9 research outputs found
Nano-Imprinted Thin Films of Reactive, Azlactone-Containing Polymers: Combining Methods for the Topographic Patterning of Cell Substrates with Opportunities for Facile Post-Fabrication Chemical Functionalization
Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) were used to characterize changes in nanoscale structure that occur when ultrathin polyelectrolyte multilayers (PEMs) are incubated in aqueous media. The PEMs investigated here were fabricated by the deposition of alternating layers of plasmid DNA and a hydrolytically degradable polyamine onto a precursor film composed of alternating layers of linear poly(ethylene imine) (LPEI) and sodium poly(styrene sulfonate) (SPS). Past studies of these materials in the context of gene delivery revealed transformations from a morphology that is smooth and uniform to one characterized by the formation of nanometer-scale particulate structures. We demonstrate that in-plane registration of LSCM and AFM images acquired from the same locations of films fabricated using fluorescently labeled polyelectrolytes allows the spatial distribution of individual polyelectrolyte species to be determined relative to the locations of topographic features that form during this transformation. Our results suggest that this physical transformation leads to a morphology consisting of a relatively less disturbed portion of film composed of polyamine and DNA juxtaposed over an array of particulate structures composed predominantly of LPEI and SPS. Characterization by scanning electron microscopy and energy-dispersive X-ray microanalysis provides additional support for this interpretation. The combination of these different microscopy techniques provides insight into the structures and dynamics of these multicomponent thin films that cannot be achieved using any one method alone, and could prove useful for the further development of these assemblies as platforms for the surface-mediated delivery of DNA
Characterization of Degradable Polyelectrolyte Multilayers Fabricated Using DNA and a Fluorescently-Labeled Poly(β-amino ester): Shedding Light on the Role of the Cationic Polymer in Promoting Surface-Mediated Gene Delivery
Polyelectrolyte multilayers (PEMs) fabricated from cationic
polymers
and DNA have been investigated broadly as materials for surface-mediated
DNA delivery. One attractive aspect of this âmultilayeredâ
approach is the potential to exploit the presence of cationic polymer
âlayersâ in these films to deliver DNA to cells more
effectively. Past studies demonstrate that these films can promote
transgene expression in vitro and in vivo, but significant questions
remain regarding roles that the cationic polymers could play in promoting
the internalization and processing of DNA. Here, we report physicochemical
and in vitro cell-based characterization of DNA-containing PEMs fabricated
using fluorescently end-labeled derivatives of a degradable polycation
(polymer <b>1</b>) used in past studies of surface-mediated
transfection. This approach permitted simultaneous characterization
of polymer and DNA in solution and in cells using fluorescence-based
techniques, and provided information about the locations and behaviors
of polymer <b>1</b> that could not be obtained using other methods.
LSCM and flow cytometry experiments revealed that polymer <b>1</b> and DNA released from film-coated objects were both internalized
extensively by cells and that they were colocalized to a significant
extent inside cells (e.g., âź58% of DNA was colocalized with
polymer). Fluorescence anisotropy measurements of solutions containing
partially eroded films were also consistent with the presence of aggregates
of polymer <b>1</b> and DNA in solution (e.g., after release
from surfaces, but prior to internalization by cells). Our results
support the view that polymer <b>1</b>, which is incorporated
into these materials as âlayersâ rather than as part
of optimized, preformed âpolyplexesâ, can act to promote
or enhance surface-mediated DNA delivery. More broadly, our results
suggest opportunities to improve the delivery properties of DNA-containing
PEMs by incorporation of additional âlayersâ of other
conventional cationic polymers designed to address specific intracellular
barriers to transfection, such as endosomal escape, more effectively
Reactive Polymer Multilayers Fabricated by Covalent Layer-by-Layer Assembly: 1,4-Conjugate Addition-Based Approaches to the Design of Functional Biointerfaces
We report on conjugate addition-based approaches to the
covalent
layer-by-layer assembly of thin films and the post-fabrication functionalization
of biointerfaces. Our approach is based on a recently reported approach
to the âreactiveâ assembly of covalently cross-linked
polymer multilayers driven by the 1,4-conjugate addition of amine
functionality in polyÂ(ethyleneimine) (PEI) to the acrylate groups
in a small-molecule pentacrylate species (5-Ac). This process results
in films containing degradable β-amino ester cross-links and
residual acrylate and amine functionality that can be used as reactive
handles for the subsequent immobilization of new functionality. Layer-by-layer
growth of films fabricated on silicon substrates occurred in a supra-linear
manner to yield films âź750 nm thick after the deposition of
80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM)
suggested a mechanism of growth that involves the reactive deposition
of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared
(IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate
addition without formation of amide functionality. Additional experiments
demonstrated that acrylate-containing films could be postfunctionalized
via conjugate addition reactions with small-molecule amines that influence
important biointerfacial properties, including water contact angles
and the ability of film-coated surfaces to prevent or promote the
attachment of cells <i>in vitro</i>. For example, whereas
conjugation of the hydrophobic molecule decylamine resulted in films
that supported cell adhesion and growth, films treated with the carbohydrate-based
motif d-glucamine resisted cell attachment and growth almost
completely for up to 7 days in serum-containing media. We demonstrate
that this conjugate addition-based approach also provides a means
of immobilizing functionality through labile ester linkages that can
be used to promote the long-term, surface-mediated release of conjugated
species and promote gradual changes in interfacial properties upon
incubation in physiological media (e.g., over a period of at least
1 month). These covalently cross-linked films are relatively stable
in biological media for prolonged periods, but they begin to physically
disintegrate after âź30 days, suggesting opportunities to use
this covalent layer-by-layer approach to design functional biointerfaces
that ultimately erode or degrade to facilitate elimination
Polyelectrolyte Multilayers Promote Stent-Mediated Delivery of DNA to Vascular Tissue
We
report an approach to deliver DNA to vascular tissue <i>in vivo</i> using intravascular stents coated with degradable,
DNA-containing polyelectrolyte multilayers (PEMs). Ionically cross-linked
multilayers âź120 nm thick were fabricated layer-by-layer on
the surfaces of balloon-mounted stainless steel stents using plasmid
DNA and a hydrolytically degradable polyÂ(β-amino ester) (polymer <b>1</b>). Characterization of stents coated using a fluorescently
end-labeled analog of polymer <b>1</b> revealed film erosion
to be uniform across the surfaces of the stents; differential stresses
experienced upon balloon expansion did not lead to faster film erosion
or dose dumping of DNA in areas near stent joints when stents were
incubated in physiologically relevant media. The ability of film-coated
stents to transfer DNA and transfect arterial tissue <i>in vivo</i> was then investigated in pigs and rabbits. Stents coated with films
fabricated using fluorescently labeled DNA resulted in uniform transfer
of DNA to sub-endothelial tissue in the arteries of pigs in patterns
corresponding to the locations and geometries of stent struts. Stents
coated with films fabricated using polymer <b>1</b> and plasmid
DNA encoding EGFP resulted in expression of EGFP in the medial layers
of stented tissue in both pigs and rabbits two days after implantation.
The results of this study, combined with the modular and versatile
nature of layer-by-layer assembly, provide a polymer-based platform
that is well suited for fundamental studies of stent-mediated gene
transfer. With further development, this approach could also prove
useful for the design of nonviral, gene-based approaches for prevention
of complications that arise from the implantation of stents and other
implantable interventional devices