Elsevier

Biomaterials

Volume 27, Issue 14, May 2006, Pages 2820-2828
Biomaterials

Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells

https://doi.org/10.1016/j.biomaterials.2005.12.022Get rights and content

Abstract

Labeling of cells with particles for in-vivo detection is interesting for various biomedical applications. The objective of this study was to evaluate the feasibility and efficiency labeling of cells with polymeric particles without the use of transfection agents. We hypothesized that surface charge would influence cellular uptake. The submicron particles were synthesized by the miniemulsion process. A fluorescent dye which served as reporter was embedded in these particles. The surface charge was varied by adjusting the amount of copolymerized monomer with amino group thus enabling to study the cellular uptake in correlation to the surface charge. Fluorescent-activated cell sorter (FACS) measurements were performed for detecting the uptake of the particles or attachment of particles in mesenchymal stem cells (MSC), and the three cell lines HeLa, Jurkat, and KG1a. These cell lines were chosen as they can serve as models for clinically interesting cellular targets. For these cell lines—with the exception of MSCs—a clear correlation of surface charge and fluorescence intensity could be shown. For an efficient uptake of the submicron particles, no transfection agents were needed. Confocal laser scanning microscopy and transmission electron microscopy (TEM) revealed differences in subcellular localization of the particles. In MSCs and HeLa particles were mostly located inside of cellular compartments resembling endosomes, while in Jurkat and KG1a, nanoparticles were predominantly located in clusters on the cell surface. Scanning electron microscopy showed microvilli to be involved in this process.

Introduction

Materials in the submicron level are increasingly used for biomedical applications exploiting specific effects on this level. As an example the superparamagnetic effect of iron oxide particles makes them interesting contrast agents in magnetic resonance tomography [1]. These particles are also used as non-viral vehicles for gene therapy [2], [3], [4], drug delivery [5], [6], [7], immunization [8], [9], [10], [11] and detoxification [12].

Understanding the interactions of these materials with cells is crucial for improving their behaviour in-vivo and in-vitro. Especially uptake into cells and degradation in intracellular compartments is of high importance. In order to increase the rate of intracellular uptake of polymeric particles transfection agents can be used [13]. These transfection agents are mostly cationic, positively charged molecules [14]. These are toxic and not approved for clinical use. Hence applications in human trials and therapeutic interventions are prohibited up to now. For small molecules and macromolecules, routes of uptake into cells have been described [15], [16]. Particles in the range of a few to several hundred nanometers are much larger and conditions and mechanisms of uptake involving several possible mechanisms like pinocytosis, non-specific endocytosis, receptor-mediated endocytosis, and for larger particles phagocytosis have not been studied thoroughly [17]. The exact mechanism of receptor-mediated endocytosis, for instance, is not completely understood, although some receptors for receptor-mediated endocytosis like opsonin, lectin and scavenger receptors are known [18].

The surface of the particles should be the determining factor for cellular uptake [19], [20] but studies that vary the amount of surface charge in a wide range are lacking. Therefore we studied the uptake of submicron-sized polymeric particles as a function of the amount of surface charge on these particles without using any transfection agent. The aim of our study was to design polymeric particles with defined biologically active surfaces for an efficient uptake without the need for transfection agents. This should help to elucidate the mechanism of uptake. Furthermore we were interested in differences in the interaction of submicron particles with a variety of cell lines as these are important for in-vitro and in-vivo applications. As cell lines we chose HeLa as an adherent cell line which is well established in cell culture labs, mesenchymal stem cells (MSC) for their potential in regenerative medicine, KG1a as model for CD34+ hematopoetic stem cells and Jurkat as model for T cells.

The polymeric particles were synthesized by the process of miniemulsion followed by polymerization of the monomer in the miniemulsion preparation. This type of heterophase polymerization enables to label the polymeric particles with any fluorescent dye as reporter and to functionalize the surface of the particles [21], [22]. By using different co-monomers and by varying the amount of the co-monomers in the miniemulsions we were able to obtain different densities of amino groups on the surface of the particles (for details see [23]). As a reporter, a fluorescent dye (N-(2,6-diisopropylphenyl)-perylene-3,4-dicarbonacidimide (PMI)) was embedded into the particles.

Section snippets

Cell culture

HeLa cells were kept in DMEM supplemented with 10% FCS, 100 units penicillin and 100 μg/mL streptomycin, 2 mm l-glutamine (all from Invitrogen, Germany). MSCs were generated from bone marrow aspirations or explanted hips after obtaining informed consent. The study was approved by the ethics committee of the University Ulm, Germany. Primary human MSCs were generated as previously described [24] and kept in α-MEM (Cambrex, Belgium) supplemented with 20% FCS, 100 units penicillin and 100 μg/mL medium

Results and discussion

Miniemulsion is a process in which the particle size can be tailored by the choice of surfactant, monomer, co-monomer and process parameters like the time of ultrasonication. It yields submicron-sized particles with a narrow size distribution. Furthermore by adding monomers with functional groups—like amino groups here—the surface characteristics can be embedded in the polymer in contrast to absorbed molecules. The polymeric particles synthesized had a wide range of positively charged groups on

Summary and conclusions

It was shown that submicron-sized polymeric particles functionalized with cationic groups on their surface enhanced the uptake of particles without using transfection agents compared to uncharged particles into HeLa cells and MSCs. In HeLa and MSCs this labeling of the cells resulted in intracellular uptake into compartments resembling endosomes while cytoplasmatic localization was not clearly observed. In Jurkat and KG1a the fluorescent particles were attached to the cell membrane in clusters

Acknowledgments

In the Institute of Clinical Transfusion Medicine and Immunogenetics, Ulm, we would like to thank G. Baur and T. Becker for their assistance with the MSC cell cultures and in the Central Facility for Electron Microscopy G. Kräutle, E. Schmidt and R. Weih for their expert technical assistance with the TEM and SEM.

This work was supported by a Bausteinförderung from the University Clinic of Ulm (P871) and the German Research Foundation (DFG; LA1013-3 and MA3271-1).

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