Efficient delivery of quantum dots in live cells by gold nanoparticle mediated photoporation
Ranhua Xionga,b, Freya Jorisa, Ine De Cocka, Jo Demeestera, Stefaan C. De Smedta, Andre G. Skirtachb.c.d, Kevin Braeckmansa,b *
aLaboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium; bCentre for Nano- and Biophotonics, Harelbekestraat 72, 9000 Ghent, Belgium; cDepartment of Molecular Biotechnology, Ghent University, 9000 Ghent, Belgium; dMax-Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany
ABSTRACT
There is considerable interest in using Quantum Dots (QDs) as fluorescent probes such for cellular imaging due to unique advantages in comparison with conventional molecular dyes. However, cytosolic delivery of QDs into live cells remains a major challenge. Here we demonstrate highly efficient delivery of PEG-coated QDs into live cells by means of laser-induced vapour nanobubbles. Using this procedure we succeeded in high-throughput loading of ~80% of cells while maintaining a cell viability of ~85%.
Keywords:QDs, photoporation, vapor nanobubble, gold nanoparticle
- INTRODUCTION
Quantum Dots (QDs) as fluorescent probes have attracted considerable interest in many biological and biomedical applications, especially cellular imaging[1-12]. In comparison with conventional molecular dyes, QDs have several unique advantages such as broad absorption with narrow emission spectra, high resistance against photobleaching, and size-tunable fluorescent emission[13-16]. To date, QDs have been successfully applied to label fixed and permeabilized cells, or to label membrane proteins in living cells[5, 15, 17, 18]. To extend their use for more general subcellular labeling of living cells, efficient cytosolic delivery into live cells is required, which remains a major challenge[8, 12]. Recently, gold nanoparticle (AuNP) medicated photoporation was shown to be an efficient, non-toxic approach to deliver macromolecules from tens of kDa to hundreds of kDa in live cells[19, 20]. By irradiating AuNPs attached the cell membrane with pulsed laser light of sufficient energy, ‘explosive’ water vapour nanobubbles (VNB) can be formed around these AuNPs. When the thermal energy of the AuNP is consumed, the VNB violently collapses and causes local damage to the cell membrane by high-pressure shock waves. Using this approach we demonstrated that large macromolecules, such as 500 kDa FITC-dextran, could be introduced into the cytoplasm with negligible cytotoxicity[20]. Considering that the size of PEGylated QDs is similar to the hydrodynamic radius of 500 kDa dextran, here we have evaluated if laser-induced VNB mediated membrane poration can also efficiently deliver QDs in cells with low toxicity.
2.MATERIALS AND METHODS
2.1 Materials
530 (+/-10) nm CdSeS/ZnS Fluorescent nanocrystals coated with thiol oligomer and surface functionalized with -COOH groups (PEG-coated QDs) with 10 nmol in 1 mL H2O or PBS were purchased from AC Diagnostics, Inc. (#CAQD-530-P-1, AC Diagnostics, Inc., Fayetteville, AR, USA). Cationic AuNPs of 70 nm were purchased from NanoPartz (#C2159, Nanopartz Inc., Loveland, CO, USA). These AuNPs had a zeta potential of 30 mV as measured by dynamic light scattering (NanoSizer, Malvern, UK). Calcein red AM (#C34851, CellTrace, Belgium) for cell viability quantification is obtained from Invitrogen.
*; phone +32 (0)9 264 80 98; fax +32 (0)9 264 81 89;
2.2 Cell experiments
HeLa cells (1.5×104 cells/well) were grown in cell medium of DMEM/F-12 with 2 mM glutamine, 10% heat-inactivated fetal bovine serum (FBS, Hyclone) and 100 U/mL penicillin/streptomycine, in 96 wells (#655892, Greiner Bio-One, Germany) at 37° in a humidified atmosphere containing 5% CO2 for 24 hours before laser treating. For laser treatment, the cells were incubated with AuNPs for 30 min at a fixed concentration of 1.77×1010 particles/mL, which corresponds to 8 particles/cell on average[20]. Following incubation with AuNPs, the cells were washed to remove any remaining free AuNPs in solution. Just prior to the laser scanning treatment, the solution of QDs was added to the cells. After the laser treatment, the cells were washed and supplied with fresh cell medium. CellTrace® Calcein red AM was added to the samples for 45 min incubation at room temperature to stain living cells for quantifying cell viability. The prepared cell samples were imaged by confocal microscopy (C1-si, Nikon, Japan).
2.3Generation and detection of AuNP heating and VNB formation
A setup was made to generate and detect the generation of VNB[20]. A pulsed laser (~7 ns) tuned at a wavelength of
561 nm (OpoletteTM HE 355 LD, OPOTEK Inc., Faraday Ave, CA, USA) was used for illumination of AuNPs to
generate VNBs. The setup has time-response and light scattering modes for detecting VNB formation.
- RESULTS AND DISCUSSION
Figure 1. (a) Schematic overview of QD delivery into cells by AuNP mediated photoporation: 1. AuNPs are added to cells in culture for adsorption to the cell membrane. 2. Nonadherent AuNPs are removed in a washing step. 3. QDs are added to the cells just prior to the laser treatment. 4. Using an electronic sample stage, the entire sample is scanned through the laser beam so that all cells are treated. (b) AuNPs adsorption to cells can be verified by confocal microscopy in reflection mode (some of the AuNPs are indicated by arrows) due to their excellent scattering of light as shown by the intensity profile in the insert. (c) VNBs can be monitored as a sudden transient change in transmitted light detected by a photodetector (left panel), or as bright spots of scattered light in darkfield microscopy (VNBs indicated by red arrows). The green circle indicates the laser-irradiated area. The right panel shows the cells after VNB treatment.
As shown in Fig.1a, the experimental procedure of intracellular delivery of QDs by AuNP mediated photoporation includes a number of sequential steps. First, AuNPs are incubated with the cells for adsorption onto the cell membrane. In this work we used positively charged AuNPs with 70 nm, which were incubation with HeLa cells for half an hour at 37 °C. As described in previous work of ours[20], the number of AuNP that are adsorbed to the cells can be quantified from confocal images in reflection mode (Fig.1b). Next, the nonadherent AuNPs are removed and cell medium containing QDs is added to the cells just prior to starting the laser illumination procedure. After that, all cells are treated with a single pulse of laser light (7 ns @ 561 nm) by scanning the sample through the laser beam with an electronic sample stage. VNB formation can be detected by a change in transmitted intensity of a CW laser focused on the sample, or by dark-field microscopy (Fig.1c). Finally, the cells are washed to remove the remaining extracellular QDs in the solution and cell viability is quantified by a cell viability probe of Calcein red AM. In order to quantify the delivery efficiency and toxicity, the cells are imaged by confocal microscopy.
Figure 2. Confocal images showing the delivery of PEG-coated QDs in the green channel (first row) and the viability of HeLa cells labeled with calcein red AM in the red channel (second row). An overlay of green and red images is shown in the bottom row. The first of column is the negative control (cells treated with 2.08 J/cm2 laser light but without AuNPs), the second and third column show the delivery efficiency of QDs as a function of laser fluence and the number of treatment cycles (N). The field of view is 410 µm by 410 µm.
As is clear from Fig. 2, cells could be successfully loaded with QDs with little or no cytotoxicity by AuNP mediated photoporation. At a low laser fluence of 0.26 J/cm2 the AuNPs become heated, but not enough to form VNBs. Membrane pores can be formed that way, but they are smaller than those generated by VNBs, which explains the low loading efficiency. Instead, by increasing the laser fluence above the VNB generation threshold to 2.08 J/cm2, efficient delivery of QDs is indeed obtained.By repeating the VNB loading procedure a second time, the loading efficiency could be substantially improved further. Based on similar images, the QD loading efficiency and toxicity was quantified as a function of the laser fluence (Fig. 3). These results show that a single cycle of VNB-induced photoporation results in ~60% positive cells and a fluorescence per cell that is markedly higher than in the control samples (where some QD uptake may have occurred by endocytic processes). By repeating the entire loading procedure a second time, >80% of the cells were loaded with QDs and with an even higher fluorescence per cell. At the same time, the viability of cells remained as high as ~85%. From these results, we can conclude that PEG-coated QDs can be efficiently delivered into live cells with low toxicity by VNB-induced photoporation.
Figure 3. Efficiency of QD delivery and cell viability as a function of laser fluence as quantified by image processing of confocal images. Black bars are the percentage of QD positive cells, gray bars are the percentage of live cells and green bars are the mean fluorescence intensity per cell (MFI). NC is the number of loading cycles. The data shown are the result from three independent experiments.
4.CONCLUSIONS
In summary, we have demonstrated that AuNP mediated photoporation is an efficient method for delivering PEG-coated QDs into live cells with low toxicity, thus paving the way towards the use of QDs for labelling of subcellular structures in living cells.
REFERENCES
[1]M. Y. Han, X. H. Gao, J. Z. Su et al., “Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules,” Nature Biotechnology, 19(7), 631-635 (2001).
[2]E. Klarreich, “Biologists join the dots,” Nature, 413(6855), 450-452 (2001).
[3]S. Pathak, S. K. Choi, N. Arnheim et al., “Hydroxylated quantum dots as luminescent probes for in situ hybridization,” Journal of the American Chemical Society, 123(17), 4103-4104 (2001).
[4]W. C. W. Chan, D. J. Maxwell, X. H. Gao et al., “Luminescent quantum dots for multiplexed biological detection and imaging,” Current Opinion in Biotechnology, 13(1), 40-46 (2002).
[5]X. Y. Wu, H. J. Liu, J. Q. Liu et al., “Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots (vol 21, pg 41, 2003),” Nature Biotechnology, 21(4), 452-452 (2003).
[6]X. H. Gao, Y. Y. Cui, R. M. Levenson et al., “In vivo cancer targeting and imaging with semiconductor quantum dots,” Nature Biotechnology, 22(8), 969-976 (2004).
[7]I. L. Medintz, H. T. Uyeda, E. R. Goldman et al., “Quantum dot bioconjugates for imaging, labelling and sensing,” Nature Materials, 4(6), 435-446 (2005).
[8]X. Michalet, F. F. Pinaud, L. A. Bentolila et al., “Quantum dots for live cells, in vivo imaging, and diagnostics,” Science, 307(5709), 538-544 (2005).
[9]M. P. H. Bruchez, C. Z.; , [Quantum dots: applications in biology] Humana Press, Totowa(2007).
[10]A. M. Smith, H. W. Duan, A. M. Mohs et al., “Bioconjugated quantum dots for in vivo molecular and cellular imaging,” Advanced Drug Delivery Reviews, 60(11), 1226-1240 (2008).
[11]S. C. Shanmugavel Chinnathambi, Singaravelu Ganesan, Nobutaka Hanagata, “Silicon Quantum Dots for Biological Applications,” Advanced Healthcare Materials, (2013).
[12]N. M. Xuewen He, “An overview of recent advance of quantum dots for biomedical applications,” Colloids and Surfaces B: Biointerfaces, (2014).
[13]M. Bruchez, M. Moronne, P. Gin et al., “Semiconductor nanocrystals as fluorescent biological labels,” Science, 281(5385), 2013-2016 (1998).
[14]W. C. W. Chan, and S. M. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science, 281(5385), 2016-2018 (1998).
[15]U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot et al., “Quantum dots versus organic dyes as fluorescent labels,” Nature Methods, 5(9), 763-775 (2008).
[16]G. Patterson, M. Davidson, S. Manley et al., “Superresolution Imaging using Single-Molecule Localization,” Annual Review of Physical Chemistry, Vol 61, 61, 345-367 (2010).
[17]J. K. Jaiswal, H. Mattoussi, J. M. Mauro et al., “Long-term multiple color imaging of live cells using quantum dot bioconjugates,” Nature Biotechnology, 21(1), 47-51 (2003).
[18]M. Howarth, W. H. Liu, S. Puthenveetil et al., “Monovalent, reduced-size quantum dots for imaging receptors on living cells,” Nature Methods, 5(5), 397-399 (2008).
[19]E. Y. Lukianova-Hleb, D. S. Wagner, M. K. Brenner et al., “Cell-specific transmembrane injection of molecular cargo with gold nanoparticle-generated transient plasmonic nanobubbles,” Biomaterials, 33(21), 5441-5450 (2012).
[20]R. Xiong, K. Raemdonck, K. Peynshaert et al., “Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells,” ACS Nano, 8(6), 6288-96 (2014).