Protamine ion complex with light-regulated hemostatic performance and preparation method thereof

Abstract

The present application relates to the technical fields of hemostatic materials and stimuli-responsive materials, and mainly relates to a protamine ion complex with light-regulated hemostatic performance and a preparation method thereof.The protamine ion complex with light-regulated hemostatic performance is obtained by mixing protamine, quaternary ammonium salt containing an azobenzene group and sodium dodecyl sulfate in an aqueous solution, and then performing oscillation, centrifugal separation and drying treatment.The protamine ion complex with light-regulated hemostatic performance prepared by the present application can undergo photo-induced phase change at room temperature.The protamine ion complex with light-regulated hemostatic performance prepared by the present application has good biocompatibility, can release protamine rapidly under ultraviolet irradiation under physiological conditions, inhibit heparin activity, and achieve rapid hemostasis.The protamine ion complex with light-regulated hemostatic performance prepared by the present application can be applied in the field of hemostatic materials.

Description

Technical Field

[0001] This invention relates to the field of hemostatic materials and stimulus-responsive materials, and mainly to a protamine ion complex with photo-regulated hemostatic properties and its preparation method. Background Technology

[0002] Heparin is a widely used anticoagulant drug, primarily used to treat and prevent thrombosis. In practice, accurate dosage control is crucial; excessive use can easily cause side effects such as bleeding, thrombocytopenia, and osteoporosis. Furthermore, for the body, rapidly clearing or reducing the concentration of heparin in the body is essential when coagulation is required.

[0003] In terms of molecular structure, heparin is a polysaccharide compound with a linear structure, containing a large number of sulfonate and carboxyl groups. Heparin has the highest negative charge density among biological macromolecules, and its anticoagulant function depends on the electrostatic and hydrogen bonding forces formed between its negatively charged groups and antithrombin. Therefore, theoretically, positively charged functional materials can act as inhibitors of heparin, accelerating blood clotting. Based on this mechanism, various material systems have been developed to clear heparin from the blood, including polymeric materials, self-assembled materials, and synthetic proteins. However, the only heparin inhibitor currently approved for clinical use is protamine, making the development of protamine-based heparin inhibitor materials even more important.

[0004] Stimulus-response mechanisms are widely used in the design and fabrication of smart materials. With the aid of controllable external stimuli, smart materials can undergo controllable property changes. Compared to other external stimuli, photostimulation offers advantages such as good spatiotemporal controllability, no byproducts, and minimal damage, making photoresponsive materials a focus of attention. For protamine, the molecule itself does not contain functional groups that respond to photostimulation. To develop protamine into a photoresponsive material, it needs to be combined with photosensitive molecules. In research on photoresponsive materials, azobenzene is one of the most widely used photosensitive molecules. Under ultraviolet light irradiation, azobenzene changes from a planar trans structure to a non-planar cis structure, while visible light can induce the molecule to revert to the planar trans structure. This photoisomerization of azobenzene converts absorbed light energy into molecular mechanical energy, enabling various functional materials to achieve photomodulation of their physicochemical properties. Currently, no photoresponsive protamine materials based on azobenzene photoisomerization have been reported, which limits the development and application of protamine in the field of smart drug materials. By utilizing the photoisomerization of azobenzene, it is possible to controllably expose the positively charged functional groups of protamine molecules, potentially achieving photomodulated inhibition of heparin's anticoagulant function. Therefore, developing protamine materials with photomodulated hemostatic properties and achieving controllable photomodulation of blood clotting time at room temperature is of great significance for expanding the application of protamine in basic research and hemostatic materials. Summary of the Invention

[0005] The purpose of this invention is to provide a protamine ion complex with photo-regulated hemostatic properties and its preparation method.

[0006] The objective of this invention can be achieved through the following technical solutions: A protamine ion complex with photo-regulated hemostatic properties is synthesized from protamine, a quaternary ammonium salt containing an azophenyl group, and sodium dodecyl sulfonate. The protamine is a salmon protamine sulfate with a molecular weight of 5100 g / mol and containing 22 arginine residues. The quaternary ammonium salt containing an azophenyl group is N,N-dimethyl-N-cyclohexyl-4-(4-((4-n-octoxyphenyl)diazeninyl)phenoxy)n-butylammonium bromide.

[0007] As a further aspect of the present invention: at room temperature, a quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate are first dissolved in water to prepare an aqueous solution of dual surfactants. Then, the aqueous solution of dual surfactants is mixed with the aqueous solution of protamine in a sample tube. The mixture is shaken and centrifuged, the supernatant is discarded, and the aggregated layer material at the bottom of the sample tube is collected. The aggregated layer material is freeze-dried to obtain a protamine ion complex with photo-regulated hemostatic properties.

[0008] As a further aspect of the present invention: the concentration of the protamine aqueous solution is 1-3 mM, the concentration of the quaternary ammonium salt containing the azophenyl group in the aqueous solution of the dual surfactant is 1-3 mM, and the concentration of sodium dodecyl sulfonate is 1.5-4.5 mM.

[0009] As a further aspect of the present invention: the molar ratio of protamine to the quaternary ammonium salt containing an azophenyl group is 1:22, and the molar ratio of protamine to sodium dodecyl sulfonate is 1:33.

[0010] As a further aspect of the present invention: the shaking time of the mixture is 3 minutes, and the freeze-drying time of the condensed layer material is 10 hours.

[0011] The beneficial effects of this invention are: 1. The protamine ion complex with photo-regulated hemostatic properties obtained in this invention is an ion complex formed by protamine, a quaternary ammonium salt containing an azophenyl group, and sodium dodecyl sulfonate through electrostatic interaction.

[0012] 2. The protamine ion complex with photo-regulated hemostatic properties obtained by the present invention can be completely transformed from a crystal into a disordered fluid under ultraviolet light stimulation.

[0013] 3. The protamine ion complex with photo-regulated hemostatic properties obtained by the present invention can rapidly release protamine in aqueous solution under ultraviolet light stimulation, thereby reducing the heparin concentration in the system.

[0014] 4. The protamine ion complex with photo-regulated hemostatic properties obtained by this invention has good biocompatibility and low cytotoxicity and hemolytic toxicity.

[0015] 5. The protamine ion complex with photo-regulated hemostatic properties obtained by the present invention can release protamine to clear heparin under physiological conditions through ultraviolet light irradiation, effectively accelerating the coagulation time. Attached Figure Description

[0016] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0017] Figure 1 Comparison of X-ray small-angle scattering spectra of the protamine ion complex with photo-modulated hemostatic properties described in Example 1 before and after ultraviolet light irradiation; Figure 2 The comparison of the time it takes for the protamine ion complex with photo-regulated hemostatic properties described in Example 1 to reach saturation in an aqueous solution system under ultraviolet light irradiation and without light irradiation. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0019] At room temperature, an aqueous solution of a dual surfactant was first prepared by dissolving a quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate in water, wherein the concentrations of the quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate were 2 mM and 3 mM, respectively. Then, 10.0 mL of the aqueous solution of the dual surfactant was mixed with 364.0 μL of an aqueous solution of salmon protamine sulfate with a concentration of 2.5 mM in a sample tube. The mixture was shaken for 3 minutes and then centrifuged at a relative centrifugal force of 6124 g for 10 minutes. The supernatant was discarded, and the condensed layer at the bottom of the sample tube was freeze-dried for 10 hours to obtain the protamine ion complex with photo-regulated hemostatic properties described in this invention.

[0020] The protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 is shown in the X-ray small-angle scattering (XAP) spectra at room temperature without UV irradiation and after 5 minutes of UV irradiation, as follows: Figure 1 As shown, according to Figure 1 It was found that the protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 can completely transform from a crystal structure to a disordered fluid structure under ultraviolet light irradiation.

[0021] The protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 was compared in terms of the time it took for the protamine concentration to reach saturation in an aqueous solution system under UV irradiation and without light irradiation at room temperature. Figure 2 As shown, according to Figure 2 The results show that the protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 can rapidly release protamine under ultraviolet light irradiation.

[0022] The protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 was tested for cytotoxicity and hemolytic toxicity. The protamine ion complex with photo-regulated hemostatic properties showed low biotoxicity and good biocompatibility.

[0023] The protamine ion complex with photo-regulated hemostatic properties prepared in Example 1 was tested in a biological experiment. The test showed that the protamine ion complex with photo-regulated hemostatic properties significantly accelerated clotting time under ultraviolet light irradiation, achieving rapid hemostasis. In the biological experiment, the hemostasis time required by the protamine ion complex with photo-regulated hemostatic properties under ultraviolet light irradiation was 40% of that without the use of any drugs.

[0024] Comparative Example 1: At room temperature, an aqueous solution of a dual surfactant was first prepared by dissolving a quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate in water, wherein the concentrations of the quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate were 2 mM and 10 mM, respectively. Then, 10.0 mL of the aqueous solution of the dual surfactant was mixed with 364.0 μL of an aqueous solution of salmon protamine sulfate with a concentration of 2.5 mM in a sample tube. The mixture was shaken for 3 minutes and then centrifuged at a relative centrifugal force of 6124 g for 10 minutes. The supernatant was discarded, and only a small amount of substance was obtained at the bottom of the sample tube. The small amount of substance at the bottom of the sample tube was freeze-dried for 10 hours to obtain the ionic complex of protamine. The test showed that the ionic complex of protamine did not have photoresponsive properties.

[0025] Comparative Example 2: At room temperature, an aqueous solution of a dual surfactant was first prepared by dissolving a quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate in water, wherein the concentrations of the quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate were 8 mM and 2 mM, respectively. Then, 10.0 mL of the aqueous solution of the dual surfactant was mixed with 364.0 μL of an aqueous solution of salmon protamine sulfate with a concentration of 2.5 mM in a sample tube. The mixture was shaken for 3 minutes and then centrifuged at a relative centrifugal force of 6124 g for 10 minutes. The supernatant was discarded, and only a small amount of substance was obtained at the bottom of the sample tube. The small amount of substance at the bottom of the sample tube was freeze-dried for 10 hours to obtain the ionic complex of protamine. The test showed that the ionic complex of protamine did not have photoresponsive properties.

[0026] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A protamine ion complex with photo-regulated hemostatic properties, characterized in that, Its synthetic raw materials include protamine sulfate, a quaternary ammonium salt containing an azophenyl group, and sodium dodecyl sulfate. The protamine sulfate is derived from salmon and has a molecular weight of 5100 g / mol, containing 22 arginine residues. The quaternary ammonium salt containing an azophenyl group is... N,N -dimethyl- N -cyclohexyl-4-(4-((4-n-octyloxyphenyl)diazenin)phenoxy)n-butylammonium bromide; The preparation method of the protamine ion complex with photo-regulated hemostatic properties includes: at room temperature, firstly, a quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate are dissolved in water to prepare an aqueous solution of dual surfactants, wherein the concentrations of the quaternary ammonium salt containing an azophenyl group and sodium dodecyl sulfonate are 2 mM and 3 mM, respectively. Then, 10.0 mL of the aqueous solution of dual surfactants and 364.0 μL of an aqueous solution of 2.5 mM protamine sulfate derived from salmon are mixed in a sample tube. The mixture is shaken and then centrifuged at a relative centrifugal force of 6124 g for 10 minutes. The supernatant is discarded, and the condensed layer at the bottom of the sample tube is freeze-dried to obtain the protamine ion complex with photo-regulated hemostatic properties of the present invention.

2. The protamine ion complex with photo-regulated hemostatic properties as described in claim 1, characterized in that, The mixing liquid in the preparation method is shaken for 3 minutes, and the freeze-drying time of the condensed layer material is 10 hours.

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