Novel Strategy for Microsphere-Mediated DNA Transfection

Abstract

A new approach for microsphere-mediated delivery of plasmid DNA has been developed and successfully evaluated. Basic molecular biology techniques were used to linearize and functionalize plasmid DNA by aminomodification, enabling efficient conjugation to carboxy-functionalized microspheres. A T cell hybridoma line was successfully transfected as determined by the efficient expression of a biologically relevant YFP fusion protein. Moreover, our data identified microsphere-mediated delivery of plasmid DNA as a noninvasive, nontoxic, and efficient gene delivery method with the potential to be applied to transfection-resistant, nondividing primary cells, including naïve T cells.

Tracking Cell Proliferation Using a Nanotechnology-Based Approach

“…Specifically surface-modified polystyrene nanoparticles are homogeneous, exhibit a low polydispersity index, and form stable colloids in biological fluids. These nanodevices have been extensively used as systems for in vitro applications [7][8][9]. Furthermore, these NPs enable solid phase multistep chemistries to become compatible with different bio-orthogonal strategies [10,11].…”

“…Furthermore, these NPs enable solid phase multistep chemistries to become compatible with different bio-orthogonal strategies [10,11]. Easy entry in a broad range of cell types including adherent, suspensions and primary cell lines has been reported [8][9][10][11][12]. On the other hand, gene-expression profiling studies showed that these NPs did not induce any significant alteration in nanofected cell transcriptomes [13].…”

Polymeric Microspheres as Protein Transduction Reagents

“…Disulfide linkers have been used effectively in the delivery of nucleic acids to cells (27, 30) and in releasing protein from nanocapsules (31). Such linkers permit intracellular release from a carrier because the cytoplasm of mammalian cells is maintained as a reducing environment, containing high concentrations of reduced glutathione (tripeptide γ-glutamyl-cysteinyl-glycine), which cleaves disulfide bonds to generate free thiols.…”

“…In contrast, the recent demonstrations that polystyrene microspheres can carry a variety of molecular cargoes with them into the cytoplasm (4, 5, 7, 26, 27) make them particularly exciting as potential vectors for delivering functional proteins and/or protein complexes. β-Galactosidase retains its activity when delivered via this route (7), confirming the potential of microspheres to act as generic protein-delivery vehicles.…”

Biocompatible Polymer Nanoparticles for Intra-cellular Applications

“…Cellular processes then transported the pDNA to the nucleus resulting in the expression of the protein. [22] Another approach would be the delivery of synthetic siRNA into cells which has increasing applications due to the ability to switch off a genes without the necessity of being transported into the nucleus. [23] Microspheres functionalized with a double stranded siRNA were shown to efficiently silence green fluorescent protein (eGFP) expressed in human cervical cancer (HeLa) cells.…”