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Cell membrane-permeable and cytocompatible phospholipid polymer nanoprobes conjugated with molecular beacons

机译：与分子信标共轭的细胞膜可渗透性和细胞相容性磷脂聚合物纳米探针

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Introduction: Real-time monitoring the dynamic distribution of mRNA in living cells could aid in the early detection of cell pathogenesis, accurate clinical diagnoses and effective treatments. To overcome the limitations of traditional methods, a noninvasive probe with a high degree of sensitivity as well as high spatial and temporal resolution is required. Amphiphilic 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers contain extremely hydrophilic phosphorylcholine groups and have plasma membrane-inspired structure can penetrate into living cell through a simple diffusion process. Molecular beacons (MBs) with DNA hairpin structure that are widely used as fluorescent probes for the detection of complementary intra- and extracellular molecules in real time. Highly permeable MPC polymers conjugated with MBs could be used to rapidly label biomolecules and allow the extended monitoring of intracellular events. Materials and Methods: The water-soluble amphiphilic phospholipid polymer, poly[MPC-co-n-butyl methacrylate (BMA)-co-N-succinimidyloxycarbonyl tetra(ethylene glycol) methacrylate (PENHS)] (PMBS), was synthesized through conventional radical polymerization method (Figure 1). PMBS was conjugated with MBs to form nanoprobes by a chemical reaction between ester group of N-hydroxysuccinimide and amine group of the MBs. The morphology of PMBS in serum-free DMEM was determined by measurement of surface tension of polymer solution. Cellular uptake of the nanoprobes by the HeLa cells was observed with a laser scanning confocal microscopy. The single-stranded DNA-binding proteins and target mRNA fragment were used to evaluate the stability and specificity of nanoprobe, respectively. Results and Discussion: PMBS polymer with higher composition of hydraphobic BMA unit can strongly reduce the surface tension of serum-free DMEM, which remained constant at concentrations greater than 0.5 mg/mL This result indicates that the PMBS361 (composition, MPC: BMA: PENHS = 3:6:1) formed stable polymer aggregates at concentrations above 0.5 mg/mL. Laser scanning confocal microscopy results show that the red fluorescence of Cy3 molecules from PMBS361-MB nanoprobe was detected in cell cytoplasm after 2 h, colocalizing well with MitoTracker, while fluorescence of other probe can not be observed inside cell (Figure 2). Thus, aggregate formation is considered to be a predominant factor contributing to the ability of these probes to penetrate the cell membrane. Conjugation of PMBS to MB can significantly increase the stability without causing any damage to the specificity of MB. Conclusion: Cell-membrane-permeable and cytocompatible nanoprobes composed of PMBS and MBs were synthesized successfully as a noninvasive tool for monitoring intracellular biomolecules in living cells. The nanoprobes have high target specificity and resistance to nonspecific degradation by surrounding proteins, and they effectively penetrated the cytoplasm, permitting the real time visualization of mRNA in HeLa cells. Thus, membrane-penetrating, amphiphilic, phospholipid-based polymers can be combined with nano/sub-nano-scale oligonucleotide MBs to generate highly sensitive nanoprobes that can be used to deepen our understanding of basic cellular processes and could also be applied toward the early detection, accurate clinical diagnosis, and effective treatment of diseases in the future.
机译：简介：实时监测活细胞中mRNA的动态分布有助于早期发现细胞发病机制，准确的临床诊断和有效的治疗方法。为了克服传统方法的局限性，需要具有高度灵敏度以及高时空分辨率的无创探针。两亲性2-甲基丙烯酰氧基乙基磷酰胆碱（MPC）聚合物包含极亲水的磷酰胆碱基团，并具有质膜激发的结构，可通过简单的扩散过程渗入活细胞。具有DNA发夹结构的分子信标（MBs）被广泛用作实时检测互补细胞内和细胞外分子的荧光探针。与MBs共轭的高渗透性MPC聚合物可用于快速标记生物分子，并允许对细胞内事件进行扩展监测。材料和方法：通过常规自由基合成水溶性两亲性磷脂聚合物聚[MPC-甲基丙烯酸正丁酯（BMA）-N-琥珀酰亚胺基氧基羰基甲基丙烯酸乙二醇酯（PENHS）]（PMBS）。聚合方法（图1）。通过N-羟基琥珀酰亚胺的酯基和MBs的胺基之间的化学反应，将PMBS与MBs共轭以形成纳米探针。通过测量聚合物溶液的表面张力来确定无血清DMEM中PMBS的形态。用激光扫描共聚焦显微镜观察到HeLa细胞对纳米探针的细胞摄取。用单链DNA结合蛋白和靶mRNA片段分别评估纳米探针的稳定性和特异性。结果与讨论：疏水性BMA单元组成较高的PMBS聚合物可显着降低无血清DMEM的表面张力，当浓度大于0.5 mg / mL时，其保持恒定。该结果表明PMBS361（组成，MPC：BMA：PENHS = 3：6：1）形成浓度高于0.5 mg / mL的稳定聚合物聚集体。激光扫描共聚焦显微镜结果表明，PMBS361-MB纳米探针中Cy3分子的红色荧光在2 h后在细胞质中被检测到，与MitoTracker共定位良好，而在细胞内未观察到其他探针的荧光（图2）。因此，聚集体形成被认为是促成这些探针穿透细胞膜的能力的主要因素。 PMBS与MB的结合可以显着提高稳定性，而不会损害MB的特异性。结论：成功地合成了由PMBS和MBs组成的细胞膜可渗透性和细胞相容性纳米探针，作为监测活细胞中细胞内生物分子的非侵入性工具。纳米探针具有高的靶标特异性和对周围蛋白质非特异性降解的抗性，并且可以有效地穿透细胞质，从而可以实时观察HeLa细胞中的mRNA。因此，可以将渗透膜，两亲，基于磷脂的聚合物与纳米/亚纳米级寡核苷酸MB结合使用，以产生高度敏感的纳米探针，这些探针可以用于加深我们对基本细胞过程的理解，也可以应用于早期检测，准确的临床诊断以及将来对疾病的有效治疗。

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