Gene / drug co-loaded liposome and its preparation method and use

Abstract

The present invention provides a gene / drug co-loaded liposome and a preparation method and use thereof. The liposome of the present invention comprises a lipid membrane, a small molecule drug and a gene; the lipid membrane is a cationic lipid membrane; the small molecule drug is a STAT3 pathway inhibitor; the gene targets the JAK / STAT3 signaling pathway. The co-loaded liposome provided by the present invention can simultaneously load genes and small molecule drugs targeting the key signaling pathway JAK / STAT3 in psoriasis, deliver the gene and the small molecule drug into the cell together, block the STAT3 signaling pathway from the gene and protein levels respectively, exert a synergistic effect, have good biocompatibility and safety, and achieve the purpose of better treating psoriasis.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, in particular to a gene / drug co-loaded liposome and a preparation method and application thereof. Background Art

[0002] Psoriasis is a chronic, recurrent, inflammatory skin disease. Its pathogenesis is still unclear. It is generally believed to be caused by genetic, environmental, immune and other factors. Psoriasis seriously affects the quality of life of patients and increases the risks of psychological diseases, cardiovascular diseases, metabolic syndrome, psoriatic arthritis and other diseases. The incidence of psoriasis in my country is about 0.5%, and there may be more than 7 million psoriasis patients.

[0003] There are currently three main forms of treatment for psoriasis: topical treatment; phototherapy; systemic treatment. The choice of treatment method is based on the severity of psoriasis. Mild psoriasis is usually treated topically, such as topical application of creams, foams or gels containing glucocorticoids or vitamin D3 analogs; if the effect is not good, phototherapy is used. Moderate to severe psoriasis requires systemic treatment, commonly oral or injectable methotrexate, cyclosporine, acitretin, etc. Methotrexate, cyclosporine, etc. lack selectivity, and when used systemically to treat psoriasis, they often cause systemic side effects. In recent years, my country has approved the marketing of some biologics for the treatment of psoriasis, including tumor necrosis factor α (TNF-α) antagonists, interleukin 12 / 23 (IL-12 / 23) antagonists, and interleukin 17A (IL-17A) antagonists. Biologics need to be administered subcutaneously, and patients have poor compliance and it is difficult to adhere to long-term treatment. Therefore, there is an urgent need to develop new, safe, effective, and easy-to-use therapies to support the unmet clinical needs of psoriasis treatment.

[0004] Among the various treatments for psoriasis, except for retinoic acid, the purpose of the remaining methods is to fight inflammation, slow down the renewal rate of epidermal keratinocytes and smooth plaques. The JAK-STAT signaling pathway plays an important role in the signal transduction of multiple cell chemical pathways such as growth, survival, and differentiation. Among them, JAK / STAT3 is one of the key signaling pathways in the occurrence and development of psoriasis. As an important transcription factor, STAT3 plays a key role in the onset of psoriasis by participating in important pathological processes such as Th17 cell differentiation, excessive proliferation and abnormal differentiation of keratinocytes, interaction with inflammatory cells, and dermal vascular hyperplasia. In recent years, clinical trial results have shown that JAK kinase inhibitors have good efficacy and safety in the treatment of psoriasis. For example, the JAK targeted inhibitor tofacitinib is currently in the phase III clinical trial stage. However, there are relatively few studies on STAT3 at present, which needs further exploration.

[0005] The combined use of gene therapy and small molecule drugs can bring out the synergistic effect of the two, that is, play a regulatory role at the gene level and protein level respectively. Gene / drug co-loaded preparations can deliver both to the same cell at the same time, which can enhance the therapeutic effect while helping to reduce the dosage of small molecule drugs, reduce the adverse reactions of chemical drugs, and improve the low efficiency of single gene transfection. In addition, co-delivery based on nanoplatforms can also reduce the number of dosing times and improve patient compliance. The combined administration of genes and chemotherapeutic drugs has received widespread attention in the treatment of multidrug resistance of tumors. Multidrug resistance therapeutic genes can be delivered to tumor cells through appropriate carriers, and the purpose of treatment can be achieved by downregulating the expression of resistance-related proteins or silencing other genes that regulate tumor function. Studies have shown that the combined use of small molecule drugs and gene therapy with the help of nanocarriers can bring out the synergistic effect of the two, which is a promising treatment strategy.

[0006] However, the co-delivery of genes and small molecules still faces many challenges: (1) Gene drugs and small molecule drugs have different physical and chemical properties and mechanisms of action, mainly manifested in the hydrophilicity and high instability of gene drugs, while small molecule drugs have different hydrophilicity and lipophilicity. (2) Gene drugs generally need to enter the cytoplasm or nucleus to exert their effects, while small molecule drugs need to exert their effects inside or outside the cell due to different targets. (3) It is necessary to control the time interval between the release of small molecule drugs and gene drugs. For example, small interfering RNA (siRNA) is often used to reverse multidrug resistance. In addition, chemotherapy drugs should be released after siRNA silences resistance-related genes to obtain the best synergistic effect, but how to accurately control the release of different substances at different time points is still a huge challenge in current research. (4) Although researchers have designed many types of vectors, low transfection efficiency and difficulty in endosome / lysosome escape still severely limit the clinical use of siRNA. (5) The requirement of co-delivering two types of drugs increases the complexity of vector design. When synthesizing complex vectors, multiple raw materials are required, and the safety of each raw material needs to be evaluated to reduce vector toxicity or off-target effects. In order to meet the above challenges, it is of great significance to find a nanodrug preparation that can achieve the co-delivery of small molecule drugs and gene drugs, with high stability, high transfection efficiency, good biocompatibility and safety. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a liposome that simultaneously carries small molecule drugs and gene drugs to solve the problems of poor stability, low biocompatibility and safety, poor delivery efficiency and inability to accurately control release of existing carriers.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a gene / drug co-loaded liposome, the liposome comprising a lipid membrane, a small molecule drug and a gene; the lipid membrane is a cationic lipid membrane; the small molecule drug is a STAT3 pathway inhibitor; and the gene targets the JAK / STAT3 signaling pathway.

[0010] The liposome described in the present invention is a closed vesicle with a lipid bilayer structure, and the small molecule drug is encapsulated in the lipid bilayer. Liposomes are formed by self-assembly of amphiphilic lipid molecules, and the lipid bilayer enables the liposome to have a relatively independent internal aqueous phase. Therefore, when used as a drug delivery carrier, hydrophilic or lipophilic drug molecules can be loaded in the internal aqueous phase or bilayer of the liposome, respectively. Compared with other carriers, liposomes have excellent biocompatibility and safety, high drug loading capacity, and can simultaneously meet the co-loading requirements of hydrophilic and hydrophobic drugs.

[0011] The cationic lipid membrane of the present invention contains at least one cationic lipid, and the gene is combined with the cationic lipid by electrostatic interaction. The cationic lipid can efficiently combine nucleic acid drugs with opposite charges, such as siRNA, by electrostatic interaction, and mediate the escape of the endosomal body by the proton sponge effect, thereby transporting the siRNA to the cytoplasm to specifically degrade the mRNA and ensure the transfection efficiency.

[0012] The gene described in the present invention is selected from small interfering RNA, small RNA, messenger RNA or DNA targeting the JAK / STAT3 signaling pathway. Small interfering RNA (siRNA) is a double-stranded RNA with a length of about 20-25 nucleotides. siRNA can integrate to form an RNA-induced silencing complex and guide it to target the target RNA, preventing the target gene from being translated into a functional protein, thereby taking effect at the gene level. However, siRNA is a negatively charged water-soluble substance that is easily degraded by nucleases, has a short blood circulation time, and cannot diffuse into the cytoplasm to exert its effect. Therefore, siRNA must be transported by a carrier to enter the cytoplasm to exert its effect. In some preferred embodiments of the present invention, the gene is STAT3 siRNA.

[0013] The small molecule drug in the present invention is selected from any one or more of inulin lactone, isoinulin lactone, and other selective STAT3 inhibitors. In some embodiments of the present invention, the small molecule drug is inulin lactone. Inulin lactone (ALA) is a sesquiterpene lactone compound extracted from inulin, with a molecular formula of C 15 H 20 O2. Studies have shown that inulin has multiple pharmacological effects such as anti-tumor, antibacterial, and anti-inflammatory. Inulin has an inhibitory effect on STAT3, but because it is insoluble in water, conventional preparations are difficult to reach an effective therapeutic concentration.

[0014] The particle size of the liposomes in the present invention ranges from 140 to 600 nm, preferably, it may be less than 200 nm, or may be 150 to 250 nm, or may be 250 to 300 nm, or may be 300 to 500 nm, and more preferably, it may be less than 200 nm.

[0015] In some embodiments of the present invention, the molar ratio of the small molecule drug to the lipid molecule in the liposome is 0.025 to 0.2:1, preferably 0.1:1.

[0016] In some embodiments of the present invention, the molar ratio of nitrogen atoms in the cationic lipid to phosphate groups in the gene in the liposome is 1 to 20:1, preferably 6:1.

[0017] In some embodiments of the present invention, the gene is a double-stranded RNA and / or shRNA, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA dimer, and the sequence of the first strand is the same as the target sequence in the STAT3 gene, and the nucleotide sequence of the first strand is as shown in SEQ ID NO:1.

[0018] Cationic lipid membrane described in the present invention comprises cationic lipid, neutral phospholipid, cholesterol and PEGylated phospholipid.Phospholipid and cholesterol are the materials constituting lipid nanocarrier, and both are amphipathic molecules.Therefore, the hydrophobic structure region formed by hydrophobic groups (mainly fatty acid hydrocarbon chain of phospholipid and cholesterol steroid structure) and the hydrophilic structure region consisting of phosphate head and polyethylene glycol are naturally present in lipid nanocarrier.Such structural feature provides possibility for the co-carrying of hydrophobic small molecule compound and nucleic acid drug.

[0019] In some embodiments of the present invention, the cationic lipid is selected from any one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride, dioleoylpropyl trimethylammonium chloride, 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl potassium.

[0020] In some embodiments of the present invention, the neutral phospholipid is selected from any one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine, dioleoylphosphatidylethanolamine, dimyristoylphosphatidylcholine, and dipalmitoylphosphatidylcholine.

[0021] In some embodiments of the present invention, the PEGylated phospholipid is selected from any one or more of distearoylphosphatidyl acetamide-polyethylene glycol, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, and methoxy-PEG-2-myristyl acid stannoyl-3-phosphatidylethanolamine. More preferably, it is distearoylphosphatidyl acetamide-polyethylene glycol 2000. The introduction of PEGylated phospholipids can increase the steric hindrance between nanoparticles, reduce liposome aggregation, and enhance stability.

[0022] In some preferred embodiments of the present invention, the encapsulation efficiency of the gene / drug co-loaded liposomes is 71.2%.

[0023] The second aspect of the present invention provides a method for preparing gene / drug co-loaded liposomes, comprising the following steps:

[0024] Step 1, preparing cationic liposomes loaded with small molecule drugs;

[0025] Step 2: co-incubating the gene with the cationic liposome obtained in step 1 to obtain a gene / drug co-loaded lipid complex;

[0026] Step 3: dialyze the gene / drug co-loaded lipid complex obtained in step 2 to obtain gene / drug co-loaded liposomes.

[0027] In some embodiments of the present invention, the cationic liposomes loaded with small molecule drugs in step 1 are prepared by a thin film dispersion method. Specifically, the following steps are included: S1, adding cationic lipids, neutral phospholipids, cholesterol and pegylated phospholipids to ethanol to prepare a lipid mixture; S2, adding small molecule drugs to the lipid mixture prepared in S1, evaporating under reduced pressure to remove ethanol, obtaining a lipid film containing small molecule drugs, adding ultrapure water for hydration, and obtaining a lipid suspension; S3, extruding the lipid suspension several times with a liposome extruder to obtain a lipid complex loaded with small molecule drugs; S4, transferring the lipid complex obtained in S3 to a dialysis bag for dialysis to remove free drugs, and obtaining cationic liposomes loaded with small molecule drugs.

[0028] In some embodiments of the present invention, the molar ratio of the small molecule drug to the lipid molecule in step 1 is 0.025 to 0.2: 1. In some preferred embodiments of the present invention, the molar ratio of the small molecule drug to the lipid molecule in step 1 is 0.1:1.

[0029] In some embodiments of the present invention, the incubation temperature in step 2 is room temperature and the time is 10 to 30 minutes. In some preferred embodiments of the present invention, the incubation temperature in step 2 is room temperature and the time is 20 minutes.

[0030] In some embodiments of the present invention, the molar ratio of nitrogen atoms in the cationic lipid to phosphate groups in the gene in step 2 is 1 to 20: 1. In some preferred embodiments of the present invention, the molar ratio of nitrogen atoms in the cationic lipid to phosphate groups in the gene in step 2 is 6:1.

[0031] In some embodiments of the present invention, the dialysis bag cutoff used for dialysis in step three is 10KD, and the dialysis medium used is ultrapure water.

[0032] The third aspect of the present invention provides a use of the above gene / drug co-loaded liposome in the preparation of a small molecule drug and a gene drug co-delivery preparation. Preferably, the small molecule drug is inulin lactone, and the gene drug is STAT3 siRNA.

[0033] The fourth aspect of the present invention provides a use of the above-mentioned gene / drug co-loaded liposome in the preparation of a drug for treating psoriasis. The gene / drug co-loaded liposome provided by the present invention can be used as a co-delivery drug delivery system for combining STAT3 siRNA with scutellaria lactone as a STAT3 inhibitor, on the one hand, using siRNA to specifically silence STAT3 mRNA, and on the other hand, using scutellaria lactone, a small molecule inhibitor, to synergistically act on STAT3, respectively blocking the STAT3 signaling pathway at the gene and protein levels, thereby achieving the purpose of treating psoriasis.

[0034] The gene / drug co-loaded liposomes of the present invention have at least one of the following functions: 1) inhibiting the excessive proliferation of keratinocytes; 2) reducing the area of ​​erythema in psoriatic skin; 3) reducing the degree of scaling in psoriatic skin; 4) reducing the degree of wrinkling in psoriatic skin; 5) reducing the epidermal thickness of psoriatic skin; 6) inhibiting the excessive immune response of psoriasis.

[0035] The carrier used in the gene / drug co-carrying liposome system provided by the present invention is a liposome formed by cationic lipids, cholesterol, neutral phospholipids and polyethylene glycol phospholipids. Wherein, cationic lipids can efficiently combine nucleic acid drugs with opposite charges by electrostatic action, and mediate endosome escape by proton sponge effect, so as to deliver siRNA to the cytoplasm to specifically degrade mRNA, and ensure transfection efficiency; there is a hydrophobic structure region formed by a hydrophobic group (mainly a fatty acid hydrocarbon chain of phospholipids and a cholesterol steroid structure), and a hydrophilic structure region composed of a phosphate head and polyethylene glycol in the liposome, and such structural features provide the possibility for the co-carrying of hydrophobic small molecule drugs and nucleic acid drugs; meanwhile, the introduction of polyethylene glycol phospholipids can increase the steric hindrance between nanoparticles, reduce liposome aggregation, and enhance stability. Therefore, the carrier can realize the co-delivery of gene / drug, achieve the purpose of coordinated regulation of gene level and protein level, enhance the curative effect, and reduce adverse reactions. In addition, the liposome composition is highly similar to skin phospholipids, can promote the penetration of drugs and reach the site of action, and significantly improve the delivery efficiency.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The present invention proposes a lipid nanocarrier-based liposome that co-loads poorly soluble drugs and gene drugs, which has better stability, excellent biocompatibility and safety, and can efficiently deliver small molecule compounds and genes into cells to exert a synergistic effect. At the same time, the selected carrier material ensures the lysosomal escape and transfection efficiency of siRNA, solving the challenge of co-delivery of genes and small molecule drugs.

[0038] 2. The present invention targets the gene / small molecule drug co-loaded liposomes of the key signaling pathway JAK / STAT3 in psoriasis. On the one hand, STAT3 siRNA is used to specifically silence STAT3 mRNA. On the other hand, the small molecule inhibitor scutellariae lactone is used to synergistically act on STAT3 siRNA to block the STAT3 signaling pathway at the gene and protein levels, respectively. Good results have been achieved in the treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the specificity spectrum of the HPLC method for inulin lactone, where A is ultrapure water; B is blank cationic liposomes; C is blank solvent; and D is inulin lactone standard solution.

[0040] Figure 2 This is the HPLC standard curve of inulin lactone.

[0041] Figure 3 The encapsulation efficiency of scutellaria baicalensis lactone in liposomes under different lipid formulations and at different drug-lipid ratios.

[0042] Figure 4 Particle size and electric potential of gene / drug co-loaded liposomes at different nitrogen-phosphorus ratios.

[0043] Figure 5 Agarose gel retardation experiment was used to evaluate the binding of gene / drug co-loaded liposomes to siRNA.

[0044] Figure 6 The storage stability and morphology of gene / drug co-loaded liposomes at different nitrogen-phosphorus ratios are characterized, where A is the change in particle size; B is the change in potential; C is the change in encapsulation efficiency; and D is the transmission electron micrograph of the gene / drug co-loaded liposomes.

[0045] Figure 7 In vitro release of scutellaria lactone from different preparations.

[0046] Figure 8 The effect of different concentrations of scutellariae lactone on the proliferation of HaCaT cells.

[0047] Fig. 9 The effect of blank liposomes at different concentrations on the proliferation of HaCaT cells.

[0048] Fig.10 The effects of different drug-loaded preparations on the proliferation of HaCaT cells.

[0049] Fig.11 The effects of different drug-loaded preparations on the back skin of psoriasis model mice.

[0050] Fig.12 The effect of different drug-loaded preparations on the PASI score of psoriasis model mice.

[0051] Fig.13 The effect of different drug-loaded preparations on the body weight of psoriasis model mice.

[0052] Fig.14 The effects of different drug-loaded preparations on liver function in psoriasis model mice.

[0053] Fig.15 The effects of different drug-loaded preparations on renal function in psoriasis model mice.

[0054] Fig.16 The effect of different drug-loaded preparations on the spleen coefficient of psoriasis model mice.

[0055] Fig.17 Pathological sections of mouse skin after treatment with different drug-loaded preparations.

[0056] Fig.18 The effects of different drug-loaded preparations on the epidermal thickness of psoriasis model mice.

[0057] Fig.19 The effects of different drug-loaded preparations on cell proliferation in the skin tissue of psoriasis model mice. DETAILED DESCRIPTION

[0058] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0059] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0060] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0061] The present invention uses STAT3 siRNA to specifically silence STAT3 mRNA on the one hand, and uses a small molecule inhibitor, scutellaria lactone, as a small molecule drug to synergistically act on STAT3 on the other hand, uses small unilamellar liposomes as carriers, and encapsulates the small molecule drug in the bilayer of the small unilamellar liposomes. STAT3 siRNA is combined with cationic lipids in the lipid membrane through electrostatic interaction, so as to prepare co-loaded liposomes of gene / small molecule drugs targeting the key signal pathway JAK / STAT3 in psoriasis, realize the orderly release of gene drugs and small molecule drugs, respectively block the STAT3 signal pathway at the gene and protein levels, and achieve the purpose of treating psoriasis.

[0062] The abbreviations involved in the following embodiments are as follows:

[0063]

[0064] The definitions of the terms involved are as follows:

[0065] Encapsulation efficiency: the ratio of the amount of drug encapsulated in liposomes to the total amount of drug put into the liposome solution.

[0066] Drug-lipid ratio: the molar ratio of drug to lipid in liposomes.

[0067] Nitrogen to phosphorus ratio: the molar ratio of nitrogen atoms in cationic lipids to phosphate groups in nucleic acids in liposomes.

[0068] The STAT3 siRNA sequences used are as follows:

[0069]

[0070] Example 1: HPLC Detection Method of Inula lactone

[0071] Inula lactone contains conjugated double bonds in its structure and has strong signal absorption in the ultraviolet region. This example establishes and verifies a high performance liquid chromatography (HPLC) detection method for inula lactone.

[0072] 1.1 Chromatographic conditions

[0073] Mobile phase: water and acetonitrile, volume ratio of 40:60; flow rate of 1 mL / min; time of 12 min; column temperature of 35 °C; detection wavelength of DAD detector of 210 nm; injection volume of 20 μL; column model Agilent ZORBAX SB-C184.6*150 mm5 μm.

[0074] 1.2 Preparation of standard stock solution and standard solution

[0075] Accurately weigh 5 mg of solid powder of inulin lactone, transfer it to a 5 mL volumetric flask, add ultrapure water to dissolve and dilute to volume to obtain the standard stock solution of inulin lactone, with a final concentration of 1.0 mg / mL. Add an appropriate amount of standard stock solution to the volumetric flask, and dilute it with ultrapure water to obtain a series of standard solutions with concentration gradients of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL and 10 μg / mL.

[0076] 1.3 HPLC detection methodology validation

[0077] 1) Specificity: Under the chromatographic conditions, ultrapure water, standard solution of scutellaria lactone, and blank cationic liposomes were loaded onto the machine for HPLC detection. The chromatograms were recorded to exclude the influence of impurities and other substances on the detection method and to investigate the specificity of the method.

[0078] 2) Linearity: Under the present chromatographic conditions, standard solutions of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL and 10 μg / mL of scutellaria lactone were tested on the machine, and the chromatogram and peak area were recorded. The solution concentration and the corresponding peak area were linearly regressed using the least squares method, and a fitting equation was established to determine the linear range of the detection concentration. The test results are shown in Table 1.

[0079] Table 1 HPLC detection values ​​of different concentrations of scutellaria lactone

[0080]

[0081] The standard curve was established according to the results in Table 1. Figure 2 The standard curve of scutellaria lactone is Y=Y=57.42*X-1.584(R=6), R 2 =0.9999, good fitting, and the linear range is 0.5-10.0 μg / mL.

[0082] 3) Precision: Under the chromatographic conditions, take the standard solution of scutellaria lactone, test it continuously on the machine, record the peak area of ​​six tests, and examine the precision.

[0083] Table 2 Investigation on the precision of HPLC method for detecting the content of scutellaria lactone

[0084]

[0085] 4) Recovery rate: Under the present chromatographic conditions, standard solutions of high, medium and low concentrations (10.0 μg / mL, 5.0 μg / mL, 1.0 μg / mL) were respectively taken for HPLC detection, the concentration of the test product was calculated, and the recovery rate of the established method was examined.

[0086] Table 3 Accuracy of HPLC method for detecting the content of scutellaria lactone

[0087]

[0088] like Figure 1 As shown in Tables 1, 2, and 3, the established HPLC detection method for inulin lactone has good specificity, and blank solvent and liposomes have no interference with the determination. The RSD of the precision experiment is 0.172%, indicating that the method has good repeatability, and the recovery rate of the HPLC detection method is within the range of 100±5%, which meets the requirements. In summary, the established HPLC detection method for inulin lactone has good specificity, good linear relationship, and the precision and recovery results meet the requirements, which is suitable for the quantitative detection of inulin lactone.

[0089] Example 2: Preparation and performance determination of ALAlip

[0090] 2.1 Preparation of Inulin lactone liposomes

[0091] (1) The ethanol stock solutions of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (CHOL), and distearoylphosphatidyl acetamide-polyethylene glycol (2000)-amino (DSPE-PEG2000) were mixed in a 5 mL round-bottom flask to obtain a lipid mixture (DOTAP / DSPC / CHOL / DSPE-PEG2000–50:10:37.5:2.5 mol %).

[0092] (2) Add 10 mg / mL of inulin (dissolved in ethanol) to the lipid mixture in (1) to achieve 0, 2.5%, 5%, 10% or 20% of the total lipid moles. Remove the ethanol by evaporation under reduced pressure to obtain a lipid film containing inulin. Add ultrapure water and hydrate at 40°C for 30 minutes to obtain a lipid suspension; use a liposome extruder (Lipexextruder, Northern Lipid) to extrude the lipid suspension through polycarbonate membranes (200 nm, 100 nm and 80 nm) at 50°C in sequence, each extruded several times, to obtain inulin liposomes.

[0093] 2.2 Performance determination of liposomes containing scutellaria lactone

[0094] 2.2.1 Determination of particle size and potential

[0095] Take 50 μL of the prepared inulin lactone liposomes, add 950 μL of ultrapure water and dilute 20 times, measure the average particle size and Zeta potential (Malvern Zetasizer, Malvern, UK), and repeat the measurement three times for each sample.

[0096] 2.2.2 Determination of encapsulation efficiency

[0097] Take the dialyzed inulin lactone liposomes, add methanol at a volume ratio of 1:9, vortex mix for 1 minute, centrifuge at 10000 rpm for 10 minutes, take the supernatant, and determine the concentration of inulin lactone by the HPLC method established in Example 1. Calculate the encapsulation efficiency of inulin lactone. The formula is as follows:

[0098] Encapsulation efficiency = (amount of drug measured in liposomes) ÷ (total amount of drug injected) × 100%

[0099] The results of the test are as follows Figure 3As shown in the figure, the results show that as the drug-lipid ratio increases, the encapsulation rate of ALA in liposomes decreases. When the drug-lipid ratio is 0.1, the encapsulation rate is about 70%, and when the drug-lipid ratio is 0.2, the encapsulation rate drops sharply to 42%, indicating that the ALA that can be encapsulated by liposomes under this prescription condition has reached the maximum limit. Therefore, the drug-lipid ratio is preferably 0.1.

[0100] Example 3: Preparation and performance determination of ALA-STAT3 co-loaded liposomes

[0101] 3.1 Preparation of silerolactone / si-STAT3 co-loaded liposomes

[0102] (1) Before use, the si-STAT3 powder was taken out of the -30°C refrigerator, returned to room temperature, and centrifuged briefly at high speed. An appropriate amount of DEPC water (pH = 4) was added to dissolve the powder to obtain a 20 μM stock solution, which was then dispensed into enzyme-free centrifuge tubes and transferred to a -30°C refrigerator for storage.

[0103] (2) The ALA single-loaded nanoparticles (inulin lactone liposomes) preferably obtained in Example 2 were mixed with si-STAT3 at different N / P ratios and incubated at room temperature and 400 rpm for 20 minutes to obtain inulin lactone / si-STAT3 co-loaded liposomes.

[0104] 3.2 Performance determination of ALA-STAT3 lip

[0105] The characterization method in Example 2 was used to determine the particle size, potential, ALA encapsulation efficiency, and siRNA binding capacity of the co-loaded liposomes at different N / P ratios.

[0106] 3.2.1 Determination of particle size and potential

[0107] The particle size determination results are as follows Figure 4 As shown, compared with blank cationic liposomes, after the introduction of 10% molar ratio of ALA, the particle size and PDI of the liposomes did not change significantly, and were distributed around 180nm, and the PDI was less than 0.1, indicating that the preparation had good uniformity. When N / P was 0, 1, and 2, the particle size of each group was concentrated around 170nm. When N / P was 4 and 5, the particle size increased sharply to more than 400nm, and the PDI increased to 0.2-0.4, indicating that the stability of the nanoparticles was poor in this state. When N / P was 6-20, the particle size gradually stabilized, distributed around 200nm, and the PDI was less than 0.1.

[0108] The measurement results of potential are as follows Figure 5 As shown, the potential measurement results show that with the increase of N / P, the potential gradually increases and gradually tends to be stable when N / P is greater than 5.

[0109] 3.2.2 Agarose gel electrophoresis

[0110] 2% agarose gel electrophoresis was used to separate free si-STAT3 and si-STAT3 encapsulated in the preparation. Preparations with different nitrogen-phosphorus ratios were prepared into loading solutions with a final concentration of 20 ng / μL of si-STAT3. The T suffix of each group indicated that 1% Triton was added to the group to rupture the liposomes and expose all si-STAT3, so as to determine the degradation of si-STAT3. The electrophoresis solution was 0.5×TBE, and the electrophoresis was performed at a voltage of 100V for 15 minutes.

[0111] Agarose gel electrophoresis results Figure 5 As shown, the results indicate that when N / P is higher than 4, the preparation can fully bind to si-STAT3.

[0112] Therefore, considering the data of particle size, PDI, potential and si-STAT3 encapsulation rate, N / P is preferably 6, at which point the composite nanoformulation has good stability and uniformity and completely encapsulates si-STAT3.

[0113] Example 4: Storage stability and morphology characterization of scutellaria lactone / si-STAT3 co-loaded liposomes

[0114] 4.1 Storage stability test

[0115] (1) The above-mentioned composite lipid carriers with different N / P ratios (inulin lactone / si-STAT3 co-loaded liposomes) were stored in a refrigerator at 4°C. On the 1st and 15th days of preparation, the particle size and potential of each group of preparations were measured using the method in Example 2, and each test was repeated 3 times.

[0116] (2) We selected liposomes containing silymarin / si-STAT3 and ALA alone with N / P=6 and investigated the storage stability of ALA loading at 4°C for 15 days when the drug-lipid ratio was 0.1 in both groups.

[0117] The results are shown in Table 4 and Figure 6 As shown in A to C, there was no significant change in particle size, PDI, potential, and ALA drug loading in each group after storage in a 4°C refrigerator for 15 days, indicating that the co-loaded liposome preparation had good stability.

[0118] Table 4 Stability of ALA / si-STAT3 co-loaded liposomes with different nitrogen-phosphorus ratios

[0119]

[0120]

[0121] 4.2 Morphological characterization

[0122] In the Vitrobot Mark IV cryo-sample preparation system, 3 μL of ALA-STAT3 co-loaded liposomes were dripped onto the surface of the copper mesh, the excess solution was absorbed with filter paper, and the liposomes were quickly immersed in liquid ethane. The morphology of the liposomes was observed using a biological field emission transmission electron microscope (Talos F200S G2 200 kV). The voltage was 200 kV, the Cryo-TEM system and the low-does mode were selected, and the observation was performed within the magnification range of 115-480000.

[0123] The morphological results of ALA-STAT3 lip are as follows Figure 6 As shown in D. The co-loaded liposomes are all small unilamellar liposomes with a diameter of about 170nm, which is consistent with the results of dynamic light scattering. Since ALA is a hydrophobic structure, according to the principle of like dissolves like, scutellaria lactone is mainly loaded in the hydrophobic region of the lipid membrane. Electron microscopy results show that the shape of some co-loaded liposomes is non-spherical, which may be related to the fact that the insertion of ALA interferes with the arrangement of the lipid bilayer.

[0124] Example 5: In vitro release study of liposomes loaded with or co-loaded with scutellaria lactone

[0125] The in vitro drug release rate of ALA lip and ALA-STAT3 lip was measured using an air shaker. The experimental steps are as follows: First, the prepared reagent was placed in a constant temperature oscillator and shaken at 100 pm and 37°C. Samples were taken at predetermined time points (1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours), and centrifuged at 1000 pm for 15 minutes, and the supernatant was removed. Finally, the precipitate was dissolved with an appropriate amount of methanol, and its peak area at 210 nm was determined by HPLC, and compared with the peak area of ​​the solution just prepared to obtain the release curve of scutellaria lactone.

[0126] In terms of in vitro release, Figure 7 As shown, the in vitro release curves of ALA lip and ALASTAT3 lip are similar, and their release rates quickly rise to about 20% in the first 2 hours, and then basically maintain a slow release until the 96th hour, with a cumulative release of about 90%. The results show that the prepared ALA-STAT3-Lip can be continuously and slowly released within 4 days, has high stability, and is suitable for local transdermal administration.

[0127] Example 6: Cytotoxicity of Inulin / si-STAT3 Co-loaded Liposomes

[0128] 6.1 Cell culture

[0129] HaCaT is a human immortalized keratinocyte cell line that has good in vitro differentiation and proliferation capabilities and is used in many studies. It is often used as a reproducible model for in vitro studies of psoriasis. HaCaT is an adherent cell cultured in a constant temperature incubator at 37°C and 5% CO2. The complete culture medium is prepared with DMEM cell culture medium and supplemented with 5% FBS and 1% double antibody. When cells are passaged, an appropriate amount of 0.25% trypsin-EDTA is added for digestion, and an appropriate amount of complete culture medium is added to terminate the digestion. Collect the cell suspension, centrifuge at low speed, resuspend the cell pellet in fresh culture medium, and passage at a ratio of 1:3.

[0130] 6.2 Detection of cell viability by CCK-8 method

[0131] 1) When the cell density reaches above 80%, digest and collect the cells and inoculate them into 96-well plates.

[0132] 2) After the cell density reaches the requirement, subsequent drug administration is performed and the culture is continued.

[0133] 3) After the drug incubation is completed, the medium is changed, and 10% volume ratio of CCK8 solution is added to the fresh culture medium. After mixing, 100 μL is added to each well of the 96-well plate.

[0134] 4) Transfer the well plate to an incubator and incubate for 2 hours, and measure the absorbance at 450 nm using an ELISA reader. Calculate the relative activity or proliferation capacity of the cells according to the formula in the instruction manual.

[0135] Figure 8 The results of the cell proliferation experiment showed that when the ALA concentration exceeded 10 μM, it had a significant inhibitory effect on the proliferation of HaCaT and could significantly inhibit the growth of HaCaT cells within 48 hours. Fig. 9 The results showed that the total lipid concentration of blank cationic liposomes exceeded 200 μg / ml and could significantly inhibit the proliferation of HaCaT cells. The concentration to be used in subsequent in vitro experiments was about 100 μg / mL. Based on the results of this experiment, it can be seen that within this concentration range, the preparation will not have a significant effect on cell activity and has good safety. Fig.10 The results showed that the results of proliferation inhibition by different preparations indicated that si-STAT3 had no effect on HaCaT proliferation, and the combined use of si-STAT3 and ALA significantly enhanced the inhibitory effect on HaCaT proliferation.

[0136] Example 7: In vivo therapeutic effect of scutellaria lactone / si-STAT3 co-loaded liposomes

[0137] The mouse psoriasis model induced by imiquimod (IMQ) is a widely used experimental model. IMQ is a Toll-like receptor (TLR) 7 / 8 agonist that activates the innate immune system and promotes or aggravates psoriasis-like skin lesions, including erythema, scaling, and epidermal thickening. The model is quick and simple to establish, with low cost and easy to replicate. In addition, commercially available calcipotriol betamethasone ointment is often used as a positive control drug for the treatment of psoriasis in studies (calcipotriol betamethasone ointment has been shown to be useful in topical transdermal anti-psoriatic treatments).

[0138] According to the common method in the literature, the mouse psoriasis model was established using commercially available IMQ. 8-week-old male BALB / c mice were randomly divided into 12 groups, 5 mice in each group, and the specific groups are shown in Table 5.

[0139] Table 5 Animal experiment groups and dosage

[0140]

[0141]

[0142] Before the experiment, the hair on the back of the mice was shaved with a pet-specific hair trimmer. The shaved area was 2*3cm. The fine and soft hair that was difficult to remove was cut with scissors. Depilatory cream was applied to completely eradicate the hair, and the back skin surface was scrubbed with saline to avoid the effects of the depilatory cream. 2 Apply 62.5 mg of imiquimod cream daily to the skin for 7 days to build up the inflammatory symptoms of psoriasis.

[0143] Except for the control group and the model group, each group was given 125 μl of the corresponding preparation 4 h after IMQ modeling, and the concentration of 125 mg / cm was applied to the back of the mice in the positive control group. 2 (calcipotriol betamethasone ointment) for 7 days. The therapeutic effect of each group of preparations was evaluated by the following indicators. The experimental materials are shown in Table 6:

[0144] Table 6 Reagents and consumables

[0145]

[0146]

[0147] (1) Inflammation and psoriasis area and severity index (PASI score) on the back of mice:

[0148] The PASI score is widely used in clinical practice to assess the condition of psoriasis patients, and the assessment indicators are erythema, scaling, thickness and area. Erythema refers to the area of ​​​​the rash that appears on the back skin of mice after the application of imiquimod, scaling refers to the degree of scaling of the skin after the onset of inflammatory symptoms, and thickness refers to the degree of thickening of the skin after suffering from psoriasis symptoms. The assessment index range is 0-4 points, 0 points means no obvious lesions, 1 point means mild lesions, 2 points means moderate lesions, 3 points means obvious lesions, and 4 points means that the lesions continue to worsen on the basis of 3 points. The sum of the above three scores (0-12 points) is used to assess the severity of psoriasis inflammation. Starting from the day of the experiment, the PASI score was recorded every day for a total of 7 days. The specific standards for PASI scoring are shown in Table 7:

[0149] Table 7 PASI score (mice)

[0150]

[0151] After seven days of continuous modeling and treatment, there was no significant change in the back of the mice in the blank group, while the back skin of the mice in the model group was red, with more erythema, scaling and wrinkling, which are typical symptoms of psoriasis, indicating that the modeling was successful. After treatment with calcipotriol-betamethasone ointment, the erythema, scaling and wrinkling of the positive control group were significantly alleviated. The specific back inflammation conditions are shown in Fig.11 .

[0152] The results of the study showed that ( Fig.12 ), low, medium and high concentrations of scutellaria lactone liposomes can reduce the PASI score of mice, and the PASI score decreases with increasing concentration. The combination of ALA+STAT3 at low concentration (48μM ALA+ and 1nmolSTAT3 siRNA) is more effective than single-drug treatment at the same dose, indicating that the therapeutic effect of STAT3 siRNA and scutellaria lactone is enhanced after combined use.

[0153] (2) Changes in mouse body weight: The body weight of mice was recorded on the first and last day of drug administration to determine the effect of the preparation on the normal growth of mice. Fig.13 The results showed that after IMQ modeling, the body weight of mice in each group had no significant change compared with the blank group, but after treatment with positive drugs, the body weight of mice decreased by 17.3%, indicating that the drug had a large side effect on mice, while the safety and effectiveness of the scutellaria lactone preparations in each group were good.

[0154] (3) Mouse liver and kidney function: On the day of sampling, 200 μL of orbital venous blood was collected from each mouse after isoflurane anesthesia and dripped into an EDTA anticoagulant tube. The tube wall was flicked to mix the blood and EDTA evenly to prevent blood coagulation and precipitation. The sample was left at room temperature for 2 hours and centrifuged at 3500 r / min for 10 minutes. After centrifugation, the serum was located on the upper layer and was light yellow and transparent. It was collected in a new centrifuge tube for subsequent biochemical testing. The liver function indicators ALT and AST and the kidney function indicator CREA-S in the serum were detected using an automatic biochemical analyzer, and the remaining part was transferred to a -80°C refrigerator for use.

[0155] Fig.14 and Fig.15 The analysis results showed that there was no significant difference in ALT, AST, and CREA-S between the mice in each group treated with the drug-loaded preparation and those in the normal group, indicating that each group of preparations had good safety.

[0156] (4) Mouse organ coefficient: On the 8th day of the experiment, mice were humanely killed, and the spleen of the mice was isolated and washed with PBS. The excess liquid was absorbed with filter paper and the wet weight was weighed. The morphology of the spleen in each group was recorded, and the organ coefficient of the above organs was calculated using the following formula:

[0157] Organ coefficient = (mouse organ mass) / mouse body weight*100%.

[0158] Fig.16 The results showed that 7 days after IMQ modeling, the spleen coefficient of mice increased significantly, low-concentration liposomes of scutellaria lactone could not reduce the spleen coefficient of mice, and STAT3 siRNA single liposomes could significantly reduce the spleen coefficient (P < 0.05). The combined use of the two enhanced the effect of inhibiting immune response (P < 0.01).

[0159] (5) Skin staining and pathological sections: On the eighth day of the experiment, mice in each group were killed by ether anesthesia. The skin of the dorsal modeling area was cut off and fixed with tin foil to prevent curling. The skin was immediately placed in a 4% paraformaldehyde tissue fixative solution with a volume of more than ten times and soaked for 24 hours. Then, the mouse skin that was transparentized with xylene was embedded in paraffin, cut into thin slices, and transferred into a xylene-ethanol mixture with a volume ratio of 1:1. After 5 minutes, different concentrations of ethanol-water solution were added. Subsequently, the solution was stained with hematoxylin stain, rinsed with running water, and stained with 0.5% eosin stain. Then, it was dehydrated with different concentrations of ethanol-water solution and treated with xylene. Finally, an appropriate amount of neutral gum was immediately added to fix the skin slices, and a thin cover glass was covered to exclude the outside air. After the slices were made, the skin tissue was observed with an upright fluorescence microscope, mainly to observe the uniformity of the arrangement of the epidermal cells, and the thickness of the epidermal layer was recorded with a ruler.

[0160] Fig.17 and Fig.18The results of HE staining of skin cross-sections showed that the epidermis (the outermost dark red structure) was significantly thickened after modeling, and the epidermis extended downward in a spike-like shape, indicating that psoriasis-like symptoms in mice were successfully induced. At the same time, after treatment with different drug-loaded preparations, the thickness of the epidermis in each group decreased significantly, indicating that the treatment effect was good.

[0161] (6) Immunohistochemical staining:

[0162] Ki67 is an intracellular antigen molecule that is mainly involved in intracellular proliferation activities and can be easily detected during the mitosis phase of cells. Therefore, Ki67 is often used to mark active cells in a proliferative state. In experiments, the specific binding of antigens and antibodies can be used to locate these molecules, and the proliferation state of tissues can be observed intuitively. According to the degree of color development of positive markers, they are divided into: light yellow, indicating weak positivity; brown-yellow, indicating moderate positivity; brown-black, indicating strong positivity.

[0163] The immunohistochemical experimental steps include: 1) dewaxing and hydration; 2) antigen repair; 3) incubating with 3% hydrogen peroxide at room temperature for 10 minutes, washing 3 times with PBS; 4) adding 5% BSA solution to seal for 20 minutes, 37°C, washing 3 times with PBS, and shaking off excess liquid; 5) adding primary antibody and incubating at 4°C overnight; 6) washing 3 times with room temperature PBS, adding 1 drop of polymer enhancer, and incubating at room temperature for 20 minutes; 7) removing PBS, adding horseradish peroxidase, and incubating at room temperature for 30 minutes; 8) washing 3 times with PBS, adding DAB color developing solution, and stopping the reaction when obvious brick red appears; 9) washing with distilled water, counter-staining with hematoxylin for 2 minutes, and differentiation with hydrochloric acid and alcohol; 10) dehydration, transparency, sealing, and microscopic examination.

[0164] The results of immunohistochemistry showed that ( Fig.19 ), the epidermis was significantly thickened after IMQ modeling, and a large area of ​​brown-yellow appeared in the visual field, indicating that Ki67 increased significantly and cells overproliferated, which is a typical psoriasis-like symptom. The thickness of the epidermis decreased in each group after treatment, and the brown-yellow color decreased significantly, indicating that different preparations had an inhibitory effect on cell overproliferation. The expression of Ki67 after ALA and si-STAT3 combined treatment was lower than that of STAT3 siRNA and ALA liposome (48μM) monotherapy group, indicating that the combined treatment enhanced the cell proliferation inhibitory effect of the drug and reduced the required dose of small molecule drugs.

[0165] The above experimental results show that the gene / drug co-loaded liposomes provided by the present invention can inhibit the excessive proliferation of keratinocytes, reduce the PASI score of psoriasis patients, reduce the epidermal thickness of psoriasis skin, and inhibit the excessive immune response of psoriasis, and have good application prospects in the development of psoriasis treatment drugs.

[0166] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A gene / drug co-loaded liposome, characterized in that: The liposome comprises a lipid membrane, a small molecule drug and a gene; the lipid membrane is a cationic lipid membrane; the small molecule drug is selected from scutellaria lactone; the gene is selected from a small interfering RNA targeting a JAK / STAT3 signaling pathway; the liposome is a closed vesicle having a lipid bilayer structure, and the small molecule drug is encapsulated in the lipid bilayer; the small interfering RNA is a double-stranded RNA, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand complement each other to form an RNA dimer, and the sequence of the first strand is the same as the target sequence in the STAT3 gene, and the nucleotide sequence of the first strand is as shown in SEQ ID NO:1; the cationic lipid membrane includes (2,3-dioleoyl-propyl)-trimethylammonium chloride, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol and distearoylphosphatidylacetamide-polyethylene glycol 2000-amino, and the molar percentages thereof are 50:10:37.5:2.5; the small interfering RNA is bound to (2,3-dioleoyl-propyl)-trimethylammonium chloride through electrostatic interaction; the molar ratio of the inulin lactone to the lipid molecule is 0.1:1; the molar ratio of the nitrogen atom in the (2,3-dioleoyl-propyl)-trimethylammonium chloride to the phosphate group in the gene is 6:

1.

2. A method for preparing gene / drug co-loaded liposomes as claimed in claim 1, characterized in that: The steps include: Step 1, preparing cationic liposomes loaded with small molecule drugs; Step 2: co-incubating the gene with the cationic liposome obtained in step 1 to obtain a gene / drug co-loaded lipid complex; Step 3: dialyze the gene / drug co-loaded lipid complex obtained in step 2 to obtain gene / drug co-loaded liposomes.

3. The method for preparing gene / drug co-loaded liposomes according to claim 2, characterized in that: Also includes one or more of the following features: (a) In step 1, cationic liposomes loaded with small molecule drugs are prepared by a thin film dispersion method; (b) in step 1, the molar ratio of the small molecule drug to the lipid molecule is 0.1:1; (c) In step 2, the incubation temperature is room temperature and the incubation time is 10 to 30 min; (d) The dialysis bag used for dialysis in step 3 has a cutoff volume of 10 KD and the dialysis medium used is ultrapure water.

4. Use of the gene / drug co-loaded liposome as claimed in claim 1 or the gene / drug co-loaded liposome prepared by the preparation method as claimed in claim 2 or 3 in the preparation of small molecule drugs and gene drug co-delivery preparations.

5. Use of the gene / drug co-loaded liposome as claimed in claim 1 or the gene / drug co-loaded liposome prepared by the preparation method as claimed in claim 2 or 3 in the preparation of drugs for treating psoriasis.

6. The use according to claim 5, characterized in that The psoriasis treatment drug has at least one of the following functions: 1) Inhibit excessive proliferation of keratinocytes; 2) Reduce the area of ​​rashes on psoriasis skin; 3) Reduce the degree of scaling of psoriatic skin; 4) Reduce the wrinkling of psoriatic skin; 5) Reduce epidermal thickness in psoriatic skin; 6) Inhibit the excessive immune response of psoriasis.

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