Biofilm-targeting self-assembled composite nanosystem, preparation method and application thereof

Abstract

The application discloses a biofilm-targeted self-assembled composite nano system, a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. The active ingredient of the biofilm-targeted self-assembled composite nano system is composed of an organic ligand, metal ions and a gel matrix. The self-assembled composite nano system has double targeting properties. Not only can the self-assembled composite nano system efficiently target an infection site and stay at the infection site for up to 15 days, but also can target adhesion and effectively penetrate a biofilm to produce active oxygen, so that the problem that conventional therapeutic drugs are difficult to penetrate the biofilm due to the obstruction of the biofilm barrier is overcome. Meanwhile, the self-assembled composite nano system promotes the immune response of a host, so that the treatment effect on biofilm infection is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a biofilm-targeted self-assembled composite nano system, a preparation method and application thereof. BACKGROUND

[0002] Due to the rapid emergence of drug-resistant bacteria, a large number of public health problems have been caused, resulting in huge public medical and financial burdens. The main reason for the high mortality rate of bacterial infections is drug resistance, and biofilm plays an important role in bacterial resistance to antibiotics. Biofilm not only hinders the penetration of antibiotics into the biofilm, thereby preventing the antibiotics from killing the bacteria in the biofilm; at the same time, the low-oxygen living environment in the biofilm enables the bacteria in the biofilm to change from the wild type to the small colony variant through phenotypic switching, because the metabolism level is lower and the expression of virulence factors is lower than that of the normal phenotype, the host innate immune system is difficult to detect the presence of SCV through the secretion of toxins and pro-inflammatory factors, so that it can evade the recognition of the host immune system and the killing of antibacterial drugs; and when it detaches from the original infection site and re-infects other cells, it will quickly recover to the fully toxic wild type. In addition, bacteria can directly attack host immune cells through secreted factors (nucleases, hemolysins, etc.) to inhibit the clearance of the host immune response. The biofilm barrier not only hinders the antibacterial effect of antibiotics and host immune factors on bacteria, but also serves as a source of acute infection foci, ready to release pathogenic planktonic bacteria to cause recurrent infections in the host. Due to the limitations of dosage and multidrug resistance, even antimicrobial drugs are ineffective in eradicating biofilm infections, and there is an urgent need to develop new treatment strategies with high efficiency against biofilm and less likely to develop drug resistance.

[0003] Photodynamic therapy is a promising strategy for antimicrobial infection. It mainly uses photosensitizers to produce active oxygen and other antibacterial active substances under suitable wavelength light, has great potential to destroy bacterial cell walls and DNA and induce bacterial apoptosis or necrosis, and is less likely to develop drug resistance. However, the photodynamic efficiency of clinically approved photosensitizers (porphyrin and its derivatives) is limited by their water solubility. The planar stacking of the photosensitizers in water media leads to aggregation, resulting in a lower absorption coefficient in the red light region of the absorption spectrum. Metal-organic frameworks with large specific surface area, small size and regularity can form a controllable and ordered coordination of crystalline porous structure through metal nodes and organic linkers, and due to the synergistic effect of metal-linker bridging units and periodic structure, the optimized photonic function and light trapping ability of porphyrin can be achieved.

[0004] Amino acids are essential nutrients for the host, and have good water solubility and biocompatibility. And in the process of bacterial infection, it can trigger or promote the host immune response in the initial inflammation of infection and the tissue recovery process after infection clearance. In the early stage of infection, the host needs a large amount of amino acids to resist bacterial infection by producing pro-inflammatory factors such as iNOS and TNF-alpha; and when the bacteria are cleared, the host needs to use amino acids to produce anti-inflammatory factors such as Arg-1 and IL-4 to promote tissue repair at the infection site.

[0005] Nanogels are widely used as drug carriers due to their good physicochemical properties (drug controlled release and affinity for aqueous solutions), excellent colloidal stability, high intracellular internalization properties, and inertness in blood. Natural polymers have less toxicity, good biocompatibility and biodegradability. Incorporating therapeutic agents into natural gel matrices can protect active compounds from degradation, enhance absorption, improve therapeutic effect and reduce dosing frequency. And some natural gel matrices have targeted adhesion to bacteria, which can enhance the efficacy of therapeutic agents.

[0006] In the administration route of drugs, intravenous injection can avoid first-pass effect, fast onset, high bioavailability and other advantages, but due to the non-specific distribution of antibacterial drugs in the body, the clearance of the mononuclear macrophage system in the blood and the barrier effect of the biofilm on the antibacterial drugs, long-acting precise treatment of biofilm infection cannot be achieved. At present, the most effective treatment method for radical cure of biofilm in clinical practice is to remove the infected tissue through surgery, which leads to low patient compliance and high treatment cost. Therefore, a new strategy is urgently needed to improve the targeting of biofilm so as to efficiently and precisely treat biofilm infection. SUMMARY

[0007] The present application provides a biofilm-targeted self-assembled composite nanosystem, a preparation method and applications thereof, which solves the problem that antibacterial drugs are difficult to eradicate biofilm due to the inability to target biofilm infection sites and penetrate biofilm, and the self-assembled composite nanosystem can release and stay for a long time at the infection site, synergize photodynamic and immunity to enhance its anti-biofilm activity, and promote the recovery of infected tissue, providing a new treatment strategy for biofilm infection treatment.

[0008] To achieve the above-mentioned purpose, the present application provides a biofilm-targeted self-assembled composite nanosystem, raw materials of which include organic ligands, metal ions and gel matrix;

[0009] The organic ligand comprises organic ligand A and organic ligand B; the organic ligand A is a photosensitizer type ligand; and the organic ligand B is an amino acid type ligand. The photosensitizer type ligand is used to generate a photodynamic effect to kill the biofilm, the amino acid type ligand is used to improve the host immune response and promote the treatment of the biofilm by the nano system, and the gel matrix is used to target the adhered biofilm and promote the penetration of the nano system to the biofilm, so that the nano system kills the bacteria in the biofilm and avoids recurrent infection.

[0010] The active ingredient of the biofilm-targeting self-assembled composite nano system of the application is composed of an organic ligand, a metal ion and a gel matrix. The self-assembled composite nano system has dual targeting properties. Not only can it efficiently target the infection site and stay at the infection site for up to 15 days, but also can target the adhered biofilm and effectively penetrate the biofilm to generate reactive oxygen, overcoming the difficulty that conventional therapeutic drugs cannot easily penetrate the biofilm due to the barrier of the biofilm. Thus, the treatment effect on biofilm infection is improved.

[0011] Further, the mass ratio of the organic ligand A to the organic ligand B is 1-3:1-2.

[0012] Further, the organic ligand A comprises at least one of hemin, meso-tetra(4-carboxyphenyl) porphyrin, 5,10,15,20-tetra(4-aminophenyl) porphyrin and benzoic acid.

[0013] The organic ligand B comprises at least one of arginine and lysine.

[0014] The metal ion comprises at least one of trivalent iron ion, calcium ion and zirconium ion.

[0015] The gel matrix comprises at least one of gelatin, guar gum and carob bean gum.

[0016] Further, the organic ligand A is meso-tetra(4-carboxyphenyl) porphyrin or benzoic acid.

[0017] The organic ligand B is arginine.

[0018] The metal ion is calcium ion or zirconium ion.

[0019] The gel matrix is gelatin.

[0020] A preparation method of the biofilm-targeting self-assembled composite nano system, comprising the following steps:

[0021] Dissolve the organic ligand A in an organic solvent (preferably N, N-dimethylformamide), add metal ions, ultrasonic mixing, stirring to prepare metal organic framework A; then dissolve the organic ligand B in water, add the metal organic framework A, prepare the metal organic framework B by ligand replacement; finally, dissolve the gel matrix and add it to the metal organic framework B, stir to prepare the biofilm-targeted self-assembled composite nanosystem.

[0022] Further, the preparation method of the biofilm-targeted self-assembled composite nanosystem specifically comprises the following steps:

[0023] (1) Dissolve the organic ligand A in N, N-dimethylformamide, add metal ions, ultrasonic mixing for 1 min, the temperature is 60-90 DEG C, the stirring speed is 500 r / min, and the time is 8-24 h to obtain the metal organic framework A;

[0024] (2) Dissolve the organic ligand B in water, add the metal organic framework A, the temperature is 60 DEG C, the stirring speed is 500 r / min, and the time is 24 h to prepare the metal organic framework B by ligand replacement;

[0025] (3) Dissolve the gel matrix and add it to the metal organic framework B, the temperature is 40 DEG C, the stirring speed is 500 r / min, and the time is 24 h to prepare the biofilm-targeted self-assembled composite nanosystem.

[0026] Further, the mass percentage contents of the metal organic framework B, the metal ions and the gel matrix are 48-80%, 6-10% and 12-42% respectively.

[0027] The biofilm-targeted self-assembled composite nanosystem is applied to the preparation of a biofilm-targeted self-assembled composite nanosystem product.

[0028] The biofilm-targeted self-assembled composite nanosystem is applied to the preparation of an anti-infection drug.

[0029] Compared with the prior art, the biofilm-targeted self-assembled composite nanosystem has the following advantages and technical effects:

[0030] The biofilm-targeted self-assembled composite nanosystem has photodynamic therapy activity, can target the biofilm infection site, can target the adhesion to the biofilm and penetrate the biofilm, and can accurately and efficiently treat the bacterial biofilm infection.

[0031] The biofilm-targeting self-assembled composite nanosystem of the present application is prepared from a metal organic framework and a nanogel technology, and solves the problem of recurrent infection caused by the difficulty of penetrating the biofilm. The self-assembled composite nanosystem realizes dual targeting of the infection site and the biofilm, precisely and efficiently targets adhesion to the biofilm, and can stay at the infection site for up to 15 days, reducing the complexity of frequent drug administration, and improving the treatment efficiency of the nanosystem at the infection site. At the same time, photodynamic therapy in combination with immunity can regulate the immune response of the host to promote the clearance of bacteria by the host and the recovery of the tissue, and provides a new strategy for biofilm infection treatment. BRIEF DESCRIPTION OF DRAWINGS

[0032] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and serve as an explanation of the illustrative embodiments of the present application and their description, and do not constitute improper limitations on the present application. In the drawings:

[0033] Figure 1 It is a physical diagram of the self-assembled composite nanosystem in Test Example 1;

[0034] Figure 2 It is an electron microscope image of the self-assembled composite nanosystem in Test Example 1;

[0035] Figure 3 It is a diagram of the targeting of the biofilm by the organic metal framework A, the organic metal framework B and the self-assembled composite nanosystem in Test Example 1;

[0036] Figure 4 It is a wound imprint of the mouse biofilm infection model in Test Example 2;

[0037] Figure 5 It is a diagram of the targeting of the infection site by the organic metal framework A, the organic metal framework B and the self-assembled composite nanosystem in Test Example 2;

[0038] Figure 6 It is a diagram of the targeting fluorescence intensity of the infection site by the organic metal framework A, the organic metal framework B and the self-assembled composite nanosystem in Test Example 2;

[0039] Figure 7 It is a diagram of the regulation effect of the organic metal framework A, the organic metal framework B and the self-assembled composite nanosystem on the pro-inflammatory factors secreted by RAW264.7 in Test Example 3. DETAILED DESCRIPTION

[0040] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0041] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0043] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0044] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0045] The components of the biofilm-targeting self-assembled composite nanosystem of the present application are composed of organic ligands, metal ions and gel matrix, which has dual targeting properties of targeting bacterial infection sites and biofilms, and solves the problem that it is difficult to eradicate biofilms due to the inability to target biofilm infection sites and penetrate biofilms. Moreover, the self-assembled composite nanosystem can be released for long-term residence at the infection site, reducing the complexity of frequent administration, while synergizing photodynamic and immunity to enhance its anti-biofilm activity, and promoting the recovery of infected tissues, providing a new treatment strategy for biofilm infection treatment.

[0046] The hemin, meso-tetra(4-carboxyphenyl)porphyrin and 5,10,15,20-tetra(4-aminophenyl)porphyrin in the embodiments of the present application are purchased from Shanghai Teng Sai Biological Technology Co., Ltd.; arginine and lysine are purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; benzoic acid, ferric chloride (providing ferric ions) and calcium chloride (providing calcium ions) are purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; guar gum, gelatin, carob bean gum and zirconyl chloride octahydrate (providing zirconium ions) are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0047] Composition and yield of metal-organic framework A

[0048] 1.1 Composition and preparation of metal-organic framework A

[0049] The composition of metal-organic framework A is shown in Tables 1-1 to 1-3.

[0050] Table 1-1 Composition of metal-organic framework A, group 1

[0051]

[0052] Table 1-2 Composition of metal-organic framework A, group 2

[0053]

[0054] Table 1-3 Composition of metal-organic framework A, group 3

[0055]

[0056]

[0057] Preparation steps:

[0058] The organic ligand A was dissolved in 50 mL of N’N-dimethylformamide, and the metal ion was added and mixed under ultrasonic for 1 min, the temperature was 60°C and 90°C respectively, the stirring speed was 750 r / min, and the time was 8 h, to obtain metal-organic framework A;

[0059] 1.2 Yield of metal-organic framework A

[0060] Table 2 Yield of metal-organic framework A

[0061]

[0062] As shown in Table 2, the composition of the three metal-organic frameworks has a higher yield under the condition of 90°C than under the condition of 60°C, and the yield of group 2 is more advantageous, so the metal-organic framework A is synthesized under the condition of 90°C with group 2.

[0063] Example 2 Composition, photodynamic and immunomodulatory efficiency of metal-organic framework B

[0064] 2.1 Composition 1 and preparation of metal-organic framework B

[0065] The composition 1 of metal-organic framework B is shown in Tables 3-1 to 3-2.

[0066] Table 3-1 Composition 1 of metal-organic framework B, group 1

[0067]

[0068] Table 3-2 Composition 1 of metal-organic framework B Group 2

[0069]

[0070] Preparation steps:

[0071] Dissolve organic ligand B in water, add prepared metal-organic framework A (Group 2 in Example 1 synthesized at 90°C), temperature 60°C, stirring speed 500 r / min, time 24 h, prepare metal-organic framework B by ligand replacement;

[0072] 2.2 Photodynamic and immunomodulatory efficiency of metal-organic framework B

[0073] Table 4 Photodynamic efficiency of metal-organic framework B

[0074]

[0075] As shown in Table 4, there is no significant difference in immunomodulation between Group 1 and Group 2, but the photodynamic efficiency of Group 1 is much higher than that of Group 2, so further optimization is selected in Group 1.

[0076] 2.3 Composition 2 and preparation of metal-organic framework B

[0077] The composition 2 of metal-organic framework B is shown in Tables 5-1 to 5-4.

[0078] Table 5-1 Composition 2 of metal-organic framework B Group 1

[0079]

[0080] Table 5-2 Composition 2 of metal-organic framework B Group 2

[0081]

[0082] Table 5-3 Composition 2 of metal-organic framework B Group 3

[0083]

[0084] Table 5-4 Composition 2 of metal-organic framework B Group 4

[0085]

[0086] Preparation steps:

[0087] Dissolve organic ligand B in water, add prepared metal-organic framework A (Group 2 in Example 1 synthesized at 90°C), temperature 60°C, stirring speed 500 r / min, time 24 h, prepare metal-organic framework B by ligand replacement;

[0088] 2.4 Photodynamic and immunomodulatory efficiency of metal-organic framework B

[0089] Table 6 Photodynamic and immunomodulatory efficiency of metal-organic framework B

[0090]

[0091] As shown in Table 6, the photodynamic efficiency of Groups 1 to 4 gradually decreased, while the immunomodulatory efficiency of Group 2 was the best. In terms of the comprehensive photodynamic and immunomodulatory efficiency, Group 2, i.e., the metal-organic framework B of the self-assembled composite nanosystem of Example 3, was preferred.

[0092] Composition, photodynamic and immunomodulatory efficiency of the self-assembled composite nanosystem of Example 3

[0093] 3.1 Composition 1 of the self-assembled composite nanosystem and preparation

[0094] The composition 1 of the self-assembled composite nanosystem is shown in Tables 7-1 to 7-6.

[0095] Table 7-1 Composition 1 of the self-assembled composite nanosystem, Group 1

[0096]

[0097] Table 7-2 Composition 1 of the self-assembled composite nanosystem, Group 2

[0098]

[0099] Table 7-3 Composition 1 of the self-assembled composite nanosystem, Group 3

[0100]

[0101] Table 7-4 Composition 1 of the self-assembled composite nanosystem, Group 4

[0102]

[0103]

[0104] Table 7-5 Composition 1 of the self-assembled composite nanosystem, Group 5

[0105]

[0106] Table 7-6 Composition 1 of the self-assembled composite nanosystem, Group 6

[0107]

[0108] Preparation method:

[0109] The gel matrix was dissolved in water at 40°C to obtain a gel matrix solution (same below) and the metal salt was dissolved in water to obtain a corresponding metal ion solution (same below). The metal-organic framework B was added to the gel matrix solution and the metal ion solution in sequence at a temperature of 40°C, a stirring speed of 500 r / min, and for a time of 24 h to obtain the self-assembled composite nanosystem.

[0110] 3.2 Photodynamic and immunomodulatory efficiency of the self-assembled composite nanosystem

[0111] Table 8 Photodynamic and immunomodulatory efficiency of the composite nanosystem

[0112]

[0113] As shown in Table 8, Groups 1 to 6 have no significant difference in photodynamic efficiency, but Groups 1 and 2 have stronger immunomodulatory effects, and Group 2 is superior to Group 1, so further optimization is selected in Group 2.

[0114] 3.3 Composition 2 and preparation of the self-assembled composite nanosystem

[0115] Composition 2 of the self-assembled composite nanosystem is shown in Tables 9-1 to 9-4

[0116] Table 9-1 Composition 2 of the self-assembled composite nanosystem, Group 1

[0117]

[0118] Table 9-2 Composition 2 of the self-assembled composite nanosystem, Group 2

[0119]

[0120] Table 9-3 Composition 2 of the self-assembled composite nanosystem, Group 3

[0121]

[0122] Table 9-4 Composition 2 of the self-assembled composite nanosystem, Group 4

[0123]

[0124] Preparation method:

[0125] The metal-organic framework B was added to the gel matrix solution and the metal ion solution at a temperature of 40°C, a stirring speed of 500 r / min, and for a time of 24 h to obtain the self-assembled composite nanosystem.

[0126] 3.4 Photodynamic and immunomodulatory efficiency of the self-assembled composite nanosystem

[0127] Table 10 Photodynamic and immunomodulatory efficiency of the self-assembled composite nanosystem

[0128]

[0129]

[0130] The photodynamic efficiency of Groups 1 to 4 gradually increases, while the immunomodulatory efficiency of Group 2 is optimal, and the comprehensive photodynamic and immunomodulatory efficiency is optimal.

[0131] Preparation of self-assembled composite nanosystem, transmission electron microscope observation and biofilm targeting of test example 1

[0132] 1.1 Preparation of self-assembled composite nanosystem

[0133] Table 11 Composition of self-assembled composite nanosystem

[0134]

[0135] Preparation method:

[0136] (1) Dissolve meso-tetra(4-carboxyphenyl)porphine and benzoic acid in 50 mL of N,N-dimethylformamide according to the mass percentage, add metal ions and mix by ultrasonic for 1 min, the temperature is 90°C, the stirring speed is 500 r / min, and the time is 8 h, to prepare metal organic framework A;

[0137] (2) Dissolve arginine in water and add to the metal organic framework A prepared in step (1), the temperature is 60°C, the stirring speed is 500 r / min, and the time is 24 h, to prepare metal organic framework B;

[0138] (3) Add the dissolved gelatin solution and calcium chloride solution to the metal organic framework B prepared in step (2) in sequence, the temperature is 40°C, the stirring speed is 500 r / min, and the time is 24 h, to prepare the self-assembled composite nanosystem.

[0139] 1.2 Transmission electron microscope observation of self-assembled composite nanosystem

[0140] (1) Drug: 3.3 self-assembled composite nanosystem prepared in Group 2 of Group 2

[0141] (2) Instrument: Transmission electron microscope

[0142] (3) Test steps

[0143] Transmission electron microscope observation.

[0144] The composite nanosystem is configured into a solution of 10 μg / mL, 2 μL of which is placed on a copper mesh, and then dried at room temperature after being negatively stained with sodium phosphotungstate (2 wt%), and observed under a transmission electron microscope.

[0145] 1.3 Targeting of biofilm by self-assembled composite nanosystem, metal-organic framework A and metal-organic framework B

[0146] (1) Drug: self-assembled composite nanosystem (abbreviated as nanosystem group) prepared in group 2 of composition 2 and metal-organic framework A obtained in step group 1 of 1.1 and metal-organic framework B obtained in group 2 of composition 2 of 2.4.

[0147] (2) Test reagent: DAPI, physiological saline, trypticase soy broth.

[0148] (3) Strain: Staphylococcus aureus (B1-1).

[0149] (3) Instrument: confocal microscope.

[0150] (4) Test steps:

[0151] Targeting of biofilm.

[0152] Staphylococcus aureus biofilm was cultured in trypticase soy broth (TSB), and an appropriate amount of recovered B1-1 bacterial liquid was used to adjust the bacterial concentration to 10 8 CFU / mL, 1 mL of the bacterial liquid was added to a confocal dish, and the dish was cultured in a 37°C incubator for 24 h to form a biofilm. The culture medium was aspirated and washed with physiological saline three times to obtain a mature biofilm. Then, an equal volume and concentration of nanosystem, nanosystem A and nanosystem B were added to the biofilm for different incubation times (0 h, 1 h, 2 h). After incubation, the culture medium was discarded and washed with physiological saline three times. Then, 200 μL of DAPI was added for staining at 37°C for 25 min, and finally washed with physiological saline three times to remove free DAPI. Then, the stained biofilm was imaged using a STORM super-resolution laser confocal scanning microscope, and Z-stacks were compiled into 3D images.

[0153] 1.4 Results

[0154] The test results of the self-assembled composite nanosystem are shown in Figure 1 The transmission electron microscopy images of the self-assembled composite nanosystem are shown in Figure 2

[0155] The targeting of biofilm by the self-assembled composite nanosystem, metal-organic framework A and metal-organic framework B is shown in Figure 3 Compared with metal-organic framework A and metal-organic framework B, more self-assembled composite nanosystems adhered to the surface of the biofilm when incubated for 1 h. When incubated for 2 h, more self-assembled composite nanosystems entered the interior of the biofilm compared with metal-organic framework A and metal-organic framework B. ​

[0156] Infection site targeting of test example 2 organic metal framework A, organic metal framework B and self-assembled composite nanosystem

[0157] 2.1 Test method

[0158] According to the test needs, first subcutaneously inject 10 8 CFU / mL of S. aureus suspension 100 μL to form a mouse subcutaneous biofilm infection film, and verify success or failure by wound imprint. Then intravenously inject organic metal framework A, organic metal framework B and self-assembled composite nanosystem into the biofilm infection mouse. After different injection times, the mouse is placed into an imaging dark box platform after intraperitoneal injection of chloral hydrate, the software controls the lifting of the platform to a suitable field of view, and a photograph is taken. Each treatment group is set up in triplicate.

[0159] 2.2 Results

[0160] The wound imprint chart of the biofilm infection model is shown in Figure 4 .

[0161] The targeting of organic metal framework A, organic metal framework B and self-assembled composite nanosystem to the infection site is shown in Figure 5 and Figure 6 (MOFA is organic metal framework A, MOFB is organic metal framework B, and Nanosystem is self-assembled composite nanosystem). After intravenous injection of organic metal framework A, organic metal framework B and self-assembled composite nanosystem, the biofilm infection site of the mouse appears fluorescence of organic metal framework A, organic metal framework B and self-assembled composite nanosystem, and the infection site reaches the highest fluorescence intensity after 1 day of injection, but the fluorescence intensity of the composite nanosystem is 8.39 times and 3.36 times that of organic metal framework A and organic metal framework B. Thereafter, with the passage of time, organic metal framework A, organic metal framework B and self-assembled composite nanosystem gradually decrease at the infection site, and organic metal framework A and organic metal framework B stay at the infection site for 7 days and 11 days, respectively, while self-assembled composite nanosystem stays at the infection site for 15 days.

[0162] Test example 3 in vitro immunomodulatory test of organic metal framework A, organic metal framework B and self-assembled composite nanosystem

[0163] During the infection process, the host needs to mobilize immune cells to resist bacterial infection by producing pro-inflammatory factors, and macrophages are one of the immune cells and important cells for the host to clear away foreign pathogenic microorganisms and regulate immune response. Therefore, mouse monocyte macrophage RAW264.7 was selected as a model cell to establish a biofilm infection model and detect pro-inflammatory factors.

[0164] 3.1 Test method

[0165] RAW264.7 cells were first infected with Staphylococcus aureus for 12 hours, and then co-incubated with 8 μg / mL of organic metal framework A, organic metal framework B and self-assembled composite nanosystems for 2 hours. They were then irradiated with a 660 nm laser for 30 minutes or not, and then incubated for another 6 hours. The cells were broken and proteins were extracted. The concentrations of pro-inflammatory factors were detected using TNF-α and iNOS detection kits. Three replicates were set for each treatment group.

[0166] 3.2 Results

[0167] like Figure 7 As shown (Control is the control group infected with biofilm but not treated with drugs, MOFA+D is the group with metal organic framework A but not irradiated with light, MOFA+L is the group with metal organic framework A and irradiated with light, MOFB+D is the group with metal organic framework B but not irradiated with light, MOFB+L is the group with metal organic framework B and irradiated with light, Nanosystem+D is the group with self-assembled composite nanosystem but not irradiated with light, Nanosystem+L is the group with self-assembled composite nanosystem and irradiated with light), compared with the control group and MOFB Compared with MOFA+D, the Nanosystem+L group significantly increased the expression of pro-inflammatory factors iNOS and TNF-α (p < 0.001). At the same time, compared with the MOFA+D group, the expression of TNF-α (p < 0.01) and iNOS (p < 0.001) was significantly increased. Compared with the MOFA+L, MOFB+L, and Nanosystem+D groups, the expression of TNF-α was significantly increased (p < 0.05) and the expression of iNOS was significantly increased (p < 0.001). Therefore, the self-assembled composite nanosystem has a significantly better regulatory effect on the pro-inflammatory factors iNOS and TNF-α than the organic metal framework A and organic metal framework B.

[0168] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A biofilm-targeted self-assembling composite nanosystem, characterized in that, The raw material is composed of an organic ligand, a metal ion and a gel matrix; The organic ligand is composed of an organic ligand A and an organic ligand B; the organic ligand A is a photosensitizer type ligand; and the organic ligand B is an amino acid type ligand; The organic ligand A is meso-tetra(4-carboxyphenyl) porphyrin and benzoic acid; The organic ligand B is arginine; The metal ion is calcium ion and zirconium ion; The gel matrix is gelatin; In the biofilm-targeting self-assembled composite nano system, the mass percentage of meso-tetra(4-carboxyphenyl) porphyrin is 3%, the mass percentage of benzoic acid is 29%, the mass percentage of arginine is 24%, the mass percentage of zirconium ion is 4%, the mass percentage of calcium ion is 8%, and the mass percentage of gelatin is 32%.

2. A method of preparing the biofilm-targeted self-assembling composite nanosystem according to claim 1, characterized by, The method comprises the following steps: The organic ligand A is dissolved in an organic solvent, zirconium ions are added and mixed by ultrasonic, and stirring is performed to prepare a metal organic framework A; then the organic ligand B is dissolved in water, the metal organic framework A is added, and a metal organic framework B is prepared by ligand replacement; finally, the gel matrix and calcium ions are dissolved and added to the metal organic framework B, and stirring is performed to prepare the biofilm-targeting self-assembled composite nano system.

3. The method for preparing the biofilm-targeted self-assembled composite nanosystem according to claim 2, characterized in that: The method comprises the following steps: (1) The organic ligand A is dissolved in N,N-dimethylformamide, zirconium ions are added and mixed by ultrasonic for 1 min, the temperature is 60-90℃, the stirring speed is 500 r / min, and the time is 8-24 h to obtain the metal organic framework A; (2) The organic ligand B is dissolved in water, and the metal organic framework A is added, the temperature is 60℃, the stirring speed is 500 r / min, and the time is 24 h to prepare the metal organic framework B by ligand replacement; (3) The gel matrix and calcium ions are dissolved and added to the metal organic framework B, the temperature is 40℃, the stirring speed is 500 r / min, and the time is 24 h to prepare the biofilm-targeting self-assembled composite nano system.

4. The biofilm-targeting self-assembled composite nano system of claim 1 is used for preparing an anti-infection drug.

Images