ABSTRACT
The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.
Subject(s)
Drug Carriers/chemical synthesis , Drug Delivery Systems , Microspheres , Benzopyrans , Drug Carriers/chemistry , Fluorescent Dyes , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/pharmacokinetics , HeLa Cells , Humans , Indoles , Microscopy, Fluorescence , Particle Size , Polymers/chemical synthesis , Polymers/chemistryABSTRACT
Amino functionalized cross-linked polystyrene microspheres of well defined sizes (0.2-2 mum) have been prepared and shown to be efficient and controllable delivery devices, capable of transporting anything from small dye molecules to bulky proteins into cells. However, the specific mechanism of cellular entry is largely unknown and widely variant from study to study. As such, chemical, biological and microscopic methods are used to elucidate the mechanism of cellular uptake for polystyrene microspheres of 0.2, 0.5 and 2 mum in mouse melanoma cells. Uptake is found to be wholly unreliant upon energetic processes, while lysosomal and endosomal tracking agents failed to show co-localisation with lysosomes/endosomes, suggesting a non-endocytic uptake pathway. To further explore the consequences of microsphere uptake, gene expression profiling is used to determine if there is a transcriptional response to "beadfection" in both murine and human cells. None of the common transcriptional responses to enhanced endocytosis are observed in beadfected cells, further supporting a non-endocytic uptake mechanism. Furthermore, the microspheres are noted to have a limited interaction with cells at a transcriptional level, supporting them as a non-toxic delivery vehicle.
Subject(s)
Drug Delivery Systems/methods , Gene Expression Profiling/methods , Microspheres , Polystyrenes/pharmacokinetics , Adenosine Triphosphate/metabolism , Animals , Caveolae/metabolism , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cholesterol/metabolism , Clathrin/metabolism , Endosomes/metabolism , Fluorescein/metabolism , Humans , Lysosomes/metabolism , Mice , Microscopy, Confocal , Microtubules/metabolism , Pinocytosis , Polystyrenes/administration & dosage , Polystyrenes/chemistry , Polystyrenes/pharmacology , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , TemperatureABSTRACT
Embryonic stem (ES) cells are in vitro cell lines that can differentiate into all lineages of the fetus and the adult. Despite the versatility of genetic manipulation in murine ES cells, these approaches are time-consuming and rely on inefficient transient cellular delivery systems that can only be applied to undifferentiated ES cell cultures. Here we describe a polystyrene microsphere-based system designed to efficiently deliver biological materials into both undifferentiated and differentiating ES cells. Our results demonstrate that these microspheres can be successfully employed for simultaneous cellular labeling and controlled transfer of various cargos such as fluorophores, proteins and nucleic acids into ES cells without any significant toxicity or loss of pluripotency. This versatile delivery system is also effective in other stem cell lines derived from early embryos, trophoblast and neural stem cells.
Subject(s)
Drug Carriers/chemistry , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Proteins/pharmacokinetics , RNA, Small Interfering/pharmacokinetics , Transfection/methods , Animals , Biocompatible Materials/chemistry , Cells, Cultured , Contrast Media , Materials Testing , Mice , Microspheres , Proteins/administration & dosage , RNA, Small Interfering/administration & dosageABSTRACT
Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that beta-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.
Subject(s)
Drug Carriers/chemistry , Neurons/enzymology , Stem Cells/enzymology , Transfection/methods , beta-Galactosidase/pharmacokinetics , Animals , Cells, Cultured , Humans , Mice , MicrospheresABSTRACT
200 and 500 nm polymeric microspheres have been conjugated to siRNA targeted against EGFP expressed in human cervical cancer (HeLa) cells and shown to efficiently silence protein expression over 72 h, without detrimental cytotoxicity. Furthermore, with use of an independent Cy5 tracking label on the siRNA-laden microsphere, silencing of EGFP could be assessed by selecting only those cells that contained the delivery vehicle (and thus the siRNA) generating a more accurate picture of microsphere-induced gene silencing.
