Oligonucleotides with consecutive alkylated phosphate units: aggregation characteristics and drug transport into living cells

Abstract

We prepared DNA amphiphiles with modified phosphate units, and characterized their properties of aggregate formation. The oligodeoxynucleotides with five consecutive hydrophobic phosphate groups aggregated to encapsulate hydrophobic nile red dye, and the resulting aggregates efficiently delivered the hydrophobic nile red as a virtual drug into living cells.

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Artificial DNA has been used as a building block for the construction of nanoscale structures.^1,2 The controllable hybridization features of DNA has been applied to form appropriate shapes with two- or three-dimensional structures, which have been used as biological devices and as nonbiological engineering materials. Among the artificial DNAs, the DNA-based amphiphiles have attracted attention because of their utility and have been employed in the field of DNA biotechnology, such as the antisense strategy and drug delivery.^3–5

Most of the conventional DNA amphiphiles consist of oligonucleotides and an alkyl chain, which function as hydrophilic and hydrophobic components, respectively.^5,6 In aqueous solution, amphiphiles form aggregates and exert their inherent functions. The DNA amphiphiles described previously were valuable for the construction of aggregates and nanostructures; however, the complicated procedures necessary for their preparation and difficulties in additional modification of the aggregates remain problems that need to be solved. In this study, we developed a new class of DNA amphiphiles and aggregates, which were useful for drug encapsulation and drug delivery to living cells. Using modification methods of oligodeoxynucleotides (ODNs), which were reported previously by us⁷ or others,⁸ we constructed the hydrophobic component of the amphiphile via alkylation of the phosphate group of ODNs. We not only synthesized DNA amphiphiles easily via automated DNA synthesis, but also achieved the enhancement of hydrophobicity at the alkylated phosphate unit by introducing a benzyl group using the facile click chemistry.^7–9 We designed and prepared ODNs that possessed five consecutive alkylated phosphate groups to form amphiphiles that retained the basic DNA skeleton. The resulting aggregate showed an advantageous feature as a carrier that was able to deliver hydrophobic molecules into living cells.

ODNs that possessed an alkylated phosphate group (ODN 1) were prepared by automated DNA synthesis using the conventional phosphoramidite method.⁷ The crude ODNs were purified by reversed-phase HPLC and incorporation of the alkyl chain into the ODNs was confirmed by ESI mass spectrometry. The structure of the synthesized ODN 1 is shown in Fig. 1A. As ODN 1 had an acetylene group at the end of alkyl chain, we performed a click reaction of ODN 1 to prepare an amphiphilic ODN with additional hydrophobicity. The cycloaddition was performed between ODN 1 possessing five acetylene groups and benzyl azide in the presence of CuSO₄ and tris-(benzyltriazolyl)methyl amine (TBTA) using sodium ascorbate as a reducing agent in an aqueous solution containing 20% t-BuOH. As shown in Fig. 1B and C, efficient reaction proceeded to form ODN 2, and the product was purified by HPLC. The incorporation of benzyl groups at all acetylene groups was confirmed by ESI mass spectrometry.

Fig. 1 (A) Click reaction of ODN possessing acetylene unit (ODN 1) in the presence of benzyl azide. (B and C) HPLC profiles for click reaction between ODN 1 (60 μM) and benzyl azide (360 μM) was performed in the presence of CuSO₄ (12 μM), Na ascorbate (60 μM) and TBTA (12 μM). (B) Before the reaction; (C) after click reaction for 21 h at ambient temperature.

We initially evaluated the fluorescence emission of nile red in the presence of ODNs, to characterize their aggregation in aqueous solution. Nile red is a water-insoluble fluorescent dye; therefore, it shows no emission in aqueous suspension. On the other hand, aggregate formation of amphiphilic molecules and encapsulation of nile red into the aggregates to dissolve the dyes results in fluorescence emission, even in aqueous solution. Thus, we verified the aggregation of ODNs by monitoring fluorescence emission. As shown in Fig. 2, we observed moderate fluorescence emission of nile red around 640 nm in the presence of ODN 1, indicating that ODN 1 formed aggregates in aqueous solution and encapsulated nile red molecules. It should be noted that fluorescence emission in the presence of ODN 2, which possessed a benzyl group at the phosphate unit, was higher than that observed in the presence of ODN 1. To estimate the encapsulation efficiency, we measured absorption spectra after the formation of aggregates (200 μM) in the presence of nile red (10 μM). The analysis of the spectra revealed that the amount of nile red encapsulated in the ODN 1 and ODN 2 aggregates were 0.68 μM and 1.91 μM, respectively (Fig. S1†). Thus, ODN 2 formed aggregates that had a higher capability of encapsulating nile red, probably because it contained a highly hydrophobic benzyl group. In a separate experiment, we also measured the fluorescence emission of nile red in the presence of normal ODN dT₁₁; however, we observed negligible fluorescence. Therefore, it is likely that the hydrophobic alkyl chains and benzyl group at phosphate groups are responsible for the formation of aggregates that encapsulate hydrophobic nile red molecules.

Fig. 2 Formation of aggregates from ODNs and encapsulation of nile red. (A) Fluorescence spectra of nile red (10 μM) in aqueous solution containing phosphate Na buffer (10 mM, pH 7.0) in the presence of ODN 1 (200 μM, dotted line) ODN 2 (200 μM, solid line), or dT₁₁ (200 μM, dashed line). The fluorescence spectra were measured at 580 nm excitation. (B) Representative plot of fluorescence intensity of nile red vs. concentration of ODNs to determine critical aggregation concentration (CAC). The plot for ODN 2 was shown.

Next, we measured the critical aggregation concentration (CAC) of ODNs by monitoring fluorescence emission in the presence of increasing concentrations of ODNs. A sudden increase in fluorescence intensity at the concentration of 166 and 143 μM was observed for ODN 1 and ODN 2, respectively, and these concentrations were assigned as the CACs of the amphiphiles. The evidence that ODN 2 aggregate had a lower CAC value compared with ODN 1 aggregate indicates that the high hydrophobicity of ODN 2 facilitates aggregation.

We performed an additional measurement of the size of the aggregates consisted of ODNs by means of dynamic light scattering (DLS). Fig. 3 shows representative spectra of ODNs in 10 mM phosphate buffer. From the sample of ODN 1, we observed the formation of two sizes of aggregate with a diameter of ca. 154 nm and 3 nm, respectively. Interestingly, the quite small aggregate with a diameter below 10 nm was formed from ODN 1. Consecutive linear alkyl chain at phosphate group may induce a formation of small aggregate,^6a but an exact shape of the small aggregate was unknown at present. On the other hand, ODN 2 formed single size aggregate with a diameter of ca. 128 nm. The hydrophobic part of ODNs affected the size of the aggregates in an obvious fashion.

Fig. 3 DLS experiments were conducted using aqueous solution of ODNs containing phosphate Na buffer (10 mM, pH 7.0). (A) Aggregate consisted of ODN 1. (B) Aggregate consisted of ODN 2.

Based on the properties of ODNs described above, we also attempted to demonstrate the transportation of nile red as a virtual drug into living cells using a human cell line of lung carcinoma A549. For the assessment of cellular penetration, A549 cells were cultured for 2 h in the presence of the aggregates of ODN 1 or ODN 2, which encapsulated nile red, and were subsequently subjected to microscopic observation after the washing of the cells by PBS. As shown in Fig. 4, clear fluorescence of nile red was observed in the cytoplasm of A549 cells, that were incubated with both aggregates consisted of ODN 1 and ODN 2 possessing hydrophobic substituents. The fluorescence intensity of nile red in the cells that were incubated with ODN 2 aggregate was twice as strong as that observed in the cells that were incubated with ODN 1 aggregate, indicating that the aggregate consisted of ODN 2 had better potency as a drug carrier. The evidence that negligible fluorescence was observed in the cells, which were incubated in the absence of ODNs, led the conclusion that the aggregates consisted of DNA amphiphiles can deliver hydrophobic drugs into living cells.

Fig. 4 Emission images of A549 cells as incubated with aggregates of ODNs (200 μM) encapsulating nile red (1 μM). The cells were incubated with the aggregate for 2 h at 37 °C and then the medium was replaced by PBS. After the replacement, the images were immediately taken by means of microscopy (excitation at 560 nm and emission at 630 nm). The images were obtained from the cells incubated in the absence of ODNs (A), in the presence of ODN 1 aggregate (B), or in the presence of ODN 2 aggregate (C). Upper stand: bright field images. Lower stand: fluorescence images. (D) Fluorescence intensity at 630 nm obtained from the cells incubated with aggregates encapsulating nile red. Each error bars represents the SD calculated from arbitrary four cells.

In summary, we prepared ODNs that possessed a hydrophobic group at phosphate units and characterized their aggregation properties. Among the ODNs used in this study, ODN 2, in which benzyl units were incorporated by click reaction, showed a preferable aggregation feature because of its elevated hydrophobicity and high capacity for encapsulating drugs. Because the aggregate of ODN 2 facilitated the transfer of hydrophobic molecules into living cells, they are a potentially good drug carrier.

There is an increasing number of studies on the design and development of DNA-type biological materials, because DNA is a highly tunable biocompatible polymer. The future directions of this study will be a control of the size of aggregates and a modification of its surface, because the transport of the object into the specified cells depends on its size and characteristics of its surface. In addition, we will apply the aggregates to in vivo studies to establish the practical drug carrier systems.

Acknowledgements

This work is partly supported by the Adaptable and Seamless Technology Program through target driven R&D (A-STEP) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra47662f

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