Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging - PubMed (original) (raw)

. 2008 Jul 16;130(28):9006-12.

doi: 10.1021/ja800086u. Epub 2008 Jun 21.

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Proton-sponge coated quantum dots for siRNA delivery and intracellular imaging

Maksym V Yezhelyev et al. J Am Chem Soc. 2008.

Abstract

We report the rational design of multifunctional nanoparticles for short-interfering RNA (siRNA) delivery and imaging based on the use of semiconductor quantum dots (QDs) and proton-absorbing polymeric coatings (proton sponges). With a balanced composition of tertiary amine and carboxylic acid groups, these nanoparticles are specifically designed to address longstanding barriers in siRNA delivery such as cellular penetration, endosomal release, carrier unpacking, and intracellular transport. The results demonstrate dramatic improvement in gene silencing efficiency by 10-20-fold and simultaneous reduction in cellular toxicity by 5-6-fold, when compared directly with existing transfection agents for MDA-MB-231 cells. The QD-siRNA nanoparticles are also dual-modality optical and electron-microscopy probes, allowing real-time tracking and ultrastructural localization of QDs during delivery and transfection. These new insights and capabilities represent a major step toward nanoparticle engineering for imaging and therapeutic applications.

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Figures

Figure 1

Figure 1

Rational design of proton-sponge coated quantum dots and their use as a multifunctional nanoscale carrier for siRNA delivery and intracellular imaging. (a) Chemical modification of polymer-encapsulated QDs to introduce tertiary amine groups, and adsorption of siRNA on the particle surface by electrostatic interactions. (b) Schematic diagram showing the steps of siRNA-QD in membrane binding, cellular entry, endosomal escape, capturing by RNA binding proteins, loading to RNA-induced silencing complexes (RISC), and target degradation. (c) Schematic illustration of the proton-sponge effect showing the involvement of the membrane protein ATPase (proton pump), osmotic pressure buildup, and organelle swelling and rupture. For optimized silencing efficiency and cellular toxicity, the QD surface layer is composed of 40% (molar) carboxylic acids and 60% tertiary amines. The optimal number of siRNA molecules per particle is approximately 2 (as shown in the diagram).

Figure 2

Figure 2

Size, charge, and optical properties of proton-sponge coated QDs. (a) Optical absorption and emission spectra; (b) core size measured by transmission electron microscopy (TEM); (c) hydrodynamic size measured by dynamic light scattering; and (d) surface charges of unmodified and proton-sponge coated QDs (green 545 nm and red 620 nm) as measured by gel electrophoresis. The current separation resolution is not sufficient to show motility difference between the green and red QDs, because the sizes of polymer-coated QDs are greatly determined by the polymer layer. Nevertheless, the gel image clearly shows the opposite surface charge between original and modified QDs. With longer running distance, the differential motility between multiple colors should be distinguishable. For the red QD of 6 nm core (measured by TEM), the proton-sponge dots have hydrodynamic diameters of ~13 nm before siRNA binding and 17 nm after siRNA binding. They are positively charged with a zeta potential of +19.4 mV before siRNA binding and +8.5 mV after siRNA binding.

Figure 3

Figure 3

Comparison of gene silencing efficiencies between proton-sponge coated QDs and commercial transfection reagents by western blotting. The level of cyclophilin B protein expression was reduced to 1.81%±1.47% by QDs, to 13.45%±9.48% by Lipofectamine, to 30.58%±6.90% by TransIT, and to 51.00%±11.46% by JetPEI (corresponding to efficiency improvements of 7.4, 16.9, and 28.2 fold, respectively). The quantitative values were obtained from the western blot (inset). On the average, the proton-sponge coated QDs are 18 times as efficient as the three transfection agents commonly used.

Figure 4

Figure 4

Comparison of cellular toxicity between proton-sponge coated QDs and commercial transfection reagents using the SRB assay. (a) Cytotoxicity data obtained from QDs and three transfection reagents (Lipofectamine 2000, TransIT, and JetPEI) at their optimal transfection efficiencies (100 nM for QDs, see Methods for details). Data points were obtained at 24 hours, and the proton-sponge coated QDs were nearly non-toxic to MDAMB-231 cells. (b) Cellular toxicity data as a function of transfection time obtained from QDs and conventional reagents at siRNA concentrations for optimal transfection efficiency. Note that the QD-based agents performed especially well for extended transfection times. (c) Dose-dependent toxicity data for QDs and conventional agents. The x-axis indicates the fold of siRNA concentration relative to the optimal concentration for transfection. The QD agents performed much better than other transfection agents when the siRNA concentrations were 5-10 times higher than the optimal concentration.

Figure 5

Figure 5

Time-dependent fluorescence imaging of QD-siRNA nanoparticle conjugates and their entry and transport in living cells. Fluorescence micrographs of MDA-MB 231 cells obtained at (a) 15 min, and (b) 4 hours after the addition of QD-siRNA. The images show that at early incubation, the QD fluorescence is limited to the cell membrane, whereas extended incubation allows cytoplasm localization.

Figure 6

Figure 6

Ultrastructural localization of QDs inside MDA-MB-231 cells by transmission electron microscopy (TEM). (a) TEM micrograph showing the process of siRNA-QD endocytosis and the formation of an endosome just about to enter the cell. (b) TEM micrograph showing a large multivesicular structure, QD clustering, and QD attachment to the inner vesicle membrane.

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