CN110563979A - Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof - Google Patents
Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof Download PDFInfo
- Publication number
- CN110563979A CN110563979A CN201910898304.7A CN201910898304A CN110563979A CN 110563979 A CN110563979 A CN 110563979A CN 201910898304 A CN201910898304 A CN 201910898304A CN 110563979 A CN110563979 A CN 110563979A
- Authority
- CN
- China
- Prior art keywords
- protein
- mixed solution
- film
- nanofilm
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7007—Drug-containing films, membranes or sheets
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K5/00—Use of organic ingredients
- C08K5/36—Sulfur-, selenium-, or tellurium-containing compounds
- C08K5/37—Thiols
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Manufacturing & Machinery (AREA)
- Endocrinology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Diabetes (AREA)
- Inorganic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a protein nano-film based on an exchange reaction of sulfydryl and a disulfide bond and application thereof. The nano film can be prepared in a large area, is simple in preparation method, has strong mechanical strength without crosslinking, has good stability, optical transmittance and adhesion, and can be adhered to various substrates. The nano film can be used for encapsulating molecules with different sizes, can keep the activity of the molecules and realize controllable release.
Description
Technical Field
The invention relates to a two-dimensional protein nano film with better mechanical strength, which is prepared by exchange reaction of sulfydryl and disulfide bonds and has the characteristic of being used for encapsulating and releasing active biomolecules with different sizes.
Background
Two-dimensional organic and biological films have attracted increasing research interest, and in recent years, films with excellent physical and chemical properties have promising applications in electronics, energy, biosensors, and smart materials. However, proteins or polypeptides exhibit excellent water solubility and safety compared to insoluble cellulose membranes, and for natural examples, such as S-layer cells rely only on mild water molecule glycoprotein supramolecular organization, but have low assembly efficiency, slow speed, limited dual structural and functional control capability, and delayed development of synthetic alternatives; most of the silk fibroin or amyloid fiber is only limited to structure and form control, the film has low strength, is easy to scatter and is fragile, the strength needs to be improved through crosslinking of glutaraldehyde and the like, and the silk fibroin or amyloid fiber is obtained through toxic templates or extreme conditions. In this respect, large area synthesis of protein thin films is a key challenge, especially in view of ease of preparation, low cost, large scale preparation and further functional expansion, such as: controllable release of the functional molecule from the surface.
Disclosure of Invention
The invention aims to provide a protein nano-film which has simple preparation method, excellent stability, optical permeability and adhesiveness, and provides a new application for the protein nano-film.
Aiming at the purposes, the protein nano-film adopted by the invention is obtained by reducing protein containing disulfide bonds by using a reducing agent containing sulfhydryl groups and carrying out exchange reaction between the sulfhydryl groups and the disulfide bonds.
The specific preparation method of the protein nano film comprises the following steps: adjusting the pH of a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a sulfhydryl-containing reducing agent at 0.5-50 mg/mL by using NaOH to 7-10, and then uniformly mixing the pH with a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a disulfide bond-containing protein at 0.5-50 mg/mL according to a volume ratio of 1:1 to obtain a mixed solution; dripping the mixed solution on the surface of a base material, and clearly forming a layer of protein nano film on a gas-liquid interface of the base material; or the substrate is reversed on the surface of the mixed solution, and a layer of protein nano-film is formed on the solid-liquid interface of the reversed substrate and the mixed solution.
In the preparation method, preferably, 2-15 mg/mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a reducing agent containing sulfydryl is adjusted to pH 8-9 by NaOH, and then is uniformly mixed with 2-15 mg/mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of protein containing disulfide bonds in a volume ratio of 1:1 to obtain a mixed solution; dripping the mixed solution on the surface of a base material, and clearly forming a layer of protein nano film on a gas-liquid interface of the base material; or the substrate is reversed on the surface of the mixed solution, and a layer of protein nano-film is formed on the solid-liquid interface of the reversed substrate and the mixed solution.
In the above production method, the mass ratio of the disulfide bond-containing protein to the thiol-group-containing reducing agent is more preferably 1:0.5 to 2, and the mass ratio of the disulfide bond-containing protein to the thiol-group-containing reducing agent is more preferably 1:1 to 1.5.
The base material is any one of metal, metal oxide (magnesium, aluminum, gold, silver, platinum, nickel, copper, titanium and indium tin oxide films), inorganic non-metallic material (silicon, glass, quartz, mica and porcelain), organic high polymer material (polymethyl methacrylate, polypropylene, polyethylene terephthalate, polypropylene, polycarbonate and photosensitive polyimide) and daily necessities (wood, mobile phones, medical instruments and paper).
The protein nano film of the invention is used as a carrier in the encapsulation of molecules or particles with the size of more than 1nm, such as: micromolecular rhodamine, methyl orange, doxorubicin hydrochloride, middle molecular polypeptide, insulin, horse radish peroxidase, cytochrome C, macromolecular bovine serum albumin and gold nanoparticles.
The invention has chemoselectivity and site specificity reaction with specific protein in the sulfydryl-disulfide bond exchange reaction, further induces the protein to be gathered on a gas-liquid interface or a solid-liquid interface to form a two-dimensional protein film or coating, and has the functions of encapsulating and releasing functional molecules. The method not only provides a non-toxic strategy for preparing the protein film on various surfaces, but also encapsulates and releases the protein on the surfaces under the condition of not losing the activity of functional molecules, and has important significance in the fields of biosensing, diagnosis, biomaterial science and tissue engineering.
The invention has the following beneficial effects:
1. The invention utilizes the reducing agent containing sulfydryl to effectively reduce the disulfide bond of the protein to induce the rapid self-assembly of the protein (within minutes), and obtains the two-dimensional protein nano film with controllable area and various functions. The two-dimensional protein nano film has adjustable thickness, excellent optical transparency, good adhesion and strength, can be adhered to various substrate surfaces (including antifouling surfaces), and has good chemical stability and mechanical stability.
2. The protein nano film can be used as a carrier to encapsulate molecules or particles with the size of more than 1nm, such as drug molecules, enzymes and the like, has wide universality, can realize higher activity retention rate of encapsulated molecules, and even though the surrounding environment has slight change; and simultaneously, the controllable release of the encapsulated molecules can be realized.
3. The preparation method of the protein nano film is simple and rapid, and is easy to realize large-area preparation.
Drawings
FIG. 1 is an optical picture of the protein nano-film prepared in example 2.
FIG. 2 is an atomic force microscope photograph of the protein nano-film prepared in example 2.
FIG. 3 is a fluorescent microscope photograph of the protein nano-film encapsulated insulin prepared in example 2.
FIG. 4 is a graph of activity of protein nanofilm encapsulated insulin and horseradish peroxidase prepared in example 2.
FIG. 5 is a graph showing the controlled release of insulin after insulin is encapsulated in the protein nano-film prepared in example 2.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Example 1
At room temperature, adding 50mg of L-cysteine into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 50mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 2
At room temperature, adding 70mg of L-cysteine into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 70mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop (see the figure 1-2).
Example 3
At room temperature, adding 100mg of L-cysteine into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 100mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 4
At room temperature, adding 150mg of L-cysteine into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 150mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 5
At room temperature, adding 70mg of mercaptoethanol into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L sodium hydroxide aqueous solution, adding 70mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 6
At room temperature, adding 70mg of glutathione into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L sodium hydroxide aqueous solution, adding 70mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 7
At room temperature, adding 70mg of dithiothreitol into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 70mg of lysozyme into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet within 60min or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet and the mixed solution drop.
Example 8
At room temperature, adding 70mg of L-cysteine into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 70mg of lactalbumin into 10mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and (3) dropwise adding the mixed solution on the surface of a glass sheet or reversely buckling the glass sheet on the surface of the mixed solution drop, and clearly forming a layer of protein nano-film on the gas-liquid interface of the glass sheet or forming a layer of protein nano-film on the solid-liquid interface of the reversely buckled glass sheet contacted with the mixed solution drop.
Example 9
Example 2 application of protein Nanofilm as Carrier for encapsulating rhodamine B
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; adding a rhodamine B aqueous solution into the obtained mixed solution to ensure that the concentration of the rhodamine B in the mixed solution is respectively 0.05mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL and 2mg/mL, then reversely buckling a 18mm multiplied by 18mm glass sheet on the surface of the solution, and forming a layer of protein nano film for encapsulating the rhodamine B at a solid-liquid interface of the reversely buckled glass sheet and the solution within 60 min. According to the formula: (m)1-m2) Calculating the drug loading density, m, of the drug1To encapsulate the initial mass of the molecule, m2Is the mass of unencapsulated molecules, and S is the area of the substrate. The maximum drug loading density of rhodamine B is calculated to be 0.045 mu g/cm2。
Example 10
Example 2 application of protein Nanofilm as Carrier for encapsulating doxorubicin hydrochloride
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and adding an aqueous solution of doxorubicin hydrochloride into the obtained mixed solution to ensure that the concentrations of the doxorubicin hydrochloride in the mixed solution are respectively 0.05mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL and 2mg/mL, then inversely buckling a 18mm x 18mm glass sheet on the surface of the solution, and forming a layer of protein nano-film for encapsulating the doxorubicin hydrochloride at a solid-liquid interface where the inversely buckled glass sheet is contacted with the solution within 60 min. The calculated drug loading density of the doxorubicin hydrochloride is 0.0546 mu g/cm2。
Example 11
Application of protein nano-film of example 2 as carrier to encapsulate methyl orange
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; adding methyl orange aqueous solution into the obtained mixed solution to ensure that the concentration of methyl orange in the mixed solution is respectively 0.05mg/mL, 0.1mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL and 2mg/mL, then, reversely buckling a 18mm multiplied by 18mm glass sheet on the surface of the solution, and forming a layer of protein nano film for encapsulating the methyl orange at a solid-liquid interface of the reversely buckled glass sheet contacted with the solution within 60 min. The calculated medicine loading density of the methyl orange is 0.04 mu g/cm2。
Example 12
Example 2 application of protein Nanoplain as Carrier to encapsulate leucine-rich amelogenin peptides
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and adding a leucine-rich amelogenin peptide aqueous solution into the obtained mixed solution to enable the concentrations of the leucine-rich amelogenin peptide in the mixed solution to be 0.2mg/mL, 0.25mg/mL, 0.28mg/mL, 0.3mg/mL, 0.32mg/mL, 1mg/mL and 1.5mg/mL respectively, then reversely buckling a 18mm multiplied by 18mm glass sheet on the surface of the solution, and forming a layer of protein nano film for encapsulating the leucine-rich amelogenin peptide at a solid-liquid interface where the reversely buckled glass sheet is contacted with the solution within 60 min. The calculated medicine carrying density of the leucine-rich amelogenin peptide is 0.27 mu g/cm2。
Example 13
Example 2 use of protein nanofilm as vehicle to encapsulate insulin
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; to the resulting mixed solution was added an aqueous insulin solution so that the concentrations of insulin in the mixed solution were 0.2mg/mL, 0.25mg/mL, 0.28mg/mL, 0.3mg/mL, 0.32mg/mL, 0.45mg/mL, 0.85mg/mL, respectively, and then an 18mm × 18mm glass slide was inverted on the surface of the solution to form a layer of an insulin-encapsulating protein nano-film at the solid-liquid interface where the inverted glass slide was in contact with the solution within 60min (see FIG. 3). The calculated drug loading density of the insulin is 0.253 mu g/cm2。
The protein nano-film encapsulating the insulin is placed in 10mL phosphate buffer solution with pH value of 7.2-7.4, and the activity of the released insulin after 72h is detected by a double antibody sandwich method, and the result is shown in FIG. 4. The results show that the activity retention rate of the insulin reaches 87%.
The protein nano-film encapsulating insulin was placed in 10mL of phosphate buffer solution with pH 7.2-7.4, and the amount of released insulin was measured at different times, and the results are shown in fig. 5. The results show that the controllable release of insulin is realized after the protein nano film encapsulates the insulin.
Example 14
Example 2 application of protein Nanofilm as Carrier for encapsulation of cytochrome C
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; adding a cytochrome C aqueous solution into the obtained mixed solution to ensure that the concentration of cytochrome C in the mixed solution is 0.2mg/mL, 0.25mg/mL, 0.28mg/mL, 0.3mg/mL, 0.32mg/mL, 0.45mg/mlL and 0.85mg/mL respectively, then reversely buckling an 18mm x 18mm glass sheet on the surface of the solution, and forming a layer of protein nano film for encapsulating cytochrome C at a solid-liquid interface of the reversed glass sheet and the solution within 60 min. The calculated drug loading density of cytochrome C is 0.25 mug/cm2。
Example 15
Example 2 application of protein Nanofilm as Carrier for encapsulating Horseradish peroxidase
at room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; adding horseradish peroxidase aqueous solution to the obtained mixed solution to make the concentration of horseradish peroxidase in the mixed solution to be 0.2mg/mL, 0.25mg/mL, 0.28mg/mL, 0.3mg/mL, 0.32mg/mL, 0.45mg/mL, 0.85mg/mL, and then reversely buckling the 18mm × 18mm glass plate on the surface of the solution, fixing the reversed glass plate in contact with the solution within 60minThe liquid interface forms a layer of protein nano-film encapsulating horseradish peroxidase. The calculated drug loading density of the horseradish peroxidase is 0.258 mu g/cm2。
The protein nano-film encapsulating the horseradish peroxidase is placed in 10mL of phosphate buffer solution with the pH value of 7.2-7.4, and the activity of the horseradish peroxidase released after 72h is detected by a 3,3',5,5' -tetramethylbenzidine body color development method, and the result is shown in FIG. 4. The results show that the activity retention rate of insulin reaches 91%.
Example 16
Example 2 application of protein Nano-film as Carrier encapsulating bovine serum Albumin
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; and adding an aqueous solution of horseradish peroxidase into the obtained mixed solution to ensure that the concentration of the horseradish peroxidase in the mixed solution is respectively 0.55mg/mL, 1.1mg/mL, 1.6mg/mL, 2.2mg/mL and 3.275mg/mL, then, reversely buckling a 18mm multiplied by 18mm glass sheet on the surface of the solution, and forming a layer of protein nano-film for encapsulating the horseradish peroxidase at a solid-liquid interface of the reversely buckled glass sheet contacted with the solution within 60 min. The calculated drug loading density of the horseradish peroxidase is 0.3626 mu g/cm2。
Example 17
Example 2 use of protein nanofilm as support to encapsulate gold nanoparticles
At room temperature, adding 7mg of L-cysteine into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, adjusting the pH value to be 8-10 by using 5mol/L of sodium hydroxide aqueous solution, adding 7mg of lysozyme into 1mL of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.4, and then uniformly mixing the two solutions to obtain a mixed solution; adding 200 mu L of 50 mu g/mL gold nanoparticle (10nm) aqueous solution into the obtained mixed solution, then reversely buckling a 18mm multiplied by 18mm glass sheet on the surface of the solution, and forming a layer of protein nano-film for encapsulating the gold nanoparticles at a solid-liquid interface of the reversely buckled glass sheet and the solution in 60 min.
Claims (10)
1. A protein nano-film based on exchange reaction of sulfydryl and disulfide bonds is characterized in that: the protein nano-film is obtained by reducing protein containing disulfide bonds by a reducing agent containing sulfhydryl groups and carrying out exchange reaction between the sulfhydryl groups and the disulfide bonds.
2. The protein nanofilm of claim 1, wherein: the reducing agent containing sulfhydryl group is any one of cysteine, glutathione, mercaptoethanol and dithiothreitol.
3. The protein nanofilm of claim 1, wherein: the protein containing the disulfide bond is lysozyme and lactalbumin.
4. The protein nanofilm according to any one of claims 1 to 3, characterized in that it is prepared by the following method: adjusting the pH of a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a sulfhydryl-containing reducing agent at 0.5-50 mg/mL by using NaOH to 7-10, and then uniformly mixing the pH with a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a disulfide bond-containing protein at 0.5-50 mg/mL according to a volume ratio of 1:1 to obtain a mixed solution; dripping the obtained mixed solution on the surface of a base material, and clearly forming a layer of protein nano film on a gas-liquid interface of the base material; or the substrate is reversed on the surface of the mixed solution, and a layer of protein nano-film is formed on the solid-liquid interface of the reversed substrate and the mixed solution.
5. The protein nanofilm of claim 4, characterized in that it is prepared by the following method: adjusting the pH of a 2-15 mg/mL 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of a sulfhydryl-containing reducing agent to 8-9 by using NaOH, and then uniformly mixing the pH of the buffer solution with 2-15 mg/mL 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution of protein containing disulfide bonds in a volume ratio of 1:1 to obtain a mixed solution; dripping the obtained mixed solution on the surface of a base material, and clearly forming a layer of protein nano film on a gas-liquid interface of the base material; or the substrate is reversed on the surface of the mixed solution, and a layer of protein nano-film is formed on the solid-liquid interface of the reversed substrate and the mixed solution.
6. The protein nanofilm of claim 4, wherein: the mass ratio of the protein containing the disulfide bond to the reducing agent containing the sulfydryl is 1: 0.5-2.
7. The protein nanofilm of claim 6, wherein: the mass ratio of the protein containing the disulfide bond to the reducing agent containing the sulfydryl is 1: 1-1.5.
8. The protein nanofilm of claim 4 or 5, wherein: the base material is any one of metal or metal oxide, inorganic non-metal material, organic high polymer material and daily necessities.
9. Use of the protein nanofilm of claim 1 as a carrier for encapsulating molecules and particles having a size of 1nm or more.
10. Use of the protein nanofilm as a carrier in the encapsulation of molecules or particles with a size above 1nm, according to claim 7, characterized in that: the molecules or particles with the size of more than 1nm comprise rhodamine B, doxorubicin hydrochloride, methyl orange, leucine-rich amelogenin peptide, insulin, horse radish peroxidase, cytochrome C, bovine serum albumin and gold nanoparticles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910898304.7A CN110563979B (en) | 2019-09-23 | 2019-09-23 | Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910898304.7A CN110563979B (en) | 2019-09-23 | 2019-09-23 | Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110563979A true CN110563979A (en) | 2019-12-13 |
| CN110563979B CN110563979B (en) | 2022-10-21 |
Family
ID=68781746
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910898304.7A Active CN110563979B (en) | 2019-09-23 | 2019-09-23 | Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110563979B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111802385A (en) * | 2020-02-29 | 2020-10-23 | 陕西师范大学 | Protein product capable of fixing target and removing according to need |
| CN112574578A (en) * | 2020-11-19 | 2021-03-30 | 陕西师范大学 | Protein/polysaccharide composite nano film and application thereof in preventing conductive coating from generating cracks |
| CN114470149A (en) * | 2022-01-26 | 2022-05-13 | 陕西师范大学 | Antibacterial solution with size-controllable protein-based nanoparticles |
| CN114469750A (en) * | 2022-01-28 | 2022-05-13 | 陕西师范大学 | Mineralized material for treating tooth sensitivity and promoting enamel epitaxial growth and application thereof |
| CN114736610A (en) * | 2022-05-10 | 2022-07-12 | 陕西师范大学 | Structural color material and preparation method thereof |
| CN115216034A (en) * | 2021-04-16 | 2022-10-21 | 复旦大学 | Silk protein/lysozyme transferable protein nano film or coating and preparation thereof |
| CN115286958A (en) * | 2022-08-31 | 2022-11-04 | 陕西师范大学 | Biomass-based antifouling material and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030035820A1 (en) * | 1997-11-26 | 2003-02-20 | Southwest Research Institute And Keraplast Technologies, Ltd. | Keratin containing implant material |
| CN106867000A (en) * | 2017-03-16 | 2017-06-20 | 苏州佰锐生物科技有限公司 | A kind of method for promoting keratolysis and enhancing keratin material intensity |
| CN108379228A (en) * | 2018-02-28 | 2018-08-10 | 湖南大学 | A kind of albumin nanoparticle and its preparation method and application of package pharmacological active substance |
| CN108854599A (en) * | 2018-05-14 | 2018-11-23 | 陕西师范大学 | A kind of dialysis membrane and its application based on crosslinking lysozyme |
| CN109674741A (en) * | 2017-10-19 | 2019-04-26 | 中国农业大学 | Pharmaceutical carrier and preparation method thereof |
-
2019
- 2019-09-23 CN CN201910898304.7A patent/CN110563979B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030035820A1 (en) * | 1997-11-26 | 2003-02-20 | Southwest Research Institute And Keraplast Technologies, Ltd. | Keratin containing implant material |
| CN106867000A (en) * | 2017-03-16 | 2017-06-20 | 苏州佰锐生物科技有限公司 | A kind of method for promoting keratolysis and enhancing keratin material intensity |
| CN109674741A (en) * | 2017-10-19 | 2019-04-26 | 中国农业大学 | Pharmaceutical carrier and preparation method thereof |
| CN108379228A (en) * | 2018-02-28 | 2018-08-10 | 湖南大学 | A kind of albumin nanoparticle and its preparation method and application of package pharmacological active substance |
| CN108854599A (en) * | 2018-05-14 | 2018-11-23 | 陕西师范大学 | A kind of dialysis membrane and its application based on crosslinking lysozyme |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111802385A (en) * | 2020-02-29 | 2020-10-23 | 陕西师范大学 | Protein product capable of fixing target and removing according to need |
| CN111802385B (en) * | 2020-02-29 | 2021-08-31 | 西安曼特思生物科技有限公司 | Protein product capable of fixing target and removing according to need |
| CN113545996A (en) * | 2020-02-29 | 2021-10-26 | 西安曼特思生物科技有限公司 | Protein product capable of fixing target and removing according to need |
| CN113545996B (en) * | 2020-02-29 | 2023-11-17 | 西安曼特思生物科技有限公司 | Protein product capable of fixing target object and being removed according to need |
| CN112574578A (en) * | 2020-11-19 | 2021-03-30 | 陕西师范大学 | Protein/polysaccharide composite nano film and application thereof in preventing conductive coating from generating cracks |
| CN115216034A (en) * | 2021-04-16 | 2022-10-21 | 复旦大学 | Silk protein/lysozyme transferable protein nano film or coating and preparation thereof |
| CN115216034B (en) * | 2021-04-16 | 2023-08-29 | 复旦大学 | Silk protein/lysozyme transferable protein nano film or coating and preparation thereof |
| CN114470149A (en) * | 2022-01-26 | 2022-05-13 | 陕西师范大学 | Antibacterial solution with size-controllable protein-based nanoparticles |
| CN114470149B (en) * | 2022-01-26 | 2024-01-23 | 陕西师范大学 | Antibacterial liquid with controllable-size protein-based nanoparticles |
| CN114469750A (en) * | 2022-01-28 | 2022-05-13 | 陕西师范大学 | Mineralized material for treating tooth sensitivity and promoting enamel epitaxial growth and application thereof |
| CN114736610A (en) * | 2022-05-10 | 2022-07-12 | 陕西师范大学 | Structural color material and preparation method thereof |
| CN115286958A (en) * | 2022-08-31 | 2022-11-04 | 陕西师范大学 | Biomass-based antifouling material and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110563979B (en) | 2022-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110563979B (en) | Protein nano-film based on exchange reaction of sulfydryl and disulfide bond and application thereof | |
| Dinc et al. | Recent advances on core–shell magnetic molecularly imprinted polymers for biomacromolecules | |
| Xu et al. | Stimuli-responsive molecularly imprinted polymers: versatile functional materials | |
| Dai et al. | High-capacity binding of proteins by poly (acrylic acid) brushes and their derivatives | |
| Lau et al. | Direct write protein patterns for multiplexed cytokine detection from live cells using electron beam lithography | |
| US20190360933A1 (en) | Metal-organic frameworks as protective coatings and for enhancing sensitivity of biodiagnostic chips | |
| Prieto-Simon et al. | Biomolecule immobilization in biosensor development: tailored strategies based on affinity interactions | |
| Luo et al. | based radial chromatographic immunoassay for the detection of pathogenic bacteria in milk | |
| US8465972B2 (en) | Nanoparticulate cell culture surface | |
| Zhand et al. | Metal–organic framework-enhanced ELISA platform for ultrasensitive detection of PD-L1 | |
| CN101791439A (en) | Construction method of medical titanium alloy implant surface growth factor delivery system | |
| Biasotto et al. | ZnO nanorods-gold nanoparticle-based biosensor for detecting hepatitis C | |
| Pan et al. | Nanomaterials‐Based Surface Protein Imprinted Polymers: Synthesis and Medical Applications | |
| Ziółkowski et al. | Studies on the development of electrochemical immunosensor for detection of diphtheria toxoid | |
| Zhao et al. | Denatured proteins show new vitality: Green synthesis of germanium oxide hollow microspheres with versatile functions by denaturing proteins around bubbles | |
| Lamaoui et al. | Key advances in MIP-based sensors applied for cancer and cardiovascular biomarkers detection | |
| KR100887530B1 (en) | In Situ Preparation of Substrates with Dispersed Gold Nanoparticles | |
| JP5409671B2 (en) | Affinity hydrogel and its label-independent detection method | |
| Zhao et al. | Accurate Immobilization of Antibody‐Functionalized Peptide Nanotubes on Protein‐Patterned Arrays by Optimizing their Ligand–Receptor Interactions | |
| JP4547324B2 (en) | Protein recognition structure, protein recognition substrate, and production method thereof | |
| CN108505023A (en) | A kind of preparation method of self-supporting two-dimensional metallic film using protein welding | |
| Zhang et al. | Multifunctional oligomer immobilized on quartz crystal microbalance: a facile and stabilized molecular imprinting strategy for glycoprotein detection | |
| JP6179521B2 (en) | Substance measurement sensor | |
| Rai et al. | Molecularly imprinted polymers for drug delivery and biomedical applications | |
| Mao et al. | Recent progress in protein imprinting technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |