Manganese dioxide albumin nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a manganese dioxide albumin nano-composite and a preparation method and application thereof, the manganese dioxide albumin nano-composite is successfully synthesized in a water phase by utilizing a biomineralization strategy and using albumin as a biological template, and a CXCR4 antagonist can be further loaded to obtain a CXCR4 antagonistic nano-composite. The manganese dioxide albumin nanocomposite has good biocompatibility and biodegradability, provides a new thought for diagnosis and treatment of hepatic fibrosis with a safe and efficient drug delivery strategy, and has great potential in the aspect of clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a manganese dioxide-albumin nanocomposite targeting hepatic stellate cells, and a preparation method and application thereof. Background Art

[0002] Liver fibrosis is a dynamic process in which excessive deposition of extracellular matrix (ECM) such as collagen and fibrin forms fibrous scars after liver damage. Among them, hepatic stellate cells (HSCs) play a vital role. When the liver is stimulated by various forms of damage, HSCs will be activated from a quiescent state to an activated state, leading to collagen fiber deposition and scar formation, which is the core step in the occurrence of fibrosis. At present, there is no approved treatment for direct and effective reversal of advanced fibrosis, and the development of drugs for liver fibrosis is still urgent. Therefore, it is necessary to develop new and effective strategies to reverse HSCs to a quiescent state and reduce collagen accumulation to slow down the process of fibrosis.

[0003] Studies have shown that many cytokines, growth factors and inflammatory chemokines are involved in the activation of HSCs in patients with cirrhosis and carbon tetrachloride (CCl 4 In a mouse liver fibrosis model induced by CXCR4, it was found that CXCR4 is a transmembrane protein overexpressed on the surface of activated HSCs. It binds to the matrix-derived chemokine CXCL12, activates the CXCR4 / CXCL12 pathway, promotes the activation and proliferation of HSCs, and leads to the occurrence of fibrosis. Therefore, inhibiting the CXCR4 / CXCL12 pathway is a feasible solution for reversing activated HSCs to a quiescent state.

[0004] Cellular senescence is one of the key stages in the life cycle of cells and is involved in the disease process of many diseases. Cellular senescence is an anti-proliferative process, which refers to an irreversible cell cycle arrest state triggered by various cellular and environmental stresses (such as telomere shortening, DNA damage, mitochondrial dysfunction, etc.). cGMP-AMP (cGAMP) synthase (cGAS) is a cytoplasmic DNA sensor that is closely related to DNA damage. The activation of the cGAS-STING signaling pathway is considered to be one of the driving factors of cell senescence. Mn 2+As an effective cGAS-STING agonist, HSCs that can be aged and activated during the process of liver fibrosis show typical senescence-associated secretory phenotype (SASP), including remodeling of ECM, such as upregulating matrix metalloproteinases (MMPs) to alleviate collagen accumulation in fibrosis. SASP can also recruit natural killer cells (NK cells), macrophages, etc., mediate the timely clearance of senescent cells by immune cells to promote the regression of fibrosis and restore liver homeostasis. Using the natural anti-aging mechanism of the biological body, inducing the senescence of activated HSCs and timely eliminating senescent cells, and degrading collagen deposition is an effective strategy to safely and efficiently alleviate liver fibrosis.

[0005] Biomineralization refers to the process by which organisms construct life functional materials by regulating the nucleation, orientation, growth and assembly of inorganic minerals and coordinating organic-inorganic composite assembly. It has application potential in biomedical fields such as vaccine improvement, tumor treatment, blood transportation and arthritis treatment. As an endogenous substance, albumin has good biocompatibility, high drug loading, long half-life, and passive targeting to organs such as liver and kidney. It is a drug carrier with great application prospects. Therefore, this application designs and utilizes the biomineralization strategy, using albumin as a biological template to prepare manganese dioxide albumin nanocomplexes, giving manganese dioxide nanoparticles biocompatibility, and combining albumin with high negative charge to adsorb drugs to achieve precise targeted delivery in vivo. Summary of the invention

[0006] One of the objects of the present invention is to provide a manganese dioxide albumin nanocomposite prepared from albumin and a metal salt; The albumin is one or a mixture of bovine serum albumin (BSA), human serum albumin (HSA) or chicken ovalbumin (OVA); The metal salt is dimethyl manganese salt, which is selected from one or a mixture of manganese chloride, manganese sulfate, manganese fluoride or manganese bromide.

[0007] Furthermore, the manganese dioxide-albumin nanocomposite is adsorbed with active drugs.

[0008] Furthermore, the active drug is a CXCR4 antagonist selected from one or a mixture of Plerixafor (AMD3100), Mavorixafor (AMD070), USL-311 or PTX-9908.

[0009] In a specific embodiment of the present invention, the albumin is bovine serum albumin; the divalent manganese compound is manganese chloride; and the CXCR4 antagonist is Plerixafor (AMD3100). Figure 1 As shown, under alkaline conditions, the conformation of albumin changes, and MnO 2 It is encapsulated inside to form a nanocomplex, and then the CXCR4 antagonist AMD3100 is adsorbed through the electrostatic effect of the high-density negative charge of albumin.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned manganese dioxide albumin nanocomposite, the preparation process of which is: mixing an albumin solution and a metal salt solution, adjusting the pH of the mixed solution to alkaline, incubating and then performing ultrasound to obtain a manganese dioxide albumin nanocomposite.

[0011] Furthermore, the pH of the mixed solution is adjusted to alkaline, and then the active drug is added and incubated.

[0012] In a specific embodiment of the present invention, bovine serum albumin and MnCl 2 ·4H 2 The mass ratio of O is (0.25-4):1, preferably (0.5-2):1; MnCl 2 ·4H 2 The mass ratio of O to bovine serum albumin is (0.25-4):1, preferably (0.5-2):1, and more preferably 1:1; the pH of the mixed solution is adjusted to an alkaline pH of 9-12.

[0013] The third object of the present invention is to provide the use of the above-mentioned manganese dioxide albumin nanocomposite in the preparation of drugs for treating liver fibrosis.

[0014] The present invention designs a manganese dioxide albumin nanocomposite targeting hepatic stellate cells. The nanocomposite uses albumin as a biomineralization template, successfully synthesizes a manganese dioxide albumin nanocomposite in an aqueous phase, and simultaneously loads a CXCR4 antagonist to obtain a CXCR4 antagonist nanocomposite. The obtained nanocomposite has a suitable size, good water dispersibility and biocompatibility. The biomineralization strategy utilizes the abundant metal binding sites on the albumin molecule, which has a strong affinity with metal ions, thereby performing in situ crystal growth; then the high-density negative charge carried by albumin is used to electrostatically adsorb the CXCR4 antagonist small molecule drug. The drug is targeted to hepatic stellate cells that highly express the CXCR4 receptor, and then MnO 2 Drugs and manganese ions are released in response to acidic conditions in lysosomes. CXCR4 antagonists reverse activated HSCs to a quiescent state, while the cGAS-STING pathway activates senescent activated HSCs, degrades collagen accumulation, and slows the progression of liver fibrosis.

[0015] The manganese dioxide-albumin nanocomplex of the present invention has good biocompatibility and biodegradability, provides a new approach for the diagnosis and treatment of liver fibrosis with a safe and efficient drug delivery strategy, and has great potential in clinical transformation.

[0016] The manganese dioxide-albumin nanocomplex of the present invention is simple to prepare, easy to operate, and has high stability. It is enriched in activated hepatic stellate cells to achieve precise release of the drug, thereby further inhibiting the CXCR4 / CXCL12 pathway, activating the cGAS-STING pathway, and exerting an anti-fibrosis effect.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The manganese dioxide albumin nanocomposite prepared by the present invention has a simple preparation process, is easy to operate, has high stability, and is easy for clinical transformation; 2. The manganese dioxide albumin nanocomposite prepared by the present invention has high targeting to hepatic stellate cells, specifically delivers manganese and drugs, prolongs the accumulation time in the liver in vivo, and provides a research idea for targeted albumin nanoformulations; 3. The manganese dioxide protein nanocomplex prepared by the present invention can be enriched in hepatic stellate cells, reverse the phenotype of hepatic stellate cells, inhibit cell activation and proliferation, induce senescence of activated hepatic stellate cells, degrade collagen accumulation, and thus delay the process of liver fibrosis. This indicates that it is a good anti-fibrosis nanocomposite with potential medical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the preparation of manganese dioxide protein nanocomplex in the example.

[0019] Figure 2 The results of the BSA dosage screening in Example 1 are shown.

[0020] Figure 3 MnCl in Example 1 2 ·4H 2 O dosage screening results.

[0021] Figure 4 This is the particle size distribution result of the manganese dioxide albumin nanocomposite prepared in Example 3.

[0022] Figure 5 This is the particle size distribution result of the manganese dioxide albumin nanocomposite prepared in Example 4.

[0023] Figure 6 This is the particle size distribution result of the manganese dioxide albumin nanocomposite prepared in Example 5.

[0024] Figure 7This is the particle size distribution result of AMB NPs in Example 2.

[0025] Figure 8 This is the transmission electron microscopy morphology of AMB NPs in Example 2.

[0026] Fig. 9 This is the valence distribution diagram of the metal element manganese in AMB NPs in Example 2.

[0027] Fig.10 The particle size stability results of AMB NPs in Example 2 in aqueous solution and 10% FBS culture medium (4°C and 37°C).

[0028] Fig.11 This is the manganese ion acid response release curve of AMB NPs in Example 2.

[0029] Fig.12 These are the cytotoxicity results of MB NPs and AMB NPs on qHSC and aHSC in Example 2.

[0030] Fig.13 These are the cytotoxicity results of MB NPs and AMB NPs on HUVEC and LO2 in Example 2.

[0031] Fig.14 These are the results of the plate cloning assay of MB NPs and AMB NPs on HSC cells in Example 2. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example 1

[0035] 1. Screening of bovine serum albumin (BSA) dosage The manganese dioxide protein nanocomplex was prepared by the aqueous solvent method. Specifically, 19.8 mg MnCl 2 ·4H 2 O crystal powder and 78.8 mg BSA powder were dissolved in 2 mL purified water respectively, and then the BSA solution was placed in a 37 °C water bath and MnCl was slowly added dropwise.2 ·4H 2 O aqueous solution, by mass ratio BSA: MnCl 2 ·4H 2 O=1:4, 1:2, 1:1, 2:1, 4:1 were reacted respectively. After stirring for 5 min, the mixture was transferred to an ice bath and 0.1 M NaOH solution was added to adjust the pH to 10. The solution turned brown. Stirring and incubation continued for 4 hours to allow the nanocomplex to grow. The mixed solution was taken out and ultrasonically treated with an ultrasonic power of 20%, an ultrasonic time of 1 s, a rest time of 2 s, an ultrasonic temperature of 4°C, and an ultrasonic treatment of 12 min. After the ultrasonic treatment, the solution was centrifuged at high speed to remove free BSA and MnO. 2 , speed of 12000 rpm, time of 10 min, temperature of 4°C. After centrifugation, the supernatant was discarded, purified water was added for resuspending, and ultrasonic treatment was continued. The ultrasonic power was 20%, the ultrasonic time was 1 s, the rest time was 2 s, the ultrasonic temperature was 4°C, and the ultrasonic time was 4 min. After the ultrasonic treatment, the manganese dioxide albumin nanocomposite MnO 2 -BSANPs (MB NPs).

[0036] like Figure 2 As shown, when BSA:MnCl 2 ·4H 2 When the feed ratio of O is 1:2~2:1, the particle size of the obtained manganese dioxide protein nanocomplexes is about 150 nm, the PDI is less than 0.2, and the particle size is uniform.

[0037] 2. MnCl 2 ·4H 2 O dosage screening Manganese dioxide albumin nanocomposite was prepared by aqueous solvent method. Specifically, 78.8 mg MnCl 2 ·4H 2 O crystalline powder and 19.8 mg BSA powder were dissolved in 2 mL purified water, respectively, according to the mass ratio of MnCl 2 ·4H 2 The reaction was carried out with the feed ratio of O:BSA = 1:4, 1:2, 1:1, 2:1 and 4:1 respectively. The reaction operation was the same as “Screening of the amount of bovine serum albumin (BSA)”.

[0038] like Figure 3 As shown, when MnCl 2 ·4H 2 When the feed ratio of O:BSA was 1:2~2:1, the particle size of the prepared manganese dioxide albumin nanocomposite was about 150 nm, the PDI was about 0.2, and the particle size was appropriate and uniform.

[0039] When MnCl 2 ·4H 2 When the feed ratio of O:BSA was 1:1, the particle size of MB NPs was appropriate and uniform, and this feed ratio was used for subsequent characterization experiments. Example 2

[0040] Preparation of manganese dioxide albumin nanocomposite loaded with CXCR4 antagonist. Manganese dioxide albumin nanocomposite was prepared by aqueous solvent method. Specifically, 19.7 mg BSA powder and 19.8 mg MnCl 2 ·4H 2 O crystalline powder was dissolved in 2 mL of purified water, 4 mg of AMD3100 powder was weighed and dissolved in 2 mL of methanol, and then the BSA solution was placed in a 37°C water bath and MnCl was slowly added dropwise. 2 ·4H 2 O aqueous solution, stirred for 5 min, then transferred to an ice bath, 0.1 M NaOH solution was added to adjust the pH to 10, and the solution turned brown; AMD3100 solution was added and stirred for 4 hours to allow the nanocomplex to grow. The mixed solution was taken out and ultrasonicated with an ultrasonic power of 20%, an ultrasonic time of 1 s, a rest time of 2 s, an ultrasonic temperature of 4°C, and an ultrasonic time of 12 min. After the ultrasonication, the solution was centrifuged at high speed to remove free BSA and MnO 2 , speed of 12000 rpm, time of 10 min, temperature of 4°C. After centrifugation, the supernatant was discarded, purified water was added for resuspending, and ultrasonic treatment was continued. The ultrasonic power was 20%, the ultrasonic time was 1 s, the rest time was 2 s, the ultrasonic temperature was 4°C, and the ultrasonic time was 4 min. After the completion of the ultrasonic treatment, the manganese dioxide albumin nanocomposite AMD3100@BSA-MnO loaded with CXCR4 antagonist was prepared. 2 NPs (AMB NPs). Example 3

[0041] Weigh 19.7 mg human serum albumin (HSA) powder and 19.8 mg MnCl 2 ·4H 2 O crystalline powder was dissolved in 2 mL of purified water. 4 mg of Mavorixafor (AMD070) powder was weighed and dissolved in 2 mL of ethanol, and then the HSA solution was placed in a 37°C water bath and MnCl was slowly added dropwise. 2 ·4H 2O aqueous solution, stirred for 5 min, then transferred to an ice bath, 0.1 M NaOH solution was added to adjust the pH to 10, and the solution turned brown; AMD070 solution was added and stirred for 4 hours to allow the nanocomposite to grow. The mixed solution was taken out and ultrasonicated in an ice bath, and the unreacted substances were removed by high-speed centrifugation, and ultrasonicated again in an ice bath to obtain the final nanocomposite. Figure 4 As shown, the particle size of the nanocomposite is 167.0±1.99 nm. Example 4

[0042] Weigh 13.2 mg of egg ovalbumin (OVA) powder and 15.1 mg of manganese sulfate powder and dissolve them in 2 mL of purified water. Weigh 4 mg of AMD3100 powder and dissolve it in 2 mL of ethanol. Then place the OVA solution in a 37°C water bath and slowly add MnCl 2 ·4H 2 O aqueous solution, stirred for 5 min, then transferred to an ice bath, 0.1 M NaOH solution was added to adjust the pH to 10, and the solution turned brown; AMD3100 solution was added and stirred for 4 hours to allow the nanocomposite to grow. The mixed solution was taken out and ultrasonicated in an ice bath, and the unreacted substances were removed by high-speed centrifugation, and ultrasonicated in an ice bath again to obtain the final nanocomposite. Figure 5 As shown, the particle size of the nanocomposite is 151.1±1.33 nm. Example 5

[0043] Weigh 19.7 mg of bovine serum albumin (BSA) powder and 21.5 mg of manganese bromide powder and dissolve them in 2 mL of purified water. Weigh 4 mg of USL311 powder and dissolve it in 2 mL of dimethyl sulfoxide (DMSO). Then place the BSA solution in a 37°C water bath and slowly add MnCl 2 ·4H 2 O aqueous solution, stirred for 5 min, then transferred to an ice bath, 0.1 M NaOH solution was added to adjust the pH to 10, and the solution turned brown; USL311 solution was added and stirred and incubated for 4 hours to allow the nanocomposite to grow. The mixed solution was taken out and ultrasonicated in an ice bath, and the unreacted substances were removed by high-speed centrifugation, and ultrasonicated again in an ice bath to obtain the final nanocomposite. Figure 6 As shown, the particle size of the nanocomposite is 167.8±6.10 nm.

[0044] The manganese dioxide albumin nanocomposite prepared in the above Example 2 was tested below.

[0045] 1. Particle size potential determination of manganese dioxide albumin nanocomposite AMB NPs were dispersed in deionized water, and the particle size distribution and potential of AMB NPs were evaluated by Malvern particle size analyzer. Figure 7 As shown, the particle size of the prepared manganese dioxide albumin nanocomposite was 126.2±1.11 nm, the PDI was 0.164, the potential was -17.2±0.85 mV, and the particle size was uniform.

[0046] 2. Morphology determination of manganese dioxide-albumin nanocomposite The morphology of the particles was observed under a high-resolution transmission electron microscope. Figure 8 As shown in Figure 3, the morphology of AMB NPs is spherical.

[0047] 3. Elemental analysis of manganese dioxide-albumin nanocomposite X-ray photoelectron spectroscopy (XPS) was used to detect the valence distribution of metal ions in AMB NPs. Fig. 9 As shown in Figure 2, there are four main peaks in XPS, namely Mn2p (641.37 eV), O1s (530.87 eV), N1s (399.87 eV) and C1s (284.87 eV). Among them, the Mn2p spectrum shows that Mn(IV) is dominant, indicating that BSA successfully encapsulates MnO 2 .

[0048] 4. Stability study of manganese dioxide-albumin nanocomplex AMB NPs were dispersed in deionized water and DMEM medium containing 10% FBS at a manganese concentration of 5 mM, respectively, and placed at 4°C and 37°C, respectively. The solution was taken out at fixed time points and dispersed in deionized water. The particle size distribution of AMB NPs was evaluated by Malvern particle size analyzer. Fig.10 As shown, AMB NPs had good stability in aqueous solution and DMEM medium containing 10% FBS at 4°C and 37°C for 14 days, respectively.

[0049] 5. Investigation of the acid dissociation of manganese dioxide-albumin nanocomplexes MnO 2 Mn can be released under acidic conditions 2+ , Mn was detected by dialysis 2+ Cumulative release. Specifically: a dialysis bag with a molecular weight of 1000Da was selected, and the dialysis medium was a PBS solution of pH 5.0 and pH 7.0. 1 mL of AMB NPs was drawn into the dialysis bag. The dialysis system was 30 mL, and the mixture was incubated on a shaker at 37°C. 1 mL of dialysate was taken out of the dialysis bag at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 12 h, and fresh PBS was added. The manganese content of the dialysate was detected by ICP-MS. Fig.11 As shown in Figure 2, pH 5.0 showed a significant release compared with pH 7.4, with more than 90% released after 2 h. The acidic environment of lysosomes triggered the release of Mn 2+ Quick release.

[0050] 6. Biosafety of MnO2-Albumin Nanocomplexes The cytotoxicity of manganese dioxide-albumin nanocomplexes to qHSC-T6 (quiescent state), aHSC-T6 (activated state), LO2, and HUVEC was evaluated by MTT assay. 4 ) were seeded in a 96-well plate and incubated in an incubator for 24 h to allow them to adhere to the wall. Then the culture medium was discarded, and culture medium containing MB NPs and AMB NPs with Mn concentrations corresponding to 0, 1.25, 2.5, 5, 10, 20, 50, 100, 200, and 400 μM was added. After incubation for 24 h, the drug-containing culture medium was discarded, and 20 μL of MTT (5 mg / mL) culture medium was added to each well and incubated for 4 h. The culture medium was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The absorbance of the sample at 490 nm was measured by an enzyme reader, and the cell survival rate was calculated. Fig.12 As shown in the figure, qHSC-T6 and aHSC-T6 were incubated with MB NPs without obvious toxicity; aHSC-T6 incubated with AMB NPs at the same concentration showed lower viability compared with qHSC-T6; this indicates that AMB NPs have a stronger inhibitory effect on the proliferation of aHSC-T6 and have no effect on the viability of qHSC-T6 cells. Fig.13 As shown, the survival rates of HUVEC and LO2 of AMB NPs at a dose of 100 μM were higher than 90%, indicating that the preparation of the present invention has good biosafety.

[0051] 7. Plate cloning assay of MnO2-albumin nanocomplexes on HSC-T6 cells HSC-T6 cells in the logarithmic growth phase were digested and inoculated into 6-well plates (400-1000 cells / well). After the cells were stable, the drugs were administered. PBS, TGF-β (10 ng / ml), MB NPs (Mn 2+ 50 μM, TGF-β 10 ng / ml), AMD3100 (0.1 mM, TGF-β 10 ng / ml), AMB NPs (Mn 2+ The medium was replaced with 50 μM and TGF-β was 10 ng / ml) every 3 days and the culture was continued until the number of cells in a single clone was greater than 50. After the cloning was completed, the cells were washed with PBS, fixed, and stained with crystal violet. The cells were washed with PBS several times and dried, and photographed. Fig.14As shown in the results, AMB NPs can significantly inhibit the proliferation of HSC-T6, indicating that inhibition of the CXCR4 / CXCL12 pathway reverses the activated state to the quiescent state, and activation of the cGAS-STING axis drives senescence of activated state cells. Both inhibit the proliferation of HSCs and thus delay the progression of liver fibrosis.

Claims

1. A manganese dioxide albumin nanocomposite, characterized in that: Prepared from albumin and metal salts; The albumin is one or a mixture of bovine serum albumin, human serum albumin or chicken ovalbumin; The metal salt is dimethyl manganese salt, which is selected from one or a mixture of manganese chloride, manganese sulfate, manganese fluoride or manganese bromide.

2. The manganese dioxide albumin nanocomposite according to claim 1, characterized in that: The manganese dioxide albumin nanocomposite is loaded with active drugs.

3. The manganese dioxide albumin nanocomposite according to claim 2, characterized in that: The active drug is a CXCR4 antagonist, selected from one or a mixture of Plerixafor, Mavorixafor, USL-311 or PTX-9908.

4. A method for preparing the manganese dioxide-albumin nanocomposite according to claim 1, characterized in that: The albumin solution and the metal salt solution are mixed, the pH value of the mixed solution is adjusted to alkaline, and ultrasound is performed after incubation to prepare a manganese dioxide albumin nanocomposite.

5. The preparation method according to claim 4, characterized in that: After adjusting the pH of the mixture to alkaline, the active drug is added and incubated.

6. The preparation method according to claim 4 or 5, characterized in that: The step of adjusting the pH of the mixed solution to alkaline is to adjust the pH to 9-12.

7. Use of the manganese dioxide-albumin nanocomposite according to any one of claims 1 to 3 in the preparation of a drug for treating liver fibrosis.

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