CN107158401B - Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof - Google Patents

Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof Download PDF

Info

Publication number
CN107158401B
CN107158401B CN201610128330.8A CN201610128330A CN107158401B CN 107158401 B CN107158401 B CN 107158401B CN 201610128330 A CN201610128330 A CN 201610128330A CN 107158401 B CN107158401 B CN 107158401B
Authority
CN
China
Prior art keywords
calcium
polyethylene glycol
modified
microemulsion
particle
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.)
Active
Application number
CN201610128330.8A
Other languages
Chinese (zh)
Other versions
CN107158401A (en
Inventor
赵静
王志勇
黄萍
杨磊
何成宜
陈志英
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.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
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 Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to CN201610128330.8A priority Critical patent/CN107158401B/en
Publication of CN107158401A publication Critical patent/CN107158401A/en
Application granted granted Critical
Publication of CN107158401B publication Critical patent/CN107158401B/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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Optics & Photonics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention provides a calcium-based nano-drug delivery particle modified by polyethylene glycol, and a preparation method and application thereof. The particle size of the polyethylene glycol modified calcium-based nano-drug delivery particle provided by the invention is 20-200 nm; the polyethylene glycol modified calcium-based nano drug delivery particle comprises a first target delivery object and a calcium-based particle wrapped with the first target delivery object, wherein the surface of the calcium-based particle is modified with polyethylene glycol, the first target delivery object is a biological drug, a chemical drug or a second target delivery object wrapped with a nucleic acid fragment, and the second target delivery object wrapped with the nucleic acid fragment is a cationic polymer, a polypeptide, a polyamino acid or a transfection reagent wrapped with, combined with or blended with the nucleic acid fragment. The calcium-based nano-drug delivery particle modified by polyethylene glycol provided by the invention has the advantages that the particle size of the nano-particle is controllable in the preparation process, the surface of the calcium-based particle is protected by the polyethylene glycol chain, so that the in vivo circulation time is prolonged, and the preparation cost is low, the toxicity is low, and the preparation method is safe and effective.

Description

Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a calcium-based nano-drug delivery particle modified by polyethylene glycol and a preparation method and application thereof.
Background
Gene therapy holds promise for cure of many serious acquired and congenital diseases. However, the lack of a safe, efficient and stably expressed gene delivery system is the biggest obstacle to the entry of gene therapy into the clinic. Viral vectors are a common class of gene vectors, which use viral infection of a host cell to introduce a foreign gene into the host cell. Generally, an Adenovirus Vector (AV), an adeno-associated virus vector (AAV), a retrovirus, and the like are included. Although the viral vector has high transfection efficiency, the loading gene capacity of the viral vector is limited, and the immunogenicity has potential biological safety hazards, so that the clinical application of the viral gene vector is difficult to advance. In recent years, the appearance of minicircleDNA (mcDNA) has become a large bright spot in gene vectors, and minicircleDNA is a circular expression cassette, which is a product obtained by removing the bacterial DNA framework of a standard plasmid by a DNA recombination technology, i.e., minicircleDNA only contains a gene expression cassette and has no external bacterial framework sequence. However, since naked DNA is rapidly cleared in physiological environments (mainly by degradation by various enzymes in the body) and does not enter target nuclei efficiently, a suitable delivery system is still required to deliver the minicircle DNA to the target tissue or organ.
Common gene delivery systems include calcium silicate, cationic liposomes, cationic polymers (polyethyleneimine PEI), and the like. Such delivery systems have low immunogenicity, large gene loading capacity, but low transfection efficiency, greatly limiting their clinical application. Therefore, the key to the clinical application of gene medicine is to develop a gene delivery system with high efficiency, good biocompatibility and targeting. Calcium silicon base (CaO. SiO)2The basic) biological material is a novel biological active material which is started in the last thirty years, the introduction of calcium element enables the basic material to have better biological activity, biocompatibility and biodegradability than the traditional silicon-based material, the basic material can be gradually converted into bone-like apatite under the environment of simulated body fluid, and the basic material is a giant material with huge biological activity, biocompatibility and biodegradabilityInorganic biomaterials with large application prospect have been widely applied in the fields of drug loading, bone repair, tooth repair and the like, and calcium silicate as a gene vector is rarely reported. Calcium silicate particles prepared by a traditional precipitation method have poor granularity controllability, are easy to agglomerate in an aqueous solution, and have poor dispersibility. The invention prepares monodisperse calcium silicate nano-particles by a microemulsion method, wraps micro-ring DNA in the calcium silicate particles, modifies polyethylene glycol (PEG) outside, and utilizes a hydration layer formed by the polyethylene glycol (PEG) to protect the DNA from being removed by a reticuloendothelial system (RES) of an organism and degradation of various DNase in the in-vivo delivery process, thereby realizing the targeted delivery of the micro-ring DNA.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a calcium-based nano-drug delivery particle modified by polyethylene glycol and a preparation method and application thereof.
In a first aspect, the invention provides a calcium-based nano-drug delivery particle modified by polyethylene glycol, wherein the particle size of the calcium-based nano-drug delivery particle modified by polyethylene glycol is 20-200 nm; the polyethylene glycol modified calcium-based nano drug delivery particle comprises a first target delivery object and a calcium-based particle wrapped with the first target delivery object, wherein the surface of the calcium-based particle is modified with polyethylene glycol, the first target delivery object is a biological drug, a chemical drug or a second target delivery object wrapped with a nucleic acid fragment, and the second target delivery object wrapped with the nucleic acid fragment is a cationic polymer, a polypeptide, a polyamino acid or a transfection reagent wrapped with, combined with or blended with the nucleic acid fragment.
As used herein, "nucleic acid fragment" includes natural or synthetic DNA fragments, RNA fragments, mRNA, siRNA, shRNA and the like.
Optionally, the biological drug includes, but is not limited to, one or more of a nucleic acid, a polypeptide, a protein, and a vaccine; the chemical drugs include but are not limited to one or more of anti-tumor small molecule drugs, fluorescein and lymph tracer.
Preferably, the drug loading rate of the calcium-based nano drug delivery particle modified by polyethylene glycol is 0.001-0.85, and further preferably 0.05-0.85.
As used herein, the "drug loading" is the mass ratio of the encapsulated drug to the polyethylene glycol-modified calcium-based nano-drug delivery particle.
Optionally, in the first target delivery, the encapsulated drug is a biologic or a chemical; in the second target delivery, the encapsulated drug is a nucleic acid fragment.
Preferably, the calcium base is a biodegradable calcium salt including, but not limited to, calcium silicate, calcium phosphate, calcium citrate, or calcium carbonate.
Preferably, the cationic polymer (e.g., polyethyleneimine), polypeptide, polyamino acid, or transfection reagent encapsulating, binding, or blending the nucleic acid fragments may each be independently selected from the cationic polymers, polypeptides, polyamino acids, transfection reagents exemplified in table 1:
TABLE 1
Figure BDA0000936206220000021
Figure BDA0000936206220000031
The chinese and english references in table 1 are shown in table 2:
TABLE 2
Figure BDA0000936206220000032
Further preferably, the second target delivery object coated with the nucleic acid fragments is prepared by mixing and incubating a cationic polymer (such as polyethyleneimine), a polypeptide, a polyamino acid or a commercial transfection reagent with a solution containing the nucleic acid fragments according to a nitrogen-phosphorus ratio of 0.1-100: 1 (preferably according to a nitrogen-phosphorus ratio of 10-20: 1) (preferably for 5-40 min).
As used herein, the "nitrogen to phosphorus ratio" is the ratio of the number of moles of amino groups in a cationic polymer (e.g., polyethyleneimine), polypeptide, polyamino acid, or commercial transfection reagent to the number of moles of phosphate groups in a solution containing the nucleic acid fragments.
Further preferably, the second target delivery object carrying the nucleic acid fragments is prepared by mixing and incubating a polyethyleneimine solution or polyethyleneimine derivative (PEICL) and a solution containing the nucleic acid fragments according to a nitrogen-phosphorus ratio of 0.1-100: 1 for 5-40 min, wherein the molecular weight of the polyethyleneimine is 600-70000 daltons; the molecular weight of the polyethyleneimine derivative (PEICL) is 1000-70000 daltons (preferably 34000-70000 daltons), the structural formula of the polyethyleneimine derivative (PEICL) is shown as a formula I, wherein x, y and z are non-negative integers, and x, y and z are not zero;
Figure BDA0000936206220000041
even more preferably, the polyethyleneimine has a molecular weight of 25000 daltons.
Preferably, the particle size of the polyethylene glycol modified calcium-based nano drug delivery particle is 20-40 nm.
In a second aspect, the present invention provides a method for preparing a calcium-based nano drug delivery particle modified by polyethylene glycol, comprising the following steps:
(1) uniformly mixing the first target delivery substance solution and the first soluble calcium salt solution, adding the mixture into the organic phase, and stirring for 20-60 min to prepare a microemulsion A; wherein the first target delivery object is a biological drug, a chemical drug or a second target delivery object coated with a nucleic acid fragment; the organic phase is prepared by mixing a surfactant and an alkane compound with 6-12 carbon atoms (preferably in a volume ratio of 25-40: 60-75), wherein the surfactant is one or more of polyethylene glycol nonylphenyl ether (Igepal CO-520), polyethylene glycol octylphenyl ether (Triton X-100), sodium bis (2-ethylhexyl) succinate sulfonate (AOT) and Sodium Dodecyl Sulfate (SDS); the second target delivery object coated with the nucleic acid fragments is a cationic polymer, polypeptide, polyamino acid or transfection reagent coated, combined or blended with the nucleic acid fragments, wherein the ratio of the mole number of amino groups in the cationic polymer, polypeptide, polyamino acid or transfection reagent to the mole number of phosphate groups in the nucleic acid fragments is 0.1-100: 1;
(2) adding a soluble salt solution into the microemulsion A obtained in the step (1), and stirring for 2-4 h to obtain a microemulsion B; the soluble salt solution comprises but is not limited to a soluble silicon salt solution, a soluble phosphate solution, a soluble citrate solution or a soluble carbonate solution, wherein the molar ratio of silicon in the soluble silicon salt solution, phosphorus in the soluble phosphate solution, citrate in the soluble citrate solution or carbonate in the soluble carbonate solution to calcium in the first soluble calcium salt solution in the step (1) is 0.01-10: 1 (preferably 0.43-3.5);
(3) adding a phosphoric acid-polyethylene glycol aqueous solution into the microemulsion B obtained in the step (2), and stirring for 2-4 hours to obtain a microemulsion C, wherein the ratio of the mass of phosphoric acid-polyethylene glycol in the phosphoric acid-polyethylene glycol aqueous solution to the amount of the calcium element in the first soluble calcium salt solution in the step (1) is 0.001-100: 1 (preferably 0.008-100);
(4) removing the organic phase in the microemulsion C obtained in the step (3), centrifuging and washing, and dispersing with water and/or ethanol to obtain the calcium-based nano-drug delivery particle modified by polyethylene glycol; wherein the particle size of the calcium-based nano-drug delivery particle modified by the polyethylene glycol is 20-200 nm; the polyethylene glycol modified calcium-based nano drug delivery particle comprises a first target delivery object and a calcium-based particle wrapped with the first target delivery object, wherein the surface of the calcium-based particle is modified with polyethylene glycol, the first target delivery object is a biological drug, a chemical drug or a second target delivery object wrapped with a nucleic acid fragment, and the second target delivery object wrapped with the nucleic acid fragment is a cationic polymer, a polypeptide, a polyamino acid or a transfection reagent wrapped with, combined with or blended with the nucleic acid fragment.
In the second aspect of the present invention, a method for preparing polyethylene glycol modified calcium-based nano drug delivery particles is provided, wherein the second target delivery object is formed by encapsulating nucleic acid fragments with electropositive cationic polymers, polypeptides, polyamino acids or transfection reagents to form small particles of several tens to hundreds of nanometers, so as to provide a soft template for the subsequent growth of calcium-based drugs; further, the size of the prepared calcium-based nanoparticles can be adjusted by adjusting the volume of the aqueous system and the organic phase. Still further, the second aspect of the present invention provides a method for preparing a calcium-based nano-drug delivery particle modified with polyethylene glycol, wherein the size of the prepared calcium-based nano-particle can be adjusted by adjusting the amount of the nucleic acid fragment and the nitrogen-phosphorus (N/P) ratio of the nucleic acid fragment to the cationic polymer.
Optionally, in step (1), the biological drug includes, but is not limited to, one or more of nucleic acids, polypeptides, proteins, and vaccines; the chemical drugs include, but are not limited to, one or more of anti-tumor small molecule drugs, fluorescein, and lymphatic tracers.
Preferably, in the step (1), the nucleic acid is wrapped, combined or blended with a cationic polymer (such as polyethyleneimine), polypeptide, polyamino acid or transfection reagent containing nucleic acid fragments, and the cationic polymer, polypeptide, polyamino acid and transfection reagent can be respectively and independently selected from the cationic polymers, polypeptides, polyamino acids and transfection reagents listed in table 1.
Further preferably, in the second target delivery material coated with nucleic acid fragments, the ratio of the number of moles of amino groups in a cationic polymer (e.g., polyethyleneimine), a polypeptide, a polyamino acid, or a commercial transfection reagent to the number of moles of phosphate groups in a solution containing nucleic acid fragments is 10-20: 1.
Optionally, the second target delivery object coated with the nucleic acid fragments is prepared by mixing and incubating a cationic polymer (such as polyethyleneimine), a polypeptide, a polyamino acid or a commercial transfection reagent with a solution containing the nucleic acid fragments (preferably for 5-40 min).
Further preferably, the second target delivery object carrying the nucleic acid fragments is prepared by mixing and incubating a polyethyleneimine solution or polyethyleneimine derivative (PEICL) and a solution containing the nucleic acid fragments according to a nitrogen-phosphorus ratio of 0.1-100: 1 for 5-40 min, wherein the molecular weight of the polyethyleneimine is 600-70000 daltons; the molecular weight of the polyethyleneimine derivative (PEICL) is 1000-70000 daltons (preferably 34000-70000 daltons), the structural formula of the polyethyleneimine derivative (PEICL) is shown as a formula I, wherein x, y and z are non-negative integers, and x, y and z are not zero;
Figure BDA0000936206220000061
even more preferably, the polyethyleneimine has a molecular weight of 25000 daltons.
Preferably, in the step (1), the concentration of the first soluble calcium salt solution is 1-1000 mM.
Preferably, in step (1), the first soluble calcium salt solution includes, but is not limited to, one or more of calcium chloride, calcium nitrate and calcium bromide.
Preferably, in the step (1), the organic phase is prepared by mixing cyclohexane and polyethylene glycol nonylphenyl ether according to the volume ratio of 7: 3.
Preferably, in the step (1), the ratio of the total volume of the first target delivery substance solution and the first soluble calcium salt solution to the volume of the organic phase is 0.01-0.05: 1, and more preferably 0.01-0.02: 1.
Preferably, in the step (2), the volume ratio of the soluble salt solution to the microemulsion A obtained in the step (1) is 0.01-0.1: 1, and more preferably 0.02-0.1: 1.
Preferably, in the step (2), the soluble silicon salt includes, but is not limited to, one or more of ethyl orthosilicate, sodium silicate and potassium silicate.
Preferably, in the step (2), the concentration of silicate in the soluble silicate solution is 1 to 1000 mM.
Preferably, in the step (2), the soluble phosphate includes, but is not limited to, one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, and ammonium phosphate.
Preferably, in the step (2), the concentration of phosphate in the soluble phosphate solution is 1 to 1000 mM.
Preferably, in the step (2), the soluble citrate includes, but is not limited to, one or more of sodium citrate, calcium citrate and potassium citrate.
Preferably, in the step (2), the concentration of citrate in the soluble citrate solution is 1-1000 mM.
Preferably, in the step (2), the soluble carbonate includes, but is not limited to, one or more of sodium carbonate, sodium bicarbonate and potassium carbonate.
Preferably, in the step (2), the concentration of carbonate in the soluble carbonate solution is 1-1000 mM.
Preferably, in the step (2), the soluble salt solution is added into the microemulsion A obtained in the step (1), and the microemulsion B is prepared by stirring for 2-4 hours, and specifically comprises the following steps:
adding a soluble salt solution into the microemulsion A obtained in the step (1), stirring for 2-4 h, then adding a second soluble calcium salt solution, and stirring for 15-18 h to obtain a microemulsion B; wherein the molar ratio of the calcium element in the second soluble calcium salt solution to the calcium element in the first soluble calcium salt solution in the step (1) is 0.1-10: 1 (more preferably 0.45-8: 1).
Further preferably, the first soluble calcium salt solution and the second soluble calcium salt solution are each independently selected from one or more of calcium chloride, calcium nitrate and calcium bromide.
Further preferably, the concentration of the second soluble calcium salt solution is 1 to 1000 mM.
Further preferably, in the step (2), the soluble salt solution is added into the microemulsion a obtained in the step (1), and the microemulsion B is prepared by stirring for 2-4 hours, which specifically comprises:
adding a soluble salt solution into an organic solution, stirring for 1-2 h, adding into the microemulsion A obtained in the step (1), stirring for 2-4 h, adding a second soluble calcium salt solution, and stirring for 15-18 h to obtain a microemulsion B; the organic solution is prepared by mixing a surfactant and an alkane compound with 6-12 carbon atoms (preferably in a volume ratio of 25-40: 60-75), wherein the surfactant is one or more of polyethylene glycol nonylphenyl ether (Igepal CO-520), polyethylene glycol octylphenyl ether (Triton X-100), sodium bis (2-ethylhexyl) succinate sulfonate (AOT) and Sodium Dodecyl Sulfate (SDS); the molar ratio of the calcium element in the second soluble calcium salt solution to the calcium element in the first soluble calcium salt solution in the step (1) is 0.1-10: 1 (more preferably 0.45-8: 1).
Further preferably, in the step (2), the volume ratio of the organic solution to the organic phase in the step (1) is 1: 1.
Preferably, in the step (3), the volume ratio of the phosphoric acid-polyethylene glycol aqueous solution to the microemulsion B obtained in the step (2) is 0.002-0.03: 1.
Preferably, in the step (3), the molecular weight of the phosphoric acid-polyethylene glycol is 600-12000 daltons.
Further preferably, the molecular weight of the phospho-polyethylene glycol is 2000 daltons.
Preferably, in the step (3), the phosphoric acid-polyethylene glycol includes, but is not limited to, monophosphoric acid polyethylene glycol, diphosphonic acid polyethylene glycol, and maleimide phosphoric acid polyethylene glycol, wherein the monophosphoric acid polyethylene glycol has a structural formula shown in formula ii, n is a non-negative integer and is not zero,
Figure BDA0000936206220000071
the structural formula of the diphosphonic acid polyethylene glycol is shown as a formula III, n is a non-negative integer and is not zero,
Figure BDA0000936206220000081
the structural formula of the maleimide phosphoric acid polyethylene glycol is shown as a formula IV, n is a non-negative integer and is not zero,
Figure BDA0000936206220000082
further preferably, the phosphoric acid-polyethylene glycol is a compound shown as a formula II.
Preferably, in the step (3), the concentration of the phosphoric acid-polyethylene glycol aqueous solution is 1-1000 mM.
Preferably, in the step (3), the molar ratio of the phosphoric acid-polyethylene glycol in the phosphoric acid-polyethylene glycol aqueous solution to the calcium element in the first soluble calcium salt solution is 0.001-100: 1 (more preferably 0.008-100).
It is understood that, in the step (3), the polyethylene glycol end in the phospho-polyethylene glycol may be connected with a ligand including, but not limited to, folic acid, RGD (the "RGD" according to the present invention is composed of arginine, glycine and aspartic acid) and analogs thereof, polypeptide, galactose, transferrin or antibody, and in the step (4), the polyethylene glycol end in the polyethylene glycol modified calcium-based nano-drug delivery particle may be connected with a ligand including, but not limited to, folic acid, RGD and analogs thereof, polypeptide, galactose, transferrin or antibody.
Preferably, in the step (4), the particle size of the calcium-based nano drug delivery particle modified by polyethylene glycol is 20-40 nm.
Preferably, in the step (4), the drug loading rate of the calcium-based nano drug delivery particle modified by polyethylene glycol is 0.001-0.85 (more preferably 0.05-0.85).
It is understood that in the first target delivery, the encapsulated drug is a biological drug or a chemical drug; in the second target delivery, the encapsulated drug is a nucleic acid fragment.
Preferably, the polyethylene glycol modified calcium-based nano drug delivery particle according to the first aspect of the present invention is prepared by using the preparation method of the polyethylene glycol modified calcium-based nano drug delivery particle according to the second aspect.
In a third aspect, the present invention also provides a pharmaceutical composition comprising the polyethylene glycol-modified calcium-based nano-drug delivery particle according to the first aspect.
In a fourth aspect, the present invention also provides a method for preparing the polyethylene glycol-modified calcium-based nano-drug delivery particle according to the first aspect or the polyethylene glycol-modified calcium-based nano-drug delivery particle according to the second aspect, for use in preparing a drug.
Preferably, the use of a method of preparation of a polyethylene glycol modified calcium-based nano-drug delivery particle according to the first aspect or a polyethylene glycol modified calcium-based nano-drug delivery particle according to the second aspect for the preparation of a targeted delivery drug.
The invention has the beneficial effects that:
(1) compared with other inorganic nanoparticles, the particle size of the polyethylene glycol modified calcium-based nano-drug delivery particle provided by the invention is effectively controlled, is below 200nm, can form monodisperse particles in ethanol or water, and has stable dispersion property and no agglomeration; the surfaces of the calcium-based particles are protected by polyethylene glycol (PEG) chains, so that the particles can be prevented from being phagocytized by a reticuloendothelial system too early, the time of in vivo circulation is prolonged, and the calcium-based particles are used for in vivo transfection research;
(2) the size of the prepared calcium-based nanoparticles can be adjusted by adjusting the amount of the nucleic acid fragments, the nitrogen-phosphorus (N/P) ratio of the cationic polymer to the nucleic acid fragments, the volume of an aqueous system, the volume of an organic phase and other conditions;
(3) optionally, the other end of polyethylene glycol (PEG) is connected with ligands with different targeting properties, so that the particles can have specific targeting properties;
(4) the calcium-based nano drug delivery particle modified by polyethylene glycol provided by the invention has the advantages of low preparation cost, low toxicity, safety and effectiveness.
Drawings
Fig. 1-2 is a Transmission Electron Microscope (TEM) photograph (fig. 1) and a particle size distribution diagram (fig. 2) of the phosphate-PEG modified calcium silicate-minicircle DNA nanoparticle provided in the example of the present invention;
FIG. 3 is a fluorescent (A) and white light (B) images of in vitro co-culture of phospho-PEG modified calcium silicate-minicircle DNA (eGFP) nanoparticles and 293T cell line provided in the examples of the present invention;
FIG. 4 is a Transmission Electron Microscope (TEM) photograph (A) and a particle size distribution chart (B) of nanoparticles formed by coating the luteolin with polyethylene glycol (PEG) modified calcium silicate according to an embodiment of the present invention;
fig. 5 is a Transmission Electron Microscope (TEM) photograph (a) and a particle size distribution chart (B) of nanoparticles formed by coating an IR820 dye with polyethylene glycol (PEG) modified calcium silicate provided in the examples of the present invention.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
The structural formula of PEICL adopted by the embodiment of the invention is shown as a formula I, wherein x, y and z are non-negative integers, and x, y and z are not zero;
Figure BDA0000936206220000101
the phosphoric acid-polyethylene glycol adopted in the embodiments 1-4 and 6-17 of the invention is monophosphoric acid-polyethylene glycol, the structural formula of which is shown in a formula II, wherein n is a non-negative integer and is not zero,
Figure BDA0000936206220000102
example 1 preparation of calcium silicate-minicircle DNA nanoparticles without polyethylene glycol (PEG) modification
An embodiment of the present invention provides a method for preparing calcium silicate-minicircle DNA nanoparticles without polyethylene glycol (PEG) modification, comprising the following steps:
(1) mu.l of DNA solution (0.5. mu.g/. mu.l) was mixed well with 10. mu.l of PEI25K cationic polymer solution (1.3. mu.g/. mu.l) and incubated for 30 minutes; then, 80 μ l of calcium chloride solution (500mM, pH10.0) is added, and after being uniformly mixed by blowing with a liquid-transfering gun, the mixture is dropwise added into 5ml of organic phase (the organic phase is prepared by mixing cyclohexane and polyethylene glycol nonyl phenyl ether 520(Igepal CO-520) according to the volume ratio of 7/3) under stirring, and the mixture is stirred for 1 hour to form microemulsion A;
(2) dropwise adding Tetraethoxysilane (TEOS) into the microemulsion A obtained in the step (1) while stirring, and stirring for one hour to form microemulsion B;
(3) dropwise adding 100 mu l of calcium chloride solution (500mM, pH10.0) into the microemulsion B obtained in the step (2) under stirring, and stirring for 18 hours to obtain microemulsion C; and (3) evaporating to remove cyclohexane in the microemulsion C by using a rotary evaporator, centrifuging for 15 minutes at 14000g, washing with absolute ethyl alcohol for three times, and dispersing in water to obtain the calcium silicate-micro-ring DNA nanoparticles.
1. And (3) particle size measurement:
dissolving polyethylene glycol (PEG) -calcium silicate-micro-ring DNA nano-particles prepared in the embodiment of the invention in 1ml double distilled water, carrying out ultrasonic treatment for 30s, observing the particles by using a transmission electron microscope, and determining the particle size by using a Malvern dynamic light scattering particle size analyzer; the measurement results are shown in table 1.
2. And (3) determination of drug loading capacity: weighing 1mg of the calcium silicate-minicircle DNA nanoparticles obtained in the step (4), calculating the weight loss at the temperature of about 900 ℃ to obtain the drug loading capacity, wherein the heating rate is 10 ℃/min, and the atmosphere is flowing air atmosphere; drug loading is the mass of DNA/total mass of nanoparticles.
The experimental results are as follows:
(1) the calcium silicate-micro-ring DNA nano-particles without modification of polyethylene glycol (PEG) prepared by the embodiment of the invention have the average particle size of 20.36 nm;
(2) the drug loading rate of the calcium silicate-micro-ring DNA nano-particles without polyethylene glycol (PEG) modification prepared by the embodiment of the invention is 0.102.
Example 2 preparation of polyethylene glycol (PEG) -modified calcium silicate-minicircle DNA nanoparticles
An embodiment of the present invention provides a method for preparing calcium silicate-minicircle DNA nanoparticles modified with polyethylene glycol (PEG), comprising the following steps:
(1) mu.l of DNA solution (0.5. mu.g/. mu.l) was mixed with 10. mu.l of polyethyleneimine (PEI25K) solution (1.3. mu.g/. mu.l) and incubated for 30 minutes; adding 80 μ l of calcium chloride solution (500mM, pH10.0), mixing with a pipette, adding 5ml of organic phase (prepared by mixing cyclohexane and polyethylene glycol nonylphenyl ether 520(Igepal CO-520) at a volume ratio of 7/3) dropwise under stirring, and stirring for one hour to form microemulsion A;
(2) dropwise adding 500 mu l of Tetraethoxysilane (TEOS) into the microemulsion A obtained in the step (1) while stirring, stirring for 4 hours, dropwise adding 10 mu l of calcium chloride solution (500mM, pH10.0) while stirring, and stirring for 18 hours to obtain a microemulsion B;
(3) dropwise adding 15 μ l of phosphoric acid-PEG (molecular weight of phosphoric acid-PEG is 2000 daltons) water solution (100 μ g/μ l) into the microemulsion B obtained in the step (2), and stirring for 4 hours to obtain microemulsion C;
(4) and (3) evaporating to remove cyclohexane in the microemulsion C by using a rotary evaporator, centrifuging for 15 minutes at 14000g, washing with absolute ethyl alcohol for three times, washing with high-purity water for two times, and dispersing in water to obtain the polyethylene glycol (PEG) -calcium silicate-micro-ring DNA nanoparticles.
1. And (3) particle size measurement:
after dissolving the polyethylene glycol (PEG) -calcium silicate-micro-ring DNA nano-particles prepared in the embodiment of the invention in 1ml double distilled water, carrying out ultrasonic treatment for 30s, observing the particles by using a transmission electron microscope, and determining the particle size by using a Malvern dynamic light scattering particle size analyzer.
2. Transfection assay comprising the following steps:
resuscitating and culturing human kidney epithelial cell line (293T), respectively and uniformly inoculating 104 cells per well in a 96-well plate before transfection, culturing with DMEM medium containing 10% fetal bovine serum, changing to 40 mu L of blank medium without FBS after the cells reach 70% -80% fusion, 37 ℃, and 5% CO2Incubating for 2-6 hours, respectively and sequentially adding 10 μ l of the calcium silicate-minicircle DNA nanoparticles (each hole contains 0.5 μ g of DNA) modified by the polyethylene glycol (PEG) obtained in the step (5), gently blowing and sucking up and down by using a pipette, uniformly mixing, and carrying out 5% CO treatment at 37 DEG C2After incubation for 6 hours under these conditions, the medium was changed with 10% fetal calf serum and incubated at 37 ℃ with 5% CO2The culture was continued under the conditions, and the Green Fluorescent Protein (GFP) expressed in the cells was observed every 24 hours with an inverted fluorescence microscope.
3. And (3) determination of drug loading capacity: weighing 1mg of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in the step (4), calculating the weight loss at the temperature of 900 ℃ to obtain the drug loading capacity, wherein the heating rate is 10 ℃/min, and the atmosphere is flowing air atmosphere; drug loading is the mass of DNA/total mass of nanoparticles.
The experimental results are as follows:
(1) fig. 1-2 are a Transmission Electron Microscope (TEM) photograph (fig. 1) and a particle size distribution chart (fig. 2) of the phosphate-PEG modified calcium silicate-minicircle DNA nanoparticle prepared in the example of the present invention. As shown in FIG. 1, the particle size of the calcium silicate-minicircle DNA nanoparticle modified by phosphoric acid-PEG prepared in the embodiment of the invention is 20-30 nm. As shown in fig. 2, the average particle size of the calcium silicate-minicircle DNA nanoparticle modified by phosphate-PEG prepared in the example of the present invention was 23.90 nm.
(2) FIG. 3 is a fluorescent (A) and white light (B) images of in vitro co-culture of phospho-PEG modified calcium silicate-minicircle DNA (eGFP) nanoparticles and 293T cell line provided in the examples of the present invention. As can be seen from fig. 3, the calcium silicate-minicircle DNA nanoparticle modified with polyethylene glycol (PEG) obtained in step (5) of example 2 has a certain transfection effect on 293T cells.
(3) The drug loading rate of the calcium silicate-micro-ring DNA nano-particles modified by the phosphoric acid-PEG prepared by the embodiment of the invention is 0.275.
Examples 3 to 4
To further illustrate the advantageous effects of the present invention, the experimental procedure of example 2 was repeated to replace the concentrations and volumes of the respective drugs in steps (1) to (4) of example 2 with those of example 3 shown in table 1, respectively, to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles.
To further illustrate the advantageous effects of the present invention, the experimental procedure of example 2 was repeated to replace the concentrations and volumes of the respective drugs in steps (1) to (4) of example 2 with those of example 4 shown in table 3, respectively, to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles.
To further illustrate the advantageous effects of the present invention, the procedure of example 2 was repeated to replace Tetraethoxysilane (TEOS) in step (2) of example 2 with a sodium silicate solution having a concentration of 500mM and a volume of 100ul (pH9.0) to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles.
The average particle size and drug loading of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 3 to 5 were measured according to the particle size measurement method and drug loading measurement method of example 2, respectively.
The experimental results are as follows:
(1) the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 3 to 4 had average particle diameters of 20.4 nm and 85.6nm, respectively.
(2) The drug loading rates of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 3 to 4 were 0.05 and 0.85, respectively.
TABLE 3 dosage of the drugs used in each of the steps of examples 3-4
Figure BDA0000936206220000131
Figure BDA0000936206220000141
Note: the "\\" in Table 3 indicates that no relevant information needs to be provided in the table.
Example 5
To further illustrate the advantageous effects of the present invention, the procedure of example 2 was repeated, Tetraethoxysilane (TEOS) in step (2) of example 2 was replaced with a sodium silicate solution having a concentration of 500mM and a volume of 100ul, and phosphoric acid-polyethylene glycol in step (3) of example 2 was replaced with polyethylene glycol diphosphate to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles; wherein the structural formula of the diphosphonic acid polyethylene glycol is shown as a formula III, n is a non-negative integer and is not zero,
Figure BDA0000936206220000142
the average particle size and drug loading of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in this example were measured according to the particle size measurement method and drug loading measurement method of example 2.
The experimental results are as follows:
(1) the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in the examples of the present invention have an average particle size of 37.7nm, respectively.
(2) The drug loading rates of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in the embodiments of the present invention are 0.366, respectively.
Examples 6-7 Effect of Nitrogen phosphorus (N/P) ratio on particle size and drug load
To further illustrate the beneficial effects of the present invention, further study the effect of nitrogen phosphorus (N/P) ratio of polyethyleneimine cationic polymer and DNA on the particle size of polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles, the experimental procedure of example 2 was repeated to replace 10ul PEI25K solution (1.3 μ g/ul) in step (1) of example 2 with 5ul PEI25K solution (1.3 μ g/ul) (as shown in example 6 of table 4), to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles.
To further illustrate the beneficial effects of the present invention, further study the effect of nitrogen phosphorus (N/P) ratio of polyethyleneimine cationic polymer and DNA on the particle size of polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles, the experimental procedure of example 2 was repeated to replace 10ul PEI25K solution (1.3 μ g/ul) in step (1) of example 2 with 1ul PEI25K solution (1.3 μ g/ul) (as shown in example 7 of table 4) to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles, and the particle size of the nanoparticles was obtained.
The average particle size and drug loading of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in example 6 and example 7 were measured according to the particle size measurement method and drug loading measurement method of example 2, respectively.
The experimental results are as follows:
(1) as shown in table 4, the average particle diameters of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 2, 6, and 7 of the present invention are 23.90, 110.23, and 1040.50nm, respectively, and as the nitrogen-phosphorus ratio (i.e., the ratio of N/P, the number of moles of amino groups of polyethyleneimine to the number of moles of phosphate groups of DNA) increases, the effect of polyethyleneimine on compressing DNA macromolecules is more significant, and the mixture of PEI and DNA provides a soft template effect for mineralized calcium silicate, so that the average particle diameter of the formed polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles decreases.
(2) As shown in table 4, the drug loading rates of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 2, 6, and 7 of the present invention were 0.275, 0.305, and 0.224, respectively, which indicates that the nitrogen-phosphorus ratio has little influence on the drug loading rate.
TABLE 4 influence of nitrogen phosphorus (N/P) ratio on particle size and drug loading
Figure BDA0000936206220000151
Examples 8-9 Effect of amount of organic phase on particle size and drug Loading
To further illustrate the beneficial effects of the present invention, further considering the effect of the amount of organic phase on the particle size of the polyethylene glycol (PEG) -calcium silicate-micro-ring DNA nanoparticles, the experimental procedure of example 2 was repeated to replace 5ml of organic phase in step (1) of example 2 with 2.5ml of organic phase (as shown in example 8 of table 5), to obtain polyethylene glycol (PEG) -calcium silicate-micro-ring DNA nanoparticles.
To further illustrate the beneficial effects of the present invention, further considering the effect of the amount of organic phase on the particle size of polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles, the experimental procedure of example 2 was repeated to replace 5ml of organic phase in step (1) of example 2 with 1ml of organic phase (as shown in example 9 of table 5), to obtain polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles.
The average particle size and drug loading of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in example 6 and example 7 were measured according to the particle size measurement method and drug loading measurement method of example 2, respectively.
The experimental results are as follows:
(1) as shown in table 5, the average particle diameters of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 2, 8, and 9 were 23.90, 175.11, and 788.52nm, respectively; as the volume of the organic phase decreases (the volume of the aqueous phase increases relatively), the radius of the water droplets in the microemulsion formed during the reaction increases, and the particle size of the product increases.
(2) As shown in table 5, the drug loading rates of the polyethylene glycol (PEG) -calcium silicate-minicircle DNA nanoparticles obtained in examples 2, 8, and 9 of the present invention were not much different, and were 0.275 and 0.314, respectively; in example 9 of the present invention, the drug loading was reduced to 0.102 because the volume of the organic phase was too small (i.e., the volume of the aqueous phase was too large) and the drug loaded in the product during collection was leaked.
TABLE 5 influence of the amount of organic phase on particle size and drug load
Examples Volume/ml of organic phase Average particle diameter/nm Drug loading
Example 2 5 23.90 0.245
Example 8 2.5 175.11 0.314
Example 9 1 788.52 0.102
Example 10 preparation of polyethylene glycol (PEG) -modified calcium silicate coated calcein nanoparticles
An embodiment of the invention provides a preparation method of calcium chlorophyll nanoparticles wrapped by calcium silicate modified by polyethylene glycol (PEG), which comprises the following steps:
(1) mixing 5 μ l calcein solution (saturated water solution) with 100 μ l calcium chloride solution (calcium chloride concentration is 500mM, pH10.0) uniformly; dropwise adding the mixed solution into 5ml of an organic phase (prepared by mixing cyclohexane and polyethylene glycol nonyl phenyl ether 520(Igepal CO-520) according to a volume ratio of 7/3) under stirring, and stirring for 1h to form a microemulsion A;
(2) stirring 10 mu l of Tetraethoxysilane (TEOS) in the microemulsion A obtained in the step (1) for 4 hours, then dropwise adding 80 mu l of calcium chloride solution (the concentration of calcium chloride is 500mM, the pH value is 10.0) while stirring, and stirring for 18 hours to obtain a microemulsion B;
(3) dripping 15 mul (100 mug/mul) of phosphoric acid-PEG aqueous solution into the microemulsion B obtained in the micro step (2), and stirring for 4 hours to obtain microemulsion C;
(4) and (3) evaporating and removing cyclohexane in the microemulsion C obtained in the micro step (3) by using a rotary evaporator, centrifuging for 15 minutes at 14000g, washing with absolute ethyl alcohol for three times, washing with high-purity water for two times, and dispersing in water to obtain the polyethylene glycol (PEG) -calcium silicate-calcein nanoparticles.
The polyethylene glycol (PEG) -calcium silicate-calcein nanoparticles obtained in step (4) were tested for particle size and drug loading according to the methods for determining particle size and drug loading in example 2.
The experimental results are as follows:
(1) particle size: fig. 4 is a Transmission Electron Microscope (TEM) photograph (a) and a particle size distribution chart (B) of nanoparticles formed by coating calcein with polyethylene glycol (PEG) modified calcium silicate provided in the examples of the present invention. As shown in fig. 4(a), the particle size of the nanoparticle formed by wrapping calcein with calcium silicate modified with polyethylene glycol (PEG) prepared in the embodiment of the present invention is 30 to 40 nm. As shown in fig. 4(B), the calcium chlorophyll is coated with polyethylene glycol (PEG) modified calcium silicate prepared in the example of the present invention to form nanoparticles having an average particle size of 34.1 nm.
(2) Drug loading rate: the drug loading rate of the nanoparticles formed by coating calcein with polyethylene glycol (PEG) modified calcium silicate prepared in the embodiment of the invention is 0.112.
Example 11 preparation of polyethylene glycol (PEG) -modified calcium silicate-coated calcium IR820 dye nanoparticles
The embodiment of the invention provides a preparation method of calcium silicate coated calcium IR820 dye nanoparticles modified by polyethylene glycol (PEG), which comprises the following steps:
(1) mu.l (1. mu.g/. mu.l) of IR820 aqueous solution was mixed well with 100. mu.l of calcium chloride solution (500mM, pH 10.0); dropwise adding the mixed solution into 5ml of an organic phase (prepared by mixing cyclohexane and polyethylene glycol nonyl phenyl ether 520(Igepal CO-520) according to a volume ratio of 7/3) under stirring, and stirring for one hour to form a microemulsion A;
(2) stirring 100 mu l of tetraethyl orthosilicate (TEOS) in the microemulsion A obtained in the step (1) for 4 hours, dropwise adding 80 mu l of calcium chloride solution (500mM, pH10.0) while stirring, and stirring for 18 hours to obtain a microemulsion B;
(3) dripping 1 mul of phosphoric acid-polyethylene glycol (PEG) water solution (10 mug/mul) into the microemulsion B obtained in the step (2), and stirring for 4 hours to obtain microemulsion C;
(4) and (3) evaporating cyclohexane in the microemulsion C obtained in the step (3) by using a rotary evaporator, centrifuging for at least 15 minutes at 14000g, washing with absolute ethyl alcohol for three times, washing with high-purity water for two times, and dispersing in water to obtain the polyethylene glycol (PEG) -calcium silicate-IR 820 nano-particles.
The polyethylene glycol (PEG) -calcium silicate-calcein nanoparticles obtained in step (4) were tested for particle size and drug loading according to the methods for determining particle size and drug loading in example 2.
The experimental results are as follows:
(1) particle size: fig. 5 is a Transmission Electron Microscope (TEM) photograph (a) and a particle size distribution chart (B) of nanoparticles formed by coating an IR820 dye with polyethylene glycol (PEG) modified calcium silicate provided in the examples of the present invention. As shown in fig. 5(a), the particle size of the nanoparticle formed by coating the IR820 dye with polyethylene glycol (PEG) modified calcium silicate prepared in the embodiment of the present invention is 30 to 40 nm. As shown in fig. 5(B), the average particle size of the nanoparticle formed by coating the IR820 dye with polyethylene glycol (PEG) modified calcium silicate prepared in the embodiment of the present invention is 31.1 nm.
(2) Drug loading rate: the drug loading rate of the nanoparticles formed by coating the IR820 dye with the polyethylene glycol (PEG) modified calcium silicate prepared by the embodiment of the invention is 0.144.
Example 12-13 preparation of phosphoric acid-polyethylene glycol (PEG) -modified calcium phosphate-minicircle DNA-nanoparticles
Embodiment 12 of the present invention provides a method for preparing calcium silicate-minicircle DNA-nanoparticles modified with phosphoric acid-polyethylene glycol (PEG), comprising the following steps:
(1) mu.l of DNA solution (1. mu.g/. mu.l) was mixed well with 5. mu.l of PEICL34K cationic polymer solution (3.8. mu.g/. mu.l) and incubated for 30 minutes; then adding 90 μ l of calcium chloride solution (100mM, pH9.0), and mixing well with pipette gun; dropwise adding the mixture into 5ml of an organic phase (prepared by mixing cyclohexane and polyethylene glycol nonyl phenyl ether 520(Igepal CO-520) according to a volume ratio of 7/3) under stirring, and stirring for one hour to form a microemulsion A;
(2) dropwise adding 100 mu l of disodium hydrogen phosphate solution (100mM, pH9.0) into 5ml of an organic phase (the organic phase is prepared by mixing cyclohexane and polyethylene glycol nonylphenyl ether 520(Igepal CO-520) according to the volume ratio of 7/3) while stirring, stirring for one hour, dropwise adding the solution into the microemulsion A obtained in the step (1) while stirring, stirring for 4 hours, dropwise adding 20 mu l of calcium chloride solution (100mM, pH9.0) while stirring, and stirring for 18 hours to obtain microemulsion B;
(3) dropwise adding 20 mu l of phosphoric acid-polyethylene glycol (1 mu g/mu l) water solution into the microemulsion B obtained in the step (2) under stirring, and stirring for 4 hours to obtain a microemulsion C;
(4) evaporating to remove cyclohexane in the microemulsion C by using a rotary evaporator, centrifuging for 15 minutes at 14000g, washing with absolute ethyl alcohol for three times, washing with high-purity water for two times, and dispersing in water to obtain polyethylene glycol (PEG) -calcium phosphate-micro-ring DNA nanoparticles, wherein the obtained solution is light yellow brown.
To further illustrate the beneficial effects of the present invention, the above experimental steps (1) - (4) were repeated, and the concentration and volume of each drug in the steps (1) - (4) were respectively replaced with the concentration and volume of each drug as shown in table 6, to obtain polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles.
TABLE 6 dosage of the drug used in each step of example 13
Figure BDA0000936206220000181
Figure BDA0000936206220000191
Note: "\\" in Table 6 indicates that no relevant information need be provided in the table.
The polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles obtained in step (4) were tested for particle size and drug loading according to the methods for determining particle size and drug loading of example 2.
The experimental results are as follows:
(1) the average particle diameters of the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared in the embodiments 12 and 13 of the present invention are 21.44 nm and 35.23nm, respectively.
(2) The drug loading rates of the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared by the embodiment of the invention are 0.235 and 0.344 respectively.
Example 14-15 preparation of phosphoric acid-polyethylene glycol (PEG) -modified calcium citrate-DNA nanoparticles
Example 14 to repeat the experimental steps (1) to (4) of example 12, the disodium hydrogen phosphate solution (ph9.0) in the experimental step (2) of example 12 was replaced with a sodium citrate solution (ph11.5) to obtain calcium citrate-DNA nanoparticles modified with phosphoric acid-polyethylene glycol (PEG).
Example 15 to repeat the experimental procedure of example 13, the disodium hydrogen phosphate solution (ph9.0) in example 13 was replaced with a sodium citrate solution (ph11.5) to obtain phosphoric acid-polyethylene glycol (PEG) -modified calcium citrate-DNA nanoparticles.
The polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles obtained in examples 14 and 15 were tested for particle size and drug loading according to the methods for determining particle size and drug loading of example 2.
The experimental results are as follows:
(1) the average particle diameters of the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared in examples 14 and 15 of the present invention were 30.40 nm and 29.25nm, respectively.
(2) The drug loading rates of the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared in examples 14 and 15 of the present invention were 0.307 and 0.115, respectively.
Example 16-17 Phosphate (PEG) -modified calcium carbonate-DNA nanoparticles
Example 16 to repeat the experimental steps (1) to (4) of example 12, the disodium hydrogen phosphate solution (ph9.0) in the experimental step (2) of example 12 was replaced with a sodium carbonate solution (ph11.5) to obtain phosphoric acid-polyethylene glycol (PEG) -modified calcium citrate-DNA nanoparticles.
Example 17 to repeat the experimental procedure of example 13, the disodium hydrogen phosphate solution (ph9.0) in example 13 was respectively replaced with a sodium carbonate solution (ph11.5) to obtain phosphoric acid-polyethylene glycol (PEG) -modified calcium citrate-DNA nanoparticles.
The polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles obtained in examples 16 and 17 were tested for particle size and drug loading according to the methods for determining particle size and drug loading of example 2.
The experimental results are as follows:
(1) the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared by the embodiment of the invention have average particle diameters of 30.12 nm and 38.55nm respectively.
(2) Drug loading rate: the drug loading rates of the polyethylene glycol (PEG) -calcium phosphate-minicircle DNA nanoparticles prepared by the embodiment of the invention are 0.785 and 0.256 respectively.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (12)

1. A preparation method of calcium-based nano drug delivery particles modified by polyethylene glycol is characterized by comprising the following steps:
(1) uniformly mixing the first target delivery substance solution and the first soluble calcium salt solution, adding the mixture into the organic phase, and stirring for 20-60 min to prepare a microemulsion A; the organic phase is prepared by mixing a surfactant and an alkane compound with 6-12 carbon atoms, wherein the surfactant is one or more of polyethylene glycol nonyl phenyl ether, polyethylene glycol octyl phenyl ether, sodium bis (2-ethylhexyl) succinate sulfonate and sodium dodecyl sulfate;
(2) adding a soluble salt solution into the microemulsion A obtained in the step (1), and stirring for 2-4 h to obtain a microemulsion B; the soluble salt solution comprises a soluble silicon salt solution, and the molar ratio of silicon element in the soluble silicon salt solution to calcium element in the first soluble calcium salt solution in the step (1) is 0.01-10: 1;
(3) adding a phosphoric acid-polyethylene glycol aqueous solution into the microemulsion B obtained in the step (2), and stirring for 2-4 h to obtain a microemulsion C, wherein the ratio of the mass of phosphoric acid-polyethylene glycol in the phosphoric acid-polyethylene glycol aqueous solution to the amount of the calcium element in the first soluble calcium salt solution in the step (1) is 0.001-100: 1;
(4) removing the organic phase in the microemulsion C obtained in the step (3), centrifuging and washing, and dispersing with water and/or ethanol to obtain the calcium-based nano-drug delivery particle modified by polyethylene glycol; wherein the particle size of the calcium-based nano-drug delivery particle modified by the polyethylene glycol is 20-200 nm; the polyethylene glycol modified calcium-based nano drug delivery particle comprises the first target delivery object and a calcium-based particle wrapped with the first target delivery object, wherein the surface of the calcium-based particle is modified with phosphoric acid-polyethylene glycol, and the first target delivery object is a biological drug or a chemical drug; the calcium-based particles comprise calcium silicate.
2. The method of claim 1, wherein the biological agent comprises a second target delivery material coated with a nucleic acid fragment, and the second target delivery material coated with a nucleic acid fragment is a cationic polymer, a polypeptide or a transfection reagent coated, combined or blended with a nucleic acid fragment.
3. The method of claim 2, wherein the ratio of the number of moles of amino groups in the cationic polymer, polypeptide, or transfection reagent to the number of moles of phosphate groups in the nucleic acid fragment is 0.1 to 100: 1.
4. The method for preparing polyethylene glycol-modified calcium-based nano drug delivery particles according to claim 2, wherein in the step (1), the cationic polymer comprises branched and chain polyethyleneimine, a modified polyethyleneimine, polylysine, polyamidoamine dendrimer and its derivatives, polypropyleneimine dendrimer and its derivatives, spermine or chitosan; the polypeptide comprises protamine, hemoglobin or albumin; the transfection reagent comprises X-tremagene HP DNA transfection reagent, Lipofectamine 2000 and EndofectinTMSerial transfection reagents, JetPEI transfection reagents, or engien series transfection reagents.
5. The method for preparing polyethylene glycol-modified calcium-based nano drug delivery particles according to claim 1, wherein in the step (1), the ratio of the total volume of the first target delivery substance solution and the first soluble calcium salt solution to the volume of the organic phase is 0.01-0.05: 1.
6. The preparation method of the polyethylene glycol-modified calcium-based nano drug delivery particle according to claim 1, wherein in the step (2), the volume ratio of the soluble salt solution to the microemulsion A obtained in the step (1) is 0.01-0.1: 1.
7. The preparation method of the polyethylene glycol-modified calcium-based nano drug delivery particle according to claim 1, wherein in the step (2), the soluble salt solution is added into the microemulsion A obtained in the step (1), and the microemulsion B is prepared by stirring for 2-4 h, and specifically comprises the following steps:
adding a soluble salt solution into the microemulsion A obtained in the step (1), stirring for 2-4 h, then adding a second soluble calcium salt solution, and stirring for 15-18 h to obtain a microemulsion B; wherein the molar ratio of the calcium element in the second soluble calcium salt solution to the calcium element in the first soluble calcium salt solution in the step (1) is 0.1-10: 1.
8. The preparation method of the polyethylene glycol-modified calcium-based nano drug delivery particle according to claim 1, wherein in the step (2), the soluble salt solution is added into the microemulsion A obtained in the step (1), and the microemulsion B is prepared by stirring for 2-4 h, and specifically comprises the following steps:
adding a soluble salt solution into an organic solution, stirring for 1-2 h, adding into the microemulsion A obtained in the step (1), stirring for 2-4 h, adding a second soluble calcium salt solution, and stirring for 15-18 h to obtain a microemulsion B; the organic solution is prepared by mixing a surfactant and an alkane compound with 6-12 carbon atoms, and the molar ratio of calcium element in the second soluble calcium salt solution to calcium element in the first soluble calcium salt solution in the step (1) is 0.1-10: 1.
9. The method for preparing polyethylene glycol-modified calcium-based nano-drug delivery particles according to claim 1, wherein in the step (3), the polyethylene glycol end of the phosphoric acid-polyethylene glycol is connected with a ligand, and the ligand comprises folic acid, RGD and analogues thereof, polypeptide, galactose, transferrin or antibody.
10. The calcium-based nano drug delivery particle modified by polyethylene glycol is obtained by the preparation method of any one of claims 1 to 9, and the particle size of the calcium-based nano drug delivery particle modified by polyethylene glycol is 20-200 nm; the polyethylene glycol modified calcium-based nano drug delivery particle comprises a first target delivery object and a calcium-based particle wrapped with the first target delivery object, wherein the surface of the calcium-based particle is modified with phosphoric acid-polyethylene glycol, and the first target delivery object is a biological drug or a chemical drug; the calcium-based particles comprise calcium silicate.
11. A pharmaceutical composition comprising the polyethylene glycol-modified calcium-based nano-drug delivery particle of claim 10.
12. Use of a method of preparation of a polyethylene glycol-modified calcium-based nano-drug delivery particle according to claim 10 or a polyethylene glycol-modified calcium-based nano-drug delivery particle according to any one of claims 1 to 9 for the preparation of a medicament.
CN201610128330.8A 2016-03-07 2016-03-07 Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof Active CN107158401B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610128330.8A CN107158401B (en) 2016-03-07 2016-03-07 Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610128330.8A CN107158401B (en) 2016-03-07 2016-03-07 Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN107158401A CN107158401A (en) 2017-09-15
CN107158401B true CN107158401B (en) 2020-11-17

Family

ID=59849239

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610128330.8A Active CN107158401B (en) 2016-03-07 2016-03-07 Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN107158401B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108904820A (en) * 2018-08-03 2018-11-30 苏州大学 A kind of diphosphonate nano material and preparation method thereof and application
US20210169083A1 (en) * 2019-12-09 2021-06-10 Corning Incorporated Alkali-resistant calcium phosphate/nucleic acids hybrid carrier for pest control and method to produce the particles

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104147614A (en) * 2013-05-15 2014-11-19 深圳先进技术研究院 Nano particles modified by alendronic acid functionalized polyethylene glycol and preparation method of nano particles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104147614A (en) * 2013-05-15 2014-11-19 深圳先进技术研究院 Nano particles modified by alendronic acid functionalized polyethylene glycol and preparation method of nano particles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Gene delivery with bisphosphonate-stabilized calcium phosphate nanoparticles;Elisabeth V. Giger et al.;《Journal of Controlled Release》;20101124;第150卷;第87-93页 *

Also Published As

Publication number Publication date
CN107158401A (en) 2017-09-15

Similar Documents

Publication Publication Date Title
Noureddine et al. Engineering of monosized lipid-coated mesoporous silica nanoparticles for CRISPR delivery
Choi et al. Glutamine-chitosan modified calcium phosphate nanoparticles for efficient siRNA delivery and osteogenic differentiation
Ding et al. A self-assembled RNA-triple helix hydrogel drug delivery system targeting triple-negative breast cancer
Sokolova et al. Functionalisation of calcium phosphate nanoparticles by oligonucleotides and their application for gene silencing
Tahara et al. Chitosan-modified poly (D, L-lactide-co-glycolide) nanospheres for improving siRNA delivery and gene-silencing effects
WO2017197726A1 (en) Bone-targeted gene carrier, preparation method therefor and application thereof
Li et al. A novel cationic liposome formulation for efficient gene delivery via a pulmonary route
CN106890343B (en) Targeting polypeptide nano gene vector compound
Xin et al. Preparation of internalizing RGD-modified recombinant methioninase exosome active targeting vector and antitumor effect evaluation
CN114099533A (en) Nucleic acid drug delivery system, preparation method, pharmaceutical composition and application
CN103550783A (en) Nucleic acid drug targeting delivery system and preparation method thereof
Qiu et al. Preparation and application of calcium phosphate nanocarriers in drug delivery
Zhang et al. The effect of serum in culture on RNAi efficacy through modulation of polyplexes size
Huang et al. Therapeutic nanosystems co-deliver anticancer drugs and oncogene SiRNA to achieve synergetic precise cancer chemo-gene therapy
CN104862337B (en) Polyanionic polymer/calcium phosphate nano genes delivery system and preparation method
CN110151697A (en) A kind of nucleic acid drug delivery system and preparation method thereof
CN107158401B (en) Polyethylene glycol modified calcium-based nano-drug delivery particle and preparation method and application thereof
CN107867677B (en) One-dimensional calcium phosphate nano/micron material and preparation method and application thereof
Fröhlich et al. Peptide-and polymer-based delivery of therapeutic RNA
CA3074248A1 (en) Biodegradable multilayer nanocapsules for the delivery of biologically active agents in target cells
Wang et al. Redox triggered disassembly of frame-guided assemblies
CN104974343A (en) Modified polyethyleneimine and application thereof in the preparation of gene transfection vector reagent
US20190307901A1 (en) Method for enhanced nucleic acid transfection using a peptide
He et al. A novel gene carrier based on amino-modified silica nanoparticles
CN114848831A (en) Coated nano preparation and preparation method and application of carrier thereof

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