A Molecular Beacon Approach to Measuring the DNA Permeability of Thin Films

A new method for determining the permeability of thin films has been developed. A molecular beacon immobilized inside a porous silica particle that is subsequently encapsulated within a thin film can be used to determine the size of DNA that can permeate through the film. Using this technique, it has been determined that over 3 h, molecules larger than 4.7 nm do not permeate 15-nm thick polyelectrolyte multilayers and after 75 h molecules larger than 6 nm were excluded. This technique has applications for determining the permeability of films used for controlled drug and gene delivery

Manipulating the Salt and Thermal Stability of DNA Multilayer Films via Oligonucleotide Length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature (Tm) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 °C for the 20-mer films to 53 °C for the 60-mer films. Further, an alternating trend in Tm of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher Tm (44−49 °C) than films with an outermost TC layer (ca. 38 °C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films

Probing the Dynamic Nature of DNA Multilayer Films Using Förster Resonance Energy Transfer

DNA films are of interest for use in a number of areas, including sensing, diagnostics, and as drug/gene delivery carriers. The specific base pairing of DNA materials can be used to manipulate their architecture and degradability. The programmable nature of these materials leads to complex and unexpected structures that can be formed from solution assembly. Herein, we investigate the structure of DNA multilayer films using Förster resonance energy transfer (FRET). The DNA films are assembled on silica particles by depositing alternating layers of homopolymeric diblocks (polyA₁₅G₁₅ and polyT₁₅C₁₅) with fluorophore (polyA₁₅G₁₅-TAMRA) and quencher (polyT₁₅C₁₅-BHQ2) layers incorporated at predesigned locations throughout the films. Our results show that DNA films are dynamic structures that undergo rearrangement. This occurs when the multilayer films are perturbed during new layer formation through hybridization but can also take place spontaneously when left over time. These films are anticipated to be useful in drug delivery applications and sensing applications

DNA Multilayer Films on Planar and Colloidal Supports: Sequential Assembly of Like-Charged Polyelectrolytes

Multilayer films comprising solely negatively charged polyelectrolytes were sequentially assembled based on DNA hybridization. Films prepared from alternating layers of two-block homopolymeric nucleotides (polyA20G20/polyT20C20) grew linearly with increasing layer number, as verified by quartz crystal microgravimetry, UV−vis spectrophotometry and optical microscopy. Urea treatment of the films induced morphological changes, while exposure to low ionic strength solutions resulted in film disassembly. DNA multilayer films were also formed on silica particles, and DNA hollow capsules were obtained following dissolution of the template core

Influence of Salt Concentration on the Assembly of DNA Multilayer Films

DNA multilayer films are promising candidates for a plethora of applications, including sensing, diagnostics, and drug/gene delivery. Fabricated solely from DNA, the use of salt in forming DNA multilayers is crucial in promoting and maintaining hybridization of complementary base pairs by minimizing the repulsive forces between the oligonucleotides and preventing disassembly of the layers once formed. Herein, we examine the role of salt on the assembly of DNA films assembled from oligonucleotides composed of two homopolymeric diblocks (polyAnGn and polyTnCn) in salt concentrations ranging from 0.1 to 2 M. Using quartz crystal microgravimetry (QCM) and flow cytometry, we show that films assembled at high salt concentrations (2 M salt) exhibit a different morphology and are denser than those assembled from lower (1 M salt) salt solutions. Formation of the T·A*T triplex in solution and within the DNA film was also studied using circular dichroism (CD) and QCM, respectively. DNA films assembled using oligonucleotides of various lengths (20- to 60-mer) at high salt concentration (2 M salt) showed no significant influence on the film growth. This work shows that salt plays an important role in the assembly and final morphology of DNA multilayer films, hence enabling films with different properties to be tailored

Tuning the Formation and Degradation of Layer-by-Layer Assembled Polymer Hydrogel Microcapsules

Engineered polymer capsules are finding widespread importance in the delivery of encapsulated toxic or fragile drugs. The effectiveness of polymer capsules as therapeutic delivery vehicles is often dependent on the degradation behavior of the capsules because it is often necessary to release the encapsulated drugs at specific times and in certain locations. Herein we investigate the parameters that govern the formation and degradation of a recently introduced new class of polymer hydrogel capsules based on disulfide cross-linked poly(methacrylic acid). We report a new and efficient method for the synthesis of thiol-functionalized poly(methacrylic acid) (PMASH), the main component of the capsules. Polymeric capsules were synthesized by the layer-by-layer deposition of PMASH and poly(vinylpyrrolidone) (PVPON) on silica particle templates, followed by cross-linking the PMASH layers and removing PVPON and the template particles. The disulfide cross-links provided a redox-active trigger for degradation that was initiated by a cellular concentration of glutathione. We demonstrate that increasing the degree of PMASH thiol modification affords direct control over the thickness of the polymer film and the degradation rate of the polymer capsules. Furthermore, the degradation rate of the PMASH capsules was independent of film thickness, suggesting a bulk erosion process

Approaches to Quantifying and Visualizing Polyelectrolyte Multilayer Film Formation on Particles

Colloidal particles prepared by using the layer-by-layer technique are increasingly finding application in diagnostics, drug delivery, and sensing. Herein, we outline methods for applying three established techniques, confocal laser scanning microscopy (CLSM), flow cytometry, and differential interference contrast (DIC) microscopy, to characterize ultrathin films of poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) assembled on silica particles. Both CLSM and flow cytometry require the use of fluorescently labeled polyelectrolytes (PEs). The film homogeneity can be assessed using CLSM, while flow cytometry allows analysis at unparalleled speed (thousands of particles per second) with unprecedented sensitivity (<0.5 fg of adsorbed polymer) of polydispersed particles of different size (∼300 nm to tens of micrometers). Using CLSM and flow cytometry measurements, in conjunction with quartz crystal microgravimetry measurements on planar supports, allows quantification of PSS/PAH layer buildup on the particles. Furthermore, flow cytometry and DIC microscopy were used to unequivocally distinguish between silica-core PSS/PAH-shell particles and hollow PSS/PAH capsules obtained following core removal. The techniques outlined here are not limited to measuring PE deposition on solid particles but, in principle, are equally applicable to quantifying the adsorption of other materials (such as DNA, proteins, or nanoparticles) on a variety of particulate systems, including hollow capsules, emulsions, and cells

Probing the Permeability of Polyelectrolyte Multilayer Capsules via a Molecular Beacon Approach

Application of polyelectrolyte multilayer (PEM) capsules as vehicles for the controlled delivery of substances, such as drugs, genes, pesticides, cosmetics, and foodstuffs, requires a sound understanding of the permeability of the capsules. We report the results of a detailed investigation into probing capsule permeability via a molecular beacon (MB) approach. This method involves preparing MB-functionalized bimodal mesoporous silica (BMSMB) particles, encapsulating the BMSMB particles within the PEM film to be probed, and then incubating the encapsulated BMSMB particles with DNA target sequences of different lengths. Permeation of the DNA targets through the capsule shell causes the immobilized MBs to open due to hybridization of the DNA targets with the complementary loop region of the MBs, resulting in an increase in the MB fluorescence. The assay conditions (BMSMB particle concentration, MB loading within the BMS particles, DNA target concentration, DNA target size, pH, sodium chloride concentration) where the MB−DNA sensing process is effective were first examined. The permeability of DNA through poly(sodium 4-styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) multilayer films, with and without a poly(ethyleneimine) (PEI) precursor layer, was then investigated. The permeation of the DNA targets decreases considerably as the thickness of the PEM film encapsulating the BMSMB particles increases. Furthermore, the presence of a PEI precursor layer gives rise to less permeable PSS/PAH multilayers. The diffusion coefficients calculated for the DNA targets through the PEM capsules range from 10-19 to 10-18 m2 s-1. This investigation demonstrates that the MB approach to measuring permeability is an important new tool for the characterization of PEM capsules and is expected to be applicable for probing the permeability of other systems, such as membranes, liposomes, and emulsions

Optically Characterized DNA Multilayered Assemblies and Phenomenological Modeling of Layer-by-Layer Hybridization

Layer-by-layer assemblies based on deoxyribonucleic acid (DNA) hybridization have potential for various bio- and nanotechnology applications because of their programmability, biodegradability, and ability to control the structure of the assemblies on the nanometer scale. Herein, we investigate the growth and salt stability of DNA films by the optical technique dual polarization interferometry and numerically model the film buildup. The DNA films were assembled by sequentially depositing pairs of oligonucleotides comprised of two different block sequences onto the surface. The oligonucleotides used in the assembly of the different films were as follows: a homopolymeric diblock pair of AxGx/TxCx (x = 15, 20, or 30), a homopolymeric diblock pair of A15G15/C15T15 in which the orientation of the T15C15 diblock was reversed (C15T15), and a random diblock pair of X15Y15/X′15Y′15. The characteristics of the layer growth were highly dependent on the type of the oligonucleotide pair used: the mass of DNA deposited followed a linear, stepwise, or saturated growth with increasing layer deposition. The layer growth of each film was numerically modeled by taking into account the effective hybridization rate and the effective dissociation rate of the oligonucleotides. The proposed modeling offers a framework for molecularly designing oligonucleotide pairs to obtain DNA multilayer films with desired physicochemical properties (thickness, density, stability)

A Mechanism for Forming Large Fluorescent Organo-Silica Particles: Potential Supports for Combinatorial Synthesis

The ability to track multiple compounds through a combinatorial synthesis on solid support particles can be a challenging exercise. A novel solution to this problem is to use the optical characteristics of each support particle to identify the biomolecule synthesized on its surface. To achieve this, we have synthesized a new class of porous, thiol-functionalized supports in a two-step process that used 3-mercaptopropyl trimethoxysilane as the monomer. The monomer was hydrolyzed and polymerized in an acidic solution, which formed an emulsion that was subsequently cross-linked with either ammonia (NH3) or methylamine (CH3NH2). The synthetic process and resulting organo-silica particles were characterized using silicon NMR, scanning electron microscopy techniques, and fluorescence microscopy. Furthermore, thin sections of the porous beads were successfully produced and analyzed via transmission electron microscopy. By controlling the reaction conditions during the synthesis, we achieved a variety of particle morphologies, including hollow particles, particles with macropores on the surface, and particles with a highly porous interior. The mechanism for forming and controlling the morphology of these particles is described here. Also described is the unique process of incorporating fluorescence dyes using combinatorial methods. This enabled the synthesis of a highly optically diverse population of particles, which could be produced over a small number of reactions. Flow cytometry was used to demonstrate the diversity of fluorescence signatures possessed by these encoded particles