Enhancing radiotherapy by lipid nanocapsule-mediated delivery of amphiphilic gold nanoparticles to intracellular membranes - PubMed (original) (raw)

. 2014 Sep 23;8(9):8992-9002.

doi: 10.1021/nn502146r. Epub 2014 Aug 20.

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Enhancing radiotherapy by lipid nanocapsule-mediated delivery of amphiphilic gold nanoparticles to intracellular membranes

Yu-Sang Yang et al. ACS Nano. 2014.

Abstract

Amphiphilic gold nanoparticles (amph-NPs), composed of gold cores surrounded by an amphiphilic mixed organic ligand shell, are capable of embedding within and traversing lipid membranes. Here we describe a strategy using crosslink-stabilized lipid nanocapsules (NCs) as carriers to transport such membrane-penetrating particles into tumor cells and promote their transfer to intracellular membranes for enhanced radiotherapy of cancer. We synthesized and characterized interbilayer-crosslinked multilamellar lipid vesicles (ICMVs) carrying amph-NPs embedded in the capsule walls, forming Au-NCs. Confocal and electron microscopies revealed that the intracellular distribution of amph-NPs within melanoma and breast tumor cells following uptake of free particles vs Au-NCs was quite distinct and that amph-NPs initially delivered into endosomes by Au-NCs transferred over a period of hours to intracellular membranes through tumor cells, with greater intracellular spread in melanoma cells than breast carcinoma cells. Clonogenic assays revealed that Au-NCs enhanced radiotherapeutic killing of melanoma cells. Thus, multilamellar lipid capsules may serve as an effective carrier to deliver amphiphilic gold nanoparticles to tumors, where the membrane-penetrating properties of these materials can significantly enhance the efficacy of frontline radiotherapy treatments.

Keywords: amphiphilic nanoparticles; biological TEM; cell-penetrating nanoparticles; glycocalyx; gold nanoparticles; multilamellar lipid vesicles; radiotherapy.

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Figures

Figure 1

Figure 1

TEM and Cryo-TEM micrographs of Au-NCs with encapsulated OVA. (a–b) Au-NCs were dried on grids followed by negative staining with phosphotunstic acid and TEM imaging. Scale bars 200 nm (a), 100 nm (b). (c) Cryo-TEM of Au-NCs in water (scale bar 100 nm). (d) Gold to lipid mass ratios as a function of NC diameter measured for a preparation of nanocapsules divided into 4 different size fractions. Error bars show the std. dev. of each size fraction.

Figure 2

Figure 2

Uptake of amph-NPs and Au-NCs by B16F10 melanoma cells. (a) Flow cytometry analysis of bodipy-labeled gold NP uptake after 24 hr incubation with 250 nM Au particles (free amph-NPs or equivalent amounts of amph-NPs loaded in ICMV capsules). (b, c) B16F10 cells were incubated with 250 nM Au (green) as free amph-NPs (b) or loaded in ICMV capsules (c) and then imaged after 3 or 24 hr by confocal microscopy. ICMVs were co-labeled by encapsulated fluorescent ovalbumin protein (red, c). (Scale bars 20 um).

Figure 3

Figure 3

Uptake of amph-NPs and Au-NCs by 4T1 breast carcinoma cells. (a) Flow cytometry analysis of bodipy-labeled gold NP uptake after 24 hr incubation with 250 nM Au particles (free amph-NPs or equivalent amounts of amph-NPs loaded in ICMV capsules). (b, c) 4T1 cells were incubated with 250 nM Au (green) as free amph-NPs (b) or loaded in ICMV capsules (c) and then imaged after 3 or 24 hr by confocal microscopy. ICMVs were co-labeled by encapsulated fluorescent ovalbumin protein (red, c). (Scale bars 20 um).

Figure 4

Figure 4

Thin-sectioned TEM images of B16F10 melanoma cells incubated with free amph-NPs or Au-NCs for 24 hr. (a,b) amph-NPs (scale bars 250 nm(a), 100 nm (b)). (c, d) Au-NCs (scale bars 100 nm). Line arrows in (a) highlight amph-NPs in endosomes; Block arrows in (a) highlight amph-NPs dispersed in the cytosol.

Figure 5

Figure 5

Thin-sectioned TEM images of 4T1 breast cancer cells incubated with free amph-NPs (a, b, c) or Au-NCs (d) for 24 hr (scale bars (a,b) 100 nm, (c, d) 500 nm). Arrows highlight amph-NPs dispersed among endosomal and intracellular membranes.

Figure 6

Figure 6

(a) Confocal images of the glycocalyx of B16F10 and 4T1 cells labeled with AF555-Wheat Germ Agglutinin (WGA). (b) Flow cytometry quantification of WGA-labeled tumor cells. ***, P=0.0007 by unpaired t test.

Figure 7

Figure 7

Clonogenic assay assessing radiosensitization promoted by amph-NPs and Au-NCs. B16F10 or 4T1 cells were incubated with amph-NPs (250 nM), Au-NCs (250 nM amph-NPs), or media alone for 24 hr at 37°C, and then irradiated with 4 Gy. (a) Growth of B16F10 tumor cell colonies assayed 7 days following 4 Gray of 137Cs γ irradiation, with or without addition of amph-NPs or Au-NCs. (b) Surviving fraction of B16F10 and 4T1 cells (normalized to untreated, unirradiated cells) following irradiation with or without added amph-NPs or Au-NCs. ***, P < 0.001 by ANOVA.

Scheme 1

Scheme 1

Au-NC synthesis. (i) Dried lipid films composed of 50% DOPC 50% MPB-PE rehydrated with buffer containing amph-NPs and OVA protein., forming (ii) gold-embedded/protein-encapsulated liposomes. (iii) Anionic liposomes fused by addition of calcium. (iv) Stacked lipid bilayers were crosslinked by addition of DTT, and (v) outer surfaces PEGylated.

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