CN112826943A - Protein nano-carrier, carrier loaded with targeting substance, preparation method and application - Google Patents

Protein nano-carrier, carrier loaded with targeting substance, preparation method and application Download PDF

Info

Publication number
CN112826943A
CN112826943A CN202110046195.3A CN202110046195A CN112826943A CN 112826943 A CN112826943 A CN 112826943A CN 202110046195 A CN202110046195 A CN 202110046195A CN 112826943 A CN112826943 A CN 112826943A
Authority
CN
China
Prior art keywords
protein
sirna
solution
carrier
rna
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
Application number
CN202110046195.3A
Other languages
Chinese (zh)
Other versions
CN112826943B (en
Inventor
郭英姝
曹秀萍
尚鑫鑫
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Biotech Biotechnology Co.,Ltd.
Original Assignee
Qilu University of Technology
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Qilu University of Technology filed Critical Qilu University of Technology
Priority to CN202110046195.3A priority Critical patent/CN112826943B/en
Publication of CN112826943A publication Critical patent/CN112826943A/en
Application granted granted Critical
Publication of CN112826943B publication Critical patent/CN112826943B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • 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
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nanotechnology (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medical Informatics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a protein nano-carrier, a carrier loaded with a targeting substance, a preparation method and application, belonging to the technical field of nano-materials; the protein nano-carrier takes aptamer-modified iron-deficient protein as a shell and is embedded with influenza virus hemagglutinin. The protein nano-carrier has good biocompatibility, lower immunogenicity and higher transfer efficiency, and can selectively stimulate the iron deficiency protein-tfr 1 expression cells. The pH sensitivity of ferritin allows for the release of influenza virus hemagglutinin. With the help of influenza virus hemagglutinin, the targeting substance loaded by the protein nano-carrier can escape from lysosomes, get rid of the fate of degradation, release a large amount of targeting substance into cytoplasm, and ensure the integrity and effectiveness of the targeting substance. In addition, the ferritin-deficient nano shell has no obvious toxic or side effect, and a new method is provided for safer and more effective targeted delivery.

Description

Protein nano-carrier, carrier loaded with targeting substance, preparation method and application
Technical Field
The invention relates to the technical field of nano materials, in particular to a protein nano carrier, a carrier loaded with a targeting substance, a preparation method and application.
Background
Cancer has been one of the major causes of human life and health hazards. Chemotherapy is a commonly used cancer treatment method in clinical practice. Although chemotherapy has a certain treatment effect, it lacks targeting, and brings great pain to patients due to damage to normal tissue cells. At present, the nano material provides a new idea for targeted delivery of drugs. The nano material carries small molecule drugs to enter cancer cells, the loss of the drugs in the in vivo circulation process is reduced by the unique size advantage, and the treatment efficiency of the drugs is improved to a certain extent. In addition, the existence of the nano-carrier also improves the water solubility and the stability of the small molecule drug.
The nanocarriers disclosed in the prior art are primarily metal nanoparticles, such as gold nanoparticles. However, the metal nanoparticles have the problems of slow metabolism and easy accumulation in vivo, and the liposome is large and uneven in volume and still has many hidden troubles in the aspect of biosafety.
Disclosure of Invention
The invention aims to provide a protein nano-carrier, a carrier loaded with a targeting substance, a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a protein nano-carrier, which takes aptamer modified iron-deficient protein as a shell and is embedded with influenza virus hemagglutinin.
Preferably, the nucleic acid aptamer comprises the nucleic acid AS1411 aptamer.
The invention also provides application of the protein nano-carrier in the scheme in preparation of a targeted delivery carrier.
The invention also provides a carrier loaded with a targeting substance, which comprises the protein nano-carrier and a combination body, wherein the combination body is embedded in the shell of the protein nano-carrier, and the combination body is a combination body of double-chain siRNA and nuclear localization signal peptide.
Preferably, the double-stranded siRNA is obtained by base complementary pairing of a first RNA and a second RNA; the nucleotide sequence of the first RNA is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the second RNA is shown as SEQ ID NO: 2, respectively.
Preferably, the preparation method of the double-stranded siRNA comprises the following steps: mixing the first RNA solution and the second RNA solution, and carrying out base complementary pairing reaction to obtain double-stranded siRNA; the time of the base complementary pairing reaction is 5-6 h; the temperature of the base complementary pairing reaction is 20-30 ℃.
The invention also provides a preparation method of the carrier in the scheme, which comprises the following steps:
1) mixing double-stranded siRNA, nuclear localization signal peptide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution, and carrying out a binding reaction to obtain a binding reaction product, wherein the binding reaction product comprises a combination of the siRNA and the nuclear localization signal peptide and is named as siRNA/NLS;
2) dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; mixing the combined reaction product, an influenza virus hemagglutinin solution and the dissociated iron-deficient protein to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure, wherein the core-shell structure is named as Apn/siRNA/NLS/HA;
3) mixing the Apn/siRNA/NLS/HA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and NHS solution to obtain a mixed solution, and performing first dialysis on the mixed solution to obtain a first dialysate; the cut-off molecular weight of a dialysis membrane adopted by the first dialysis is 12-14 KD;
4) and mixing the first dialysate with the aptamer solution of the nucleic acid AS1411, and modifying to obtain a modified product, wherein the modified product comprises the carrier.
Preferably, the time of the combination reaction in the step 1) is 5-6 h; the temperature of the bonding reaction is 20-30 ℃.
Preferably, after the assembling, the method further comprises sequentially performing enzymolysis on the assembled mixture by using RNase A and proteinase K.
Preferably, the mixing time in the step 3) is 1.5-2.5 h.
The invention provides a protein nano-carrier, which takes aptamer modified iron-deficient protein as a shell and is embedded with influenza virus hemagglutinin. In the invention, the aptamer has targeting property, and the protein nano-carrier enters cells at a target region of the aptamer, so that the transfer efficiency is high. The iron-deficient protein shell can embed a target substance, the acidic environment of lysosomes causes the iron-deficient protein shell to be damaged, and the embedded target substance and influenza virus hemagglutinin are released. Influenza virus hemagglutinin can damage the lysosome membrane, so that the target substance escapes from lysosome, and the integrity of the target substance is maintained to the maximum extent. The ferritin-deficient nano shell can selectively stimulate ferritin-tfr 1 expression cells, and has no obvious toxic or side effect, so that the protein nano carrier has good biocompatibility and lower immunogenicity.
Drawings
FIG. 1 is a schematic diagram showing the process of drug release and loading of the protein nanocarrier of the invention;
FIG. 2 is a transmission electron microscope image of a ferritin-deficient protein;
FIG. 3 is a transmission electron microscope image of a protein nanocarrier according to an embodiment of the invention;
FIG. 4 shows the size change of ferritin and protein nanocarriers in the examples of the present invention;
FIG. 5 shows Zeta potential changes of ferritin and protein nanocarriers in the examples of the present invention;
FIG. 6 shows the detection of siRNA release in ferritin-deficiency proteins at pH5.5 and pH7.4, respectively, at 37 ℃;
FIG. 7 shows the toxic effect of ferritin-deficiency, ferritin-deficiency/siRNA/Apt, Apn/siRNA/nuclear localization signal peptide/Apt, ferritin-deficiency/siRNA/HA/Apt and Apn/siRNA/NLS/HA/Apt on MCF-7 cells;
FIG. 8 shows the fluorescent detection of intracellular ferritin deficiency/Cy 5 siRNA/NLS/HA/Apt; wherein a represents MCF-7 cells, b represents MCF-7 cells + iron deficiency protein/Cy 5 siRNA/NLS/HA/Apt;
FIG. 9 is a confocal microscope image of a cell; group A is characterized in that the ferritin is incubated with cells for 6h, group B is incubated with the cells for 6h by using Apn/Cy5-siRNA/NLS/HA/Apt, and group C is incubated with the cells for 12h by using Apn/Cy 5-siRNA/NLS/HA/Apt.
Detailed Description
The invention provides a protein nano-carrier, which takes aptamer modified iron-deficient protein as a shell and is embedded with influenza virus hemagglutinin.
In the present invention, the iron-deficient protein is preferably purchased from sigma.
In the present invention, the nucleic acid aptamer preferably includes the nucleic acid AS1411 aptamer. In the invention, the nucleotide sequence of the aptamer of the nucleic acid AS1411 is shown AS SEQ ID NO: 4, the nucleic acid AS1411 aptamer can accurately target and locate MCF-7 breast cancer cells; the amino acid sequence of the influenza virus Hemagglutinin (HA) is shown as SEQ ID NO: and 6.
In the present invention, the ratio of the mass of the ferritin to the molar amount of the aptamer of nucleic acid AS1411 is preferably (8-12) g: 0.1mmol, more preferably 10 g: 0.1 mmol; the ratio of the mass of the iron-deficiency protein to the molar amount of the influenza virus hemagglutinin is preferably (8-12) g: 0.1mmol, more preferably 10 g: 0.1 mmol.
In the present invention, the protein nanocarrier is preferably prepared by the following method:
s1, dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; mixing an influenza virus hemagglutinin solution and a dissociated ferritin to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure named as Apn/HA;
s2, mixing the Apn/HA, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and the NHS solution to obtain a mixed solution, and performing first dialysis on the mixed solution to obtain a first dialysate; the cut-off molecular weight of a dialysis membrane adopted by the first dialysis is 12-14 KD;
and S3, mixing the first dialysate with the nucleic acid AS1411 aptamer solution, and modifying to obtain a modified product, wherein the modified product comprises a protein nano-carrier.
Firstly, dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; mixing an influenza virus hemagglutinin solution and a dissociated ferritin to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure named as Apn/HA
In the present invention, the step of dissociating the ferritin at a pH of 2 to 2.5 preferably comprises adding 0.1M hydrochloric acid to the ferritin to lower the pH of the solution to 2 to 2.5, thereby dissociating the ferritin. In the present invention, the solvent of the influenza virus hemagglutinin solution is preferably sterilized water. In the invention, the volume ratio of the influenza virus hemagglutinin solution to the dissociation iron-deficiency protein is preferably (0.5-1.5): (0.5 to 1.5), and more preferably 1: 1; the concentration of the influenza virus hemagglutinin solution is preferably 0.1-0.15 mM; the concentration of the dissociated iron-deficiency protein is preferably 8-12 mg/mL, and more preferably 10 mg/mL. In the present invention, the mixing time is preferably 10 to 20min, and more preferably 15 min. In the present invention, the Apn/HA is preferably stored at 20 ℃ for 60 min.
After Apn/HA is obtained, the Apn/HA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and NHS solution are mixed to obtain a mixed solution, and first dialysis is performed on the mixed solution to obtain a first dialysate; the cut-off molecular weight of a dialysis membrane adopted in the first dialysis is 12-14 KD. In the present invention, the mixing time is preferably 1.5 to 2.5 hours, and more preferably 2 hours. In the present invention, the reagent used in the first dialysis is preferably physiological saline. In the practice of the present invention, fresh physiological saline solution is used instead every 8 hours.
After the first dialysate is obtained, the first dialysate and the nucleic acid AS1411 aptamer solution are mixed for modification to obtain a modified product, wherein the modified product comprises the protein nano-carrier. In the present invention, the concentration of the aptamer solution of the nucleic acid AS1411 is preferably 0.1-0.15 mM. In the present invention, the solvent of the nucleic acid AS1411 aptamer solution is preferably sterilized water. After obtaining the modified product, the invention preferably further comprises performing a second dialysis on the modified product, and collecting a second dialysate, wherein the second dialysate contains the protein nanocarriers. In the present invention, the second dialysis serves to remove the free nucleic acid AS1411 aptamer.
The invention also provides application of the protein nano-carrier in the scheme in preparation of a targeted delivery carrier.
The invention also provides a carrier loaded with a targeting substance, which comprises the protein nano-carrier and a combination body, wherein the combination body is embedded in the shell of the protein nano-carrier, and the combination body is a combination body of double-chain siRNA and nuclear localization signal peptide. In the present invention, the double-stranded siRNA is used to silence breast cancer cells for tumor suppression; the double stranded siRNA is introduced into the nucleus under the localization of the nuclear localization signal peptide.
In the invention, the double-stranded siRNA is obtained by base complementary pairing of a first RNA and a second RNA; the nucleotide sequence of the first RNA is shown as SEQ ID NO: 1, specifically: 5' -AAGCGGUCGGCGCGGGAACCAAAAAAA- (CH)2)3-NH2-3'; the nucleotide sequence of the second RNA is shown as SEQ ID NO: 2, specifically: 3 '-UUCGCCAGCCGCGCCCUUGGU-5'.
In the invention, the amino acid sequence of the nuclear localization signal peptide is shown as SEQ ID NO: 5, respectively.
In the present invention, the method for preparing the double-stranded siRNA preferably comprises the steps of: and mixing the first RNA solution and the second RNA solution, and carrying out base complementary pairing reaction to obtain the double-stranded siRNA. In the present invention, the concentration of the first RNA solution and the concentration of the second RNA solution are independently preferably 0.5 to 1.5mM, and more preferably 1 mM. In the present invention, the volume ratio of the first RNA solution to the second RNA solution is preferably (2-3): (2-3). In the present invention, the solvent of the first RNA solution and the second RNA solution is sterilized water or DEPC water. In the invention, the time of the base complementary pairing reaction is preferably 5-6 h; the temperature of the base complementary pairing reaction is preferably 20 to 30 ℃, and more preferably 25 ℃.
The invention also provides a preparation method of the carrier in the scheme, which comprises the following steps:
1) mixing the double-stranded siRNA, the nuclear localization signal peptide, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and the N-hydroxysuccinimide solution, and carrying out a binding reaction to obtain a binding reaction product, wherein the binding reaction product contains a binding body of the siRNA and the nuclear localization signal peptide and is named as siRNA/NLS;
2) dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; mixing the combined reaction product, an influenza virus hemagglutinin solution and the dissociated iron-deficient protein to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure, wherein the core-shell structure is named as Apn/siRNA/NLS/HA;
3) mixing the Apn/siRNA/NLS/HA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and NHS solution to obtain a mixed solution, and performing first dialysis on the mixed solution to obtain a first dialysate; the cut-off molecular weight of a dialysis membrane adopted by the first dialysis is 12-14 KD;
4) and mixing the first dialysate with the aptamer solution of the nucleic acid AS1411, and modifying to obtain a modified product, wherein the modified product comprises the carrier.
Firstly, mixing double-stranded siRNA, nuclear localization signal peptide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution, and carrying out a binding reaction to obtain a binding reaction product, wherein the binding reaction product comprises a combination of the siRNA and the nuclear localization signal peptide and is named as siRNA/NLS. In the invention, the time of the combination reaction is preferably 5-6 h; the temperature of the binding reaction is preferably 20-30 ℃. In the present invention, the solvent of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and the N-hydroxysuccinimide solution is preferably sterilized water. The molar concentration ratio of the double-stranded siRNA, the nuclear localization signal peptide, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and the N-hydroxysuccinimide solution used in the synthesis process is (0.8-10): (0.8-1): (18-20): (18-20).
After a binding reaction product is obtained, dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; and mixing the combined reaction product, the influenza virus hemagglutinin solution and the dissociated ferritin to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure named as Apn/siRNA/NLS/HA. In the present invention, the step of dissociating the ferritin at a pH of 2 to 2.5 preferably comprises adding 0.1M hydrochloric acid to the ferritin to lower the pH of the solution to 2 to 2.5, thereby dissociating the ferritin. In the present invention, the solvent of the influenza virus hemagglutinin solution is preferably sterilized water or DEPC water. In the invention, the volume ratio of the binding reaction product, the influenza virus hemagglutinin solution and the dissociation iron-deficiency protein is preferably (2-3): (0.5-1.5): (0.5 to 1.5); the concentration of the influenza virus hemagglutinin solution is preferably 0.1-0.15 mM; the concentration of the dissociated iron-deficiency protein is preferably 8-12 mg/mL, and more preferably 10 mg/mL. In the invention, after the assembling, the method preferably further comprises the steps of sequentially carrying out enzymolysis on the assembled mixture by using RNaseA and proteinase K; the reaction concentration of the RNase A is preferably 0.5 mg/ml; the reaction concentration of the proteinase K is preferably 0.5 mg/ml; the temperature of the enzymolysis is preferably 37 ℃; the time for enzymolysis by RNaseA is preferably 25-35 min, and more preferably 30 min; the time for enzymolysis by adopting protease K is preferably 25-35 min, and further preferably 30 min; in the present invention, RNase A digests all free double-stranded siRNA outside the iron-deficient protein shell, and proteinase K degrades RNase. In the present invention, the mixing time is preferably 10 to 20min, and more preferably 15 min. In the present invention, the Apn/siRNA/NLS/HA is preferably preserved at 20 ℃ for 60 min.
After Apn/siRNA/NLS/HA is obtained, the Apn/siRNA/NLS/HA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and NHS solution are mixed to obtain mixed solution, and first dialysis is performed on the mixed solution to obtain first dialysate; the cut-off molecular weight of a dialysis membrane adopted in the first dialysis is 12-14 KD. In the present invention, the mixing time is preferably 1.5 to 2.5 hours, and more preferably 2 hours. In the present invention, the reagent used in the first dialysis is preferably physiological saline. In the practice of the present invention, fresh physiological saline solution is used instead every 8 hours.
After the first dialysate is obtained, the first dialysate and the nucleic acid AS1411 aptamer solution are mixed and modified to obtain a modified product, wherein the modified product contains the vector and is named AS Apn/siRNA/NLS/HA/Apt. In the present invention, the concentration of the aptamer solution of the nucleic acid AS1411 is preferably 0.1-0.15 mM. In the present invention, the solvent of the nucleic acid AS1411 aptamer solution is preferably sterilized water or DEPC water. After obtaining the modified product, the invention preferably further comprises performing a second dialysis on the modified product, and collecting a second dialysate, wherein the second dialysate contains the protein nanocarriers. In the present invention, the second dialysis serves to remove the free nucleic acid AS1411 aptamer.
In the present invention, the process of drug release and loading of a targeting agent loaded carrier is schematically illustrated in fig. 1. The rise and fall in pH is a process that triggers the dissociation and recombination of Apn, and can occur both intracellularly and extracellularly. Outside the cell, by adjusting the pH; when the vector passes through lysosomes in cells, the lysosomes are acidic in vivo, and the pH decreases and rises as the vector escapes from the lysosomes. At the target region of the aptamer of nucleic acid AS1411, the protein nanocarrier enters the cell. The acidic environment of the lysosome (pH drop) results in the disruption of the ferritin nanocage and the release of the siRNA and nuclear localization signal peptide conjugate and influenza virus hemagglutinin. The influenza virus hemagglutinin can damage a lysosome membrane, so that a combination of siRNA and a nuclear localization signal peptide escapes from a lysosome, a large amount of siRNA is released into cytoplasm, and the integrity and the effectiveness of the siRNA are maintained to the maximum extent. The siRNA is introduced into the nucleus under nuclear localization signal peptide localization. Gene therapy will be performed in the genetic information base of the nucleus, and may be more effective in suppressing or even killing cancer cells. In the whole process, the siRNA contained in the ferritin-deficient nano shell better retains the pharmacological activity, and the ferritin-deficient shell has no obvious toxic or side effect, so that a new method is provided for safer and more effective targeted delivery.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Materials and reagents
DNA oligonucleotides were synthesized and purified by Shanghai three photobiotechnology, Inc., and the sequences of DNA and RNA oligonucleotides are shown in Table 1. 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-Dimethylmethylaminopyridine (DMAP) were purchased from Aladdin. Ferritin is obtained from sigma aldrich (st. louis, missouri). 2- (4-aminophenyl) -6-indolcarbamide hydrochloride (DAPI) and 3,3' -octacosyloxycarbocyanine (DIO) were purchased from Beyotime. A 300 mesh copper mesh was purchased from prositech. Throughout the experiment, Sartorius ultrapure water (18.2M Ω cm) was used.
2. Instrument for measuring the position of a moving object
The product was characterized by Transmission Electron Microscopy (TEM) (JEM-2100, JEOL). All samples were recorded on an F-4600 fluorescence spectrophotometer (Hitachi) and flow cytometer (Beckman Kurt). The average particle size and zeta potential were measured by Dynamic Light Scattering (DLS) and confocal fluorescence imaging studies were performed using a Laser Scanning Confocal Microscope (LSCM) (Nikon C2 plus) with objective lens (× 20).
TABLE 1 sequence Listing of nucleotides and Polypeptides
Figure BDA0002897270220000091
3. Preparation of siRNA/NLS
To synthesize siRNA/NLS, 20. mu.L of 1mM RNA 1(TK1Pro-siRNA sense) and 20. mu.L of 1mM RNA 2(TK1-Pro-siRNA antisense) were mixed and reacted at room temperature for 6h, and reacted with 20. mu.L of 1mM NLS under the action of 10. mu.L of 2mM EDC and 10. mu.L of 2mM NHS for 6 h. And extracting RNA from polyacrylamide gel by using a multi-gel RNA extraction kit. After RNA separation by polyacrylamide gel electrophoresis, the gel containing the target RNA fragment was excised and crushed with two pieces of RNase-free glass. The gel was soaked in a buffer containing EDTA and transferred to a filtration column to remove gel debris. After addition of fresh binding buffer, the gel was transferred to a Hibind spin column and bound to RNA centrifugation. After a brief wash, the RNA was washed with DEPC water.
4. Preparation of Apn/siRNA/NLS/HA/Apt
0.1M hydrochloric acid was added to the ferritin to lower the pH of the solution to 2.0 and dissociate the ferritin. Add 20. mu.L siRNA/NLS and 10. mu.L 0.1mM influenza hemagglutinin to 10. mu.L 10 mg. multidot.mL-1In (3) iron deficiency protein. The solution was mixed for 15min, then 0.1M sodium hydroxide was added to raise the pH to 7.4 and encapsulate the siRNA/NLS and HA within the ferritin. The mixture was stored at 20 ℃ for 60 min. The treatment with 0.5mg/ml RNase A at 37 ℃ for 30min followed by 0.5mg/ml proteinase K at 37 ℃ for 30 min. RNase A digests all free siRNA except for iron-deficient proteins, and proteinase K degrades RNase. Apn/siRNA/NLS/HA was added to 10. mu.L of 2mM EDC and 10. mu.L of 2mM NHS for 2 h. The resulting solution was then transferred to dialysis bags (molecular weight cut-off 12-14 kD) and dialyzed against 600ml of 0.9% NaCl solution for 24h (replaced every 8h with fresh 0.9% NaCl solution) to remove free EDC and NHS. mu.L of 0.1mM aptamer (AS1411) was added to Apn/siRNA/NLS/HA to form Apn/siRNA/NLS/HA/Apt. The solution was then transferred to a dialysis bag to remove free aptamers.
Cell culture
MCF-7 cells were incubated at 37 ℃ with 5% CO2In the environment of (1), in the culture medium10% heat-inactivated fetal bovine serum, penicillin (50 units/mL), streptomycin (50. mu.g/mL) were added.
Characterization of Apn/siRNA/NLS/HA/Apt
We observed ferritin deficiency and Apn/siRNA/NLS/HA/Apt by transmission electron microscopy, indicating that the two substances have uniform particle size and good dispersibility (FIGS. 2 and 3). Dynamic light scattering measurements showed that the mean particle size of the ferritin-deficient nanocages was 10.57mM, while the mean particle size of the Apn/siRNA/NLS/HA/Apt increased to 13.13mM (FIG. 4), which is consistent with the results obtained by transmission electron microscopy. Meanwhile, we also detected zeta potentials of iron-deficient protein and Apn/siRNA/NLS/HA/Apt. It was found that the ferritin-deficient potential was reduced from-5.02 mV to-9.87 mV after modification (FIG. 5).
Compared to normal tissues, the tumor microenvironment has a higher acidic pH, and cancer cells strongly depend on glycolysis rather than oxidative phosphorylation to consume energy to increase biosynthesis, resulting in increased acidity of lactate production. Ferritin-deficient nanocages can break down into protein subunits and release encapsulated molecules in an acidic environment. We evaluated the performance of Apn/siRNA/NLS/HA/Apt to selectively release the targeting substance in the tumor microenvironment under simulated physiological conditions (PBS, pH7.4) and acidic environment (acetate buffer, pH 5.5). The release of the target substance is verified by an in vitro fluorescence intensity method. Apn/siRNA/NLS/HA/apt3h, 6h, 9h, 12h, 15h and 24h were first incubated at pH5.5, pH7.4 and 37 ℃ and the release of siRNA was then monitored by measuring the fluorescence intensity of the solution. The results are shown in FIG. 6. As can be seen from fig. 6, the protein nanocarrier of the present invention can efficiently release the target substance when pH is lowered.
Cytotoxicity assays
MCF-7 cells were seeded in 96-well plates at a density of 1X 10 per well4And (4) cells. After 12h, cells were treated with ferritin-deficient protein, ferritin-deficient protein/siRNA/Apt, Apn/siRNA/nuclear localization signal peptide/Apt, ferritin-deficient protein/siRNA/HA/Apt, and Apn/siRNA/NLS/HA/Apt, respectively. After treatment, the cells were detected with a microplate reader and the absorbance recorded. The results show that the survival rate of MCF-7 cells incubated by ferritin is higher (>96 percent) and fully proves that the ferritin-deficient nano-scaleThe cage has good biocompatibility. The ferritin/siRNA has certain toxicity to cells, but the toxicity of the ferritin/siRNA/Apt group is obviously lower than that of the ferritin/siRNA/Apt group due to the lack of targeting and escape capability. Compared with the control group, the cytotoxicity of the Apn/siRNA/nuclear localization signal peptide/Apt and the iron-deficient protein/siRNA/HA/Apt is obviously higher than that of the former two groups, but still lower than that of the Apn/siRNA/NLS/HA/Apt group. The survival rate of the cells treated by the Apn/siRNA/NLS/HA/Apt is reduced to 38 percent, which shows that the influenza virus hemagglutinin and the nuclear localization signal peptide play important roles in lysosome escape and siRNA translocation to the nucleus, and the siRNA also HAs good therapeutic effect on tumor cells, thereby being a promising therapeutic strategy (figure 7).
Flow cytometry assay
Cell uptake experiments were performed under flow cytometry and fluorescence microscopy. Cell density of 1X 105The MCF-7 cells of (A) were isolated and collected in serum-free colorless DMEM medium. The cells were then cultured at 37 ℃. Next, a conjugate of ferritin-deficient protein/Cy 5-siRNA and nuclear localization signal peptide/HA/Apt was added and cultured for 8 h. The residue containing the cell particles was again placed in DMEM medium (1mg/mL) containing trypsin and cultured for another 1 min. Finally, the suspension was resuspended in PBS by centrifugation and analyzed by flow cytometry. The larger the peak value, the stronger the fluorescence intensity. From fig. 8, it can be found that the peak position is significantly shifted and the fluorescence intensity is significantly increased.
Feasibility study of combination of ferritin/Cy 5-siRNA and nuclear localization signal peptide/HA/Apt for cell fluorescence imaging
MCF-7 cells were seeded on glass substrates at a density of 8000 cells per plate under a fluorescence microscope and incubated for 12 h. In the Apn/siRNA/NLS/HA/Apt uptake studies, cells were treated with a conjugate of Apn/Cy5-siRNA and nuclear localization signal peptide/HA/Apt for 6h and 12h, respectively, and cells were treated with ferritin for 6 h. MCF-7 cells were then washed 3 times with PBS. Then, nuclei were labeled with DAPI and cell membranes were labeled with DIO. MCF-7 cells were washed 3 times with PBS. Finally, the slides were mounted and viewed directly with a Laser Scanning Confocal Microscope (LSCM). As can be seen from FIG. 9, the ferritin-deficient protein of group A HAs substantially no fluorescence, and the conjugated product of Apn/Cy5-siRNA and nuclear localization signal peptide/HA/Apt of group B shows strong fluorescence, which can be distinguished from normal cells, and the imaging effect is very good. The fluorescence of the conjugate of the Apn/Cy5-siRNA and the nuclear localization signal peptide/HA/Apt is stable, the C picture shows that the conjugate of the Apn/Cy5-siRNA and the nuclear localization signal peptide/HA/Apt still HAs obvious fluorescence after the Apn/Cy5-siRNA and the nuclear localization signal peptide are incubated for 12 hours, and the good targeting capability and the stable imaging effect of the conjugate of the Apn/Cy5-siRNA and the nuclear localization signal peptide/HA/Apt are fully proved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of Qilu Industrial science
<120> protein nano-carrier, carrier loaded with targeting substance, preparation method and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aagcggucgg cgcgggaacc aaaaaaa 27
<210> 2
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
uucgccagcc gcgcccuugg u 21
<210> 3
<211> 27
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aagcggucgg cgcgggaacc aaaaaaa 27
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggtggtggtg gttgtggtgg tggtgg 26
<210> 5
<211> 34
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Cys Gly Gly Gly Gly Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly
1 5 10 15
Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Pro Lys Lys Lys Arg
20 25 30
Lys Val
<210> 6
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly
1 5 10 15
Met Ile Asp Gly Trp Tyr Gly
20

Claims (10)

1. A protein nano-carrier takes aptamer modified iron-deficient protein as a shell, and is embedded with influenza virus hemagglutinin.
2. The protein nanocarrier of claim 1, wherein the nucleic acid aptamer comprises a nucleic acid AS1411 aptamer.
3. Use of the protein nanocarrier of claim 1 or 2 in the preparation of a targeted delivery vehicle.
4. A targeting substance-loaded carrier comprising the protein nanocarrier of claim 1 or 2 and a conjugate, wherein the conjugate is embedded in a shell of the protein nanocarrier, and the conjugate is a conjugate of double-stranded siRNA and a nuclear localization signal peptide.
5. The vector of claim 4, wherein the double stranded siRNA is derived from base complementary pairing of a first RNA and a second RNA; the nucleotide sequence of the first RNA is shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the second RNA is shown as SEQ ID NO: 2, respectively.
6. The vector according to claim 5, wherein the preparation method of the double-stranded siRNA comprises the following steps: mixing the first RNA solution and the second RNA solution, and carrying out base complementary pairing reaction to obtain double-stranded siRNA; the time of the base complementary pairing reaction is 5-6 h; the temperature of the base complementary pairing reaction is 20-30 ℃.
7. A method for preparing the carrier of any one of claims 4 to 6, comprising the steps of:
1) mixing double-stranded siRNA, nuclear localization signal peptide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and N-hydroxysuccinimide solution, and carrying out a binding reaction to obtain a binding reaction product, wherein the binding reaction product comprises a combination of the siRNA and the nuclear localization signal peptide and is named as siRNA/NLS;
2) dissociating the iron-deficiency protein under the condition that the pH value is 2-2.5 to obtain dissociated iron-deficiency protein; mixing the combined reaction product, an influenza virus hemagglutinin solution and the dissociated iron-deficient protein to obtain a mixed solution, adjusting the pH value of the mixed solution to 7.4-7.6, and assembling to obtain a core-shell structure, wherein the core-shell structure is named as Apn/siRNA/NLS/HA;
3) mixing the Apn/siRNA/NLS/HA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and NHS solution to obtain a mixed solution, and performing first dialysis on the mixed solution to obtain a first dialysate; the cut-off molecular weight of a dialysis membrane adopted by the first dialysis is 12-14 KD;
4) and mixing the first dialysate with the aptamer solution of the nucleic acid AS1411, and modifying to obtain a modified product, wherein the modified product comprises the carrier.
8. The preparation method according to claim 7, wherein the time of the combination reaction in the step 1) is 5-6 h; the temperature of the bonding reaction is 20-30 ℃.
9. The method of claim 7, wherein the assembling further comprises subjecting the assembled mixture to enzymatic hydrolysis using RNase A and proteinase K in sequence.
10. The preparation method of claim 7, wherein the mixing time in the step 3) is 1.5-2.5 h.
CN202110046195.3A 2021-01-14 2021-01-14 Protein nano-carrier, carrier loaded with targeting substance, preparation method and application Active CN112826943B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110046195.3A CN112826943B (en) 2021-01-14 2021-01-14 Protein nano-carrier, carrier loaded with targeting substance, preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110046195.3A CN112826943B (en) 2021-01-14 2021-01-14 Protein nano-carrier, carrier loaded with targeting substance, preparation method and application

Publications (2)

Publication Number Publication Date
CN112826943A true CN112826943A (en) 2021-05-25
CN112826943B CN112826943B (en) 2022-06-03

Family

ID=75928113

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110046195.3A Active CN112826943B (en) 2021-01-14 2021-01-14 Protein nano-carrier, carrier loaded with targeting substance, preparation method and application

Country Status (1)

Country Link
CN (1) CN112826943B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113470751A (en) * 2021-06-30 2021-10-01 南方科技大学 Protein nanopore amino acid sequence screening method, protein nanopore and application of protein nanopore
CN114984217A (en) * 2022-06-21 2022-09-02 南京林业大学 Preparation method and application of aptamer ferritin nanoparticles co-loaded with paclitaxel and manganese phthalocyanine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110327308A (en) * 2019-07-02 2019-10-15 中国药科大学 A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA
CN111249469A (en) * 2020-03-20 2020-06-09 临沂大学 Peptide nanoparticle capable of escaping lysosome and preparation method and application thereof
CN111939138A (en) * 2019-05-17 2020-11-17 南京林业大学 Method for preparing taxol nano-drug by taking apoferritin as carrier and recovering taxol

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111939138A (en) * 2019-05-17 2020-11-17 南京林业大学 Method for preparing taxol nano-drug by taking apoferritin as carrier and recovering taxol
CN110327308A (en) * 2019-07-02 2019-10-15 中国药科大学 A kind of recombination apoferritin nanocages and preparation method thereof being loaded with siRNA
CN111249469A (en) * 2020-03-20 2020-06-09 临沂大学 Peptide nanoparticle capable of escaping lysosome and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NA LI ET AL.: ""Nuclear-targeted siRNA delivery for long-term gene silencing"", 《CHEM. SCI.》 *
NA LI ET AL.: ""Nuclear-targeted siRNA delivery for long-term gene silencing"", 《CHEM. SCI.》, vol. 8, no. 2017, 19 January 2017 (2017-01-19), pages 2816 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113470751A (en) * 2021-06-30 2021-10-01 南方科技大学 Protein nanopore amino acid sequence screening method, protein nanopore and application of protein nanopore
CN114984217A (en) * 2022-06-21 2022-09-02 南京林业大学 Preparation method and application of aptamer ferritin nanoparticles co-loaded with paclitaxel and manganese phthalocyanine

Also Published As

Publication number Publication date
CN112826943B (en) 2022-06-03

Similar Documents

Publication Publication Date Title
Wen et al. Utilizing viral nanoparticle/dendron hybrid conjugates in photodynamic therapy for dual delivery to macrophages and cancer cells
Gonçalves et al. Enhanced targeting of invasive glioblastoma cells by peptide-functionalized gold nanorods in hydrogel-based 3D cultures
Oh et al. Electrostatic charge conversion processes in engineered tumor-identifying polypeptides for targeted chemotherapy
Nuñez-Rivera et al. Brome mosaic virus-like particles as siRNA nanocarriers for biomedical purposes
Tesarova et al. Taking advantage of cellular uptake of ferritin nanocages for targeted drug delivery
CN112826943B (en) Protein nano-carrier, carrier loaded with targeting substance, preparation method and application
JP2011524446A (en) Chitosan oligosaccharide fatty acid graft product modified with polyglycol, its preparation method and use thereof
CN107184987B (en) Lipoic acid modified targeted integrin alpha v beta 3 nano-polypeptide carrier and preparation method and application thereof
Yang et al. Magainin II modified polydiacetylene micelles for cancer therapy
CN106890343B (en) Targeting polypeptide nano gene vector compound
Li et al. Dual-therapeutics-loaded mesoporous silica nanoparticles applied for breast tumor therapy
Tanziela et al. Cancer-exocytosed exosomes loaded with bio-assembled AgNCs as smart drug carriers for targeted chemotherapy
Gao et al. A progressively targeted gene delivery system with a pH triggered surface charge-switching ability to drive angiogenesis in vivo
CN113633625B (en) Nano-drug of hybrid membrane loaded oxidative phosphorylation inhibitor and preparation method thereof
CN107129522B (en) Lipoic acid modified inherent disordered protein nano-carrier and preparation method and application thereof
CA2994809A1 (en) Silica-based biomolecule carrier, pharmaceutical composition comprising the same, preparation method and use thereof
Cheng et al. Synthesis of biotin-modified galactosylated chitosan nanoparticles and their characteristics in vitro and in vivo
Tao et al. A novel biocompatible, simvastatin-loaded, bone-targeting lipid nanocarrier for treating osteoporosis more effectively
Siringan et al. Interactions between fullerene derivatives and biological systems
Zhang et al. Constructing a nanocage-based universal carrier for delivering TLR-activating nucleic acids to enhance antitumor immunotherapy
Jia et al. Messenger nanozyme for reprogramming the microenvironment of rheumatoid arthritis
CN111249469B (en) Peptide nanoparticle capable of escaping lysosome and preparation method and application thereof
CN114642736A (en) Blood-brain barrier penetrating drug delivery system and preparation method and application thereof
Yu et al. Microenvironment-responsive DNA-conjugated albumin nanocarriers for targeted therapy
Wan et al. Rapid preparation of hyperbranched β-CD functionalized hydroxyapatite based on host-guest reaction for cell imaging and drug delivery

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
TR01 Transfer of patent right

Effective date of registration: 20221222

Address after: Room 3115, No. 135, Ward Avenue, Ping'an Street, Changqing District, Jinan, Shandong 250300

Patentee after: Shandong Jiqing Technology Service Co.,Ltd.

Address before: 250353 University Road, Changqing District, Ji'nan, Shandong Province, No. 3501

Patentee before: Qilu University of Technology

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20231207

Address after: Room 901, 9th Floor, Building 10, Courtyard 2, Kechuang 6th Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing, 100176

Patentee after: Beijing Biotech Biotechnology Co.,Ltd.

Address before: Room 3115, No. 135, Ward Avenue, Ping'an Street, Changqing District, Jinan, Shandong 250300

Patentee before: Shandong Jiqing Technology Service Co.,Ltd.

TR01 Transfer of patent right