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