CN115671045B - Non-liver-targeting nucleic acid nano preparation and preparation method and application thereof - Google Patents
Non-liver-targeting nucleic acid nano preparation and preparation method and application thereof Download PDFInfo
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- CN115671045B CN115671045B CN202211741557.1A CN202211741557A CN115671045B CN 115671045 B CN115671045 B CN 115671045B CN 202211741557 A CN202211741557 A CN 202211741557A CN 115671045 B CN115671045 B CN 115671045B
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- polyethylene glycol
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a non-liver-targeting nucleic acid nano preparation and a preparation method and application thereof. The preparation method is carried out in a microchannel reactor, and then the product is obtained by evaporating organic solvent, concentrating, freeze-drying and drying. The nucleic acid nano preparation can be prepared in a large-scale, continuous and intelligent manner based on a microchannel reactor, and has the advantages of targeting property and high stability. The preparation method can be used for preparing a large amount of the traditional Chinese medicine composition, can be stored for a long time, can be applied to the treatment of various diseases such as tumors, virus infection and the like, and has huge clinical application potential.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a non-liver-targeting nucleic acid nano preparation continuously prepared based on a microchannel reactor and a preparation method and application thereof.
Background
In recent years, nucleic acid drugs such as siRNA and the like have the advantages of high target specificity and the like, and the effect and the prospect in disease treatment are increasingly prominent. The use of nucleic acid drugs such as siRNA to regulate target spots such as genes and proteins and tumor microenvironment has received extensive attention from basic research and clinical transformation. For example, the siRNA drugs ALN-VSP02 and TKM-080301 targeting vascular endothelial growth and Polo-like kinase 1 gene for treatment of liver cancer have completed phase I clinical trials. In addition, the 2 mRNA vaccines against the new coronavirus were approved one after another and showed great potential in the prevention of viral infectious diseases. Nevertheless, there are still many problems in the application of nucleic acid drugs in vivo, such as targeting, instability in vivo, toxic and side effects, etc.
The nano-carriers currently under research and clinically applied to nucleic acid drugs can be mainly classified into the following categories: 1. lipid nano-carriers. The siRNA delivery carrier constructed based on lipid materials realizes the successful clinical transformation of the first siRNA nucleic acid medicament Patisiran, and is also an in vivo delivery carrier which is depended by two mRNA vaccines aiming at the new coronavirus. 2. A polymer nano-carrier. CALLA-01, a cyclodextrin polymer (CDP) -based water-soluble polycation nanoparticle-entrapped siRNA developed by Calando pharmaceutical company in the United states, was the first polymeric nanocarrier for tumor-targeted siRNA delivery clinical trials. 3. The delivery carrier is coupled, and the bioavailability and the curative effect of the drug molecule are improved by chemically modifying the material, for example, siRNA is coupled with polymer, polypeptide, antibody and the like for application. Three acetylgalactosamine modified siRNA chains are utilized by Alnylam pharmaceutical company, and siRNA drugs ALN-TTRsc, ALN-PCS and ALN-AT3 based on the delivery technology are approved to be clinically applied to treat liver diseases such as thyroid protein amyloidosis, hypercholesterolemia and hemophilia respectively.
A large number of basic research and clinical test results show that the current nucleic acid nano-carrier preparation still faces a great promotion space in clinical application. On one hand, the LNP system which is clinically approved is mainly enriched at the liver part after administration, is suitable for the disease type of administration aiming at the liver part and is difficult to expand to diseases of other parts; even the subcutaneous LNP-based mRNA vaccine will concentrate on the liver site, causing autoimmune hepatitis. On the other hand, the clinical transformation-oriented nano drug carrier material and delivery system need to follow the basic principle of drug development of "safety, effectiveness and controllable quality". Therefore, a non-liver-targeting nucleic acid nano-drug delivery system is constructed by utilizing clinical medicines or auxiliary materials with medicinal prospects, a large-scale, continuous and intelligent preparation technology platform based on the system is established, a production process capable of accurately regulating and controlling the performance of the nucleic acid nano-drug is formed, a quality evaluation system of standardized preparations is established, the bioavailability and the drug effect of clinical nucleic acid are improved, and the system becomes an urgent demand for research and development of nucleic acid drugs.
In the previous research, the inventor prepares the siRNA-encapsulating nano-carrier by a double emulsification method by using clinically approved pharmaceutic adjuvants of polyethylene glycol-polylactic acid and cationic lipid. The method specifically comprises the following steps: dissolving polyethylene glycol-polylactic acid and cationic lipid in organic phase dichloromethane (250 μ L), adding aqueous solution (50 μ L) of siRNA, and performing primary emulsification through an ultrasonic probe; then adding water phase (1.0 mL), emulsifying carrier to obtain siRNA-encapsulated nano preparation. The nano preparation can efficiently entrap nucleic acid drugs and realize in-vivo nucleic acid drug delivery. However, it should be noted that the transfection efficiency of the nano-preparation is not good enough: the cellular level needs to be under 200-300 nM conditions, so that efficient target gene silencing can be realized, and the equivalent efficacy of a commercial transfection reagent Lipofectamine can be achieved. In the invention, a nucleic acid drug delivery system based on three components of polyethylene glycol-polylactic acid, cationic lipid and ionizable lipid is developed, the composition of the three components can obviously improve the transfection effect of the nucleic acid drug, and the effect equivalent to that of Lipofectamine can be achieved at the concentration of 2-50 nM. In addition, unlike the double emulsification process used with the two components, the preparation process of the present invention is primarily prepared by a microchannel reactor. The microchannel reactor is a device with micron-sized structural components as cores for reaction, mixing, separation and the like, the channel size is generally 10-1000 microns, fluid can flow and combine in the reactor in a specific physical state, and the microchannel reactor has the characteristics of continuous reaction and the like and can realize high yield. The technology is not currently used for the preparation of nucleic acid drugs.
Disclosure of Invention
Based on the above, the invention aims to provide a non-liver-targeting nucleic acid nano preparation which can be prepared on a large scale, continuously and intelligently based on a microchannel reactor, and a preparation method and application thereof.
The invention provides a non-liver-targeting nucleic acid nano preparation, which is prepared by taking an aqueous solution of nucleic acid as an internal aqueous phase, and taking polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-glycolic acid) copolymer, cationic lipid and ionizable lipid material as organic phases; the cationic lipid to nucleic acid N/P ratio is less than 12, and the ionizable lipid to nucleic acid N/P ratio is greater than 1.5.
Preferably, the nucleic acid-containing aqueous solution is used as an internal aqueous phase, polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid and ionizable lipid are dissolved in an organic solvent to be used as an organic phase, DEPC water is used as an external aqueous phase, and the reaction is carried out in a microchannel reactor.
The nucleic acid may be siRNA (small interfering RNA)), mRNA (messenger RNA).
In the invention, ionizable lipid is added into two components of polyethylene glycol-polylactic acid and cationic lipid as a third component, the composition of the three components can obviously improve the transfection effect of nucleic acid drugs, and the effect equivalent to Lipofectamine can be achieved at the concentration of 2-50 nM.
The second aspect of the invention provides a method for preparing the nucleic acid nano preparation in a large scale, which specifically comprises the steps of utilizing a microchannel reactor and the like to build a large-scale production platform for the nucleic acid nano medicine preparation, realizing continuous preparation and online quality control of nano medicines, introducing an automatic and intelligent control technology, improving the production process of the nano medicine preparation, and forming a whole set of original key technology reserves from research to production.
A preparation method of the nucleic acid nano preparation comprises the following steps: (1) continuously preparing a microchannel reactor: in a microchannel reactor, the internal aqueous phase and the organic phase respectively enter a reaction plate 1 through a channel for mixing reaction to obtain a mixture, then enter a reaction plate 2, and the external aqueous phase enters the reaction plate 2 through the channel to be mixed with the mixture to obtain a nucleic acid nano preparation solution;
(2) Removing the organic solvent;
(3) Concentrating;
(4) And (4) carrying out freeze-drying technology on the concentrated nanoparticle solution obtained in the step (3) to obtain the nucleic acid nano preparation.
In a third aspect of the present invention, there is provided a use of the nucleic acid nanoformulation as described above in the treatment of a disease.
The application of the nucleic acid nano preparation in preparing a medicament for preventing or treating tumors.
The application of the nucleic acid nano preparation in preparing a medicament for preventing or treating virus infection.
The nucleic acid nano preparation is prepared by nucleic acid medicines such as siRNA, mRNA and the like, polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic acid-glycolic acid) copolymer, cationic lipid and ionizable lipid material, has good stability, high encapsulation efficiency and drug loading rate of the nucleic acid medicines and high transfection efficiency, and can protect the nucleic acid medicines from degradation.
According to the invention, a large-scale, continuous and intelligent preparation technology platform for obtaining the nano-medicament is established for the first time, a production process capable of accurately regulating and controlling the performance of the nucleic acid nano-medicament is formed, the nucleic acid preparation can be rapidly, efficiently and massively produced, and a quality evaluation system of a standardized preparation is established, so that the technology for preparing the nucleic acid medicament on line is realized. The invention creatively applies the microchannel reactor to continuously and intelligently prepare nucleic acid nano preparation medicaments for the first time, provides a convertible and intelligent preparation technology for the fields of nucleic acid medicinal preparations such as siRNA, mRNA and the like, and has huge clinical application potential.
Drawings
FIG. 1 is a schematic view of a process flow for preparing a nucleic acid nanoformulation; wherein, A is the emulsification reaction of the micro-channel reactor, B is the organic solvent of the rotary foaming, C is the tangential flow concentration, and D is the freeze-drying preparation.
FIG. 2 is a schematic diagram of a nucleic acid nanoformulation and characterization of the nanoformulation after reconstitution; wherein A is a nucleic acid nano preparation flow chart, B is a freeze-dried nano preparation picture, C is a particle size distribution diagram of the PIC/siRNA nano preparation, and D is a particle size distribution diagram of the PIC/mRNA nano preparation.
FIG. 3 is a schematic diagram showing the stability of the nucleic acid nano-preparation in a culture medium containing 10% serum, wherein A is a graph of the change in the particle size of PCI/siRNA, and B is a graph of the change in the particle size of PCI/mRNA.
FIG. 4 is a schematic illustration of the effect of siRNA nucleic acid nanoformants in silencing genes in tumor cells; wherein, A is a graph of the effect of the siRNA nucleic acid nano preparation on reducing the expression of CD47 mRNA, and B is an electron microscope graph of the siRNA nucleic acid nano preparation.
FIG. 5 is a schematic diagram of the results of the effect of various PCI/siRNA nanoparticles on the target gene knockout efficiency in example 6.
Figure 6 is a graph of the efficacy of the nanoformulation to deliver siRNA to inhibit tumor growth.
FIG. 7 is a graph showing the in vivo expression effect of the nano-formulation delivery of Luci-mRNA.
FIG. 8 is a graph showing the expression effect of loaded Luci-mRNA nano-preparations with different components on the main organs in vivo after intravenous injection.
FIG. 9 is the nuclear magnetic hydrogen spectrum of ionizable lipid BHEM-DBA.
FIG. 10 is the nuclear magnetic hydrogen spectrum of ionizable lipid BHEM-APMP.
FIG. 11 is the nuclear magnetic hydrogen spectrum of ionizable lipid BHEM-EAA.
FIG. 12 is the nuclear magnetic hydrogen spectrum of the ionizable lipid BHEM-AEA.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Experimental procedures not specifying specific conditions in the following examples are generally published in 2013 according to conventional conditions, for example, molecular Cloning: A Laboratory Manual, fourth edition, master code of Green and Sambrook, or according to conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs
The meaning is generally understood by those skilled in the art. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a nucleic acid nano preparation based on a three-component system of polyethylene glycol-polylactic acid, cationic lipid and ionizable lipid, which is prepared in a large-scale, continuous and intelligent manner by utilizing a microchannel reactor, and a preparation method and application thereof. Different from the effect of the nucleic acid drug delivered by lipid nano-carrier mainly in liver, the nano-system prepared by the invention can deliver the nucleic acid drug to different organs such as liver, lung, spleen and the like through component regulation.
In some embodiments of the present invention, a microchannel reactor is provided, which can realize a large-scale, continuous and intelligent preparation technology of a nucleic acid nano-preparation by continuous sample injection of a microchannel reactor pump, and the specific technical process includes the microchannel reactor, rotary evaporation of an organic solvent, tangential flow concentration, and freeze-drying of the nucleic acid nano-preparation by a freeze dryer, wherein the microchannel reactor is a microchannel reactor established on continuous flow.
The invention relates to a non-liver-targeting nucleic acid nano preparation, which is prepared from an aqueous solution of nucleic acid, polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid and ionizable lipid material as organic phases, wherein the N/P (molar ratio of amino nitrogen to phosphate groups) ratio of the cationic lipid to the nucleic acid is less than 12, and the N/P ratio of the ionizable lipid to the nucleic acid is more than 1.5.
In some embodiments, the N/P ratio of the cationic lipid to the nucleic acid is less than or equal to 12, or less than or equal to 10, preferably 2 to 12, and the N/P ratio of the ionizable lipid to the nucleic acid is greater than or equal to 1.5, or greater than or equal to 3, preferably 3 to 10.5; more preferably, the N/P ratio of the cationic lipid to the nucleic acid is 4 to 8, and the N/P ratio of the ionizable lipid to the nucleic acid is 4.5 to 9. The N/P of the proper cationic lipid and ionizable lipid and nucleic acid enables the nano-preparation to effectively load the drug, and has high knocking efficiency and good silencing effect. In some embodiments, the nucleic acid nanoformulation is prepared by mixing an aqueous solution of nucleic acid as an internal aqueous phase, polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer, cationic lipid, ionizable lipid dissolved in an organic solvent as an organic phase, and DEPC water as an external aqueous phase in a microchannel reactor.
In some embodiments, the microchannel reactor-based reaction is performed by first mixing the internal aqueous phase containing the nucleic acid drug and the organic phase containing the cationic lipid and the ionizable lipid in the first reaction plate, and performing the reaction to obtain a mixed solution. Further, the mixed solution and an organic phase of a polymer (such as polyethylene glycol-polylactic acid) are mixed in a second reaction plate and added into an aqueous phase, so as to obtain a nucleic acid-loaded nano preparation solution.
Or mixing the inner water phase containing nucleic acid medicine and the organic phase containing cationic lipid, ionizable lipid and polymer (polyethylene glycol-polylactic acid) in the first reaction plate, and adding the mixture into the outer water phase to obtain the nucleic acid loaded nano preparation solution.
In some of these embodiments, the nucleic acid is an siRNA or mRNA.
In some embodiments, the siRNA or mRNA is a tumor therapy or antiviral infection therapy.
In some of these embodiments, the cationic lipid material may be dimethyl-2, 3-dioleyloxypropyl-2- (2-spermicarbonamido) ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyl-2, 3-dioleyloxypropyl ammonium bromide (DOTMA), trimethyl-2, 3-dioleyloxypropyl ammonium bromide (DOTAP), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORI), dimethyl-2-hydroxyethyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE), dimethyl-3-hydroxypropyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE-HP), dimethyl-4-hydroxybutyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE-RIE), dimethyl-5-hydroxypentyl-2, 3-dioleyloxypropyl ammonium bromide (DORIE-HP), dimethyldioctadecyloxypropylammonium bromide (DORIE-2, 3-dioctadecylpropyl ammonium bromide (DORIE-2, 3-bis-hydroxyethylammonium bromide (DORIE-2, 3-bis-octadecyloxypropyl ammonium bromide (DORIE-2, 3-bis-3-hydroxyethylammonium bromide (DORIE), 3-Ditetradecyloxypropylammonium (DMRIE), N- (2-sperminoyl) -N ', N' -dioctadecyl glycinamide (DOGS), 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), 3 beta- [ N- (N ', N' -dimethylaminoethyl) carbamoyl ] cholesterol (DC-Chol), lipid poly-L-lysine (LPLL), stearylamine (SA).
More preferably, the cationic lipid in step (1) is at least one selected from DOTMA, DOTAP, DORI, DSRIE, DOGS, and DOSC, and is most preferably DOTAP.
In some embodiments, the N/P ratio of the cationic lipid to the nucleic acid is 2 to 12, and the N/P ratio of the ionizable lipid to the nucleic acid is 3 to 10.5.
In some preferred embodiments, the mass ratio of the cationic lipid to the ionizable lipid in the organic phase is 1:0.5-1.5, more preferably 1:0.8-1.2, more preferably 1:1.
in some embodiments, the nucleic acid drug includes small interfering RNA (siPD-L1, siCD 47) targeting PD-L1 and CD47, mRNA, and the like.
In some embodiments, the particle size of the nucleic acid nano preparation after being reconstituted ranges from 50 nm to 500 nm, and preferably ranges from 50 nm to 200 nm.
In some of these embodiments, the ionizable lipid is Dlin-MC3-DMA, SM-102, ALC-0315, dlin-KC2-DMA, and may be any of the following ionizable lipids:
BHEM-DBA：
；
BHEM-APMP：
BHEM-EAA：
/>
BHEM-AEA：
。
the synthesis of these ionizable lipids is as follows.
BHEM-DBA: triethylamine (2.25 g,22.28 mmol) and N, N-dibutylethylenediamine (2.11 g,12.25 mmol) were dissolved in 10mL of chloroform, stirred for 30 min, and then dropped into a cholesteryl chloroformate solution (5 g,11.14 mmol) dissolved in 40mL of chloroform while cooling. After the addition, stirring was continued at room temperature for 12 h. The compound was purified by flash column chromatography system to obtain BHEM-DBA lipid compound (5.13 g, 78.7%).
The main data of nuclear magnetic hydrogen spectra of BHEM-DBA are shown in FIG. 9.
BHEM-APMP: triethylamine (2.25 g,22.28 mmol) and N- (3-aminopropyl) morpholine (1.77 g,12.25 mmol) were dissolved in 10mL of chloroform, stirred for 30 min, and then dropped into a cholesteryl chloroformate solution (5 g,11.14 mmol) dissolved in 40mL of chloroform in a cold bath. After the addition, stirring was continued at room temperature for 12 h. The compound was purified by flash column chromatography system to give BHEM-APMP lipid compound (4.51 g, 70.7%).
The main data of nuclear magnetic hydrogen spectra of BHEM-APMP are shown in FIG. 10.
BHEM-EAA: triethylamine (2.25 g,22.28 mmol) and 2, 2-n-propylethylenediamine (1.76 g,12.25 mmol) were dissolved in 10mL of chloroform, stirred for 30 min, and then dropped into a cholesteryl chloroformate solution (5 g,11.14 mmol) dissolved in 40mL of chloroform while cooling. After the addition, stirring was continued at room temperature for 12 h. The compound was purified by flash column chromatography to give BHEM-EAA lipid compound (4.33 g, 71.9%).
The main data of nuclear magnetic hydrogen spectra of BHEM-EAA are shown in FIG. 11.
BHEM-AEA: triethylamine (2.25 g,22.28 mmol) and 2- (azetidin-1 yl) ethylamine (1.23 g,12.25 mmol) were dissolved in 10mL of chloroform, stirred for 30 min, and then added dropwise to a cholesteryl chloroformate solution (5 g,11.14 mmol) dissolved in 40mL of chloroform, while cooling. After the addition, stirring was continued at room temperature for 12 h. The compound was purified by flash column chromatography system to give BHEM-AEA lipid compound (3.78 g, 62.7%).
The main data of nuclear magnetic hydrogen spectra of BHEM-AEA are shown in FIG. 12.
In some embodiments, the organic solvent is chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, acetone, etc., preferably ethanol, acetonitrile.
In some of these embodiments, the polymer is a polyethylene glycol modified aliphatic and/or polyester aliphatic polyester.
In some embodiments, the polyethylene glycol-modified aliphatic polyester is at least one of polyethylene glycol-modified polylactide (PEG-PLA) and polyethylene glycol-modified poly (glycolide-co-lactide) (PEG-PLGA).
In some embodiments, the aliphatic polyester has a molecular weight in the range of 1000 to 11000 daltons.
In some embodiments, the polyethylene glycol has a molecular weight in the range of 1000 to 10000 daltons.
In some embodiments, the aliphatic polyester is poly (glycolide-co-lactide) with a ratio of LA/GA in a range from 95/5 to 50/50, specifically in a range from 95/5, 85/15, 75/25, 50/50, or any two specific ratios.
In some embodiments, the present invention relates to a method for preparing the nucleic acid nano preparation based on the microchannel reactor, which comprises the following steps:
(1) In a microchannel reactor, the internal aqueous phase and the organic phase respectively enter a reaction plate 1 through a channel for mixing reaction to obtain a mixture, then enter a reaction plate 2, and the external aqueous phase enters the reaction plate 2 through the channel to be mixed with the mixture to obtain a nucleic acid nano preparation solution;
(2) And (2) transferring the solution obtained in the step (1) to a rotary evaporator, gradually increasing the pressure in a water bath at about 37 ℃, and evaporating the organic solvent in the solution to obtain an initial nucleic acid nano preparation solution.
(3) Concentrating the nucleic acid nano preparation solution obtained in the step (2) through tangential flow to obtain a concentrated nucleic acid nano preparation solution.
(4) And (4) adding the concentrated nucleic acid nano preparation solution in the step (3) into a freeze-drying protective agent with the final concentration of 10 to 20% (w/v), and carrying out vacuum low-temperature freeze drying to obtain the nucleic acid nano preparation.
In some embodiments, the aqueous siRNA solution or aqueous mRNA solution of the inner aqueous phase in step (1) is dissolved in DEPC-treated ultrapure water.
In some of the embodiments, the organic solvent in step (1) is chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, acetone, etc.
In some of these embodiments, the external aqueous phase of step (1) is DEPC-treated ultrapure water.
In some of these embodiments, the concentration of nucleic acid in the aqueous nucleic acid solution is from 700. Mu.g/mL to 900. Mu.g/mL.
In some of these embodiments, the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer concentration is 8 mg/mL to 12mg/mL in the organic phase.
In some of these embodiments, the cationic lipid concentration is 0.4mg/mL to 0.6mg/mL in the organic phase.
In some of these embodiments, the ionizable lipid concentration is between 0.4mg/mL and 0.6mg/mL in the organic phase.
In some of these embodiments, the volume ratio of the inner aqueous phase, organic phase, outer aqueous phase is from 1.
In some embodiments, the flow rate of the inner aqueous phase is 0.1 mL/min to 100 mL/min, more preferably 0.1 mL/min to 10mL/min.
In some embodiments, the flow rate of the organic phase is 1.0 mL/min to 100 mL/min, and more preferably 1.0 mL/min to 40mL/min.
In some embodiments, the flow rate of the outer organic phase is 5.0 mL/min to 100 mL/min, more preferably 5.0 mL/min to 50mL/min.
In the actual preparation process, the flow rate can be adjusted accordingly as required, and according to the volume of the reaction, the flow rate is generally 1.
In some embodiments, the microchannel reactor of step (1) mainly comprises: control panel, three pumps (from left to right are pump 1, pump 2, pump 3 respectively), three pipeline (correspond pipeline 1, pipeline 2, pipeline 3 of pump 1, pump 2, pump 3 respectively), two microchannel reaction plates and 1 output pipeline.
In some embodiments, the method step of preparing a technology by a microchannel reactor in step (1) comprises: (1) opening a micro-channel switch, and controlling the switch by a constant temperature panel; the microchannel reactor pump is connected with an ethanol solution, a flushing pump and a pipeline; air of 3 pumps was removed by syringe; then connecting organic solvent (chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran and acetone) at a pump 1 and a pump 2, and flushing the pumps and pipelines; pump 3 connects DEPC water, flush pump and tubing. (2) Setting the flow rate of a pump 1 to be 0.1 to 10mL/min as an internal water phase (siRNA water solution); setting the flow rate of the pump 2 to be 1.0-40 mL/min, and taking the pump as an organic phase (cationic lipid, ionizable lipid and polymer organic solvent solution); setting the flow rate of a pump 3 to be 5.0-100 mL/min, and taking the pump as an external water phase (DEPC ultrapure water); the three-phase volume ratio is adjusted by the mobile phase velocity as required. (3) Taking down the pipeline 1 connected with the pump 1, discharging the organic solvent remained in the pipeline, and sucking the prepared siRNA solution by using an injector (provided with a special injection needle head); injecting the siRNA solution into the pipeline 1, reserving air columns with corresponding volumes at the two ends of the pipeline 1, then reconnecting the pipeline 1 to the middle of the pump 1 and the reaction plate, starting the pump 1 (connecting the organic solvent solution) to push the siRNA solution column to a position about 3 cm away from the inlet of the reaction plate, and suspending the pump 1. (4) Connecting the pump 2 with an organic phase, starting the pump 2, starting the pump 1 after about 80s (timing by using a timer), and starting the inner water phase and the organic phase to enter the reaction plate 1 after about 1 min to start mixing; (5) the mixed solution in the reaction plate 1 is pushed to the reaction plate 2 by using the pump 1 and the pump 2, meanwhile, the pump 3 is opened, the mixed solution in the reaction plate 1 enters the reaction plate 2 and is mixed with the external water phase for the second time, and the mixed liquid can be seen in the reaction plate 2, namely the nucleic acid nano preparation solution.
In some of these embodiments, the rotary evaporator of step (2) includes, but is not limited to, a parallel rotary evaporator, and the organic solvent is removed to obtain the initial nucleic acid nanoformulation solution.
In some embodiments, the method for concentrating the initial nucleic acid nanoformulation solution of step (3) comprises: pass through 300 kDa envelope, concentrate to the desired volume, 10mL above. Other conventional methods may also be used as long as the nucleic acid nanoformulation solution can be concentrated.
In some embodiments, the lyophilizing process of the nucleic acid nano-preparation by the lyophilizer in step (4) includes adding a lyoprotectant with a final concentration of, for example, 9-11 wt% to the concentrated nucleic acid nano-preparation solution, and lyophilizing under vacuum to obtain the nucleic acid nano-preparation capable of long-term storage. Specifically, the lyoprotectant may be sucrose, but is not limited thereto, and may also be glucose, trehalose, mannitol, and the like.
The present invention will be described in further detail with reference to specific examples.
Sources of raw materials used in the following examples:
cationic lipid DOTAP: (2, 3-Dioleyloxypropyl) trimethylammonium chloride available from Avanti Polar Lipids;
ionizable lipids: dlin-MC3-DMA, SM-102, ALC-0315, dlin-KC2-DMA, available from Avanti Polar Lipids; in addition, the following ionizable lipids are included:
BHEM-DBA、BHEM-APMP、BHEM-EAA、BHEM-AEA。
poly (glycolide-co-lactide) Polymer (PLGA): LA/GA ratio of 75/25, purchased from the bio-technology company of the large handle of the Jinan Dai.
The molecular weight range of polyethylene glycol (PEG) is 1000 to 10000 daltons.
Lipofectamine ™ RNAiMAX Transfection Reagent, cat # 13778075, available from Thermo corporation;
EasyPure
RNA Kit, cat # ER101-01, available from general gold;
PrimeScript RT reagent Kit, cat # RR036A, available from TaKaRa;
FastStart Essential DNA Green Master, cat # 06924204001, available from Roche;
mouse HBsAg ELISA kit, cat # LT-120009, available from blueprint Biotechnology Ltd of yellow Stone;
mouse HBeAg ELISA kit, cat # LT-120011, purchased from blueprint Biotech, inc. of Huangshi;
sirnas were purchased from bexin biotechnology, suzhou, ltd; mRNA was purchased from synfeina biotechnology limited;
organic solvent solution, purchased from chemical reagents of national drug group, ltd;
other reagents not described were used as they were.
Instrument model and company used in the examples:
micro-channel reactor: corning Lab reactor system, corning corporation, usa;
rotating the evaporator: model RV10 auto control, IKA, germany;
minimate tangential flow ultrafiltration system and 300 kDa envelope: model OAPMP220, PALL, USA;
development type freeze dryer: the model is LyoStar TM SP Scientific, USA;
nano-particle size and Zeta-potentiometer: model no ZSE, malvern, uk;
flow cytometry analysis: model number BD FACSCelesta, BD company, usa;
qPCR instrument: model number Roche Light Cycler 96, roche USA.
The present invention will be described in further detail with reference to specific examples.
Example 1 Process flow for preparation of nucleic acid Nanodiulation
The microchannel reactor shown in fig. 1A mainly comprises: control panel, three pump (from left to right respectively be pump 1, pump 2, pump 3), three charge-in pipeline (correspond respectively pump 1, pump 2, pipeline 1, pipeline 2, pipeline 3 of pump 3), two microchannel reaction plates (front and back respectively are reaction plate 1 and reaction plate 2) and 1 output tube.
The method steps of mixing by a microchannel reactor include: (1) opening a micro-channel switch, and controlling the switch by a constant temperature panel; the microchannel reactor pump is connected with an ethanol solution, a flushing pump and a pipeline; air of 3 pumps was removed by syringe; then connecting the organic solvent with the pump 1 and the pump 2, and flushing the pump and the pipeline; pump 3 connects DEPC water, flush pump and tubing. (2) Setting the pump 1 as an internal water phase (siRNA aqueous solution or mRNA aqueous solution, nucleic acid concentration is 800 mug/mL); setting the pump 2 as an organic phase (polyethylene glycol modified poly (glycolide-co-lactide) (PEG-PLGA), cationic lipid and ionizable lipid are dissolved in acetonitrile, and the concentration is 10.0 mg/mL); pump 3 was set to an external aqueous phase (DEPC ultrapure water); the three-phase volume ratio (1. (3) Taking down the pipeline 1 connected with the pump 1, discharging the organic solvent remained in the pipeline, and sucking the prepared siRNA solution by using an injector (provided with a special injection needle head); injecting the siRNA solution into the pipeline 1, reserving air columns with corresponding volumes at the two ends of the pipeline 1, then reconnecting the pipeline 1 to the middle of the pump 1 and the reaction plate, starting the pump 1 (connecting the organic solvent solution) to push the siRNA solution column to a position about 3 cm away from the inlet of the reaction plate, and suspending the pump 1. (4) Connecting the pump 2 with an organic phase, starting the pump 2, starting the pump 1 after about 80s (timing by using a timer), starting the inner water phase and the organic phase to enter the reaction plate 1 after about 1 min, starting a mixing reaction, and observing the reaction plate 1 to see a mixed solution; (5) the mixed solution in the reaction plate 1 is pushed to the reaction plate 2 by using the pump 1 and the pump 2, and simultaneously, the pump 3 is opened, and the mixed solution in the reaction plate 1 enters the reaction plate 2 to be secondarily mixed with the external water phase.
The mixed solution was collected using a 500 mL round-bottomed flask (observing the initial discharge time and the complete discharge time of the mixed solution in the reaction plate 2 to avoid non-mixed liquid before and after collection), and as shown in FIG. 1B, a rotary evaporator was connected to remove the organic solvent to obtain an initial nucleic acid nano-formulation solution.
As shown in FIG. 1C, the initial nucleic acid nanoformulation solution was concentrated to the desired volume using tangential flow through a 300 kDa envelope. Further, a sucrose solution with a concentration of 50% is prepared, as shown in fig. 1D, a sucrose solution with a final concentration of 10% is added to the concentrated nucleic acid nano-preparation solution, and the nucleic acid nano-preparation is obtained by freeze sublimation drying under a vacuum condition. The prepared nano systems loaded with siRNA and mRNA are named as PCI/siRNA and PCI/mRNA respectively.
Example 2 nucleic acid Nanopamulation schematic and nucleic acid Nanopamulation characterization after lyophilization and reconstitution
As shown in fig. 2A, the nucleic acid nano-formulation was prepared from lipid/polymer nucleic acid nanoparticles in the same manner as in example 1. After adding the lyoprotectant, the mixture is subpackaged into 10mL freeze-drying bottles (2 mL/bottle) and is subjected to freeze-drying, and the freeze-drying step comprises the following steps: (1) checking the air pressure of an air and nitrogen cylinder to ensure that the air and nitrogen are enough, wherein the nitrogen is at least more than 3 MPa, opening an air valve, regulating a partial pressure valve to be more than 0.8 MPa, and regulating the air to be 0.5 MPa and the nitrogen to be 0.7 MPa according to an internal pressure valve in a purification room; (2) the freeze dryer is opened, the sample is placed in the sample tray, and the rubber cover is in a half-covered state. The sample tray is pushed into the freeze-drying chamber, the tray is pulled out, and the freeze-drying chamber door and the lower condensation chamber door are closed; (3) the lyophilization software was automatically turned on and logged in, and the lyophilization program was adjusted on the freeze dry page to begin lyophilization. Observing a status bar to ensure good air tightness and the environment temperature to be lower than 30 ℃; (4) in the freeze-drying process, when the difference between PVG (vacuum degree containing humidity) and the set vacuum degree is not more than 3, a temperature rising step can be carried out (-28 ℃ and the subsequent steps), and the corresponding time and steps can be adjusted on a freeze dry page; (5) after the freeze-drying is finished, firstly, nitrogen is filled into the semi auto page (in cooperation with the opening of a filling pipeline) to 500 degrees, the tray is lifted, the cover is pressed, then the filling is closed, the vacuum is released, and the cabin door can be opened after the complete release. The sample was quickly removed and capped. As shown in fig. 2B, the nucleic acid nano-formulation appeared white, uniform in size and uniform in quality as a powder after freeze-drying.
Characterization of nucleic acid nanopreparations after reconstitution: 1mL of DEPC-treated ultrapure water was added to the lyophilized siRNA nanoformulation (PIC/siRNA) and mRNA nanoformulation (PIC/mRNA) for reconstitution. The particle size of the nano-preparation is measured by Dynamic Light Scattering (DLS), and as shown in FIG. 2C and FIG. 2D, the particle size of the siRNA nano-preparation and the particle size of the mRNA nano-preparation after reconstitution are about 136.7 nm and 166.0 nm, respectively.
Example 3 stability characterization of nucleic acid Nanodiulation in media containing 10% serum
The prepared nucleic acid nano preparation comprises PCI/siRNA and PCI/mRNA, and is diluted to 0.5 mg/mL by ultrapure water, added with 10% serum respectively and sealed; incubation in a constant temperature shaking table at 37 ℃ is carried out, as shown in FIGS. 3A and 3B, and the particles are respectively taken out at 0 h,1 h,2 h,4 h,6 h,12 h,24 h,48 h and 72 h and are detected by a Dynamic Light Scattering (DLS) instrument, and the results show that the particle sizes of the PCI/siRNA and the PCI/mRNA are not obviously changed within the observation time and the particle size distribution does not fluctuate greatly, which indicates that the aqueous solution of the nucleic acid nano-preparation of the invention can keep the stability of the hydration radius within 72 hours.
Example 4 optimization study of PCI/siRNA nanoparticle formulation
In order to examine the ratio among the lipids DOTAP, dlin-MC3-DMA and the copolymer and the influence of the molecular weight of the copolymer on the nanoparticle encapsulation efficiency, the size and the potential, the experiment firstly carried outBy immobilising mPEG 2K -PLGA 2K (the mPEG block has a molecular weight of 2000, and the PLA block has a molecular weight of 2000,) and the influence of the ratio of DOTAP to Dlin-MC3-DMA on the nanoparticles is researched by respectively changing the dosage of DOTAP and Dlin-MC 3-DMA. As shown in Table 1, the ratio of DOTAP significantly affects the loading efficiency of siRNA, and when the N/P ratio of DOTAP/siRNA (the molar ratio of amino nitrogen of DOTAP to phosphate group of nucleic acid drug) is 2 or more, nucleic acid drug can be loaded effectively, while the higher the ratio of Dlin-MC3-DMA, the lower the dispersion degree of particles.
Table 1, the effects of the amounts of DOTAP and Dlin-MC3-DMA on the encapsulation efficiency (E.E.), the amount of nucleic acid (Loading Content), the particle Diameter (Diameter), the distribution (PDI) and the potential (Zeta potential) of the prepared nucleic acid nanocarrier.
By fixing the feeding amount of DOTAP and Dlin-MC3-DMA, mPEG is changed 2K -PLGA 2K The influence of the addition amount of the copolymer on the property of the nanoparticles is explored. As shown in Table 2, the amount of the copolymer to be charged does not greatly affect the particle entrapment efficiency, particle size, and other properties.
TABLE 2 mPEG 2K -PLGA 2K The influence of the amount of the added materials on the encapsulation efficiency (E.E.) of the prepared nucleic acid nano-carrier, the amount of the nucleic acid (Loading Content), the particle Diameter (Diameter), the distribution (PDI) and the potential (Zeta potential).
The molecular weight of the copolymer is changed through the dosage of DOTAP, dlin-MC3-DMA and the copolymer, so as to explore the influence of the molecular weight of the copolymer on the nanoparticles. As shown in tables 3 and 4, the molecular weight of the copolymer has little influence on the properties such as particle size and dispersibility of the nanoparticles.
Table 3, effect of mPEG block molecular weight (PLGA block molecular weight 3K, kept constant) in mPEG-PLGA polymer on encapsulation efficiency (e.e.), nucleic acid amount (Loading Content), and particle size (Diameter), distribution (PDI), and potential (Zeta potential) of the prepared nucleic acid nanocarrier.
Table 4, effect of molecular weight of PLGA block in mPEG-PLGA polymer (mPEG block molecular weight is 2K, remains unchanged) on encapsulation efficiency (e.e.), amount of nucleic acid (Loading Content), and particle size (Diameter), distribution (PDI), and potential (Zeta potential) of the prepared nucleic acid nanocarrier.
Example 5 Effect of two cationic lipids in PCI/siRNA nanoparticles on target Gene knockout efficiency
In order to examine the influence of the ratio of cationic lipid to ionizable lipid in the lipid/polymer hybrid nanoparticle of the present invention on the knockout efficiency, the experiment uses siRNA with the knockout target as CD47 as a model to examine the knockout of CD47 protein in B16-F10 cells.
As described in example 1, the preparation steps of the nucleic acid nanoformulation include: the pump 1 is that the inner water phase is siRNA water solution, the nucleic acid concentration is 800 mug/mL; setting the pump 2 as organic phase acetonitrile containing polymer, cationic lipid and ionizable lipid, wherein the concentration of PEG-PLGA is 10 mg/mL, the concentration of cationic lipid DOTAP is 0.5 mg/mL, and the concentration of ionizable lipid Dlin-MC3-DMA is 0.5 mg/mL; setting a pump 3 as an external water phase (DEPC ultrapure water); the three-phase flow rates are respectively 0.1 mL/min, 1.0 mL/min and 5.0 mL/min; prepared according to the method in example 1 to obtain the nano preparation PCI/siRNA DOTAP/MC3 。
According to the similar method, the Dlin-MC3-DMA is replaced by BHEM-APMP and BHEM-EAA, the concentration is unchanged, and the prepared nano preparation is PCI/siRNA DOTAP/APMP 、PCI/siRNA DOTAP/EAA 。
In addition, in the preparation process, the organic phase only adds one lipid of DOTAP (with the concentration of 0.5 mg/mL), dlin-MC3-DMA (with the concentration of 0.5 mg/mL), BHEM-APMP (with the concentration of 0.5 mg/mL) and BHEM-EAA (with the concentration of 0.5 mg/mL) besides PEG-PLGA, and other conditions are kept unchanged, so that the obtained nano preparation is PC/siRNA DOTAP 、PC/siRNA MC3 、PC/siRNA APMP 、PC/siRNA EAA 。
Accordingly, nanoparticle PC/siRNA assembled by only one cationic lipid was prepared DOTAP 、PC/siRNA MC3 、PC/siRNA APMP 、PC/siRNA EAA/siRNA 。
By using the prepared nano-particle PCI/siRNA DOTAP/MC3 、PCI/siRNA DOTAP/APMP 、PCI/siRNA DOTAP/EAA 、PC/siRNA DOTAP 、PC/siRNA MC3 、PC/siRNA APMP 、PC/siRNA EAA Co-incubation with B16F10 cells, wherein siRNA transfection concentrations for each group were 50 nM; after the incubation for 6 h, the culture medium is replaced by fresh serum-containing medium and is continued for 24 h, then the cells are collected, and the expression of CD47 mRNA is detected by using a q-PCR detection method.
And (4) experimental conclusion: nanoparticles prepared with only one cationic lipid were not effective in reducing CD47 mRNA expression at 50 nM concentration, and were effective in reducing CD47 mRNA expression when the cationic lipid was co-involved in assembling nanoparticles with ionizable lipids (fig. 4A); in addition, the nanoparticles prepared were of a solid spherical structure (fig. 4B).
Example 6 Effect of PCI/siRNA nanoparticles of different formulations on the target Gene knockout efficiency
In order to examine the effect of the ratio of cationic lipid to ionizable lipid in the lipid/polymer hybrid nanoparticle of the present invention on the knockout efficiency, the experiment uses siRNA with the knockout target as GFP as a model to examine the knockout of GFP protein in B16-F10 cells stably expressing GFP.
24 kinds of nano-particle PC prepared by PEG-PLGA, cationic lipid DOTAP and ionizable lipid Dlin-MC3-DMA are preparedI/siGFP DOTAP/MC3 The method specifically comprises the following steps: the aqueous phase was prepared by diluting 0.45 nmol of siGFP in 72. Mu.L of DEPC water. The components forming the oil phase of the particles are respectively mPEG 2K -PLGA 2K (150 mg/mL in DMSO), DOTAP (10 mg/mL in ethanol), and Dlin-MC3-DMA (10 mg/mL in ethanol). The oil phase configuration of the various particles is as follows, PCI 4-0 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—0 μL）、MC3PCI 4-1.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—1.82 μL）、MC3 PCI 4-3 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—3.63 μL）、MC3 PCI 4-4.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—5.45 μL）、MC3 PCI 4-6 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—7.26 μL）、MC3 PCI 4-7.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—9.075 μL）、MC3 PCI 4-9 （mPEG 2K -PLGA 2K —8 μL、DOTAP—5.28 μL、Dlin-MC3-DMA—10.89 μL）、PCI 8-0 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—0 μL）、MC3 PCI 8-1.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—1.82 μL）、MC3 PCI 8-3 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—3.63 μL）、MC3 PCI 8-4.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—5.45 μL）、MC3 PCI 8-6 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—7.26 μL）、MC3 PCI 8-7.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—9.075 μL）、MC3 PCI 8-9 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—10.89 μL）、MC3 PCI 8-10.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—10.56 μL、Dlin-MC3-DMA—12.71 μL）、PCI 12-0 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—0 μL）、MC3 PCI 12-1.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—1.82 μL）、MC3 PCI 12-3 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—3.63 μL）、MC3 PCI 12-4.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—5.45 μL）、MC3 PCI 12-6 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—7.26 μL）、MC3 PCI 12-7.5 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—9.075 μL）、MC3 PCI 12-9 （mPEG 2K -PLGA 2K —8 μL、DOTAP—15.84 μL、Dlin-MC3-DMA—10.89 μL）、MC3 PCI 12-10.5 （mPEG 2K -PLGA 2K 8. Mu.L, DOTAP-15.84. Mu.L, dlin-MC 3-DMA-12.71. Mu.L). The aqueous phase was mixed with the oil phase separately and shaken on a Votex for 20 s. Standing at room temperature for 15 min.
In the nanoparticles prepared above, the subscripts indicate the N/P ratio of DOTAP, dlin-MC3-DMA and siRNA, such as MC3 PCI 4-1.5 It is shown that the N/P of DOTAP and siRNA is 4, and the N/P of Dlin-MC3-DMA and siRNA is 1.5. In addition, PCI 4-0 、PCI 8-0 、PCI 12-0 It indicates that the N/P of DOTAP and siRNA is 4, 8 and 12, but Dlin-MC3-DMA lipid is not doped.
The particles prepared above were incubated with B16F10 cells stably expressing GFP at a concentration of 2nMsiGFP, and after 24 h the silencing efficiency of GFP was assessed by flow cytometry.
As shown in FIG. 5, the content of Dlin-MC3-DMA is proportional to the target gene knockdown efficiency: as shown in FIG. 5, the silencing effect was gradually increased when the N/P ratio of the Dlin-MC3-DMA lipid to siRNA was 1.5 or higher, and the effect was best when 4.5 or higher was reached. The content of DOTAP is inversely proportional to the knockdown efficiency of the target gene: compared with the nanoparticles with the N/P ratio of the DOTAP lipid to the siRNA being 4 and 8, the knocking efficiency of the nanoparticles is obviously reduced when the N/P ratio of the DOTAP lipid to the siRNA is 12, and the knocking efficiency is poor when the N/P ratio of the DOTAP lipid to the siRNA is more than 12.
Example 7 study of tumor-inhibiting efficacy of PCI/siRNA nanoparticles
In order to examine the effect of the lipid/polymer hybrid nanoparticle delivery siRNA of the invention on treating diseases, the experiment takes siRNA with the target spot being knocked out as CD47 and PD-L1 as a model, and examines the inhibition condition of B16-F10 tumor.
A total of three nanoparticles PCI/siRNA prepared from PEG-PLGA, cationic DOTAP and ionizable lipid Dlin-MC3-DMA are prepared DOTAP/MC3 Wherein, PCI/siCD47 DOTAP/MC3 The preparation method is shown as example 4 and specifically comprises the following steps: as described in example 1, the preparation steps of the nucleic acid nanoformulation include: the pump 1 is an internal water phase which is siRNA (siCD 47) water solution aiming at CD47 genes, and the nucleic acid concentration is 800 mug/mL; setting the pump 2 as organic phase acetonitrile containing polymer, cationic lipid and ionizable lipid, wherein the concentration of polymer PEG-PLGA is 10 mg/mL, the concentration of cationic lipid DOTAP is 0.5 mg/mL, and the concentration of ionizable lipid Dlin-MC3-DMA is 0.5 mg/mL; setting a pump 3 as an external water phase (DEPC ultrapure water); the three-phase flow rates are respectively 0.1 mL/min, 1.0 mL/min and 5.0 mL/min; prepared according to the method in example 1 to obtain the nano preparation PCI/siCD47 DOTAP/MC3 。
PCI/siNC DOTAP/MC3 And PCI/siPD-L1 DOTAP/MC3 Replacing siRNA only with non-functional siRNA (siNC) and siRNA against PD-L1 gene (siPD-L1); PCI/siCD47+ siPD-L1 DOTAP/MC3 Is PCI/siCD47 and siPD-L1 DOTAP/MC3 Mixing; the administration dose of siRNA was 0.25 OD once every two days, and the injection mode was tail vein injection.
And (4) experimental conclusion: PCI/siCD47 alone by tail vein injection DOTAP/MC3 Or sipD-L1 DOTAP/MC3 Can effectively inhibit tumor growth, and can be injected with two kinds of nanoparticles of PCI/siCD47 DOTAP/MC3 And sipD-L1 DOTAP/MC3 When the system is used, the tumor growth can be more effectively inhibited, and the nano system has good effect of delivering siRNA in vivo (figure 6).
Example 8 mRNA-loaded composition nanoparticle in vivo transfection efficiency validation
In order to examine the in vivo mRNA transport efficiency of the nanoparticles of the present invention, mRNA delivery and expression efficiency of two administration modes, subcutaneous injection and intramuscular injection, were examined using luciferase-expressing mRNA (Luci-mRNA) as a model in the experiment. The experiment shows that the nanoparticle PCI/Luci-mRNA is prepared from PEG-PLGA, cationic DOTAP and ionizable lipid Dlin-MC 3-DMA; the preparation is analogous to example 1: the preparation method comprises the following steps: the pump 1 is an internal water phase Luci-mRNA aqueous solution with the nucleic acid concentration of 800 mug/mL; setting the pump 2 as organic phase acetonitrile containing polymer, cationic lipid and ionizable lipid, wherein the concentration of PEG-PLGA is 10 mg/mL, the concentration of cationic lipid DOTAP is 0.5 mg/mL, and the concentration of ionizable lipid Dlin-MC3-DMA is 0.5 mg/mL; setting a pump 3 as an external water phase (DEPC ultrapure water); the three-phase flow rates are respectively 0.1 mL/min, 1.0 mL/min and 5.0 mL/min; prepared according to the method in example 1 to obtain the nano preparation PCI/Luci-mRNA.
Mice were imaged in vivo at different time points after subcutaneous or intramuscular injection with the constructed PCI/Luci-mRNA, and the expression efficiency of mLuc-NPs at 6 h,12 h,24 h,48 h after immunization was evaluated by bioluminescence. The experiment was performed in 2 groups (subcutaneous and intramuscular administration groups) in total, with 2 mice per group. For the subcutaneous group, each mouse was injected subcutaneously with 20. Mu.g of mLuc-NPs, and for the intramuscular group, each mouse was injected intramuscularly with 20. Mu.0 of PCI/Luci-mRNA; mice were anesthetized by intraperitoneal injection of 125 μ L of sodium pentobarbital (1%) at 6 h,12 h,24 h, and 48 h after immunization; luciferase protein expression was read by in vivo bioluminescence using an In Vivo Imaging System (IVIS) following tail vein injection of 200. Mu.L luciferase substrate luciferin (15 mg/mL). Experiments show that: the cationic lipid-hybridized nanoparticles mLuc-NPs prepared by the above method can successfully deliver mRNA into the body and achieve high expression of target protein, whether administered subcutaneously or intramuscularly (fig. 7).
Also using Luci-mRNA as a model, we further examined the transfection efficiency of the mRNA-loaded nanoparticles after intravenous injection. After intravenous injection of the constructed PCI/Luci-mRNA into the immunized animal, the mice were subjected to in vivo imaging of the animal at 6 h, and the expression efficiency of the PCI/Luci-mRNA after immunization was evaluated by bioluminescence. Each mouse was injected intravenously with 10. Mu.g of PCI/Luci-mRNA. At 6 h after immunization, mice were anesthetized by intraperitoneal injection of 125 μ L sodium pentobarbital (1%); luciferase protein expression was read by in vivo bioluminescence using an In Vivo Imaging System (IVIS) following tail vein injection of 200. Mu.L luciferase substrate luciferin (15 mg/mL). Experiments show that: after intravenous injection, luciferase protein was mainly expressed in spleen and lung, and the expression level in liver was low (fig. 8).
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (19)
1. A non-liver-targeting nucleic acid nano preparation is characterized in that the nucleic acid nano preparation is prepared by taking a nucleic acid aqueous solution as an internal aqueous phase, and taking polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-glycolic acid) copolymer, cationic lipid and ionizable lipid as organic phases; the N/P ratio of the cationic lipid to the nucleic acid is 4 to 8, and the N/P ratio of the ionizable lipid to the nucleic acid is 4.5 to 9, or 10.5; the cationic lipid is trimethyl-2, 3-dioleoyloxypropylammonium bromide, (2, 3-dioleoyloxypropyl) trimethylammonium chloride, dimethyl-2-hydroxyethyl-2, 3-dioleoyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2, 3-dioctadecyloxypropylammonium bromide, N- (2-sperminoyl) -N ', N' -dioctadecyl glycinamide, 1, 2-dioleoyl-3-succinyl-sn-glycerocholine ester; the ionizable lipid is selected from at least one of: dlin-MC3-DMA, dlin-KC2-DMA,
BHEM-DBA：
、
BHEM-APMP：
、
BHEM-EAA：
、
BHEM-AEA：
，
The mass ratio of the cationic lipid to the ionizable lipid in the organic phase is 1:0.5-1.5.
2. The non-liver-targeting nucleic acid nano-preparation according to claim 1, wherein the preparation is prepared by taking an aqueous solution of nucleic acid as an internal aqueous phase, dissolving polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid), cationic lipid and ionizable lipid in an organic solvent as an organic phase, taking DEPC water as an external aqueous phase, and reacting in a microchannel reactor.
3. The non-liver-targeted nucleic acid nanoformulation of claim 1, wherein the nucleic acid is siRNA or mRNA.
4. The non-liver-targeted nucleic acid nanoformulation of claim 1, wherein the cationic lipid is (2, 3-dioleyloxypropyl) trimethylammonium chloride.
5. The non-liver-targeted nucleic acid nanoformulation of claim 1, wherein the ionizable lipid is at least one selected from the group consisting of: dlin-MC3-DMA, dlin-KC2-DMA,
BHEM-APMP：
、
BHEM-EAA：
。
6. The non-liver-targeted nucleic acid nano-formulation according to claim 1, wherein the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer is a polyethylene glycol modified polylactide or a polyethylene glycol modified poly (glycolide-co-lactide).
7. The non-liver-targeted nucleic acid nanoformulation according to claim 6, wherein the ratio of LA/GA in the polylactide or poly (glycolide-co-lactide) is in the range of 95/5 to 50/50.
8. The non-liver-targeted nucleic acid nano-formulation according to claim 6 or 7, wherein the molecular weight of the polyethylene glycol is in the range of 1000 to 10000 daltons.
9. The non-liver-targeted nucleic acid nanoformulation of claim 1, wherein the N/P ratio of cationic lipid to nucleic acid is 4.5, 6, or 7.5.
10. The non-liver-targeted nucleic acid nanoformulation according to any one of claims 1-7, wherein the mass ratio of the cationic lipid to the ionizable lipid in the organic phase is 1:0.8-1.2.
11. The non-liver-targeting nucleic acid nanoformulation according to any one of claims 1 to 7, wherein the organic solvent is chloroform, dichloromethane, dimethylsulfoxide, dimethylformamide, ethanol, acetonitrile, tetrahydrofuran, or acetone.
12. The non-liver-targeted nucleic acid nano-preparation according to any one of claims 1 to 7, wherein the particle size of the nucleic acid nano-preparation after reconstitution is in the range of 50 nm to 200 nm.
13. A method for preparing a nucleic acid nanoformulation according to any one of claims 1 to 12, comprising the steps of: (1) continuously preparing a microchannel reactor: in a microchannel reactor, the internal aqueous phase and the organic phase respectively enter a reaction plate 1 through channels for mixing reaction to obtain a mixture, and then enter a reaction plate 2; the external water phase enters the reaction plate 2 through the channel and is mixed with the mixture to obtain a nucleic acid nano preparation solution;
(2) Removing the organic solvent;
(3) Concentrating to obtain a concentrated nucleic acid nano particle solution;
(4) And (3) freeze-drying the nucleic acid nano-particle solution to obtain the nucleic acid nano-preparation.
14. The method according to claim 13, wherein the concentration of the nucleic acid in the internal aqueous phase is 700 μ g/mL to 900 μ g/mL; and/or in the organic phase, the concentration of the polyethylene glycol-polylactic acid or polyethylene glycol-poly (lactic-co-glycolic acid) copolymer is 8 mg/mL-12 mg/mL; and/or the cationic lipid concentration is 0.4mg/mL to 0.6mg/mL; and/or the ionizable lipid concentration is between 0.4mg/mL and 0.6mg/mL.
15. The method according to claim 13 or 14, wherein the volume ratio of the inner aqueous phase, the organic phase and the outer aqueous phase is 1.
16. The method for preparing the nucleic acid nanoparticles of claim 13 or 14, further comprising adding a lyoprotectant to the concentrated nucleic acid nanoparticles solution to a final concentration of 10% to 20% w/v.
17. The method of claim 16, wherein the lyoprotectant is sucrose or glucose, trehalose, or mannitol.
18. The method according to claim 13 or 14, wherein the removing of the organic solvent is by rotary evaporation; and/or the concentrating is tangential flow concentrating.
19. Use of the nucleic acid nanoformulation according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of a tumor.
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|---|---|---|---|
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