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)[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 hydrophobic 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[1],[2].
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.
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (No. 2306) from Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References:
[1] Xiaojie Lin, Tomohiro Konno, Kazuhiko Ishihara. Biomacromolecules 15 (2014) 150-157.
[2] Xiaojie Lin, Kazuhiko Ishihara. Journal of Biomaterials Science-Polymer Edition 25 (2014) 1461-1478.