WO2014056414A1 - 一种还原刺激响应型基因载体系统及其制备和应用 - Google Patents

一种还原刺激响应型基因载体系统及其制备和应用 Download PDF

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WO2014056414A1
WO2014056414A1 PCT/CN2013/084652 CN2013084652W WO2014056414A1 WO 2014056414 A1 WO2014056414 A1 WO 2014056414A1 CN 2013084652 W CN2013084652 W CN 2013084652W WO 2014056414 A1 WO2014056414 A1 WO 2014056414A1
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solution
gene
dna
reduction
sensitive
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PCT/CN2013/084652
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English (en)
French (fr)
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顾忠伟
聂宇
何一燕
程刚
谢丽
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四川大学
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Priority claimed from CN201210380312.0A external-priority patent/CN102899343B/zh
Priority claimed from CN201310242451.1A external-priority patent/CN103305549B/zh
Application filed by 四川大学 filed Critical 四川大学
Priority to EP13845441.8A priority Critical patent/EP2907876B1/en
Priority to US14/434,758 priority patent/US9707303B2/en
Publication of WO2014056414A1 publication Critical patent/WO2014056414A1/zh

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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • 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
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/95—Protection of vectors from inactivation by agents such as antibodies or enzymes, e.g. using polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Definitions

  • the invention belongs to the field of biomedical materials, and in particular relates to a nanoparticle gene carrier.
  • Gene therapy is a promising treatment for the treatment of hereditary diseases as well as tumors and has been greatly advanced over the past decade.
  • one of the bottlenecks in gene therapy is the delivery of genes.
  • Nucleic acids are negatively charged biomacromolecules, so stable and efficient synthetic carriers are usually positively charged.
  • a number of cationic lipid and polymer carrier systems have been developed that bind to DNA and promote gene transfection in vitro.
  • PEI Polyethylenimine
  • viruses are nanoscale particles with a core-shell structure composed of the inner core of the compressed genome and the coated protein shell.
  • the virus is stable outside the cell, which can recognize the signal provided by the cell, and carry out the stepwise molecular transformation according to the change of the in vivo or cellular microenvironment to initiate the disassembly response.
  • the design of the non-viral gene vector provides a good design. Enlightenment.
  • the dual-response design also proves that "artificial viruses” with both “shell de-masking” and “nuclear decompression” are more sensitive, but they do not really mimic the process of masking and decompressing the procedural response of the virus.
  • the level of reducing agents such as glutathione (GSH) in tumor tissues is about 4 times higher than that of normal tissues (1 to 5 ⁇ mol/L), reaching a level of about 20 ⁇ mol/L (Cancer Research.
  • a pseudoviral gene vector designed with multiple stimuli can intelligently control the release of genes and the targeted killing of tumor cells.
  • PEI Polyethyleneimine
  • OEI OEI with a molecular weight of 800 Da, although it has almost no cytotoxicity, does not compress DNA very well and has low transfection efficiency.
  • the applicant first proposed the gene carrier with gradient reduction stimuli responsiveness, and it is expected to develop a carrier with good stability and biodegradability into a non-toxic and safe small molecule, and at the same time have multiple stimuli responsiveness such as reduction, and A gene vector with high transfection efficiency.
  • the present invention provides a ternary complex nanoparticle gene vector having reduced stimuli responsiveness and a preparation method thereof, and is used for gene therapy of gene transfection, tumor, asthma and cardiovascular diseases in vitro.
  • a gene carrier system consisting of a masking system, a cationic polymer material and a plasmid DNA, the cationic polymer material and the plasmid DNA are combined to form a binary composite particle, and the shielding system is shielded to the binary composite by electrostatic action On the surface of the object, ternary composite particles are formed, and the masking system contains a reduction sensitive bond.
  • both the masking system and the cationic material contain a reducing sensitive bond to form a nanoparticle gene carrier having multiple reduction stimuli response characteristics.
  • the strategy of multiple reduction stimuli response is closer to the de-masking and decompression process of viral vectors, which can improve the efficiency of gene delivery and is far superior to single response.
  • the reduction sensitive bond is at least one of a disulfide bond or a double selenium bond, such that the material undergoes degradation under reducing conditions.
  • the masking system and the cationic material may both contain a disulfide bond or both contain a double selenium bond, or one may contain a disulfide bond, and the other may contain a double selenium bond, and each may also contain a disulfide bond and a double selenium bond.
  • the masking system contains a disulfide bond
  • the cationic material contains a double selenium bond.
  • the gene carrier system of this structure can make a real-time response through the change of molecular chemical structure under the condition of extracellular and different reducing agent concentration in the tumor, and realize the gradient reduction stimulation response.
  • the ternary complex carrier can make the ternary complex carrier stable in the environment of extracellular low GSH concentration, circulate for a long time, and protect DNA from deoxyribonuclease (DNase) degradation in serum; when reaching the target tumor tissue, due to tumor tissue slightly High GSH concentration,
  • DNase deoxyribonuclease
  • the disulfide bond that causes the outer layer (in the masking body) to be more sensitive is first broken (or partially broken), which is more conducive to endocytosis of the composite particles; after entering the cell, the intracellular GSH concentration is up to 1000 times higher than the extracellular When it reaches 1 ⁇ 10mmol/L, at this time, the relatively stable double selenium bond (in the cationic material) is also broken, the cationic material is degraded, and the DNA is released, which is favorable for DNA into the nucleus, thereby realizing high expression of the gene.
  • DNase deoxyribonuclease
  • the masking system contains a double selenium bond.
  • the gene carrier of this structure can better protect DNA from degradation by DNase in serum and avoid the phenomenon of coagulation caused by negatively charged serum proteins in serum.
  • the outer layer (in the shielding body) uses a relatively more stable double selenium bond, which makes the ternary complex carrier more stable in the environment of extracellular low GSH concentration, and the circulation time is longer. When reaching the target tumor tissue, the tumor tissue is slightly higher. The GSH concentration still needs to be contacted for a longer period of time or until it enters the cell, and then the cell is completely disassembled after the cell is introduced, thereby releasing the DNA and realizing the gene transfection. It can also be mixed with a carrier containing a disulfide bond on the outer layer to achieve a better overall effect.
  • the masking system may be a modification of a carboxyl group on at least one glucuronic acid unit on the glycosaminoglycan, and the obtained sugar having a reduction-sensitive disulfide bond or a double selenium bond and having a terminal carboxyl group at the end may be obtained.
  • Aminoglycan derivatives Glycosaminoglycan, a kind of heteropolysaccharide, is divided into: hyaluronic acid, chondroitin 4-sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate. Wait.
  • glycosaminoglycan It is a long-chain polysaccharide composed of repeating disaccharide units.
  • One of its disaccharide units is aminohexose (glucosamine or aminogalactose), so it is called glycosaminoglycan; the other is uronic acid.
  • Glycosaminoglycans are an important component of the interstitial cells. Most of the interstitial cells are glycosaminoglycans, so they have good biocompatibility and degradability.
  • the glycosaminoglycan used in the masking system of the present invention has a molecular weight of 5 to 2000 kDa.
  • the glycosaminoglycan in the present invention is at least one of hyaluronic acid, chondroitin 4-sulphate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate.
  • the masking system is a hyaluronic acid derivative having a reduction-sensitive disulfide bond and a terminal carboxyl group by modifying a carboxyl group on at least one glucuronic acid unit on hyaluronic acid ( HA-SS-COOH).
  • HA-SS-COOH hyaluronic acid
  • the cationic material is composed of a compound containing a disulfide bond or a double selenium bond and a polyethyleneimine, a polypropylene imine, a spermine, an amino acid polypeptide, a peptide dendrimer or a peptide-containing dendrimer. At least one of a cationic lipid material or other lipid material (such as a cationic lipid polymer, etc.) is crosslinked.
  • the periphery of the cationic material is rich in amino groups, and is convenient to crosslink with a compound containing a disulfide bond or a double selenium bond, and the compound containing a disulfide bond or a double selenium bond is a disulfide bond or a double selenium bond.
  • the polyethyleneimine, polypropyleneimine, spermine, amino acid polypeptide or lipid material has a molecular weight of 200 to 4000 Da.
  • the compound having a disulfide bond or a double selenium bond is crosslinked with polyethyleneimine or polypropyleneimine or spermine to form a cationic material:
  • DT is polyethyleneimine or polypropyleneimine or spermine.
  • the cationic material has low cytotoxicity, high transfection efficiency, and controllable molecular weight.
  • Oleimide (OEI-SeSe x ), the structure is as follows:
  • the structure of the cation ion material is
  • R 2 is an amino acid group, a saturated/unsaturated hydrocarbon group or At least one of , and at least one is an amino acid group
  • X, Y, ⁇ is ⁇ , 0 or S
  • o, p, q are each independently 1 or 0
  • the saturated/unsaturated hydrocarbon group is preferably a fluorenyl group, an alkenyl group, an aromatic group More preferably, it is a C10-C20 fluorenyl group, an alkenyl group, or an aryl group-containing amino acid derivative, and the amino acid group is preferably a lysine, arginine or histidine group.
  • the structural formula of the cationic lipid material containing the peptide dendrimer is:
  • the plasmid DNA described in the gene vector system of the present invention uses plasmid DNA expressed in eukaryotic cells. It may be a eukaryotic recombinant expression plasmid containing a reporter gene or a cytokine gene or a tumor suppressor gene.
  • the invention also provides a method for preparing the above gene carrier system, the specific steps of which are as follows:
  • HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mM, 5% glucose) to prepare a DNA solution at a concentration of 0.1 mg/mL; Soluble in HBG buffer, formulated into a concentration of 0.1 - 10 mg / mL of solution A; the masking system dissolved in HBG buffer, formulated into a concentration of 0.01 - 1 mg / mL of solution B;
  • the solution A obtained in the above step was mixed with the DNA solution, and after incubation at room temperature for 20 minutes, a binary complex was obtained, and then solution B was added thereto, and after incubation at room temperature for 20 minutes, a ternary complex was obtained.
  • HA-Cys cystamine-grafted hyaluronic acid
  • PBS phosphate buffered saline
  • DTT dithiothreitol
  • HA-SH thiolated hyaluronic acid
  • PBS phosphate buffered saline
  • the invention also provides the application of the above gene carrier system: combining the gene carrier with superparamagnetic nanoparticles (MNP) and/or a fat-soluble drug to form a composite carrier or drug for in vitro gene transfection, tumor, Gene therapy for asthma or cardiovascular disease, combination therapy with genetic and chemical drugs, and integration of disease diagnosis and treatment.
  • MNP superparamagnetic nanoparticles
  • the present invention also provides a composite carrier system comprising a gene carrier in combination with magnetic nanoparticles, which may be water soluble or fat soluble.
  • the magnetic nanoparticles and genes are dispersedly distributed in the gene vector.
  • the magnetic nanoparticles are at least one of triiron tetroxide, gamma ferric oxide or ferrite magnetic nanoparticles doped with manganese, cobalt or zinc.
  • the magnetic nanoparticles are negatively charged, and have an average particle size scale of 5 to 50 nm and a surface potential of between -5 and -50 mV.
  • the magnetic nanoparticles are surface-modified with a negatively-charged organic material
  • the negatively-charged organic material may be at least one of a carboxysilicone coupling agent, an amino acid dendrimer, tartaric acid, citric acid, oxalic acid, and acetic acid.
  • the composite carrier can respond to the applied magnetic field and concentrate to the target site under the guidance of an external magnetic field to realize the magnetic targeting function.
  • the present invention also provides a composite drug comprising the gene carrier and a pharmaceutically active ingredient.
  • the drug may be encapsulated in the cationic material together with the plasmid DNA to achieve simultaneous release of the plasmid DNA and the drug, and at the same time achieve the purpose of gene therapy and drug treatment; the drug may also be encapsulated in the masking system or simultaneously Encapsulated in the masking system and cationic materials to achieve a phased release of the drug and DNA.
  • the invention also provides a composite carrier system in which the gene carrier is simultaneously combined with a drug and a magnetic nanoparticle. The functions of the three are integrated in a carrier system.
  • the invention also provides a gene vector application, which is used for gene therapy of in vitro gene transfection, tumor, asthma or cardiovascular disease, and combined treatment of gene and chemical medicine, and integration of disease diagnosis and treatment.
  • the gene carrier of the present invention uses a hyaluronic acid derivative as a shielding body, which does not affect the ability of the cationic polymer to recombine DNA on the one hand, and enhances the gene carrier and cell surface by utilizing the targeting function of hyaluronic acid on the other hand.
  • the binding efficiency of the receptor and the ability to maintain endosomal detachment by HA receptor-mediated endocytosis, promotes the uptake of the PEI/DNA/HA-SS-COOH ternary complex by the cells.
  • the terminal carboxyl group can be combined with a large amount of amino groups on the surface of the composite particle formed by the cationic polymer and the plasmid DNA to achieve the shielding effect and reduce the toxicity. And avoiding the aggregation caused by serum albumin, escaping the clearance of the reticuloendothelial system, thereby reducing the cytotoxicity of the cationic polymer and increasing the stability of the serum; on the other hand, after entering the cell, under the action of the intracellular reducing environment The reduction of the sensitive disulfide bond will break, thereby unblocking, exposing the loaded gene, and improving its ability to escape from the endosomes, thereby greatly improving the transfection efficiency.
  • the gene carrier of the present invention introduces a reduction-sensitive response masking layer having a targeting function into a gene carrier system by electrostatic binding, and can control over-shadowing by adjusting the masking ratio (HA-SS-COOH/DNA).
  • the dyeing efficiency is reduced, and the specific ligand is used to enhance the specific recognition and transmission of the target tissue by the gene carrier, and the carried DNA environment is responsively released into the target cells.
  • the gene carrier having multiple reduction stimuli responsiveness according to the present invention adopts a strategy of multiple reduction stimulation response of the book, which is closer to the de-masking and decompression process of the viral vector, and the gene transfection efficiency is obviously superior to other single stimulation responses or Traditional gene vector for non-intelligent response.
  • the reduced stimuli-responsive cationic material (such as OEI-SeSe x ) containing a double selenium bond according to the present invention can form a positively charged nano-scale binary complex particle with surface (OEI-SeSe) by electrostatic interaction with plasmid DNA.
  • x /DNA bin a ry polypl exeS abbreviated as DSe).
  • the reduction stimuli-responsive masking system (such as HA-SS-COOH) containing a disulfide bond of the present invention, the carboxyl group at the end thereof can be combined with the positive charge remaining on the surface of the DSe by electrostatic interaction to form a surface having a very low positive charge or It is a negatively charged ternary complex (OEI-SeSe x /DNA/HA-SS-COOH ternary polyplexes, abbreviated as DSeS).
  • DSeS negatively charged ternary complex
  • the reduction stimuli response masking system containing the disulfide bond of the present invention can achieve the function of shielding positive charges, reduce toxicity and avoid aggregation caused by serum albumin, escape the scavenging effect of the reticuloendothelial system, thereby reducing cations.
  • the cytotoxicity of the polymer increases the stability of the serum; on the other hand, after reaching the tumor tissue site or entering the cell, under the action of the reducing environment, the more sensitive disulfide bond will be broken, the shielding effect is removed, and the internal DSe is exposed to improve its ability to escape from the endosomes, which is beneficial to improve transfection efficiency.
  • the gene vector system of the present invention responds in real time by changes in molecular chemical structure under conditions of extracellular and intracellular concentrations of reducing agents in the tumor. Gradient reduction stimuli responsiveness allows the OEI-SeSe x /DNA/HA-SS-COOH ternary complex to remain stable in an extracellular low GSH concentration environment, circulate for long periods of time, and protect DNA from DNase degradation in serum.
  • the more sensitive disulfide bond of the outer layer is first broken (or partially broken), which is more conducive to endocytosis of the complex particles; after entering the cell,
  • the concentration of intracellular GSH is up to 1000 times higher than that of extracellular to 1 to 10 mmol/L.
  • the relatively stable double selenium bond is also broken, degrading into low molecular weight OEI, releasing DNA, which is beneficial to DNA nucleation. Thereby achieving high expression of genes.
  • the method for preparing the gene vector of the present invention is simple in operation and convenient for large-scale production.
  • the gene vector of the present invention can be conveniently used for gene therapy of gene transfection, tumor, asthma and cardiovascular diseases in vitro.
  • Figure 1 is a schematic view showing the structure and synthesis process of one of the shielding systems of the present invention.
  • Figure 2 is an agarose gel electrophoresis pattern of PEI/DNA/HA-SS-COOH complex, in which there are eight lanes from left to right, the first is naked DNA; the second to seventh are PEI/DNA /HA-SS-COOH complex, wherein the mass ratio of HA-SS-COOH/DNA is 10, 6, 3, 2, 1 and 0.5, respectively; the eighth channel is PEI/DNA complex.
  • Figure 3 shows the cell viability of B 16 cells transfected 24 hours after different concentrations of PEI/DNA/HA-SS-COOH complex.
  • Figure 4 is a graph showing the transfection efficiency of green fluorescent protein particles to B 16 cells by PEI/DNA complex and PEI/DNA/HA-SS-COOH (HA-SS-COOH/DNA mass ratio 1) complex, respectively.
  • Figure 5 is a structural formula of a reduction stimuli response masking system containing a disulfide bond of the present invention.
  • Figure 6 is a structural formula of a reducing stimuli-responsive cationic material containing a double selenium bond according to the present invention.
  • Fig. 7 is a schematic view showing the ternary complex nanoparticle gene carrier according to Embodiment 11 of the present invention.
  • Figure 8 is a chromatogram measured by GPC.
  • Figure 9 is the cell viability of HepG2 cells transfected 24 hours after different transfection complexes.
  • Figure 10 is a graph showing the transfection efficiency of green fluorescent protein particles to HepG2 cells by different transfection complexes (DP; DSe; DPS DSeS).
  • Figure 11 is a different transfection complex as described in Example 18 (control: untransfected HepG2 cells, DP: cation/DNA/masking ternary complex of the invention, MDP-cc: cation of the invention /DNA/shield + MNP quaternary complex, MF means placing a magnet under the culture plate after transfection) respectively mediated the transfection efficiency of green fluorescent protein particles to HepG2 cells, where A is a fluorescence micrograph and B is a flow Cytometry test results.
  • Figure 12 is a photograph of tumor tissue taken from each experimental group 21 days after in vivo treatment as described in Example 19, with 3 replicates per group.
  • Figure 13 is a graph showing the statistical results of the volume of tumors taken at various time points in the in vivo treatment experiment described in Example 19 (eight parallel samples were set for each time point in each group).
  • DSe A binary complex of OEI-SeSe x and DNA.
  • DPS a ternary complex of PEI 25kDa, DNA and HA-SS-COOH.
  • DSeS a ternary complex of OEI-SeSe x , DNA and HA-SS-COOH.
  • Example 1 Preparation of a reduction-sensitive masking system with a targeting function
  • hyaluronic acid was dissolved in phosphate buffer (PBS) at pH 6 6.8, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt was added.
  • the acid salt (ED HC1) and 1-hydroxybenzotriazole (HOBT) were stirred and reacted at room temperature for 2 hours to activate the carboxyl group.
  • Add cystamine dihydrochloride (Cys) stir, and react the above reaction solution at room temperature overnight. After the reaction, the reaction product was dialyzed against a dialysis bag with a retention of 3500 for 48 hours, and lyophilized to obtain cystamine grafted.
  • Hyaluronic acid (HA-Cys) The graft ratio of cystamine in the product (HA-Cys) was calculated by nuclear magnetic spectroscopy, and the results are shown in Table 1.
  • HA-Cys cystamine-grafted hyaluronic acid
  • PBS phosphate buffered saline
  • DTT dithiothreitol
  • HC1 hydrochloric acid
  • NaCl sodium chloride
  • HA-SH Thiolated hyaluronic acid
  • hyaluronic acid with molecular weights of 5k Da, 40k Da, 100 kDa, 1000 kDa and 2000 kDa were also selected for the preparation test.
  • the results showed that the hyaluronic acid of each molecular weight could be successfully modified to obtain disulfide.
  • Example 2 Preparation of a gene vector system with a reducing functional masking system with targeting function
  • the plasmid DNA was dissolved in sterile HBG buffer to prepare DNA solution A at a concentration of 0.1 mg/mL; the cationic polymer gene carrier polyethyleneimine (PEI) was dissolved in sterile HBG buffer (4-hydroxyethyl) Peptazine ethanesulfonic acid 20 mmol, pH 7.4, 5% glucose), formulated as a PEI solution B at a concentration of 0.01-1 mg/mL; a reduction-sensitive masking system with a targeted function (HA-SS-COOH) It is dissolved in sterile HBG buffer and formulated into a HA-SS-COOH solution C at a concentration of 0.01-1 mg/mL.
  • PEI cationic polymer gene carrier polyethyleneimine
  • DP does not contain a masking system 1.2: 1 116.25 27.50 DPS30-0.5 HA-SS-COOH 30 1.2: 1: 0.5 155.20 13.97
  • a series of ternary complexes were successfully prepared by using the hyaluronic acid derivatives of different molecular weights prepared in Example 1 as shielding systems, respectively. Particles.
  • B16 cells in logarithmic growth phase were taken within 24 hours prior to transfection, diluted with DMEM medium after trypsinization, and seeded in 96-well culture plates at a density of 1 > ⁇ 10 4 cells per well, placed at 5% (volume) Percentage of C0 2 , the culture was continued to 80-90% confluence in an incubator at 37 ° C.
  • the culture medium in the cell culture plate that was added the previous day was aspirated, and washed twice with PBS.
  • Add the genome-transfected complex particles and DMEM medium containing 10% (mass/volume percentage) of calf serum to a final volume of 0.1 mL, continue to culture for 24 hours; add 10 MTT solution at a concentration of 5 mg/mL.
  • a sample is the absorbance value of the transfected cell sample well, and A ⁇ ntol is the absorbance value of the cell control well that does not interact with the transfection complex, and each set of experiments is repeated six times.
  • Figure 3 shows the cell viability of B 16 cells transfected 24 hours after different concentrations of PEI/DNA/HA-SS-COOH complex. It can be seen from Fig. 3 that the PEI/DNA/HA-SS-COOH ternary complex is much less toxic to B 16 cells than the PEI/DNA complex, and the cell survival rate is above 80%, and the gene carrier system of the present invention can be seen. Low cytotoxicity.
  • Example 5 Determination of the efficiency of in vitro transfection of B 16 cells by green fluorescent protein granules mediated by a gene vector system with a targeted reduction-sensitive masking system
  • B 16 cells Mouse melanoma B16 cells were cultured in a culture medium containing 10% (mass/volume percent) fetal bovine serum at 5% (by volume) C0 2 at 37 ° C. Cultivate in an incubator for 24 hours;
  • B16 cells in logarithmic growth phase were digested with try digestively diluted with DMEM medium, and seeded in a 6-well culture plate at a density of 4 ⁇ 10 5 cells per well, placed in a 5% (volume percent) C0 2
  • the culture was continued to 80-90% confluence in an incubator at 37 ° C.
  • the culture medium in the cell culture plate that was added the previous day was aspirated, washed twice with PBS, and then genomically transfected.
  • HA-SS-COOH has hyaluronic acid (HA) properties and can interact with the CD44 receptor on the surface of B16 cells to help PEI/DNA/HA-SS-COOH ternary complex particles endocytosis; After the cells, the disulfide bond of the reduction response breaks, helping to detach the masking system, exposing the positive charge of PEI and exerting its proton pumping effect, thereby promoting the improvement of transfection efficiency.
  • HA hyaluronic acid
  • Example 6 Determination of in vitro transfection efficiency of B16 cells by a luciferase plasmid mediated by a gene vector system with a targeting-reactive reduction-sensitive masking system
  • B 16 cells were seeded at 4 ⁇ 10 5 cells/well in a 6-well plate, and cultured in a 5% C0 2 cell culture incubator at 37 ° C for 24 hours. When transfected, the cells cultured the previous day were aspirated. The culture medium in the plate was washed twice with PBS, and the complex particles containing luciferase DNA listed in Table 3 were added and serum-free or containing 10%, 50% (mass/volume percentage) of calf serum. DMEM medium to a final volume of 2 mL, continue to culture for 24 hours;
  • the invention utilizes a PEI/DNA/HA-SS-COOH ternary complex with a reducing-sensitive masking gene gene delivery system with a targeting function to improve the performance of the gene carrier under the conditions of serum-free, 10% serum and 50% serum, respectively.
  • the transfection efficiency of B16 cells was increased by 14 times, 538 times and 130 times.
  • HepG2 cells were taken from human liver tumor cells in a medium containing 10% (mass/volume percentage) fetal calf serum, containing 5% (by volume) C0 2 at 37 °C. Cultivate in an incubator for 24 hours;
  • HepG2 cells were seeded at 4 ⁇ 10 5 cells/well in a 6-well plate, and cultured in a YTC, 5% C0 2 cell culture incubator for about 24 hours. When transfected, the cells in the cell culture plate that was added the previous day were discarded. The culture medium was washed twice with PBS, and the granulocyte-containing complex particles listed in Table 4 and the serum-free or DMEM medium containing 10%, 50% (mass/volume percentage) of calf serum were added. The final volume is 2 mL and the culture is continued for 24 hours.
  • the invention utilizes a PEI/DNA/HA-SS-COOH ternary complex with a reducing-sensitive masking gene gene delivery system with a targeting function to improve the performance of the gene carrier under the conditions of serum-free, 10% serum and 50% serum, respectively.
  • the transfection efficiency of HepG2 cells was increased by 13 times, 28 times and 33 times.
  • Example 8 Preparation of a gene vector (OEI-SS/DNA/HA-SS-COOH) having dual reduction stimuli responsiveness
  • the plasmid DNA was dissolved in a sterile HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mmol, 5% dextrose) to prepare a DNA solution at a concentration of 0.1 mg/mL; the disulfide bond was cross-linked.
  • Polyethyleneimine (OEI-SS) is dissolved in HBG buffer and formulated into OEI-SS solution at a concentration of 0.1-100 mg/mL; the masking system containing disulfide bond (HA-SS-COOH) is dissolved in HBG In the buffer, a solution of 0.01-1 mg/mL of HA-SS-COOH was prepared.
  • the plasmid DNA was dissolved in a sterile HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mmol, 5% dextrose) to prepare a DNA solution at a concentration of 0.1 mg/mL; the double selenium bond was cross-linked.
  • Polyethyleneimine (OEI-SeSe x ) is dissolved in HBG buffer and formulated into OEI-SeSe x solution at a concentration of 0.1-10 mg/mL; the shielding system containing HA+SeSe-COOH is dissolved.
  • a HA-SeSe-COOH solution having a concentration of 0.01-1 mg/mL was prepared.
  • the plasmid DNA was dissolved in a sterile HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mmol, 5% glucose) to prepare a DNA solution at a concentration of 0.1 mg/mL; the double selenium bond was cross-linked.
  • Polyethyleneimine (OEI-SeSe x ) is dissolved in HBG buffer and formulated into OEI-SeSe x solution at a concentration of 0.1-10 mg/mL; the masking system containing disulfide bond (HA-SS-COOH) is dissolved.
  • a HA-SS-COOH solution having a concentration of 0.01-1 mg/mL was prepared.
  • OEI-SeSe x solution solution and the plasmid DNA solution were mixed, and the mixed solution was incubated at room temperature for 20 minutes to obtain an OEI-SeSe x /DNA binary complex. Further, a HA-SS-COOH solution was added, and the resulting mixed solution was incubated at room temperature for 20 minutes to obtain a gene carrier OEI-SeSe x /DNA/HA-SS-COOH ternary complex having multiple reduction stimuli responsiveness.
  • the plasmid DNA was dissolved in a sterile HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mmol, 5% glucose) to prepare a DNA solution at a concentration of 0.1 mg/mL; the double selenium bond was cross-linked.
  • Polyethyleneimine (OEI-SeSe x ) is dissolved in HBG buffer and formulated into OEI-SeSe x solution at a concentration of 0.1-10 mg/mL; a masking system containing disulfide bonds (HA-SS-COOH) ) Dissolved in HBG buffer and formulated into a HA-SS-COOH solution at a concentration of 0.01-1 mg/mL.
  • the polyethyleneimine (OEI-SeSe x ) solution cross-linked with different concentrations of double selenium bonds and the plasmid DNA solution are mixed at a certain mass ratio, and the mixed solution is incubated at room temperature for 20 minutes to obtain OEI-SeSe x /DNA.
  • Binary complex Further, different concentrations of HA-SS-COOH solution were added, and the resulting mixed solution was incubated at room temperature for 20 minutes to obtain a gene vector OEI-SeSe x /DNA having both double-sulphur bond and double-selenium bond with gradient-reduction dual-stimulation responsiveness.
  • /HA-SS-COOH ternary complex This OEI-SeSe x /DNA/HA-SS-COOH ternary complex was used for the next toxicity and transfection experiments.
  • Table 5 The composition and properties of the ternary composite particles prepared according to the above formula are shown in Table 5.
  • the OEI 8QQ -SeSe x handling certain time (4 h or 8 h) were treated with different concentrations of GSH (10 ⁇ or 100 ⁇ ), gel permeation chromatography (GPC Method)
  • GPC Method gel permeation chromatography
  • the molecular weight is measured, and the chromatogram is shown in Fig. 8.
  • the parameters of the GPC equipment are as follows: Waters 2690D HPLC, ultrahydrogel 120 and ultrahydrogel 1000 column in series, refractive index detector; mobile phase: 0.1 mol/L sodium formate buffer (pH 2.8), flow rate 1.0 ml/min, column temperature 35 ° C;
  • the molecular weight was calculated using polyethylene glycol as a standard substance.
  • FIG 8 is a GPC chromatogram measured using, respectively, from the bottom up: PEI 25k polyethyleneimine having a molecular weight of 25kDa; OEI 8QQ -SeSe x Se double bond is crosslinked polyethylenimine; OEI 800 -SeSe after OEI 8QQ -SeSe x 100 ⁇ GSH was treated 4 h;; after OEI 8QQ -SeSe x 10 ⁇ GSH after treatment 8 h; 10 ⁇ GSH after treatment by x 4 h OEI 8QQ a molecular weight of 800 Da oligonucleotide polyethyleneimine amine.
  • the disulfide bond is also broken with time due to the 10 ⁇ level of reducing agent. It is indicated that under the same 10 ⁇ level of reducing agent, the double selenium bond is more stable than the disulfide bond; at higher levels of reducing agent, the double selenium bond and the disulfide bond are disconnected. It can be seen that the OEI-SeSe x /DNA/HA-SS-COOH ternary complex carrier of the present invention has gradient reduction stimuli response performance.
  • HepG2 cells were taken from human liver tumor cells in a culture medium containing 10% (mass/volume percent) fetal bovine serum at 5% (by volume) C0 2 at 37 ° C. Incubate for 24 hours in an incubator.
  • HepG2 cells in logarithmic growth phase were digested with DMEM medium after trypsinization, and seeded in 96-well culture plates at a density of 1 > ⁇ 10 4 cells per well, placed at 5% (volume) Percentage of C0 2 , the culture was continued to 80-90% confluence in an incubator at 37 ° C.
  • the culture medium in the cell culture plate added the previous day was aspirated, and washed twice with PBS.
  • Transfection complex particles and DMEM medium containing 10% (mass/volume percent) fetal bovine serum were added to a final volume of 0.1 mL, and culture was continued for 24 hours;
  • a sample is the absorbance value of the transfected cell sample well, and A ⁇ ntol is the absorbance value of the cell control well that does not interact with the transfection complex, and each set of experiments is repeated six times.
  • Figure 9 is the cell viability of different transfection complexes after 24 hours of transfection of HepG2 cells.
  • the dual stimuli-responsive DSeS ternary complex is much less toxic to HepG2 than the single nuclear stimulation response DSe and the single shell stimuli response DPS complex, with a HA-SS-COOH/DNA mass ratio of 2
  • the DSeS cell survival rate is above 80%, and it can be seen that the double-stimulated response gene vector of the present invention has low cytotoxicity.
  • Example 14 Determination of in vitro transfection efficiency of green fluorescent protein granules to HepG2 cells by OEI-SeSe x /DNA/HA-SS-COOH ternary complex gene vector with gradient reduction stimuli response
  • the HepG2 cells in logarithmic growth phase were digested with trypsin and diluted with DMEM medium.
  • the cells were seeded at a density of 4> ⁇ 10 5 cells per well in a 6-well culture plate at 5% (volume percent).
  • C0 says 2 , the culture is continued to 80-90% fusion in the incubator at 37 °C.
  • the culture solution in the cell culture plate added the previous day is aspirated, washed twice with PBS, and then transferred.
  • Dye the complex particles and DMEM medium containing 10% (mass/volume percent) fetal bovine serum to a final volume of 2 mL. After 4 hours, replace the fresh medium containing 10% fetal bovine serum and continue to culture for 44 hours. .
  • Figure 10 shows the transfection efficiency of green fluorescent protein particles to HepG2 cells by different transfection complexes (DP; DSe; DPS; and DSeS), respectively.
  • DP as a standard for gene transfection gold, DSE for single nuclear reduction stimuli response, and DPS for single stimuli response, and DSeS for dual stimuli response, mediated by green fluorescent protein granules after transfection of HepG2 cells Fluorescent photo.
  • Example 15 Determination of in vitro transfection efficiency of luciferase plasmid to HepG2 cells by OEI-SeSe x /DNA/HA-SS-COOH ternary complex gene vector with gradient reduction stimuli response
  • HepG2 cells were seeded at 4 ⁇ 10 5 cells/well in a 6-well plate, and cultured in a 5% C0 2 cell culture incubator at 37 ° C for 24 hours. When transfected, the cell culture plate added the previous day was aspirated. The medium was washed twice with PBS, and the luciferase DNA-containing transfection complex particles listed in Table 2 and fresh DMEM medium containing 10% (mass/volume percent) fetal bovine serum were added. After a final volume of 2 mL, 4 hours later, fresh medium containing 10% fetal bovine serum was replaced and culture was continued for 20 hours.
  • the present invention utilizes the OEI-SeSe x /DNA/HA-SS-COOH (DSeS* ) ternary complex gene vector with gradient reduction stimuli response performance to significantly improve the performance of the gene vector, and the transfection efficiency of HepG2 cells is respectively higher than that of the HepG2 cells.
  • the gold standard DP(*) the DSe(*) of the single nuclear stimulation response and the DPS(*) of the single shell stimulation response were increased by 197.2 times, 95.4 times and 43 times.
  • Example 16 Determination of in vitro transfection efficiency of HepG2 cells by ternary complex gene vector with reduced stimuli-responsive glycosaminoglycan masking system mediated by luciferase plasmid
  • the hyaluronic acid raw material was replaced with 4-chondroitin sulfate, 6-chondroitin sulfate, dermatan sulfate, and acetyl sulfate.
  • One of glycosaminoglycans such as heparin, heparin and keratan sulfate gives a glycosaminoglycan derivative having a disulfide bond modification and a terminal carboxyl group as a masking system having a reduction-sensitive property.
  • plasmid DNA in sterile water or sterile HBG buffer (4-hydroxyethylpiperazineethanesulfonic acid 20 mM, 5% glucose) to prepare a DNA solution at a concentration of 0.1 mg/mL; (Tested by polyethyleneimine, polypropyleneimine, spermine, amino acid peptide and lipid, respectively) dissolved in HBG buffer to prepare a solution A with a concentration of 0.1-10 mg/mL; the above disulfide bond The modified glycosaminoglycan derivative having a terminal carboxyl group is dissolved in HBG buffer to prepare a solution B having a concentration of 0.01-1 mg/mL;
  • the solution A obtained in the above step was mixed with the DNA solution, and after incubation at room temperature for 20 minutes, a binary complex was obtained, and then the solution B was added thereto, and after incubation at room temperature for 20 minutes, a ternary complex having a reduction-sensitive property was obtained. .
  • the obtained vector was selected for cell transfection efficiency measurement (the method is the same as in Example 7), and the binary complex DP containing no masking system and the ternary complex containing non-reducing sensitive masking system (not glycosaminoglycan)
  • the disulfide bond modification was used as a control.
  • the results showed that the reduction-sensitive glycosaminoglycan derivative other than hyaluronic acid was used as a masking system, and the in vitro transfection efficiency of luciferase plasmid to HepG2 cells was significantly higher than that of the two.
  • the control materials were similar to those in Example 7.
  • Example 17 Masking system and cationic material ternary complex gene vector with simultaneous stimuli response characteristics mediated luciferase plasmid assay for in vitro transfection efficiency of HepG2 cells
  • the hyaluronic acid raw material was replaced with one of glycosaminoglycans such as 4-chondroitin sulfate, 6-chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and keratan sulfate.
  • glycosaminoglycan derivative having a disulfide bond modification and a terminal carboxyl group is obtained as a masking system having a reduction-sensitive property.
  • Cross-linking a dicarboxylic acid or a diene containing a disulfide bond or a double selenium bond with a cationic material such as polyethyleneimine, polypropyleneimine, spermine, amino acid polypeptide or lipid to obtain a cationic carrier material containing a reduction-sensitive bond .
  • the cross-linking may be carried out by a conventional physical cross-linking or chemical cross-linking method. For a specific method, reference may be made to the Chinese Patent Application Publication No. CN 02604H0.
  • the sensitive cationic material is dissolved in HBG buffer to prepare a solution A with a concentration of 0.1-10 mg/mL;
  • the above-mentioned reduction-sensitive masking system is dissolved in HBG buffer to prepare a concentration of 0.01-1 mg/mL.
  • Solution B The solution A obtained in the above step is mixed with the DNA solution, and after incubation at room temperature for 20 minutes, a binary complex is obtained, and then solution B is added, and after incubation at room temperature for 20 minutes, a multi-reduction-sensitive property is obtained. Ternary complex.
  • the obtained vector was selected for cell transfection efficiency determination (the same method as in Example 15), and the binary complex DP and the non-reduction-sensitive ternary complex (without glycosaminoglycan and cationic materials) were respectively contained without a masking system. Without the reduction-sensitive key modification), the results showed that the reduction-sensitive glycosaminoglycan derivative other than hyaluronic acid was used as a masking system, and the luciferase plasmid mediated the in vitro transfection efficiency of HepG2 cells was significantly higher than that. Two control materials, and the transfection efficiency of the gene vector having a heavy reduction-sensitive property prepared in Example 16 were higher, similar to the results in Example 15.
  • Example 18 Preparation of a composite vector system and its in vitro transfection efficiency of HepG2 cells mediated by luciferase plasmid
  • the therapeutic plasmid DNA is dissolved in a sterile HBG buffer to prepare a DNA solution having a concentration of 0.1 mg/mL; the dicarboxylic acid or diene containing a disulfide bond or a double selenium bond is combined with polyethyleneimine and polypropylene.
  • a cationic material such as an imine, a spermine, an amino acid polypeptide or a lipid is cross-linked to obtain a cationic carrier material containing a reduction-sensitive bond dissolved in a HBG buffer to prepare an A solution having a concentration of 0.1-10 mg/mL;
  • the glycosaminoglycan derivative masking system modified with a bond and having a carboxyl group at the end is dissolved in HBG buffer to prepare a B solution having a concentration of 0.01-1 mg/mL.
  • the above A solution and the plasmid DNA solution were mixed while magnetic nanoparticles were added, and the mixed solution was incubated at room temperature for 20 minutes to obtain a cationic material/DNA/magnetic nanoparticle ternary complex.
  • B solution is added (magnetic nanoparticles can be added again in this step), and the resulting mixed solution is incubated at room temperature for 20 minutes to obtain a composite carrier of the combined magnetic nanoparticles.
  • the composite carrier of the combined magnetic nanoparticles is capable of responding to an applied magnetic field.
  • the above A solution and the plasmid DNA solution are mixed, and the drug is added at the same time, and the mixed solution is incubated at room temperature for 20 minutes to obtain a cationic material/DNA/drug ternary complex, and then the B solution is added (this step can be repeated again) After the drug was added, the resulting mixed solution was incubated at room temperature for 20 minutes to obtain a composite drug of the combination drug.
  • a solution and the plasmid DNA solution are mixed, and the mixed solution is incubated at room temperature for 20 minutes to obtain a cationic material/DNA binary complex, and then the B solution is added while adding the drug, and the resulting mixed solution is incubated at room temperature. After a minute, a composite carrier of another combination drug was obtained.
  • the composite carrier of the combined magnetic nanoparticles and drug is capable of responding to an applied magnetic field.
  • the bright spot is the successfully transfected cells
  • B is the result of flow cytometry
  • the control group is untransfected HepG2 cells
  • the DP group is transfected with the cation/DNA/screening ternary complex of the present invention.
  • the MDP-cc group is transfected with the quaternary complex of the cation/DNA/shield+MNP of the present invention, and the MF indicates that the magnet is placed under the culture plate after transfection, and the results obtained by the two detection methods are basically the same: After transfection for 10 minutes, the transfection efficiency of the magnetic nanoparticle-containing group was improved compared with the non-magnetic nanoparticle group. After 4 hours of transfection, the enhancement effect was more obvious. Under the action of an external magnetic field, only transfection was performed for 10 minutes. The post-transfection efficiency was significant for all other experimental groups.
  • Example 19 In vivo therapeutic effect of a composite carrier system
  • mice with good growth in logarithmic growth phase were digested with 0.25% trypsin, added with PBS buffer to form a single cell suspension, and subcutaneously inoculated with 2 X 10 6 cells in the right hind leg of the mouse. Cells, inoculated in a volume of 50 ⁇ .
  • 42 mice with similar tumor volumes and a size of about 100 mm 3 were selected as experimental models (two of which were spare).
  • Group A Regular tail vein injection of phosphate buffer (PBS);
  • Group B Periodically injecting the cation/DNA/masking ternary complex solution of the present invention into the tail vein;
  • Group C regular tail vein injection of the cation / DN said A / shielding + MNP quaternary complex solution
  • Group D periodic injection of the cation/DNA/screening + doxorubicin quaternary complex solution of the present invention
  • Group E The cation/DNA/masking + doxorubicin + MNP quaternary complex solution of the present invention was periodically injected into the tail vein.
  • the injection was performed every three days, and the tumor size was measured using vernier calipers on 0, 3, 6, 9, 12, 15, 18, and 21 days (8 parallel samples per time point in each group), and the mice were sacrificed on the 21st day.
  • the tumors were taken out (three parallel samples were taken from each group), and the results are shown in Fig. 12 and Fig. 13.
  • the materials of the B, C, D, and E groups have tumor volume growth relative to the PBS group.
  • Significant inhibition the inhibition of tumor volume growth of each group from small to large in order to group E>0 group>0 group>8 group>eight groups.

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Abstract

本发明公开了一种含有靶向功能的还原敏感遮蔽体系的基因载体系统及其制备方法和在基因治疗领域的应用。该基因载体系统由具备靶向功能的还原敏感遮蔽体系、阳离子高分子材料和质粒DNA组成；阳离子高分子材料和质粒DNA复合形成复合物颗粒，具有靶向功能的还原敏感遮蔽体系通过静电作用遮蔽到复合物表面，可以降低载体的毒性，并成功将负载的基因物质转移到细胞内，实现基因物质的表达，完成转染过程，并且可以提高基因转染的靶向性，同时提高基因转染效率。

Description

一种还原剌激响应型基因载体系统及其制备和应用
技术领域
本发明属于生物医学材料领域， 具体涉及纳米粒子基因载体。
背景技术
在治疗遗传性疾病以及肿瘤方面，基因治疗是一个很有前途的治疗手段，在过去的十年中得到极大地推进。 然而， 基因治疗的瓶颈之一是基因的传递。 核酸是带负电的生物大分子， 因此稳定和高效的合成载体通常 是带正电的。 人们开发了许多的阳离子脂质和聚合物载体系统， 与 DNA静电结合， 并用在体外促进基因 转染。
聚乙烯亚胺 (PEI)是非病毒类载体中受到关注最多的一个阳离子聚合物，它已被广泛地作为黄金基准的聚合 物载体， 但是 PEI固有的细胞毒性和很高的表面正电荷， 限制其在体内的应用。 随着对 PEI基因载体转染 过程的逐渐认识， 对 PEI/DNA进行遮蔽的策说略得到了发展， 开发出多种多样的遮蔽材料， 如非离子型的 亲水性的聚乙二醇 (PEG)、 带负电荷的脂质体和一些可降解的聚合物等。 这些遮蔽材料或与 PEI共价结合， 或与 PEI/DNA复合物静电作用结合， 从而屏蔽正电荷， 降低毒性， 防止盐和血清白蛋白引起聚集， 甚至 达到肿瘤靶向性。 但是由于这些遮蔽材料与基因载体结合书牢固， 在进入细胞后不易脱落， 依然将运载的基 因紧紧包裹， 使其难以发挥作用， 从而大大降低了基因转染的效率。
因此， 开发出既具有较低的细胞毒性， 有具有较高的转染效率的基因载体是行业亟需解决的技术问题。 发明内容
申请人研究发现， 在治疗遗传性疾病以及肿瘤方面， 病毒虽然是目前最有效的基因载体， 但是其不可忽视 的安全隐患限制了病毒载体的临床应用。 大多数病毒是由压缩基因组的内核和包被的蛋白质壳层所组成的 具有核壳结构的纳米尺度颗粒。 病毒在细胞外稳定存在， 其可以识别细胞所提供的信号， 并依据的体内或 细胞微环境的变化而进行逐步的分子转化从而启动解组装响应的特点为非病毒基因载体的设计提供了很 好的启迪。 人们也设计出模拟病毒解组装响应的核壳型 "人造病毒"， 如特定的酶、 酸碱度或还原环境而 引起单一式响应的壳的去遮蔽或核的解压缩过程。 双响应式设计也证明， 同时具备 "壳的去遮蔽"和 "核 的解压缩"的 "人造病毒"更为敏感， 但它们都没有真正模仿到病毒的程序性响应的去遮蔽和解压缩过程。 肿瘤组织中的还原剂如谷胱甘肽 (GSH)的水平比正常组织 （1〜5微摩尔/升） 的高 4倍左右， 达到约 20微 摩尔 /升的水平 （Cancer Research. 2002;62:307-12.) ; 细胞内的还原剂总浓度更高， 相当于正常组织的 1000 倍， 达到了 1〜 10毫摩尔 /升的水平 （Adv Drug Deliver Rev. 2003;55: 199-215.)。 针对这些不同组织不同部 位的不同还原环境， 设计多重刺激响应的拟病毒基因载体， 可以智能化的控制基因的释放表达和对于肿瘤 细胞的靶向杀伤性。
分子量为 25kDa的聚乙烯亚胺 (PEI)是非病毒类载体中被广泛地作为黄金基准的聚合物载体，但是 PEI固有 的细胞毒性和很高的表面正电荷， 限制其在体内的应用。分子量为 800Da的 OEI，虽然几乎没有细胞毒性， 但不能很好压缩 DNA， 转染效率较低。
因此， 申请人首次提出了具有梯度还原刺激响应性的基因载体的设想， 期望开发出具有良好稳定性并能生 物降解为无毒安全小分子的载体， 又同时具有还原等多重刺激响应性， 且具有较高转染效率的基因载体。 本发明提供了一种具有还原刺激响应性的三元复合物纳米粒子基因载体及其制备方法， 并将其用于体外基 因转染、 肿瘤、 哮喘和心血管疾病的基因治疗。
本发明通过以下技术方案来实现：
一种基因载体系统，由遮蔽体系、阳离子高分子材料和质粒 DNA组成，所述阳离子高分子材料和质粒 DNA 复合形成二元复合物颗粒，所述遮蔽体系通过静电作用遮蔽到所述二元复合物表面，形成三元复合物颗粒， 所述遮蔽体系中含有还原敏感键。
作为可选方式， 所述遮蔽体系和阳离子材料中都含有还原敏感键， 构成具有多重还原刺激响应特性的纳米 粒子基因载体。 采用多重还原刺激响应的策略， 更接近病毒载体的去遮蔽和解压缩过程， 可以提高基因输 送的效率， 并远优于单一式响应。
作为可选方式， 所述还原敏感键为双硫键或双硒键中的至少一种， 使得材料在还原条件下会发生降解。 所 述遮蔽体系和阳离子材料中可以都含有双硫键或都含有双硒键， 也可以一个含双硫键， 另一个含双硒键， 还可以各自都同时含有双硫键和双硒键。
作为可选方式， 所述遮蔽体系中含有双硫键， 所述阳离子材料中含有双硒键。 这种结构的基因载体系统能 够在肿瘤的细胞外和细胞内不同还原剂浓度的条件下， 通过分子化学结构的变化作出实时响应， 实现梯度 还原刺激响应。 可以使得三元复合物载体在细胞外低 GSH浓度的环境中保持稳定， 长时间循环， 并保护 DNA免受血清中的脱氧核糖核酸酶 (DNase)降解； 到达目标肿瘤组织时， 由于肿瘤组织稍高的 GSH浓度， 导致外层 （遮蔽体中） 更为敏感的双硫键首先断开 （或部分断开）， 更有利于复合物颗粒的内吞； 进入细 胞后，胞内 GSH浓度比胞外高至 1000倍达到 1〜10毫摩尔 /升，此时，较为稳定的双硒键（阳离子材料中） 也断开， 阳离子材料降解， 释放出 DNA, 有利于 DNA入核， 从而实现基因的高表达。
作为可选方式， 所述遮蔽体系中含有双硒键。 这种结构的基因载体能更好的保护 DNA, 使其免受血清中 的脱氧核糖核酸酶 （DNase) 降解， 以及避免血清中负电荷的血清蛋白造成的聚沉现象而形成血栓。 外层 (遮蔽体中） 采用相对更稳定的双硒键， 使得三元复合物载体在细胞外低 GSH浓度的环境中更稳定， 循 环时间更长， 到达目标肿瘤组织时， 在肿瘤组织稍高的 GSH浓度下仍然需要接触更长的时间或直到进入 细胞后才会断开， 进入细胞后， 整个三元复合物体系彻底解组装， 从而释放出 DNA, 实现基因转染。 还 可以与外层含双硫键的载体混合使用， 已到达更好的综合效果。
作为可选方式， 所述遮蔽体系可以为对糖胺聚糖上的至少一个葡萄糖醛酸单元上的羧基进行修饰， 得到的 具有还原敏感的双硫键或双硒键并且末端仍是羧基的糖胺聚糖衍生物。糖胺聚糖（glycosaminoglycan) , 为 杂多糖的一种， 糖胺聚糖分为： 透明质酸、 4-硫酸软骨素、 6-硫酸软骨素、 硫酸皮肤素、 硫酸乙酰肝素、 肝素和硫酸角质素等。 它是由重复的二糖单位说构成的长链多糖， 其二糖单位之一是氨基己糖（氨基葡萄糖 或氨基半乳糖）， 故称糖胺聚糖； 另一个是糖醛酸。 糖胺聚糖是细胞间质的重要组成部分， 细胞间质绝大 部分都是糖胺聚糖， 因此具有良好的生物相容性和可降解性。
作为可选方式， 本发明中所述遮蔽体系中所用的糖胺聚糖的分子量为 5-2000 kDa。
作为可选方式， 本发明中所述糖胺聚糖为透明质酸、 4-硫书酸软骨素、 6-硫酸软骨素、 硫酸皮肤素、 硫酸乙 酰肝素、 肝素和硫酸角质素中的至少一种。
作为可选方式， 所述遮蔽体系为通过对透明质酸上的至少一个葡萄糖醛酸单元上的羧基进行修饰， 得到的 具有还原敏感的双硫键并且末端仍是羧基的透明质酸衍生物 （HA-SS-COOH)。 结构示意图如下：
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Figure imgf000004_0001
作为可选方式， 所述阳离子材料由含双硫键或双硒键的化合物与聚乙烯亚胺、 聚丙烯亚胺、 精胺、 氨基酸 多肽、 肽类树状分子或含肽类树状分子的阳离子脂质材料或其他脂质材料（如阳离子脂质聚合物等） 中的 至少一种交联而成。 所述阳离子材料的外围都含有丰富的氨基， 便于与含双硫键或双硒键的化合物交联， 所述含双硫键或双硒键的化合物为含双硫键或双硒键的二羧酸或二烯。 所述聚乙烯亚胺、 聚丙烯亚胺、 精 胺、 氨基酸多肽或脂质材料的分子量为 200〜4000 Da。
作为可选方式， 所述由含双硫键或双硒键的化合物与聚乙烯亚胺或聚丙烯亚胺或精胺交联而成阳离子材料 结构式为：
Figure imgf000005_0001
η= 1〜10， χ , y> l, DT为聚乙烯亚胺或聚丙烯亚胺或精胺。 所述阳离子材料细胞毒性低， 转染效率高， 分子量可控。 说
烯亚胺 (OEI-SeSex)， 结构示意图如下： 书
Figure imgf000005_0002
其中 n= 1 〜 10， X 1。
阳 离 子 脂 质 材 料 结 构 式 为
Figure imgf000005_0003
烃基或氨基酸基团， R2 为氨基酸基团、 饱和 /不饱和烃基或
Figure imgf000005_0004
， 和 中至少一个为氨基酸基团， X、 Y、 Ζ为 ΝΗ、 0或 S， o、 p、 q 分别独立为 1或 0; 所述饱和 /不饱和烃基优选垸基、 烯基、 芳香基， 更优选为 C10-C20的垸基、 的烯基、 含芳香基的氨基酸衍生物， 所述氨基酸基团优选为赖氨酸、 精氨酸或组氨酸基团。
作为可选方式， 所述含肽类树状分子的阳离子脂质材料结构式为：
说
作为可选方式， 本发明所述基因载体系统中所述的质粒 DNA采用在真核细胞表达的质粒 DNA。 可以是含 报告基因或细胞因子基因或抑癌基因的真核重组表达质粒。
作为可选方式， 本发明所述基因载体系统中遮蔽体系与质粒 DNA的质量比例为 0. 1 : 1 〜 50: 1， 阳离 子材料与质粒 DNA的质量比例为 0. 1 : 1 〜 50: 1。 书
本发明还提供了制备上述基因载体系统的方法， 其具体步骤如下：
将质粒 DNA溶于灭菌水或无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖） 中， 配制成 浓度为 0.1 mg/mL的 DNA溶液； 将阳离子材料溶于 HBG缓冲液中， 配制成浓度为 0.1 - 10 mg/mL的溶液 A; 将遮蔽体系溶于 HBG缓冲液中， 配制成浓度为 0.01 - 1 mg/mL的溶液 B;
将上述步骤中得到的溶液 A与 DNA溶液混合，在室温下孵育 20分钟后，得到二元复合物，再加入溶液 B, 在室温下孵育 20分钟后， 得到三元复合物。
作为一种可选方式， 所述基因载体系统的方法的具体步骤如下：
( 1 ) 具有靶向功能的还原敏感遮蔽体系的制备：
1 )将透明质酸溶于 pH 6.8的磷酸盐缓冲液（PBS)中，加入 1- (3-二甲氨丙基) -3-乙基碳二亚胺盐酸盐（EDC •HC1) 和 1-羟基苯并三唑 （HOBT)， 搅拌， 活化羧基； 加入胱胺二盐酸盐 （Cys)， （作为优选， 透明质酸
(HA) 和胱胺二盐酸盐的质量比在 1 : 30 - 30: 1之间） 搅拌， 室温下反应 12小时， 反应结束后将反应产 物进行透析， 冷冻干燥， 得到胱胺接枝的透明质酸 （HA-Cys) ;
2)将胱胺接枝的透明质酸（HA-Cys)溶于 pH 8.5的磷酸盐缓冲液（PBS)中，加入过量的二硫苏糖醇（DTT) , 在室温下反应 4小时后，用盐酸（HC1 )调节 pH值到 3. 5，然后加入氯化钠（NaCl)至终浓度为 5% (w/v) , 随后， 用乙醇沉淀， 再复溶于水， 离心， 冷冻干燥即可得到巯基化的透明质酸 （HA-SH) ;
3 ) 巯基化的透明质酸 （HA-SH) 溶于磷酸盐缓冲液 （PBS ) 中， 与过量的 3-巯基丙酸在室温下反应过夜， 反应结束后将反应产物进行透析， 冷冻干燥， 得到二硫键修饰且末端为羧基的透明质酸（HA-SS-COOH)；
(2 ) 具有靶向功能的还原敏感遮蔽体系的基因载体系统的制备：
将质粒 DNA溶于灭菌水或无菌 HBG缓冲液中， 获得 DNA溶液； 将阳离子聚合物基因载体溶于 HBG缓 冲液中， 得到溶液 A; 将具有靶向功能的还原敏感遮蔽体系 （HA-SS-COOH) 溶于 HBG缓冲液中， 得到 溶液 B; 将 DNA溶液和溶液 B复合得到基因载体和质粒 DNA的复合物颗粒溶液， 在室温下放置 20分钟 后， 加入溶液 B， 得到所述基因载体系统。
本发明还提供了上述基因载体系统的应用： 将所述基因载体与超顺磁性纳米粒子 （MNP ) 和 /或脂溶性的 药物联合制成复合载体或药物， 用于体外基因转染、 肿瘤、 哮喘或心血管疾病的基因治疗、 基因与化学 药物疗联合治疗、 疾病诊疗一体化。
本发明还提供了由基因载体与磁性纳米粒子联合的复合载体系统， 所述磁性纳米粒子可以是水溶性的也可 以是脂溶性的。 作为优选， 所述磁性纳米粒子和基因弥散地分布于所述基因载体中。 作为优选， 所述磁性 纳米粒子为四氧化三铁，伽马三氧化二铁或掺有锰、钴或锌的铁氧磁性纳米粒子中的至少一种。作为优选， 所述磁性纳米粒子为负电性的， 其平均粒径尺度在 5〜 50 nm之间，其表面电位在 -5〜 -50 mV之间。 作为优 选， 所述磁性纳米粒子表面修饰有负电性有机材料， 所述负电性有机材料可以是羧基硅垸偶联剂、 氨基酸 类树状分子、 酒石酸、 柠檬酸、 草酸、 乙酸中的至少一种。 通过加入磁性纳米粒子使复合载体能响应外加 磁场， 并在外加磁场的引导下集中到目标部位， 实现磁靶向功能。
本发明还提供了一种复合药物， 包括所述基因载体和药物有效成分。 所述药物可以与所述质粒 DNA—起 包裹在所述阳离子材料， 实现质粒 DNA与药物同时释放， 同时达到基因治疗和药物治疗的目的； 所述药 物也可以包裹在所述遮蔽体系中或同时包裹在遮蔽体系和阳离子材料中，实现药物与 DNA的分阶段释放。 本发明还提供了所述基因载体与药物和磁性纳米粒子三者同时复合的复合载体系统。 将三者的功能集成在 一个载体系统中。 本发明还提供了一种所述基因载体应用， 将其用于体外基因转染、 肿瘤、 哮喘或心血管疾病的基因治疗以 及基因与化学药物疗联合治疗、 疾病诊疗一体化。
本说明书中公开的所有特征， 或公开的所有方法或过程中的步骤， 除了互相排斥的特征和 /或步骤以外， 均 可以以任何方式组合。
本发明的有益效果：
1. 本发明所述基因载体采用透明质酸衍生物作为遮蔽体， 一方面不会影响阳离子聚合物对 DNA的复合能 力， 另一方面利用透明质酸的靶向功能提高了基因载体与细胞表面受体的结合效率， 并通过 HA受体介导 的细胞内吞作用维持内涵体脱离的能力， 促进 PEI/DNA/HA-SS-COOH三元复合物被细胞摄取。
2. 本发明所述基因载体的遮蔽体系， 一方面其末端的羧基可以与阳离子聚合物和质粒 DNA形成的复合物 颗粒表面带有的大量氨基通过静电作用相结合，达到遮蔽的作用，降低毒性及避免血清白蛋白引起的聚集， 逃脱网状内皮系统的清除作用， 从而减少阳离子聚合物的细胞毒性、 增加血清稳定性； 另一方面， 在进入 细胞后， 在胞内还原性环境的作用下， 还原敏感的二硫键会发生断裂， 从而解除屏蔽作用， 使装载的基因 暴露出来， 提高其从内涵体逃逸的能力， 从而说大大提高转染效率。
3. 本发明所述基因载体， 利用静电结合的方法将具有靶向功能的还原敏感响应遮蔽层引入基因载体体系， 可以通过调节遮蔽比例 （HA-SS-COOH/DNA)， 控制过分遮蔽对转染效率的降低， 同时利用靶向配体提高 基因载体对目标组织的特异性识别和传递， 并将运载的 DNA环境响应地释放在靶细胞内。
4. 本发明所述具有多重还原刺激响应性的基因载体， 采用书多重还原刺激响应的策略， 更接近病毒载体的去 遮蔽和解压缩过程， 其基因转染效率明显优于其他单一式刺激响应或非智能响应的传统基因载体。
5. 本发明所述含有双硒键的还原刺激响应阳离子材料（如 OEI-SeSex)， 能与质粒 DNA通过静电作用形成 表面带有正电荷的纳米尺度的二元复合物颗粒 （OEI-SeSex/DNA binary polyplexeS，简写为 DSe)。 本发明所 述含有双硫键的还原刺激响应遮蔽体系 （如 HA-SS-COOH) , 其末端的羧基可通过静电络合作用与 DSe表 面剩余的正电荷结合， 形成表面具有很低正电荷或是负电荷的三元复合物（OEI-SeSex/DNA/HA-SS-COOH ternary polyplexes, 简写为 DSeS)。
6. 本发明所述含有双硫键的还原刺激响应遮蔽体系， 一方面可以达到屏蔽正电荷的作用， 降低毒性及避免 血清白蛋白引起的聚集， 逃脱网状内皮系统的清除作用， 从而减少阳离子聚合物的细胞毒性、 增加血清稳 定性； 另一方面， 在到达肿瘤组织部位或进入细胞后， 在还原性环境的作用下， 更敏感的双硫键会发生断 裂， 解除屏蔽作用， 使内部的 DSe暴露出来， 提高其从内涵体逃逸的能力， 从而有利于提高转染效率。
7. 本发明所述基因载体系统在肿瘤的细胞外和细胞内不同还原剂浓度的条件下，通过分子化学结构的变化 作出实时响应。 梯度还原刺激响应性可以使得 OEI-SeSex/DNA/HA-SS-COOH三元复合物载体在细胞外低 GSH浓度的环境中保持稳定，长时间循环，并保护 DNA免受血清中的 DNase降解；到达目标肿瘤组织时， 由于肿瘤组织稍高的 GSH浓度， 导致外层更为敏感的双硫键首先断开（或部分断开）， 更有利于复合物颗 粒的内吞； 进入细胞后， 胞内 GSH浓度比胞外高至 1000倍达到 1〜10毫摩尔 /升， 此时， 较为稳定的双硒 键也断开， 降解为低分子量的 OEI, 释放出 DNA, 有利于 DNA入核， 从而实现基因的高表达。
8. 本发明所述基因载体的制备方法操作简单、 便于大规模生产。
9. 本发明所述基因载体， 能够很方便地用于体外基因转染、 肿瘤、 哮喘和心血管疾病的基因治疗。
附图说明
图 1本发明所述遮蔽体系中的一种的结构和合成过程示意图。
图 2是 PEI/DNA/HA-SS-COOH复合物的琼脂糖凝胶电泳图，其中：从左至右共八条泳道，第一道为裸 DNA; 第二道至第七道为 PEI/DNA/HA-SS-COOH复合物，其中 HA-SS-COOH/DNA的质量比分别为 10、 6、 3, 2、 1 和 0.5时的电泳图； 第八道为 PEI/DNA复合物。
图 3是不同浓度的 PEI/DNA/HA-SS-COOH复合物对 B 16细胞转染 24小时后的细胞存活率。
图 4是 PEI/DNA复合物和 PEI/DNA/HA-SS-COOH (HA-SS-COOH/DNA质量比为 1 )复合物分别介导绿色 荧光蛋白质粒对 B 16细胞转染效率图。
图 5本发明所述含有双硫键的还原刺激响应遮蔽体系的结构式。
图 6本发明所述含有双硒键的还原刺激响应阳离子材料的结构式。
图 7本发明实施例 11所述三元复合物纳米粒子基因载体示意图。
图 8是采用 GPC测定的色谱图。
图 9是不同转染复合物对 HepG2细胞转染 24小时后的细胞存活率。
图 10是不同转染复合物（DP; DSe; DPS DSeS)分别介导绿色荧光蛋白质粒对 HepG2细胞转染效率图。 图 11是实施例 18所述不同转染复合物（control: 未经转染的 HepG2细胞， DP: 本发明所述阳离子 /DNA/ 遮蔽体三元复合物， MDP-cc: 本发明所述阳离子 /DNA/遮蔽体 +MNP的四元复合物， MF表示转染后在培 养板下方放置磁铁）分别介导绿色荧光蛋白质粒对 HepG2细胞转染效率图， 其中 A为荧光显微镜照片， B 为流式细胞仪检测结果。 图 12是实施例 19所述体内治疗 21天后各实验组取出的肿瘤组织的照片， 每组 3个平行样。 图 13是实施例 19所述体内治疗实验中各时间点取出的肿瘤的体积的统计结果图 （每组每个时间点各设 8 个平行样）。
具体实施方式
为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实施例， 对本发明进行进一步详细 说明。 下列实施例选用透明质酸和聚乙烯亚胺作为示范， 本领域技术人员很容易将其推广到其他材料。 应 当理解， 此处所描述的具体实施例仅仅用以解释本发明， 并不用于限定本发明。
简写说明： 一
DP: PEI 25kDa和 DNA的二元复合物， 质量比为 1.2/1。
DSe: OEI-SeSex和 DNA的二元复合物。
DPS: PEI 25kDa、 DNA和 HA-SS-COOH的三元复合物。
DSeS: OEI-SeSex、 DNA和 HA-SS-COOH的说三元复合物。
实施例 1： 具有靶向功能的还原敏感遮蔽体系的制备
按照表 1所述的投料比例， 将透明质酸溶于 pH 6 6.8的磷酸盐缓冲液 （PBS) 中， 加入 1- (3-二甲氨丙基) -3- 乙基碳二亚胺盐酸盐（ED HC1)和 1-羟基苯并三唑（HOBT)， 搅拌， 室温下反应 2小时活化羧基。 加入 胱胺二盐酸盐 （Cys)， 搅拌， 上述反应溶液在室温下反应书过夜， 反应结束后将反应产物用截留量为 3500 的透析袋透析 48小时， 冻干， 得到胱胺接枝的透明质酸 （HA-Cys)。 通过核磁图谱计算产物 （HA-Cys) 中胱胺的接枝率， 结果见表 1。
将不同比例胱胺接枝的透明质酸（HA-Cys)溶于 pH 8.5的磷酸盐缓冲液（PBS) 中， 加入 5倍过量的二硫 苏糖醇 （DTT)， 在室温下反应 4小时后， 用盐酸 （HC1) 调节 pH值到 3.5， 然后加入氯化钠 （NaCl) 至 终浓度为 5% (w/v 随后， 用乙醇沉淀， 再复溶于水， 离心， 冻干即可得到巯基化的透明质酸（HA-SH)。 用 Ellman方法 （见 Anal Biochem. 1985; 145: 200-4. ) 计算产物 (HA-SH) 中 SH所占的比例， 结果见表 1。 不同比例巯基化的透明质酸 （HA-SH) 溶于磷酸盐缓冲液 （PBS) 中， 与 100倍过量的 3-巯基丙酸在室温 下反应过夜， 反应结束后将反应产物用截留量为 3500的透析袋透析 48小时， 冻干， 得到二硫键修饰且末 端为羧基的透明质酸衍生物 （HA-SS-COOH)。 用 Ellman方法计算产物 （HA-SS-COOH) 中 S-S所占的 比例， 结果见表 1。
Figure imgf000008_0001
在本实施例中还分别选取了分子量为 5k Da、 40k Da、 100 kDa、 1000 kDa和 2000 kDa的透明质酸进行制 备试验， 结果显示各分子量的透明质酸均能成功改性， 得到二硫键修饰且末端为羧基的透明质酸衍生物。 实施例 2： 具有靶向功能的还原敏感遮蔽体系的基因载体系统的制备
将质粒 DNA溶于无菌 HBG缓冲液中， 配制成浓度为 0.1 mg/mL的 DNA溶液 A; 将阳离子聚合物基因载 体聚乙烯亚胺 （PEI) 溶于无菌 HBG缓冲液 （4-羟乙基哌嗪乙磺酸 20 毫摩尔， pH 7.4， 5% 葡萄糖） 中， 配制成浓度为 0.01-1 mg/mL的 PEI溶液 B; 将具有靶向功能的还原敏感遮蔽体系（HA-SS-COOH)溶于无 菌 HBG缓冲液中， 配制成浓度为 0.01-1 mg/mL的 HA-SS-COOH溶液 C。
将不同浓度的阳离子聚合物 PEI溶液和质粒 DNA水溶液以质量比为 1.2: 1混合， 混合后的水溶液在室温 下孵育 20分钟后，得到 PEI/DNA复合物。再加入不同浓度的 HA-SS-COOH溶液，水溶液在室温下孵育 10 分钟后， 得到具有靶向功能的还原敏感遮蔽体系的基因载体系统 PEI/DNA/HA-SS-COOH三元复合物。 此 PEI/DNA/HA-SS-COOH三元复合物用于下一步的电泳、 转染和毒性实验。 按照上述方法制备的三元复合 物颗粒的组成及性能如表 2所示。
表 2 PEI/DNA/HA-SS-COOH三元复合物的组成和性能
三元复合物编号 HA-SS-COOH种类 PEI:DNA:HA-SS-COOH的 三元复合物粒 三元复合物表 质量比 径 (nm) 面电荷 (mV)
DP 不含遮蔽体系 1.2： 1 116.25 27.50 DPS30-0.5 HA-SS-COOH 30 1.2： 1： 0.5 155.20 13.97
DPS30-2 HA-SS-COOH 30 1.2： 1： 2 169.30 6.72
DPS30-5 HA-SS-COOH 30 1.2： 1： 5 145.20 -23.40
DPS45-2 HA-SS-COOH 45 1.2： 1： 2 175.54 6.71
DPS65-2 HA-SS-COOH 65 1.2： 1： 2 179.12 6.77
DPS65-0.1 HA-SS-COOH 65 0.1： 1： 0.1 498.00 -41.80
DPS65-50 HA-SS-COOH 65 50： 1： 50 150.70 23.40 在本实施例中还分别采用实施例 1中制备的具有不同分子量透明质酸衍生物作为遮蔽体系成功制备了一系 列三元复合物颗粒。
实施例 3： 利用凝胶电泳鉴定复合物颗粒的稳定性
取 5 0.1 mg/mL的 DNA溶液与 3 μ 0.2 mg/mL的 PEI溶液混合，在室温下孵育 20分钟，然后加入 5 μL 不同浓度的 HA-SS-COOH溶液， 使 HA-SS-说COOH/DNA的质量比分别为 10、 6、 3、 2、 1和 0.5， 并在室 温下孵育 10分钟后， 利用凝胶电泳阻滞实验检测复合物颗粒在不同量的 HA-SS-COOH遮蔽体系加入后的 稳定性。
图 2的电泳结果表明， 加入的 HA-SS-COOH透明质酸遮蔽体系的量达到 DNA量的 10倍时也不会破坏阳 离子聚合物与 DNA形成的复合物。 书
实施例 4: 细胞存活率检测
转染前 24小时内， 取对数生长期 B16细胞， 胰酶消化后用 DMEM培养基稀释， 按每孔 1 >< 104细胞的密度 接种于 96孔培养板， 置于含 5% (体积百分数） 的 C02， 温度为 37°C的孵箱中继续培养至 80-90%融合， 转染时， 吸去前一天加注的细胞培养板中的培养液， 用 PBS洗涤两次后， 加入基因组转染的复合物颗粒和 含有 10% (质量 /体积百分数） 的小牛血清的 DMEM培养基至终体积 O. l mL, 继续培养 24小时； 加入 10 浓度为 5 mg/mL 的 MTT溶液 （3- (4， 5-二甲基噻唑 -2 ) -2， 5-二苯基四氮唑溴盐） 在 37°C孵 育 4小时， 加入 150 L DMSO (二甲基亚砜）。 然后用酶标仪 （Bio-Rad) 测试每孔的吸光度值 A， 测试 波长选用 492 nm。 细胞存活率按下公式计算：
细胞存活率 （％) = (Asample/Acontro！) 100
Asample是转染后的细胞样品孔的吸光度值， A∞ntol是不与转染复合物作用的细胞对照孔的吸光度值， 每组 实验重复六次。
图 3是不同浓度的 PEI/DNA/HA-SS-COOH复合物对 B 16细胞转染 24小时后的细胞存活率。 由图 3可见， PEI/DNA/HA-SS-COOH三元复合物对 B 16细胞毒性远远低于 PEI/DNA复合物， 细胞存活率在 80%以上， 可见本发明的基因载体系统具有较低的细胞毒性。
实施例 5: 利用具有靶向功能的还原敏感遮蔽体系的基因载体系统介导绿色荧光蛋白质粒对 B 16细胞的体 外转染效率的测定
B 16细胞的培养： 取小鼠黑色素瘤细胞 B16细胞， 在含有 10% (质量 /体积百分数） 的胎牛血清的培养液 中， 在含 5% (体积百分数） C02， 温度为 37°C的培养箱中培养 24小时；
转染前， 取对数生长期 B16细胞， 胰酶消化后用 DMEM培养基稀释， 按每孔 4χ 105细胞的密度接种于 6 孔培养板， 置于含 5% (体积百分数） 的 C02， 温度为 37°C的培养箱中继续培养至 80-90%融合， 转染时， 吸去前一天加注的细胞培养板中的培养液， 用 PBS洗涤两次后， 加入基因组转染的复合物颗粒以及含有 10% (质量 /体积百分数） 的小牛血清的 DMEM培养基至终体积 2ml， 继续培养 48个小时；
体外转染效率的测定： 取出培养板， 用倒置荧光显微镜照相； 图 4分别显示了无功能性 HA-SS-COOH作 为遮蔽体系的 PEI/DNA和有遮蔽体系的 PEI/DNA/HA-SS-COOH两种基因载体介导绿色荧光蛋白质粒对 B 16细胞转染后的荧光照片。从照片中绿色荧光蛋白表达的情况可见， HA-SS-COOH作为遮蔽体系， 很大 程度的提高了绿色荧光蛋白质粒在 B16细胞中的表达。 这是由于 HA-SS-COOH具有透明质酸（HA) 的特 性， 可以和 B16细胞表面的 CD44受体作用， 帮助 PEI/DNA/HA-SS-COOH三元复合物粒子内吞； 同时粒 子进入细胞后， 还原响应的二硫键断裂， 帮助遮蔽体系的剥离， 使得暴露出 PEI的正电荷， 发挥其质子泵 效应， 从而促进转染效率的提高。
实施例 6： 利用具有靶向功能的还原敏感遮蔽体系的基因载体系统介导荧光素酶质粒对 B16细胞的体外转 染效率的测定
B 16细胞的培养： 同实施例 4的方法。
在 6孔板上以 4χ 105细胞 /孔接种 B 16细胞， 在 37°C， 5%的 C02的细胞培养箱中培养 24小时左右， 转染 时，吸去前一天加注的细胞培养板中的培养液，用 PBS洗涤两次后，加入表 3中所列的含有荧光素酶 DNA 的复合物颗粒以及无血清或者含有 10%， 50% (质量 /体积百分数）的小牛血清的 DMEM培养基至终体积 2 mL， 继续培养 24个小时；
体外转染效率的测定： 取出培养板， 吸去培养液， PBS洗涤细胞 2次， 然后加入含 1%的 Triton X-100的裂 解液， 使细胞裂解后用 Promega公司的荧光素酶检测试剂盒检测。 结果如表 4所示。
本发明利用具有靶向功能的还原敏感遮蔽体系基因载体系统 PEI/DNA/HA-SS-COOH三元复合物， 提高基 因载体的性能， 分别在无血清， 10%血清及 50%血清的条件下， 其对 B16细胞的转染效率相应提高了 14 倍， 538倍及 130倍。
表 3具有靶向功能的还原敏感遮蔽体系的基因载体系统介导荧光素酶质粒对 B16的体外转染效率表
说
Figure imgf000010_0001
转染效率的测定
HepG2细胞的培养： 取人肝肿瘤细胞 HepG2细胞， 在含书有 10% (质量 /体积百分数） 的胎牛血清的培养液 中， 在含 5% (体积百分数） C02， 温度为 37°C的培养箱中培养 24小时；
在 6孔板上以 4χ 105细胞 /孔接种 HepG2细胞， 在 YTC， 5%的 C02的细胞培养箱中培养 24小时左右， 转 染时，吸弃前一天加注的细胞培养板中的培养液，用 PBS洗涤两次后，加入表 4中所列的含有荧光素酶 DNA 的复合物颗粒以及无血清或者含有 10%， 50% (质量 /体积百分数）的小牛血清的 DMEM培养基至终体积 2 mL， 继续培养 24个小时；
体外转染效率的测定： 取出培养板， 吸去培养液， PBS洗涤细胞 2次， 然后加入含 1%的 Triton X-100的裂 解液， 使细胞裂解后用 Promega公司的荧光素酶检测试剂盒检测。 结果如表 4所示。
表 4具有靶向功能的还原敏感遮蔽体系的基因载体系统介导荧光素酶质粒对 HepG2的体外转染效率表
Figure imgf000010_0002
本发明利用具有靶向功能的还原敏感遮蔽体系基因载体系统 PEI/DNA/HA-SS-COOH三元复合物， 提高基 因载体的性能，分别在无血清， 10%血清及 50%血清的条件下，其对 HepG2细胞的转染效率相应提高了 13 倍， 28倍及 33倍。
实施例 8: 具有双重还原刺激响应性的基因载体 （OEI-SS/DNA/HA-SS-COOH) 的制备
将质粒 DNA溶于无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖）中，配制成浓度为 0.1 mg/mL的 DNA溶液；将双硫键交联的聚乙烯亚胺（OEI-SS)溶于 HBG缓冲液中，配制成浓度为 0.1-lO mg/mL 的 OEI-SS溶液；将含有双硫键的遮蔽体系（HA-SS-COOH)溶于 HBG缓冲液中，配制成浓度为 0.01-1 mg/mL 的 HA-SS-COOH溶液。
将上述 OEI-SS溶液溶液和质粒 DNA溶液混合，混合后的溶液在室温下孵育 20分钟后，得到 OEI-SS/DNA 二元复合物。 再加入 HA-SS-COOH溶液， 所得混合溶液在室温下孵育 20分钟后， 得到遮蔽体系和阳离子 材料同时含有双硫键的具有多重还原刺激响应性的基因载体 OEI-SS/DNA/HA-SS-COOH三元复合物。 实施例 9: 具有双重还原刺激响应性的基因载体 （OEI-SeSex/DNA/HA-SeSe-COOH) 的制备
将质粒 DNA溶于无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖） 中， 配制成浓度为 0.1 mg/mL的 DNA溶液； 将双硒键交联的聚乙烯亚胺（OEI-SeSex)溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的 OEI-SeSex溶液； 将含有双硒键的遮蔽体系 （HA-SeSe-COOH) 溶于 HBG缓冲液中， 配制成浓 度为 0.01-1 mg/mL的 HA-SeSe-COOH溶液。
将上述 OEI- SeSex溶液溶液和质粒 DNA溶液混合， 混合后的溶液在室温下孵育 20分钟后， 得到 OEI- SeSex/DNA二元复合物。 再加入 HA-SeSe-COOH溶液， 所得混合溶液在室温下孵育 20分钟后， 得到遮蔽 体系和阳离子材料同时含有双硒键的具有多重还原刺激响应性的基因载体 OEI-SeSex /DNA/HA- SeSe- COOH三元复合物。
- - - 将质粒 DNA溶于无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖）中，配制成浓度为 0.1 mg/mL的 DNA溶液； 将双硒键交联的聚乙烯亚胺（OEI-SeSex)溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的 OEI-SeSex溶液； 将含有双硫键的遮蔽体系 （HA-SS-COOH) 溶于 HBG缓冲液中， 配制成浓度 为 0.01-1 mg/mL的 HA-SS-COOH溶液。
将上述 OEI- SeSex溶液溶液和质粒 DNA溶液混合， 混合后的溶液在室温下孵育 20分钟后， 得到 OEI- SeSex/DNA二元复合物。 再加入 HA-SS-COOH溶液， 所得混合溶液在室温下孵育 20分钟后， 得到具有多 重还原刺激响应性的基因载体 OEI-SeSex/DNA/HA- SS-COOH三元复合物。
体外基因转染试验显示实施例 8-10所述的三种基因载体基因转染效率均高于只含有一重还原刺激响应性 的基因载体。
实施例 11： 具有梯度还原刺激响应性的基因载体的制备
将质粒 DNA溶于无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖）中，配制成浓度为 0.1 mg/mL的 DNA溶液； 将双硒键交联的聚乙烯亚胺（OEI-SeSex)溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的 OEI-SeSex溶液； 将含有双硫键的遮说蔽体系 （HA-SS-COOH) 溶于 HBG缓冲液中， 配制成浓度 为 0.01-1 mg/mL的 HA-SS-COOH溶液。
将不同浓度的双硒键交联的聚乙烯亚胺 （OEI-SeSex) 溶液和质粒 DNA溶液以一定质量比混合， 混合后的 溶液在室温下孵育 20分钟后 , 得到 OEI-SeSex/DNA二元复合物。 再加入不同浓度的 HA-SS-COOH溶液， 所得混合溶液在室温下孵育 20分钟后 , 得到同时含有双书硫键和双硒键具有梯度还原双重刺激响应性的基 因载体 OEI-SeSex/DNA/HA-SS-COOH三元复合物。此 OEI-SeSex/DNA/HA-SS-COOH三元复合物用于下一 步的毒性和转染等实验。 按照上述方 ¾ί制备的三元复合物粒子的组成及性能如表 5所示。
表 5. OEI-SeSex/DNA/HA-SS-COOH三元复合物的组成和性能
Figure imgf000011_0001
实施例 12: 双硒键的还原刺激响应性能测试
为了验证双硒键的还原刺激响应性能， 将 OEI8QQ-SeSex分别用不同浓度 GSH ( 10 μΜ或 100 μΜ ) 处理一定 的时间 （4 h或 8 h) 后， 用凝胶渗透色谱法 （GPC法） 测其分子量， 色谱图见图 8。 GPC设备参数如下： Waters 2690D HPLC, ultrahydrogel 120 及 ultrahydrogel 1000柱串联， 折射率检测器； 流动相： 0.1 摩尔 /升 的甲酸钠缓冲液 （pH 2.8)， 流速 1.0 毫升 /分， 柱温 35° C; 以聚乙二醇为标准物质计算分子量。
图 8是采用 GPC测定的色谱图， 从下往上分别是： PEI25k是分子量为 25kDa的聚乙烯亚胺； OEI8QQ-SeSex 是双硒键交联的聚乙烯亚胺； OEI800-SeSex经 10 μΜ GSH处理 4 h后； OEI8QQ-SeSex经 10 μΜ GSH处理 8 h 后； OEI8QQ-SeSex经 100 μΜ GSH处理 4 h后； OEI8QQ是分子量为 800 Da的寡聚乙烯亚胺。 由图 8可见， 本实施例中选用与 PEI25k分子量相当的 OEI8QQ-SeSex， 经过 10 μΜ GSH处理 4 h或 8 h后， 其分子量均不发 生改变， 而当用更高浓度的 GSH ( ΙΟΟ μΜ) 处理 4 h后， 分子量变小至 OEI8QQ。 说明 OEI8QQ-SeSex中的双 硒键在 lO M GSH环境下是比较稳定的， 而在 100 μΜ的高浓度下， 双硒键会断开， 使 OEI8QQ-SeSex降解 为小分子的 OEI8QQ片段。 而文献报道 （Aaps Journal. 2009; 11： 445-455 )， 双硫键在 10μΜ水平的还原剂 作用下也会随着时间的延长而断开。 说明在相同 10 μΜ水平的还原剂下， 双硒键要比双硫键更稳定； 在更 高水平的还原剂下， 双硒键和双硫键都会断开。 可见， 本发明所述 OEI-SeSex/DNA/HA-SS-COOH三元复 合物载体具有梯度还原刺激响应性能。
实施例 13: 细胞存活率检测
HepG2细胞的培养： 取人的肝肿瘤细胞 HepG2细胞， 在含有 10% (质量 /体积百分数） 的胎牛血清的培养 液中， 在含 5% (体积百分数） C02， 温度为 37°C的培养箱中培养 24小时。
转染前 24小时内， 取对数生长期 HepG2细胞， 胰酶消化后用 DMEM培养基稀释， 按每孔 1 >< 104细胞的密 度接种于 96孔培养板， 置于含 5% (体积百分数） 的 C02， 温度为 37°C的培养箱中继续培养至 80-90%融 合， 转染时， 吸去前一天加注的细胞培养板中的培养液， 用 PBS洗涤两次后， 加入转染复合物颗粒和含有 10% (质量 /体积百分数） 的胎牛血清的 DMEM培养基至终体积 0.1 mL， 继续培养 24小时；
然后加入 10 浓度为 5 m /mL 的 MTT溶液 （3- (4， 5-二甲基噻唑 -2 ) -2， 5-二苯基四氮唑溴盐） 在 37 °C孵育 4小时， 加入 150 L DMSO (二甲基亚砜）。 然后用酶标仪 （Bio-Rad) 测试每孔的吸光度值 A, 测试波长选用 492 nm。 细胞存活率按下公式计算：
细胞存活率 （％) = (Asample/Acontro！) 100
Asample是转染后的细胞样品孔的吸光度值， A∞ntol是不与转染复合物作用的细胞对照孔的吸光度值， 每组 实验重复六次。
图 9是不同转染复合物对 HepG2细胞转染 24小时后的细胞存活率。 由图 9可见， 双重刺激响应的 DSeS 三元复合物对 HepG2细胞毒性远远低于单一核刺激响应的 DSe和单一壳刺激响应的 DPS复合物， 在 HA- SS-COOH/DNA质量比为 2， 不同的 OEI-SeSex/DNA质量比的情况下， DSeS细胞存活率在 80%以上， 可 见本发明的双重刺激响应的基因载体具有较低的细胞毒性。
实施例 14： 具有梯度还原刺激响应的 OEI-SeSex/DNA/HA-SS-COOH三元复合物基因载体介导绿色荧光蛋 白质粒对 HepG2细胞的体外转染效率的测定
转染前， 取对数生长期 HepG2细胞， 胰酶消化后用 DMEM培养基稀释， 按每孔 4>< 105细胞的密度接种于 6 孔培养板， 置于含 5% (体积百分数） 的 C0说2， 温度为 37°C的培养箱中继续培养至 80-90%融合， 转染时， 吸去前一天加注的细胞培养板中的培养液， 用 PBS洗涤两次后， 加入转染复合物颗粒以及含有 10% (质量 /体积百分数） 的胎牛血清的 DMEM培养基至终体积 2 mL， 4小时后， 更换新鲜的含 10%胎牛血清的培养 基， 继续培养 44个小时。
体外转染效率的测定： 取出培养板， 用倒置荧光显微镜照书相； 图 10是不同转染复合物（DP; DSe; DPS; 和 DSeS )分别介导绿色荧光蛋白质粒对 HepG2细胞转染效率图。 分别显示了作为基因转染金标准的 DP， 单 一核还原刺激响应的 DSe和单一壳刺激响应的 DPS, 以及双重刺激响应的 DSeS四种基因载体介导绿色荧 光蛋白质粒对 HepG2细胞转染后的荧光照片。
从照片中绿色荧光蛋白表达的情况可见， 具有双重梯度还原刺激响应性能的 DSeS基因载体系统， 很大程 度的提高了绿色荧光蛋白质粒在 HepG2细胞中的表达。 这是由于具有双硫键的 HA-SS-COOH作为遮蔽体 系， 在粒子进入细胞后， 还原响应的双硫键断裂， 帮助遮蔽体系的剥离， 使得暴露出 OEI-SeSex的正电荷， 发挥其质子泵效应， 帮助粒子从内涵体中逃逸出来， 进一步双硒键的断裂可以促进 DNA的释放， 进而明 显的提高了基因转染效率。
实施例 15: 具有梯度还原刺激响应的 OEI-SeSex/DNA/HA-SS-COOH三元复合物基因载体介导荧光素酶质 粒对 HepG2细胞的体外转染效率的测定
在 6孔板上以 4χ 105细胞 /孔接种 HepG2细胞， 在 37°C， 5%的 C02的细胞培养箱中培养 24小时左右， 转 染时，吸去前一天加注的细胞培养板中的培养液，用 PBS洗涤两次后，加入表 2中所列的含有荧光素酶 DNA 的转染复合物颗粒以及含有 10% (质量 /体积百分数） 的胎牛血清的新鲜 DMEM培养基至终体积 2 mL， 4 小时后， 更换新鲜的含 10%胎牛血清的培养基， 继续培养 20个小时。
体外转染效率的测定： 取出培养板， 吸去培养液， PBS洗涤细胞 2次， 然后加入含 1%的 Triton X-100的裂 解液，使细胞裂解后用 Promega公司的荧光素酶检测试剂盒检测相对荧光强度，相应的总蛋白量用 Thermo 公司的 BCA试剂盒检测， 最后转染结果表示为 RLU/mg protein, 结果如表 6所示。
本发明利用具有梯度还原刺激响应性能的 OEI-SeSex/DNA/HA-SS-COOH(DSeS* ) 三元复合物基因载体， 显著提高基因载体的性能， 其对 HepG2细胞的转染效率分别比作为金标准的 DP(*)， 单一核刺激响应的 DSe(*)和单一壳刺激响应的 DPS(*)， 相应提高了 197.2倍， 95.4倍及 43倍。
表 6具有梯度还原刺激响应性能的基因载体系统介导荧光素酶质粒对 HepG2的体外转染效率表
Figure imgf000012_0001
实施例 16： 具有还原刺激响应糖胺聚糖遮蔽体系的三元复合物基因载体介导荧光素酶质粒对 HepG2细胞 的体外转染效率的测定
按照实施例 1中所述的方法， 将透明质酸原料换成 4-硫酸软骨素、 6-硫酸软骨素、 硫酸皮肤素、 硫酸乙酰 肝素、 肝素和硫酸角质素等糖胺聚糖中的一种， 得到二硫键修饰且末端为羧基的糖胺聚糖衍生物， 作为具 有还原敏感特性的遮蔽体系。
将质粒 DNA溶于灭菌水或无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖） 中， 配制成 浓度为 0.1 mg/mL的 DNA溶液； 将阳离子材料 （分别选取聚乙烯亚胺、 聚丙烯亚胺、 精胺、 氨基酸多肽 和脂质进行试验） 溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的溶液 A; 将上述二硫键修饰且末端 为羧基的糖胺聚糖衍生物溶于 HBG缓冲液中， 配制成浓度为 0.01-1 mg/mL的溶液 B ;
将上述步骤中得到的溶液 A与 DNA溶液混合，在室温下孵育 20分钟后，得到二元复合物，再加入溶液 B, 在室温下孵育 20分钟后， 得到具有还原敏感特性的三元复合物。
选取所得载体进行细胞转染效率测定， （方法与实施例 7相同）， 分别以不含遮蔽体系的二元复合物 DP和 含非还原敏感的遮蔽体系的三元复合物 （不对糖胺聚糖进行二硫键修饰） 为对照， 结果显示， 采用透明质 酸以外的还原敏感性的糖胺聚糖衍生物作为遮蔽体系，介导荧光素酶质粒对 HepG2细胞的体外转染效率明 显高于两个对照材料， 与实施例 7中的结果相似。
实施例 17:遮蔽体系和阳离子材料同时具有还说原刺激响应特性的三元复合物基因载体介导荧光素酶质粒对 HepG2细胞的体外转染效率的测定
按照实施例 1中所述的方法， 将透明质酸原料换成 4-硫酸软骨素、 6-硫酸软骨素、 硫酸皮肤素、 硫酸乙酰 肝素、 肝素和硫酸角质素等糖胺聚糖中的一种， 得到二硫键修饰且末端为羧基的糖胺聚糖衍生物， 作为具 有还原敏感特性的遮蔽体系。 书
将含双硫键或双硒键的二羧酸或二烯与聚乙烯亚胺、 聚丙烯亚胺、 精胺、 氨基酸多肽或脂质等阳离子材料 进行交联得到含有还原敏感键的阳离子载体材料。 所述交联可以选择常用的物理交联或化学交联方法， 具 体方法可参考公开号为 CN 02604H0中国专利申请。
将质粒 DNA溶于灭菌水或无菌 HBG缓冲液（4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5%葡萄糖） 中， 配制成 浓度为 0.1 mg/mL的 DNA溶液； 将上述还原敏感的阳离子材料溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的溶液 A; 将上述还原敏感的遮蔽体系溶于 HBG缓冲液中， 配制成浓度为 0.01-1 mg/mL的溶液 B; 将上述步骤中得到的溶液 A与 DNA溶液混合，在室温下孵育 20分钟后，得到二元复合物，再加入溶液 B, 在室温下孵育 20分钟后， 得到具有多重还原敏感特性的三元复合物。
选取所得载体进行细胞转染效率测定， （方法与实施例 15相同）， 分别以不含遮蔽体系的二元复合物 DP和 非还原敏感的三元复合物 （对糖胺聚糖和阳离子材料都不进行还原敏感键修饰） 为对照， 结果显示， 采用 透明质酸以外的还原敏感性的糖胺聚糖衍生物作为遮蔽体系，介导荧光素酶质粒对 HepG2细胞的体外转染 效率明显高于两个对照材料， 且比实施例 16中制备的具有一重还原敏感特性的基因载体的转染效率更高, 与实施例 15中的结果相似。
实施例 18： 复合载体系统的制备及其介导荧光素酶质粒对 HepG2细胞的体外转染效率的测定
将治疗性质粒 DNA溶于无菌 HBG缓冲液中， 配制成浓度为 0.1 mg/mL的 DNA溶液； 将含双硫键或双硒 键的二羧酸或二烯与聚乙烯亚胺、 聚丙烯亚胺、 精胺、 氨基酸多肽或脂质等阳离子材料进行交联得到含有 还原敏感键的阳离子载体材料溶于 HBG缓冲液中， 配制成浓度为 0.1-10 mg/mL的 A溶液； 将二硫键修饰 且末端为羧基的糖胺聚糖衍生物遮蔽体系溶于 HBG缓冲液中， 配制成浓度为 0.01-1 mg/mL的 B溶液。 将上述 A溶液和质粒 DNA溶液混合， 同时加入磁性纳米粒子， 混合后的溶液在室温下孵育 20分钟后， 得 到阳离子材料 /DNA/磁性纳米粒子三元复合物。 再加入 B溶液 （该步骤中还可以再次加入磁性纳米粒子）， 所得混合溶液在室温下孵育 20分钟后， 得到联合磁性纳米粒子的复合载体。 所述联合磁性纳米粒子的复 合载体能够响应外加磁场。 或将上述 A溶液和质粒 DNA溶液混合， 同时加入药物， 混合后的溶液在室温 下孵育 20分钟后，得到阳离子材料 /DNA/药物三元复合物，再加入 B溶液 (该步骤中还可以再次加入药物 ) , 所得混合溶液在室温下孵育 20分钟后， 得到联合药物的复合载体。
或将上述 A溶液和质粒 DNA溶液混合， 混合后的溶液在室温下孵育 20分钟后， 得到阳离子材料 /DNA二 元复合物， 再加入 B溶液， 同时加入药物， 所得混合溶液在室温下孵育 20分钟后， 得到另一种联合药物 的复合载体。
还可以在所述基因载体的制备过程中既加入磁性纳米颗粒又加入药物， 得到同时联合磁性纳米粒子和药物 的复合载体。 所述联合磁性纳米粒子和药物的的复合载体能够响应外加磁场。
在上述各实施例中所述的制备基因载体的过程中， 在将 DNA、 阳离子材料和遮蔽体系的溶液进行混合时， 加入磁性纳米粒子和 /或相应的药物 （如阿霉素、 紫杉醇、 5-氟尿嘧啶， 甲氨蝶呤、 顺铂等）， 制备出既具 有还原敏感特性又具有磁响应性， 或既具有还原敏感特性又具有药物治疗效果， 或同时具有这三种特性的 多功能基因载体。体外基因转染结果显示： 所述多功能基因载体与不加磁性纳米粒子和 /或相应的药物的载 体的基因转染效率相当， 值得注意的是： 含有磁性纳米粒子的多功能载体在外加磁场的辅助作用下 （在培 养板下方放置磁铁） 可以获得更高的转染效率。 还采用流式细胞仪检测了磁纳米粒和外加磁场对基因转染 的促进作用。 图 11通过荧光显微照片和流式细胞仪检测结果显示了对比结果， 其中 A为荧光显微镜照片 (亮点为成功转染的细胞）， B为流式细胞仪检测结果， control组为未经转染的 HepG2细胞， DP组为采 用本发明所述阳离子 /DNA/遮蔽体三元复合物转染， MDP-cc组是采用本发明所述阳离子 /DNA/遮蔽体 +MNP的四元复合物转染， MF表示转染后在培养板下方放置磁铁， 两种检测手段得到的结果基本一致： 在转染 10分钟之后， 含磁纳米粒组的转染效率较不含磁纳米粒组就有所提高， 转染 4小时后， 提高作用 更加明显， 在外加磁场的作用下， 仅转染 10分钟后的转染效率就显著其他所有实验组。
实例 19. 复合载体系统的体内治疗效果
构建小鼠肿瘤模型： 取对数生长期生长状态良好的 HepG2细胞， 用 0.25%胰蛋白酶消化， 加入 PBS缓冲液 配成单个细胞悬液， 在小鼠右侧后腰皮下接种 2 X 106个细胞， 接种的体积为 50 μί。 接种一周后， 选择肿 瘤体积相近， 大小约为 100 mm3的小鼠 42只为实验模型 （其中两只为备用）。
随机分为 5组， 每组 8只：
A组： 定期尾静脉注射磷酸缓冲液 （PBS) ;
B组： 定期尾静脉注射本发明所述阳离子 /DNA/遮蔽体三元复合物溶液；
C组： 定期尾静脉注射本发明所述阳离子 /DN说A/遮蔽体 +MNP四元复合物溶液；
D组： 定期尾静脉注射本发明所述阳离子 /DNA/遮蔽体 +阿霉素四元复合物溶液；
E组： 定期尾静脉注射本发明所述阳离子 /DNA/遮蔽体 +阿霉素 +MNP五元复合物溶液。
每三天注射一次， 分别在 0、 3、 6、 9、 12、 15、 18、 21天采用游标卡尺测量肿瘤尺寸 （每组每个时间点 设 8个平行样）， 并第 21天处死小鼠取出肿瘤照相 （每组书取 3个平行样）， 结果如图 12和图 13所示， 由 图中可见， 相对于 PBS组， B、 C、 D、 E组的材料对肿瘤体积增长均有明显的抑制作用， 各组材料对肿瘤 体积增长的抑制作用由小到大依次为 E组>0组>0组>8组>八组。

Claims

权 利 要 求 书
1. 一种还原剌激响应型基因载体系统， 由遮蔽体系、 阳离子高分子材料和质粒 DNA组成， 其特征在于： 所述阳离子高分子材料和质粒 DNA复合形成二元复合物颗粒， 所述遮蔽体系 通过静电作用遮蔽到所述二元复合物表面， 形成三元复合物颗粒， 所述遮蔽体系中含有还原 敏感键。
2. 如权利要求 1所述的基因载体系统， 其特征在于： 所述遮蔽体系和阳离子材料中都含有还 原敏感键。
3. 如权利要求 1或 2所述的基因载体系统， 其特征在于： 所述还原敏感键为双硫键或双硒键 中的至少一种。
4. 如权利要求 2所述的基因载体系统， 其特征在于： 所述遮蔽体系中含有双硫键， 所述阳离 子材料中含有双硒键。
5. 如权利要求 1-4中任意一个所述的基因载体系统， 其特征在于： 所述阳离子材料为双硫键 或双硒键交联的聚乙烯亚胺、 聚丙烯亚胺、 精胺、 氨基酸多肽、 肽类树状分子或含肽类树状 分子的阳离子脂质材料中的至少一种。
6. 如权利要求 1-4中任意一个所述的基因载体系统， 其特征在于： 所述遮蔽体系为对糖胺聚 糖上的至少一个葡萄糖醛酸单元上的羧基进行修饰， 得到的具有还原敏感的双硫键或双硒键 并且末端仍是羧基的糖胺聚糖衍生物。
7. 如权利要求 6所述的基因载体系统， 其特征在于： 所述遮蔽体系是通过对透明质酸上的至 少一个葡萄糖醛酸单元上的羧基进行修饰， 得到的具有还原敏感的二硫键并且末端仍是羧基 的透明质酸衍生物。
8. 如权利要求 1-7中任意一个所述的基因载体系统的制备方法，其特征在于，具体步骤如下：
1)将质粒 DNA溶于灭菌水或无菌 HBG缓冲液（ 4-羟乙基哌嗪乙磺酸 20 毫摩尔， 5% 葡萄糖） 中， 配制成浓度为 O.l mg/mL的 DNA溶液； 将阳离子材料溶于 HBG缓冲液中， 配制成浓度 为 0.1〜10 mg/mL的溶液 A; 将遮蔽体系溶于 HBG缓冲液中， 配制成浓度为 0.01〜1 mg/mL 的溶液 B;
2) 将上述步骤中得到的溶液 A与 DNA溶液混合， 在室温下孵育 20分钟后， 得到二元复合 物， 再加入溶液 B， 在室温下孵育 20分钟后， 得到三元复合物。
9. 如权利要求 7中所述的基因载体系统的制备方法， 其特征在于， 具体步骤如下：
( 1 ) 具有靶向功能的还原敏感遮蔽体系的制备：
1 )将透明质酸溶于 pH 6.8的磷酸盐缓冲液（PBS ) 中， 加入 1- (3-二甲氨丙基) -3-乙基碳二亚 胺盐酸盐 （EDC*HC1) 和 1-羟基苯并三唑 （HOBT)， 搅拌， 活化羧基； 加入胱胺二盐酸盐
(Cys), 搅拌， 室温下反应 12小时， 反应结束后将反应产物进行透析， 冷冻干燥， 得到胱胺 接枝的透明质酸 （HA-Cys) ;
2) 将胱胺接枝的透明质酸 （HA-Cys) 溶于 pH 8.5的磷酸盐缓冲液 （PBS ) 中， 加入过量的 二硫苏糖醇 （DTT)， 在室温下反应 4小时后， 用盐酸（HC1 )调节 pH值到 3.5， 然后加入氯 化钠 （NaCl) 至终浓度为 5% (w/v), 随后， 用乙醇沉淀， 再复溶于水， 离心， 冷冻干燥即 可得到巯基化的透明质酸 （HA-SH) ;
3 )巯基化的透明质酸 （HA-SH)溶于磷酸盐缓冲液 （PBS) 中， 与过量的 3-巯基丙酸在室温 下反应过夜， 反应结束后将反应产物进行透析， 冷冻干燥， 得到二硫键修饰且末端为羧基的 透明质酸 （HA-SS-COOH) ;
(2) 具有靶向功能的还原敏感遮蔽体系的基因载体系统的制备：
将质粒 DNA溶于灭菌水或无菌 HBG缓冲液中， 获得 DNA溶液； 将阳离子聚合物基因载体 溶于 HBG缓冲液中， 得到溶液 A; 将具有靶向功能的还原敏感遮蔽体系（HA-SS-COOH)溶 于 HBG缓冲液中， 得到溶液 B; 将 DNA溶液和溶液 B复合得到基因载体和质粒 DNA的复 合物颗粒溶液， 在室温下放置 20分钟后， 加入溶液 B， 得到所述基因载体系统。
10. 一种复合载体系统， 其特征在于， 由权利要求 1-7中任意一个所述的基因载体系统与 磁性纳米粒子和 /或药物有效成分复合而成。
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