Generic Delivery of Payload of Nanoparticles Intracellularly via Hybrid Polymer Capsules for Bioimaging Applications

Towards the goal of development of a generic nanomaterial delivery system and delivery of the ‘as prepared’ nanoparticles without ‘further surface modification’ in a generic way, we have fabricated a hybrid polymer capsule as a delivery vehicle in which nanoparticles are loaded within their cavity. To this end, a generic approach to prepare nanomaterials-loaded polyelectrolyte multilayered (PEM) capsules has been reported, where polystyrene sulfonate (PSS)/polyallylamine hydrochloride (PAH) polymer capsules were employed as nano/microreactors to synthesize variety of nanomaterials (metal nanoparticles; lanthanide doped inorganic nanoparticles; gadolinium based nanoparticles, cadmium based nanoparticles; different shapes of nanoparticles; co-loading of two types of nanoparticles) in their hollow cavity. These nanoparticles-loaded capsules were employed to demonstrate generic delivery of payload of nanoparticles intracellularly (HeLa cells), without the need of individual nanoparticle surface modification. Validation of intracellular internalization of nanoparticles-loaded capsules by HeLa cells was ascertained by confocal laser scanning microscopy. The green emission from Tb3+ was observed after internalization of LaF3:Tb3+(5%) nanoparticles-loaded capsules by HeLa cells, which suggests that nanoparticles in hybrid capsules retain their functionality within the cells. In vitro cytotoxicity studies of these nanoparticles-loaded capsules showed less/no cytotoxicity in comparison to blank capsules or untreated cells, thus offering a way of evading direct contact of nanoparticles with cells because of the presence of biocompatible polymeric shell of capsules. The proposed hybrid delivery system can be potentially developed to avoid a series of biological barriers and deliver multiple cargoes (both simultaneous and individual delivery) without the need of individual cargo design/modification

Surface characterization of nanoparticles-loaded PSS/PAH capsules.

<p>Scanning electron micrograph of (a) Au nanoparticles-loaded PSS/PAH capsules and (b) blank PSS/PAH capsules.</p

Uptake of nanoparticles-loaded PSS/PAH capsules by HeLa cells.

<p>(<b>a</b>) Confocal laser scanning microscopy (CLSM) image of HeLa cells after uptake of LaF₃:Tb³⁺(5%) nanoparticles-loaded PSS/PAH capsule. Inset shows high magnification image of a HeLa cell, which has internalized Au nanoparticles-loaded capsule. Actin cytoskeleton of cells was stained with FITC-Phalloidin (green). The outer PAH layer of the capsules is labeled with RITC (red) and (<b>b</b>) efficiency of uptake of Au nanoparticles-loaded PSS/PAH capsules and blank PSS/PAH capsules by HeLa cells as investigated by flow cytometry. Efficiency of uptake is represented by percentage positive cells i.e. cells associated (both internalized and bound) with capsules.</p

Optical properties of PSS/PAH capsules co-loaded with two types of nanoparticles.

<p>(a) Photoluminescence emission spectrum, (b) UV-Vis absorption spectrum and (c) EDX analysis of PSS/PAH capsules co-loaded with Au and LaVO₄:Eu³⁺(5%) nanoparticles.</p

Optical properties of nanoparticles-loaded PSS/PAH capsules.

<p>(a) UV-Vis absorbance spectra of (1) Ag and (2) Au nanoparticles-loaded PSS/PAH capsules and (3) blank PSS/PAH capsules; (b) photoluminescence emission spectra of (1) GdF₃:Tb³⁺(5%), (2) LaF₃:Tb³⁺(5%) and (3) LaVO₄:Eu³⁺(5%) nanoparticles-loaded PSS/PAH capsules [Excitation wavelength used was 488 nm for GdF₃:Tb³⁺(5%) and LaF₃:Tb³⁺(5%) and 465 nm for LaVO₄:Eu³⁺(5%)].</p

Validation of nanoparticle presence inside the PSS/PAH capsules.

<p>Representative TEM image of (a) Au, (b) Ag, (c) LaVO₄:Eu³⁺(5%), (d) LaF₃:Tb³⁺(5%), (e) CdS, and (f) GdF₃:Tb³⁺(5%) nanoparticles-loaded PSS/PAH capsules. (Inset in each figure shows the respective nanoparticles-loaded PSS/PAH capsule at lower magnification.).</p

<i>In vitro</i> biocompatibility of nanoparticles-loaded PSS/PAH capsules.

<p>MTT assay results of HeLa cells, blank PSS/PAH capsules, GdF₃:Tb³⁺(5%), LaF₃:Tb³⁺(5%), and Au nanoparticles-loaded PSS/PAH capsules on incubation with HeLa cells.</p

PSS/PAH capsules loaded with different shapes of nanoparticles and co-loaded with two types of nanoparticles.

<p>TEM image of PSS/PAH capsules loaded with (a) gold nanoprism, (b) gold nanorods, (c) multifaceted gold nanostructures and (d) Au and LaVO₄:Eu³⁺(5%) nanoparticles (co-loaded, black and white arrow show LaVO₄:Eu³⁺(5%) and Au nanoparticles respectively.).</p

CLSM images showing the characteristic emission of Tb³⁺ from the HeLa cell internalized LaF₃:Tb³⁺(5%) nanoparticles-loaded PSS/RITC-PAH capsule.

<p>(a) Composite image of bright field and fluorescent signals arising from LaF₃:Tb³⁺(5%) nanoparticles (green emission from Tb³⁺) and RITC-PAH (red); and (b) composite image of fluorescent signals arising from LaF₃:Tb³⁺(5%) nanoparticles (characteristic green emission from Tb³⁺) and RITC-PAH (red) showing internalized LaF₃:Tb³⁺(5%) nanoparticles-loaded capsules.</p