Lentiviral transfection enhanced nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses lentivirus transfection enhanced nanoparticles as well as a preparation method and application thereof, and belongs to the field of biomedical engineering. According to the invention, macromolecular polyethylene glycol with anions and a surfactant effect and micromolecular spermidine with cations and a cell phagocytosis enhancement inducing effect are used as raw materials for preparing the lentivirus transfection enhanced nanoparticles; nanoparticles with uniform particle size and high dispersity are prepared through electrostatic adsorption and low-temperature and low-speed self-assembly. After lentivirus is loaded on the nano-particles, lentivirus transfection enhanced nano-particles are obtained. The lentiviral transfection enhanced nanoparticles provided by the invention have very high transfection efficiency on tissue cells with relatively low cell membrane fluidity or relatively long growth cycle, have good biocompatibility, and can rapidly discharge cells after transfection, so that interference of empty carriers of the nanoparticles on subsequent research is reduced; therefore, the efficiency and accuracy of gene editing operation are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and specifically relates to a lentiviral transfection enhanced nanoparticle and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Lentiviruses are often used in biological and biomedical research to edit cell genes in order to achieve the goals of knocking out, overexpressing or recombining target genes. However, due to the natural barrier function of the cell membrane and the negative potential of its surface, negatively charged lentiviruses often have difficulty passing through the cell membrane on their own. Currently, lentiviruses are often loaded onto nanoparticles with cell membrane penetration to improve the efficiency of lentiviral transfection.

[0004] The basic principle of lentiviral transfection enhancement nanoparticles is to improve the transfection efficiency of lentivirus in target cells through the physical properties of nanoparticles. These nanoparticles can enrich viral particles on the cell surface, increase the contact opportunities between viruses and cells, and then promote viral infection of cells, thus providing strong support for gene editing. Lentivirus transfection enhancement nanoparticles mainly include polymer nanoparticles, lipid nanocarriers, protein complexes, cell penetrating peptide nanocarriers, etc.

[0005] Although there are many commercial lentiviral transfection enhancement nanoparticles, such as polybrene, it is still difficult for existing lentiviral transfection enhancement nanoparticles to achieve high transfection efficiency for some tissues or cells with poor cell membrane fluidity or long growth cycles (such as osteoblasts and bone tissue, etc.), which greatly affects the gene editing process. At the same time, polybrene also has the problem of high biological toxicity. Therefore, the development of a lentiviral transfection enhancement nanoparticle with a wider range of applications, higher transfection efficiency, and lower biological toxicity is an urgent problem to be solved in the field of gene editing and nanomaterials. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a lentiviral transfection enhancing nanoparticle and a preparation method and application thereof. The nanoparticles prepared by synthesis based on polyethylene glycol and spermidine provided by the present invention have extremely high binding force and penetration force on tissues or cells with low cell membrane fluidity and long growth cycle (such as osteoblasts and bone tissue), thereby significantly enhancing the transfection efficiency of the carried lentivirus.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] A first aspect of the present invention provides a nanoparticle, wherein the nanoparticle is prepared by self-assembly of polyethylene glycol (PEG) and spermidine.

[0009] For specific cell populations with low cell membrane fluidity or long growth cycles such as osteoblasts, the current commercial lentiviral transfection enhancement nanoparticles generally have the problem of low transfection efficiency, that is, the number of cells infected per unit time is limited, which leads to tedious and lengthy cell continuous passage and screening after lentiviral transfection. In response to this problem, the present invention innovatively proposes to use anionic macromolecular polyethylene glycol with surfactant effect and cationic small molecule spermidine with inducing cell phagocytosis enhancement as raw materials for preparing lentiviral transfection enhancement nanoparticles, and prepare nanoparticles with uniform particle size and high dispersion through electrostatic adsorption and low-temperature and low-speed self-assembly; the hydrophilicity of polyethylene glycol is used to ensure the water solubility and stability of nanoparticles; spermidine is used to induce physiological autophagy and endocytosis of cells to enhance the efficiency of lentiviral transfection. The present invention has been shown through a large number of experiments that compared with a number of commercial lentiviral transfection enhancement nanoparticles currently used, polyethylene glycol-spermidine nanoparticles increase the lentiviral transfection efficiency by 80% per unit time, and the stability of continuous transfection is increased by 5 times.

[0010] Although both polyethylene glycol and spermidine have high biocompatibility, when they enter cells as components of nanoparticles, they will inevitably trigger a series of stress responses in cells. In particular, polyethylene glycol, as a biologically inert macromolecule, often induces immune rejection reactions in cells. In order to ensure the actual effect of the lentivirus transfected into the cells, it is necessary to minimize the impact of nanoparticles on cells as much as possible to reduce interference with the gene editing effect. The present invention proposes for the first time to use spermidine as a "soothing agent" for the immune rejection reaction of polyethylene glycol cells, and to use spermidine as a natural polyamine to moderately reduce the increased level of oxidative stress caused by polyethylene glycol in cells; spermidine is used to promote physiological autophagy to accelerate the discharge of polyethylene glycol in cells. At the same time, since spermidine is a small molecule active substance and participates in a variety of intracellular metabolic activities, it can be completely metabolized and converted a few hours after entering the cell, and will not affect the normal development of subsequent cell experiments such as gene editing effects and target gene effects.

[0011] In some embodiments of the present invention, the molecular weight of the polyethylene glycol is 4000-8000.

[0012] In some embodiments of the present invention, the particle size of the nanoparticles is 200-250 nm.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned nanoparticles, comprising:

[0014] dissolving polyethylene glycol and spermidine in water to obtain a mixed solution;

[0015] The mixed solution was stirred at 0-5° C. and 100-300 rpm for 1-3 h, and centrifuged to obtain nanoparticles.

[0016] In some embodiments of the present invention, in the mixed solution, the concentration of polyethylene glycol is 1-20 mg / mL, and the concentration of spermidine is 0.5-10 μM.

[0017] Although polyethylene glycol has high hydrophilicity, when it undergoes affinity adsorption with cations, it often causes sedimentation due to too fast a polymerization rate, thereby affecting the uniformity and stability of the prepared nanoparticles. The present invention has been proved through a large number of experiments that the polymerization rate of polyethylene glycol and spermidine can be effectively controlled by adopting a low-temperature and low-speed self-assembly method, that is, the reaction temperature is 0-5°C and the stirring speed is 100-300rpm, especially under the conditions of a reaction temperature of 4°C and a stirring speed of 200rpm, thereby preparing nanoparticles with uniform particle size, high dispersion and long-term stability.

[0018] The nanoparticles prepared by the synthesis of polyethylene glycol and spermidine provided by the present invention have extremely high binding and penetration forces for tissues or cells with low cell membrane fluidity and long growth cycles (such as osteoblasts and bone tissue), and can significantly enhance the transfection efficiency of the carried lentivirus. Therefore, the third aspect of the present invention provides an application of the above-mentioned nanoparticles or the nanoparticles prepared by the above-mentioned preparation method in the preparation of products that improve the transfection efficiency of lentiviruses.

[0019] In some embodiments of the present invention, the product is a kit, a transfection enhancer or a vector for improving the efficiency of lentiviral transfection.

[0020] In some embodiments of the present invention, the product is used for lentiviral transfection of cells or tissues with poor cell membrane fluidity; and / or lentiviral transfection of cells or tissues with a long growth cycle.

[0021] In some embodiments of the present invention, the cells or tissues are osteoblasts and bone tissue.

[0022] A fourth aspect of the present invention provides a method for improving the efficiency of lentiviral transfection, wherein the above-mentioned nanoparticles or nanoparticles prepared by the above-mentioned preparation method are loaded with lentivirus, and gene transfection is performed on a target sample in an in vitro experiment.

[0023] In some embodiments of the present invention, the target sample includes tissues or cells with poor cell membrane fluidity or long growth cycle.

[0024] Preferably, the target sample is an osteoblast or a bone tissue sample.

[0025] A fifth aspect of the present invention provides a lentivirus transfection enhancing nanoparticle, which is a nanoparticle loaded with a lentivirus;

[0026] In the lentiviral transfection enhanced nanoparticles, the titer of the nanoparticles is 900-1100TU, and the titer of the lentivirus is ≤10 6 TU;

[0027] The nanoparticles are the above-mentioned nanoparticles or nanoparticles prepared by the above-mentioned preparation method.

[0028] The lentiviral transfection enhancing nanoparticles provided by the present invention have high biocompatibility, are non-cytotoxic, and have a simple and efficient preparation method. In particular, they can significantly improve the transfection efficiency of lentiviruses on cells with low cell membrane fluidity or long growth cycles, thereby greatly improving the gene editing effect.

[0029] In some embodiments of the present invention, the particle size of the lentiviral transfection enhancing nanoparticles is 300-350 nm.

[0030] In some embodiments of the present invention, the lentivirus is a lentivirus that overexpresses ALDH6A1.

[0031] A sixth aspect of the present invention provides a method for preparing the above-mentioned lentiviral transfection enhancing nanoparticles, comprising:

[0032] The lentivirus is mixed with the above-mentioned nanoparticles or the nanoparticles prepared by the above-mentioned preparation method to achieve loading of the lentivirus into the nanoparticles.

[0033] It should be noted that the molar ratio of the present invention is lentivirus: nanoparticles

[0034] =10:1~1000000:1 for loading, which can achieve efficient transfection of cells by lentivirus while ensuring good stability and dispersion of polyethylene glycol-spermidine nanoparticles.

[0035] A seventh aspect of the present invention provides a use of the above-mentioned lentiviral transfection enhancing nanoparticles in the preparation of a lentiviral transfection product.

[0036] In some embodiments of the present invention, the product is a medicine, a kit or a transfection reagent.

[0037] In some embodiments of the present invention, the product is an injectable preparation.

[0038] In some embodiments of the present invention, the product is used for lentiviral transfection of cells or tissues with poor cell membrane fluidity and / or lentiviral transfection of cells or tissues with a long growth cycle.

[0039] In some embodiments of the present invention, the product is used for lentiviral transfection of osteoblasts and / or lentiviral transfection of bone tissue.

[0040] In some embodiments of the present invention, the product is used for lentiviral transfection of bone tissue around implants.

[0041] The beneficial effects of the present invention are:

[0042] The lentiviral transfection enhancement nanoparticles provided by the present invention can significantly improve the transfection efficiency of the internally loaded lentivirus to the cells, especially for the improvement of the transfection efficiency of tissues or cells with low cell membrane fluidity or long growth cycle. At the same time, compared with the currently commercialized lentiviral transfection enhancement reagents, the lentiviral transfection enhancement nanoparticles provided by the present invention have outstanding advantages such as higher biosafety, wider application range, and stronger stability. In addition, the formula of the lentiviral transfection enhancement nanoparticles provided by the present invention can achieve rapid metabolism of the components in the cell (spermidine) or rapid excretion outside the cell (polyethylene glycol), without interfering with the subsequent experiments such as gene editing effects and target gene effect detection, which greatly improves the operational efficiency of using lentivirus to edit the target gene in the cell. In summary, the lentiviral transfection enhancement nanoparticles provided by the present invention are safe and reliable, and can significantly improve the efficiency of lentivirus carrying target genes to transfect cells and tissues, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0044] Figure 1 Characterization of the physicochemical properties and stability of the lentiviral transfection-enhancing nanoparticles prepared in Example 1 of the present invention; wherein A is the appearance of the nanoparticles (PS) prepared from polyethylene glycol and spermidine and the nanoparticles loaded with viruses (PS@Virus) under a transmission electron microscope; B is the particle size of PS and PS@Virus; C is the surface potential of PS and PS@Virus; D is the confidence value (PDI) when measuring the average particle size of PS and PS@Virus; E is the stability of PS and PS@Virus under different temperature conditions; F is the stability of PS and PS@Virus under different buffer conditions;

[0045] Figure 2 The nanoparticles prepared in Example 1 of the present invention were loaded with different titers of lentivirus and the in vitro and in vivo toxicity to cells and animals, as well as the transfection efficiency compared with that of commercial transfection enhancement reagents; wherein A is the nanoparticle titer of 103 TU, the CCK8 detection results of osteoblast proliferation activity loaded with different titers of lentivirus; B is when the titer of nanoparticles is 10 3 TU, the effect of loading different titers of lentivirus and injecting it into the knee joint of mice on the weight change of mice; C is the comparison of osteoblast transfection efficiency without using transfection enhancement reagent, using commercial transfection enhancement reagent, and using lentiviral transfection enhancement nanoparticles, as well as the comparison of the transcription of target gene after transfection for multiple consecutive days;

[0046] Figure 3 The effect of the lentiviral transfection enhanced nanoparticles prepared in Example 1 of the present invention loaded with the target gene fragment (overexpressing ALDH6A1) on the proliferation activity and biological activity of osteoblasts; wherein, A is the effect of nanoparticles (PS) and nanoparticles (PS@Virus) loaded with lentivirus overexpressing ALDH6A1 gene fragments in high-fat culture environment (high-fat, HF) on the cell proliferation activity after infecting osteoblasts using the CCK8 method; B is the effect of nanoparticles (PS) and nanoparticles (PS@Virus) loaded with lentivirus overexpressing ALDH6A1 gene fragments in high-fat culture environment (high-fat, HF) on the cell proliferation activity after infecting osteoblasts using EdU staining; C is Figure 3 Statistical analysis of EdU staining results in Figure B;

[0047] Figure 4 The effect of the nanoparticles (PS@Virus) loaded with the lentivirus overexpressing the ALDH6A1 gene fragment prepared in Example 1 of the present invention on the redox homeostasis of osteoblasts when applied in a high-fat environment; wherein A is the effect of the lentivirus overexpressing ALDH6A1 on the ROS level in osteoblasts detected by DCFH-DA fluorescence staining; B is the Figure 4 Figure A shows the statistical analysis of DCFH-DA fluorescence staining results; Figure C shows the effect of lentivirus overexpressing ALDH6A1 on ROS levels in osteoblast mitochondria using Mito-SOX fluorescence staining; Figure D shows the effect of lentivirus overexpressing ALDH6A1 on ROS levels in osteoblast mitochondria Figure 4 Statistical analysis of Mito-SOX fluorescence staining results in Figure C;

[0048] Figure 5Schematic diagram of the setting and operation instructions for the in vivo animal experiment grouping in Experimental Example 1 of the present invention; wherein, the in vivo animal experiment is divided into 2 groups: a control treatment group (HF-PS group) in which mice were fed a high-fat diet to form hyperlipidemia and nanoparticles were locally injected into the knee joint capsule, and a treatment group (HF-PS@Virus group) in which mice were fed a high-fat diet to form hyperlipidemia and nanoparticles loaded with a lentivirus overexpressing ALDH6A1 were locally injected into the knee joint capsule. Mice in each group were implanted with a titanium implant with a length of 3 mm and a diameter of 1 mm at the distal end of the femur, and received an injection treatment every 5 days until the mice were killed 30 days later and the corresponding specimens were obtained;

[0049] Figure 6 The results of the in vivo experiment in Experimental Example 1 of the present invention; A is the change of ALDH6A1 expression in the tissue around the implant after local injection of nanoparticles loaded with lentivirus overexpressing ALDH6A1 into the knee joint capsule of normal mice and hyperlipidemic mice by immunofluorescence staining of tissue sections; B is Figure 5 A shows the results of immunofluorescence staining; C shows the changes in bone integration of mouse implants; D shows the changes in bone integration of mouse implants. Figure 5 Statistical analysis of the percentage of new bone to total peri-implant volume in C. DETAILED DESCRIPTION

[0050] The present invention provides a lentiviral transfection enhancing nanoparticle and a preparation method and application thereof, which are used to solve the problem in the prior art that for tissue cells with poor cell membrane fluidity or long growth cycle (such as osteoblasts and bone tissue, etc.), the existing lentiviral transfection enhancing nanoparticles are still difficult to achieve high transfection efficiency.

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0052] The raw materials and reagents used in the following examples are all commercially available or homemade.

[0053] The "lentivirus overexpressing ALDH6A1" used in the following experiments was synthesized by Shanghai GeneCare Gene Medical Technology Co., Ltd., and its structure is consistent with the lentivirus overexpressing ALDH6A1 prepared by Wang Yanan (Wang Yanan, Oral Clinical Medicine. Study on the role and mechanism of ALDH6A1 in improving implant bone integration in hyperlipidemic mice by regulating the ROS / Wnt / β-catenin pathway [D]. Shandong University [2024-11-25].).

[0054] Example 1

[0055] A preparation method of lentiviral transfection enhanced nanoparticles comprises the following steps:

[0056] First, 10 mg / mL of PEG6000 and 1 μM of spermidine were mixed and vortexed at 1000 rpm for 1 min to obtain a mixed solution.

[0057] Secondly, the mixed solution was placed at 4°C and stirred continuously at 200 rpm for 2 h.

[0058] Next, the thoroughly mixed solution was centrifuged at 4°C and 12,000 rpm for 1 h, and the supernatant was discarded to obtain nanoparticles self-assembled at low temperature and low speed based on polyethylene glycol and spermidine, abbreviated as PS.

[0059] Finally, the lentivirus overexpressing ALDH6A1 was mixed with the nanoparticles by gently blowing and mixing with a pipette at a molar ratio of lentivirus: nanoparticle = 100:1 to 1000000:1 to achieve loading of the lentivirus into the nanoparticles. The obtained lentiviral transfection-enhanced nanoparticles are abbreviated as PS@Virus. In the obtained lentiviral transfection-enhanced nanoparticles, the nanoparticle titer is 1000TU, and the lentivirus titer is 10 5 TU, 10 6 TU, 10 7 TU, 10 8 TU, 10 9 TU.

[0060] Experimental Example 1

[0061] The nanoparticles PS and the virus-loaded nanoparticles PS@Virus used in this experimental example were prepared by Example 1.

[0062] 1. Characterization of Lentiviral Transfection Enhanced Nanoparticles

[0063] 1.1 Characterization of physical and chemical properties of lentiviral transfection-enhanced nanoparticles

[0064] Detection method:

[0065] Transmission electron microscope (TEM) was used to detect the morphology, particle size, dispersion and other properties of the nanoparticles, and statistical analysis was performed.

[0066] Malvern laser particle size analyzer was used to detect the average particle size, surface potential, confidence value and other data of nanoparticles, and statistical analysis was performed.

[0067] The prepared nanoparticles PS and virus-loaded nanoparticles PS@Virus were placed at 4°C or 37°C at the same time, and the retention of nanoparticles was measured for 10 consecutive days to reflect the stability of the nanoparticles under different temperature conditions.

[0068] PS and PS@Virus were placed in α-MEM medium and high-fat medium (HF) at the same time, and the retention of nanoparticles was measured for 10 consecutive days to reflect the stability of nanoparticles under different buffer conditions.

[0069] The formula of high-fat medium (HF) is as follows: DMEM finished medium + 500 μM oleic acid + 500 μM palmitic acid + 10% FBS. The DMEM medium is: Dulbecco's modified eagle medium (DMEM, Biological Industries, Israel).

[0070] Experimental results:

[0071] like Figure 1 As shown in Figure A, the nanoparticles PS prepared based on polyethylene glycol and spermidine and the nanoparticles PS@Virus loaded with lentivirus have good dispersion, with average particle sizes of 216±15.8nm and 318±43.3nm, respectively. Figure 1 B); Figure 1 As shown in Figure 1C and Figure 1D, the surface potentials of both PS and PS@Virus nanoparticles are positive and the confidence values ​​of the data measured by the Malvern laser particle size analyzer meet the requirements (PDI < 0.5). Figure 1 As shown in Figure 1E and Figure 1F, the nanoparticle PS is relatively stable in different temperatures and buffers, and the degradation amount does not exceed 20% within 10 days; although the stability of the nanoparticle PS@Virus loaded with lentivirus has decreased, it still retains more than 75% within 10 days, and there is no significant statistical difference between the retention amount of the nanoparticle PS and that of the nanoparticle PS.

[0072] The above results indicate that nanoparticles PS and nanoparticles PS@Virus loaded with lentivirus have good stability at both storage temperature (refrigerator, 4°C) and working temperature (body temperature, 37°C), and the particle size is appropriate and meets the basic requirements for penetrating the cell membrane.

[0073] 1.2 Testing of biocompatibility and transfection efficiency of nanoparticles loaded with lentivirus overexpressing ALDH6A1

[0074] Detection method:

[0075] The titers of the lentivirus used were 0TU, 10 5 TU, 10 6 TU, 10 7 TU, 10 8TU nanoparticles were transfected into osteoblasts, and the CCK8 method was used to detect the effects of each concentration gradient on the proliferation activity of osteoblasts to reflect the cytotoxicity of nanoparticles with different lentiviral loading titers.

[0076] The titers of the lentivirus used were 0TU, 10 5 TU, 10 6 TU, 10 7 TU, 10 8 TU, 10 9 TU nanoparticles were transfected into the knee joint capsule tissue of mice, and the changes in mouse body weight were continuously detected on days 1, 4, 7, and 14 after transfection to reflect the animal toxicity of nanoparticles loaded with different lentiviral titers.

[0077] The lentivirus overexpressing ALDH6A1 was loaded onto a commercial transfection enhancement reagent, recorded as the Commercialization@Virus group; the lentivirus overexpressing ALDH6A1 was loaded onto the nanoparticles provided by the present invention, recorded as the PS@Virus group; the unloaded lentivirus overexpressing ALDH6A1 was used as a negative control, recorded as the Virus group; and the qRT-PCR method was used to detect the transcription level of ALDH6A1 in osteoblasts on the 1st, 3rd, and 5th day after the transfection operation to reflect the transfection efficiency of the nanoparticles loaded with the lentivirus overexpressing ALDH6A1.

[0078] Experimental results:

[0079] like Figure 2 As shown in Figure A, different titers of lentivirus-loaded nanoparticles transfected cells had significant differences in the effects on the proliferation activity of osteoblasts. In short, compared with the control group (NC group) not transfected with lentivirus, the empty PS vector of nanoparticles not loaded with lentivirus had good biocompatibility, and the titer of lentivirus loaded with nanoparticles was 10 6 There is no obvious cytotoxicity in TU. Figure 2 As shown in Figure B, the effects of different titers of lentivirus-loaded nanoparticles on the body weight of mice after transfection of knee joint tissues were significantly different. In short, compared with the control group (NC group) not transfected with lentivirus, the nanoparticles without lentivirus-loaded empty vector PS and the loaded lentivirus titers at 10 6 There was no significant difference in the body weight of the experimental groups within TU. The above in vivo and in vitro experiments showed that the titer of lentivirus should be controlled at 10 6 TU or less, while significantly improving the transfection efficiency, it also has good biocompatibility.

[0080] like Figure 2As shown in C, the results of qRT-PCR showed that the transcription level of ALDH6A1 in osteocytes of the PS@Virus group was significantly higher than that of the other two groups on days 1, 3, and 5 after transfection; and compared with the transfection efficiency of the lentivirus overexpressing ALDH6A1 using a commercial transfection enhancement reagent, the lentivirus transfection enhancement nanoparticles provided by the present invention loaded with the lentivirus overexpressing ALDH6A1 can achieve a higher and more lasting transfection effect. The above results show that compared with transfecting cells with lentivirus alone (Virus group) or transfecting cells with lentivirus loaded with a commercial transfection enhancement reagent (polybrene) (Commercialization@Virus group), loading the lentivirus onto the nanoparticles provided by the present invention (PS@Virus group) significantly improves the transfection efficiency of the target gene.

[0081] 2. Effect of lentivirus-loaded nanoparticles overexpressing ALDH6A1 on the biological activity remodeling of osteoblasts in a high-fat environment

[0082] 2.1 Effects of lentiviral nanoparticles overexpressing ALDH6A1 on osteoblast proliferation and apoptosis

[0083] Detection method:

[0084] Firstly, osteoblasts transfected with empty nanoparticle vectors were incubated in high-fat culture medium, recorded as the HF-PS group, and osteoblasts transfected with nanoparticles carrying lentivirus overexpressing ALDH6A1 were incubated in high-fat culture medium, recorded as the HF-PS@Virus group; secondly, the CCK8 method was used to detect the effect of nanoparticles transfected with lentivirus overexpressing ALDH6A1 on the proliferation activity of remodeled osteoblasts; finally, the EdU fluorescence staining method was used to detect the effect of nanoparticles transfected with lentivirus overexpressing ALDH6A1 on the physiological activity of remodeled osteoblasts, and statistical analysis was performed.

[0085] Experimental results:

[0086] According to the in vitro and in vivo biosafety evaluation data in “1.2 Biocompatibility and transfection efficiency testing of nanoparticles loaded with lentivirus overexpressing ALDH6A1”, this experiment was performed with a lentivirus titer of 10 6 TU used nanoparticles loaded with lentivirus overexpressing ALDH6A1 (HF-PS@Virus group) and empty vectors without lentivirus (HF-PS group) to transfect osteoblasts. CCK8 detection results showed that in a high-fat culture environment, the proliferation activity of osteoblasts in the HF-PS@Virus group was significantly enhanced compared with the HF-PS group ( Figure 3EdU staining results showed that nanoparticles transfected with lentivirus overexpressing ALDH6A1 significantly reduced the apoptotic rate of osteoblasts ( Figure 3 B and C).

[0087] 2.2 Detection of the effect of lentiviral nanoparticles overexpressing ALDH6A1 on ROS levels in osteoblasts

[0088] Detection method:

[0089] Firstly, osteoblasts transfected with empty nanoparticle vectors were incubated in high-fat culture medium, recorded as the HF-PS group, and osteoblasts transfected with nanoparticles carrying lentivirus overexpressing ALDH6A1 were incubated in high-fat culture medium, recorded as the HF-PS@Virus group; secondly, DCFH-DA fluorescence staining was used to detect the effect of nanoparticles transfected with lentivirus overexpressing ALDH6A1 on the ROS level in osteoblasts in a high-fat environment, and statistical analysis was performed; finally, Mito-SOX fluorescence staining was used to detect the effect of nanoparticles transfected with lentivirus overexpressing ALDH6A1 on the ROS level in the mitochondria of osteoblasts in a high-fat environment, and statistical analysis was performed.

[0090] Experimental results:

[0091] like Figure 4 As shown in A and B, the results of DCFH-DA fluorescence staining showed that compared with the HF-PS group, the ROS level in osteoblasts under high-fat environment was significantly reduced after transfection with nanoparticles loaded with lentivirus overexpressing ALDH6A1; at the same time, the results of Mito-SOX staining also showed that compared with the HF-PS group, the ROS level in osteoblast mitochondria under high-fat environment was also significantly reduced after transfection with nanoparticles loaded with lentivirus overexpressing ALDH6A1 ( Figure 4 C and D).

[0092] The above results indicate that nanoparticles loaded with lentivirus overexpressing ALDH6A1 can efficiently introduce lentivirus vectors overexpressing ALDH6A1 into osteoblasts, and utilize overexpression of ALDH6A1 to significantly reduce the level of oxidative stress in cells and mitochondria, promote the recovery of osteoblast proliferation activity, and thereby reshape the biological activity of osteoblasts in a high-fat environment.

[0093] 3. Effect of lentivirus-loaded nanoparticles overexpressing ALDH6A1 on implant bone integration in hyperlipidemic mice

[0094] 3.1 Construction of hyperlipidemia mouse model and experimental grouping

[0095] First, C57 mice were fed with a high-fat diet for 30 days until their blood lipid levels significantly increased to 3 times that of mice fed with a normal diet, and they were defined as hyperlipidemic mice. Second, C57 mice fed with a normal diet and the above-mentioned high-fat diet-induced hyperlipidemia mice were used for animal experiments and divided into two groups: a control treatment group (HF-PS group) in which mice were fed with a high-fat diet to form hyperlipidemia and nanoparticles were injected locally into the knee joint capsule, and a treatment group (HF-PS@Virus group) in which mice were fed with a high-fat diet to form hyperlipidemia and nanoparticles loaded with lentivirus overexpressing ALDH6A1 were injected locally into the knee joint capsule. Mice in each group were implanted with a titanium implant with a length of 3 mm and a diameter of 1 mm in the distal femur and received an injection treatment every 5 days until the mice were killed 30 days later and the corresponding specimens were obtained ( Figure 5 ).

[0096] Continuous monitoring of physiological indicators such as body weight of each group of mice showed that repeated local injection of lentivirus or empty vector had no adverse effects on mice, that is, the lentiviral transfection-enhancing nanoparticles provided by the present invention had good in vivo safety.

[0097] 3.2 Effect of lentiviral nanoparticles overexpressing ALDH6A1 on implant bone integration in hyperlipidemic mice

[0098] Detection method:

[0099] First, the femoral specimens were fixed with paraformaldehyde and dehydrated with ethanol gradient. Second, the titanium femoral implants were removed and embedded in paraffin to obtain 5 μm thick tissue sections. Third, the expression of ALDH6A1 in the tissue sections was detected by immunofluorescence staining, and the effect of local injection of nanoparticles overexpressing ALDH6A1 lentivirus on increasing the expression of ALDH6A1 in the tissue around the implant was analyzed. Finally, microcomputed tomography (Micro-CT) was used to evaluate the implant bone integration and the proportion of new bone around the titanium implant in the remaining mouse femoral specimens, and then the effect of nanoparticles overexpressing ALDH6A1 lentivirus on improving the bone integration level under hyperlipidemia was analyzed.

[0100] Experimental results:

[0101] like Figure 6As shown in Figure A, the results of immunofluorescence staining of mouse femoral tissue sections showed that the expression of ALDH6A1 around the implants of mice in the control treatment group (HF-PS group) that received local injection of nanoparticles into the knee joint capsule was significantly reduced, while the expression of ALDH6A1 in the bone tissue around the implants of mice in the treatment group (HF-PS@Virus group) that received local injection of nanoparticles loaded with lentivirus overexpressing ALDH6A1 was significantly increased ( Figure 6 (B) The above results indicate that nanoparticles loaded with lentivirus overexpressing ALDH6A1 can successfully introduce lentivirus into bone tissue and efficiently transfect into tissue cells.

[0102] like Figure 6 As shown in C and D, the results of evaluating the bone integration of implants in each group of mice by micro computed tomography (Micro-CT) showed that the bone integration rate of mice in the HF-PS@Virus group was significantly improved compared with that in the HF-PS group (8.8±1.6% vs 16.6±2.2%). The above results indicate that local injection of nanoparticles loaded with lentivirus overexpressing ALDH6A1 can effectively enhance the intensity of osteogenic activity on the implant surface under hyperlipidemia and ultimately improve the bone integration effect.

[0103] In summary, in this embodiment, transfection-enhanced nanoparticles loaded with lentivirus overexpressing ALDH6A1 were successfully developed, which have good in vivo biocompatibility and can efficiently introduce the lentivirus loaded in the nanoparticles into tissue cells to increase the expression of ALDH6A1 in the tissue around the implant, thereby significantly reshaping the biological activity of osteoblasts under hyperlipidemia, and ultimately improving the implant bone integration effect under hyperlipidemia.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanoparticle, characterized in that: The nanoparticles are prepared from polyethylene glycol and spermidine through self-assembly.

2. The nanoparticle according to claim 1, characterized in that The molecular weight of the polyethylene glycol is 4000-8000; Preferably, the particle size of the nanoparticles is 200-250 nm.

3. A method for preparing the nanoparticles according to any one of claims 1 to 2, characterized in that: include: dissolving polyethylene glycol and spermidine in water to obtain a mixed solution; The mixed solution was stirred at 0-5° C. and 100-300 rpm for 1-3 h, and centrifuged to obtain nanoparticles.

4. The preparation method according to claim 3, characterized in that: In the mixed solution, the concentration of polyethylene glycol is 1-20 mg / mL, and the concentration of spermidine is 0.5-10 μM.

5. Use of the nanoparticles according to any one of claims 1 to 2 or the nanoparticles prepared by the preparation method according to claim 3 or 4 in preparing a product that improves lentiviral transfection efficiency.

6. The use according to claim 5, characterized in that The product is a kit, a transfection enhancer or a vector for improving the efficiency of lentiviral transfection; Preferably, the product is used for lentiviral transfection of cells or tissues with poor cell membrane fluidity; and / or lentiviral transfection of cells or tissues with a long growth cycle; Preferably, the cells or tissues are osteoblasts and bone tissue.

7. A method for improving lentiviral transfection efficiency, characterized in that: The nanoparticles according to any one of claims 1 to 2 or the nanoparticles prepared by the preparation method according to claim 3 or 4 are loaded with lentivirus, and gene transfection is performed on the target sample in an in vitro experiment; Preferably, the target sample includes tissues or cells with poor cell membrane fluidity or long growth cycle; Preferably, the target sample is an osteoblast or a bone tissue sample.

8. A lentiviral transfection enhancing nanoparticle, characterized in that: It is a nanoparticle loaded with lentivirus; In the lentiviral transfection enhanced nanoparticles, the titer of the nanoparticles is 900-1100TU, and the titer of the lentivirus is ≤10 6 TU; The nanoparticles are the nanoparticles according to any one of claims 1 to 2 or the nanoparticles prepared by the preparation method according to claim 3 or 4; Preferably, the particle size of the lentiviral transfection enhancing nanoparticles is 300-350 nm; Preferably, the lentivirus is a lentivirus that overexpresses ALDH6A1.

9. A method for preparing the lentiviral transfection-enhancing nanoparticles according to claim 8, characterized in that: include: The lentivirus is mixed with the nanoparticles described in any one of claims 1-2 or the nanoparticles prepared by the preparation method described in claim 3 or 4 to achieve loading of the lentivirus into the nanoparticles.

10. Use of the lentiviral transfection enhancing nanoparticles according to claim 8 in preparing lentiviral transfection products; Preferably, the product is a medicine, a kit or a transfection reagent; Preferably, the product is used for lentiviral transfection of cells or tissues with poor cell membrane fluidity and / or lentiviral transfection of cells or tissues with a long growth cycle; Preferably, the product is used for lentiviral transfection of osteoblasts and / or lentiviral transfection of bone tissue; Preferably, the product is used for lentiviral transfection of bone tissue around implants.

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