CN112237633B - PEI/ON compound and preparation method and application thereof - Google Patents
PEI/ON compound and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- 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
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Abstract
The invention relates to the field of biological medicine, in particular to a PEI/ON complex, a preparation method and application thereof. The invention provides a PEI/ON complex, which comprises an oligonucleotide fragment, wherein polyethyleneimine is loaded ON the oligonucleotide fragment. The PEI/ON complex provided by the invention can effectively reduce the expression level of HBsAg in cell supernatant in an in vitro cell experiment, so that the PEI/ON complex can be used for recovering virus-specific immune response of patients, provides a new potential path for the development of anti-HBV treatment, and has good industrialization prospect.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a PEI/ON complex, a preparation method and application thereof.
Background
Hepatitis b is a transfection disease that is a serious hazard to human life and health caused by infection with hepatitis b virus (Hepatitis B virus, HBV). It is estimated that over 2.9 million people worldwide have chronic HBV infection and are at risk of dying from HBV-related cirrhosis and hepatocellular carcinoma complications. In HBV carriers, HBV infected hepatocytes produce a large number of non-infectious (globular or filamentous) cells into the peripheral blood circulation, typically 1000 larger than infectious Dane particles: 1 to 10,000:1. overproduction of subviral particles (SVP) comprising only the envelope glycoprotein (HBsAg) and host-derived lipids can lead to immune tolerance and to chronicity of HBV infection. Current clinical antiviral strategies are mainly using nucleoside drugs and interferon-based therapies, but rarely completely eliminate HBsAg from the blood to achieve a functional cure of HBV, i.e., HBsAg negative. This highlights how to reduce HBsAg levels is an important issue in developing a therapeutic strategy for chronic hepatitis b virus infection.
Oligonucleotide (ON) -based therapies are now becoming potential strategies for the treatment of viral infections, tumors and genetic diseases. Over the last several decades, many different types of therapeutic oligonucleotides have been developed, including antisense oligonucleotides, small interfering RNAs (sirnas) that bind to RNA, anti-gene oligonucleotides that bind to DNA and nucleic acid aptamers that bind to proteins, decoy oligonucleotides, and CpG oligonucleotides.
On the other hand, successful delivery of exogenous genes or oligonucleotides into cells is also a prerequisite for their effective use in medical therapy.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a PEI/ON complex, a preparation method and use thereof, for solving the problems in the prior art.
To achieve the above and other related objects, one aspect of the present invention provides a PEI/ON complex comprising an oligonucleotide fragment having a polyethyleneimine supported thereon.
In some embodiments of the invention, the oligonucleotide fragment has a length of 10 to 50nt, preferably 15 to 45nt, more preferably 25 to 45nt.
In some embodiments of the invention, the polynucleotide sequence of the oligonucleotide fragment comprises a sequence as set forth in one of SEQ ID NOS.1-4.
In some embodiments of the invention, the oligonucleotide fragment is a DNA fragment and/or an RNA fragment.
In some embodiments of the invention, the oligonucleotide fragment is single-stranded and/or double-stranded.
In some embodiments of the invention, the polyethyleneimine has a weight average molecular weight of 20K to 30kDa and a number average molecular weight of 8K to 12K.
In some embodiments of the present invention, the PEI/ON complexes are loaded with less than or equal to 400nM per 1ug of PEI, preferably 100-300 nM per 1ug of PEI, more preferably 150-250 nM per 1ug of PEI.
In some embodiments of the invention, the polyethyleneimine is supported on the oligonucleotide fragment by electrostatic binding.
In another aspect, the present invention provides a method for preparing the PEI/ON complex, comprising: polyethyleneimine is loaded onto the oligonucleotide fragment to provide the PEI/ON complex.
In some embodiments of the invention, the method of loading polyethyleneimine on an oligonucleotide fragment specifically comprises: the oligonucleotide fragment and polyethyleneimine are co-incubated in the presence of a solvent.
In another aspect the present invention provides the use of a PEI/ON complex as described above for the preparation of a medicament or kit for:
a) Hepatitis b surface antigen inhibitors; and/or the number of the groups of groups,
b) Anti-hepatitis b virus; and/or the number of the groups of groups,
c) Treating diseases associated with hepatitis B virus or with abnormal expression of hepatitis B surface antigen.
In some embodiments of the invention, the disease is selected from hepatitis b.
Drawings
FIG. 1 is a schematic representation showing the results of the preparation and identification of PEI/ON complexes in example 1 of the present invention.
FIG. 2 shows a schematic representation of the detection of transfected HepAD38 cells with PEI/ON complexes according to example 2 of the present invention.
FIG. 3 shows a schematic representation of the detection of cytotoxicity of HepAD38 by PEI/ON complexes in example 3 of the present invention.
FIG. 4 is a graph showing the results of HBsAg reduction by PEI/ON complexes at various concentrations in example 4 of the present invention.
FIG. 5 is a schematic representation showing the results of the reduction of HBsAg by complexes of oligonucleotides of different lengths with PEI in example 5 of the present invention.
FIG. 6 is a schematic representation showing the results of HBsAg reduction by complexes of randomly double-stranded oligonucleotides of different lengths with PEI in example 6 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention more apparent, the present invention will be further described in detail with reference to the following examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the disclosure of the present specification.
In a first aspect the invention provides a PEI/ON complex comprising an oligonucleotide fragment having a polyethyleneimine supported thereon. The PEI/ON complex provided by the invention is formed by loading polyethyleneimine ON an oligonucleotide fragment, and the formed complex can be used for effectively transfecting cells, so that the expression level of HBsAg in cell supernatant can be reduced or eliminated, and the PEI/ON complex can be used for reducing the expression level of hepatitis B surface antigen (HBsAg) or resisting Hepatitis B Virus (HBV). Specifically, the nonspecific action of PEI as a surfactant and a lipid-altering agent in the PEI/ON complex may affect the lipid metabolism involved in SVP, thereby possibly interfering with the assembly of SVP in hepatocytes, blocking the synthetic and secretory pathways, and thus exhibiting a down-regulation of the secreted HBsAg antigen detection level.
In the PEI/ON complexes provided by the present invention, the length of the oligonucleotide fragment is generally not too long or too short, which may result in a decrease in the activity of the PEI/ON complex. For example, the length of the oligonucleotide fragment may be 10 to 50nt, 10 to 15nt, 15 to 20nt, 20 to 25nt, 25 to 30nt, 30 to 35nt, 35 to 40nt, 40 to 45nt, or 45 to 50nt, preferably 15 to 45nt, and more preferably 25 to 45nt. The change in PEI/ON complex activity may be related to the amount of charge carried by the nucleic acid sequence, the longer the oligonucleotide fragment, the more negative charge the oligonucleotide fragment carries. For example, oligonucleotide fragments below 10nt may carry too little negative charge to form a stable complex with PEI to enter the cell and function accordingly. While too long oligonucleotide fragments, while having a sufficient amount of negative charge, presumably too long oligonucleotides may form complexes (nanoparticles) with PEI that are too large in size, rather degrading transfection efficiency, resulting in too small an amount of complexes into the cell that is insufficient to produce the corresponding effect, or the physicochemical properties of such complexes that are formed are such that they are not able to perform the corresponding effect. The sequence of the oligonucleotide fragment may be any of various random sequences, and may be a DNA fragment, an RNA fragment, a single-stranded oligonucleotide fragment, or a double-stranded oligonucleotide fragment. In a specific embodiment of the invention, the oligonucleotide fragment may be single stranded and the polynucleotide sequence may comprise a sequence as shown in one of SEQ ID NO. 1-5. In another embodiment of the invention, the oligonucleotide fragment may be double stranded, wherein the polynucleotide sequence of one strand may comprise a sequence as shown in one of SEQ ID NO. 6-15.
In the PEI/ON complexes provided by the present invention, polyethyleneimine is typically required to have a suitable molecular weight, as PEI of too low a molecular weight may not possess efficient transfection properties, and PEI of relatively high molecular weight may be more suitable for transfection. For example, the weight average molecular weight of the loaded polyethyleneimine may be 20K-30 kDa, 20K-22 kDa, 22K-24 kDa, 24K-26 kDa, 26K-28 kDa, or 28K-30 kDa. For another example, the number average molecular weight of the loaded polyethyleneimine may be 8K to 12K, 8K to 9K, 9K to 10K, 10K to 11K, or 11K to 12K.
In the PEI/ON complex provided by the invention, a proper proportion is usually required between PEI and ON, and the proportion between PEI and ON is usually not too high or too low, otherwise the activity of the PEI/ON complex can be reduced. For example, in the PEI/ON complex, every 1ug of PEI can be loaded at 400nM or less, 50-100 nM, 100-150 nM, 150-200 nM, 200-250 nM, 250-300 nM, 300-350 nM, or 350-400 nM ON, preferably every 1ug of PEI is loaded at 100-300 nM ON, more preferably every 1ug of PEI is loaded at 150-250 nM ON.
In the PEI/ON complexes provided by the present invention, PEI can be bound to negatively charged oligonucleotide fragments, typically electrostatically, to form amphiphilic binary complexes having a hydrophobic core (partially neutralized DNA) and a hydrophilic shell (cationic polymer chains).
The second aspect of the present invention provides a method for preparing the PEI/ON complex according to the first aspect of the present invention, comprising: polyethyleneimine is loaded onto the oligonucleotide fragment to provide the PEI/ON complex. The person skilled in the art may select a suitable method for loading polyethyleneimine on an oligonucleotide fragment, for example, may comprise: the oligonucleotide fragment and polyethyleneimine are co-incubated in the presence of a solvent.
In a third aspect, the present invention provides the use of a PEI/ON complex provided in the first aspect of the invention for the preparation of a medicament or kit which may be used for: a) Hepatitis b surface antigen (HBsAg) inhibitors. The hepatitis B surface antigen inhibitor generally refers to a substance that can inhibit the expression and/or function of hepatitis B surface antigen. For example, the hepatitis B surface antigen inhibitor may be partially inhibited, i.e., reduce the expression and/or function of hepatitis B surface antigen, or may be fully inhibited, i.e., completely eliminate the expression and/or function of hepatitis B surface antigen. The PEI/ON complex provided by the invention can be used for effectively transfecting cells, has a certain inhibition effect ON the secretion of HBsAg, can reduce or eliminate the expression level of HBsAg in cell supernatant, and has obviously enhanced inhibition effect along with the increase of the concentration of oligonucleotides, and has no obvious toxic effect ON cells in a certain working concentration range, so that the PEI/ON complex can be used as a hepatitis B surface antigen inhibitor and is used as a drug or a kit for reducing the expression level of hepatitis B surface antigen (HBsAg) in cells.
The medicament or kit may also be used for: b) anti-Hepatitis B Virus (HBV). As described above, the PEI/ON complex provided by the invention has a certain inhibition effect ON the secretion of HBsAg, can reduce or eliminate the expression level of HBsAg in cell supernatant, and the reduction of the expression level of HBsAg generally means down-regulation of the surface antigen amount in peripheral blood circulation, is expected to improve the state of body immunity, and can be used for resisting hepatitis B virus.
The medicament or kit may also be used for: c) Treating diseases associated with hepatitis B virus or with abnormal expression of HBsAg. As described above, the PEI/ON complex provided by the invention has a certain inhibition effect ON the secretion of HBsAg, can reduce or eliminate the expression level of HBsAg in cell supernatant, and effectively reduces the activity of hepatitis B virus, so that the PEI/ON complex can be used as a medicament or a kit for treating diseases related to hepatitis B virus or related to abnormal expression of HBsAg, such as hepatitis B and the like.
In the medicine or the kit provided by the invention, the PEI/ON complex can be used as a single active ingredient or can be combined with other active ingredients to jointly form the active ingredient for the application.
The drug nucleoside (nucleotide) analogues disclosed in the prior art can effectively reduce HBV DNA, but cannot cure hepatitis B functionally (defined as continuous disappearance of HBsAg), and the PEI/ON complex provided by the invention can effectively reduce the expression level of HBsAg in cell supernatant in an in vitro cell experiment, so that the drug nucleoside (nucleotide) analogues can be used for recovering virus-specific immune response of patients, provide a new potential approach for development of anti-HBV treatment, and have good industrialization prospect.
The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art literature and see, in particular, sambrook et al MOLECULAR CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989and Third edition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman and a.p. wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (p.b. becker, ed.) Humana Press, totowa,1999, etc.
Example 1
Preparation and identification of PEI/ON Complex:
different volumes of 100uM of 40nt oligonucleotide aqueous solution (0.5ul,1.0ul,1.5ul,2.0ul,SEQ ID NO.1, single stranded fragment) were mixed with 2. Mu.l of PEI aqueous solution (concentration 1ug/ul, average Mw-25000) in 50. Mu.l of opti-MEM, incubated for 15min (room temperature) and then 450ul of opti-MEM were added to give final concentrations of oligonucleotides (100 nM,200nM,300nM,400 nM) for use in transfecting cells.
20ul of the prepared PEI/ON complex was pipetted onto a 0.8% agarose gel and the result of the electrophoresis is shown in FIG. 1, wherein LANE1-4 are the products of incubation of oligonucleotides with PEI at different concentrations, LANE5 is a maker, and LANE6-9 is opti-MEM containing only oligonucleotides at different concentrations. As can be seen from FIG. 1, the electrophoretic migration of the oligonucleotides in the PEI/ON complex is blocked as the concentration of the oligonucleotides in the complex increases.
ACACACACACACACACACACACACACACACACACACACAC(SEQ ID NO.1)
Example 2
Detection of PEI/ON Complex transfected HepAD38 cells:
HepAD38 cell strain is cultured under normal condition, when the cell fusion degree reaches 90%, the culture solution is firstly poured out, the culture solution is washed for 1 time by using sterile PBS buffer solution, 0.25% pancreatin and 0.2% EDTA solution are added, the cells are fully digested for 3 minutes at 37 ℃ and digested for 3 minutes, the culture solution is added to gently blow into single cell suspension, and then the single cell suspension is passaged at a ratio of 1:4, and the culture is continued. For cell culture, all media contained 10% FBS (fetal bovine serum), 100IU/mL penicillin and 100mg/mL streptomycin, and were placed in an incubator at 37℃and 5% CO2, and cultured under saturated humidity. HepAD38 cells are a liver cell cancer line expressed by HBV genes, cultured in DMEM/F12 medium supplemented with 10% FBS and G418 (GIBCO) at a final concentration of 100 mg/L. After the cells grow fully, digesting again, counting by a cell counting plate, adding culture solution to dilute into a cell suspension with a certain concentration, inoculating into a 12-hole cell culture plate, placing at 37 ℃ and 5% CO 2 Culturing for 24h, and performing experiments after cells are grown into a monolayer by adherence.
The prepared oligonucleotide was labeled with a fluorescent group of FTTC (Fluorescein Isothiocyanate ), 1ul of the FTTC-labeled oligonucleotide was pipetted (100. Mu.M concentration) and incubated with 2ug of PEI (ug/ul) at room temperature for 15min, and then added to serum-free cell culture medium (100 nM final concentration of oligonucleotide ON in PEI/ON complex, slightly different preparation method, 40nt of ON (AC repeat) with FITC fluorescent tag, according to example 1, were usedTo observe whether the ON complexes enter the cell), placed at 37℃with 5% CO 2 Incubator, transfection for 5h. Then, the serum-free transfection solution was aspirated, and the cell culture solution containing 10% FBS was added thereto, and the mixture was left at 37℃with 5% CO 2 Incubator 48h. After medium exchange, green fluorescent signal was observed under fluorescent microscope, positive cells emitted bright green fluorescence, indicating successful transfection of PEI/ON complex into cells, and the results are shown in FIG. 2.
Example 3
Detection of cytotoxicity of the PEI/ON Complex against HepAD 38:
the inhibition of HepAD38 cell growth by the PEI/ON complex was tested by reference to the CCK-8 assay kit. After digestion of the logarithmic phase of HepAD38, the cells were counted by a cell counter and diluted to a concentration of 1X10 in culture 4 Cell suspensions of individual cells/mL were seeded into 96-well cell culture plates at 100. Mu.l per well, 37℃and 5% CO 2 Culturing for 24h. After cell attachment growth to a monolayer, concentration gradient sets (PEI/ON complex preparation method referred to in example 1) were set up with final concentrations of oligonucleotides 100nM,200nM,300nM,400nM in the wells of the cell culture solution, 3 multiplex wells were set up for each concentration, and a blank control set (culture solution without test complex) was set up. After 5h transfection with PEI/ON complex, 10. Mu.LCCK-8 solution was added to each well (note that no bubbles were generated in the wells, which would affect the reading of OD values). The plates were incubated in the incubator for 1 hour. The absorbance at 450nm was measured with a microplate reader and the OD450 values of the different groups were compared to reflect the number of living cells, and the results are shown in fig. 3. As can be seen from FIG. 3, the PEI/ON complex had no inhibitory effect ON HepAD38 cell growth.
Example 4
Different concentrations of PEI/ON complex reduced HBsAg:
different volumes of 100uM of 40nt oligonucleotide (40 nt of repeated AC sequence, SEQ ID NO. 1) were transfected with 2ug of PEI and the effect experiments of secretion of HBsAg and HBeAg by HepAD38 cells were performed. The logarithmic phase of HepAD38 cell suspension (5 xl0 5 cell/mL) was inoculated into a 12-well plate (lml broth/well), after culturing for 24 hours, the broth was aspirated, and 500ul of serum-free culture containing various concentrations of PEI/ON complex to be tested was addedThe nutrient solution was transfected (obtained as in example 1), and after 5 hours, the transfection solution was aspirated, replaced with 1ml of 10% fresh culture solution, and cultured for 48 hours, and the cell supernatant was collected. The experimental concentration was set with 3 duplicate wells and a blank (culture without test complex) was set. Finally, the expression level of HBsAg and HBeAg in the supernatant of HepAD38 cells was measured, and the inhibition rate of PEI/ON complex ON secretion of HBsAg and HBeAg was calculated, and the results are shown in FIG. 4, wherein P is expressed as follows<0.05 is a statistically significant difference, P is represented by:<0.01 represents P<0.001. The measurement result shows that the PEI/ON complex has a certain inhibition effect ON the secretion of HBsAg after the HepAD38 cell strain is transfected, and the inhibition effect is enhanced but not in a dose-dependent relationship with the increase of the concentration of the oligonucleotide, but has no obvious inhibition effect ON the secretion of HBeAg.
Example 5
Complexes of oligonucleotides of different lengths with PEI reduced HBsAg:
HepAD38 cells were cultured at 5X10 5 Cell density of cells/well cells were seeded in 12-well plates. PEI/ON complexes corresponding to oligonucleotides of different lengths (10 nt, 20nt, 30nt, 40nt, SEQ ID NO. 2-5, respectively) and concentrations (100 nM,200nM,300nM,400 nM) were prepared and the preparation method of PEI/ON complexes was described in example 1.
The prepared PEI/ON complex was added to HepAD38 cells, placed in an incubator at 37℃with 5% CO2, and after 5h incubation and transfection, the culture broth was aspirated, replaced with 1ml of 10% fresh culture broth, and cultured for 48h, and the cell supernatant was collected. The secretion levels of HBsAg and HBeAg were measured, and the complex experimental group was provided with 3 biological wells, and quantitative measurements were performed on HBsAg and hepatitis b virus e antigen (HBeAg) using the Abbott archetect immunoassay system (Abbott Laboratories), as shown in fig. 5, where P <0.05 is a significant statistical difference and P <0.01. As can be seen from FIG. 5, the results showed that the PEI/ON (10 nt) complex was not effective in reducing the levels of HBsAg and HBeAg after transfection of HepAD38 cells, except for the 20nt, 30nt and 40nt length oligonucleotides, and that the relatively long oligonucleotides inhibited better. Whereas for a 20nt oligonucleotide a higher ON ratio relative to PEI is more pronounced for HBsAg inhibition, for both 30nt and 40nt length oligonucleotides an ON ratio that is too high or too low relative to PEI is less pronounced for HBsAg inhibition.
ACACACACAC(SEQ ID NO.2)
ACACACACACACACACACAC(SEQ ID NO.3)
ACACACACACACACACACACACACACACAC(SEQ ID NO.4)
ACACACACACACACACACACACACACACACACACACACAC(SEQ ID NO.5)
Example 6
The complex formed by double-stranded oligonucleotides of random sequence with PEI reduces HBsAg:
1ul volume of 100uM of random double-stranded oligonucleotides (double stranded oligonucleotide, dsON, five samples total, polynucleotide sequences for each sample detailed in Table 1) was mixed with 2ul PEI (1 ug/ul) in 50ul opti-MEM and incubated for 15min (room temperature), and then 450ul opti-MEM was added to give a final oligonucleotide concentration of 200nM for cell transfection.
HepAD38 cells were cultured at 5X10 5 Cell density of cells/well was inoculated into 12-well plate, after culturing for 24 hours, the culture broth was aspirated, and transfection was performed by adding 500ul of serum-free culture broth containing PEI/dsON complex to the opti-MEM, after standing in the incubator for 5 hours, the transfection broth was aspirated, and 1ml of 10% fresh culture broth was changed to culture for 48 hours, and the cell supernatant was collected. The experimental concentration was set with 3 duplicate wells and a blank (culture without test complex) was set. HBsAg and HBeAg were measured, counted and plotted, and the results are shown in FIG. 6, wherein P is represented by<0.05 is a statistically significant difference, P is represented by:<0.01 represents P<0.001. As can be seen from FIG. 6, the oligonucleotide fragment may be of various random sequences, and when the oligonucleotide fragment is a double-stranded fragment, it also has a very significant inhibitory effect on secretion of HBsAg, but no significant inhibitory effect on secretion of HBeAg.
TABLE 1
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
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Claims (7)
1. A PEI/ON complex comprising an oligonucleotide fragment, wherein the oligonucleotide fragment is loaded with polyethyleneimine, and the polynucleotide sequence of the oligonucleotide fragment is shown as SEQ ID NO. 3; the preparation method of the PEI/ON compound comprises the following steps: mu.l of 100. Mu.M 20nt oligonucleotide in water was mixed with 2. Mu.l of PEI in water at a concentration of 1ug/ul and average Mw-25000 in 50. Mu.l of opti-MEM, incubated at room temperature for 15 minutes, and then 450ul of opti-MEM was added thereto at a final concentration of 400nM.
2. A PEI/ON complex comprising an oligonucleotide fragment, wherein the oligonucleotide fragment is loaded with polyethyleneimine, and the polynucleotide sequence of the oligonucleotide fragment is shown as SEQ ID NO. 4; the preparation method of the PEI/ON compound comprises the following steps: mu.l of 100. Mu.M 30nt oligonucleotide in water was mixed with 2. Mu.l of PEI in water at a concentration of 1ug/ul and average Mw-25000 in 50. Mu.l of opti-MEM, incubated at room temperature for 15 minutes, and then 450ul of opti-MEM was added thereto at a final concentration of 200nM.
3. The PEI/ON complex according to claim 1 or 2, wherein said polyethylenimine is supported by electrostatic binding to an oligonucleotide fragment.
4. The method for preparing a PEI/ON complex according to claim 1 or 2, comprising: polyethyleneimine is loaded onto the oligonucleotide fragment to provide the PEI/ON complex.
5. The method according to claim 4, wherein the method for loading polyethyleneimine on an oligonucleotide fragment comprises: the oligonucleotide fragment and polyethyleneimine are co-incubated in the presence of a solvent.
6. Use of the PEI/ON complex according to claim 1 or 2 in the manufacture of a medicament or a kit for:
a) Hepatitis b surface antigen inhibitors; and/or the number of the groups of groups,
b) Anti-hepatitis b virus; and/or the number of the groups of groups,
c) Treating diseases associated with hepatitis B virus or with abnormal expression of hepatitis B surface antigen.
7. The use according to claim 6, wherein the disease is selected from hepatitis b.
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Citations (4)
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| US6013240A (en) * | 1994-07-13 | 2000-01-11 | Rhone-Poulenc Rorer Sa | Nucleic acid containing composition, preparation and uses of same |
| WO2006119619A1 (en) * | 2005-05-06 | 2006-11-16 | Replicor Inc. | Oligonucleotides inhibiting cell proliferation |
| CN101084232A (en) * | 2004-10-19 | 2007-12-05 | 里普利科股份有限公司 | Antiviral oligonucleotides |
| WO2008058457A1 (en) * | 2006-11-16 | 2008-05-22 | Wei Dong | A biodegradable crosslinked polyethyleneimine and its uses |
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| DE19743135A1 (en) * | 1997-09-30 | 1999-04-01 | Hoechst Marion Roussel De Gmbh | Biologically compatible low molecular weight polyethyleneimines |
| AU2002348163A1 (en) * | 2001-11-02 | 2003-05-19 | Intradigm Corporation | Therapeutic methods for nucleic acid delivery vehicles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6013240A (en) * | 1994-07-13 | 2000-01-11 | Rhone-Poulenc Rorer Sa | Nucleic acid containing composition, preparation and uses of same |
| CN101084232A (en) * | 2004-10-19 | 2007-12-05 | 里普利科股份有限公司 | Antiviral oligonucleotides |
| WO2006119619A1 (en) * | 2005-05-06 | 2006-11-16 | Replicor Inc. | Oligonucleotides inhibiting cell proliferation |
| WO2008058457A1 (en) * | 2006-11-16 | 2008-05-22 | Wei Dong | A biodegradable crosslinked polyethyleneimine and its uses |
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Effective date of registration: 20240416 Address after: 250012 No. 107, Wenhua West Road, Shandong, Ji'nan Patentee after: QILU HOSPITAL OF SHANDONG University Country or region after: China Address before: No. 220, Handan Road, Yangpu District, Shanghai 200025 Patentee before: Lin Junyu Country or region before: China |