CN108956540A - A kind of SPR method of quick screening charge reversal type cationic gene carriers - Google Patents
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 37
- 125000002091 cationic group Chemical group 0.000 title claims abstract description 34
- 239000000969 carrier Substances 0.000 title claims abstract description 30
- 238000012216 screening Methods 0.000 title claims abstract description 17
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000011156 evaluation Methods 0.000 claims abstract description 16
- 239000003642 reactive oxygen metabolite Substances 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
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- 239000002105 nanoparticle Substances 0.000 claims description 14
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- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 238000004401 flow injection analysis Methods 0.000 claims description 4
- 150000002978 peroxides Chemical class 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims 2
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract 1
- 230000003578 releasing effect Effects 0.000 abstract 1
- 230000000007 visual effect Effects 0.000 abstract 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 17
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 7
- 239000010931 gold Substances 0.000 description 7
- 229910052737 gold Inorganic materials 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000002926 oxygen Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
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Abstract
Description
技术领域technical field
本发明涉及基因载体性能的评估方法技术领域，尤其涉及一种快速筛选电荷反转型阳离子基因载体的SPR方法。The invention relates to the technical field of evaluation methods for the performance of gene carriers, in particular to an SPR method for rapidly screening charge-reversal cationic gene carriers.
背景技术Background technique
基因治疗是目前治疗遗传性疾病的有效方法,但是DNA体积大和负电荷的性质影响了细胞膜的摄取，并且静脉注射的游离DNA会被血液中的血清核酸酶快速降解,以上缺点都限制了基因治疗在临床的应用。针对基因治疗存在的问题，人们正在积极的寻找有效的基因载体来进行药物传递。非病毒基因载体由于具有良好的生物相容性、安全和可以大批量生产等优点受到人们的关注。在众多基因载体中阳离子载体由于其具有低毒性和转染效率高的特点相比于其他非病毒基因载体来说被更广泛的用于基因治疗的研究。但是阳离子载体/DNA复合物纳米颗粒是热力学稳定的，通常难于解离，这极大的阻碍了在细胞中对DNA的释放，影响转染效率。于是有研究学者设计出了能够在活性氧簇作用下发生从正电荷向负电荷转变的阳离子载体，该种载体在肿瘤细胞等活性氧簇相对较高的病变细胞中发生快速的电荷反转，释放DNA，从而解决阳离子载体转染效率低的问题。然而，电荷反转型阳离子载体热稳定性以及对DNA的负载及释放研究如果在细胞或组织中进行，不仅需要较高的实验条件和复杂的实验步骤，并且不能在线、实时的对其进行监测和观察，增加了实验研究的时间，不能再较短的时间内对载体的性能进行评价。因此，建立一种能够在体外在线、快速、实时评价载体对DNA负载及释放能力的研究方法对于缩短基因载体性能评价时间，建立快速筛选性能优良的载体的方法，加速基因药物的研发意义重大。Gene therapy is currently an effective method for the treatment of genetic diseases, but the large size of DNA and the nature of negative charges affect the uptake of cell membranes, and the free DNA injected intravenously will be rapidly degraded by serum nucleases in the blood, all of which limit gene therapy. In clinical application. Aiming at the problems of gene therapy, people are actively looking for effective gene carriers for drug delivery. Non-viral gene vectors have attracted people's attention due to their good biocompatibility, safety and mass production. Among many gene carriers, cationic carriers are more widely used in gene therapy research than other non-viral gene carriers due to their low toxicity and high transfection efficiency. However, cationic carrier/DNA complex nanoparticles are thermodynamically stable and are usually difficult to dissociate, which greatly hinders the release of DNA in cells and affects the transfection efficiency. Therefore, some researchers have designed a cationic carrier that can change from positive charge to negative charge under the action of reactive oxygen species. This kind of carrier undergoes rapid charge reversal in diseased cells with relatively high reactive oxygen species such as tumor cells. Release DNA, thereby solving the problem of low transfection efficiency of cationic carriers. However, if the thermal stability of the charge-reversal cation carrier and the loading and release of DNA are carried out in cells or tissues, it not only requires high experimental conditions and complicated experimental steps, but also cannot be monitored online and in real time. And observation, increase the time of experimental research, can not evaluate the performance of the carrier in a shorter time. Therefore, establishing a research method that can evaluate the DNA load and release capacity of the carrier in vitro online, quickly and in real time is of great significance for shortening the performance evaluation time of the gene carrier, establishing a method for quickly screening the carrier with excellent performance, and accelerating the research and development of the gene drug.
表面等离子体共振仪(SPR)由于具有待测物免标记、灵敏度高、实时监测反应、背景干扰小等优点被广泛用于物理、化学、生物及生命科学等领域。本发明利用SPR技术能够在线快速监测膜上物质结构变化的特点，建立了一种体外在线、快速评价阳离子基因载体性能的传感方法。Surface plasmon resonance (SPR) has been widely used in the fields of physics, chemistry, biology, and life sciences due to its advantages of label-free, high sensitivity, real-time monitoring of reactions, and low background interference. The invention utilizes the characteristic that the SPR technology can quickly monitor the change of the material structure on the membrane, and establishes a sensing method for in vitro online and rapid evaluation of the performance of the cationic gene carrier.
发明内容Contents of the invention
基于背景技术存在的技术问题，本发明提供了一种免标记、灵敏、在线的研究电荷反转型阳离子基因载体对DNA运载及释放的SPR方法，利用这种方法可以快速在线的检测载体与任何DNA的结合与解离，解决影响DNA释放与转染效率的阳离子载体的热力学稳定性问题。该方法具有简单、快速、可以在线检测的优点。Based on the technical problems existing in the background technology, the present invention provides a label-free, sensitive and online SPR method for studying the delivery and release of DNA by charge-reversal cationic gene carriers. This method can be used to quickly detect the carrier and any The combination and dissociation of DNA solves the problem of thermodynamic stability of cationic carriers that affect DNA release and transfection efficiency. The method has the advantages of being simple, fast and capable of on-line detection.
本发明之所以能够实现上述目的，其特征在于：Why the present invention can realize above-mentioned object, it is characterized in that:
1.在线评价电荷反转型阳离子基因载体对DNA的负载及释放能力，对载体进行初步评价和筛选。1. Online evaluation of the charge-reversal cationic gene carrier's ability to load and release DNA, and conduct preliminary evaluation and screening of the carrier.
2.在线评价载体/DNA复合纳米粒子在金膜表面的固定以及在不同浓度活性氧物质作用下对DNA的释放能力，为载体在细胞或是活体中的应用提供保障。2. Online evaluation of the immobilization of the carrier/DNA composite nanoparticles on the surface of the gold film and the ability to release DNA under the action of different concentrations of reactive oxygen species provide guarantee for the application of the carrier in cells or in vivo.
3.基于不同原理的电荷反转型阳离子基因载体的性能均可用此方法进行评价，从而对载体进行筛选。3. The performance of charge-reversal cationic gene carriers based on different principles can be evaluated by this method, so as to screen the carriers.
本发明的技术方案如下：Technical scheme of the present invention is as follows:
一种快速筛选电荷反转型阳离子基因载体的SPR方法，包括以下步骤：A kind of SPR method of rapid screening charge reversal cationic gene carrier, comprises the following steps:
A、在修饰了一定浓度电荷反转型阳离子基因载体的金膜表面流动注射不同浓度的DNA，利用实验室自行搭建的差分型表面等离子体基元共振仪，在线实时监测载体对DNA的捕捉情况，根据不同浓度DNA作用下SPR信号的变化，评价载体对DNA的负载能力；A. Flow inject different concentrations of DNA on the surface of the gold film modified with a certain concentration of charge-reversal cationic gene carrier, and use the differential surface plasmon resonance instrument built by the laboratory to monitor the capture of DNA by the carrier in real time. , according to the change of SPR signal under the action of different concentrations of DNA, evaluate the load capacity of the carrier to DNA;
B、将阳离子载体与DNA在一定的N/P比条件下合成的载体/DNA复合物纳米粒子；B. Carrier/DNA composite nanoparticles synthesized by cationic carrier and DNA under certain N/P ratio conditions;
C、在修饰了一定浓度载体/DNA复合物纳米粒子的金膜表面流动注射不同浓度的过氧化氢，根据不同浓度过氧化氢作用下SPR信号的变化，评价纳米粒子对DNA的释放情况。C. Flow injection of different concentrations of hydrogen peroxide on the surface of the gold film modified with a certain concentration of carrier/DNA complex nanoparticles, and evaluate the release of nanoparticles to DNA according to the change of SPR signal under the action of different concentrations of hydrogen peroxide.
优选的，所述的过氧化氢可以替换为任何活性氧物质。如过氧化物、生物酶、人工酶、含氧自由基等。Preferably, the hydrogen peroxide can be replaced by any reactive oxygen species. Such as peroxides, biological enzymes, artificial enzymes, oxygen-containing free radicals, etc.
优选的，基于不同原理的电荷反转型阳离子基因载体的性能均可用此方法进行筛选。Preferably, the performance of charge-reversal cationic gene carriers based on different principles can be screened by this method.
本发明的快速筛选电荷反转型阳离子基因载体的SPR方法，在线评价电荷反转型阳离子基因载体对DNA的负载及释放能力，对载体进行初步评价和筛选；在线评价载体/DNA复合纳米粒子在金膜表面的固定以及在不同浓度活性氧物质作用下对DNA的释放能力，为载体在细胞或是活体中的应用提供前期保障；基于不同原理的电荷反转型阳离子基因载体的性能均可用此方法进行评价，从而对载体进行筛选。The SPR method for rapidly screening the charge-reversal cationic gene carrier of the present invention evaluates the load and release capacity of the charge-reversal cationic gene carrier to DNA on-line, and initially evaluates and screens the carrier; online evaluation carrier/DNA composite nanoparticle The immobilization of the surface of the gold film and the ability to release DNA under the action of different concentrations of active oxygen species provide early guarantee for the application of the carrier in cells or in vivo; the performance of the charge-reversal cationic gene carrier based on different principles can be used. Methods were evaluated to screen vectors.
本发明的有益之处在于：The benefits of the present invention are:
1)电荷反转型阳离子基因载体对DNA负载及释放的SPR在线研究方法，无需标记样品，操作简单，检测结果快速直观。1) The SPR online research method of DNA loading and release by charge-reversal cationic gene carriers does not need to label samples, the operation is simple, and the detection results are fast and intuitive.
2)通过该方法可以在线、快速的研究电荷反转型阳离子基因载体对DNA的负载以及在活性氧物质作用下对DNA的释放作用，对载体的性能进行更加直观的评价。用过氧化氢或酶等模拟体内的活性氧簇，在线研究载体在不同浓度的活性氧物质作用下对DNA的释放效果。2) Through this method, the loading of DNA by the charge-reversal cationic gene carrier and the release of DNA under the action of reactive oxygen species can be studied online and rapidly, and the performance of the carrier can be more intuitively evaluated. Use hydrogen peroxide or enzymes to simulate reactive oxygen species in the body, and study the release effect of the carrier on DNA under the action of different concentrations of reactive oxygen species online.
3)该方法适用于基于不同原理的电荷反转型阳离子基因载体性能的研究，是一种简单、快速的在线研究电荷反转型阳离子载体对药物的运载及释放的方法，为药物载体研究提供了一种新的药物载体评价方法，有助于药物载体的快速筛选。3) This method is suitable for the study of the performance of charge-reversal cationic gene carriers based on different principles. It is a simple and fast online method for studying the drug delivery and release of charge-reversal cationic carriers, and provides a good source for drug carrier research. A new evaluation method for drug carriers was developed, which is helpful for the rapid screening of drug carriers.
本发明用过氧化氢或酶等模拟体内的活性氧簇，在线研究载体在不同浓度的活性氧物质作用下对DNA的释放效果。旨在帮助评价基因载体的性能，缩短基因载体性能评价时间，建立快速筛选性能优良的载体的方法，加速基因药物的研发。本方法无需标记样品，实验步骤简单，检测快，现象直观，是一种体外快速评价基因载体性能的理想研究方法。The invention uses hydrogen peroxide or enzymes to simulate the active oxygen clusters in the body, and studies online the release effect of the carrier on the DNA under the action of active oxygen species of different concentrations. The aim is to help evaluate the performance of gene carriers, shorten the evaluation time of gene carrier performance, establish a method for rapid screening of carriers with excellent performance, and accelerate the research and development of gene drugs. The method does not need to label samples, the experimental steps are simple, the detection is fast, and the phenomenon is intuitive. It is an ideal research method for rapidly evaluating the performance of gene carriers in vitro.
附图说明Description of drawings
图1：固定载体后，流动注射不同浓度的DNA的SPR信号图；Figure 1: After immobilizing the carrier, the SPR signal diagram of flow injection of different concentrations of DNA;
图2：合成的载体/DNA复合纳米粒子的SEM表征图；Figure 2: SEM characterization of the synthesized carrier/DNA composite nanoparticles;
图3：在金膜上固定载体/DNA复合纳米粒子后对DNA释放的SPR信号图。Figure 3: SPR signal map of DNA release after immobilization of carrier/DNA composite nanoparticles on gold membrane.
具体实施方式Detailed ways
实施例1：Example 1:
在修饰了一定浓度电荷反转型阳离子基因载体的金膜表面流动注射不同浓度的过氧化氢，利用实验室自行搭建的差分型表明等离子体基元共振仪，在线实时监测载体对DNA的捕捉情况。跟据不同浓度DNA作用下SPR信号的变化，评价载体对DNA的负载能力。相应的SPR信号曲线图如图1所示。Flow inject different concentrations of hydrogen peroxide on the surface of the gold film modified with a certain concentration of charge-reversal cationic gene carrier, and use the differential type display plasmon resonance instrument built by the laboratory to monitor the capture of DNA by the carrier in real time . According to the change of SPR signal under the action of different concentrations of DNA, the load capacity of the carrier to DNA was evaluated. The corresponding SPR signal curve is shown in Fig. 1 .
实施例2：Example 2:
首先将载体与DNA在一定的N/P比条件下合成载体/DNA复合物纳米粒子，之后在修饰了一定浓度纳米粒子的金膜表面流动注射不同浓度的过氧化氢，例如10-5M-10-3M，在线观察不同浓度过氧化氢作用下的SPR信号变化，通过比较信号的大小来研究纳米粒子对DNA的释放与过氧化氢浓度的关系，从而在线、直观的评价药物载体在不同活性氧浓度的细胞中对基因药物的释放能力。相应的纳米粒子表征图如图2所示；SPR信号曲线如图3所示。First, the carrier and DNA are synthesized under a certain N/P ratio to synthesize carrier/DNA composite nanoparticles, and then flow inject different concentrations of hydrogen peroxide on the surface of the gold film modified with a certain concentration of nanoparticles, such as 10 -5 M- 10 -3 M, observe the SPR signal changes under the action of different concentrations of hydrogen peroxide online, and study the relationship between the release of nanoparticles to DNA and the concentration of hydrogen peroxide by comparing the size of the signal, so as to evaluate the drug carrier online and intuitively in different concentrations. Reactive oxygen concentration in cells for gene drug release capacity. The corresponding nanoparticle characterization diagram is shown in Figure 2; the SPR signal curve is shown in Figure 3.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.
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