Growth factor release from a chemically modified elastomeric poly(1,8-octanediol-co-citrate) thin film promotes angiogenesis in vivo.

The ultimate success of in vivo organ formation utilizing ex vivo expanded "starter" tissues relies heavily upon the level of vascularization provided by either endogenous or artificial induction of angiogenic or vasculogenic events. To facilitate proangiogenic outcomes and promote tissue growth, an elastomeric scaffold previously shown to be instrumental in the urinary bladder regenerative process was modified to release proangiogenic growth factors. Carboxylic acid groups on poly(1,8-octanediol-co-citrate) films (POCfs) were modified with heparan sulfate creating a heparan binding POCf (HBPOCf). Release of proangiogenic growth factors vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF-1) from HBPOCfs demonstrated an approximate threefold increase over controls during a 30-day time course in vitro. Atomic force microscopy demonstrated significant topological differences between films. Subcutaneous implantation of POCf alone, HBPOCf, POCf-VEGF, and HBPOCf-VEGF within the dorsa of nude rats yielded increased vascular growth in HBPOCf-VEGF constructs. Vessel quantification studies revealed that POCfs alone contained 41.1 ± 4.1 vessels/mm², while HBPOCf, POCf-VEGF, and HBPOCF-VEGF contained 41.7 ± 2.6, 76.3 ± 9.4, and 167.72 ± 15.3 vessels/mm², respectively. Presence of increased vessel growth was demonstrated by CD31 and vWF immunostaining in HBPOCf-VEGF implanted areas. Data demonstrate that elastomeric POCfs can be chemically modified and possess the ability to promote angiogenesis in vivo.

Agarose-assisted dip-pen nanolithography of oligonucleotides and proteins.

This paper describes a method for the direct transfer of biomolecules encapsulated within a viscous fluid matrix by dip-pen nanolithography (DPN). The method relies on the use of agarose as a "universal" carrier that is compatible with many types of biomolecules including proteins and oligonucleotides. Agarose-assisted DPN allows one to generate nanoarrays of such materials on activated glass substrates with the same deposition rates for different biomolecules, which will greatly expand future capabilities for parallel, multiplexed biomolecule deposition. The fluidity of the matrix may be systematically varied to control the deposition process, resulting in an additional parameter affecting deposition rates besides tip-substrate contact-time and humidity. Agarose-assisted DPN results in extremely fast biomolecule patterning with typical contact times less than 1 s. Feature sizes as small as 50 nm are demonstrated. The biorecognition properties of both protein and oligonucleotide structures are characterized by studying their reactivity with fluorophore-labeled antibody and complementary oligonucleotide sequences, respectively.

An Electrochemical Approach to and the Physical Consequences of Preparing Nanostructures from Gold Nanorods with Smooth Ends.

We have developed a method to smooth the end sections of nanowires and nanograps generated via the On-Wire Lithography process and studied these rods with optical spectroscopies and theoretical modeling (Discrete Dipole Approximation). The first step of the smoothing process is a reductive one aimed at controlling the diffusion and migration of metal ions to the growing nanorod surface by adjusting the applied potential and concentration of the metal ions in the growth solution. A second oxidative smoothing step, based in part on the energetic differences between topologically rough and smooth surfaces, is used to further smooth the nanorod. The RMS roughness can be reduced over five fold to approximately 5 nm. The properties of these smoothed rods were investigated by empirical and theoretical methods, where it was found the smoothed rods have sharper plasmon resonances and decreased SERS intensity.

Transfer printing of DNA by "click" chemistry.

This paper describes a straightforward procedure to immobilize oligonucleotides on glass substrates in well-defined micropatterns by microcontact printing with a dendrimer-modified stamp. The oligonucleotides are efficiently immobilized by "click" chemistry induced by microcontact printing. Acetylene-modified oligonucleotides were treated with an azide-terminated glass slide under the confinement of the dendrimer-modified stamp, without the use of a Cu(I) catalyst. The immobilization is an irreversible, covalent, and one-step reaction that results in stable attachment of the oligonucleotides. Oligonucleotides with the acetylene-modification at the 5' terminus hybridize selectively with full-length, complementary targets. Strands with more than one acetylene linker do not hybridize with complementary strands.

Dendrimer-mediated transfer printing of DNA and RNA microarrays.

This paper describes a new method to replicate DNA and RNA microarrays. The technique, which facilitates positioning of DNA and RNA with submicron edge resolution by microcontact printing (muCP), is based on the modification of poly(dimethylsiloxane) (PDMS) stamps with dendrimers ("dendri-stamps"). The modification of PDMS stamps with generation 5 poly(propylene imine) dendrimers (G5-PPI) gives a high density of positive charge on the stamp surface that can attract negatively charged oligonucleotides in a "layer-by-layer" arrangement. DNA as well as RNA is transfer printed from the stamp to a target surface. Imine chemistry is applied to immobilize amino-modified DNA and RNA molecules to an aldehyde-terminated substrate. The labile imine bond is reduced to a stable secondary amine bond, forming a robust connection between the polynucleotide strand and the solid support. Microcontact printed oligonucleotides are distributed homogeneously within the patterned area and available for hybridization. By using a robotic spotting system, an array of hundreds of oligonucleotide spots is deposited on the surface of a flat, dendrimer-modified stamp that is subsequently used for repeated replication of the entire microarray by microcontact printing. The printed microarrays are characterized by homogeneous probe density and regular spot morphology.

Solid-supported monolayers and bilayers of amphiphilic beta-cyclodextrins.

This paper describes the adsorption and spreading of beta-cyclodextrin (CD) vesicles on hydrophobic and hydrophilic substrates, which involves a transition from bilayer vesicles to planar molecular monolayers or bilayers. On substrates that are patterned with self-assembled monolayers by microcontact printing (muCP), the CD vesicles preferentially adsorb on hydrophobic areas instead of hydrophilic (nonionic) areas, and on cationic areas instead of hydrophilic (nonionic) areas. Supported monolayers of amphiphilic cyclodextrins CD1 and CD2 were obtained by adsorption of CD vesicles to hydrophobic substrates, and supported bilayers of amphiphilic cyclodextrins CD1 and CD2 were prepared by adsorption of CD vesicles on cationic substrates. Contact angle goniometry, atomic force microscopy and confocal fluorescence microscopy (CFM) were used to analyze the supported CD layers. The fluidity of the supported CD layers was verified using fluorescence recovery after photobleaching experiments. The supported layers function as a supramolecular platform that can bind suitable guest molecules through inclusion in the CD host cavities. Additionally, the CD host layers were patterned with fluorescent guest molecules by supramolecular muCP on the supported CD layers. The host-guest interactions were investigated with CFM and fluorescence resonance energy transfer experiments.

Surface modification of elastomeric stamps for microcontact printing of polar inks.

Chemical modification of the surface of a stamp used for microcontact printing (microCP) is interesting for controling the surface properties, such as the hydrophilicity. To print polar inks, plasma polymerization of allylamine (PPAA) was employed to render the surface of poly(dimethylsiloxane) (PDMS), polyolefin plastomers (POP), and Kraton elatomeric stamps hydrophilic for long periods of time. A thin PPAA film of about 5 nm was deposited on the stamps, which increased the hydrophilicity, and which remained stable for at least several months. These surface-modified stamps were used to transfer polar inks by microCP. The employed microCP schemes are as follows: (a) a second generation of dendritic ink having eight dialkyl sulfide end groups to fabricate patterns on gold substrates by positive microCP, (b) fluorescent guest molecules on beta-cyclodextrin (beta-CD) printboards on glass employing host-guest recognition, and (c) Lucifer Yellow ethylenediamine resulting in covalent patterning on an aldehyde-terminated glass surface. All experiments resulted in an excellent performance of all three PPAA-coated stamp materials to transfer the polar inks from the stamp surface to gold and glass substrates by microCP, even from aqueous solutions.

Directional movement of dendritic macromolecules on gradient surfaces.

A gradient-driven methodology has been developed to manipulate the movement of dendritic macromolecules. Poly(propyleneimine) dendrimers, labeled with rhodamine B, are attached to glass substrates via multiple imine bonds. The dendrimers are able to move on the surface by the hydrolysis and re-formation of these imine bonds. In the absence of an external stimulus, this random movement results in a two-dimensional diffusion on the substrate. We are able to bias the movement of these nanoparticles by means of an aldehyde gradient on the glass substrate.

Covalent microcontact printing of proteins for cell patterning.

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An aldehyde-terminated monolayer on glass or on gold was obtained by the reaction between an amino-terminated monolayer and terephthaldialdehyde. The aldehyde monolayer was employed as a substrate for the direct microcontact printing of bioengineered, collagen-like proteins by using an oxidized poly(dimethylsiloxane) (PDMS) stamp. After immobilization of the proteins into adhesive "islands", the remaining areas were blocked with amino-poly(ethylene glycol), which forms a layer that is resistant to cell adhesion. Human malignant carcinoma (HeLa) cells were seeded and incubated onto the patterned substrate. It was found that these cells adhere to and spread selectively on the protein islands, and avoid the poly(ethylene glycol) (PEG) zones. These findings illustrate the importance of microcontact printing as a method for positioning proteins at surfaces and demonstrate the scope of controlled surface chemistry to direct cell adhesion.

Reversible covalent patterning of self-assembled monolayers on gold and silicon oxide surfaces.

This paper describes the generation of reversible patterns of self-assembled monolayers (SAMs) on gold and silicon oxide surfaces via the formation of reversible covalent bonds. The reactions of (patterned) SAMs of 11-amino-1-undecanethiol (11-AUT) with propanal, pentanal, decanal, or terephthaldialdehyde result in dense imine monolayers. The regeneration of these imine monolayers to the 11-AUT monolayer is obtained by hydrolysis at pH 3. The (patterned) monolayers were characterized by Fourier transform infrared reflection absorption spectroscopy, X-ray photoelectron spectroscopy, contact angle and electrochemical measurements, and atomic force microscopy. Imines can also be formed by microcontact printing of amines on terephthaldialdehyde-terminated substrates. Lucifer Yellow ethylenediamine was employed as a fluorescent amine-containing marker to visualize the reversible covalent patterning on a terephthaldialdehyde-terminated glass surface by confocal microscopy. These experiments demonstrate that with reversible covalent chemistry it is possible to print and erase chemical patterns on surfaces repeatedly.

Atomic force microscopy assisted immobilization of lipid vesicles.

We report on a new approach to direct the immobilization of unilamellar lipid vesicles on substrate-supported lipid bilayers in a spatially confined manner. The adsorption of vesicles from solution is limited to areas of disorder in the bilayers, which is induced by scanning a pattern in situ with an atomic force microscopy (AFM) tip using high imaging forces. Lines of vesicles with a length exceeding 25 microm and a width corresponding to that of a single surface-immobilized vesicle have been fabricated. The adsorbed vesicles are effectively immobilized and do not desorb spontaneously. However, AFM with forces of several nanoNewtons allows one to displace vesicles selectively. The novel methodology described, which may serve as a platform for research on proteins incorporated in the lipid bilayers comprising the vesicles, does not require chemical labeling of the vesicles to guide their deposition.