CN116271183B - Medical europium-containing absorption pad and preparation method thereof - Google Patents
Medical europium-containing absorption pad and preparation method thereof Download PDFInfo
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/20—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing organic materials
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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Abstract
The invention provides a medical europium-containing absorption pad and a preparation method thereof, wherein the medical europium-containing absorption pad contains a ternary assembly antibacterial agent, and the ternary assembly antibacterial agent comprises the following components in percentage by mole as follows: 1-2:5-10 PK-14, la-doped Eu multi-metal oxygen cluster and Spd. The ternary assembly antibacterial agent of the invention uses EuLaW 10 After being assembled with polypeptide and biogenic amine in a grading way, the modified fluorescent dye has high-efficiency disinfection performance on staphylococcus aureus, and loads a binary assembly EuLaW 10 The disinfection performance of the Spd is improved by 6.5 times compared with that of the original absorption pad, and the load ternary assembly EuLaW 10 the/Spd/PK-14 ratio was 36 times higher compared to the unloaded pad.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a medical europium-containing absorption pad and a preparation method thereof.
Background
The function of the skin is to provide a physical barrier between the internal organs and the external environment, protecting the body from pathogens, radiation, temperature changes and moisture loss. Skin lesions impair their function for a variety of reasons, including burns, trauma, surgery or lacerations. Thus, proper wound care is important for preventing infections and further related complications that may lead to impaired wound healing and may increase morbidity and mortality. For this reason, how to avoid the wound from being further infected by external bacteria becomes a hot spot in the next discussion.
Multimetal oxides (POMs) are a very unique metal oxygen cluster, typically formed by the condensation of oxygen and transition metals. POMs have nanosize, stable structure, high charge and excellent optical, electrical and magnetic properties. Polyoxometallates are widely used in the catalytic field due to their good redox and photooxidation properties. The multimetal oxide is a green catalyst and plays a role in various fields. Due to its special structure and excellent physicochemical properties, polyoxymethylene is widely used not only in industrial catalysis, functional materials and biomedical fields. As one of the few highly effective low-toxic inorganic drugs, multimetal oxides are receiving a great deal of attention in the biomedical field. According to the literature, POMs have antibacterial, antiviral, anticancer properties.
The absorbent pad is widely applied to wound repair with small area, but most of the absorbent pads on the market only play a role in stopping bleeding, and have little bacterial isolation effect and sterilization effect on the outside, so the addition of the POMs antibacterial component can promote the protection of the absorbent pad on the wound and prevent the damaged skin from being inflamed due to external infection.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects in the prior art, and provides a medical europium-containing absorption pad and a preparation method thereof.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
a medical europium-containing absorption pad which contains a ternary assembly antibacterial agent; the ternary assembly antibacterial agent comprises the following components in percentage by mole: 1-2:5-10 PK-14, la-doped Eu multi-metal oxygen cluster and Spd.
Preferably, the molar concentration ratio of PK-14, la-doped Eu multi-metal oxygen cluster to Spd is 1:1:5.
further, the La-doped Eu multi-metal oxygen cluster is Na x [Eu 0.5 La 0.5 W 10 O 36 ]•yH 2 O, wherein x is 8, 9 or 10, and y is 30, 31, 32, 33, 34 or 35.
Preferably, x is 9 and y is 32.
Further, the La-doped Eu multi-metal oxygen cluster is prepared by a method comprising the following steps: na is mixed with 2 WO 4 ·2H 2 Dissolving O in distilled water, adjusting pH to 7.0-7.5, adding Eu (NO) 3 ) 3 •6H 2 O and La (NO) 3 ) 3 •6H 2 O, heating and stirring, and cooling to room temperature to obtain crystallized Na x [Eu 0.5 La 0.5 W 10 O 36 ]•yH 2 O, filtering and drying filter residues to obtain the product; the Na is 2 WO 4 ·2H 2 O and Eu (NO) 3 ) 3 •6H 2 O and La (NO) 3 ) 3 •6H 2 The molar ratio of O is 1:5.5-6:5.5-6.
Further, the ternary assembly antibacterial agent is prepared by a method comprising the following steps:
step 1: building a binary assembly: uniformly mixing La-doped Eu multi-metal oxygen cluster and Spd in MES-NaOH buffer solution with pH value of 6-7, and incubating at room temperature for 5-15 min to obtain EuLaW 10 A Spd binary assembly;
step 2: building a ternary assembly: to said EuLaW 10 Adding PK-14 into Spd, and incubating at room temperature for 5-15 min to obtain EuLaW 10 Ternary assembly antibacterial agent of/Spd/PK-14.
The medical europium-containing absorption pad comprises the following steps: and immersing the base fabric in the ternary assembly antibacterial agent, and then taking out and drying to obtain the medical europium-containing absorption pad.
Further, the temperature of the dipping step is 35-40 ℃ and the time is 10-16 hours.
Further, the concentration of the ternary assembly antimicrobial agent is 20-100 mu M.
The polyacid is composed of metal-oxyacid anions, and spermidine Spd and polypeptide PK-14 serving as cations can be stably constructedThereby being sufficiently attached to the surface of the polyacid to make the triplet assembly more stable. Doping of La causes EuW 10 The antibacterial agent has better temperature sensitivity, and the antibacterial component still maintains good antibacterial performance at low temperature.
Compared with the prior art, the invention has the following advantages:
the ternary assembly antibacterial agent of the invention uses EuLaW 10 After being assembled with polypeptide and biogenic amine in a grading way, the modified fluorescent dye has high-efficiency disinfection performance on staphylococcus aureus, and loads a binary assembly EuLaW 10 The disinfection performance of the Spd is improved by 6.5 times compared with that of the original absorption pad, and the load ternary assembly EuLaW 10 the/Spd/PK-14 ratio was 36 times higher compared to the unloaded pad.
The medical europium-containing absorption pad provides a novel concept of the rare earth-containing inorganic-biological antibacterial absorption pad, and can promote the rare earth metal and metal amine or polypeptide assembly to be used for developing a novel medical disinfection absorption pad.
The medical europium-containing absorption pad solves the problem of bacterial breeding caused by the fact that the medical absorption pad cannot be sterilized, and reduces the risk of further infection of wounds.
Drawings
FIG. 1 shows the preparation of different EuLaW at three different pH values according to example 2 of the present invention 10 Is a fluorescence intensity map of (2);
FIG. 2 shows the EuLaW constructed with different amounts of Spd according to example 3 of the present invention 10 Fluorescence intensity plot of the Spd binary assembly;
FIG. 3 shows the different concentrations of PK-14 constructed triad EuLaW according to example 4 of the present invention 10 Fluorescence intensity profile of/Spd/PK-14;
FIG. 4 is a flow cytometer detection diagram of a blank control according to example 6 of the present invention;
FIG. 5 is a flow cytometer detection view of a binary assembly according to example 6 of the present invention;
FIG. 6 is a flow cytometer detection view of the ternary assembly of example 6 of the present invention;
FIG. 7 is a statistical chart of the apoptosis rate of the flow cell detection bacteria according to example 6 of the present invention;
FIG. 8 is a chart showing the antibacterial property at 10℃of example 7 of the present invention: 8-A is the experimental group and 8-B is the control group.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
Spermidine (Spd), 2',7' -dichlorofluorescein diacetate (DCFH-DA) and crystal violet were purchased from ala Ding Huaxue limited.
PK-14 is the sequence of N-terminal amino acid 488-501 of HPV type 11L 1 protein; HPV type 11L 1 protein was obtained from NCBI website (https:// www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax. Cgi) and ID number 345408.
Na 9 [EuLaW 10 O 36 ]•32H 2 O, hereinafter referred to as EuLaW 10 。
2- (N-Morpholino) ethanesulfonic acid (MES), fluorescein isothiocyanate (Annexin V-FITC) and sodium hydroxide (NaOH) were purchased from beijing chemical plant (beijing, china), proteinase K, propidium iodide and triton x-100 were purchased from Sigma, all chemicals were used without further treatment, and distilled water (ρ=18.2M Ω.cm,25 ℃) was from Millipore milli-Q water purification system. In addition, 10.0 mM MES-NaOH buffer with pH=6.0 was prepared from 10.0 mM MES and NaOH using distilled water. Preparation of EuLaW in 2.0. 2.0 mM aqueous solution 10 And Spd stock solution, and stored in dark (4 ℃) and then diluted to the required concentration according to different experimental requirements. Luria Broth (LB) was purchased from Sigma-Aldrich, wicklow, ireland). Staphylococcus aureus s.aureus is from beijing four-ring biopharmaceutical limited.
The present invention will be described in detail with reference to examples.
Example 1 Na 9 [EuLaW 10 O 36 ]•32H 2 O(EuLaW 10 ) Is prepared and purified
26.8 mmol Na 2 WO 4 •2H 2 O was dissolved in 20 mL distilled water to prepare a total of 3 batches, each batch was treated with acetic acid (CH 3 COOH) to adjust the pH to 7.0-7.5. Will contain 1.2 mmol Eu (NO) 3 ) 3 •6H 2 O and 1.2 mmol La (NO) 3 ) 3 •6H 2 Dropwise adding mixed water solution of O2 mL into the above solution, stirring at 80-90deg.C, and cooling to room temperature to obtain crystallized Na 9 [EuLaW 10 O 36 ]•32H 2 O, after filtration, is dried in air to obtain Na 9 [EuLaW 10 O 36 ]•32H 2 O. Is marked as EuLaW 10 。
Example 2 pH value vs Na 9 [EuLaW 10 O 36 ]•32H 2 O(EuLaW 10 ) Influence of (2)
26.8 mmol Na 2 WO 4 •2H 2 O was dissolved in 20 mL distilled water to prepare a total of 3 batches, each batch was treated with acetic acid (CH 3 COOH) to a pH of 7.0, 10.0, 13.0. Will contain 1.2 mmol Eu (NO) 3 ) 3 •6H 2 O and 1.2 mmol La (NO) 3 ) 3 •6H 2 Dropwise adding mixed water solution of O2 mL into the above solution, stirring at 80-90deg.C, and cooling to room temperature to obtain crystallized Na 9 [Eu 0.5 La 0.5 W 10 O 36 ]•32H 2 O, after filtration, is dried in air to obtain Na 9 [Eu 0.5 La 0.5 W 10 O 36 ]•32H 2 O. Is denoted as (EuLaW) 10 -7)、(EuLaW 10 -10)、(EuLaW 10 -13)。
As shown in fig. 1, the signal is obtained by a pair (EuLaW 10 -7)、(EuLaW 10 -10)、(EuLaW 10 -13) it can be observed that EuLaW decreases with decreasing pH value 10 The fluorescence intensity of (2) is gradually increased, namely: euLaW 10 -7＞EuLaW 10 -10＞EuLaW 10 -13。
Example 3 construction of EuLaW at different concentrations 10 Spd binary assembly
Different volumes of 0, 0.125, 0.25, 0.5, 1, 1.5, 2.5, 5, 12.5, 25, 30. Mu.L of Spd were added to 2.0 mM EuLaW 10 Is buffered in MES-NaOH (10.0 mM, pH=6.5) and stirred vigorously. The final volume of each set of mixed solutions was fixed at 1.0 mL. After incubation for 10 min at room temperature, euLaW is constructed 10 As shown in FIG. 2, the fluorescence spectrum results show that the fluorescence emission intensity of the mixed system is enhanced with increasing concentration of Spd, and the fluorescence emission intensity of the system is strongest at 591 and nm when the Spd addition amount of 2.0 mM is 25. Mu.L.
EXAMPLE 4 construction of EuLaW at different concentrations 10 Ternary assembly of/Spd/PK-14
In the case of binary assembly EuLaW prepared in example 2 10 MES-NaOH (10.0. 10.0 mM, pH=6.5) buffer at a concentration of 0, 5.00, 10.0, 15.0, 25.0, 35.0. Mu.M PK-14 was added to the Spd (50. Mu.M/50. Mu.M) buffer to give a final volume of 1.0 mL. After incubation at room temperature for 10 min, fluorescence spectrum changes were recorded at an excitation wavelength of 500 nm, and as shown in FIG. 3, the fluorescence generation intensity of the ternary assembly system reached the maximum when the concentration of PK-14 was 35.0. Mu.M.
EXAMPLE 5 preparation of medical absorbent pad
The absorbent pad is prepared by using non-woven fabrics which are common in the market as a base fabric and adopting an in-situ deposition mode, and the specific implementation scheme is as follows: 100. Mu.M EuLaW respectively 10 Spd and EuLaW 10 The dispersion of/Spd/PK-14 was immersed in a base fabric at 37℃and 12. 12 h. Then taking out the absorption pad, and drying to obtain the load EuLaW 10 Spd and EuLaW 10 Sterilized absorbent pad of/Spd/PK-14, and a single nonwoven fabric as a blank absorbent pad.
Example 6 flow cytometry
MRSA single colony was inoculated into Luria-Bertani (LB) agar medium, and cultured at 37℃and 180 rpm for 14-16 h to obtain suspensions of different types of bacteria with optical density of 600 nm (OD 600 ) Is used to evaluate E.coli growth, the OD is controlled in this invention 600 =0.4-0.6。
LB liquid culture solution is used as the environment of human wound epidermis, and is sterilized at high temperature. Placing 0.2. 0.2 mL high-temperature sterilized LB culture solution into EP tube, and injecting Staphylococcus aureus suspension (OD 600 =0.2), the three absorbent pads in example 5 were cut into 2×2 mm pieces, respectively 2 Is added to the EP tube and incubated 24 h.
The EP tube after the incubation was removed from the plate, and proteinase K (682.6. Mu.M) was added to the plate at 5. Mu. L, triton-X-100. Mu.L and digested at 50℃for 15min; 800. Mu.L of 0.1. 0.1M PBS buffer was added; 10000 Centrifuging at rpm for 5min, removing fat layer and supernatant with microsyringe, and retaining precipitate; fluorescein isothiocyanate (Annexin V-FITC) was added at 5. Mu.L. After 15 minutes, an additional 5 μl of Propidium Iodide (PI) was added, excitation light of 488 nm was used, and the signal of 535 nm was recorded.
Flow cytometry results on staphylococcus aureus as shown in figures 4-7, the number of staphylococcus aureus apoptosis was increased with the addition of the absorbent pad loaded with the binary and ternary assemblies. Compared with the blank, the binary assembly EuLaW is loaded 10 The disinfecting absorption pad of Spd has the apoptosis rate of staphylococcus aureus which is 6.5 times of that of a blank, and is loaded with a ternary assembly EuLaW 10 The disinfection absorption pad of/Spd/PK-14 is 36 times that of the blank. Description of the binary assembly EuLaW 10 Spd is also a ternary assembly EuLaW 10 The Spd/PK-14 has an inhibitory effect on the growth of staphylococcus aureus in wounds.
Example 7 EuLaW 10 Antibacterial property measurement of/Spd/PK-14 ternary assembly
Adding 10 g peptone, 5 g yeast extract and 170.9 mmol sodium chloride into 1L water, stirring, sterilizing in a high temperature sterilizing pot, standing, cooling to room temperature, sealing peripheral opening with sealing film, and placing into a heat-insulating refrigerator at 4deg.C
Combining the three components of the raw materials to form EuLaW 10 Suspension of/Spd/PK-14 in Staphylococcus aureus (OD 600 =0.2) were mixed. After incubation at 37℃for 3 h, the 0.2: 0.2 mL diluted bacterial suspension was plated on LB agar, incubated at 10℃for 18: 18 h, and plated on standard platesThe viability of the bacteria was determined by counting.
As shown in FIG. 8, wherein Panel B is a blank and Panel A is an experimental group, it is evident that the three-component assembly EuLaW was observed by agar plates at a low temperature of 10 ℃C 10 The Spd/PK-14 has good inhibition performance on the growth of staphylococcus aureus. Thus, the polyacid EuW after doping La 10 Has better temperature sensitivity, so that the growth of bacteria can be inhibited under the condition of low temperature.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. A medical europium-containing absorption pad which is characterized in that: the medical europium-containing absorption pad contains a ternary assembly antibacterial agent; the ternary assembly antibacterial agent comprises the following components in percentage by mole: 1-2:5-10 PK-14, la-doped Eu multi-metal oxygen cluster and Spd; the PK-14 is the sequence of N-terminal amino acid 488-501 of HPV 11 type L1 protein; the Eu multi-metal oxygen cluster doped with La is Na x [Eu 0.5 La 0.5 W 10 O 36 ]•yH 2 O, wherein x is 8, 9 or 10, and y is 30, 31, 32, 33, 34 or 35.
2. The medical europium-containing absorption pad according to claim 1, wherein: the molar concentration ratio of PK-14 to La-doped Eu multi-metal oxygen cluster to Spd is 1:1:5.
3. the medical europium-containing absorption pad according to claim 1, wherein: and x is 9, and y is 32.
4. The medical europium-containing absorption pad according to claim 1, wherein: the La-doped Eu multi-metal oxygen cluster is prepared by a method comprising the following steps: na is mixed with 2 WO 4 ·2H 2 O is dissolved in distilled water to regulate pH is 7.0-7.5, eu (NO) is added thereto 3 ) 3 •6H 2 O and La (NO) 3 ) 3 •6H 2 O, heating and stirring, and cooling to room temperature to obtain crystallized Na x [Eu 0.5 La 0.5 W 10 O 36 ]•yH 2 O, filtering and drying filter residues to obtain the product; the Na is 2 WO 4 ·2H 2 O and Eu (NO) 3 ) 3 •6H 2 O and La (NO) 3 ) 3 •6H 2 The molar ratio of O is 1:5.5-6:5.5-6.
5. The medical europium-containing absorption pad according to claim 1, wherein: the ternary assembly antibacterial agent is prepared by a method comprising the following steps:
step 1: building a binary assembly: uniformly mixing La-doped Eu multi-metal oxygen cluster and Spd in MES-NaOH buffer solution with pH value of 6-7, and incubating at room temperature for 5-15 min to obtain EuLaW 10 A Spd binary assembly;
step 2: building a ternary assembly: to said EuLaW 10 Adding PK-14 into Spd, and incubating at room temperature for 5-15 min to obtain EuLaW 10 Ternary assembly antibacterial agent of/Spd/PK-14.
6. The medical europium-containing absorbent pad of any one of claims 1-5, which is characterized in that: the method comprises the following steps: and immersing the base fabric in the ternary assembly antibacterial agent, and then taking out and drying to obtain the medical europium-containing absorption pad.
7. The europium-containing medical absorbent pad according to claim 6, characterized in that: the temperature of the dipping step is 35-40 ℃ and the time is 10-16 hours.
8. The europium-containing medical absorbent pad according to claim 6, characterized in that: the concentration of the ternary assembly antibacterial agent is 20-100 mu M.
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| CN113016821B (en) * | 2021-03-12 | 2021-11-23 | 广东药科大学 | Antibacterial nano liquid drop and application thereof |
| CN113583673B (en) * | 2021-08-03 | 2022-04-19 | 吉林大学 | Fluorescent probe based on polyoxometallate and assembly thereof and application of fluorescent probe in spermine detection |
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