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Cytotoxicity of Polyelectrolyte Microcapsules Encoded with Semiconductor Nanocrystals

Polyelectrolyte microcapsules are promising carriers of drugs and diagnostic agents for designing targeted and controlled delivery systems design. The use of quantum dots (QDs) as fluorescent labels in bioimaging is a promising approach to bioimaging tool development. The potential toxicity of QDsmakes their applicability as fluorescent labels in vivo questionable. Therefore, the cytotoxicity of polyelectrolytemicrocapsules encoded with semiconductor nanocrystals has been investigated.


Introduction
The use of polyelectrolyte microcapsules as a tool for targeted delivery and controlled release of pharmaceuticals, contrast agents, and fluorescent probes for in vitro and in vivo imaging is a promising approach to personalized diagnosis and treatment of various human diseases [1]. QDs are fluorescent semiconductor nanocrystals from 2 to 10 nm in diameter characterized by a wide absorption spectrum and a narrow emission spectrum, as shown in Fig. 1. A high photostability and a bright fluorescence signal make QDs advanced nanophotonic detection and imaging labels [2,3]. However, the presence of heavy metals in the cores of the best QDs and the related potential toxicity make in vivo applications of QDs a questionable issue.
Preparation of polyelectrolyte microcapsules using layer-by-layer assembly of alternatively charged polymers allows drug and fluorescent dyes to be incorporated into the polymeric wall [4]. Encapsulation of QDs into the matrix structure of an interpolymeric complex of alternatively charged polymers, which limits direct interaction of QDs with  living cells seems to be an advantageous approach to diminish the adverse effects of QDs in vivo. Here, we evaluate the in vitro cytotoxicity of diagnostic agents based on polyelectrolyte microcapsules optically encoded with semiconductor CdSe/ZnS core/shell QDs.

Preparation of polyelectrolyte microcapsules encoded with semiconductor nanocrystals
Home-made calcium carbonate microbeads were prepared and used as the matrix cores [5]. The polycation poly(allylamine hydrochloride) (PAH, Sigma-Aldrich, USA) and polyanion (sodium 4-styrensulfonate) (PSS, Sigma-Aldrich, USA) were used for polyelectrolyte layers formation. The formation of the multilayer polyelectrolyte shell on the surface of negatively charged calcium carbonate microparticles and their encoding with water-soluble CdSe/ZnS QDs were carried out by alternately applying oppositely charged layers of polymers and QDs as described earlier [6]. The polyelectrolyte microcapsules were subsequently obtained by removing the calcium carbonate core from the prepared encoded microparticles. The resultant encoded microcapsules were stored at 4 ∘ C in the dark.

Characterization of Polyelectrolyte microcapsules
The morphology, size distribution, and fluorescent properties of microcapsules were analyzed microscopically using Carl Zeiss Axio Scope A1 microscope (Carl Zeiss, Germany) and Texas Red optical filter (ex 596 nm/em 615 nm); image processing was performed using the ZEN software (Carl Zeiss, Germany).

Cytotoxicity
The toxicity of the polyelectrolyte microcapsules encoded with QDs was evaluated

Results
The prepared calcium carbonate microbeads were characterized by a slightly negative surface charge (-5.4 ± 2.5 mV) and were used as templates for deposition of alternately charged polyelectrolyte layers. The resultant microcapsules consisted of ten polyelectrolyte layers and one layer of QDs deposited between the 5th and 6th layers of PAH.
Microscopy study has shown that the size of the resultant polyelectrolyte microcapsules ranges between 3 and 6 μm. The microcapsules are spherical hollow structures whose walls are intensely fluorescent due to incorporation of QDs and can be distinguished as individual objects, as shown in Fig. 2.

Discussion
Encoding of microbeads with QDs using the layer-by-layer method based on successive application of alternately charged polyelectrolyte layers of polycation PAH and polyanion PSS and solubilized QDs ensures effective immobilization of QDs in the polymer matrix, which provides optimal fluorescence properties of the microcapsules obtained.
The prepared polyelectrolyte microcapsules themselves do not exhibit toxicity, which indicates QDs as the cause of the cell viability decrease. QD encapsulation into the polymeric wall of the microcapsules prevents the contact and limits the time of the direct exposure of cells to them, which is beneficial in terms of their in vivo applications.

Conclusion
The data show that the developed polyelectrolyte microcapsules encoded with CdSe/ZnS QDs are characterized by bright fluorescence and low cytotoxicity, which offers the opportunity for development of new systems for delivery of diagnostic and theranostic agents and drugs on their basis.