, the nanoparticles prepared by the present invention have good stability and meet the biological safety requirements;

[0025] Second, the nanoparticles prepared by the present invention have a good ability to target gout inflammation sites and can increase the accumulation of drugs in the inflammation sites;

[0026] Third, the nanoparticles prepared by the present invention have the effects of clearing urate crystals and reducing the level of reactive oxygen. At the same time, Prussian blue has good photothermal conversion efficiency and can be combined with photothermal therapy to significantly relieve acute gout and reduce the degree of swelling in the soles of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without paying creative work.

[0028] Figure 1is the particle size distribution diagram of PB, HMPB, UPB and UPB@NM;

[0029] Figure 2 Transmission electron microscopy images of PB, HMPB, and UPB@NM;

[0030] Figure 3 Figure 2 is the cytotoxicity test result of UPB@NM;

[0031] Figure 4 This is the catalase activity analysis diagram of UPB@NM;

[0032] Figure 5 This is an analysis diagram of experimental data on the adsorption of inflammatory factors by neutrophil-like cell membranes;

[0033] Figure 6 In vivo fluorescence imaging of gout mice after intravenous injection of free DiR and DiR-UPB@NM;

[0034] Figure 7 The results of cell activity detection caused by uric acid decomposition catalyzed by different preparations (HMPB, UOX, UPB and UPB@NM);

[0035] Figure 8 The results of the detection of reactive oxygen species (ROS) levels in macrophages after treatment with different preparations (UOX, HMPB and UPB@NM);

[0036] Fig. 9 The diagram shows the changes in the soles of gout mice in the control group (Control), gout model group (Model), free uricase (UOX) group, free hollow Prussian blue nanoparticles (HMPB) group, uricase-loaded hollow Prussian blue nanoparticles (UPB) group, biomimetic uricase-loaded hollow Prussian blue nanoparticles (UPB@NM) group, and biomimetic uricase-loaded hollow Prussian blue nanoparticles combined with photothermal therapy (UPB@NM+NIR) group;

[0037] Fig.10 Representative photos of mouse paws;

[0038] Fig.11 A diagram showing the experimental results of increasing cellular uptake by nanoparticles combined with photothermal therapy. DETAILED DESCRIPTION

[0039] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0040] The following describes the implementation methods of the present application through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0041] It should be understood that the following description of K in the present invention is 3 [Fe(CN) 6 ] The mass volume ratio of PVP K30 and HCl solution and the mass volume ratio of solid Prussian blue, PVP K30 and HCl solution are only preferred embodiments and should not be construed as limiting the scope of protection of the claims.

[0042] In the present invention, the abbreviations of various substances have the following meanings:

[0043] HMPB is hollow Prussian blue;

[0044] PBS is phosphate buffered saline;

[0045] UPB is hollow Prussian blue nanoparticles;

[0046] DMSO is dimethyl sulfoxide;

[0047] UPB@NM is a biomimetic hollow Prussian blue nanoparticle loaded with uricase;

[0048] DiR-UPB@NM is a fluorescently labeled biomimetic hollow Prussian blue nanoparticle loaded with uricase;

[0049] K 3 [Fe(CN) 6 ] is potassium ferrocyanide;

[0050] PVP K30 is polyvinylpyrrolidone K30;

[0051] HCl is hydrochloric acid;

[0052] MgCl 2 It is magnesium chloride.

[0053] Example 1

[0054] A method for preparing fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles comprises the following steps:

[0055] S1: Preparation of hollow Prussian blue HMPB by synthesis and acid etching

[0056] S11: 132mgK 3 [Fe(CN) 6 ] and 3g PVP K30 were dissolved in 40mL 0.01M HCl solution, and magnetically stirred for 30min to obtain a mixed solution, which was a yellow transparent liquid. The mixed solution was placed in a reactor, reacted at 80°C for 20h, cooled to room temperature, and centrifuged (12000rpm for 30min) to collect the precipitate, washed the precipitate 3 times with deionized water and ethanol respectively to obtain solid Prussian blue, and dried at room temperature;

[0057] S12: 20 mg of solid Prussian blue obtained from S11 and 100 mg of PVP K30 were dissolved in 20 mL of 1 M HCl solution, stirred for 2 h to obtain a blue mixed solution, and the mixed solution was placed in a reactor, reacted at 140 ° C for 4 h, cooled to room temperature, and centrifuged (12000 rpm for 30 min) and the precipitate was collected, and the precipitate was washed 3 times with deionized water and ethanol respectively to obtain a hollow Prussian blue HMPB.

[0058] S2: First, 5 mg of HMPB was uniformly dispersed in 5 mL of PBS to obtain an HMPB dispersion, and then 1 mL of a 1 mg / mL uricase solution dissolved in PBS was added dropwise to the HMPB dispersion. After stirring, the mixture was centrifuged at 12,000 rpm for 30 min, and the precipitate was collected to obtain hollow Prussian blue nanoparticles UPB loaded with uricase.

[0059] S3: HL-60 cells were induced to form neutrophil-like cells by dimethyl sulfoxide (DMSO), and then the neutrophil-like cell membrane was isolated and extracted by extrusion method.

[0060] HL-60 cells were cultured in RPMI-1640 medium, and the inducer DMSO was added to the medium to make the concentration of DMSO 1.25%. After 5 days of culture, the cells were collected and cultured at 2.0×10 7 The cells were suspended at a density of 100 cells / mL in TM buffer, which has a pH of 7.4 and contains 0.01 M Tris and 0.001 M MgCl 2, placed at 4°C for 30 minutes, after 5 freeze-thaw cycles in liquid nitrogen, the cells were squeezed 30 times by a micro-squeezer to destroy the cells to obtain a mixture, and the mixture was centrifuged at 10000×g and 4°C for 20 minutes to precipitate the intracellular substances, the supernatant was collected, and centrifuged at 100000×g and 4°C for 60 minutes. The final precipitate was collected as the purified neutrophil-like cell membrane and stored at -80°C for future use.

[0061] S4: The neutrophil-like membrane obtained in S3 was wrapped in the outer layer of UPB by extrusion to obtain the biomimetic hollow Prussian blue nanoparticles UPB@NM loaded with uricase

[0062] 1 mL UPB and 1 × 10 8 The cell membranes extracted from HL-60 cells were mixed and squeezed back and forth 20 times through a 400nm polycarbonate membrane using a 1mL micro-extruder. The collected preparation was the biomimetic uricase-loaded hollow Prussian blue nanoparticles UPB@NM.

[0063] S5: Take 1 μL of DiR solution with a concentration of 1 mg / mL and drop it into 1 mg / mL UPB@NM, and stir them together for 30 minutes to obtain fluorescently labeled biomimetic uricase-loaded hollow Prussian blue nanoparticles DiR-UPB@NM.

[0064] The particle sizes of Prussian blue PB, hollow Prussian blue HMPB, hollow Prussian blue nanoparticles UPB loaded with uricase, and hollow Prussian blue nanoparticles UPB@NM loaded with biomimetic uricase were measured using a Malvern nano-laser particle size analyzer, and the morphology and size of PB, HMPB, and UPB@NM were observed under a transmission electron microscope (TEM). The particle size distribution diagram and transmission electron microscope diagram are shown in Figure 2. Figure 1 and Figure 2 As shown in the figure, the average diameter of UPB@NM is 218.5nm, which is slightly increased compared with UPB (201.7nm). Figure 1 ); Figure 2 The transmission electron microscopy imaging results showed that Prussian blue showed an obvious hollow structure after acid etching, and there was an obvious thin film structure on the outer layer of Prussian blue nanoparticles after membrane wrapping.

[0065] THP-1 cells were co-incubated with UPB@NM at different concentrations for 24 h, and the cell activity was detected using CCK8 to investigate the cytotoxicity. Figure 3 The results of cytotoxicity assay showed that UPB@NM had no obvious cytotoxicity to THP-1 cells within the concentration range of 100 μM, indicating that it has good biosafety.

[0066] The different concentrations of the preparations were mixed with H 2 O2 Mix and use a dissolved oxygen meter to detect the O generated in the system. 2 , record the O generated within 10 min 2 The catalase activity of UPB@NM was investigated. Figure 4 As shown in the figure, the results of catalase activity investigation showed that UPB@NM can effectively catalyze H 2 O 2 O 2 , laying the foundation for its antioxidant effect.

[0067] Example 2 Experiment on adsorption of inflammatory factors by neutrophil membrane

[0068] Take 1 mL of 500 μg / mL UPB and UPB@NM suspensions, add PBS solutions containing TNF-α and IL-6 to make their concentrations 10 ng / mL, incubate the reaction system at 37°C for 6 h, and then high-speed centrifuge at 13000 rpm for 20 min to remove the nanoparticles. Collect the supernatant and detect it using TNF-α and IL-6 ELISA kits.

[0069] The results are as follows Figure 5 As shown, it can be seen that the TNF-αR and IL-1R proteins expressed on the surface of neutrophil-like cell membranes have the function of adsorbing inflammatory factors, so the neutrophil-like cell membranes have the dual effects of targeting and anti-inflammatory.

[0070] Example 3 Investigation of the targeting ability of UPB@NM

[0071] Gout mice were randomly divided into 2 groups (n=3) and intravenously injected with DiR and DiR-UPB@NM, respectively. At 2h, 6h, 12h, 24h, 48h and 72h after injection, the mice were anesthetized with isoflurane and imaged with IVIS. The results are shown in Figure 6 shown. Figure 6 It was shown that the fluorescence signal of the paw of the free DiR group was relatively low, while the fluorescence of the paw of the gout site of the mice in the DiR-UPB@NM group was the strongest at each time point and lasted for 72 hours, indicating that DiR-UPB@NM has good targeting ability to the site of gout inflammation.

[0072] Example 4 Investigation of UPB@NM cell-level effects

[0073] Cell activity assessment: The cells were cultured in appropriate culture medium, and different prescription preparations (HMPB, UOX, UPB and UPB@NM) were added for treatment. After treatment, appropriate amounts of Calcein-AM working solution (final concentration 2 μM) and PI working solution (final concentration 10 μg / mL) were added, and incubated at 37°C in the dark for 30 minutes. After the incubation, the cells were immediately examined using a fluorescence microscope. The results are shown in Figure 7 .

[0074] Investigation of ROS scavenging effect: PMA-induced THP-1 was pre-stimulated with 100 ng / mL LPS for 3 h, and then stimulated with 200 μg / mL LPS for 6 h to induce the formation of inflammatory macrophages. Different preparations (UOX, HMPB, UPB and UPB@NM) were added for co-incubation. After the effect, DCFH-DA was added to the cells and incubated in the dark at 37°C for 30 min. The cells were then washed with PBS to remove the unloaded DCFH-DA probes, and then imaged under a fluorescence microscope to detect the intracellular ROS level. The results are shown in Figure 8 .

[0075] The results showed that: Calcein-AM / PI staining results showed that compared with the control group, the number of dead cells increased significantly after uricase (UOX) catalyzed uric acid (UA) because uricase produced H2O2 after catalyzing the decomposition of uric acid, which caused certain damage to the cells. The activity of cells in the UPB group and UPB@NM group increased significantly, and the number of dead cells decreased because Prussian blue could remove H2O2 produced during the catalytic process and improve cell activity. The results of DCFH-DA detection of intracellular ROS showed that the ROS level in inflammatory THP-1 increased significantly, and the ROS level in the uricase (UOX) group decreased slightly. This was because uricase promoted the degradation of MSU crystals. ROS was significantly reduced after combined treatment with Prussian blue, which was mainly attributed to the powerful antioxidant effect of ROS.

[0076] Example 5 Investigation of the effect of UPB@NM on gout in vivo

[0077] Mice were randomly divided into control group (Control), gout model group (Model), free uricase (UOX) group, free hollow Prussian blue nanoparticles (HMPB) group, uricase-loaded hollow Prussian blue nanoparticles (UPB) group, biomimetic uricase-loaded hollow Prussian blue nanoparticles (UPB@NM) group, and biomimetic uricase-loaded hollow Prussian blue nanoparticles combined with photothermal therapy (UPB@NM+NIR) group. Monosodium urate crystals (MSU) were injected into the sole of the foot and then the corresponding preparations were intravenously administered. Near-infrared laser irradiation (808 nm, 1 W / cm 2 , 5min) on the right sole of the foot. The thickness of the left and right feet was measured with a vernier caliper at 2, 4, 8, 12, 24, 48, and 72 hours. The swelling degree of the sole of the foot was photographed at 48 hours. The results are shown in Fig. 9 and Fig.10 .

[0078] like Fig. 9 and Fig.10As shown in the figure, after MSU was injected into the sole of the foot, the thickness of the sole of the foot of the gout model group (Model) mice gradually increased and reached a peak at 8 hours. The effectiveness of free UOX and HMPB in reducing sole swelling is limited, mainly due to the low bioavailability and poor targeting of UOX. The UPB@NM group has a better effect in relieving sole swelling, and the degree of sole swelling is most significantly reduced after UPB@NM combined with photothermal therapy, indicating that this treatment strategy can effectively treat acute gout.

[0079] Example 6 Experiment on increasing cellular uptake by nanoparticles combined with photothermal therapy

[0080] DiI-labeled UPB@NM was co-incubated with activated and unactivated THP-1 cells. Activated THP-1 cells were induced to form crystals by prestimulation with 100 ng / mL LPS for 3 h and stimulation with 200 μg / mL monosodium urate crystals for 6 h. The photothermal therapy group was irradiated with near-infrared light (808 nm, 1 W / cm 2 , 5min), and after incubation for 8h, they were observed under a fluorescence microscope.

[0081] The experimental results are as follows Fig.11 As shown, the results of fluorescence microscopy observations showed that activated THP-1 took up more UPB@NM and had more red fluorescence than inactivated THP-1, which is beneficial for the preparation to play a role in inflammatory cells. After photothermal therapy, both activated and inactivated THP-1 had more UPB@NM uptake and more red fluorescence.

[0082] Example 7 Experiment of Nanoparticles Combined with Photothermal Therapy to Upregulate Heat Shock Protein 70 (Hsp70)

[0083] Proteomic analysis was performed on the soles of the mice in the gout model group and after treatment with UPB@NM+NIR. The results are shown in Table 1. The expression of Hspa4 and Hspa12b was significantly upregulated (pvalue<0.05), indicating that the expression of heat shock protein 70 (Hsp70) in the sole tissue was upregulated after photothermal treatment. Hsp70 plays an important regulatory role in the inflammatory response. It can affect the expression and release of inflammatory mediators by interacting with inflammation-related signaling pathways. For example, it can inhibit the activity of transcription factors such as nuclear factor κB (NF-κB), thereby reducing the expression and secretion of proinflammatory cytokines (such as IL-1, TNF-α, etc.). The upregulated expression of Hsp70 helps to alleviate the inflammatory response.

[0084] Table 1. Proteomics results

[0085]

[0086] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of fluorescently labeled biomimetic hollow Prussian blue nanoparticles loaded with uricase and near-infrared laser equipment in the preparation of a drug delivery system for treating acute gout, characterized in that: The biomimetic hollow Prussian blue nanoparticles loaded with uricase have a fluorescent marker, with hollow Prussian blue as the core and neutrophil-like membrane as the shell, and uricase is loaded in the hollow Prussian blue; the near-infrared laser device has a preset emission wavelength of 808 nm and a laser power density of 1 W / cm 2 , the action time is 5 minutes; when the fluorescent marker shows that the nanoparticles have reached the predetermined action site, the near-infrared laser device is started.

2. The use according to claim 1, characterized in that: The preparation method of the fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles is as follows: S1: Preparation of hollow Prussian blue HMPB by synthesis and acid etching; S2: first, HMPB is uniformly dispersed in PBS to obtain an HMPB dispersion, and then a 1 mg / mL uricase solution dissolved in PBS is added dropwise to the HMPB dispersion, stirred and centrifuged to obtain uricase-loaded hollow Prussian blue nanoparticles UPB; S3: HL-60 cells were induced to form neutrophil-like cells by dimethyl sulfoxide (DMSO), and then the neutrophil-like cell membrane was separated and extracted by extrusion method; S4: The neutrophil-like membrane obtained in S3 was wrapped in the outer layer of UPB by extrusion method to obtain the biomimetic hollow Prussian blue nanoparticles UPB@NM loaded with uricase; S5: Mixing DiR fluorescence with a concentration of 1 mg / mL with the UPB@NM obtained in S4, wherein the concentration of UPB@NM is 1 mg / mL, to obtain the fluorescently labeled biomimetic uricase-carrying hollow Prussian blue nanoparticles DiR-UPB@NM.

3. The use according to claim 2, characterized in that: The specific process of S1 is as follows: S11: K3[Fe(CN)6] and PVP K30 were dissolved in a 0.01M HCl solution, and a mixed solution was obtained after magnetic stirring for 30 minutes. The mixed solution was placed in a reaction kettle, reacted at 70°C-90°C for 20 hours, cooled to room temperature, centrifuged and the precipitate was collected, and the precipitate was washed with deionized water and ethanol for 3 times respectively to obtain solid Prussian blue, and dried at room temperature; S12: The solid Prussian blue and PVP K30 obtained in S11 were dissolved in a 1 M HCl solution, stirred for 2 h to obtain a blue mixed solution, placed in a reactor, reacted at 130°C-150°C for 4 h, cooled to room temperature, centrifuged and collected the precipitate, washed the precipitate three times with deionized water and ethanol respectively, to obtain a hollow Prussian blue HMPB.

4. The use according to claim 3, characterized in that: The mass volume ratio of K3[Fe(CN)6], PVP K30 and HCl solution in the S11 is 132 mg:3 g:40 mL.

5. The use according to claim 3, characterized in that: The mass volume ratio of solid Prussian blue, PVP K30 and HCl solution in the S12 is 1 mg:5 g:1 mL.

6. The use according to claim 3, characterized in that: The centrifugation conditions in S11 and S12 are both 10000 rpm-12000 rpm for 25 min-35 min.

7. The use according to claim 2, characterized in that: The specific process of S3 is as follows: HL-60 cells were cultured in RPMI-1640 medium, and the inducer DMSO was added to the medium. The concentration of DMSO was 1.25%. After 5 days of culture, the cells were collected and cultured at 2.0×10 7 The cells were suspended at a density of 10000 × g and 4 ° C. in TM buffer, wherein the pH of the TM buffer was 7.4 and contained 0.01M Tris and 0.001M MgCl2, and placed at 4 ° C. for 30 min. After 5 freeze-thaw cycles in liquid nitrogen, the cells were squeezed 25-35 times by a micro-squeezer to destroy the cells to obtain a mixture. The mixture was centrifuged at 10000 × g and 4 ° C. for 15 min-25 min to precipitate the intracellular substances, and the supernatant was collected. Centrifuged at 100000 × g and 4 ° C. for 45 min-60 min, and the final precipitate was collected as the purified neutrophil-like membrane and stored at -80 ° C. for future use.

8. The use according to claim 2, characterized in that: The specific process of S4 is as follows: 1 mL UPB and 1 × 10 8 The cell membranes extracted from HL-60 cells were mixed and squeezed back and forth through a 400nm polycarbonate membrane using a 1mL micro-extruder for 12-25 times. The collected preparation was the biomimetic uricase-loaded hollow Prussian blue nanoparticles UPB@NM.

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