Microminiature platinum cluster enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drugs for treating psoriasis, and particularly relates to a microminiature platinum cluster enzyme as well as a preparation method and application thereof. The microminiature platinum cluster enzyme comprises a platinum metal core and a cyclodextrin ligand, and the preparation method of the microminiature platinum cluster enzyme comprises the following steps: adding a Pt precursor solution into a sulfydryl-cyclodextrin solution, mixing, adding a reducing agent, and reacting to obtain the microminiature platinum cluster enzyme. The preparation method disclosed by the invention is simple and feasible, and the microminiature platinum cluster enzyme prepared by the method has the remarkable advantages of safety, effectiveness and economy in treatment and prevention of psoriasis.

Description

Technical Field

[0001] The invention belongs to the technical field of psoriasis therapeutic drugs, and specifically relates to an ultra-small platinum cluster enzyme and a preparation method and application thereof. Background Art

[0002] Psoriasis is a chronic inflammatory skin disease characterized by the appearance of red, thick plaques covered with silvery-white scales on the skin. The global prevalence is about 2%-3% and is on the rise. Psoriasis is often accompanied by other metabolic diseases such as cardiovascular disease and diabetes, which increase the health risks of the disease. Current treatment strategies are far from satisfactory and have the following main disadvantages: 1) The use of immunosuppressants (such as methotrexate) or monoclonal antibodies can effectively relieve psoriasis inflammation, but long-term use may lead to liver and kidney toxicity, increased risk of infection and increased risk of skin cancer; 2) Topical application of corticosteroids (such as betamethasone) and vitamin D analogs (such as calcipotriol) can significantly circumvent the in vivo toxicity of systemic drugs, but may cause skin irritation and atrophy; 3) The main clinical challenge in the treatment of psoriasis is the high recurrence rate of symptoms after drug discontinuation, which is as high as 90%; 4) Most existing treatment strategies require excipients or drug delivery systems, which increase the risk of side effects; 5) The use of biologics such as monoclonal antibodies to treat psoriasis is usually costly. Therefore, there is an urgent need to develop safe, effective, economical, and low-side effect drugs to treat psoriasis and effectively prevent its recurrence.

[0003] In the process of developing therapeutic drugs for psoriasis, we have dissected the pathological mechanisms of psoriasis, including persistent inflammation and explosive release of inflammatory factors, as well as uncontrolled proliferation of keratinocytes. This condition is often driven by excessive accumulation of reactive oxygen species (ROS) due to oxidation / antioxidation imbalance. A potential therapeutic strategy is to reduce the level of ROS in the affected area. However, direct consumption of antioxidants such as bilirubin and monomethyl fumarate cannot continuously eliminate ROS. Instead, the body relies on catalytic enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) to regulate ROS and maintain homeostasis, but psoriasis patients have aggravated symptoms due to the deficiency of these antioxidant enzymes. To address this problem, developing antioxidant enzymes to restore redox balance has become a promising approach for the treatment of psoriasis.

[0004] Recent advances in ultrasmall metal nanoclusters (NCs) in the biomedical field have provided opportunities to address this issue. Metal NCs with a typical core size of 1-2 nm have been widely used in sensing, imaging, biocatalysis, disease diagnosis and treatment, owing to their atomically precise size / structure, engineerable surface chemistry, interesting molecule-like properties (e.g., luminescence and chirality), and atomically tunable physicochemical properties such as multienzyme catalytic activity, as well as their excellent biocompatibility, including renal clearance (i.e., hydrodynamic diameter or HD ≤ 5.5 nm).

[0005] In light of the advantageous properties of metal NCs in biomedicine and encouraged by the clinical success of platinum (Pt) drugs, we envisioned the development of an ultrasmall platinum cluster enzyme (Pt CZ) for safe, economical, and side-effect-free psoriasis management and relapse prevention: 1) The surface of Pt CZ is engineered to have moderate epidermal penetration to ensure effectiveness at sites of epidermal damage and avoid systemic accumulation to reduce toxicity; 2) The ultrasmall size and biocompatibility properties ensure adequate biosafety and good renal clearance; 3) The ultrasmall size and high atomic utilization make it cost-effective with small doses; 4) The unique physicochemical properties of Pt CZ avoid the need for any excipients and additional drug delivery systems, ensuring a simple and reliable treatment approach.

[0006] However, the task of designing Pt CZ is extremely challenging, especially for the application of Pt CZ in the treatment of psoriasis, which emphasizes the complexity and importance of continued research efforts in this field. Current synthetic methods for synthesizing Pt nanomaterials are all large in size, which is caused by the excessive reduction ability of the reduction method used. 4+ Directly under the action of the strong reducing agent NaBH4, the initial formation of Pt will accelerate the decomposition of NaBH4 due to its super catalytic ability, resulting in too fast reduction kinetics, and finally generating Pt nanoparticles with a size between 5 and tens of nanometers. Therefore, it is a big challenge to synthesize ultra-small Pt nanomaterials, and it is even more difficult to apply these large-sized Pt nanoparticles to biomedical applications. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an ultra-small platinum cluster enzyme (Pt CZ), and also provides a preparation method thereof and an application thereof in preparing a drug for treating psoriasis and preventing the recurrence of psoriasis.

[0008] The technical solution of the present invention is as follows: An ultra-small platinum cluster enzyme comprising a platinum metal core and a cyclodextrin ligand.

[0009] The method for preparing the ultra-small platinum cluster enzyme of the present invention comprises the following steps: adding a Pt precursor solution to a thiol-cyclodextrin solution and mixing to obtain a complex solution, adding a reducing agent and reacting to obtain the ultra-small platinum cluster enzyme.

[0010] Preferably, the mercapto-cyclodextrin is (n) mercapto-β-cyclodextrin, where n=1-7.

[0011] Preferably, the Pt precursor solution is a Pt ion solution, and the Pt ion solution includes H2PtCl6 solution, K2PtCl6 solution and PtCl4 solution.

[0012] Preferably, (n) mercapto-β-cyclodextrin and Pt 4+ The molar ratio is ≥0.5:1.

[0013] Preferably, the reducing agent includes CO, H2, hypophosphite, hydrazine hydrate and tert-butyl borane.

[0014] Preferably, the solvent of the mercapto-cyclodextrin solution is an alkaline solution, preferably a NaOH solution, a KOH solution or an ammonia solution; and the solvent of the Pt precursor solution is water, preferably pure water.

[0015] It should be noted that when adding the Pt precursor solution, it needs to be added slowly to allow it to fully react to form a complex; When the complex solution changes color, stop adding the reducing agent; During the blocking reaction, stirring must be continued until a brown Pt CZ solution is obtained; Purification method of Pt CZ: The Pt CZ solution obtained by the reaction is ultrafiltered using a 3000 molecular ultrafiltration membrane, then dissolved in water, and freeze-dried to obtain a purified Pt CZ solid powder.

[0016] A preferred solution of the present invention is: prepare H2PtCl6 solution (20mM, 0.5mL) and Pt CZ synthetic stock solution (10mM, 1.5mL) of thiol-β-cyclodextrin (SH-β-CDs) solution (10mM, 1.5mL) with water and 0.1M NaOH respectively. Add 1.5mL of SH-β-CDs solution to a 20mL glass bottle containing 8mL of water under stirring, then slowly add 0.5mL of H2PtCl6 solution to the glass bottle, and stir continuously to make it fully react and mix. Pass CO gas for 5-10 minutes, after the solution changes color, seal the glass bottle and stir for 18-24 hours, the reaction is completed to obtain a brown Pt CZ solution, the Pt CZ solution obtained by the reaction is ultrafiltered using a 3000 molecular ultrafiltration membrane, then dissolved in water, and freeze-dried to obtain a purified Pt CZ solid powder.

[0017] The present invention also provides the use of the ultra-small platinum cluster enzyme in preparing a drug for treating psoriasis and preventing the recurrence of psoriasis.

[0018] When using, it needs to be prepared and used immediately. Disperse the Pt CZ solid powder in PBS buffer solution, apply or spray it on the psoriasis skin, and obvious therapeutic effect can be seen after 7 days of continuous use. The Pt CZ solid powder should be stored at 0-4℃.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing an ultra-small platinum cluster enzyme Pt ​​CZ, which is simple and feasible. The PtCZ prepared by the method has the significant advantages of safety, effectiveness and economy in the treatment and prevention of psoriasis.

[0020] Pt CZ exhibited remarkable epidermal penetration and enzymatic activities, including CAT-, SOD-, and GPx-like activities, which, along with its ultrasmall size, make Pt CZ a promising agent for the management of psoriasis. In vivo studies on imiquimod (IMQ)-induced psoriasis mice revealed its excellent preventive properties, through reduction of CD3+ and Ki67+ cell numbers and modulation of NF-κB signaling pathways to achieve immunomodulation. Notably, Pt CZ also showed complete recovery after topical application, with a therapeutic effect superior to that of calcipotriol / betamethasone dichloroacetone ointment (BC). The superior effect of Pt CZ can be attributed to the effective elimination of ROS and the inhibition of the expression of inflammatory factors such as IL-17, IL-12, IL-23, and IL-1β in psoriatic skin tissues. In addition, Pt CZ can also modulate gene expression by downregulating ROS and inflammatory genes and upregulating genes that inhibit proliferation and promote apoptosis of epidermal cells, providing a multifaceted approach to the management of psoriasis. Finally, Pt CZ showed low cytotoxicity and excellent biosafety, such as renal clearance, no organ damage, and unaltered blood biochemistry and liver function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the synthesis of Pt CZ; Figure 2 UV-vis absorption spectrum and digital photo of Pt CZ (taken under indoor light); Figure 3 Transmission electron microscope image and size distribution histogram of Pt CZ; Figure 4 The aberration-corrected HAADF-STEM image of the Pt CZ, the image of the medium-sized Pt CZ and its corresponding model; Figure 5 This is a schematic diagram of a psoriasis prevention experiment; Figure 6This is the red scaly plaque skin condition of mice; Figure 7 is the area and severity index of psoriasis in mice; Figure 8 This is a schematic diagram of a psoriasis treatment experiment; Fig. 9 This is a photo of the back skin of a mouse; Fig.10 For PASI score analysis; Fig.11 The Pt content in the skin of mice at different time periods after treatment. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to the examples. All raw materials used in the examples are commercially available unless otherwise specified.

[0023] Example 1 Synthesis and Characterization of Pt CZ The preparation method of the ultra-small platinum cluster enzyme Pt ​​CZ comprises the following steps: preparing Pt CZ synthetic stock solutions of H2PtCl6 solution (20mM, 0.5mL) and SH-β-CDs solution (10mM, 1.5mL) with water and 0.1M NaOH respectively. Add 1.5mL SH-β-CDs solution to a 20mL glass bottle containing 8mL water under stirring, then slowly add 0.5mL H2PtCl6 solution to the glass bottle, and stir continuously to make it fully react and mix. After CO gas is introduced for 5 minutes, the solution changes color, and then the glass bottle is sealed and stirred for 24 hours. The reaction is completed to obtain a brown Pt CZ solution, and the Pt CZ solution obtained by the reaction is ultrafiltered using a 3000 molecular ultrafiltration membrane, then dissolved in water, and freeze-dried to obtain a purified Pt CZ solid powder.

[0024] For the synthesis of platinum nanoclusters (Pt NCs), it was found that creating a mild reducing environment is crucial. In this study, ultrasmall SH-β-CD protected Pt CZs were synthesized via a “carbon monoxide reduction” method. Notably, the abnormal catalytic activity of the newly formed Pt accelerates the decomposition of sodium borohydride (NaBH4), leading to excessively fast reduction kinetics. Therefore, the typical NaBH4 reduction method produces not ultrasmall Pt NCs, but polydisperse Pt particles with sizes ranging from 2 to 6 nm. In order to achieve the synthesis of ultrasmall Pt CZs, as Figure 1 As shown, CO with mild reducing ability was adopted to replace NaBH4 to minimize the catalytic effect of Pt species and thus slow down the reduction kinetics of Pt CZ growth.

[0025] In brief, the selection of SH-β-CD as the protective ligand for Pt CZ was based on several considerations: First, the thiol groups in SH-β-CD have strong binding affinity and relatively large steric hindrance, which promote the formation of ultrasmall Pt CZ and ensure high atom utilization and low dosage. Second, the unique amphiphilic cavity structure of SH-β-CD, with a hydrophilic surface and a hydrophobic cavity, combined with the ultrasmall size of Pt CZ, facilitates moderate epidermal penetration. In addition, the binding of SH-β-CD to Pt enhances the production of CAT, SOD, and GPx activities. Moreover, the biocompatibility of SH-β-CD and Pt contributes to the good biosafety of Pt CZ. Subsequently, the synthesized Pt CZ exhibits a dark brown color in solution, and its UV-visible absorption spectrum has no characteristic absorption, which excludes the presence of large Pt nanoparticles with typical surface plasmon resonance absorption (see Figure 2 ).

[0026] The size of Pt CZ was evaluated using transmission electron microscopy (TEM), corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM), and inductively coupled plasma mass spectrometry (ICP-MS). Figure 3 As shown, TEM analysis confirmed that all Pt CZ particles were smaller than 2.5 nm, with an average size of approximately 1.35 nm, highlighting the efficient synthesis of ultra-small Pt CZs. The formation of such tiny Pt CZs was further confirmed by AC-HAADF-STEM (see Figure 4 ), where the size of Pt NCs ranges from about 0.87 to 2.16 nm, appearing as bright atomic precision clusters. ICP-MS data showed that the weight ratio of Pt species in Pt CZ was about 20.8%, from which it was inferred that the molar ratio of Pt to SH-β-CD was about 1.7:1. This result is similar to the peak area ratios of various metal elements to sulfur observed in XPS analysis. Overall, the average size of Pt CZ (i.e., 1.35 nm) is similar to that of Pt 51 (SH-β-CD) 30 The size and structure of the model are consistent ( Figure 4 ). It is worth noting that it is not feasible to directly obtain the atomic precision size of Pt CZ by mass spectrometry at present due to the ionization challenges caused by its neutral charge and non-uniform size distribution, which requires further research in the future with the advancement of Pt NCs synthesis and mass spectrometry technology.

[0027] Example 2 Efficacy evaluation in "prevention mode" In this example, 6- to 8-week-old Balb / c mice were shaved on day 0 and divided into one control group and three treatment groups, each containing 5 mice. From day 1 to day 6, all groups received daily topical application of 62.5 mg of IMQ cream. Thirty minutes after cream application, an additional treatment was applied to the middle area of ​​the back of the mice (15 mm × 25 mm). The treatments were as follows: (i) no additional treatment, labeled "IMQ"; gauze soaked with 200 μl of PBS containing 6 μm Pt CZs: (ii) "IMQ+Pt CZ", (iii) "IMQ+Au CZ", and (iv) "IMQ+Cu CZ". Each treatment area was then covered with a Tegaderm membrane (3M) to secure the treatment. The severity of psoriasis lesions in mice was assessed daily using the modified Psoriasis Area and Severity Index (PASI), which assesses erythema, scaling, and thickness and ranges from 0 (no symptoms) to 4 (very severe). In addition, the body weight of mice was recorded daily to monitor any systemic effects that might be induced by the treatment. On day 7, mice were humanely euthanized. After euthanasia, the treated skin sections were thoroughly rinsed three times with PBS to eliminate any residual treatment substances. The sections were then cut into 3 mm × 10 mm strips for histological and molecular analyses. Whole heart blood samples were collected under anesthesia before euthanasia to evaluate systemic inflammation or biochemical changes induced by the treatment, and other important organs were also collected for comprehensive pathological examination to evaluate any non-target effects. The sample size of each experimental group was determined by statistical power calculations performed according to Dunnett's formula.

[0028] To evaluate the effectiveness of Pt CZ in controlling psoriasis, we first investigated its ability to inhibit disease progression, which poses a significant therapeutic challenge given the high relapse rate of psoriasis, which is as high as 90%. In this study, we performed a psoriasis prevention experiment by co-administering IMQ (to induce psoriasis) and Pt CZ (as a therapeutic agent) into the dorsal skin of Balb / c mice for six consecutive days, accompanied by daily pathological assessments ( Figure 5 ).like Figure 6 As shown, mice treated with IMQ developed severe erythematous scaly plaques, demonstrating the potent ability of IMQ to trigger psoriasis flare-ups. However, these plaques were significantly improved in the groups treated with IMQ combined with Pt CZ, Au CZ, or Cu CZ. Notably, the skin conditions in the IMQ+Pt CZ groups were very similar to those in the healthy control group, indicating that Pt CZ has a superior preventive effect in preventing psoriasis progression. To further explore the preventive properties of Pt CZ, we evaluated mice daily using the Psoriasis Area and Severity Index (PASI), focusing on scaling, induration, and erythema ( Figure 7). Obviously, Au CZ, Cu CZ, and Pt CZ all alleviated IMQ-induced symptoms, with Pt CZ showing the greatest effect, followed by Au CZ and Cu CZ, throughout the entire evaluation period. In addition, during the prevention process, the changes in body weight of mice in all groups were negligible.

[0029] Example 3 Efficacy evaluation in "treatment mode" In this example, 6- to 8-week-old Balb / c mice were shaved on day 0 and divided into one control group and three treatment groups, each containing 5 mice. Each group received topical IMQ cream (62.5 mg) daily from day 1 to day 6. From day 7 to day 10, mice were treated daily with 200 μL of the following substances applied to gauze covering a psoriatic skin area (15 mm × 25 mm): (i) PBS, (ii) BC ointment (a thin layer of 20 mg Daivobet® cream), (iii) Pt CZ, (iv) Au CZ, or (v) Cu CZ. The treated area was then covered with a Tegaderm membrane. PASI scores and body weights were recorded daily from day 7 to day 10. Mice were euthanized on day 11. The treated skin area was rinsed three times with PBS and cut into 3 mm × 10 mm sections for further analysis. Whole blood and other organs were also collected for additional examinations. The size of each treatment group was determined using Dunnett's formula.

[0030] Similarly, Pt CZ also showed excellent therapeutic effects in the treatment of psoriasis. To confirm this finding, we used IMQ to induce a psoriasis model in mice for six days; then, Pt CZ was treated for five days to evaluate its therapeutic effect, with Au CZ, Cu CZ, and commercially available BC ointment as controls ( Figure 8 The body weight and dorsal skin photographs of the mice were carefully recorded throughout the treatment period ( Fig. 9 ). Notably, the body weight of mice treated with Pt CZ, Au CZ, and Cu CZ increased slightly during the treatment period, compared with the body weight of mice treated with BC ointment and PBS, which did not fluctuate significantly. Visually, the Pt CZ and BC ointment treatment groups showed comparable improvements in psoriasis symptoms, each slightly superior to the Au CZ and Cu CZ groups ( Fig. 9 This observation was further confirmed by subsequent analysis of PASI scores, see Fig.10 Finally, the residual Pt in the psoriatic skin of mice was detected and Fig.11The corresponding ICP-MS results are shown in Figure 2, and it can be concluded that Pt was essentially cleared from psoriatic skin after 48 hours, with no biosafety risk of metal accumulation. The clearance of Pt was mainly attributed to its ultra-small size, which enables it to easily penetrate the skin blood vessels and be metabolized and excreted through the blood.

Claims

1. An ultra-small platinum cluster enzyme, characterized in that: It includes a platinum metal core and a cyclodextrin ligand.

2. A method for preparing the ultrasmall platinum cluster enzyme according to claim 1, characterized in that: The following steps are involved: The ultra-small platinum cluster enzyme is obtained by adding a Pt precursor solution to a thiol-cyclodextrin solution, mixing the mixture, and adding a reducing agent to carry out a reaction.

3. The method for preparing the ultrasmall platinum cluster enzyme according to claim 2, characterized in that: The mercapto-cyclodextrin is (n) mercapto-β-cyclodextrin, where n=1-7.

4. The method for preparing the ultrasmall platinum cluster enzyme according to claim 3, characterized in that: The Pt precursor solution is a Pt ion solution.

5. The method for preparing the ultrasmall platinum cluster enzyme according to claim 4, characterized in that: Pt ion solutions include H2PtCl6 solution, K2PtCl6 solution and PtCl4 solution.

6. The method for preparing the ultrasmall platinum cluster enzyme according to claim 4, characterized in that: (n) The molar ratio of mercapto-β-cyclodextrin to Pt ions is ≥ 0.5:

1.

7. The method for preparing the ultrasmall platinum cluster enzyme according to claim 2, characterized in that: The reducing agents include CO, H2, hypophosphite, hydrazine hydrate and tert-butylamine borane.

8. The method for preparing the ultrasmall platinum cluster enzyme according to claim 2, characterized in that: The solvent of the mercapto-cyclodextrin solution is an alkaline solution, and the solvent of the Pt precursor solution is water.

9. Use of the ultrasmall platinum cluster enzyme according to claim 1 in the preparation of drugs for treating psoriasis and preventing the recurrence of psoriasis.

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