Mapping alternating current electroosmotic flow at the dielectrophoresis crossover frequency of a colloidal probe.

AC electroosmotic (ACEO) flow above the gap between coplanar electrodes is mapped by the measurement of Stokes forces on an optically trapped polystyrene colloidal particle. E²-dependent forces on the probe particle are selected by amplitude modulation (AM) of the ACEO electric field (E) and lock-in detection at twice the AM frequency. E²-dependent DEP of the probe is eliminated by driving the ACEO at the probe's DEP crossover frequency. The location-independent DEP crossover frequency is determined, in a separate experiment, as the limiting frequency of zero horizontal force as the probe is moved toward the midpoint between the electrodes. The ACEO velocity field, uncoupled from probe DEP effects, was mapped in the region 1-9 µm above a 28 µm gap between the electrodes. By use of variously sized probes, each at its DEP crossover frequency, the frequency dependence of the ACEO flow was determined at a point 3 µm above the electrode gap and 4 µm from an electrode tip. At this location the ACEO flow was maximal at ∼117 kHz for a low salt solution. This optical trapping method, by eliminating DEP forces on the probe, provides unambiguous mapping of the ACEO velocity field.

Acetalated dextran is a chemically and biologically tunable material for particulate immunotherapy.

Materials that combine facile synthesis, simple tuning of degradation rate, processability, and biocompatibility are in high demand for use in biomedical applications. We report on acetalated dextran, a biocompatible material that can be formed into microparticles with degradation rates that are tunable over 2 orders of magnitude depending on the degree and type of acetal modification. Varying the degradation rate produces particles that perform better than poly(lactic-co-glycolic acid) and iron oxide, two commonly studied materials used for particulate immunotherapy, in major histocompatibility complex class I (MHC I) and MHC II presentation assays. Modulating the material properties leads to antigen presentation on MHC I via pathways that are dependent or independent of the transporter associated with antigen processing. To the best of our knowledge, this is the only example of a material that can be tuned to operate on different immunological pathways while maximizing immunological presentation.

Mannosylated dextran nanoparticles: a pH-sensitive system engineered for immunomodulation through mannose targeting.

Biotherapeutic delivery is a rapidly growing field in need of new materials that are easy to modify, are biocompatible, and provide for triggered release of their encapsulated cargo. Herein, we report on a particulate system made of a polysaccharide-based pH-sensitive material that can be efficiently modified to display mannose-based ligands of cell-surface receptors. These ligands are beneficial for antigen delivery, as they enhance internalization and activation of APCs, and are thus capable of modulating immune responses. When compared to unmodified particles or particles modified with a nonspecific sugar residue used in the delivery of antigens to dendritic cells (DCs), the mannosylated particles exhibited enhanced antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. This represents the first demonstration of a mannosylated particulate system that enables enhanced MHC I antigen presentation by DCs in vitro. Our readily functionalized pH-sensitive material may also open new avenues in the development of optimally modulated vaccine delivery systems.

Acid-degradable cationic dextran particles for the delivery of siRNA therapeutics.

We report a new acid-sensitive, biocompatible, and biodegradable microparticulate delivery system, spermine modified acetalated-dextran (Spermine-Ac-DEX), which can be used to efficiently encapsulate siRNA. These particles demonstrated efficient gene knockdown in HeLa-luc cells with minimal toxicity. This knockdown was comparable to that obtained using Lipofectamine, a commercially available transfection reagent generally limited to in vitro use due to its high toxicity.

Ensemble method to measure the potential energy of nanoparticles in an optical trap.

A method is described for measuring the potential energy of nanoparticles in an optical trap by trapping an ensemble of particles with a focused laser beam. The force balance between repulsive osmotic and confining gradient-force pressures determines the single-particle trapping potential independent of interactions between the particles. The ensemble nature of the measurement permits evaluation of single-particle trapping energies much smaller than kBT. Energies obtained by this method are compared to those of single-particle methods as well as to theoretical calculations based on classical electromagnetic optics.

Chemoselective ligation in the functionalization of polysaccharide-based particles.

Despite the promise of precisely targeted or otherwise functionalized polymeric particulate drug delivery vehicles, typical biocompatible particles are generally not amenable to facile and selective surface modification. Herein, we report the development of a simple, mild, and chemoselective strategy for the conjugation of biologically active molecules to the surface of dextran-based microparticles. Alkoxyamine-bearing reagents were used to form stable oxime conjugates with latent aldehyde functionality present in reducing carbohydrate chain ends. We demonstrate the functionalization of dextran-based microparticles with a fluorophore as well as a cell-penetrating peptide sequence, which facilitated the delivery of cargo to nonphagocytic cells leading to a 60-fold increase in the expression of a reporter gene when plasmid DNA-loaded particles were used.

T-cell activation by antigen-loaded pH-sensitive hydrogel particles in vivo: the effect of particle size.

Polymeric carriers designed to encapsulate protein antigens have great potential for improving the efficacy of vaccines and immunotherapeutics for diseases such as cancer. We recently developed a carrier system based on polyacrylamide hydrogel microparticles cross-linked with acid-labile moieties. After being phagocytosed by antigen-presenting cells, the protein encapsulated within the carrier is released and processed for subsequent presentation of antigenic epitopes. To understand the impact of particle size on the activation of T-cells following uptake by antigen-presenting cells, particles with mean diameters of 3.5 microm and 35 nm encapsulating a model protein antigen were synthesized by emulsion and microemulsion based polymerization techniques, respectively. In vivo tests demonstrated that both sizes of particles were effective at stimulating the proliferation of T-cells and were capable of generating an antigen-specific cytotoxic T-cell response when coadministered with immunostimulatory DNA. Contrary to previous reports in the literature, our results suggest that there is no significant difference in the magnitude of T-cell activation for the two sizes of particles used in these experiments. This disparity in findings may be related to fundamental differences in material properties of the carriers used in these studies, such as the hydrophilicity of the polyacrylamide particles described here versus the hydrophobic nature of carriers investigated by other groups.

Impact of hydrogel nanoparticle size and functionalization on in vivo behavior for lung imaging and therapeutics.

Polymer chemistry offers the possibility of synthesizing multifunctional nanoparticles which incorporate moieties that enhance diagnostic and therapeutic targeting of cargo delivery to the lung. However, since rules for predicting particle behavior following modification are not well-defined, it is essential that probes for tracking fate in vivo are also included. Accordingly, we designed polyacrylamide-based hydrogel particles of differing sizes, functionalized with a nona-arginine cell-penetrating peptide (Arg(9)), and labeled with imaging components to assess lung retention and cellular uptake after intratracheal administration. Radiolabeled microparticles (1-5 microm diameter) and nanoparticles (20-40 nm diameter) without and with Arg(9) showed diffuse airspace distribution by positron emission tomography imaging. Biodistribution studies revealed that particle clearance and extrapulmonary distribution was, in part, size dependent. Microparticles were rapidly cleared by mucociliary routes but, unexpectedly, also through the circulation. In contrast, nanoparticles had prolonged lung retention enhanced by Arg(9) and were significantly restricted to the lung. For all particle types, uptake was predominant in alveolar macrophages and, to a lesser extent, lung epithelial cells. In general, particles did not induce local inflammatory responses, with the exception of microparticles bearing Arg(9). Whereas microparticles may be advantageous for short-term applications, nanosized particles constitute an efficient high-retention and non-inflammatory vehicle for the delivery of diagnostic imaging agents and therapeutics to lung airspaces and alveolar macrophages that can be enhanced by Arg(9). Importantly, our results show that minor particle modifications may significantly impact in vivo behavior within the complex environments of the lung, underscoring the need for animal modeling.

Enhanced cell penetration of acid-degradable particles functionalized with cell-penetrating peptides.

Biopharmaceuticals, such as proteins and DNA, have demonstrated their potential to prevent and cure diseases. The success of such therapeutic agents hinges upon their ability to cross complex barriers in the body and reach their target intact. In order to reap the full benefits of these therapeutic agents, a delivery vehicle capable of delivering cargo to all cell types, both phagocytic and non-phagocytic, is needed. This article presents the synthesis and evaluation of a microparticle delivery vehicle capable of cell penetration and sub-cellular triggered release of an encapsulated payload. pH-sensitive polyacrylamide particles functionalized with a polyarginine cell-penetrating peptide (CPP) were synthesized. The incorporation of a CPP into the microparticles led to efficient uptake by non-phagocytic cells in culture. In addition, the CPP-modified particles showed no cytotoxic effects at concentrations used in this study. The results suggest that these particles may provide a vehicle for the successful delivery of therapeutic agents to various cell types.

Two-photon degradable supramolecular assemblies of linear-dendritic copolymers.

Micelles of dendritic-linear copolymers have been developed to release a payload after infrared stimulus.

Osmotic Bulk Modulus of Charged Colloids Measured by Ensemble Optical Trapping.

The optical-bottle technique is used to measure osmotic bulk moduli of colloid suspensions. The bulk modulus is determined by optically trapping an ensemble of nanoparticles and invoking a steady-state force balance between confining optical-gradient forces and repulsive osmotic-pressure forces. Osmotic bulk moduli are reported for aqueous suspensions of charged polystyrene particles in NaCl solutions as a function of particle concentration and ionic strength, and are compared to those determined by turbidity measurements under the same conditions. Effective particle charges are calculated from the bulk moduli and are found to increase as a function of ionic strength, consistent with previously reported results.

Separability of electrostatic and hydrodynamic forces in particle electrophoresis.

By use of optical tweezers we explicitly measure the electrostatic and hydrodynamic forces that determine the electrophoretic mobility of a charged colloidal particle. We test the ansatz of O'Brien and White [J. Chem. Soc. Faraday II 74, 1607 (1978)] that the electrostatically and hydrodynamically coupled electrophoresis problem is separable into two simpler problems: (1) a particle held fixed in an applied electric field with no flow field and (2) a particle held fixed in a flow field with no applied electric field. For a system in the Helmholtz-Smoluchowski and Debye-Hückel regimes, we find that the electrostatic and hydrodynamic forces measured independently accurately predict the electrophoretic mobility within our measurement precision of 7%; the O'Brien and White ansatz holds under the conditions of our experiment.

In vivo studies on the effect of co-encapsulation of CpG DNA and antigen in acid-degradable microparticle vaccines.

Protein-based vaccines have been explored as a safer alternative to traditional weakened or killed whole organism based vaccination strategies and have been investigated for their ability to activate the immune system against certain cancers. For optimal stimulation of T lymphocytes, protein-based vaccines should deliver protein antigens to antigen presenting cells in the context of appropriate immunostimulatory signals, thus mimicking actual pathogens. In this report, we describe the synthesis, characterization, and biological evaluation of immunostimulatory acid-degradable microparticles, which are suitable delivery vehicles for use in protein-based vaccines and cancer immunotherapy. Using a 3' conjugation strategy, we optimized the attachment of immunostimulatory CpG DNA to our vaccine carriers and demonstrated that under acidic conditions similar to those found in endosomal compartments, these new particles were capable of simultaneously releasing a model protein antigen and a CpG DNA adjuvant. We found in an in vivo cytotoxicity assay that the co-encapsulation of ovalbumin, a model antigen, and immunostimulatory agent in the same particle led to superior cytotoxic T lymphocyte activity compared to particles coadministered with adjuvant in an unbound form. In addition, we investigated the ability of these acid-degradable particles to induce protective immunity in the MO5 murine melanoma model and found that they were effective until tumor escape, which appeared to result from a loss of antigen expression by the cancer cells due to in vivo selection pressure.

Osmotic properties of poly(ethylene glycols): quantitative features of brush and bulk scaling laws.

From glycosylated cell surfaces to sterically stabilized liposomes, polymers attached to membranes attract biological and therapeutic interest. Can the scaling laws of polymer "brushes" describe the physical properties of these coats? We delineate conditions where the Alexander-de Gennes theory of polymer brushes successfully fits the intermembrane distance versus applied osmotic stress data of Kenworthy et al. for poly(ethylene glycol)-grafted multilamellar liposomes. We establish that the polymer density and size in the brush must be high enough that, in a bulk solution of equivalent monomer density, the polymer osmotic pressure is independent of polymer molecular weight (the des Cloizeaux semidilute regime of bulk polymer solutions). The condition that attached polymers behave as semidilute bulk solutions offers a rigorous criterion for brush scaling-law behavior. There is a deep connection between the behaviors of semidilute polymer solutions in bulk and polymers grafted to a surface at a density such that neighbors pack to form a uniform brush. In this regime, two-parameter unconstrained fits of the Alexander-de Gennes brush scaling laws to the Kenworthy et al. data yield effective monomer lengths of 3.3-3.6 A, which agree with structural predictions. The fitted distances between grafting sites are larger than expected from the nominal mole fraction of poly(ethylene glycol)-lipids; the chains apparently saturate the surface. Osmotic stress measurements can be used to estimate the actual densities of membrane-grafted polymers.