CN112237633A

CN112237633A - PEI/ON compound and preparation method and application thereof

Info

Publication number: CN112237633A
Application number: CN202011138178.4A
Authority: CN
Inventors: 林君玉; 张欣欣
Original assignee: Individual
Current assignee: Qilu Hospital of Shandong University
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2021-01-19
Anticipated expiration: 2040-10-22
Also published as: CN112237633B

Abstract

The invention relates to the field of biomedicine, in particular to a PEI/ON complex and 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 a patient, provides a new potential path for development of anti-HBV treatment, and has good industrialization prospect.

Description

PEI/ON compound and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a PEI/ON complex and a preparation method and application thereof.

Background

Hepatitis B is a transfected disease caused by infection with Hepatitis B Virus (HBV) that seriously harms human life health. It is estimated that more than 2.9 million people worldwide have chronic HBV infection and are at risk of dying from HBV-associated cirrhosis and complications of hepatocellular carcinoma. In HBV carriers, HBV-infected hepatocytes produce large quantities of non-infectious (spherical or filamentous) into the peripheral circulation, typically 1000: 1 to 10,000: 1. overproduction of subviral particles (SVP) containing only envelope glycoprotein (HBsAg) and host-derived lipids can lead to immune tolerance and to chronisation of HBV infection. The current clinical antiviral strategies are mainly using nucleoside drugs and interferon-based therapies, but few are able to completely eliminate HBsAg from the blood to achieve a functional cure for HBV, i.e. HBsAg negativity. This highlights how to reduce HBsAg levels is an important issue in developing therapeutic strategies for chronic hepatitis b virus infection.

Oligonucleotide-based therapies are currently becoming a potential strategy 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 RNA (siRNA) conjugated to RNA, antigene oligonucleotides conjugated to DNA and aptamers conjugated to proteins, decoy oligonucleotides and CpG oligonucleotides.

On the other hand, successful delivery of foreign 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, it is an object of the present invention to provide a PEI/ON complex, a method for preparing the same and a use thereof, which solve the problems of the prior art.

To achieve the above and other related objects, according to one aspect of the present invention, there is provided a PEI/ON complex comprising an oligonucleotide fragment having polyethyleneimine loaded thereon.

In some embodiments of the invention, the oligonucleotide fragment has a length of 10 to 50nt, preferably 15 to 45nt, and more preferably 25 to 45 nt.

In some embodiments of the invention, the polynucleotide sequence of the oligonucleotide fragment comprises a sequence as set forth in one of SEQ ID No. 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 complex is loaded with ≦ 400nM ON per 1ug PEI, preferably 100-300 nM ON per 1ug PEI, and more preferably 150-250 nM ON per 1ug PEI.

In some embodiments of the invention, the polyethyleneimine is supported on the oligonucleotide fragments by electrostatic binding.

In another aspect, the present invention provides a method for preparing the above PEI/ON complex, comprising: supporting polyethyleneimine ON the oligonucleotide fragments to provide said PEI/ON complex.

In some embodiments of the present invention, the method for loading polyethyleneimine onto oligonucleotide fragments specifically comprises: the oligonucleotide fragments 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 defined above for the manufacture of a medicament or kit for:

a) inhibitors of hepatitis b surface antigen; and/or the presence of a gas in the gas,

b) anti-hepatitis b virus; and/or the presence of a gas in the gas,

c) treating diseases related to hepatitis B virus or 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 diagram showing the preparation and identification results of PEI/ON complexes in example 1 of the present invention.

FIG. 2 is a schematic diagram showing the detection of the PEI/ON complex transfected HepAD38 cells in example 2 of the present invention.

FIG. 3 is a schematic diagram showing the cytotoxicity of PEI/ON complexes ON HepAD38 in example 3 of the present invention.

FIG. 4 is a graph showing the results of reducing HBsAg by different concentrations of PEI/ON complexes in example 4 of the present invention.

FIG. 5 is a graph showing the result of reducing HBsAg by forming complexes of oligonucleotides of different lengths with PEI in example 5 of the present invention.

FIG. 6 is a graph showing the result of reducing HBsAg by complexes formed between random double-stranded oligonucleotides of different lengths and PEI in example 6 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

In a first aspect, the present invention provides a PEI/ON complex comprising an oligonucleotide fragment having polyethyleneimine loaded thereon. The PEI/ON complex provided by the invention is formed by loading polyethyleneimine ON an oligonucleotide fragment, and the formed complex can effectively transfect 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 effect of PEI in the PEI/ON complex as a surfactant and a lipid-altering agent may affect the lipid metabolism involved in SVP, thereby possibly interfering with the assembly of SVP in liver cells, blocking the synthesis and secretion pathway of SVP, and further showing that the detection level of secreted HBsAg antigen is reduced.

The length of the oligonucleotide fragment in the PEI/ON complex provided by the present invention is generally not too long nor 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 45 nt. The change in the activity of the PEI/ON complex may be related to the amount of charge carried by the nucleic acid sequence, with the longer the oligonucleotide fragment, the greater the amount of negative charge carried by the oligonucleotide fragment. For example, the amount of negative charge carried by oligonucleotide fragments of less than 10nt may be too small to form a stable complex with PEI and enter the cell and play a corresponding role. While oligonucleotide fragments that are too long, while having a sufficient amount of negative charge, it is speculated that the size of the complex (nanoparticle) formed by the oligonucleotide and PEI, which is too long, may be too large, and instead degrade transfection efficiency, resulting in too little complex entering the cell to be effective for the corresponding purpose, or that the physicochemical properties of the complex formed are such that it is not effective for the corresponding purpose. The sequence of the oligonucleotide fragment may be various random sequences, and it may be a DNA fragment, an RNA fragment, a single-stranded oligonucleotide fragment, or a double-stranded oligonucleotide fragment. In one embodiment of the invention, the oligonucleotide fragment may be single-stranded and the polynucleotide sequence may comprise a sequence as shown in any one of SEQ ID No.1 to 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 set forth in any one of SEQ ID Nos. 6 to 15.

In the PEI/ON complexes provided herein, polyethyleneimine generally needs to have a suitable molecular weight because PEI with too low a molecular weight may not have effective transfection properties and PEI with a relatively high molecular weight may be more suitable for transfection. For example, the weight average molecular weight of the supported polyethyleneimine may be 20K to 30KDa, 20K to 22KDa, 22K to 24KDa, 24K to 26KDa, 26K to 28KDa, or 28K to 30 KDa. For another example, the supported polyethyleneimine may have a number average molecular weight of 8K to 12K, 8K to 9K, 9K to 10K, 10K to 11K, or 11K to 12K.

In the PEI/ON composites provided herein, a suitable ratio between PEI and ON is generally desired, and the ratio between PEI and ON is generally not too high nor too low, which may result in a reduction in the activity of the PEI/ON composite. For example, in the PEI/ON complex, every 1ug of PEI can be loaded with ON 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, preferably every 1ug of PEI is loaded with ON at 100-300 nM, and more preferably every 1ug of PEI is loaded with ON at 150-250 nM.

In the PEI/ON complexes provided herein, PEI can be bound to negatively charged oligonucleotide fragments, typically by electrostatic binding, to form amphiphilic binary complexes having a hydrophobic core (partially neutralized DNA) and a hydrophilic shell (cationic polymer chain).

In a second aspect, the present invention provides a method for preparing a PEI/ON complex provided in the first aspect of the present invention, comprising: supporting polyethyleneimine ON the oligonucleotide fragments to provide said PEI/ON complex. One skilled in the art can select suitable methods for loading polyethyleneimine onto oligonucleotide fragments, and may include, for example: the oligonucleotide fragments 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 as provided in the first aspect of the invention in the manufacture of a medicament or kit for use in: 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 partially inhibit, i.e., reduce the expression and/or function of hepatitis B surface antigen, or may completely inhibit, i.e., completely eliminate the expression and/or function of hepatitis B surface antigen. The PEI/ON complex provided by the invention can effectively transfect cells, has a certain inhibition effect ON the secretion of the HBsAg, can reduce or eliminate the expression level of the HBsAg in cell supernatant, and the inhibition effect is obviously enhanced along with the increase of the concentration of the oligonucleotide, while in a certain working concentration range, the PEI/ON complex has no obvious toxic effect ON the cells, so that the PEI/ON complex can be used as a hepatitis B surface antigen inhibitor and used for a medicine or a kit for reducing the expression level of the hepatitis B surface antigen (HBsAg) in the cells.

The medicament or kit may also be for: b) against Hepatitis B Virus (HBV). As described above, the PEI/ON complex provided by the present invention has a certain inhibitory effect ON the secretion of HBsAg, and can reduce or eliminate the expression level of HBsAg in cell supernatant, and the reduction of the expression level of HBsAg usually means that the amount of surface antigen in peripheral blood circulation is down-regulated, and is expected to improve the immune state of the body, thereby being used for resisting hepatitis B virus.

The medicament or kit may also be for: c) treating diseases related to hepatitis B virus or HBsAg abnormal expression. As described above, the PEI/ON complex provided by the present invention has a certain inhibitory effect ON the secretion of HBsAg, and can reduce or eliminate the expression level of HBsAg in cell supernatant, effectively reduce the activity of hepatitis b virus, and thus can be used as a drug or a kit for treating a disease associated with hepatitis b virus or with abnormal expression of HBsAg, such as hepatitis b.

In the medicament or the kit provided by the invention, the PEI/ON complex can be used as a single effective component, and can also be combined with other active components to jointly form the effective component for the application.

The drug nucleotide (acid) analogue disclosed in the prior art can effectively reduce HBV DNA, but can not functionally cure hepatitis B (defined as continuous HBsAg disappearance), and the PEI/ON complex provided by the invention can effectively reduce the expression level of the 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 a patient, provides a new potential path for the development of anti-HBV treatment, and has 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 herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: 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; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1

Preparation and identification of PEI/ON complexes:

different volumes of 100uM 40nt oligonucleotide aqueous solution (0.5ul, 1.0ul, 1.5ul, 2.0ul, SEQ ID NO.1, single-stranded fragment) and 2. mu.l PEI aqueous solution (concentration 1ug/ul, average Mw. about.25000) were mixed in 50. mu.l opti-MEM, incubated for 15 minutes (room temperature), and then 450ul opti-MEM was added, with the final concentration of oligonucleotides (100nM, 200nM, 300nM, 400nM), and used to transfect cells.

20ul of the prepared PEI/ON complex was pipetted and electrophoresed ON 0.8% agarose gel, and the electrophoresis results are shown in FIG. 1, wherein LANE1-4 is the product of co-incubation of oligonucleotides with different concentrations and PEI, LANE5 is marker, and LANE6-9 is opti-MEM containing only oligonucleotides with 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:

culturing HepAD38 cell strain under normal condition, pouring out culture solution when cell fusion degree reaches 90%, washing with sterile PBS buffer solution for 1 time, adding 0.25% pancreatin and 0.2% EDTA solution to digest cell for 3min, digesting at 37 deg.C for 3min, adding culture solution, blowing to obtain single cell suspension, then subculturing at a ratio of 1:4, and continuing culturing. When the cells were cultured, all the media contained 10% FBS (fetal bovine serum), 100IU/mL penicillinAnd streptomycin 100mg/mL, incubated at 37 ℃ in an incubator with 5% CO2 under saturated humidity conditions. HepAD38 cell is a hepatocellular carcinoma line with HBV gene expression, cultured in DMEM/F12 medium supplemented with 10% FBS and G418(GIBCO) at a final concentration of 100 mg/L. Digesting again after the cells are full, counting by using a cell counting plate, adding a 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% CO₂The culture is carried out for 24h, and the experiment is carried out after the cells grow into a monolayer in an adherent way.

The prepared oligonucleotides were labeled with fluorescent group of FTTC (Fluorescein Isothiocyanate), 1ul volume (concentration of 100uM) of the FTTC labeled oligonucleotides and 2ug of PEI (ug/ul) were incubated at room temperature for 15min, and then added to serum-free cell culture medium (final concentration of oligonucleotide ON in PEI/ON complex is 100nM, and PEI/ON complex was prepared according to example 1, except that 40nt of ON (AC repeat) was labeled with FITC fluorescent label for observing whether ON complex entered into cells), and placed at 37 ℃ and 5% CO for 5%₂Incubator, transfection for 5 h. Then, the serum-free transfection solution was aspirated and added with a cell culture solution containing 10% FBS, and the mixture was placed at 37 ℃ under 5% CO₂And the incubator is 48 h. The culture medium was changed and the green fluorescence signal was observed under a fluorescence microscope, and the positive cells emitted bright green fluorescence, indicating that PEI/ON complexes were successfully transfected into the cells, the results are shown in FIG. 2.

Example 3

Detection of cytotoxicity of PEI/ON Complex to HepAD 38:

the inhibition effect of the PEI/ON complex ON the growth of the HepAD38 cell is detected by referring to the method of a CCK-8 detection kit. After digestion of logarithmic phase HepAD38, counting was performed with a cell counting plate, and diluted to 1X10 with culture medium⁴Cell suspension of individual cells/mL, seeded in 96-well cell culture plates at 100. mu.l/well, 37 ℃, 5% CO₂And culturing for 24 h. After the cells grew adherently into monolayers, a concentration gradient set was set up to have final concentrations of 100nM, 200nM, 300nM and 400nM of oligonucleotides in the wells of the cell culture medium (the PEI/ON complex preparation method is described in example 1), 3 duplicate wells were set up for each concentration, anda blank control (culture broth without test complex) was set. After 5h transfection of the PEI/ON complex, 10. mu.L CCK-8 solution was added to each well (care was taken not to generate bubbles in the wells which would affect the OD reading). The plates were incubated in an 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 the growth of HepAD38 cells.

Example 4

Different concentrations of PEI/ON complexes reduced HBsAg:

different volumes of 100uM 40nt oligonucleotide (40nt repetitive AC sequence, SEQ ID NO.1) were transfected with 2ug of PEI to perform an experiment on the effect of HBsAg, HBeAg secretion from HepAD38 cells. HepAD38 cell suspension in log phase (5xl 0)⁵cell/mL) was inoculated into 12-well plates (lml medium/well), after 24 hours of culture, the medium was aspirated, 500ul of serum-free medium containing various concentrations of PEI/ON complex to be tested was added for transfection (prepared in example 1), after 5 hours, the transfection solution was aspirated and replaced with 1mL of 10% fresh medium, culture was carried out for 48 hours, and cell supernatants were collected. Experimental concentrations were set at 3 replicate wells and a blank control (culture without test complex) was set. Finally, the expression amount of HBsAg and HBeAg in the supernatant of HepAD38 cells was measured, and the inhibition rate of PEI/ON complex ON HBsAg and HBeAg secretion was calculated, and the result is shown in FIG. 4, wherein P represents P<0.05 is marked with statistical difference, representing P<0.01, represents P<0.001. The measurement result shows that the PEI/ON complex has certain inhibition effect ON the secretion of the HBsAg after the transfection of the HepAD38 cell strain, and the inhibition effect is enhanced but is not in a dose-dependent relationship with the increase of the concentration of the oligonucleotide, but the PEI/ON complex does not have obvious inhibition effect ON the secretion of the HBeAg.

Example 5

Complexes of oligonucleotides of different lengths with PEI reduced HBsAg:

HepAD38 cells were treated at 5x10⁵Cell density of cells/well was seeded in 12-well plates. Preparing different lengths (10nt, 20nt, 30nt and 40nt, sequences are SEQ ID NO. 2-5) and different concentrations (concentration is 10)0nM, 200nM, 300nM, 400nM) of oligonucleotides, the PEI/ON complexes were prepared according to example 1.

Adding the prepared PEI/ON compound into HepAD38 cells, placing the cells in an incubator at 37 ℃ and 5% CO2, incubating and transfecting for 5h, removing the culture solution by suction, replacing 1ml of 10% fresh culture solution, culturing for 48h, and collecting cell supernatant. The secretion levels of HBsAg and HBeAg were measured, 3 wells were set in the complex experimental group, and the quantitative measurement of HBsAg and hepatitis B virus e antigen (HBeAg) was performed using Abbott Architect immunoassay system (Abbott Laboratories), and the results are shown in FIG. 5, wherein P <0.05 is a significant statistical difference and P < 0.01. As can be seen from FIG. 5, the results all show that, except for oligonucleotides of 20nt, 30nt and 40nt lengths, the PEI/ON (10nt) complex transfected HepAD38 cells did not effectively reduce the HBsAg and HBeAg levels, and the relatively longer oligonucleotide had better inhibitory effect. Whereas for the 20nt oligonucleotide, the higher ON ratio relative to PEI is more pronounced for the inhibitory effect ON HBsAg, whereas for the 30nt and 40nt length oligonucleotides, too high or too low an ON ratio relative to PEI weakens the inhibitory effect ON HBsAg.

ACACACACAC(SEQ ID NO.2)

ACACACACACACACACACAC(SEQ ID NO.3)

ACACACACACACACACACACACACACACAC(SEQ ID NO.4)

ACACACACACACACACACACACACACACACACACACACAC(SEQ ID NO.5)

Example 6

Complexes of double-stranded oligonucleotides of random sequence with PEI reduced HBsAg:

a total of five samples (dsON) of 100uM aqueous random double-stranded oligonucleotides (double stranded oligonucleotides) in a volume of 1ul were mixed with 2ul PEI (1ug/ul) in 50ul opti-MEM, incubated for 15 minutes (RT), and then 450ul opti-MEM was added to the mixture to a final concentration of 200nM oligonucleotides, and used to transfect cells.

HepAD38 cells were treated at 5x10⁵Cell density of cells/well seeded in 12 wellsAfter 24 hours of culture in the plate, the culture medium was aspirated, 500ul of opti-MEM serum-free culture medium containing PEI/dsON complex was added for transfection, the plate was left to stand in the incubator for 5 hours, the transfection solution was aspirated off, 1ml of 10% fresh culture medium was added, culture was carried out for 48 hours, and the cell supernatant was collected. Experimental concentrations were set at 3 replicate wells and a blank control (culture without test complex) was set. HBsAg and HBeAg were measured, and the results were counted and plotted as shown in FIG. 6, wherein P represents P<0.05 is marked with statistical difference, representing P<0.01, represents P<0.001. As can be seen from FIG. 6, the oligonucleotide fragments can be of various random sequences, and when the oligonucleotide fragments are double-stranded fragments, the oligonucleotide fragments also have very significant inhibitory effect on the secretion of HBsAg, but have no significant inhibitory effect on the secretion of HBeAg.

TABLE 1

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

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Claims

1. A PEI/ON complex comprising an oligonucleotide fragment having polyethyleneimine loaded thereon.

2. The PEI/ON complex of claim 1 wherein the oligonucleotide fragment is 10 to 50nt, preferably 15 to 45nt, more preferably 25 to 45nt in length;

and/or the polynucleotide sequence of the oligonucleotide fragment comprises a sequence shown in one of SEQ ID NO. 1-4.

3. The PEI/ON complex of claim 1 wherein the oligonucleotide fragment is a DNA fragment and/or an RNA fragment;

and/or, the oligonucleotide fragment is single-stranded and/or double-stranded.

4. The PEI/ON complex of claim 1 wherein the polyethyleneimine has a weight average molecular weight of 20K to 30kDa and a number average molecular weight of 8K to 12K.

5. The PEI/ON complex of claim 1 wherein 400nM ON per 1ug PEI, preferably 100-300 nM ON per 1ug PEI, more preferably 150-250 nM ON per 1ug PEI.

6. The PEI/ON complex of claim 1 wherein the polyethyleneimine is supported ON the oligonucleotide fragment by electrostatic binding.

7. A method of producing a PEI/ON complex according to any one of claims 1 to 6 comprising: supporting polyethyleneimine ON the oligonucleotide fragments to provide said PEI/ON complex.

8. The method of claim 7, wherein the step of supporting polyethyleneimine on the oligonucleotide fragment comprises: the oligonucleotide fragments and polyethyleneimine are co-incubated in the presence of a solvent.

9. Use of a PEI/ON complex according to any one of claims 1-6 for the preparation of a medicament or kit for:

b) anti-hepatitis b virus; and/or the presence of a gas in the gas,

10. The use according to claim 9, wherein the disease is selected from hepatitis b.