C60-based spherical nucleic acids as gene delivery vectors: Structure and Dynamics Studied by Time-Resolved Fluorescence Spectroscopy

C60-based spherical nucleic acids (SNA) with polyvalent nanostructures consisting of a relatively small core and densely packed, highly oriented oligonucleotides (ON). The affinity of SNAs for complementary nucleic acids, i.e. the ON to be delivered, is high leading to effective gene suppression. The outer sphere of the SNA-oligonucleotide complex can be modified by appropriate ligands, which are aimed to hide the inner oligonucleotide content and facilitate the bio-distribution, cell-specificity, cellular uptake and intracellular action of the complex.

The  complementary nucleic acids (CNA) used in this study.

The SNA was as synthesized via the same strategy as in:

Samples: The binding constant were measured by stepwise addition of CNA to SNA solution starting with CNA:SNA ratio 2:1  and finnishing at 14:1 ratio. PBS (phosphate buffer saline) was used as the solvent.

Absorption spectra were measured with Shimadzu UV-3600 spectrophotometer. Pure DPBS was used as a reference. 

Fluorescence and excitation spectra  were measured with FLS-1000 spectrofluorometer (Edinburgh Instruments, UK). Both spectra were corrected according to the wavelength sensitivity of the detector and the excitation source intensity.

Time-resolved fluorescence was measured using a time-correlated single photon counting (TCSPC) system (Pico-Quant GmBH, Chaussee, Germany) consisting of a PicoHarp 300 controller and a PDL 800-B driver. The samples were excited with the pulsed diode laser head LDH-P-C-485 at 483 nm at a time resolution of 130 ps. The signals were detected with a microchannel plate photomultiplier tube (Hamamatsu R2809U). The influence of the scattered excitation light was reduced with a cutoff filter (transmission > 490 nm) in front of the monitoring monochromator. Fluorescence decays were collected with a constant accumulation time in the 500−570 nm wavelength range with steps of 10 - 20 nm. The instrumental response function (IRF) was measured separately, and the decays were deconvoluted and fitted globally by applying the iterative least-squares method to the sum of 1-2 exponents:

The mean amplitude weighted lifetime were calculated using eq 2:

Confocal microscopy imaging was done with Nikon Eclipse Ti2-E microscope. All images were acquired using 20 × objective. AF488 was excited using 475 nm and DAPI using 395 nm. The cells were kept at 5% CO₂ and 37 °C during the imaging. 

Fluorescence lifetime images (FLIM) were acquired using a fluorescence lifetime microscope MicroTime-200 (PicoQuant, Germany) coupled to the inverted microscope Olympus IX-71 (Olympus, Japan). FLIM with a 100 × oil objective having NA 1.4 enabled a minimum spatial resolution of 300 nm and a maximum scan area of 80 μm × 80 μm. The pulsed laser diode LDH-P-C483 (PicoQuant, Germany) emitting at 483 nm (time resolution 120 ps) was used for fluorescence excitation and the emission was monitored using 510 nm long pass filter. During imaging the living PC-3 cells were kept at 37 °C and at 5% CO₂ using a custom-made incubator. In FLIM images the colors are based on the mean intensity weigted lifetimes at each pixel: 

FLIM images of the PC-3 cells at different times after addition of free i-ISE or SNA:CA-ISE = 1:12 complexes. The brightness of each pixel in the FLIM images correlates with the concentration of the fluorescent species and the color of each pixel correlates with the average fluorescence lifetime in the corresponding spatial location, the scale bars for both are given on the right.

Both free ISEs and SNA:ISE complexes are taken up by the cells. The average lilfetime increases from 2.3 ns in the absence of ISEs to ~2.7 ns in the presence of ISEs as its fluorescence starts to dominate the fluorescence signal. The distribution of the SNA:ISE complexes is more spot like compared with that of free ISEs, indicating that the complexes stay intact during up-take. The intense spots omitted in some images are aggregates of free ISE or SNA:ISE complexes floating in the medium. The lifetime of the spots is ~ 3.0 ns.

Both confocal microscopy and FLIM results are very recent and more thorough analysis is on-going.

These measurements were done by Iida Haapalehto and Emilia Löfman, Tampere University.

The presence of the sugar groups clearly influences the spectral properties of AF488, but do not hamper the formation of SNA:i-ISE complexes. For all sugar-derivatives the fluorescence decay curves are one-exponential, whereas for ISE it is two-exponential. Also, the absorption maximum was shifted 3 nm to the red compared to ISE and CA-ISE and during complexation the lifetimes of the short-living components first increased. Thus, it seems that AF488 interacts with alfa- and beta-bleomycin in the ground state.

For all the ISEs, the complexation seems to be complete at 12:12 ratio. For CA the association constant is nearly equal to that obtained for ISE. For alfa- and beta-ISEs the association constants are about half of that for ISE and CA-ISE. In determining the association constants, only ratios 4-12 or 8-12 could be used.  This could indicate that the complexation is co-operative and that the simple analysis of independent binding does not fit to the data.

Cell up-take experiments gave promising results and more careful studies are needed to determine where in cells the SNA and ISEs gather and at which point ISEs are detaching from the SNA-ISE complex. 

Acknowledgements

This work was supported by the Academy of Finland under Grants 311362 and 323669; Business Finland EVE ecosystem under Grants 1842/31/2019 and is supported by Academy of Finland GeneCellNano Flagship.