CN111607093A - pH sensitive nano-carrier and application thereof in gene drug delivery - Google Patents
pH sensitive nano-carrier and application thereof in gene drug delivery Download PDFInfo
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- 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
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
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- 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
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
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- A61P35/00—Antineoplastic agents
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
- C08G81/02—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers at least one of the polymers being obtained by reactions involving only carbon-to-carbon unsaturated bonds
- C08G81/024—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G
- C08G81/028—Block or graft polymers containing sequences of polymers of C08C or C08F and of polymers of C08G containing polyamide sequences
Abstract
The invention relates to the technical field of medicines, in particular to a pH sensitive nano-carrier and application thereof in a gene drug delivery system. The invention provides a pH sensitive nano-carrier, wherein the pH sensitive nano-carrier is a novel pH sensitive nano-carrier which is obtained by modifying a PEG-PHIS-PSD triblock copolymer on the surface of a cationic liposome, namely the pH sensitive nano-carrier is in adsorption connection with positive and negative charges of the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer by the cationic liposome, and the positive and negative charges of the cationic liposome and the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer are adsorbed in a physiological environment (pH 7.3-7.4). The PEG-PHIS-PSD triblock copolymer has the following structure: in the pH sensitive nano-carrier, PEG prolongs the circulation time of the pH sensitive nano-carrier in blood, PHIs achieves the escape function of lysosomes, and in addition, due to the existence of a pH sensitive PSD chain, the pH sensitive nano-carrier can quickly release gene drugs at tumor parts.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a pH sensitive nano-carrier and application thereof in a gene drug delivery system.
Background
Malignant tumors have become one of the leading diseases causing human death worldwide, and become a worldwide problem seriously threatening the quality of human life. Gene therapy (gene therapy) has the advantages of high safety, high efficiency, specificity and the like, and is more and more widely applied. However, there are several major obstacles to siRNA delivery: first, siRNA is extremely unstable in physiological environments and is rapidly degraded by ribonuclease (RNase) in serum. In addition, siRNA is a negatively charged hydrophilic macromolecule, and the negative charge on the surface of the cell membrane causes the cell membrane to have very poor penetrating power. Thereafter, siRNA enters the target cell and escapes from lysosomes into the cytoplasm to avoid phagocytic degradation of the entering lysosome, before RNAi can be mediated. Therefore, it is imperative to develop a multifunctional delivery system to deliver siRNA efficiently.
The multifunctional gene delivery vector for delivering siRNA mainly comprises liposome, cationic micelle, protein polypeptide and the like, and is widely applied due to the characteristics of low toxicity, easy modification, good biocompatibility, high gene loading rate and the like of the cationic liposome. However, the application of cationic liposome has been limited by the disadvantages of poor stability, low transfection efficiency, poor tumor targeting property and the like. Therefore, the copolymer with certain functions is modified on the surface of the liposome to improve the stability, transfection efficiency and tumor targeting of a gene delivery system.
A number of documents report significant differences in the physiological environment of tumor sites from normal tissues. Including the weakly acidic, highly concentrated reducing substance Glutathione (GSH) and high levels of Reactive Oxygen Species (ROS). When the nanocarrier enters the endosome or lysosome in endocytosed form, the pH will decrease further (pH 5.0-6.0). Therefore, according to the characteristic of low pH of tumor cells, various pH-sensitive polymer-based drug delivery carriers can be designed.
Among many pH sensitive materials, polyhistidine (philidine), phils, is distinguished by its high biocompatibility, low toxicity, and in particular by its lysosomal escape function. The imidazole ring of histidine is hydrophobic in physiological environment (pH 7.4), and when the pH is lower than pKa, the imidazole ring becomes hydrophilic due to protonation, so that a 'proton sponge effect' is realized to achieve a lysosome escape effect, and the intracellular release of the drug is increased.
Polysulfonyldimethoxypyrimidine (PSD) has the general characteristics of sulfonamides, and exhibits weak acidity. In a high pH environment due to sulfonyl groups (-SO)2NH-) has a high electronegativity, which attracts electrons of the sulfur atom and thus of the nitrogen atom, resulting in the electron cloud of the N-H bond moving towards the nitrogen atom and thus releasing a proton. The dimethoxypyrimidine group in SD is also an electron withdrawing group, which also facilitates the release of protons from the nitrogen atom. Therefore, under normal physiological pH conditions, the surface of the PSD has negative charges, and under the acidic environment of the lysosome, the surface charges of the PSD are converted from negative charges to neutral charges, so that charge reversal is realized.
Disclosure of Invention
The invention aims to provide a novel pH sensitive nano-carrier to improve the stability of gene drug delivery, improve specificity and quickly release the effect of gene drugs.
The invention realizes the aim through the following technical scheme:
the invention provides a PSD-PHIS-PEG triblock copolymer, which has the following structural formula:
wherein: n is 40 to 50.
The PEG-PHIS-PSD triblock copolymer is prepared by the following method:
(1) synthesis of Polysulfamediethoxypyrimidine (PSD): adopts a free radical initiation solvent polymerization method to synthesize a pH sensitive polymer, namely the polysulfonamide dimethoxy Pyrimidine (PSD).
(2) The product poly-sulfadimethoxine-poly-histidine (PSD-PHIs) is obtained by amidation reaction of poly-sulfadimethoxine (PSD) catalyzed by NHS and DCC.
(3) One end of carboxylated polyethylene glycol (PEG) is connected with PSD-PHIS through amidation reaction to obtain the final product of polyethylene glycol-polyhistidine-polysulfonamide dimethoxy pyrimidine (PEG-PHIS-PSD).
PSD synthesis: dissolving Sulfadimethoxine (SD) and NaOH in acetone/water, slowly adding excessive methyl acryloyl chloride (MC) dropwise, purifying with methanol/water for 3-4h, filtering, and vacuum drying for 48h to obtain SD acylated product (SDM). Dissolving SDM in Dimethylformamide (DMF), introducing nitrogen, adding AIBN, sealing, quickly dropwise adding mercaptoethylamine, stirring, continuously introducing nitrogen at 70-80 ℃ for 48h, aging in water for 3-4h, filtering, and vacuum drying for 48h to obtain PSD.
Synthesis of PHIS-PSD: dissolving Fmoc-NH-PHIs-COOH, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding DMSO solution of PSD, stirring at room temperature for 48h under the protection of nitrogen, precipitating with glacial ethyl ether, filtering, and vacuum drying to obtain Fmoc-NH-PHIs-PSD; removing Fmoc protection: dissolving Fmoc-NH-PHIs-PSD in dimethyl sulfoxide (DMSO), adding diethylamine to react for 2-3h, rotary steaming, adding glacial ethyl ether to precipitate a product, filtering, and vacuum drying to obtain PHIs-PSD powder.
Synthesis of PEG-PHIS-PSD: dissolving PEG, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding PHIS-PSD DMSO solution, stirring at room temperature for 48h under the protection of nitrogen, dialyzing with dialysis bag (MW 3500) for 48-72h, and lyophilizing to obtain PEG-PHIS-PSD powder.
The invention provides a pH sensitive nano-carrier, wherein the pH sensitive nano-carrier is a novel pH sensitive nano-carrier which is obtained by modifying a PEG-PHIS-PSD triblock copolymer on the surface of a cationic liposome, namely the pH sensitive nano-carrier is in adsorption connection with positive and negative charges of the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer by the cationic liposome, and the positive and negative charges of the cationic liposome and the polyethylene glycol-polyhistidine-polysulfonamide dimethoxypyrimidine (PEG-PHIS-PSD) triblock copolymer are adsorbed in a physiological environment (pH 7.3-7.4).
The cationic liposome at least contains (2, 3-dioleoxypropyl) trimethyl ammonium chloride (DOTAP).
Further, the cationic liposome comprises the following components: (ii) a combination of DOTAP with one or more of: soybean lecithin, cholesterol, egg yolk lecithin, etc.
Preferably a combination of DOTAP, soy lecithin, cholesterol.
Wherein, DOTAP comprises soybean lecithin: cholesterol is 4-8: 2-4: 1.
The cationic liposome is prepared by the following method: dissolving DOTAP, soybean phospholipid and cholesterol in a mixed solvent of chloroform and methanol, wherein the ratio of chloroform: the volume ratio of methanol is 3-4:1, and the organic solvent is removed by reduced pressure rotary evaporation to form a dry lipid film; adding water as water phase, hydrating, ultrasonic treating, and grading with 0.22 μm polycarbonate membrane to obtain cationic liposome (L).
Further, the invention provides a lipid nano-carrier obtained by combining a drug and a pH sensitive nano-carrier through electrostatic interaction, wherein the nano-lipid carrier is prepared through the following steps: in a physiological environment (pH7.3-7.4), the LR is formed by combining the medicament and cationic lipid with positive charge through electrostatic interaction. And then, the PEG-PHIS-PSD triblock copolymer with negative charges is adsorbed with positive and negative charges of LR, thus obtaining the novel drug-containing pH sensitive lipid nano-carrier (PHD/LR).
The medicine is gene medicine and nucleic acid medicine selected from: siRNA, miRNA and plasmid DNA, wherein the nucleic acid drug has negative charges and is combined with the pH sensitive nano-carrier with positive charges to form the drug-containing pH sensitive lipid nano-carrier under the adsorption effect of the positive charges and the negative charges.
The weight ratio of the gene medicine to the pH sensitive nano-carrier is as follows: 1-10: 1.
the structure of the liposome has higher similarity with a normal biological membrane, so that the liposome is easily degraded and metabolized by various substances in the body circulation so as to reduce the circulation time in the body. In order to solve the problem, the polyethylene glycol-polyhistidine-polysulfonamide dimethoxy pyrimidine (PEG-PHIs-PSD) triblock copolymer achieves the long circulation effect; the prepared liposome has smaller particle size (about 150nm-200nm), and can be specifically enriched in tumor tissues through an EPR effect so as to embody targeting; the gene medicine is delivered into tumor cells and phagocytized by lysosomes, and the escape effect of the lysosomes is achieved due to the proton sponge effect of the PHIs on the surfaces of the gene medicine, so that the gene medicine is released into cytoplasm; meanwhile, under the acidic environment of lysosome, the pH-sensitive PSD is changed from negative charge to neutral charge, and is separated from the surface of the cationic liposome to quickly release gene drugs, thereby playing a therapeutic role.
Compared with the prior art, the invention has the following advantages:
1) in the drug-containing pH sensitive lipid nano-carrier, the cationic lipid in the pH sensitive nano-carrier is combined with the gene drug through electrostatic interaction, so that the encapsulation rate of the gene drug in the pH sensitive nano-carrier is improved, the preparation method is simple and easy to implement, the reproducibility is good, and the stability is high;
2) in the invention, the prepared liposome has smaller particle size (about 150-200nm), and can be specifically enriched in tumor tissues through an EPR effect so as to have targeting property;
3) according to the invention, the PEG of the pH sensitive triblock copolymer can increase the circulation time of the pH sensitive triblock copolymer in blood, and the rapid lysosome escape function of PHIs can be realized.
4) Compared with blank cationic liposome, the invention modifies PSD-PHIs-PEG on the surface of the cationic liposome through electrostatic adsorption, thereby increasing the stability of the preparation, improving the specificity and enhancing the targeting property.
Drawings
FIG. 1 is a drawing showing a copolymer of example 1 of the present invention1HNMR mapping:
(A) process for preparing Polysulfonamidodimethoxine (PSD)1A HNMR map; (B) method for preparing polysulfonamide dimethoxy pyrimidine-polyhistidine (PSD-PHIS)1A HNMR map; (C) method for preparing polysulfonamide dimethoxy pyrimidine-polyhistidine-polyethylene glycol (PSD-PHIS-PEG)1A HNMR map;
FIG. 2 is an FTIR spectrum of the copolymer of example 1 of the present invention:
(A) FTIR spectrum of Polysulfonamidodimethoxine (PSD); (B) FTIR spectrum of Polysulfadimethoxine-polyhistidine (PSD-PHIs); (C) FTIR spectrum of Polysulfadimethoxine-polyhistidine-polyethylene glycol (PSD-PHIs-PEG);
FIG. 3 is a pH-sensitive pH-sensitivity study of pH-sensitive PHD/LR of example 3 of the present invention.
FIG. 4 is a TEM image of pH sensitive PHD/LR of example 4 of the present invention.
FIG. 5 is an atomic force microscope photograph of pH sensitive PHD/LR of example 4 of the present invention.
FIG. 6 is an in vitro release assay of PHD/LR of example 5 of the present invention in simulated media of different pH.
FIG. 7 is a photograph of in vitro heparin-decomplexation assay of PHD/LR of example 6 of the present invention.
FIG. 8 is a photograph of an in vitro serum stability assay of PHD/LR of example 7 of the present invention.
FIG. 9 is a graph of the change in tumor volume of PHD/LR in vivo anti-tumor experiments according to example 8 of the present invention.
FIG. 10 is a graph of the weight change of PHD/LR in mice tested for in vivo anti-tumor effect according to example 8 of the present invention.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended only to specifically describe the present invention and should not be construed as limiting the present invention.
Example 1
Synthesis of pH-sensitive triblock copolymer (PSD-PHIS-PEG)
PSD synthesis: dissolving Sulfadimethoxine (SD) and NaOH in acetone/water, slowly adding excessive methyl acryloyl chloride (MC) dropwise, purifying with methanol/water for 3-4h, filtering, and vacuum drying for 48h to obtain SD acylated product (SDM). Dissolving SDM in Dimethylformamide (DMF), introducing nitrogen, adding AIBN, sealing, quickly dropwise adding mercaptoethylamine, stirring, continuously introducing nitrogen at 70-80 ℃ for 48h, aging in water for 3-4h, filtering, and vacuum drying for 48h to obtain PSD.
Synthesis of PHIS-PSD: dissolving Fmoc-NH-PHIs-COOH, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding DMSO solution of PSD, stirring at room temperature for 48h under the protection of nitrogen, precipitating with glacial ethyl ether, filtering, and vacuum drying to obtain Fmoc-NH-PHIs-PSD; removing Fmoc protection: dissolving Fmoc-NH-PHIs-PSD in dimethyl sulfoxide (DMSO), adding diethylamine to react for 2-3h, rotary steaming, adding glacial ethyl ether to precipitate a product, filtering, and vacuum drying to obtain PHIs-PSD powder.
Synthesis of PEG-PHIS-PSD: dissolving PEG, NHS and EDC in dimethyl sulfoxide (DMSO), stirring at room temperature for 24h under the protection of nitrogen, adding PHIS-PSD DMSO solution, stirring at room temperature for 48h under the protection of nitrogen, dialyzing with dialysis bag (MW 3500) for 48-72h, and lyophilizing to obtain PEG-PHIS-PSD powder.
Wherein the molecular weight of PEG is 2000.
Using nuclear magnetic resonance spectroscopy (1H NMR), the structure of each reaction product was determined, and the structure of the sample was confirmed by dissolving an appropriate amount of the polymer powder in a deuterated reagent and using a nuclear magnetic resonance spectrometer, and the results are shown in fig. 1. The chemical shift of the methoxy group in the dimethoxypyrimidine group of the Polysulfuryldimethoxypyrimidine (PSD) is 3.72ppm (-OCH)3) The chemical shift of the amide group in the PSD was 10.17ppm (-NH-CO-). And the carboxyl proton peak on the polyhistidine is 13ppm, the characteristic peaks of the two compounds appear in the polysulfonamide dimethoxy pyrimidine-polyhistidine (PSD-PHIs), and the carboxyl proton peak does not appear, so that the successful connection of the PHIs and the PSD is proved. The proton peak of the methoxyl group of the PEG appears in the nuclear magnetic resonance hydrogen spectrum of the PEG-PHIs-PSD at 3.21ppm (-O-CH)3) and-CH2-CH2The chemical shift of the-O-group is at 2.88ppm, and the appearance of these characteristic peaks demonstrates the successful synthesis of PEG-PHIS-PSD copolymer.
The structural functional groups of each reaction product were measured by infrared spectroscopy (FTIR), and appropriate amounts of the products were taken and ground with potassium bromide powder, respectively, and the results were shown in fig. 2. 3386cm-1The single peak of (A) is a PSD characteristic absorption peak; 1635 and1591cm-1is a characteristic absorption peak of the stretching vibration of the polyhistidine carboxylic acid; 1755cm-1The peak of carboxylic acid absorption in PEG is 2889cm-1is-CH in PEG2Characteristic absorption peaks of the radicals. In the PSD-PHIs infrared spectrum, the characteristic peaks of PSD and PHIs appear, and 1635cm does not appear-1Absorption peaks of carboxylic acid groups, demonstrating successful attachment of PSD and phi. The characteristic peak of PEG appears in the infrared spectrum of the triblock copolymer PEG-PHIs-PSD, which proves the successful synthesis of the PEG-PHIs-PSD copolymer.
Example 2
Preparation of novel pH sensitive nano-carrier
Dissolving DOTAP, soybean phospholipid and cholesterol in chloroform and methanol (3:1, v/v) at a certain ratio, and performing rotary evaporation under reduced pressure to remove organic solvent to form dried lipid film; adding water for hydration, performing ultrasonic treatment, and grading with 0.22 μm polycarbonate membrane to obtain blank cationic liposome (L). In a physiological environment (pH 7.4), siRNA binds to positively charged cationic liposomes via electrostatic interactions to form LR. And then, adsorbing the positive and negative charges of the PSD-PHIs-PEG and LR with the negative charges to obtain the novel drug-containing pH sensitive lipid nano-carrier (PHD/LR).
Example 3
pH sensitivity investigation of drug-containing pH sensitive nano-carrier
Under physiological conditions, the PSD-PHIs-PEG with negative charge can mask the positive charge on the surface of the cationic liposome, so the pH sensitivity of the carrier is proved by measuring the change of zeta potential of PHD/LR, and the specific operation is as follows: the Zeta potential of the formulations was determined by dynamic light Diffraction (DLS) in DEPC water at pH5.0 and pH 7.4, incubation at 37 ℃ at 100rpm and sampling at different time points to determine the Zeta potential. The results are shown in FIG. 3. The Zeta potential of the PHD/LR preparation is rapidly increased at 5min, and after 10min, the potential of the PHD/LR preparation is almost consistent with that of the LR preparation, which indicates that the modified copolymer can be rapidly dissociated from the upper surface of LR to increase the Zeta potential of the PHD/LR preparation under the condition that the pH of the PHD/LR preparation is 5.0. This indicates that PHD/LR has significant pH sensitivity.
Example 4
Morphological Observation of PHD/LR
The particle size and shape of the PHD/LR prepared in the examples were measured by transmission electron microscopy and atomic force microscopy. As shown in FIGS. 4 and 5, the PHD/LR exhibited spherical and spheroidal shapes, with a relatively uniform distribution and particle sizes of 150nm to 200 nm.
Example 5
In vitro release experiment of PHD/LR in different pH simulation media
In vitro release experiments of PHD/LR in different pH simulated media were examined in DEPC aqueous media at pH5.0 and 7.4. The PHD/LR prepared in example 2 was placed in an ultrafiltration tube, centrifuged to remove free FAM siRNA, the sample in the ultrafiltration tube was transferred to DEPC water at pH5.0 and 7.4, shaken in a shaker at constant temperature of 37 ℃ and the release media was removed all at once into an ultrafiltration tube, centrifuged and the filtrate was recovered. The sample in the ultrafiltration tube was transferred further to the release medium and the procedure repeated. The content was calculated by a standard curve and the release curve was plotted. The cumulative percent release is calculated according to a formula.
Cumulative release percentage is cumulative release amount/total amount of PLK1-siRNA x 100%
The results are shown in FIG. 6. At pH 7.4, about 45% of FAM siRNA was released after 72h, and at pH5.0, about 80% of the drug was released from PHD/LR, demonstrating pH sensitivity of PHD/LR, which is consistent with the results of pH sensitivity studies.
Example 6
Heparin decomplexation experiment of PHD/LR
The PHD/LR preparation prepared in example 2 was taken, heparin sodium was added according to different heparin sodium/siRNA mass ratios (IU/μ g), a sucrose solution with a mass fraction of 50% was added, an appropriate amount of sample was taken for agarose gel electrophoresis experiment, and images were observed by transmitted light and photographed. The results are shown in FIG. 7. siRNA bands appeared when the LR heparin-siRNA (IU/mug) was 6, and only when the PHD/LR group was 8, indicating that the PHD-coated formulation had better heparin-resistant ability.
Example 7
Serum stability assay for PHD/LR
The PHD/LR prepared in example 2 was mixed with FBS and appropriate samples were taken at different time points and stored at-20 ℃. Adding heparin according to the heparin/siRNA mass ratio (IU/mug) of 10, adding a sucrose solution with the mass fraction of 50%, taking a proper amount of sample to perform an agarose gel electrophoresis experiment, observing and imaging by adopting transmitted light, and taking a picture. The results are shown in FIG. 8. Free siRNA began to degrade in serum 1h, and had degraded substantially completely in 3 h. The LR preparation obviously enhances the stability of siRNA in serum, and basically degrades all after 10 h. The PHD/LR coated PHD/LR preparation starts to appear weak siRNA bands at 24h, which shows that the PHD/LR has more obvious protective effect in serum.
Example 8
Antitumor experiment of pH sensitive nano carrier
Inoculating A549 cell suspension to armpit of male nude mouse, and allowing tumor to grow to 100-120mm3Groups were divided into 6 groups, randomized into 3 groups (placebo, LR and PHD/LR groups) and given different drug treatments. Each group was administered by tail vein injection at a dose of 1mg/kg siRNA once every 2 days for 4 times, and the tumor volume and the body weight of the mice were recorded. The blank control group was given the same volume of physiological saline.
The change of tumor volume is shown in fig. 9, compared with the control group, both preparation groups show obvious antitumor activity, the growth rate of the tumor volume is obviously lower than that of the control group, and the fact that the smaller particle size (<200nm) of the nano preparation can be enriched in tumor tissues through the EPR effect is shown. Compared with the LR group, the PHD/LR group has more obvious antitumor activity, and the addition of the pH sensitive triblock copolymer is proved to increase the accumulation of siRNA in cytoplasm so as to exert the antitumor activity.
The results of the weight change are shown in fig. 10, and the weight average of nude mice of each group has no obvious change, which indicates that the pH sensitive nano-carrier has no obvious toxicity in vivo.
Claims (10)
- A PSD-phi-PEG triblock copolymer having the structural formula:
n＝40～50。 - 2. the method of claim 1, wherein the PSD-PHIS-PEG triblock copolymer is prepared by the following steps,(1) synthesis of Polysulfamediethoxypyrimidine: synthesizing a pH sensitive polymer polysulfonamide dimethoxy pyrimidine by adopting a free radical initiation solvent polymerization method;(2) the polysulfanediaxine is catalyzed by NHS and DCC and then is subjected to amidation reaction with polyhistidine to obtain the product polysulfanediaxine-polyhistidine;(3) one end of carboxylated polyethylene glycol is connected with the poly-sulfadimethoxine-poly-histidine through amidation reaction, and the final product of polyethylene glycol-poly-histidine-poly-sulfadimethoxine is obtained.
- 3. A pH-sensitive nano-carrier is characterized in that the pH-sensitive nano-carrier is connected with the polyethylene glycol-polyhistidine-polysulfonamidodimethoxine triblock copolymer of claim 1 through positive and negative charge adsorption.
- 4. The pH-sensitive nanocarrier of claim 3, wherein the cationic liposome comprises at least DOTAP, and the cationic liposome can be a combination of DOTAP and one or more of the following substances: soybean lecithin, cholesterol, egg yolk lecithin, etc.
- 5. The pH-sensitive nanocarrier of claim 4, wherein the cationic liposome is a combination of DOTAP, soy lecithin, cholesterol, DOTAP: cholesterol is 4-8: 2-4: 1.
- 6. The pH-sensitive nanocarrier of claim 5, wherein the cationic liposome is prepared by the following method: dissolving DOTAP, soybean phospholipid and cholesterol in a mixed solvent of chloroform and methanol, wherein the ratio of chloroform: the volume ratio of methanol is 3-4:1, and the organic solvent is removed through reduced pressure rotary evaporation to form a dry lipid film; adding water for hydration, carrying out ultrasonic treatment, and finishing the particles of a 0.22 mu m polycarbonate film to obtain the cationic liposome.
- 7. The drug-containing pH sensitive lipid nano-carrier is characterized in that a drug and cationic lipid with positive charges are combined through electrostatic interaction to form a drug-containing cationic liposome, and a PEG-PHIs-PSD triblock copolymer with negative charges is adsorbed with the positive and negative charges of the drug-containing cationic liposome.
- 8. The drug-containing pH-sensitive lipid nanocarrier of claim 7, comprising the pH-sensitive nanocarrier of claim 1 and a drug, wherein the drug is a gene drug, a nucleic acid drug, selected from the group consisting of: siRNA, miRNA, plasmid DNA.
- 9. The drug-containing pH-sensitive lipid nanocarrier of claim 8, wherein the weight ratio of the drug to the pH-sensitive nanocarrier is: 1-10: 1.
- 10. use of the polyethylene glycol-polyhistidine-polysulfonamidodimethoxine triblock copolymer of claim 1 for the preparation of pH sensitive and lysosomal escape vehicles.
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