CN118059068A - Berberine magnetic nanoparticle for treating oral squamous cell carcinoma - Google Patents
Berberine magnetic nanoparticle for treating oral squamous cell carcinoma Download PDFInfo
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Abstract
Description
技术领域Technical Field
本发明属于医药生物技术领域，具体涉及一种治疗口腔鳞状细胞癌的小檗碱的磁性纳米颗粒。The invention belongs to the field of medical biotechnology, and in particular relates to berberine magnetic nanoparticles for treating oral squamous cell carcinoma.
背景技术Background technique
口腔癌是头颈部最常见的恶性肿瘤之一，其中最主要的病理类型为鳞状细胞癌，约占90％以上。2018年，全球口腔癌新发病例354864例，死亡病例177384例。在地区分布上，亚洲高于美洲和欧洲，受人乳头瘤病毒(papillomavirus，HPV)、咀嚼槟榔等因素影响，发病率呈逐年上升趋势。目前，以手术为主的综合序列治疗仍是口腔鳞癌的主要治疗手段。口腔癌和口咽癌的5年生存率为65％，约50％的口腔癌患者在就诊时已处于晚期(Ⅲ～Ⅳ期)，预后较差[1]。尽管在诊断技术和治疗方面有改进和创新，手术往往导致功能丧失和毁容。此外，放疗口腔癌患者的总生存率都没有得到改善，因此，迫切需要开发与临床相关的、副作用最小的高效化疗药物。[2] Oral cancer is one of the most common malignant tumors of the head and neck, of which squamous cell carcinoma is the most common pathological type, accounting for more than 90%. In 2018, there were 354,864 new cases of oral cancer and 177,384 deaths worldwide. In terms of regional distribution, Asia is higher than America and Europe. Affected by factors such as human papillomavirus (HPV) and betel nut chewing, the incidence rate has been increasing year by year. At present, comprehensive sequential treatment with surgery as the main method is still the main treatment for oral squamous cell carcinoma. The 5-year survival rate of oral cancer and oropharyngeal cancer is 65%. About 50% of oral cancer patients are in the late stage (stage III to IV) at the time of diagnosis and have a poor prognosis [1] . Despite improvements and innovations in diagnostic technology and treatment, surgery often leads to functional loss and disfigurement. In addition, the overall survival rate of oral cancer patients treated with radiotherapy has not been improved. Therefore, there is an urgent need to develop clinically relevant and highly effective chemotherapy drugs with minimal side effects. [2]
《Berberine induces FasL-related apoptosis through p38 activation inKB human oral cancer cells》[2]报道了了小檗碱的化疗前景，表明它在肝癌细胞中表现出显著的细胞毒性，但对正常细胞的细胞毒性可以忽略不计，有可能作为一种化疗药物，但副作用小，用于治疗口腔癌。"Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells" [2] reported the chemotherapeutic prospects of berberine, showing that it exhibited significant cytotoxicity in liver cancer cells, but negligible cytotoxicity to normal cells, and could potentially be used as a chemotherapeutic drug with fewer side effects for the treatment of oral cancer.
《盐酸小檗碱通过调节自噬抑制口腔鳞状细胞癌增殖的研究》[3]报道了盐酸小檗碱抑制口腔鳞状细胞癌Cal-27细胞的自噬反应，并通过抑制自噬进一步抑制其增殖。"Berberine hydrochloride inhibits the proliferation of oral squamous cell carcinoma by regulating autophagy" [3] reported that berberine hydrochloride inhibits the autophagic response of oral squamous cell carcinoma Cal-27 cells and further inhibits its proliferation by inhibiting autophagy.
小檗碱的溶解度差，在生物药剂学分类系统中属于IV类药物[4],盐酸小檗碱渗透性差，限制了其在临床上的应用[5],尚无纯小檗碱配方被批准用于任何特定疾病[4]。小檗碱口服生物利用度低主要是由于其在酸性条件下自聚集导致的溶解度差、渗透性低、P-gp(P-glycoprotein)介导的外排和肝肠代谢[4]。所以需要开发适用于治疗口腔鳞状细胞癌的小檗碱制剂以克服这些问题。Berberine has poor solubility and is classified as a Class IV drug in the biopharmaceutics classification system [4] . Berberine hydrochloride has poor permeability, which limits its clinical application [5] . No pure berberine formulation has been approved for any specific disease [4] . The low oral bioavailability of berberine is mainly due to its poor solubility caused by self-aggregation under acidic conditions, low permeability, P-gp (P-glycoprotein)-mediated efflux and hepatoenterolysis [4] . Therefore, it is necessary to develop berberine preparations suitable for the treatment of oral squamous cell carcinoma to overcome these problems.
层状双氢氧化物(LDH)纳米颗粒在药物的装载和控释方面具有潜在的应用前景。LDH是一种正水滑石，由一个金属氢氧化物层和一个通常由阴离子基团和水分子占据的层间空间组成。这些层是由水分子、阴离子基团和氢氧根层之间的氢键形成的不同类型的有机和无机阴离子可以通过离子交换和共沉淀进入氢氧化物层空间。LDH具有以下特点：易于合成，化学成分可控且灵活，具有较大的表面积。这有助于它们在吸附工艺、催化、电化学、聚合物化学、生物医学和废水处理方面的潜在应用。[6]由于LDH具有阴离子交换容量大、表面积大、毒性低、在酸性环境下降解可控制ph释放等特点，因此被用作生物相容性和ph响应性的纳米载体来提高药物在肿瘤微环境中的释放效率[6-9]。Layered double hydroxide (LDH) nanoparticles have potential applications in drug loading and controlled release. LDH is a type of orthohydrotalcite consisting of a metal hydroxide layer and an interlayer space usually occupied by anionic groups and water molecules. These layers are formed by hydrogen bonds between water molecules, anionic groups and the hydroxide layer. Different types of organic and inorganic anions can enter the hydroxide interlayer space through ion exchange and coprecipitation. LDH has the following characteristics: easy synthesis, controllable and flexible chemical composition, and large surface area. This contributes to their potential applications in adsorption processes, catalysis, electrochemistry, polymer chemistry, biomedicine, and wastewater treatment. [6] Due to its large anion exchange capacity, large surface area, low toxicity, degradation in acidic environment and pH-controlled release, LDH is used as a biocompatible and pH-responsive nanocarrier to improve the release efficiency of drugs in the tumor microenvironment [6-9] .
基于四氧化三铁的纳米颗粒在许多领域的应用已经引起了巨大的科学兴趣。四氧化三铁由于其易得性、通用性、生物相容性、可生物降解性和特殊的磁性而受到特别的研究。当暴露在快速交变的磁场中时，这些粒子释放热能，这一过程被称为磁热疗。这一过程允许对紧密定位于MNP的细胞进行加热。此外，MNP的高表面积意味着它们可以很容易地功能化，以增强其生物相容性，或固定药物，或靶向剂，如抗体。表面对癌细胞的功能化有可能产生更特异性的细胞杀伤，同时减少对健康组织的脱靶效应[10]。Nanoparticles based on ferroferric oxide have attracted great scientific interest for applications in many areas. Fiber tetroxide has been particularly studied due to its ready availability, versatility, biocompatibility, biodegradability, and unique magnetic properties. When exposed to a rapidly alternating magnetic field, these particles release thermal energy, a process known as magnetic hyperthermia. This process allows for the heating of cells that are closely located to the MNPs. In addition, the high surface area of MNPs means that they can be easily functionalized to enhance their biocompatibility or to immobilize drugs or targeting agents such as antibodies. Functionalization of the surface has the potential to produce more specific cell killing of cancer cells while reducing off-target effects on healthy tissues [10] .
在纳米颗粒表面修饰聚乙二醇可增强载体的渗透性和保留效应，可将纳米载体有效地富集于肿瘤部位，有效避免网状内皮系统对纳米载体的吞噬，显著延长纳米材料在体内的循环时间，增强药物疗效的同时减少对正常组织的毒副作用[11，12]。Modifying the surface of nanoparticles with polyethylene glycol can enhance the permeability and retention effect of the carrier, effectively enrich the nanocarrier at the tumor site, effectively prevent the phagocytosis of the nanocarrier by the reticuloendothelial system, significantly prolong the circulation time of the nanomaterials in the body, enhance the efficacy of the drug while reducing the toxic side effects on normal tissues [11, 12] .
《聚乙二醇和叶酸对层状双金属氢氧化物颗粒的表面修饰》[13]报道了:以具有伪装隐形作用的聚乙二醇(PEG)和具有靶向作用的叶酸(FA)为修饰剂,以氨丙基三甲氧基硅烷(APTMS)为连接剂,对Mg3Al-NO3层状双金属氢氧化物(LDH)进行了表面修饰,制备了LDH-PEG-FA纳米颗粒,并通过X射线衍射(XRD)、透射电子显微镜(TEM)、紫外-可见光谱(UV-Vis)、傅里叶变换红外光谱(FTIR)、粒度分布分析和元素分析等技术对其结构进行了表征.结果表明,PEG和FA的修饰量可由其原料配比调控,修饰后的产物具有良好的水再分散性,这主要源于修饰层的空间位阻效应.预期LDH-PEG-FA同时具有伪装隐形性和靶向性,可用于药物载体等领域，在该文献中LDH-PEG-FA平均粒径记载在2.5、LDH-PEG-FA的水再分散性TEM观察其平均粒径为190nm，粒度分布分析其平均粒径192nm，在没有载药的情况下空白纳米颗粒粒径就较大，表明其水再分散性仍然较差，并且未进行载药率考察。"Surface modification of layered double hydroxide particles by polyethylene glycol and folic acid" [13] reported that Mg3Al-NO3 layered double hydroxide (LDH) was surface modified with polyethylene glycol (PEG) with camouflage and stealth effects and folic acid (FA) with targeting effects as modifiers and aminopropyltrimethoxysilane (APTMS) as a linker to prepare LDH-PEG-FA nanoparticles. The structure of the nanoparticles was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), particle size distribution analysis and elemental analysis. The results showed that PE The modification amount of G and FA can be regulated by the ratio of their raw materials, and the modified product has good water redispersibility, which is mainly due to the steric hindrance effect of the modification layer. It is expected that LDH-PEG-FA has both camouflage and targeting properties, and can be used in fields such as drug carriers. In this document, the average particle size of LDH-PEG-FA is recorded at 2.5. The average particle size of LDH-PEG-FA in water redispersibility TEM observation is 190nm, and the average particle size distribution analysis shows that the average particle size is 192nm. In the absence of drug loading, the particle size of blank nanoparticles is larger, indicating that its water redispersibility is still poor, and the drug loading rate has not been investigated.
申请号201510611392.X的中国专利《一种pH敏感型Fe3O4@LDH负载甲氨蝶呤的纳米药物颗粒、制备方法及其应用》报道了：本发明公开了一种pH敏感型Fe3O4@LDH负载甲氨蝶呤的纳米药物颗粒，将LDH直接加入到Fe3O4纳米粒子的制备体系中，在Fe3O4纳米粒子形成的同时LDH经过自组装的形式包覆至Fe3O4纳米粒子的表面，生成Fe3O4@LDH载体，MTX则通过主体-客体交换技术能够有效地负载到Fe3O4@LDH载体上。本发明采用的LDH能进一步地提高Fe3O4纳米粒子的生物相容性，并且LDH自身的特殊结构能够很大程度地提高抗癌药物的负载量。本发明制备出的Fe3O4@LDH-MTX具有优异的药物缓释性能，对肿瘤细胞的抑制效果明显，具有很好的抗肿瘤效果，在靶向肿瘤治疗领域具有很大的应用潜能。该专利说明书第5段记载了甲氨蝶呤的负载量为4％～15％，负载量较低，负载量低药物可能达不到治疗剂量；从该文献的说明书附图上看粒径约200nm，较大，表明其水再分散性仍然较差；而且不具有主动靶向性，无法主动靶向肿瘤细胞。The Chinese patent application number 201510611392.X, "A pH-sensitive Fe 3 O 4 @LDH-loaded methotrexate nanoparticle, preparation method and application thereof", reports: The present invention discloses a pH-sensitive Fe 3 O 4 @LDH-loaded methotrexate nanoparticle, LDH is directly added to the preparation system of Fe 3 O 4 nanoparticles, and LDH is self-assembled and coated on the surface of Fe 3 O 4 nanoparticles while Fe 3 O 4 nanoparticles are formed, generating Fe 3 O 4 @LDH carriers, and MTX can be effectively loaded onto the Fe 3 O 4 @LDH carriers through host-guest exchange technology. The LDH used in the present invention can further improve the biocompatibility of Fe 3 O 4 nanoparticles, and the special structure of LDH itself can greatly increase the loading amount of anticancer drugs. The Fe 3 O 4 @LDH-MTX prepared by the present invention has excellent drug sustained release performance, obvious inhibitory effect on tumor cells, good anti-tumor effect, and great application potential in the field of targeted tumor treatment. Paragraph 5 of the patent specification records that the loading amount of methotrexate is 4% to 15%, which is relatively low. The drug with low loading amount may not reach the therapeutic dose; from the attached figure of the specification of the document, the particle size is about 200nm, which is relatively large, indicating that its water redispersibility is still poor; and it does not have active targeting and cannot actively target tumor cells.
申请号201710083481.0的中国专利《一种Fe3O4@LDH复合纳米材料的合成方法》报道了：本发明公开了一种Fe3O4@LDH复合纳米材料的合成方法。本发明通过一缩二乙二醇作为反应溶剂，Fe2+和Fe3+与碱溶液共沉淀反应制备得到超顺磁性Fe3O4纳米球。在保持Fe3O4纳米球均匀分散的基础上，与一缩二乙二醇溶液中的Al3+、Mg2+充分混合，然后与碱溶液发生共沉淀反应。本发明方法克服了现有技术中颗粒尺寸难以控制、颗粒分散不均、Fe3O4与LDH难以很好地形成层状复合纳米材料的缺陷，制备出比表面积大、粒径均一、形貌可调的Fe3O4@LDH层状纳米复合材料。从该专利的说明书附图上看，其Fe3O4@LDH复合纳米材料的粒径远超200nm，当纳米材料粒径＞200nm时，渗透滞留效应差，无法渗透到肿瘤深处[14]；也未对Fe3O4@LDH的分散度进行检验，无法证明该纳米颗粒分散均匀。The Chinese patent application number 201710083481.0, "A method for synthesizing Fe 3 O 4 @LDH composite nanomaterials", reports: The present invention discloses a method for synthesizing Fe 3 O 4 @LDH composite nanomaterials. The present invention uses glycol as a reaction solvent, and Fe 2+ and Fe 3+ coprecipitate with an alkaline solution to prepare superparamagnetic Fe 3 O 4 nanospheres. On the basis of keeping the Fe 3 O 4 nanospheres uniformly dispersed, they are fully mixed with Al 3+ and Mg 2+ in a glycol solution, and then coprecipitate with an alkaline solution. The method of the present invention overcomes the defects of the prior art that the particle size is difficult to control, the particles are unevenly dispersed, and Fe 3 O 4 and LDH are difficult to form a layered composite nanomaterial, and prepares a Fe 3 O 4 @LDH layered nanocomposite material with a large specific surface area, uniform particle size, and adjustable morphology. From the drawings in the patent specification, it can be seen that the particle size of the Fe 3 O 4 @LDH composite nanomaterial is far greater than 200nm. When the particle size of the nanomaterial is greater than 200nm, the penetration and retention effect is poor and it cannot penetrate deep into the tumor [14] . The dispersion of Fe 3 O 4 @LDH was not tested, and it is impossible to prove that the nanoparticles are evenly dispersed.
申请号201710083481.0的中国专利《一种HA靶向的双金属氢氧化物-超小铁纳米材料及其制备和应用》报道了：本发明涉及一种HA靶向的双金属氢氧化物-超小铁纳米材料及其制备和应用，制备：共沉淀法合成LDH-Fe3O4纳米复合材料，活化的透明质酸通过硅烷偶联剂共价键合在LDH层板表面，最后进行抗癌药物的表面负载。本发明的纳米材料颗粒分布均匀，可以在动物体内增强肿瘤部位MR成像效果。本发明的LDH-Fe3O4-HA NPs作为抗癌药物阿霉素DOX的载体，不仅具有敏感的pH响应释放特性，还可以对CD44受体表达的肿瘤细胞进行特异性识别，从而达到高效抑制肿瘤的理想化效果。该专利说明书49段记载，当DOX(甲氨蝶蛉)的含量相同时，经单纯DOX处理的黑色素瘤B16细胞的存活率都低于LDH-Fe3O4-HA/DOXNPs的存活率，当LDH-Fe3O4-HA/DOX NPs(DOX的浓度为12.5μg/mL)孵育细胞48h后，细胞存活率均在50％以下，说明LDH-Fe3O4-HA/DOX NPs虽然体外能抑制黑色素瘤B16细胞，但疗效低于DOX，从其说明书附图上也可看出这个问题，即该纳米药物的疗效与甲氨蝶蛉原料相比没有疗效优势。The Chinese patent application number 201710083481.0, "A HA-targeted double hydroxide-ultra-small iron nanomaterial and its preparation and application", reports: The present invention relates to a HA-targeted double hydroxide-ultra-small iron nanomaterial and its preparation and application, preparation: synthesizing LDH-Fe 3 O 4 nanocomposite materials by coprecipitation method, covalently bonding activated hyaluronic acid to the surface of LDH layer through silane coupling agent, and finally surface loading of anticancer drugs. The nanomaterial particles of the present invention are evenly distributed, which can enhance the MR imaging effect of tumor sites in animals. The LDH-Fe 3 O 4 -HA NPs of the present invention, as a carrier of the anticancer drug doxorubicin DOX, not only have sensitive pH response release characteristics, but also can specifically identify tumor cells expressing CD44 receptors, thereby achieving the ideal effect of efficient tumor inhibition. Paragraph 49 of the patent specification states that when the content of DOX (methotrex) is the same, the survival rate of melanoma B16 cells treated with DOX alone is lower than that of LDH-Fe 3 O 4 -HA/DOXNPs. When the cells are incubated with LDH-Fe 3 O 4 -HA/DOX NPs (DOX concentration is 12.5 μg/mL) for 48 hours, the cell survival rate is below 50%, indicating that although LDH-Fe 3 O 4 -HA/DOX NPs can inhibit melanoma B16 cells in vitro, its efficacy is lower than that of DOX. This problem can also be seen from the drawings in the specification, that is, the efficacy of the nanomedicine has no therapeutic advantage over the methotrexate raw material.
申请号202210381591.6的中国专利《一种基于大黄酸的多功能抗乳腺癌纳米药物及其制备方法和应用》报道了：本发明涉及一种基于大黄酸的多功能抗乳腺癌纳米药物及其制备方法和应用，属于抗乳腺癌纳米药物制备技术领域。本发明的纳米药物以表面修饰聚乙二醇(PEG)和叶酸(FA)的介孔聚多巴胺(MPDA)作为药物递送载体，共载大黄酸(Rhein)、柠檬酸修饰的四氧化三铁纳米颗粒(CA-Fe3O4 NPs)，用于抗乳腺癌的治疗，该纳米药物具有主动靶向和肿瘤微环境可控释药的性能。具体合成步骤包括MPDA上PEG和FA的修饰，Rhein和CA-Fe3O4 NPs的装载比例探索，最终得到兼具化疗和光热治疗功能的抗乳腺癌纳米药物。该专利说明书记载Rhein/FeO@MPDA-PEG-FA对抗癌药物大黄酸的负载量较低，仅为10.5％，负载量较低，负载量低药物可能达不到治疗剂量。The Chinese patent application number 202210381591.6, "A multifunctional anti-breast cancer nanodrug based on rhein, its preparation method and application", reports: The present invention relates to a multifunctional anti-breast cancer nanodrug based on rhein, its preparation method and application, and belongs to the technical field of preparation of anti-breast cancer nanodrugs. The nanodrug of the present invention uses mesoporous polydopamine (MPDA) with surface modified polyethylene glycol (PEG) and folic acid (FA) as a drug delivery carrier, and co-loads rhein (Rhein) and citric acid-modified ferroferric oxide nanoparticles (CA-Fe 3 O 4 NPs) for the treatment of breast cancer. The nanodrug has the properties of active targeting and controlled release of drugs in the tumor microenvironment. The specific synthesis steps include the modification of PEG and FA on MPDA, the exploration of the loading ratio of Rhein and CA-Fe 3 O 4 NPs, and finally obtain an anti-breast cancer nanodrug with both chemotherapy and photothermal therapy functions. The patent specification records that the loading amount of Rhein/FeO@MPDA-PEG-FA on the anticancer drug rhein is low, only 10.5%. The loading amount is low, and the drug with low loading amount may not reach the therapeutic dose.
由于口腔鳞状细胞癌现有的治疗手段如手术往往导致功能丧失和毁容，放射治疗不能延长患者的生存期，所以需要探索新的治疗手段。小檗碱作为一种化疗药物，副作用小，可用于治疗口腔癌，但存在溶解度差，在生物药剂学分类系统中属于IV类药物[4],盐酸小檗碱渗透性差，限制了其在临床上的应用[5],尚无纯小檗碱配方被批准用于任何特定疾病[4]，不具有主动靶向作用，无法靶向到肿瘤细胞的缺陷，另外现有其他磁性纳米制剂存在粒径较大、载药率较低和治疗作用比原料药差等缺陷，纳米颗粒的行为与其形状、大小和表面化学性质直接相关，解决这些缺陷研究适合口腔鳞状细胞癌的小檗碱纳米颗粒制剂难度大。Since existing treatments for oral squamous cell carcinoma, such as surgery, often lead to functional loss and disfigurement, and radiotherapy cannot prolong the patient's survival, new treatments need to be explored. Berberine, as a chemotherapeutic drug, has few side effects and can be used to treat oral cancer, but it has poor solubility and is classified as a Class IV drug in the biopharmaceutical classification system [4] . Berberine hydrochloride has poor permeability, which limits its clinical application [5] . No pure berberine formulation has been approved for any specific disease [4] . It does not have an active targeting effect and cannot target tumor cells. In addition, other existing magnetic nanoparticles have defects such as large particle size, low drug loading rate, and poorer therapeutic effect than raw materials. The behavior of nanoparticles is directly related to their shape, size, and surface chemical properties. It is difficult to solve these defects and study berberine nanoparticle preparations suitable for oral squamous cell carcinoma.
参考文献：references:
[1]杨玥怡,张益益,李一新,等.多学科协作诊疗模式在口腔鳞癌中的实践进展[J].中国口腔颌面外科杂志,2021,19(5):4.DOI:10.19438/j.cjoms.2021.05.017.[1] Yang Yueyi, Zhang Yiyi, Li Yixin, et al. Practical progress of multidisciplinary collaborative diagnosis and treatment model in oral squamous cell carcinoma[J]. Chinese Journal of Oral and Maxillofacial Surgery, 2021, 19(5): 4. DOI: 10.19438/j.cjoms.2021.05.017.
[2]Kim J S,Oh D,Yim M J,et al.Berberine induces FasL-relatedapoptosis through p38 activation in KB human oral cancer cells[J].OncologyReports,2015,33(4).DOI:10.3892/or.2015.3768.[2] Kim J S, Oh D, Yim M J, et al. Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells[J]. Oncology Reports, 2015, 33(4). DOI: 10.3892/or.2015.3768.
[3]肖金枝,余伟,张昊.盐酸小檗碱通过调节自噬抑制口腔鳞状细胞癌增殖的研究[J].口腔医学.2023,43(10)889-893.[3] Xiao Jinzhi, Yu Wei, Zhang Hao. Study on the effect of berberine hydrochloride on the proliferation of oral squamous cell carcinoma by regulating autophagy[J]. Journal of Stomatology. 2023, 43(10): 889-893.
[4]周剑雄,吴送姑,龚俊波,等.小檗碱的药理活性以及提升其口服生物利用度的策略[J].药学学报,2022,57(5):10.[4] Zhou Jianxiong, Wu Songgu, Gong Junbo, et al. Pharmacological activity of berberine and strategies to improve its oral bioavailability[J]. Acta Pharmaceutica Sinica, 2022, 57(5):10.
[5]SONG D，HAO J，FAN D.Biological properties and clinical applicationsof berberine[J].Front Med，2020，14(5):564－582.[5] Song D, Hao J, Fan D. Biological properties and clinical applications of berberine[J]. Front Med, 2020, 14(5): 564-582.
[6]X.Mei,W.Wang,L.Yan,T.Hu,R.Liang,D.Yan,M.Wei,D.G.Evans,X.Duan,Hydrotalcite monolayer toward high performance synergistic dual-modal imagingand cancer therapy,Biomaterials 165(2018)14-24.[6] X. Mei, W. Wang, L. Yan, T. Hu, R. Liang, D. Yan, M. Wei, D. G. Evans, X. Duan, Hydrotalcite monolayer toward high performance synergistic dual-modal imaging and cancer therapy, Biomaterials 165 (2018) 14-24.
[7]L.Yan,S.Gonca,G.Zhu,W.Zhang,X.Chen,Layered double hydroxidenanostructures and nanocomposites for biomedical applications,J.Mater.Chem.B7(2019)5583-5601.[7] L. Yan, S. Gonca, G. Zhu, W. Zhang, X. Chen, Layered double hydroxide nanostructures and nanocomposites for biomedical applications, J. Mater. Chem. B7 (2019) 5583-5601.
[8]V.K.Ameena Shirin,R.Sankar,A.P.Johnson,H.V.Gangadharappa,K.Pramod,Advanced drugdelivery applications of layered double hydroxide,Journal ofcontrolled release:official J Controlled Release 330(2021)398-426.[8] V.K.Ameena Shirin, R.Sankar, A.P.Johnson, H.V.Gangadharappa, K.Pramod, Advanced drug delivery applications of layered double hydroxide, Journal of controlled release: official J Controlled Release 330(2021)398-426.
[9]T.Hu,Z.Gu,G.R.Williams,M.Strimaite,J.Zha,Z.Zhou,X.Zhang,C.Tan,R.Liang,Layered double hydroxide-based nanomaterials for biomedicalapplications,Chem.Soc.Rev.51(2022)6126-6176.[9] T.Hu, Z.Gu, G.R.Williams, M.Strimaite, J.Zha, Z.Zhou, X.Zhang, C.Tan, R.Liang, Layered double hydroxide-based nanomaterials for biomedicalapplications, Chem.Soc.Rev.51(2022)6126-6176.
[10]N.Rahmanian,H.Hamishehkar,J.E.N.Dolatabadi,et al.Nano GrapheneOxide:A Novel Carrier for Oral Delivery of Flavonoids,Colloids and SurfacesB:Biointerfaces,123(2014)331-338.[10] N. Rahmanian, H. Hamishehkar, J. E. N. Dolatabadi, et al. Nano Graphene Oxide: A Novel Carrier for Oral Delivery of Flavonoids, Colloids and Surfaces B: Biointerfaces, 123 (2014) 331-338.
[11].FangJ,Nakamura H,Maeda H.The EPR effect:Unique features of tumorblood vessels for drugdelivery,factors involved,and limitations andaugmentation of the effect,Advanced DrugDelivery Reviews,63(2011)136-151.[11]. Fang J, Nakamura H, Maeda H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect, Advanced Drug Delivery Reviews, 63 (2011) 136-151.
[12]Kalyane,D.,et al.,Employment of enhanced permeability andretention effect(EPR):Nanoparticle-based precision tools for targetingoftherapeutic and diagnostic agent in cancer,Mater Sci Eng C Mater Biol Appl,98(2019)1252-1276.[12] Kalyane, D., et al., Employment of enhanced permeability and retention effect (EPR): Nanoparticle-based precision tools for targeting of therapeutic and diagnostic agent in cancer, Mater Sci Eng C Mater Biol Appl, 98 (2019) 1252-1276.
[13]夏志勇,杜娜,刘建强,等.聚乙二醇和叶酸对层状双金属氢氧化物颗粒的表面修饰[J].高等学校化学学报,2013.DOI:CNKI:SUN:GDXH.0.2013-03-020.[13] Xia Zhiyong, Du Na, Liu Jianqiang, et al. Surface modification of layered double hydroxide particles by polyethylene glycol and folic acid[J]. Journal of Chemistry in Universities, 2013. DOI:CNKI:SUN:GDXH.0.2013-03-020.
[14]Maeda H,Nakamura H,Fang J.The EPR effect for macromolecular drugdelivery to solid tumors:improvement of tumor uptake,lowering of systemictoxicity,and distinct tumor imaging in vivo[J].Adv Drug Deliv Rev,2013,65(1):71 -79.[14] Maeda H, Nakamura H, Fang J. The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo[J]. Adv Drug Deliv Rev, 2013, 65(1): 71 -79.
发明内容Summary of the invention
针对小檗碱水溶性差，生物利用度低、不能靶向口腔鳞状细胞癌的特性，及现有其他磁性纳米制剂粒径较大、分散度较大、载药率较低、疗效比原料药差的缺陷，本发明研制了一种用于治疗口腔鳞状细胞癌的小檗碱磁性纳米颗粒，并对其作用机制进行研究。为实现上述目的，具体技术方案如下：In view of the poor water solubility, low bioavailability and inability to target oral squamous cell carcinoma of berberine, as well as the defects of other existing magnetic nanoparticles such as large particle size, large dispersion, low drug loading rate and poor efficacy compared to raw materials, the present invention has developed a berberine magnetic nanoparticle for the treatment of oral squamous cell carcinoma and studied its mechanism of action. To achieve the above purpose, the specific technical scheme is as follows:
第一方面、合成载小檗碱的磁性纳米颗粒First aspect: Synthesis of berberine-loaded magnetic nanoparticles
(一)合成Fe3O4 NPs(I) Synthesis of Fe 3 O 4 NPs
FeCl3·6H2O和Na3Cit·2H2O溶于乙二醇中，加热搅拌，加入NaAc搅拌溶解，溶液置于Teflon衬垫上，加热，无水乙醇和去离子水洗涤除杂，收集Teflon衬垫上的Fe3O4 NPs冷冻干燥即得。FeCl 3 ·6H 2 O and Na 3 Cit·2H 2 O were dissolved in ethylene glycol, heated and stirred, NaAc was added and stirred to dissolve, the solution was placed on a Teflon pad, heated, washed with anhydrous ethanol and deionized water to remove impurities, and Fe 3 O 4 NPs on the Teflon pad were collected and freeze-dried to obtain.
(二)合成Fe3O4@LDH NPs(II) Synthesis of Fe 3 O 4 @LDH NPs
MgCl2·6H2O和AlCl3·6H2O溶解于水，加入Fe3O4 NPs超声使混合溶液均匀分散，在氮气保护下加入0.15M的NaOH溶液，磁铁吸附得到磁性产物粗品，磁性产物粗品用水加热除杂，再用磁铁吸附合成的Fe3O4@LDH NPs，水洗除杂，冷冻干燥即得。MgCl 2 ·6H 2 O and AlCl 3 ·6H 2 O were dissolved in water, Fe 3 O 4 NPs were added and ultrasonically dispersed to make the mixed solution uniformly dispersed, 0.15 M NaOH solution was added under nitrogen protection, and a crude magnetic product was obtained by magnet adsorption. The crude magnetic product was heated with water to remove impurities, and then the synthesized Fe 3 O 4 @LDH NPs were adsorbed by a magnet, washed with water to remove impurities, and freeze-dried to obtain the product.
(三)合成Fe3O4@LDH-PEG-NH2 (III) Synthesis of Fe 3 O 4 @LDH-PEG-NH 2
Fe3O4@LDH加入DMSO超声溶解，搅拌均匀后，加入EDC和NHS混匀活化羧基，避光搅拌3h，使成为活化的Fe3O4@LDH溶液；Fe 3 O 4 @LDH was dissolved by ultrasonication in DMSO and stirred evenly. EDC and NHS were added and mixed evenly to activate the carboxyl groups. The mixture was stirred for 3 h in the dark to obtain an activated Fe 3 O 4 @LDH solution.
NH2-PEG-NH2溶解于DMSO中，在持续搅拌的条件下逐滴滴加活化的Fe3O4@LDH溶液，避光磁力搅拌12h，10000r/min离心10min，弃去游离的NH2-PEG-NH2、EDC、NHS，取离心沉淀水洗三次，真空干燥，即得Fe3O4@LDH-PEG-NH2；NH 2 -PEG-NH 2 was dissolved in DMSO, and the activated Fe 3 O 4 @LDH solution was added dropwise under continuous stirring, and magnetic stirring was performed for 12 h in a dark place, and the mixture was centrifuged at 10000 r/min for 10 min to discard free NH 2 -PEG-NH 2 , EDC, and NHS, and the centrifugal precipitate was washed with water three times and vacuum dried to obtain Fe 3 O 4 @LDH-PEG-NH 2 ;
(四)合成Fe3O4@LDH/PEG-FA(IV) Synthesis of Fe 3 O 4 @LDH/PEG-FA
Fe3O4@LDH-PEG-NH2用DMSO溶解，将FA、EDC、NHS混合溶于DMSO溶液中，充分搅拌3h后，逐滴加入Fe3O4@LDH-PEG-NH2溶液中，避光磁力搅拌4h，用截留分子量为1000的透析袋清洗48h，去除游离的小分子，真空干燥得Fe3O4@LDH/PEG-FA；Fe 3 O 4 @LDH-PEG-NH 2 was dissolved in DMSO, FA, EDC, and NHS were mixed and dissolved in the DMSO solution, stirred for 3 h, and then added dropwise to the Fe 3 O 4 @LDH-PEG-NH 2 solution. The mixture was stirred magnetically for 4 h in a dark place, washed with a dialysis bag with a molecular weight cutoff of 1000 for 48 h to remove free small molecules, and dried in vacuum to obtain Fe 3 O 4 @LDH/PEG-FA.
(五)合成Fe3O4@LDH@Ber/PEG-FA(V) Synthesis of Fe 3 O 4 @LDH@Ber/PEG-FA
将Fe3O4@LDH/PEG-FA和Ber加入50％DMF中，超声混合均匀后，搅拌载药，离心，沉淀水洗，弃去游离的Ber，沉淀真空干燥，即得。Fe 3 O 4 @LDH/PEG-FA and Ber are added to 50% DMF, ultrasonically mixed, stirred to load the drug, centrifuged, washed with water, free Ber was discarded, and the precipitate was vacuum dried to obtain the product.
第二方面、合成磁性纳米颗粒工艺参数筛选和优化Second aspect: Screening and optimization of process parameters for synthesizing magnetic nanoparticles
(一)对NH2-PEG-NH2接枝在Fe3O4@LDH表面工艺参数(浓度和反应时间)筛选(I) Screening of process parameters (concentration and reaction time) for grafting NH 2 -PEG-NH 2 on Fe 3 O 4 @LDH surface
根据产物Fe3O4@LDH-PEG-NH2粒径的大小和分散度，我们选择0.5mg/mL的NH2-PEG-NH2的浓度，反应12h这一工艺参数。According to the particle size and dispersion of the product Fe 3 O 4 @LDH-PEG-NH 2 , we selected the concentration of 0.5 mg/mL NH 2 -PEG-NH 2 and the reaction time of 12 h as the process parameters.
(二)对聚乙二醇、叶酸(FA)接枝在Fe3O4@LDH表面工艺参数(浓度和反应时间)筛选(II) Screening of process parameters (concentration and reaction time) for grafting polyethylene glycol and folic acid (FA) on the surface of Fe 3 O 4 @LDH
产物Fe3O4@LDH/PEG-FA的分散度，以0.5mg/mL Fe3O4@LDH-PEG-NH2+0.5mg/mL FA，反应4h条件最优，且该条件下粒径较小，最终确定了Fe3O4@LDH-PEG-NH2浓度0.5mg/mL，叶酸(FA)浓度0.5mg/mL，反应4h条件合成磁性纳米颗粒，该工艺参数下产物Fe3O4@LDH/PEG-FA的粒径为159.14nm，分散度指数(PDI)为0.060。The dispersion of the product Fe 3 O 4 @LDH/PEG-FA was optimal when the conditions were 0.5 mg/mL Fe 3 O 4 @LDH-PEG-NH 2 +0.5 mg/mL FA and reaction time for 4 h, and the particle size was smaller under these conditions. Finally, the conditions of Fe 3 O 4 @LDH-PEG-NH 2 concentration of 0.5 mg/mL, folic acid (FA) concentration of 0.5 mg/mL and reaction time of 4 h were determined to synthesize magnetic nanoparticles. Under these process parameters, the particle size of the product Fe 3 O 4 @LDH/PEG-FA was 159.14 nm, and the dispersion index (PDI) was 0.060.
第三方面、考察载小檗碱的磁性纳米颗粒理化性能Third aspect: investigating the physical and chemical properties of berberine-loaded magnetic nanoparticles
(一)磁性纳米颗粒的晶体结构(I) Crystal structure of magnetic nanoparticles
通过x射线衍射考察了Fe3O4 NPs、Fe3O4@LDH NPs和Fe3O4@LDH@Ber/PEG-FA样品的晶体结构。The crystal structures of Fe 3 O 4 NPs, Fe 3 O 4 @LDH NPs and Fe 3 O 4 @LDH@Ber/PEG-FA samples were investigated by X-ray diffraction.
x射线衍射图(XRD谱图)结果显示，The X-ray diffraction pattern (XRD spectrum) results show that
2θ＝29.86、35.4、43.28、53.64、57.04、62.68位置处出现的衍射峰分别对应Fe3O4晶体面的(220)、(311)、(400)、(422)、(511)和(440)面；The diffraction peaks appearing at positions 2θ = 29.86, 35.4, 43.28, 53.64, 57.04 , and 62.68 correspond to the (220), (311), (400), (422), (511), and (440) planes of the Fe 3 O 4 crystal plane, respectively;
Fe3O4@LDH样品中2θ＝23.14出现弱且宽的衍射峰，对应了LDH的(006)晶体面。In the Fe 3 O 4 @LDH sample, a weak and broad diffraction peak appears at 2θ=23.14, corresponding to the (006) crystal plane of LDH.
结果表明，Fe3O4@LDH表面修饰PEG-FA后，Fe3O4@LDH/PEG-FA的特征衍射峰与Fe3O4@LDH基本一致，未出现额外的衍射峰，表明在Fe3O4@LDH表面修饰PEG-FA不影响其晶体结构。The results show that after the surface of Fe 3 O 4 @LDH was modified with PEG-FA, the characteristic diffraction peaks of Fe 3 O 4 @LDH/PEG-FA were basically consistent with those of Fe 3 O 4 @LDH, and no additional diffraction peaks appeared, indicating that the modification of PEG-FA on the surface of Fe 3 O 4 @LDH did not affect its crystal structure.
(二)磁性纳米颗粒的磁性能(II) Magnetic properties of magnetic nanoparticles
通过Squid-VSM磁测量系统检测空白磁性纳米颗粒Fe3O4、Fe3O4@LDH和Fe3O4@LDH@Ber/PEG-FA的磁性能。The magnetic properties of blank magnetic nanoparticles Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH@Ber/PEG-FA were detected by Squid-VSM magnetic measurement system.
结果：饱和磁化强度：Fe3O4为63.81emu/g，Fe3O4@LDH为52.34emu/g，，Fe3O4@LDH@Ber/PEG-FA为29.30emu/g；即饱和磁化强度：Fe3O4＞Fe3O4@LDH＞Fe3O4@LDH@Ber/PEG-FA。Results: The saturation magnetization intensity: Fe 3 O 4 is 63.81emu/g, Fe 3 O 4 @LDH is 52.34emu/g, and Fe 3 O 4 @LDH@Ber/PEG-FA is 29.30emu/g; that is, the saturation magnetization intensity: Fe 3 O 4 ＞Fe 3 O 4 @LDH＞Fe 3 O 4 @LDH@Ber/PEG-FA.
分析与结论：由于非磁性的LDH、PEG、FA的存在，Fe3O4@LDH的饱和磁化强度为52.34emu/g低于Fe3O4的63.81emu/g，载药Ber并涂层后，Fe3O4@LDH@Ber/PEG-FA的饱和磁化强度降至29.30emu/g，进一步证实了Ber成功负载在Fe3O4@LDH中，且PEG和FA成功修饰于Fe3O4@LDH的表面。Analysis and conclusion: Due to the presence of non-magnetic LDH, PEG and FA, the saturation magnetization intensity of Fe 3 O 4 @LDH is 52.34emu/g, which is lower than 63.81emu/g of Fe 3 O 4. After loading Ber and coating, the saturation magnetization intensity of Fe 3 O 4 @LDH@Ber/PEG-FA dropped to 29.30emu/g, which further confirmed that Ber was successfully loaded in Fe 3 O 4 @LDH, and PEG and FA were successfully modified on the surface of Fe 3 O 4 @LDH.
另外，上述结果还表明磁性纳米颗粒具有超顺磁性，Fe3O4@LDH@Ber/PEG-FA在体内磁靶向给药方面具有潜在的应用前景。In addition, the above results also show that magnetic nanoparticles have superparamagnetism and Fe 3 O 4 @LDH@Ber/PEG-FA has potential application prospects in in vivo magnetic targeted drug delivery.
(三)磁性纳米颗粒的载药率和体外释放曲线(III) Drug loading rate and in vitro release curve of magnetic nanoparticles
1、采用紫外-可见光谱法考察了Fe3O4@LDH@Ber/PEG-FA的载药率1. The drug loading rate of Fe 3 O 4 @LDH@Ber/PEG-FA was investigated by UV-visible spectroscopy
结果：Fe3O4@LDH@Ber/PEG-FA中小檗碱(Ber)的载药率为29.34％，Fe3O4@LDH@Ber的载药率为28.80％。Results: The drug loading rate of berberine (Ber) in Fe 3 O 4 @LDH@Ber/PEG-FA was 29.34%, and the drug loading rate of Fe 3 O 4 @LDH@Ber was 28.80%.
2、采用紫外-可见光谱法考察了Fe3O4@LDH@Ber/PEG-FA的体外释放曲线2. The in vitro release curve of Fe 3 O 4 @LDH@Ber/PEG-FA was investigated by UV-visible spectroscopy
结果：result:
(1)pH5.0条件下，Fe3O4@LDH@Ber/PEG-FA的累积释放度最大值为53.77％，Fe3O4@LDH@Ber累积释放度最大值30.41％；(1) Under pH 5.0, the maximum cumulative release of Fe 3 O 4 @LDH@Ber/PEG-FA was 53.77%, and the maximum cumulative release of Fe 3 O 4 @LDH@Ber was 30.41%;
(2)pH7.4条件下，Fe3O4@LDH@Ber/PEG-FA的累积释放量48h时为3.87％，Fe3O4@LDH@Ber的累积释放量48h时为7.02％；(2) Under pH 7.4, the cumulative release of Fe 3 O 4 @LDH@Ber/PEG-FA was 3.87% at 48 h, and the cumulative release of Fe 3 O 4 @LDH@Ber was 7.02% at 48 h;
结论：in conclusion:
(1)Fe3O4@LDH@Ber不具有pH响应性(1) Fe 3 O 4 @LDH@Ber is not pH responsive
Fe3O4@LDH@Ber在pH5.0的释放量高于pH7.4，可能与材料框架在不同pH的溶解度不同有关。The release amount of Fe 3 O 4 @LDH@Ber at pH 5.0 is higher than that at pH 7.4, which may be related to the different solubility of the material framework at different pH values.
(2)Fe3O4@LDH@Ber/PEG-FA具有pH响应性(2) Fe 3 O 4 @LDH@Ber/PEG-FA is pH responsive
Fe3O4@LDH@Ber/PEG-FA在pH7.4缓冲液环境下，48h累积释放量仅为3.87％，分析：pH7.4环境下，材料框架相对稳定，可防止过早释放；The cumulative release of Fe 3 O 4 @LDH@Ber/PEG-FA in pH 7.4 buffer solution was only 3.87% in 48 hours. Analysis: In the pH 7.4 environment, the material framework is relatively stable, which can prevent premature release;
Fe3O4@LDH@Ber/PEG-FA在pH5.0缓冲液环境下，Ber的释放量显著增高，达到53.77％，分析：PEG-FA涂层在酸性环境中可控释性，促进Ber快速释放，肿瘤环境为酸性环境，pH为5.0左右，Fe3O4@LDH@Ber/PEG-FA具有pH响应性可控制Ber在口腔癌环境中大量释放，在正常组织细胞中不释放或少释放，从而实现对Ber的可控释放，实现减毒增效的目的。In the pH 5.0 buffer environment, the release amount of Ber from Fe 3 O 4 @LDH@Ber/PEG-FA increased significantly, reaching 53.77%. Analysis: PEG-FA coating has controllable release in acidic environment, promoting rapid release of Ber. The tumor environment is acidic with a pH of about 5.0. Fe 3 O 4 @LDH@Ber/PEG-FA has pH responsiveness and can control the large-scale release of Ber in the oral cancer environment, and no or little release in normal tissue cells, thereby achieving controlled release of Ber and achieving the purpose of reducing toxicity and increasing efficacy.
第四方面、考察载小檗碱的磁性纳米颗粒对人舌鳞癌细胞的治疗作用及作用机制研究Fourthly, investigate the therapeutic effect and mechanism of berberine-loaded magnetic nanoparticles on human tongue squamous cell carcinoma cells
(一)考察人舌鳞癌细胞(CAL27细胞)对载小檗碱的磁性纳米颗粒的摄取(I) Investigating the uptake of berberine-loaded magnetic nanoparticles by human tongue squamous cell carcinoma cells (CAL27 cells)
因Ber不具有荧光性，我们将罗丹明6G(R6G)代替小檗碱(Ber)进行细胞摄取实验。Since Ber is not fluorescent, we used rhodamine 6G (R6G) instead of berberine (Ber) for cell uptake experiments.
通过荧光显微镜检测人舌鳞癌细胞对不同载R6G的磁性纳米颗粒的摄取后的荧光。The fluorescence of human tongue squamous cell carcinoma cells after taking up different R6G-loaded magnetic nanoparticles was detected by fluorescence microscopy.
结果：荧光强度Fe3O4@LDH@R6G/PEG-FA组强于Fe3O4@LDH@R6G/PEG组。Results: The fluorescence intensity of Fe 3 O 4 @LDH@R6G/PEG-FA group was stronger than that of Fe 3 O 4 @LDH@R6G/PEG group.
分析与结论：说明在Fe3O4@LDH表面加入靶向剂叶酸，通过叶酸使Fe3O4@LDH@Ber/PEG-FA与CAL27细胞表面的叶酸受体结合，促进CAL27细胞对Ber的摄取，从而增加治疗口腔癌的效果。Analysis and conclusion: It shows that adding targeting agent folic acid to the surface of Fe 3 O 4 @LDH can make Fe 3 O 4 @LDH@Ber/PEG-FA bind to the folate receptor on the surface of CAL27 cells through folic acid, promote the uptake of Ber by CAL27 cells, and thus increase the effect of treating oral cancer.
(二)CCK8法评价载小檗碱的磁性纳米颗粒对CAL27细胞的存活率的影响(II) CCK8 method to evaluate the effect of berberine-loaded magnetic nanoparticles on the survival rate of CAL27 cells
Fe3O4@LDH@Ber组和Fe3O4@LDH@Ber/PEG-FA组浓度相同时，即Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA浓度均为0、2、5、10、25、50、100μg/mL时，When the concentrations of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were the same, that is, when the concentrations of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were 0, 2, 5, 10, 25, 50, and 100 μg/mL,
Fe3O4@LDH@Ber组和Fe3O4@LDH@Ber/PEG-FA组均可抑制CAL27细胞的存活率，且具有浓度依赖性；Both the Fe 3 O 4 @LDH@Ber group and the Fe 3 O 4 @LDH@Ber/PEG-FA group could inhibit the survival rate of CAL27 cells in a concentration-dependent manner;
随浓度升高，CAL27细胞存活率不断降低，在药物浓度为100μg/mL，Fe3O4@LDH@Ber/PEG-FA组CAL27细胞存活率降低至44.94％；With the increase of concentration, the survival rate of CAL27 cells continued to decrease. When the drug concentration was 100 μg/mL, the survival rate of CAL27 cells in the Fe 3 O 4 @LDH@Ber/PEG-FA group decreased to 44.94%;
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Fe3O4@LDH@Ber组相比，浓度从低至高，P值分别为：1，0.549，0.018，0.038，0.018，0.011，0.017；即当浓度为5、10、25、50、100μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Fe3O4@LDH@Ber组；The ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was compared with that of Fe 3 O 4 @LDH@Ber group, and the P values were 1, 0.549, 0.018, 0.038, 0.018, 0.011, and 0.017 from low to high concentrations; that is, when the concentrations were 5, 10, 25, 50, and 100 μg/mL, the ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of Fe 3 O 4 @LDH@Ber group.
相同小檗碱浓度下，即小檗碱浓度均为0、5、10、25、50μg/mL时，At the same berberine concentration, that is, 0, 5, 10, 25, 50 μg/mL,
Fe3O4@LDH@Ber组降低CAL27细胞存活率的能力与Ber组相比，P值分别为：1，0.035，0.944，0.025，0.040；即当小檗碱浓度为5、25、50μg/mL时Fe3O4@LDH@Ber组降低CAL27细胞存活率的能力显著强于Ber组；The ability of Fe 3 O 4 @LDH@Ber group to reduce the survival rate of CAL27 cells was compared with that of Ber group, and the P values were 1, 0.035, 0.944, 0.025, and 0.040, respectively; that is, when the concentration of berberine was 5, 25, and 50 μg/mL, the ability of Fe 3 O 4 @LDH@Ber group to reduce the survival rate of CAL27 cells was significantly stronger than that of Ber group;
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Ber组相比，P值分别为：1，0.001，0.263，0.002，0.002；即小檗碱浓度均为5、25、50μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Ber组；The ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was compared with that of Ber group, and the P values were 1, 0.001, 0.263, 0.002, and 0.002, respectively; that is, when the berberine concentrations were 5, 25, and 50 μg/mL, the ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of Ber group;
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Fe3O4@LDH@Ber组相比，P值分别为：1，0.020，0.290，0.003，0.047；即小檗碱浓度均为5、25、50μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Fe3O4@LDH@Ber组。The ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was compared with that of Fe 3 O 4 @LDH@Ber group, with P values of 1, 0.020, 0.290, 0.003, and 0.047, respectively; that is, when the berberine concentrations were 5, 25, and 50 μg/mL, the ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of Fe 3 O 4 @LDH@Ber group.
(三)通过考察载小檗碱磁性纳米颗粒给药后CAL27细胞内ROS水平、GSH水平、H2O2水平、线粒体膜电位及BAX和Bcl-XL基因表达的变化，探索其作用机制(III) To explore its mechanism of action by investigating the changes in ROS level, GSH level, H 2 O 2 level, mitochondrial membrane potential and BAX and Bcl-XL gene expression in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
1、载小檗碱磁性纳米颗粒给药后CAL27细胞内ROS水平变化1. Changes in ROS levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
ROS在肿瘤细胞的代谢、增殖和死亡过程中起到至关重要的作用。高氧化应激状态下，ROS通过调节基因组也可以促进凋亡。ROS plays a vital role in the metabolism, proliferation and death of tumor cells. Under high oxidative stress, ROS can also promote apoptosis by regulating the genome.
通过DCFH-DA探针检测细胞内ROS水平变化。DCFH-DA可渗透入细胞内，与细胞内ROS结合从而发出绿色荧光，荧光强度越强，表示ROS生成水平越高。The DCFH-DA probe is used to detect changes in intracellular ROS levels. DCFH-DA can penetrate into cells and bind to intracellular ROS to emit green fluorescence. The stronger the fluorescence intensity, the higher the level of ROS generation.
结果：5-FU、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内ROS水平与对照组相比，均升高，且具有显著性差异。Results: Compared with the control group, the levels of ROS in CAL27 cells in the 5-FU, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA groups were significantly increased.
分析与结论：与阳性药5-FU组相比，Fe3O4@LDH负载Ber后，细胞内ROS水平降低，可能与Fe3O4@LDH过早释放小檗碱有关，在Fe3O4@LDH表面添加叶酸靶向后，CAL27细胞内ROS水平显著升高，效果好于阳性药组，表明叶酸靶向能准确将药物递送至肿瘤细胞内，增加治疗效果。Analysis and conclusion: Compared with the positive drug 5-FU group, the intracellular ROS level decreased after Fe 3 O 4 @LDH was loaded with Ber, which may be related to the premature release of berberine by Fe 3 O 4 @LDH. After adding folic acid targeting on the surface of Fe 3 O 4 @LDH, the intracellular ROS level of CAL27 cells increased significantly, and the effect was better than that of the positive drug group, indicating that folic acid targeting can accurately deliver drugs to tumor cells and increase the therapeutic effect.
2、载小檗碱磁性纳米颗粒给药后CAL27细胞内GSH水平变化2. Changes in GSH levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
GSH在肿瘤细胞的代谢、增殖和死亡过程中起到至关重要的作用。GSH plays a vital role in the metabolism, proliferation and death of tumor cells.
结果：与对照组相比，Fe3O4@LDH@Ber组CAL27细胞内GSH浓度显著降低(P<0.001)，但高于5-FU组，Fe3O4@LDH@Ber/PEG-FA组则低于Fe3O4@LDH@Ber组。Results: Compared with the control group, the intracellular GSH concentration in CAL27 cells in the Fe 3 O 4 @LDH@Ber group was significantly decreased (P<0.001), but higher than that in the 5-FU group, and lower than that in the Fe 3 O 4 @LDH@Ber/PEG-FA group.
分析：叶酸靶向后，Fe3O4@LDH@Ber/PEG-FA被CAL27细胞更多地摄取，GSH被显著消耗，减少GSH过表达对ROS的影响，从而达到治疗肿瘤的效果。Analysis: After folic acid targeting, Fe 3 O 4 @LDH@Ber/PEG-FA was taken up more by CAL27 cells, GSH was significantly consumed, and the effect of GSH overexpression on ROS was reduced, thereby achieving the effect of tumor treatment.
3、载小檗碱磁性纳米颗粒给药后CAL27细胞内BAX、Bcl-XL基因的表达变化3. Changes in the expression of BAX and Bcl-XL genes in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
结果：result:
(1)5-FU、Ber组、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组中BAX基因表达水平，与对照组相比，显著上调，P值分别为0.023，0.092，0.008，0。(1) Compared with the control group, the expression levels of BAX gene in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA groups were significantly upregulated, with P values of 0.023, 0.092, 0.008, and 0, respectively.
(2)5-FU、Ber组、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组中Bcl-XL基因表达水平，与对照组相比，显著下调，P值分别为0，0.004，0.002，0。(2) Compared with the control group, the expression levels of Bcl-XL gene in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA groups were significantly downregulated, with P values of 0, 0.004, 0.002, and 0, respectively.
分析与结论：Analysis and conclusion:
BAX是BCL-2基因家族中细胞凋亡促进基因，BAX的过度表达可拮抗BCL-2的保护效应而使细胞趋于死亡。Bcl-XL是属于Bcl-2家族的抗凋亡基因，在调控细胞凋亡、细胞自噬和肿瘤代谢等方面都具有重要作用。BAX is a cell apoptosis-promoting gene in the BCL-2 gene family. Overexpression of BAX can antagonize the protective effect of BCL-2 and cause cells to die. Bcl-XL is an anti-apoptotic gene belonging to the Bcl-2 family, which plays an important role in regulating cell apoptosis, cell autophagy and tumor metabolism.
BAX基因表达水平上调，Bcl-XL基因表达水平下调，就表明Fe3O4@LDH@Ber/PEG-FA有效促进了CAL27细胞的凋亡。The up-regulation of BAX gene expression level and the down-regulation of Bcl-XL gene expression level indicated that Fe 3 O 4 @LDH@Ber/PEG-FA effectively promoted the apoptosis of CAL27 cells.
4、载小檗碱磁性纳米颗粒给药后CAL27细胞内H2O2水平的变化4. Changes in H 2 O 2 levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
结果：result:
5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组中H2O2水平，与对照组相比，显著上调，P值均为0。The H 2 O 2 levels in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups were significantly upregulated compared with the control group, with P values of 0.
分析与结论：Analysis and conclusion:
Fe3O4@LDH/PEG-FA作为药物载体，将Ber靶向至CAL27细胞内并释放，促进肿瘤细胞内H2O2水平升高，实现药物(Ber)有效浓度堆积，并催化H2O2转化为毒性更强的·OH，加重肿瘤细胞内的氧化应激水平，从而促进肿瘤细胞调亡。Fe 3 O 4 @LDH/PEG-FA was used as a drug carrier to target Ber into CAL27 cells and release it, which promoted the increase of H 2 O 2 levels in tumor cells, achieved the effective concentration accumulation of the drug (Ber), and catalyzed the conversion of H 2 O 2 into the more toxic ·OH, aggravating the oxidative stress level in tumor cells, thereby promoting tumor cell apoptosis.
5、载小檗碱磁性纳米颗粒给药后CAL27细胞内线粒体膜电位的变化结果：5. Changes in mitochondrial membrane potential in CAL27 cells after administration of berberine-loaded magnetic nanoparticles:
对照组细胞红色荧光强度较强；阳性药组5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA给药后，CAL27细胞中红色荧光降低，绿色荧光增加。The red fluorescence intensity of the cells in the control group was stronger; after administration of 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA in the positive drug groups, the red fluorescence in CAL27 cells decreased and the green fluorescence increased.
分析与结论：Analysis and conclusion:
阳性药组5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA给药后，CAL27细胞内线粒体膜电位线粒体膜电位下降，细胞开始死亡。After administration of the positive drug groups 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA, the mitochondrial membrane potential of CAL27 cells decreased and the cells began to die.
第五方面、通过空白磁性纳米颗粒溶血试验初步考察其安全性Fifth, preliminary investigation of the safety of blank magnetic nanoparticles through hemolysis test
为验证Fe3O4@LDH/PEG-FA作为纳米药物载体的生物安全性，本实验通过模拟体内环境，将空白磁性纳米颗粒与红细胞悬液共融合，通过计算溶血率判断Fe3O4@LDH/PEG-FA的体外毒性，从而初步评估Fe3O4@LDH/PEG-FA作为静脉注射给药制剂的生物相容性和安全性。In order to verify the biosafety of Fe 3 O 4 @LDH/PEG-FA as a nanodrug carrier, this experiment simulated the in vivo environment, co-fused blank magnetic nanoparticles with red blood cell suspension, and determined the in vitro toxicity of Fe 3 O 4 @LDH/PEG-FA by calculating the hemolysis rate, thereby preliminarily evaluating the biocompatibility and safety of Fe 3 O 4 @LDH/PEG-FA as an intravenous administration preparation.
结果：Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA在25-1000ug/ml浓度范围内，红细胞溶血现象微弱，溶血率均低于5％，满足静脉给药对纳米制剂溶血毒性的要求，表明Fe3O4@LDH/PEG-FA作为纳米药物载药具有良好的生物相容性。Results: In the concentration range of 25-1000ug/ml, the hemolysis of red blood cells was weak for Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG and Fe 3 O 4 @LDH/PEG-FA, and the hemolysis rate was less than 5%, which met the requirements of hemolytic toxicity of nanoformulations for intravenous administration, indicating that Fe 3 O 4 @LDH/PEG-FA has good biocompatibility as a nanodrug carrier.
与现有技术相比，本申请的有益效果在于：Compared with the prior art, the beneficial effects of this application are:
本申请开发的载小檗碱磁性纳米颗粒Fe3O4@LDH@Ber/PEG-FA较普通的磁性纳米颗粒Fe3O4@LDH@Ber：The berberine-loaded magnetic nanoparticles Fe 3 O 4 @LDH@Ber/PEG-FA developed in this application are different from ordinary magnetic nanoparticles Fe 3 O 4 @LDH@Ber:
1、降低了分散度指数PDI，更均匀；1. Reduced the dispersion index PDI, more uniform;
2、pH5.0缓冲液环境下，小檗碱的释放量显著增高达到53.77％，在pH7.4缓冲液环境下，48h累积释放量仅为3.87％，具有pH响应性，肿瘤环境为酸性环境(pH为5.0左右)，Fe3O4@LDH@Ber/PEG-FA具有pH响应性可控制小檗碱在口腔鳞状细胞癌环境中大量释放，在正常组织细胞中不释放或少释放，从而达到减毒增效的目的；2. In the pH 5.0 buffer environment, the release of berberine increased significantly to 53.77%. In the pH 7.4 buffer environment, the cumulative release in 48 hours was only 3.87%, which was pH responsive. The tumor environment is an acidic environment (pH is about 5.0). Fe 3 O 4 @LDH@Ber/PEG-FA has pH responsiveness and can control the large-scale release of berberine in the environment of oral squamous cell carcinoma, and no or little release in normal tissue cells, thereby achieving the purpose of reducing toxicity and increasing efficacy.
3、增加了人舌鳞癌细胞(CAL27细胞)对小檗碱的摄取，从而增加小檗碱对CAL27细胞的抑制作用；3. Increased the uptake of berberine by human tongue squamous cell carcinoma cells (CAL27 cells), thereby increasing the inhibitory effect of berberine on CAL27 cells;
4、增强了降低CAL27细胞存活率的能力；4. Enhanced the ability to reduce the survival rate of CAL27 cells;
5、发现了Fe3O4@LDH@Ber/PEG-FA通过加重CAL27细胞内氧化应激损伤，诱导了线粒体功能障碍，促进了CAL27细胞的凋亡。5. It was found that Fe 3 O 4 @LDH@Ber/PEG-FA induced mitochondrial dysfunction and promoted apoptosis of CAL27 cells by aggravating oxidative stress damage in CAL27 cells.
本申请开发的Fe3O4@LDH@Ber/PEG-FA较Fe3O4@LDH@Ber/PEG增加了人舌鳞癌细胞(CAL27细胞)对小檗碱的摄取，从而增加小檗碱对CAL27细胞存活率的降低作用；Fe 3 O 4 @LDH@Ber/PEG-FA developed in this application increases the uptake of berberine by human tongue squamous cell carcinoma cells (CAL27 cells) compared to Fe 3 O 4 @LDH@Ber/PEG, thereby increasing the effect of berberine on reducing the survival rate of CAL27 cells;
另外，Fe3O4@LDH@Ber/PEG-FA的粒径较小为177.24nm，因而渗透滞留效应较好，能渗透到肿瘤深处，分散度指数PDI为0.185，较均匀；载药率较高达29.34％，为口腔鳞状细胞癌的治疗提供了较充足的剂量空间；降低人舌鳞癌细胞(CAL27细胞)存活率的作用强于小檗碱原料药。In addition, the particle size of Fe 3 O 4 @LDH@Ber/PEG-FA is relatively small, at 177.24 nm, so it has a better penetration and retention effect and can penetrate deep into the tumor. The dispersion index PDI is 0.185, which is relatively uniform. The drug loading rate is as high as 29.34%, providing a sufficient dosage space for the treatment of oral squamous cell carcinoma. The effect of reducing the survival rate of human tongue squamous cell carcinoma cells (CAL27 cells) is stronger than that of berberine raw material.
综上，载小檗碱磁性纳米颗粒Fe3O4@LDH@Ber/PEG-FA为口腔鳞状细胞癌的治疗提供了一种新的可能的治疗手段。In summary, berberine-loaded magnetic nanoparticles Fe 3 O 4 @LDH@Ber/PEG-FA provide a new possible therapeutic approach for the treatment of oral squamous cell carcinoma.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1、透射电镜观察空白磁性纳米颗粒的粒径，其中，Figure 1. Transmission electron microscopy observation of the particle size of blank magnetic nanoparticles, where
A为透射电镜下Fe3O4的粒径，A is the particle size of Fe 3 O 4 under transmission electron microscope,
B为透射电镜下Fe3O4@LDH的粒径，B is the particle size of Fe 3 O 4 @LDH under transmission electron microscopy,
C为透射电镜下Fe3O4@LDH/PEG-FA的粒径；C is the particle size of Fe 3 O 4 @LDH/PEG-FA under transmission electron microscopy;
图2、磁性纳米颗粒的分散度指数(PDI)比较的统计图；FIG2 is a statistical diagram showing a comparison of the dispersion index (PDI) of magnetic nanoparticles;
图3、Fe3O4、Fe3O4@LDH和Fe3O4@LDH@Ber/PEG-FA的X射线衍射图；Figure 3. X-ray diffraction patterns of Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH@Ber/PEG-FA;
图4、Fe3O4、Fe3O4@LDH和Fe3O4@LDH@Ber/PEG-FA的磁化曲线图；Figure 4. Magnetization curves of Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH@Ber/PEG-FA;
图5、Fe3O4、Ber、Fe3O4@LDH和Fe3O4@LDH@Ber/PEG-FA的载药量曲线；Figure 5. Drug loading curves of Fe 3 O 4 , Ber, Fe 3 O 4 @LDH and Fe 3 O 4 @LDH@Ber/PEG-FA;
图6、载小檗碱磁性纳米颗粒体外释放曲线，其中，Figure 6. In vitro release curve of berberine-loaded magnetic nanoparticles, where:
A为Fe3O4@LDH@Ber在pH5.0和pH7.4的体外释放曲线，A is the in vitro release curve of Fe 3 O 4 @LDH@Ber at pH 5.0 and pH 7.4.
B为Fe3O4@LDH@Ber/PEG-FA在pH5.0和pH7.4的体外释放曲线；B is the in vitro release curve of Fe 3 O 4 @LDH@Ber/PEG-FA at pH 5.0 and pH 7.4;
图7、空白磁性纳米颗粒对CAL27细胞活性的影响，其中，Figure 7. Effect of blank magnetic nanoparticles on the activity of CAL27 cells, where
A为不同浓度的Fe3O4、Fe3O4@LDH和Fe3O4@LDH/PEG-FA与CAL27细胞共同孵育24小时，CAL27细胞存活率，A is the survival rate of CAL27 cells after incubation of CAL27 cells with different concentrations of Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH/PEG-FA for 24 hours.
B为不同浓度的Fe3O4、Fe3O4@LDH和Fe3O4@LDH/PEG-FA与CAL27细胞共同孵育48小时，CAL27细胞存活率；B is the survival rate of CAL27 cells after incubation of CAL27 cells with different concentrations of Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH/PEG-FA for 48 hours;
图8、荧光显微镜下CAL27细胞摄取Fe3O4@LDH@R6G/PEG-FA和Fe3O4@LDH@R6G/PEG后的荧光强度，其中，Figure 8. Fluorescence intensity of CAL27 cells after taking up Fe 3 O 4 @LDH@R6G/PEG-FA and Fe 3 O 4 @LDH@R6G/PEG under a fluorescence microscope.
A为荧光显微镜下CAL27细胞摄取Fe3O4@LDH@R6G/PEG后1h、2h、4h和6h的荧光强度，A is the fluorescence intensity of CAL27 cells 1h, 2h, 4h and 6h after uptake of Fe 3 O 4 @LDH@R6G/PEG under fluorescence microscope.
B为荧光显微镜下CAL27细胞摄取Fe3O4@LDH@R6G/PEG-FA后1h、2h、4h和6h的荧光强度；B is the fluorescence intensity of CAL27 cells 1h, 2h, 4h and 6h after uptake of Fe 3 O 4 @LDH@R6G/PEG-FA under fluorescence microscope;
图9、载小檗碱磁性纳米颗粒给药后对CAL27细胞活性的影响，其中，Figure 9. Effects of berberine-loaded magnetic nanoparticles on the activity of CAL27 cells after administration,
A为0、2、5、10、25、50和100μg/mL浓度的Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA对CAL27细胞活性的影响，A is the effect of 0, 2, 5, 10, 25, 50 and 100 μg/mL concentrations of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA on the activity of CAL27 cells.
B为小檗碱浓度相同(0、5、10、25和50μg/mL)情况下的Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA对CAL27细胞活性的影响；B is the effect of Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA on the activity of CAL27 cells at the same berberine concentration (0, 5, 10, 25 and 50 μg/mL);
图10、载小檗碱磁性纳米颗粒给药后CAL27细胞内荧光情况，其中，Figure 10. Fluorescence in CAL27 cells after administration of berberine-loaded magnetic nanoparticles, where
A为对照、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内荧光显微镜下荧光，A is the fluorescence of CAL27 cells in the control, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups under fluorescence microscope.
B为对照、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内平均荧光强度统计；B is the statistics of the mean fluorescence intensity in CAL27 cells in the control, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups;
图11、载小檗碱磁性纳米颗粒给药后CAL27细胞内GSH浓度水平；Figure 11. GSH concentration levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles;
图12、载小檗碱磁性纳米颗粒给药后CAL27细胞内BAX和Bcl-XL基因表达变化，其中，Figure 12. Changes in BAX and Bcl-XL gene expression in CAL27 cells after administration of berberine-loaded magnetic nanoparticles, where
A为对照、5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内BAX基因表达量统计，A is the statistics of BAX gene expression in CAL27 cells in the control, 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups.
B为对照、5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内Bcl-XL基因量统计；B is the statistics of Bcl-XL gene quantity in CAL27 cells in control, 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups;
图13、载小檗碱磁性纳米颗粒给药后CAL27细胞内H2O2水平变化；Figure 13. Changes in H 2 O 2 levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles;
图14、载小檗碱磁性纳米颗粒给药后CAL27细胞内线粒体膜电位变化，其中，Figure 14. Changes in mitochondrial membrane potential in CAL27 cells after administration of berberine-loaded magnetic nanoparticles, where
A为对照、5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组J-monomer、J-aggregate和Merge荧光，A is the J-monomer, J-aggregate and Merge fluorescence of the control, 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups.
B为对照、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组JC-1红/绿荧光强度统计；B is the statistics of red/green fluorescence intensity of JC-1 in control, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups;
图15、空白磁性纳米颗粒的溶血情况，其中，Figure 15. Hemolysis of blank magnetic nanoparticles, where
A为阳性、阴性、25、50、100、200、400和1000μg/mL的Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA的溶血现象外观，A is the appearance of hemolysis of positive, negative, 25, 50, 100, 200, 400 and 1000 μg/mL Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG, and Fe 3 O 4 @LDH/PEG-FA.
B为阳性、阴性、25、50、100、200、400和1000μg/mL的Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA的溶血率；B is the hemolysis rate of positive, negative, 25, 50, 100, 200, 400 and 1000 μg/mL Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG, and Fe 3 O 4 @LDH/PEG-FA;
其中图1-图15中，In Figures 1 to 15,
*、**、***和****分别代表：P<0.05、P<0.01、P<0.001和P<0.0001，表示两组间相比差异具有的统计学意义。*, **, *** and **** represent P<0.05, P<0.01, P<0.001 and P<0.0001, respectively, indicating that the difference between the two groups was statistically significant.
具体实施方式Detailed ways
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
下述实施例所使用的实验方法如无特殊说明，均为常规方法。Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.
下述实施例中所有的材料、试剂等，如无特殊说明，均可从商业途径获得。All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.
实验材料Experimental Materials
1、细胞1. Cells
人舌鳞癌细胞(CAL27)，购于武汉大学中国典型培养物保藏中心Human tongue squamous cell carcinoma cells (CAL27) were purchased from China Center for Type Culture Collection, Wuhan University.
红细胞，从黑鼠新鲜血液获得Red blood cells, obtained from fresh blood of black rats
2、主要试剂2. Main reagents
FeCl3·6H2O，上海阿拉丁生化科技股份有限公司(中国)FeCl 3 ·6H 2 O, Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
柠檬酸三钠(Na3Cit·2H2O)，上海阿拉丁生化科技股份有限公司(中国)Trisodium citrate (Na 3 Cit·2H 2 O), Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
醋酸钠(NaAc)，上海阿拉丁生化科技股份有限公司(中国)Sodium acetate (NaAc), Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
无水乙醇，天津康科德科技有限公司(中国)Anhydrous ethanol, Tianjin Concord Technology Co., Ltd. (China)
MgCl2·6H2O，上海阿拉丁生化科技股份有限公司(中国)MgCl 2 ·6H 2 O, Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
AlCl3·6H2O，上海阿拉丁生化科技股份有限公司(中国)AlCl 3 ·6H 2 O, Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
二甲基亚砜(DMSO)，天津市康科德科技有限公司Dimethyl sulfoxide (DMSO), Tianjin Concord Technology Co., Ltd.
1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)，上海阿拉丁生化科技股份有限公司1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), Shanghai Aladdin Biochemical Technology Co., Ltd.
N-羟基硫代琥珀酰亚胺(NHS)，上海阿拉丁生化科技股份有限公司N-Hydroxysulfosuccinimide (NHS), Shanghai Aladdin Biochemical Technology Co., Ltd.
氨基-聚乙二醇-氨基(NH2-PEG-NH2)，上海阿拉丁生化科技股份有限公司Amino-polyethylene glycol-amino (NH 2 -PEG-NH 2 ), Shanghai Aladdin Biochemical Technology Co., Ltd.
叶酸(FA)，上海阿拉丁生化科技股份有限公司Folic acid (FA), Shanghai Aladdin Biochemical Technology Co., Ltd.
二甲基甲酰胺(DMF)，天津市康科德科技有限公司Dimethylformamide (DMF), Tianjin Concord Technology Co., Ltd.
氮气(N2)，天津永腾气体销售有限公司(中国)Nitrogen (N 2 ), Tianjin Yongteng Gas Sales Co., Ltd. (China)
氢氧化钠(NaOH)，上海阿拉丁生化科技股份有限公司(中国)Sodium hydroxide (NaOH), Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
小檗碱对照品，上海阿拉丁生化科技股份有限公司(中国)，纯度99％，-20℃保存Berberine reference substance, Shanghai Aladdin Biochemical Technology Co., Ltd. (China), purity 99%, stored at -20℃
浓盐酸，天津康科德科技有限公司(中国)Concentrated hydrochloric acid, Tianjin Concord Technology Co., Ltd. (China)
甲醇，天津康科德科技有限公司(中国)Methanol, Tianjin Concord Technology Co., Ltd. (China)
透析袋(Mw＝3500Da)，上海源叶生物科技有限公司(中国)Dialysis bag (Mw = 3500Da), Shanghai Yuanye Biotechnology Co., Ltd. (China)
10％热灭活胎牛血清，北京索莱宝科技有限公司(中国)10% heat-inactivated fetal bovine serum, Beijing Solebao Technology Co., Ltd. (China)
青霉素，北京索莱宝科技有限公司(中国)Penicillin, Beijing Solebow Technology Co., Ltd. (China)
链霉素，北京索莱宝科技有限公司(中国)Streptomycin, Beijing Solebow Technology Co., Ltd. (China)
90％DMEM培养基，Gibco赛默飞世尔科技ThermoFisher Scientific(美国)90% DMEM culture medium, Gibco ThermoFisher Scientific (USA)
4％多聚甲醛，北京索莱宝科技有限公司(中国)4% paraformaldehyde, Beijing Solebow Technology Co., Ltd. (China)
Hoechst33342染色液，北京索莱宝科技有限公司(中国)Hoechst 33342 staining solution, Beijing Solebow Technology Co., Ltd. (China)
罗丹明6G(R6G)，上海阿拉丁生化科技股份有限公司(中国)Rhodamine 6G (R6G), Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
细胞增殖试剂盒CCK8，碧云天公司(中国)Cell proliferation kit CCK8, Bio-Tech (China)
5-氟尿嘧啶(5-FU)，上海阿拉丁生化科技股份有限公司(中国)5-Fluorouracil (5-FU), Shanghai Aladdin Biochemical Technology Co., Ltd. (China)
活性氧荧光探针(DCFH-DA)，北京索莱宝科技有限公司(中国)Reactive oxygen species fluorescent probe (DCFH-DA), Beijing Solebow Technology Co., Ltd. (China)
BCA蛋白含量测定试剂盒，北京索莱宝科技有限公司(中国)BCA protein content assay kit, Beijing Solebow Technology Co., Ltd. (China)
蛋白定量(TP)测定试剂盒，南京建成生物工程研究所Protein quantitative (TP) assay kit, Nanjing Jiancheng Bioengineering Institute
还原型谷胱甘肽(GSH)测定试剂盒(微板法)，北京索莱宝科技有限公司(中国)Reduced glutathione (GSH) assay kit (microplate method), Beijing Solebow Technology Co., Ltd. (China)
按照线粒体膜电位检测试剂盒，北京索莱宝科技有限公司(中国)According to the mitochondrial membrane potential detection kit, Beijing Solebow Technology Co., Ltd. (China)
4、软件4. Software
Image J软件，NIH(National Institutes of Health,美国国家卫生研究院)Image J software, NIH (National Institutes of Health, U.S.)
5、仪器5. Instruments
实施例1、Fe3O4@LDH@Ber/PEG-FA的合成Example 1. Synthesis of Fe 3 O 4 @LDH@Ber/PEG-FA
一、合成Fe3O4 NPs1. Synthesis of Fe 3 O 4 NPs
3.25g FeCl3·6H2O和1.2g Na3Cit·2H2O溶于100mL乙二醇中，加热搅拌，加入NaAc 6g，搅拌溶解，溶液置于Teflon衬垫上，加热，无水乙醇和去离子水洗涤材料3次，除杂，收集Teflon衬垫上的Fe3O4 NPs冷冻干燥即得；3.25 g FeCl 3 ·6H 2 O and 1.2 g Na 3 Cit·2H 2 O were dissolved in 100 mL ethylene glycol, heated and stirred, 6 g NaAc was added, stirred and dissolved, the solution was placed on a Teflon pad, heated, and the material was washed three times with anhydrous ethanol and deionized water to remove impurities, and the Fe 3 O 4 NPs on the Teflon pad were collected and freeze-dried to obtain;
二、合成Fe3O4@LDH NPs2. Synthesis of Fe 3 O 4 @LDH NPs
610mg MgCl2·6H2O和241mg AlCl3·6H2O溶解于10mL水，加入99mg Fe3O4 NPs，超声使混合溶液均匀分散，在氮气保护下加入0.15M的NaOH溶液40mL，磁铁吸附得到磁性产物粗品，磁性产物粗品用40mL水加热处理(100℃，16h),再用磁铁吸附合成的Fe3O4@LDH NPs，水洗除杂，冷冻干燥即得；610 mg MgCl 2 ·6H 2 O and 241 mg AlCl 3 ·6H 2 O were dissolved in 10 mL water, 99 mg Fe 3 O 4 NPs were added, and the mixed solution was evenly dispersed by ultrasound. 40 mL of 0.15 M NaOH solution was added under nitrogen protection, and a crude magnetic product was obtained by magnet adsorption. The crude magnetic product was heated with 40 mL of water (100°C, 16 h), and the synthesized Fe 3 O 4 @LDH NPs were adsorbed by a magnet, washed with water to remove impurities, and freeze-dried to obtain;
三、合成Fe3O4@LDH-PEG-NH2 3. Synthesis of Fe 3 O 4 @LDH-PEG-NH 2
15mg的Fe3O4@LDH加入15mL的DMSO超声溶解，搅拌均匀后，加入5mg的EDC和3mg的NHS混匀活化羧基，避光搅拌3h，使成为活化的Fe3O4@LDH溶液；15 mg of Fe 3 O 4 @LDH was added to 15 mL of DMSO and dissolved by ultrasonication. After stirring evenly, 5 mg of EDC and 3 mg of NHS were added and mixed evenly to activate the carboxyl group. The mixture was stirred for 3 h in the dark to obtain an activated Fe 3 O 4 @LDH solution.
7.5mg的NH2-PEG-NH2溶解于7.5mL的DMSO中，在持续搅拌的条件下逐滴滴加活化的Fe3O4@LDH溶液15mL，避光磁力搅拌12h，10000r/min离心10min，弃去游离的NH2-PEG-NH2、EDC、NHS，取离心沉淀水洗三次，真空干燥，即得Fe3O4@LDH-PEG-NH2；7.5 mg of NH 2 -PEG-NH 2 was dissolved in 7.5 mL of DMSO, and 15 mL of activated Fe 3 O 4 @LDH solution was added dropwise under continuous stirring. The mixture was stirred magnetically for 12 h in a dark place, and centrifuged at 10000 r/min for 10 min. Free NH 2 -PEG-NH 2 , EDC, and NHS were discarded, and the centrifugal precipitate was washed with water three times and dried under vacuum to obtain Fe 3 O 4 @LDH-PEG-NH 2 ;
四、合成Fe3O4@LDH/PEG-FA4. Synthesis of Fe 3 O 4 @LDH/PEG-FA
称取30mg的Fe3O4@LDH-PEG-NH2用30ml的DMSO溶解，将15mg的FA、6.5mg的EDC、3.9mg的NHS混合于8mL的DMSO溶液中，充分搅拌3h后，逐滴加入Fe3O4@LDH-PEG-NH2溶液中，避光磁力搅拌4h，用截留分子量为1000的透析袋清洗48h，去除游离的小分子，真空干燥得Fe3O4@LDH/PEG-FA；Weigh 30 mg of Fe 3 O 4 @LDH-PEG-NH 2 and dissolve it in 30 ml of DMSO. Mix 15 mg of FA, 6.5 mg of EDC, and 3.9 mg of NHS in 8 mL of DMSO solution. After stirring for 3 h, add them dropwise to the Fe 3 O 4 @LDH-PEG-NH 2 solution. Stir magnetically in a dark place for 4 h. Wash with a dialysis bag with a molecular weight cutoff of 1000 for 48 h to remove free small molecules. Dry in vacuum to obtain Fe 3 O 4 @LDH/PEG-FA.
五、合成Fe3O4@LDH@Ber/PEG-FA5. Synthesis of Fe 3 O 4 @LDH@Ber/PEG-FA
将20mg Fe3O4@LDH/PEG-FA和20mg Ber加入50％的20mL的DMF中，超声混合均匀，搅拌24h载药，载药结束后8000r/min离心10min，弃去上清，沉淀水洗三次，真空干燥，即得Fe3O4@LDH@Ber/PEG-FA。20 mg Fe 3 O 4 @LDH/PEG-FA and 20 mg Ber were added to 20 mL of 50% DMF, mixed evenly by ultrasonication, and loaded with drugs by stirring for 24 h. After drug loading, the mixture was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, the precipitate was washed with water three times, and vacuum dried to obtain Fe 3 O 4 @LDH@Ber/PEG-FA.
实施例2、实施例1中空白磁性纳米颗粒和载小檗碱磁性纳米颗粒的粒径及分散度考察Example 2: Investigation of the particle size and dispersion of blank magnetic nanoparticles and berberine-loaded magnetic nanoparticles in Example 1
一、方法1. Methods
通过透射电镜观察样品的平均粒径，另外用动态光散射(DLS)检测样品的水动力粒径和分散度指数(PDI)，样品为实施例1中空白磁性纳米颗粒(Fe3O4、Fe3O4@LDH NPs和Fe3O4@LDH/PEG-FA)和载药磁性纳米颗粒(Fe3O4@LDH@Ber/PEG-FA)。The average particle size of the samples was observed by transmission electron microscopy, and the hydrodynamic particle size and dispersion index (PDI) of the samples were detected by dynamic light scattering (DLS). The samples were blank magnetic nanoparticles (Fe 3 O 4 , Fe 3 O 4 @LDH NPs and Fe 3 O 4 @LDH/PEG-FA) and drug-loaded magnetic nanoparticles (Fe 3 O 4 @LDH@Ber/PEG-FA) in Example 1.
二、结果2. Results
通过透射电镜观察，Fe3O4平均粒径为113.63nm的圆球体(如图1A所示)，另外，动态光散射(DLS)检测Fe3O4的水动力粒径为139.10nm，分散度为0.229；Through transmission electron microscopy, the average particle size of Fe 3 O 4 was 113.63 nm, and the spheres were as shown in Figure 1A. In addition, dynamic light scattering (DLS) detected that the hydrodynamic particle size of Fe 3 O 4 was 139.10 nm, and the dispersion was 0.229;
通过透射电镜观察，Fe3O4@LDH平均粒径为124.93nm的圆球体(如图1B所示)；另外，动态光散射(DLS)检测Fe3O4@LDH的水动力粒径为152.4nm，分散度为0.237；Through transmission electron microscopy observation, the average particle size of Fe 3 O 4 @LDH is 124.93 nm (as shown in Figure 1B); in addition, dynamic light scattering (DLS) detected that the hydrodynamic particle size of Fe 3 O 4 @LDH is 152.4 nm and the dispersion is 0.237;
通过透射电镜观察，Fe3O4@LDH/PEG-FA平均粒径为168.89nm的圆球体(如图1C所示)；另外，动态光散射(DLS)检测Fe3O4@LDH/PEG-FA的水动力粒径为168.68nm，分散度为0.181；Through transmission electron microscopy observation, the average particle size of Fe 3 O 4 @LDH/PEG-FA was 168.89 nm (as shown in Figure 1C); in addition, dynamic light scattering (DLS) detected that the hydrodynamic particle size of Fe 3 O 4 @LDH/PEG-FA was 168.68 nm and the dispersion was 0.181;
动态光散射(DLS)检测Fe3O4@LDH@Ber/PEG-FA的水动力粒径为189.83nm，分散度为0.190；Dynamic light scattering (DLS) detected that the hydrodynamic particle size of Fe 3 O 4 @LDH@Ber/PEG-FA was 189.83 nm and the dispersion was 0.190;
如图2所示，Fe3O4@LDH/PEG-FA的分散度指数PDI显著小于Fe3O4@LDH，P值为0.002。As shown in Figure 2, the dispersion index PDI of Fe 3 O 4 @LDH/PEG-FA is significantly smaller than that of Fe 3 O 4 @LDH, with a P value of 0.002.
三、分析Analysis
聚乙二醇涂层使聚合物Fe3O4@LDH@Ber/PEG-FA分散得更均匀，从而改善Fe3O4@LDH@Ber/PEG-FA的溶解度，增加药物释放。The polyethylene glycol coating made the polymer Fe 3 O 4 @LDH@Ber/PEG-FA dispersed more evenly, thereby improving the solubility of Fe 3 O 4 @LDH@Ber/PEG-FA and increasing drug release.
实施例3、NH2-PEG-NH2和FA接枝在Fe3O4@LDH表面的工艺参数筛选Example 3. Screening of process parameters for grafting NH 2 -PEG-NH 2 and FA on the surface of Fe 3 O 4 @LDH
分两步将NH2-PEG-NH2和FA接枝在Fe3O4@LDH表面，第一步先接枝NH2-PEG-NH2(聚乙二醇涂层)，第二步接枝叶酸，通过不同浓度的反应起始物以及反应时间制备成粒径较小，分散度较优的反应产物来选择最佳工艺参数。NH 2 -PEG-NH 2 and FA were grafted onto the surface of Fe 3 O 4 @LDH in two steps. In the first step, NH 2 -PEG-NH 2 (polyethylene glycol coating) was grafted, and in the second step, folic acid was grafted. The optimal process parameters were selected by preparing reaction products with smaller particle size and better dispersion through different concentrations of reaction starting materials and reaction time.
一、接枝NH2-PEG-NH2的工艺参数筛选1. Screening of process parameters for grafting NH 2 -PEG-NH 2
Fe3O4 NPs、Fe3O4@LDH NPs的合成分别与实施例1中“一、合成Fe3O4 NPs”、“二、合成Fe3O4@LDH NPs”相同，在此基础上，我们对Fe3O4@LDH NPs接枝NH2-PEG-NH2的浓度和反应时间进行筛选，其余操作同实施例1中“三、合成Fe3O4@LDH-PEG-NH2”，以获得合成Fe3O4@LDH-PEG-NH2最佳的工艺参数。The syntheses of Fe 3 O 4 NPs and Fe 3 O 4 @LDH NPs were the same as “I. Synthesis of Fe 3 O 4 NPs” and “II. Synthesis of Fe 3 O 4 @LDH NPs” in Example 1, respectively. On this basis, we screened the concentration and reaction time of Fe 3 O 4 @LDH NPs grafted with NH 2 -PEG-NH 2 , and the other operations were the same as “III. Synthesis of Fe 3 O 4 @LDH-PEG-NH 2 ” in Example 1 to obtain the optimal process parameters for synthesizing Fe 3 O 4 @LDH-PEG-NH 2 .
聚乙二醇(NH2-PEG-NH2)浓度和反应时间，按表1中条件进行了筛选。The concentration of polyethylene glycol (NH 2 -PEG-NH 2 ) and the reaction time were screened according to the conditions in Table 1 .
表1、聚乙二醇(NH2-PEG-NH2)浓度、反应时间与反应产物的粒径和分散度指数Table 1. Polyethylene glycol (NH 2 -PEG-NH 2 ) concentration, reaction time, particle size and dispersion index of the reaction product
从表1可以看出，0.5mg/mL NH2-PEG-NH2反应12h反应产物Fe3O4@LDH-PEG-NH2分散度最佳，而且粒径相对较小，所以我们选择0.5mg/mL NH2-PEG-NH2反应12h这一工艺参数。As can be seen from Table 1, the dispersion of the reaction product Fe 3 O 4 @LDH-PEG-NH 2 was the best when 0.5 mg/mL NH 2 -PEG-NH 2 was reacted for 12 h, and the particle size was relatively small, so we chose the process parameter of 0.5 mg/mL NH 2 -PEG-NH 2 for 12 h.
二、接枝叶酸(FA)的工艺参数筛选2. Screening of process parameters for grafting folic acid (FA)
Fe3O4 NPs、Fe3O4@LDH NPs的合成分别与实施例1中“一、合成Fe3O4NPs”、“二、合成Fe3O4@LDH NPs”相同，，接枝NH2-PEG-NH2的反应起始物NH2-PEG-NH2浓度0.5mg/mL，反应时间为12h，其余与实施例1中“三、合成Fe3O4@LDH-PEG-NH2”相同，我们对接枝叶酸(FA)浓度和反应时间按表2条件进行了筛选。The syntheses of Fe 3 O 4 NPs and Fe 3 O 4 @LDH NPs were the same as those in “I. Synthesis of Fe 3 O 4 NPs” and “II. Synthesis of Fe 3 O 4 @LDH NPs” in Example 1, respectively. The concentration of the reaction starting material NH 2 -PEG-NH 2 for grafting NH 2 -PEG-NH 2 was 0.5 mg/mL, and the reaction time was 12 h. The rest were the same as those in “III. Synthesis of Fe 3 O 4 @LDH-PEG-NH 2 ” in Example 1. We screened the grafting folic acid (FA) concentration and reaction time according to the conditions in Table 2.
表2、接枝叶酸浓度、反应时间与反应产物的粒径和分散度指数Table 2. Grafted folic acid concentration, reaction time, particle size and dispersion index of the reaction product
从表2可以看出，0.5mg/mL Fe3O4@LDH-PEG-NH2+0.5mg/mL FA反应4h条件，反应产物Fe3O4@LDH/PEG-FA的分散度最佳，而且粒径相对较小，所以确定了0.5mg/mL Fe3O4@LDH-PEG-NH2+0.5mg/mL FA，反应时间4小时的工艺参数接枝叶酸(FA)。As can be seen from Table 2, under the conditions of 0.5 mg/mL Fe 3 O 4 @LDH-PEG-NH 2 + 0.5 mg/mL FA reaction time of 4 h, the dispersion of the reaction product Fe 3 O 4 @LDH/PEG-FA is the best, and the particle size is relatively small. Therefore, the process parameters of 0.5 mg/mL Fe 3 O 4 @LDH-PEG-NH 2 + 0.5 mg/mL FA and reaction time of 4 hours were determined for grafting folic acid (FA).
三、最终确定的合成Fe3O4@LDH@Ber/PEG-FA最优工艺参数3. The optimal process parameters for the synthesis of Fe 3 O 4 @LDH@Ber/PEG-FA were finally determined
(一)合成Fe3O4 NPs(I) Synthesis of Fe 3 O 4 NPs
3.25g的FeCl3·6H2O和1.2g Na3Cit·2H2O溶于100mL乙二醇中，加热搅拌，加入NaAc 6g，搅拌溶解，溶液置于Teflon衬垫上，加热，无水乙醇和去离子水洗涤材料3次，除杂，收集Teflon衬垫上的Fe3O4 NPs冷冻干燥即得。3.25 g of FeCl 3 ·6H 2 O and 1.2 g of Na 3 Cit·2H 2 O were dissolved in 100 mL of ethylene glycol, heated and stirred, 6 g of NaAc was added, stirred and dissolved, the solution was placed on a Teflon pad, heated, and the material was washed three times with anhydrous ethanol and deionized water to remove impurities, and the Fe 3 O 4 NPs on the Teflon pad were collected and freeze-dried.
(二)合成Fe3O4@LDH NPs(II) Synthesis of Fe 3 O 4 @LDH NPs
610mg MgCl2·6H2O和241mg AlCl3·6H2O溶解于10mL水，加入99mg Fe3O4 NPs，超声使混合溶液均匀分散，在氮气保护下加入0.15M的NaOH溶液40mL，磁铁吸附得到磁性产物粗品，磁性产物粗品用40mL水加热处理(100℃，16h),再用磁铁吸附合成的Fe3O4@LDH NPs，水洗除杂，冷冻干燥即得。610 mg MgCl 2 ·6H 2 O and 241 mg AlCl 3 ·6H 2 O were dissolved in 10 mL water, 99 mg Fe 3 O 4 NPs were added, and the mixed solution was evenly dispersed by ultrasound. 40 mL of 0.15 M NaOH solution was added under nitrogen protection, and the crude magnetic product was adsorbed by magnet. The crude magnetic product was heated with 40 mL of water (100°C, 16 h), and the synthesized Fe 3 O 4 @LDH NPs were adsorbed by magnet, washed with water to remove impurities, and freeze-dried to obtain.
(三)合成Fe3O4@LDH-PEG-NH2 (III) Synthesis of Fe 3 O 4 @LDH-PEG-NH 2
15mg的Fe3O4@LDH加入15mL的DMSO超声溶解，搅拌均匀后，加入5mg的EDC和3mg的NHS混匀活化羧基，避光搅拌3h，使成为活化的Fe3O4@LDH溶液；15 mg of Fe 3 O 4 @LDH was added to 15 mL of DMSO and dissolved by ultrasonication. After stirring evenly, 5 mg of EDC and 3 mg of NHS were added and mixed evenly to activate the carboxyl group. The mixture was stirred for 3 h in the dark to obtain an activated Fe 3 O 4 @LDH solution.
7.5mg的NH2-PEG-NH2溶解于15mL的DMSO中，配制成0.5mg/mL的NH2-PEG-NH2的DMSO溶液，在持续搅拌的条件下逐滴滴加活化的Fe3O4@LDH溶液15mL，避光磁力搅拌12h，10000r/min离心10min，弃去游离的NH2-PEG-NH2、EDC、NHS，取离心沉淀水洗三次，真空干燥，即得Fe3O4@LDH-PEG-NH2；7.5 mg of NH 2 -PEG-NH 2 was dissolved in 15 mL of DMSO to prepare a 0.5 mg/mL NH 2 -PEG-NH 2 DMSO solution. 15 mL of activated Fe 3 O 4 @LDH solution was added dropwise under continuous stirring. The mixture was magnetically stirred for 12 h in a dark environment and centrifuged at 10000 r/min for 10 min. Free NH 2 -PEG-NH 2 , EDC and NHS were discarded. The centrifugal precipitate was washed with water three times and dried under vacuum to obtain Fe 3 O 4 @LDH-PEG-NH 2 .
(四)合成Fe3O4@LDH/PEG-FA(IV) Synthesis of Fe 3 O 4 @LDH/PEG-FA
称取30mg的Fe3O4@LDH-PEG-NH2用60mL的DMSO溶解，配制成0.5mg/mL的Fe3O4@LDH-PEG-NH2的DMSO溶液，将15mg的FA、6.5mg的EDC、3.9mg的NHS混合于30mL的DMSO溶液中，配制成FA为0.5mg/mL的DMSO溶液，充分搅拌3h后，逐滴加入Fe3O4@LDH-PEG-NH2的DMSO溶液中，避光磁力搅拌4h，用截留分子量为1000的透析袋清洗48h，去除游离的小分子，真空干燥得Fe3O4@LDH/PEG-FA；Weigh 30 mg of Fe 3 O 4 @LDH-PEG-NH 2 and dissolve it in 60 mL of DMSO to prepare a 0.5 mg/mL DMSO solution of Fe 3 O 4 @LDH-PEG-NH 2. Mix 15 mg of FA, 6.5 mg of EDC, and 3.9 mg of NHS in 30 mL of DMSO solution to prepare a 0.5 mg/mL DMSO solution of FA. After stirring for 3 h, add the mixture dropwise to the DMSO solution of Fe 3 O 4 @LDH-PEG-NH 2. Stir magnetically in a dark place for 4 h. Wash with a dialysis bag with a molecular weight cutoff of 1000 for 48 h to remove free small molecules. Dry in vacuum to obtain Fe 3 O 4 @LDH/PEG-FA.
(五)合成Fe3O4@LDH@Ber/PEG-FA(V) Synthesis of Fe 3 O 4 @LDH@Ber/PEG-FA
将20mg的Fe3O4@LDH/PEG-FA和20mg的Ber加入50％的20mL的DMF中，超声混合均匀，搅拌24h载药，载药结束后8000r/min离心10min，弃去上清，沉淀水洗三次，真空干燥，即得Fe3O4@LDH@Ber/PEG-FA。20 mg of Fe 3 O 4 @LDH/PEG-FA and 20 mg of Ber were added to 20 mL of 50% DMF, mixed evenly by ultrasonication, and loaded with drugs by stirring for 24 h. After drug loading, the mixture was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, the precipitate was washed with water three times, and vacuum dried to obtain Fe 3 O 4 @LDH@Ber/PEG-FA.
四、采用上述最优工艺参数合成Fe3O4@LDH@Ber/PEG-FA的粒径检测4. Particle size detection of Fe 3 O 4 @LDH@Ber/PEG-FA synthesized using the above optimal process parameters
通过动态光散射检测Fe3O4@LDH@Ber/PEG-FA的粒径和分散度。The particle size and dispersion of Fe 3 O 4 @LDH@Ber/PEG-FA were detected by dynamic light scattering.
结果：Fe3O4@LDH@Ber/PEG-FA的水动力粒径为177.24nm，分散度为0.185。Results: The hydrodynamic particle size of Fe 3 O 4 @LDH@Ber/PEG-FA was 177.24 nm and the dispersion degree was 0.185.
后续实验用样品均采用上述最优工艺参数合成的Fe3O4@LDH@Ber/PEG-FA样品进行。The samples used in subsequent experiments were all Fe 3 O 4 @LDH@Ber/PEG-FA samples synthesized using the above-mentioned optimal process parameters.
五、Fe3O4@LDH@Ber的合成5. Synthesis of Fe 3 O 4 @LDH@Ber
将活化好的10mL Fe3O4@LDH溶液加入含20mg Ber的50％的10mL DMF溶液中，搅拌24h载药，载药结束后8000r/min离心10min，弃去上清，沉淀水洗三次，真空干燥，即得Fe3O4@LDH@Ber。10 mL of activated Fe 3 O 4 @LDH solution was added to 10 mL of 50% DMF solution containing 20 mg of Ber, and the drug was loaded by stirring for 24 h. After the drug loading was completed, the solution was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, the precipitate was washed three times with water, and vacuum dried to obtain Fe 3 O 4 @LDH@Ber.
实施例4、通过x射线衍射考察磁性纳米颗粒的晶体结构Example 4: Investigation of the crystal structure of magnetic nanoparticles by X-ray diffraction
一、方法1. Methods
通过x射线衍射考察Fe3O4@LDH@Ber/PEG-FA、Fe3O4@LDH和Fe3O4磁性纳米颗粒的晶体结构。The crystal structures of Fe 3 O 4 @LDH@Ber/PEG-FA, Fe 3 O 4 @LDH and Fe 3 O 4 magnetic nanoparticles were investigated by X-ray diffraction.
二、结果2. Results
结果见图3，X射线衍射图(XRD谱图)，其中，The results are shown in Figure 3, X-ray diffraction diagram (XRD spectrum), where:
2θ＝29.86、35.4、43.28、53.64、57.04、62.68位置处出现的衍射峰分别对应Fe3O4晶体面的(220)、(311)、(400)、(422)、(511)和(440)面；The diffraction peaks appearing at positions 2θ = 29.86, 35.4, 43.28, 53.64, 57.04 , and 62.68 correspond to the (220), (311), (400), (422), (511), and (440) planes of the Fe 3 O 4 crystal plane, respectively;
Fe3O4@LDH样品中2θ＝23.14出现弱且宽的衍射峰，对应了LDH的(006)晶体面。In the Fe 3 O 4 @LDH sample, a weak and broad diffraction peak appears at 2θ=23.14, corresponding to the (006) crystal plane of LDH.
三、结论Conclusion
Fe3O4@LDH表面修饰PEG-FA后，Fe3O4@LDH/PEG-FA的特征衍射峰与Fe3O4@LDH基本一致，未出现额外的衍射峰，表明在Fe3O4@LDH表面修饰PEG-FA不影响晶体结构。After the surface of Fe 3 O 4 @LDH was modified with PEG-FA, the characteristic diffraction peaks of Fe 3 O 4 @LDH/PEG-FA were basically consistent with those of Fe 3 O 4 @LDH, and no additional diffraction peaks appeared, indicating that the modification of PEG-FA on the surface of Fe 3 O 4 @LDH did not affect the crystal structure.
实施例5、使用Squid-VSM磁测量系统检测磁性纳米颗粒的磁性能Example 5: Detecting the magnetic properties of magnetic nanoparticles using the Squid-VSM magnetic measurement system
一、方法1. Methods
使用Squid-VSM磁测量系统检测磁性纳米颗粒Fe3O4、Fe3O4@LDH和Fe3O4@LDH@Ber/PEG-FA的磁化曲线图。The magnetization curves of magnetic nanoparticles Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH@Ber/PEG-FA were detected using the Squid-VSM magnetic measurement system.
二、结果2. Results
结果见图4，饱和磁化强度：Fe3O4为63.81emu/g，Fe3O4@LDH为52.34emu/g，，Fe3O4@LDH@Ber/PEG-FA为29.30emu/g；饱和磁化强度：Fe3O4＞Fe3O4@LDH＞Fe3O4@LDH@Ber/PEG-FA。The results are shown in Figure 4. The saturation magnetization intensity is: Fe 3 O 4 is 63.81emu/g, Fe 3 O 4 @LDH is 52.34emu/g, and Fe 3 O 4 @LDH@Ber/PEG-FA is 29.30emu/g; the saturation magnetization intensity is: Fe 3 O 4 >Fe 3 O 4 @LDH >Fe 3 O 4 @LDH@Ber/PEG-FA.
三、分析与结论3. Analysis and Conclusion
由于非磁性LDH、PEG、FA的存在，Fe3O4@LDH的饱和磁化强度为52.34emu/g低于Fe3O4的63.81emu/g，载药Ber并涂层后，Fe3O4@LDH@Ber/PEG-FA的饱和磁化强度降至29.30emu/g，进一步证实了Ber成功负载在Fe3O4@LDH中，且PEG和FA成功修饰于Fe3O4@LDH的表面。Due to the presence of non-magnetic LDH, PEG and FA, the saturation magnetization intensity of Fe 3 O 4 @LDH is 52.34emu/g, which is lower than 63.81emu/g of Fe 3 O 4. After loading Ber and coating, the saturation magnetization intensity of Fe 3 O 4 @LDH@Ber/PEG-FA dropped to 29.30emu/g, further confirming that Ber was successfully loaded in Fe 3 O 4 @LDH and PEG and FA were successfully modified on the surface of Fe 3 O 4 @LDH.
实施例6、Fe3O4@LDH@Ber/PEG-FA的载药率和释药特性Example 6. Drug loading rate and drug release characteristics of Fe 3 O 4 @LDH@Ber/PEG-FA
一、采用紫外-可见光谱法测定Fe3O4@LDH@Ber/PEG-FA的载药率1. Determination of drug loading rate of Fe 3 O 4 @LDH@Ber/PEG-FA by UV-visible spectroscopy
(一)方法1. Methods
精确称取1mg的Fe3O4@LDH@Ber/PEG-FA装入10mL容积瓶中，加入100μL浓盐酸破坏载药配合物，再加入甲醇至10mL。Accurately weigh 1 mg of Fe 3 O 4 @LDH@Ber/PEG-FA and put it into a 10 mL volumetric bottle, add 100 μL of concentrated hydrochloric acid to destroy the drug-loaded complex, and then add methanol to 10 mL.
取小檗碱(Ber)，采用上述步骤处理，观察加入浓盐酸对Ber的影响。在345nm处用吸光度法测定了Ber的浓度。试验重复3次，计算Fe3O4@LDH@Ber/PEG-FA中Ber的载药率。Berberine (Ber) was treated with the above steps to observe the effect of adding concentrated hydrochloric acid on Ber. The concentration of Ber was determined by absorbance at 345 nm. The experiment was repeated 3 times to calculate the drug loading rate of Ber in Fe 3 O 4 @LDH@Ber/PEG-FA.
载药率(％)＝(材料中小檗碱的重量)/材料总重×100Drug loading rate (%) = (weight of berberine in the material) / total weight of the material × 100
(二)结果(II) Results
如图5所示，Fe3O4@LDH@Ber/PEG-FA中Ber的载药率为：29.34％；As shown in Figure 5, the drug loading rate of Ber in Fe 3 O 4 @LDH@Ber/PEG-FA is: 29.34%;
Fe3O4@LDH@Ber的载药率是28.80％。The drug loading rate of Fe 3 O 4 @LDH@Ber is 28.80%.
(三)结论3. Conclusion
Fe3O4@LDH@Ber/PEG-FA小檗碱的载药率较高为29.34％。The drug loading rate of berberine in Fe 3 O 4 @LDH@Ber/PEG-FA was as high as 29.34%.
二、采用紫外-可见光谱法测定载小檗碱的磁性纳米颗粒体外释放曲线2. Determination of in vitro release curve of berberine-loaded magnetic nanoparticles by UV-visible spectroscopy
模拟体内环境，考察了Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA在pH＝7.4和5的释放介质30％乙醇(v/v)的PBS中，温度37℃，48h内小檗碱的体外释放曲线。To simulate the in vivo environment, the in vitro release curves of berberine from Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were investigated in the release medium of 30% ethanol (v/v) in PBS at pH = 7.4 and 5 at 37°C for 48 h.
(一)方法1. Methods
分别取1.5mg的Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA分散于含30％乙醇(v/v)的2mL的PBS缓冲液(pH是分别为5.0或7.4)中，放入透析袋(Mw＝3500Da)。透析袋置于80mL释放介质(pH5.0或7.4的PBS缓冲液)中，37℃暗处摇匀。在预先设计的2、4、6、8、10、12、24、36、48h的时间点，吸取3mL释放介质测量吸光度，取样同时补充3mL释放介质。在345nm处用紫外-可见吸收法测定释放的Ber的浓度。试验设3个重复。1.5 mg of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were dispersed in 2 mL of PBS buffer (pH 5.0 or 7.4, respectively) containing 30% ethanol (v/v) and placed in a dialysis bag (Mw = 3500Da). The dialysis bag was placed in 80 mL of release medium (PBS buffer pH 5.0 or 7.4) and shaken at 37°C in the dark. At the pre-designed time points of 2, 4, 6, 8, 10, 12, 24, 36, and 48 h, 3 mL of release medium was drawn to measure the absorbance, and 3 mL of release medium was added while sampling. The concentration of released Ber was determined by UV-visible absorption at 345 nm. The experiment was repeated 3 times.
(二)结果(II) Results
图6A为Fe3O4@LDH@Ber在pH5.0和pH7.4条件下的体外释放曲线，其中，Figure 6A shows the in vitro release curves of Fe 3 O 4 @LDH@Ber at pH 5.0 and pH 7.4, where
pH7.4时，Fe3O4@LDH@Ber中Ber的释放相对缓慢，且释放量相对较少，48h累积释放量为7.02％；At pH 7.4, the release of Ber from Fe 3 O 4 @LDH@Ber was relatively slow and the release amount was relatively small, with a cumulative release of 7.02% in 48 h;
pH5.0时，Fe3O4@LDH@Ber中Ber的释放显著增加，累积释放量最大值为：30.41％；At pH 5.0, the release of Ber in Fe 3 O 4 @LDH@Ber increased significantly, and the maximum cumulative release was 30.41%;
图6B为Fe3O4@LDH@Ber/PEG-FA在pH5.0和pH7.4条件下的体外释放曲线，其中，Figure 6B shows the in vitro release curves of Fe 3 O 4 @LDH@Ber/PEG-FA at pH 5.0 and pH 7.4, where
pH7.4时，Fe3O4@LDH@Ber/PEG-FA中Ber的释放相对缓慢，且释放量相对较少，0-6h时释放量不断增加，随着时间增加，释放量逐渐趋于平稳，48h累积释放量为3.87％；At pH 7.4, the release of Ber from Fe 3 O 4 @LDH@Ber/PEG-FA was relatively slow, and the release amount was relatively small. The release amount increased continuously from 0 to 6 h, and gradually stabilized with time. The cumulative release amount was 3.87% in 48 h.
pH5.0时，Fe3O4@LDH@Ber/PEG-FA中Ber的释放量显著升高，48h累积释放量达到53.77％。At pH 5.0, the release of Ber from Fe 3 O 4 @LDH@Ber/PEG-FA increased significantly, and the cumulative release after 48 h reached 53.77%.
(三)分析与结论(III) Analysis and Conclusion
1、Fe3O4@LDH@Ber不具有pH响应性1. Fe 3 O 4 @LDH@Ber is not pH responsive
Fe3O4@LDH@Ber在pH5.0的释放量高于pH7.4，可能与材料框架在不同pH的溶解度不同有关。The release amount of Fe 3 O 4 @LDH@Ber at pH 5.0 is higher than that at pH 7.4, which may be related to the different solubility of the material framework at different pH values.
2、Fe3O4@LDH@Ber/PEG-FA具有pH响应性2. Fe 3 O 4 @LDH@Ber/PEG-FA is pH responsive
Fe3O4@LDH@Ber/PEG-FA在pH7.4缓冲液环境下，48h累积释放量仅为3.87％，分析：pH7.4环境下，材料框架相对稳定，可防止过早释放；The cumulative release of Fe 3 O 4 @LDH@Ber/PEG-FA in pH 7.4 buffer solution was only 3.87% in 48 hours. Analysis: In the pH 7.4 environment, the material framework is relatively stable, which can prevent premature release;
Fe3O4@LDH@Ber/PEG-FA在pH5.0缓冲液环境下，Ber的释放量显著增高，达到53.77％。In the pH 5.0 buffer environment, the release amount of Ber from Fe 3 O 4 @LDH@Ber/PEG-FA increased significantly, reaching 53.77%.
分析：肿瘤环境为酸性环境，pH为5.0左右，Fe3O4@LDH@Ber/PEG-FA具有pH响应性可控制Ber在口腔癌环境中大量释放，在正常组织细胞中不释放或少释放，从而实现对Ber的可控释放，达到减毒增效目的。Analysis: The tumor environment is acidic with a pH of about 5.0. Fe 3 O 4 @LDH@Ber/PEG-FA is pH-responsive and can control the large-scale release of Ber in the oral cancer environment, while it does not release or releases very little in normal tissue cells, thereby achieving the controlled release of Ber and achieving the purpose of reducing toxicity and increasing efficacy.
实施例7、空白磁性纳米颗粒对CAL27细胞存活率的影响Example 7: Effect of Blank Magnetic Nanoparticles on the Survival Rate of CAL27 Cells
一、方法1. Methods
(一)细胞培养1. Cell culture
CAL27细胞系在含有10％热灭活胎牛血清、1％青霉素和链霉素(100U/mL)、90％DMEM的培养基中培养，将细胞置于37℃、5％的CO2的细胞培养箱中，2天更换一次培养液。The CAL27 cell line was cultured in a medium containing 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin (100 U/mL), and 90% DMEM. The cells were placed in a cell culture incubator at 37°C and 5 % CO2, and the culture medium was replaced every 2 days.
(二)空白磁性纳米颗粒对CAL27细胞活性的影响(II) Effects of blank magnetic nanoparticles on the activity of CAL27 cells
采用CCK-8法考察不同空白纳米颗粒对CAL27细胞活性的影响。The CCK-8 method was used to investigate the effects of different blank nanoparticles on the activity of CAL27 cells.
1、将CAL27细胞以1×104个/孔的密度接种到96孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；1. CAL27 cells were seeded into 96-well plates at a density of 1×10 4 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
2、将Fe3O4、Fe3O4@LDH和Fe3O4@LDH/PEG-FA三种空白纳米颗粒，用全培养液稀释至浓度分别为0、6.125、12.5、25、50、100和200μg/mL，与CAL27细胞共孵育24h和48h。全培养液由10％FBS、90％DMEM和1％PS组成。2. Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH/PEG-FA blank nanoparticles were diluted to concentrations of 0, 6.125, 12.5, 25, 50, 100 and 200 μg/mL with whole culture medium, and incubated with CAL27 cells for 24 h and 48 h. Whole culture medium consisted of 10% FBS, 90% DMEM and 1% PS.
3、除去含空白纳米颗粒的培养基，用温PBS缓冲液洗涤细胞三次。3. Remove the culture medium containing blank nanoparticles and wash the cells three times with warm PBS buffer.
4、用100μL含10μL的CCK8的新鲜培养基(Gibco DMEM培养基)在37℃下培养2h，用酶标仪检测450nm处的吸光度。4. Incubate the cells with 100 μL of fresh culture medium (Gibco DMEM medium) containing 10 μL of CCK8 at 37°C for 2 h, and detect the absorbance at 450 nm using an enzyme reader.
二、结果2. Results
如图7所示，不同空白磁性纳米颗粒对CAL27细胞存活率的影响：24和48h，Fe3O4、Fe3O4@LDH和Fe3O4@LDH/PEG-FA不同浓度CAL27细胞存活率均大于80％。As shown in Figure 7, the effects of different blank magnetic nanoparticles on the survival rate of CAL27 cells: at 24 and 48 h, the survival rates of CAL27 cells at different concentrations of Fe 3 O 4 , Fe 3 O 4 @LDH and Fe 3 O 4 @LDH/PEG-FA were all greater than 80%.
三、结论Conclusion
在空白磁性纳米颗粒浓度为200μg/mL时，24和48h均表现出>80％的细胞存活率，表明Fe3O4@LDH/PEG-FA具有良好的生物相容性和低毒性，可作为药物载体进行后续实验。When the blank magnetic nanoparticle concentration was 200 μg/mL, the cell viability was >80% at both 24 and 48 h, indicating that Fe 3 O 4 @LDH/PEG-FA has good biocompatibility and low toxicity and can be used as a drug carrier for subsequent experiments.
实施例8、考察人舌鳞癌细胞对Fe3O4@LDH@R6G/PEG-FA的摄取Example 8: Investigation of the uptake of Fe 3 O 4 @LDH@R6G/PEG-FA by human tongue squamous cell carcinoma cells
由于Ber自身荧光强度较弱，将由荧光剂R6G代替Ber，进行癌细胞对Fe3O4@LDH@R6G/PEG-FA摄取实验的研究。Since the fluorescence intensity of Ber itself is weak, the fluorescent agent R6G will replace Ber to study the uptake experiment of Fe 3 O 4 @LDH@R6G/PEG-FA by cancer cells.
一、制备Fe3O4@LDH@R6G/PEG-FA。1. Preparation of Fe 3 O 4 @LDH@R6G/PEG-FA.
Fe3O4@LDH@R6G/PEG-FA制备方式同Fe3O4@LDH@Ber/PEG-FA。The preparation method of Fe 3 O 4 @LDH@R6G/PEG-FA is the same as that of Fe 3 O 4 @LDH@Ber/PEG-FA.
二、通过荧光显微镜观察人舌鳞癌细胞(CAL27细胞)对Fe3O4@LDH@R6G/PEG-FA和Fe3O4@LDH@R6G/PEG的摄取后的荧光2. Observation of the fluorescence of human tongue squamous cell carcinoma cells (CAL27 cells) after uptake of Fe 3 O 4 @LDH@R6G/PEG-FA and Fe 3 O 4 @LDH@R6G/PEG by fluorescence microscopy
(一)方法1. Methods
1、将CAL27细胞接种于以2×105个/孔的密度接种到12孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；1. CAL27 cells were seeded into 12-well plates at a density of 2×10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM and 1% PS;
2、Fe3O4@LDH@R6G/PEG-FA和Fe3O4@LDH@R6G/PEG分别用完全培养液稀释至200ug/mL，与CAL27细胞在37℃下共同孵育；2. Fe 3 O 4 @LDH@R6G/PEG-FA and Fe 3 O 4 @LDH@R6G/PEG were diluted to 200 ug/mL with complete culture medium and incubated with CAL27 cells at 37°C;
3、在1、2、4、6h取CAL27细胞，然后用温PBS缓冲液洗涤CAL27细胞三次，4％多聚甲醛固定30min，加入Hoechst33342孵育30min，标记细胞核。3. CAL27 cells were collected at 1, 2, 4, and 6 h, and then washed three times with warm PBS buffer, fixed with 4% paraformaldehyde for 30 min, and incubated with Hoechst33342 for 30 min to label the cell nuclei.
4、通过荧光显微镜获得视觉图像，用Image J软件对平均荧光强度进行量化。4. Visual images were obtained by fluorescence microscopy and the mean fluorescence intensity was quantified using Image J software.
(二)结果(II) Results
图8A为CAL27细胞摄取Fe3O4@LDH@R6G/PEG后的荧光强度；Figure 8A shows the fluorescence intensity of CAL27 cells after taking up Fe 3 O 4 @LDH@R6G/PEG;
图8B为CAL27细胞摄取Fe3O4@LDH@R6G/PEG-FA后的荧光强度；Figure 8B shows the fluorescence intensity of CAL27 cells after uptake of Fe 3 O 4 @LDH@R6G/PEG-FA;
CAL27细胞摄取Fe3O4@LDH@R6G/PEG-FA后的荧光强度强于摄取Fe3O4@LDH@R6G/PEG后的荧光强度。The fluorescence intensity of CAL27 cells after taking up Fe 3 O 4 @LDH@R6G/PEG-FA was stronger than that after taking up Fe 3 O 4 @LDH@R6G/PEG.
(三)分析与结论(III) Analysis and Conclusion
CAL27细胞摄取Fe3O4@LDH@R6G/PEG-FA后的荧光强度强于摄取Fe3O4@LDH@R6G/PEG后的荧光强度说明：人舌鳞癌细胞CAL27对Fe3O4@LDH@R6G/PEG-FA的摄取多于Fe3O4@LDH@R6G/PEG。The fluorescence intensity of CAL27 cells after taking up Fe 3 O 4 @LDH@R6G/PEG-FA is stronger than that after taking up Fe 3 O 4 @LDH@R6G/PEG, indicating that human tongue squamous cell carcinoma cells CAL27 take up more Fe 3 O 4 @LDH@R6G/PEG-FA than Fe 3 O 4 @LDH@R6G/PEG.
分析：在Fe3O4@LDH@R6G/PEG表面加入靶向剂叶酸，由于叶酸的受体在CAL27细胞的表面表达的量较多，通过叶酸使Fe3O4@LDH@Ber/PEG-FA与CAL27细胞表面的叶酸受体结合，促进了CAL27细胞对Ber的摄取，从而增加治疗口腔癌的效果。Analysis: The targeting agent folic acid was added to the surface of Fe 3 O 4 @LDH@R6G/PEG. Since the folic acid receptor is expressed in large quantities on the surface of CAL27 cells, folic acid enables Fe 3 O 4 @LDH@Ber/PEG-FA to bind to the folic acid receptor on the surface of CAL27 cells, promoting the uptake of Ber by CAL27 cells, thereby increasing the effect of treating oral cancer.
实施例9、CCK8法评价载小檗碱的磁性纳米颗粒对CAL27细胞的活性的影响Example 9: Evaluation of the effect of berberine-loaded magnetic nanoparticles on the activity of CAL27 cells using CCK8 method
一、方法1. Methods
(一)将CAL27细胞以1×104个/孔的密度接种到96孔板中，37℃孵育24h，弃去培养液；(A) CAL27 cells were seeded into 96-well plates at a density of 1 × 10 4 cells/well, incubated at 37°C for 24 h, and the culture medium was discarded;
(二)将Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA用培养液分别稀释至0、2、5、10、25、50和100μg/mL后与CAL27细胞共同孵育，弃去培养液；(ii) Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were diluted to 0, 2, 5, 10, 25, 50 and 100 μg/mL with culture medium, and then incubated with CAL27 cells, and the culture medium was discarded;
(三)取出CAL27细胞用PBS清洗2-3次后，CAL27细胞与100μL的CCK-8测试溶液在37℃下孵育2h；用酶标仪检测CCK-8测试溶液在450nm波长下的吸光度；(III) After the CAL27 cells were removed and washed with PBS for 2-3 times, the CAL27 cells were incubated with 100 μL of CCK-8 test solution at 37° C. for 2 h; the absorbance of the CCK-8 test solution at a wavelength of 450 nm was detected using an ELISA reader;
(四)将Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA分别用培养液稀释至Ber的浓度0、5、10、25和50μg/mL后与CAL27细胞共同孵育，弃去培养液；(iv) Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were diluted with culture medium to Ber concentrations of 0, 5, 10, 25 and 50 μg/mL, respectively, and then incubated with CAL27 cells, and the culture medium was discarded;
(五)取出CAL27细胞用PBS清洗2-3次后，CAL27细胞与100μL的CCK-8测试溶液在37℃下孵育2h；用酶标仪检测CCK-8测试溶液在450nm波长下的吸光度。(V) After the CAL27 cells were removed and washed 2-3 times with PBS, the CAL27 cells were incubated with 100 μL of CCK-8 test solution at 37° C. for 2 h; the absorbance of the CCK-8 test solution at a wavelength of 450 nm was detected using an ELISA reader.
二、结果2. Results
如图9A所示，Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA可抑制CAL27细胞的活性，且具有浓度依赖性；As shown in Figure 9A, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA could inhibit the activity of CAL27 cells in a concentration-dependent manner;
随浓度升高，CAL27细胞活性不断降低，在药物浓度为100μg/mL，Fe3O4@LDH@Ber/PEG-FA组CAL27细胞活性降低至44.94％；As the concentration increased, the activity of CAL27 cells continued to decrease. When the drug concentration was 100 μg/mL, the activity of CAL27 cells in the Fe 3 O 4 @LDH@Ber/PEG-FA group decreased to 44.94%;
Fe3O4@LDH@Ber组和Fe3O4@LDH@Ber/PEG-FA组浓度相同时，即Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA浓度均为0、2、5、10、25、50、100μg/mL时，When the concentrations of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were the same, that is, when the concentrations of Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA were 0, 2, 5, 10, 25, 50, and 100 μg/mL,
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Fe3O4@LDH@Ber组相比，浓度从低至高，P值分别为：1，0.549，0.018，0.038，0.018，0.011，0.017；即当浓度为5、10、25、50、100μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Fe3O4@LDH@Ber组；The ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was compared with that of Fe 3 O 4 @LDH@Ber group, and the P values were 1, 0.549, 0.018, 0.038, 0.018, 0.011, and 0.017 from low to high concentrations; that is, when the concentrations were 5, 10, 25, 50, and 100 μg/mL, the ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of Fe 3 O 4 @LDH@Ber group.
如图9B所示，相同小檗碱浓度下，即小檗碱浓度均为0、5、10、25、50μg/mL时，As shown in Figure 9B, at the same berberine concentration, that is, berberine concentrations of 0, 5, 10, 25, and 50 μg/mL,
Fe3O4@LDH@Ber组降低CAL27细胞存活率的能力与Ber组相比，P值分别为：1，0.035，0.944，0.025，0.040；即当小檗碱浓度为5、25、50μg/mL时Fe3O4@LDH@Ber组降低CAL27细胞存活率的能力显著强于Ber组；The ability of Fe 3 O 4 @LDH@Ber group to reduce the survival rate of CAL27 cells was compared with that of Ber group, and the P values were 1, 0.035, 0.944, 0.025, and 0.040, respectively; that is, when the concentration of berberine was 5, 25, and 50 μg/mL, the ability of Fe 3 O 4 @LDH@Ber group to reduce the survival rate of CAL27 cells was significantly stronger than that of Ber group;
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Ber组相比，P值分别为：1，0.001，0.263，0.002，0.002；即小檗碱浓度均为5、25、50μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Ber组；Compared with the Ber group, the ability of the Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was 1, 0.001, 0.263, 0.002, and 0.002, respectively; that is, when the berberine concentrations were 5, 25, and 50 μg/mL, the ability of the Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of the Ber group;
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力与Fe3O4@LDH@Ber组相比，P值分别为：1，0.020，0.290，0.003，0.047；即小檗碱浓度均为5、25、50μg/mL时，Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率的能力显著强于Fe3O4@LDH@Ber组。The ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was compared with that of Fe 3 O 4 @LDH@Ber group, with P values of 1, 0.020, 0.290, 0.003, and 0.047, respectively; that is, when the berberine concentrations were 5, 25, and 50 μg/mL, the ability of Fe 3 O 4 @LDH@Ber/PEG-FA group to reduce the survival rate of CAL27 cells was significantly stronger than that of Fe 3 O 4 @LDH@Ber group.
三、分析与结论3. Analysis and Conclusion
Fe3O4@LDH@Ber/PEG-FA组降低CAL27细胞存活率作用强于Fe3O4@LDH@Ber组，分析在FA的靶向作用下，载小檗碱的磁性纳米颗粒被CAL27细胞吞噬，使小檗碱在肿瘤细胞的酸性环境下释放，从而降低CAL27细胞的存活率。The Fe 3 O 4 @LDH@Ber/PEG-FA group had a stronger effect on reducing the survival rate of CAL27 cells than the Fe 3 O 4 @LDH@Ber group. Analysis showed that under the targeting effect of FA, berberine-loaded magnetic nanoparticles were phagocytosed by CAL27 cells, causing berberine to be released in the acidic environment of tumor cells, thereby reducing the survival rate of CAL27 cells.
实施例10、考察载小檗碱磁性纳米颗粒给药后CAL27细胞内ROS水平的变化Example 10: Investigating the changes in ROS levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
本实验通过DCFH-DA探针检测细胞内ROS水平变化。DCFH-DA可渗透入细胞内，与细胞内ROS结合从而发出绿色荧光，荧光强度越强，表示ROS生成水平越高。In this experiment, the DCFH-DA probe was used to detect changes in intracellular ROS levels. DCFH-DA can penetrate into cells and bind to intracellular ROS to emit green fluorescence. The stronger the fluorescence intensity, the higher the level of ROS generation.
一、方法1. Methods
(一)将CAL27细胞以2×105个/孔的密度接种到12孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；(i) CAL27 cells were seeded into 12-well plates at a density of 2×10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
(二)将CAL27细胞分别与PBS、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共同孵育8h，药物浓度为200ug/mL，用量为1mL，弃去上清液；(ii) CAL27 cells were incubated with PBS, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA for 8 h, respectively, with a drug concentration of 200 ug/mL and a dosage of 1 mL, and the supernatant was discarded;
(三)PBS清洗CAL27细胞2-3遍，用DCFH-DA(10μM)染色30min；PBS洗涤3次后，各组加入10ug/mL的Hoechst33342染液定位细胞核，染色30min，弃去上清液；(III) Wash CAL27 cells 2-3 times with PBS and stain with DCFH-DA (10 μM) for 30 min; After washing with PBS 3 times, add 10 μg/mL Hoechst33342 dye to locate the cell nucleus in each group, stain for 30 min, and discard the supernatant;
(四)全自动细胞成像系统检测各组细胞内荧光情况。用Image J软件计算平均荧光强度。(IV) The fully automatic cell imaging system detected the fluorescence in each group of cells, and the average fluorescence intensity was calculated using Image J software.
二、结果2. Results
如图10A所示，为荧光显微镜下，对照组、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内ROS、Hoechst33342和merge荧光。As shown in Figure 10A, ROS, Hoechst33342 and merge fluorescence in CAL27 cells in the control group, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups were observed under a fluorescence microscope.
如图10B所示，与对照组相比，5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内荧光强度显著增加，P值均为0；与5-FU组相比，Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内荧光强度显著增加，P值为0。As shown in Figure 10B, compared with the control group, the intracellular fluorescence intensity of CAL27 cells in the 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups was significantly increased, and the P value was 0; compared with the 5-FU group, the intracellular fluorescence intensity of CAL27 cells in the Fe 3 O 4 @LDH@Ber/PEG-FA group was significantly increased, and the P value was 0.
三、分析与结论3. Analysis and Conclusion
ROS在肿瘤细胞的代谢、增殖和死亡过程中起到至关重要的作用。高氧化应激状态下，ROS通过调节基因组也可以促进凋亡。ROS plays a vital role in the metabolism, proliferation and death of tumor cells. Under high oxidative stress, ROS can also promote apoptosis by regulating the genome.
由图10A可知，与空白对照组相比，5-FU、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组细胞内绿色荧光增加，表明CAL27细胞内ROS水平升高，且具有显著性差异；与阳性药5-FU组相比，Fe3O4@LDH负载Ber后，细胞内ROS水平降低，可能与Fe3O4@LDH过早释放小檗碱有关，在Fe3O4@LDH表面添加叶酸靶向后，CAL27细胞内ROS水平显著升高，效果好于阳性药组，表明通过叶酸的靶向作用能准确将药物递送至肿瘤细胞内，增加治疗效果。As can be seen from Figure 10A, compared with the blank control group, the intracellular green fluorescence of the 5-FU, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA groups increased, indicating that the intracellular ROS level of CAL27 cells increased, and there was a significant difference; compared with the positive drug 5-FU group, the intracellular ROS level decreased after Fe 3 O 4 @LDH was loaded with Ber, which may be related to the premature release of berberine by Fe 3 O 4 @LDH. After adding folic acid targeting to the surface of Fe 3 O 4 @LDH, the intracellular ROS level of CAL27 cells increased significantly, and the effect was better than that of the positive drug group, indicating that the targeting effect of folic acid can accurately deliver drugs to tumor cells and increase the therapeutic effect.
实施例11、考察载小檗碱磁性纳米颗粒给药后CAL27细胞内GSH水平的变化Example 11: Investigation of changes in GSH levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
一、方法1. Methods
(一)将CAL27细胞以5×105个/孔的密度接种到6孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；(i) CAL27 cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
(二)将CAL27细胞分别与PBS、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共同孵育8h，药物浓度为200ug/mL，用量为1mL弃去上清液；(ii) CAL27 cells were incubated with PBS, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA for 8 h, respectively, with a drug concentration of 200 ug/mL and a dosage of 1 mL, and the supernatant was discarded;
(三)用胰酶(无EDTA)将各组CAL27细胞消化，3000r/min离心5min，加入PBS清洗2-3次，离心弃去上清；(III) Digest each group of CAL27 cells with trypsin (without EDTA), centrifuge at 3000 rpm for 5 min, wash with PBS 2-3 times, and discard the supernatant after centrifugation;
(四)加入PBS，冰浴条件下破碎CAL27细胞，14000r/min离心15min，取上清，检测各组CAL27细胞内GSH水平，具体操作步骤按照蛋白定量(TP)测定试剂盒说明书测定，各组细胞蛋白含量通过BCA蛋白含量测定试剂盒进行测定。(IV) PBS was added, and CAL27 cells were disrupted under ice bath conditions. The cells were centrifuged at 14000 r/min for 15 min, and the supernatant was collected to detect the GSH level in each group of CAL27 cells. The specific operation steps were determined according to the instructions of the protein quantitative (TP) assay kit. The protein content of each group of cells was determined using the BCA protein content assay kit.
(五)Ellman'sassay测定细胞内GSH的水平变化(V) Ellman's assay to measure changes in intracellular GSH levels
根据还原型谷胱甘肽(GSH)测定试剂盒(微板法)说明书操作。The reduced glutathione (GSH) assay kit (microplate method) was used according to the instructions.
二、结果2. Results
如图11所示，与对照组相比，5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组CAL27细胞内GSH水平显著低于对照组，P值均为0。As shown in Figure 11, compared with the control group, the intracellular GSH levels of CAL27 cells in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups were significantly lower than those in the control group, with P values of 0.
三、分析与结论3. Analysis and Conclusion
GSH在肿瘤细胞的代谢、增殖和死亡过程中起到至关重要的作用。GSH plays a vital role in the metabolism, proliferation and death of tumor cells.
由图11可知，与对照组相比，Fe3O4@LDH@Ber组CAL27细胞内GSH浓度显著降低(P<0.001)，但高于5-FU组，Fe3O4@LDH@Ber/PEG-FA组则低于Fe3O4@LDH@Ber组。As shown in Figure 11, compared with the control group, the intracellular GSH concentration in the Fe 3 O 4 @LDH@Ber group was significantly reduced (P<0.001), but higher than that in the 5-FU group, while that in the Fe 3 O 4 @LDH@Ber/PEG-FA group was lower than that in the Fe 3 O 4 @LDH@Ber group.
分析：叶酸靶向后，Fe3O4@LDH@Ber/PEG-FA被CAL27细胞更多地摄取，GSH被显著消耗，减少GSH过表达对ROS的影响，从而达到治疗肿瘤的效果。Analysis: After folic acid targeting, Fe 3 O 4 @LDH@Ber/PEG-FA was taken up more by CAL27 cells, GSH was significantly consumed, and the effect of GSH overexpression on ROS was reduced, thereby achieving the effect of tumor treatment.
实施例12、考察载小檗碱磁性纳米颗粒给药后CAL27细胞内BAX、Bcl-XL基因的表达变化Example 12: Investigating the changes in the expression of BAX and Bcl-XL genes in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
一、方法1. Methods
(一)将CAL27细胞以5×105个/孔的密度接种到6孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；(i) CAL27 cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
(二)将CAL27细胞分别与PBS、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共同孵育8h，弃去上清液；(ii) CAL27 cells were incubated with PBS, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA for 8 h, and the supernatant was discarded;
(三)用PBS冲洗CAL27细胞2-3次，取CAL27细胞按照RNAsimple Total RNA Kit试剂盒说明书提取CAL27细胞总RNA；(III) Wash CAL27 cells with PBS 2-3 times, and extract total RNA from CAL27 cells according to the instructions of RNAsimple Total RNA Kit;
(三)用微量蛋白核酸分析仪检测CAL27细胞总RNA浓度，采用FastQuant RT Kit(with gDNase)试剂盒配制反转录反应体系，将获得的cDNA采用SuperReal荧光定量预混试剂盒按照两步法反应程序检测各基因的表达量，采用相对定量法处理数据；(III) The total RNA concentration of CAL27 cells was detected using a micro-protein nucleic acid analyzer, and the reverse transcription reaction system was prepared using the FastQuant RT Kit (with gDNase) kit. The obtained cDNA was used to detect the expression level of each gene using the SuperReal fluorescent quantitative premix kit according to the two-step reaction procedure, and the data were processed using the relative quantitative method;
(四)以GADPH对基因表达进行标准化，引物5.0软件用于引物序列设计；(iv) Gene expression was normalized with GADPH, and Primer 5.0 software was used for primer sequence design;
(五)采用实时荧光定量PCR检测各组CAL27细胞内BAX、Bcl-XL和GADPH基因的表达变化(V) Real-time fluorescence quantitative PCR was used to detect the expression changes of BAX, Bcl-XL and GADPH genes in CAL27 cells of each group
二、结果2. Results
如图12A所示，与对照组相比，5-FU、Ber组、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组中BAX基因表达水平显著上调，P值分别为0.023，0.092，0.008，0。As shown in Figure 12A, compared with the control group, the expression level of BAX gene in the 5-FU, Ber group, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA group was significantly upregulated, with P values of 0.023, 0.092, 0.008, and 0, respectively.
如图12B所示，与对照组相比，5-FU、Ber组、Fe3O4@LDH@Ber、Fe3O4@LDH@Ber/PEG-FA组中Bcl-XL基因表达水平显著下调，P值分别为0，0.004，0.002，0As shown in Figure 12B, compared with the control group, the expression level of Bcl-XL gene in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber, and Fe 3 O 4 @LDH@Ber/PEG-FA groups was significantly downregulated, with P values of 0, 0.004, 0.002, and 0.003, respectively.
三、分析与结论3. Analysis and Conclusion
BAX是BCL-2基因家族中细胞凋亡促进基因，BAX的过度表达可拮抗BCL-2的保护效应而使细胞趋于死亡。Bcl-XL是属于Bcl-2家族的抗凋亡基因，在调控细胞凋亡、细胞自噬和肿瘤代谢等方面都具有重要作用。BAX is a cell apoptosis-promoting gene in the BCL-2 gene family. Overexpression of BAX can antagonize the protective effect of BCL-2 and cause cells to die. Bcl-XL is an anti-apoptotic gene belonging to the Bcl-2 family, which plays an important role in regulating cell apoptosis, cell autophagy and tumor metabolism.
BAX基因表达水平上调，Bcl-XL基因表达水平下调，就表明Fe3O4@LDH@Ber/PEG-FA有效促进了CAL27细胞的凋亡。The up-regulation of BAX gene expression level and the down-regulation of Bcl-XL gene expression level indicated that Fe 3 O 4 @LDH@Ber/PEG-FA effectively promoted the apoptosis of CAL27 cells.
实施例13、考察载小檗碱磁性纳米颗粒给药后CAL27细胞内H2O2水平的变化Example 13: Investigating the changes in H 2 O 2 levels in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
一、方法1. Methods
(一)将CAL27细胞以5×105个/孔的密度接种到6孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；(i) CAL27 cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
(二)将CAL27细胞分别与PBS、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共同孵育8h，药物浓度为200ug/mL，用量为1mL，弃去上清液，；(ii) CAL27 cells were incubated with PBS, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA for 8 h, respectively, with a drug concentration of 200 ug/mL and a dosage of 1 mL, and the supernatant was discarded;
(三)用PBS冲洗CAL27细胞2-3次，取CAL27细胞，采用TMB法检测CAL27细胞内H2O2水平(III) Wash CAL27 cells with PBS 2-3 times, take CAL27 cells, and use TMB method to detect H 2 O 2 level in CAL27 cells
TMB法:加入3,3',5,5'-四甲基联苯胺(TMB)作为显色液，无色的TMB被H2O2氧化成蓝色的氧化TMB(oxTMB)，检测405nm吸光度，从而得出细胞内H2O2浓度。TMB method: 3,3',5,5'-tetramethylbenzidine (TMB) is added as a colorimetric solution, and the colorless TMB is oxidized by H 2 O 2 to blue oxidized TMB (oxTMB). The absorbance at 405 nm is detected to obtain the intracellular H 2 O 2 concentration.
二、结果2. Results
如图13所示，与对照组相比，5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组中H2O2水平显著上调，P值均为0。As shown in Figure 13, compared with the control group, the H2O2 levels in the 5-FU, Ber, Fe3O4 @ LDH @Ber and Fe3O4 @ LDH@Ber/PEG-FA groups were significantly upregulated, and the P values were all 0.
四、分析与结论4. Analysis and Conclusion
Fe3O4@LDH/PEG-FA作为药物载体，将Ber靶向至CAL27细胞内并释放，促进肿瘤细胞内H2O2水平升高，实现药物(Ber)有效浓度堆积，并催化H2O2转化为毒性更强的·OH，加重肿瘤细胞内的氧化应激水平，从而促进肿瘤细胞调亡。Fe 3 O 4 @LDH/PEG-FA was used as a drug carrier to target Ber into CAL27 cells and release it, which promoted the increase of H 2 O 2 levels in tumor cells, achieved the effective concentration accumulation of the drug (Ber), and catalyzed the conversion of H 2 O 2 into the more toxic ·OH, aggravating the oxidative stress level in tumor cells, thereby promoting tumor cell apoptosis.
实施例14、考察载小檗碱磁性纳米颗粒给药后CAL27细胞内线粒体膜电位的变化Example 14: Investigation of changes in mitochondrial membrane potential in CAL27 cells after administration of berberine-loaded magnetic nanoparticles
线粒体参与ATP合成、细胞凋亡和活性氧生成等多种细胞途径。线粒体功能障碍是癌症的主要特征之一，通过ROS介导的线粒体功能障碍可以抑制癌细胞的生长并使癌细胞凋亡。Mitochondria are involved in a variety of cellular pathways, including ATP synthesis, apoptosis, and reactive oxygen species generation. Mitochondrial dysfunction is one of the main characteristics of cancer, and ROS-mediated mitochondrial dysfunction can inhibit the growth of cancer cells and cause apoptosis of cancer cells.
线粒体膜电位是线粒体功能的一个重要参数，线粒体膜电位降低会导致某些线粒体蛋白的释放，从而导致细胞死亡。Mitochondrial membrane potential is an important parameter of mitochondrial function. A decrease in mitochondrial membrane potential can lead to the release of certain mitochondrial proteins, thereby causing cell death.
JC-1是一种广泛应用于线粒体膜电位检测的荧光探针，线粒体膜电位高时，JC-1在线粒体内聚集成聚合体，产生红色荧光，线粒体膜电位下降时，JC-1以单体形式存在，产生绿色荧光，当荧光从红色转向绿色时，意味着线粒体膜电位的下降，也间接反映出线粒体外膜的损伤程度。因此，红绿荧光的比值可进一步定量评估线粒体膜电位，从而预测细胞死亡情况。JC-1 is a fluorescent probe widely used in mitochondrial membrane potential detection. When the mitochondrial membrane potential is high, JC-1 aggregates into polymers in the mitochondria and produces red fluorescence. When the mitochondrial membrane potential decreases, JC-1 exists in monomeric form and produces green fluorescence. When the fluorescence changes from red to green, it means that the mitochondrial membrane potential has decreased, which also indirectly reflects the degree of damage to the mitochondrial outer membrane. Therefore, the ratio of red and green fluorescence can further quantitatively evaluate the mitochondrial membrane potential and thus predict cell death.
本实验通过JC-1探针法对Fe3O4@LDH@Ber/PEG-FA给药前后CAL27细胞内线粒体膜电位水平进行检测，分析Fe3O4@LDH@Ber/PEG-FA对CAL27细胞死亡的影响。In this experiment, the JC-1 probe method was used to detect the mitochondrial membrane potential level in CAL27 cells before and after the administration of Fe 3 O 4 @LDH@Ber/PEG-FA, and the effect of Fe 3 O 4 @LDH@Ber/PEG-FA on CAL27 cell death was analyzed.
一、方法1. Methods
(一)将CAL27细胞以2×105个/孔的密度接种到12孔板中，培养液为全培养液，37℃孵育24h，弃去培养液；全培养液由10％FBS、90％DMEM和1％PS组成；(i) CAL27 cells were seeded into 12-well plates at a density of 2×10 5 cells/well in a complete culture medium, incubated at 37°C for 24 h, and the culture medium was discarded; the complete culture medium consisted of 10% FBS, 90% DMEM, and 1% PS;
(二)将CAL27细胞分别与PBS、5-FU、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共同孵育24h，弃去上清液；(ii) CAL27 cells were incubated with PBS, 5-FU, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA for 24 h, and the supernatant was discarded;
(三)按照线粒体膜电位检测试剂盒说明书进行后续操作，采用细胞全自动成像系统检测各组细胞线粒体膜电位变化。(III) Follow the instructions of the mitochondrial membrane potential detection kit to perform subsequent operations and use the cell automatic imaging system to detect changes in mitochondrial membrane potential of each group of cells.
二、结果2. Results
如图14A所示，对照组细胞红色荧光强度较强；阳性药组5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA给药后，CAL27细胞中红色荧光降低，绿色荧光增加。As shown in Figure 14A, the red fluorescence intensity of the cells in the control group was stronger; after administration of 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA in the positive drug groups, the red fluorescence in CAL27 cells decreased and the green fluorescence increased.
如图14B所示，与对照组相比，5-FU、Ber、Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA组中JC-1红色荧光强度显著降低，P值均为0。As shown in Figure 14B, compared with the control group, the red fluorescence intensity of JC-1 in the 5-FU, Ber, Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA groups was significantly reduced, and the P values were all 0.
三、分析与结论3. Analysis and Conclusion
对照组细胞中JC-1聚合成聚合物，红色荧光强度较强，表明对照组细胞活性正常；阳性药组5-FU给药后，CAL27细胞中红色荧光降低，绿色荧光增加，线粒体膜电位下降，细胞开始死亡；游离Ber给药后，细胞内线粒体膜电位显著下降，多数细胞开始死亡；当CAL27细胞与Fe3O4@LDH@Ber和Fe3O4@LDH@Ber/PEG-FA共培养时，红色荧光降低，绿色荧光增加，线粒体膜电位降低，且Fe3O4@LDH@Ber/PEG-FA组细胞效果较好，表明FA靶向Ber至CAL27细胞内，促进细胞死亡。结果表明，Fe3O4@LDH@Ber/PEG-FA诱导的CAL27细胞中ROS生成与细胞线粒体膜电位障碍直接相关，ROS水平升高引发的细胞线粒体膜电位降低是诱导细胞死亡的主要原因。In the control group, JC-1 polymerized into polymers, and the red fluorescence intensity was strong, indicating that the cells in the control group were normal in activity. After the administration of 5-FU in the positive drug group, the red fluorescence in CAL27 cells decreased, the green fluorescence increased, the mitochondrial membrane potential decreased, and the cells began to die. After the administration of free Ber, the mitochondrial membrane potential in the cells decreased significantly, and most cells began to die. When CAL27 cells were co-cultured with Fe 3 O 4 @LDH@Ber and Fe 3 O 4 @LDH@Ber/PEG-FA, the red fluorescence decreased, the green fluorescence increased, the mitochondrial membrane potential decreased, and the Fe 3 O 4 @LDH@Ber/PEG-FA group had a better effect, indicating that FA targeted Ber to CAL27 cells and promoted cell death. The results showed that the ROS generation in CAL27 cells induced by Fe 3 O 4 @LDH@Ber/PEG-FA was directly related to the disorder of mitochondrial membrane potential of cells, and the decrease of mitochondrial membrane potential caused by the increase of ROS level was the main reason for the induction of cell death.
实施例15、空白磁性纳米颗粒的溶血实验Example 15 Hemolysis experiment of blank magnetic nanoparticles
为验证Fe3O4@LDH/PEG-FA作为纳米药物载体的生物安全性，本实验通过模拟体内环境，将空白磁性纳米颗粒与红细胞悬液共融合，通过计算溶血率判断Fe3O4@LDH/PEG-FA的体外毒性，从而初步评估Fe3O4@LDH/PEG-FA作为静脉注射给药制剂的生物相容性和安全性。In order to verify the biosafety of Fe 3 O 4 @LDH/PEG-FA as a nanodrug carrier, this experiment simulated the in vivo environment, co-fused blank magnetic nanoparticles with red blood cell suspension, and determined the in vitro toxicity of Fe 3 O 4 @LDH/PEG-FA by calculating the hemolysis rate, thereby preliminarily evaluating the biocompatibility and safety of Fe 3 O 4 @LDH/PEG-FA as an intravenous administration preparation.
一、方法1. Methods
(一)分别称取Fe3O4@LDH/PEG-FA、Fe3O4@LDH/PEG和Fe3O4@LDH于10mL离心管中，溶于PBS，超声溶解，配置成1mg/mL的母液；(i) Fe 3 O 4 @LDH/PEG-FA, Fe 3 O 4 @LDH/PEG and Fe 3 O 4 @LDH were weighed separately in 10 mL centrifuge tubes, dissolved in PBS, and ultrasonically dissolved to prepare a 1 mg/mL stock solution;
(二)分别将母液稀释成浓度为25、50、100、200、400和1000μg/mL的Fe3O4@LDH/PEG-FA、Fe3O4@LDH/PEG和Fe3O4@LDH的工作液；(ii) diluting the mother solution into working solutions of Fe 3 O 4 @LDH/PEG-FA, Fe 3 O 4 @LDH/PEG and Fe 3 O 4 @LDH with concentrations of 25, 50, 100, 200, 400 and 1000 μg/mL, respectively;
(三)去离子水为阴性对照组，PBS为阳性对照组；(iii) Deionized water was used as the negative control group, and PBS was used as the positive control group;
(四)取红细胞悬液加入装有1mL上述溶液的1.5mL EP管中，所有实验组在37℃摇床中震荡8h，4℃3000rpm离心10min，从每组上清液中吸取200μL溶液滴加入96孔板中，用酶标仪检测541nm处的吸光度，计算红细胞溶解率(溶血率)，每组实验平行三次。(IV) The red blood cell suspension was added to a 1.5 mL EP tube containing 1 mL of the above solution. All experimental groups were shaken in a shaker at 37°C for 8 h and centrifuged at 3000 rpm at 4°C for 10 min. 200 μL of the supernatant from each group was added dropwise to a 96-well plate. The absorbance at 541 nm was detected using an ELISA reader, and the red blood cell lysis rate (hemolysis rate) was calculated. Each experiment was performed in parallel three times.
溶解率(％)＝(实验组吸光度-阴性对照组吸光度)/(阳性对照组吸光度-阴性对照组吸光度)×100Dissolution rate (%) = (absorbance of experimental group - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) × 100
二、结果2. Results
如图15A所示，阳性对照组溶血现象强，阴性对照和Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA在25-1000ug/ml浓度范围内红细胞溶血现象微弱。As shown in Figure 15A, the positive control group showed strong hemolysis, while the negative control and Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG, and Fe 3 O 4 @LDH/PEG-FA showed weak hemolysis of erythrocytes in the concentration range of 25-1000ug/ml.
如图15B所示，阳性对照组红细胞溶解率(溶血率)100％，阴性对照组溶解率0，Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA在25-1000ug/ml浓度范围内，溶解率均低于5％。As shown in FIG. 15B , the red blood cell lysis rate (hemolysis rate) of the positive control group was 100%, and the lysis rate of the negative control group was 0. The lysis rates of Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG, and Fe 3 O 4 @LDH/PEG-FA were all lower than 5% within the concentration range of 25-1000 ug/ml.
三、分析与结论3. Analysis and Conclusion
Fe3O4@LDH、Fe3O4@LDH/PEG、Fe3O4@LDH/PEG-FA在25-1000ug/ml浓度范围内，红细胞溶血现象微弱，溶解率(溶血率)均低于5％，满足静脉给药对纳米制剂溶血毒性的要求，表明Fe3O4@LDH/PEG-FA作为纳米药物载药具有良好的生物相容性。In the concentration range of 25-1000ug/ml, the hemolysis of erythrocytes was weak for Fe 3 O 4 @LDH, Fe 3 O 4 @LDH/PEG, and Fe 3 O 4 @LDH/PEG-FA, and the dissolution rate (hemolysis rate) was less than 5%, which met the requirements for hemolytic toxicity of nanoformulations for intravenous administration, indicating that Fe 3 O 4 @LDH/PEG-FA has good biocompatibility as a nanodrug carrier.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.
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