Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo.

The translational potential of cell-based therapies is often limited by complications related to effectively engineering and manufacturing functional cells. While the use of electroporation is widespread, the impact of electroporation on cell state and function has yet to be fully characterized. Here, we use a genome-wide approach to study optimized electroporation treatment and identify striking disruptions in the expression profiles of key functional transcripts of human T cells. These genetic disruptions result in concomitant perturbation of cytokine secretion including a 648-fold increase in IL-2 secretion (P < 0.01) and a 30-fold increase in IFN-γ secretion (P < 0.05). Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vivo, with electroporated T cells failing to demonstrate sustained antigen-specific effector responses when subjected to immunological challenge. In contrast, cells subjected to a mechanical membrane disruption-based delivery mechanism, cell squeezing, had minimal aberrant transcriptional responses [0% of filtered genes misregulated, false discovery rate (FDR) q < 0.1] relative to electroporation (17% of genes misregulated, FDR q < 0.1) and showed undiminished effector responses, homing capabilities, and therapeutic potential in vivo. In a direct comparison of functionality, T cells edited for PD-1 via electroporation failed to distinguish from untreated controls in a therapeutic tumor model, while T cells edited with similar efficiency via cell squeezing demonstrated the expected tumor-killing advantage. This work demonstrates that the delivery mechanism used to insert biomolecules affects functionality and warrants further study.

Depth-profiling X-ray photoelectron spectroscopy (XPS) analysis of interlayer diffusion in polyelectrolyte multilayers.

Functional organic thin films often demand precise control over the nanometer-level structure. Interlayer diffusion of materials may destroy this precise structure; therefore, a better understanding of when interlayer diffusion occurs and how to control it is needed. X-ray photoelectron spectroscopy paired with C60(+) cluster ion sputtering enables high-resolution analysis of the atomic composition and chemical state of organic thin films with depth. Using this technique, we explore issues common to the polyelectrolyte multilayer field, such as the competition between hydrogen bonding and electrostatic interactions in multilayers, blocking interlayer diffusion of polymers, the exchange of film components with a surrounding solution, and the extent and kinetics of interlayer diffusion. The diffusion coefficient of chitosan (M = ∼100 kDa) in swollen hydrogen-bonded poly(ethylene oxide)/poly(acrylic acid) multilayer films was examined and determined to be 1.4*10(-12) cm(2)/s. Using the high-resolution data, we show that upon chitosan diffusion into the hydrogen-bonded region, poly(ethylene oxide) is displaced from the film. Under the conditions tested, a single layer of poly(allylamine hydrochloride) completely stops chitosan diffusion. We expect our results to enhance the understanding of how to control polyelectrolyte multilayer structure, what chemical compositional changes occur with diffusion, and under what conditions polymers in the film exchange with the solution.

Sustainable drag reduction in turbulent Taylor-Couette flows by depositing sprayable superhydrophobic surfaces.

We demonstrate a reduction in the measured inner wall shear stress in moderately turbulent Taylor-Couette flows by depositing sprayable superhydrophobic microstructures on the inner rotor surface. The magnitude of reduction becomes progressively larger as the Reynolds number increases up to a value of 22% at Re=8.0×10(4). We show that the mean skin friction coefficient C(f) in the presence of the superhydrophobic coating can be fitted to a modified Prandtl-von Kármán-type relationship of the form (C(f)/2)(-1/2)=Mln (Re(C(f)/2)(1/2))+N+(b/Δr)Re(C(f)/2)(1/2) from which we extract an effective slip length of b≈19 µm. The dimensionless effective slip length b(+)=b/δ(ν), where δ(ν) is the viscous length scale, is the key parameter that governs the drag reduction and is shown to scale as b(+)∼Re(1/2) in the limit of high Re.

Optimization of amine-rich multilayer thin films for the capture and quantification of prostate-specific antigen.

It is demonstrated that poly(allylamine hydrochloride)/poly(styrenesulfonate) (PAH/SPS) multilayer films can be successfully tailored for the capture and detection of small biomolecules in dilute concentrations. Based on in vitro results, these films could be potentially applied for rapid and high-throughput diagnosis of dilute biomarkers in serum or tissue. PAH presents functional amino groups that can be further reacted with desired chemistries in order to create customizable and specific surfaces for biomolecule capture. A variety of film assembly characteristics were tested (pH, molecular weight of PAH, and ionic strength) to tune the biotinylation and swelling behavior of these films to maximize detection capabilities. The resultant optimized biotinylated PAH/SPS 9.3/9.3 system was utilized in conjunction with quantum dots (Qdots) to capture and detect a dilute biomarker for prostate cancer, prostate-specific antigen (PSA). Compared to previous work, our system presents a good sensitivity for PSA detection within the clinically relevant range of 0.4-100 ng/mL.

Engineered red blood cells (activating antigen carriers) drive potent T cell responses and tumor regression in mice.

Activation of T cell responses is essential for effective tumor clearance; however, inducing targeted, potent antigen presentation to stimulate T cell responses remains challenging. We generated Activating Antigen Carriers (AACs) by engineering red blood cells (RBCs) to encapsulate relevant tumor antigens and the adjuvant polyinosinic-polycytidylic acid (poly I:C), for use as a tumor-specific cancer vaccine. The processing method and conditions used to create the AACs promote phosphatidylserine exposure on RBCs and thus harness the natural process of aged RBC clearance to enable targeting of the AACs to endogenous professional antigen presenting cells (APCs) without the use of chemicals or viral vectors. AAC uptake, antigen processing, and presentation by APCs drive antigen-specific activation of T cells, both in mouse in vivo and human in vitro systems, promoting polyfunctionality of CD8+ T cells and, in a tumor model, driving high levels of antigen-specific CD8+ T cell infiltration and tumor killing. The efficacy of AAC therapy was further enhanced by combination with the chemotherapeutic agent Cisplatin. In summary, these findings support AACs as a potential vector-free immunotherapy strategy to enable potent antigen presentation and T cell stimulation by endogenous APCs with broad therapeutic potential.

Freely suspended cellular "backpacks" lead to cell aggregate self-assembly.

Cellular "backpacks" are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a "bio-hybrid" material. Previous work has shown that these backpacks do not impair cell viability or native functions such as migration in a B and T cell line, respectively. In the current work, we show that backpacks, when added to a cell suspension, assemble cells into aggregates of reproducible size. We investigate the efficiency of backpack-cell binding using flow cytometry and laser diffraction, examine the influence of backpack diameter on aggregate size, and show that even when cell-backpack complexes are forced through small pores, backpacks are not removed from the surfaces of cells.

Monocyte-mediated delivery of polymeric backpacks to inflamed tissues: a generalized strategy to deliver drugs to treat inflammation.

Targeted delivery of drugs and imaging agents to inflamed tissues, as in the cases of cancer, Alzheimer's disease, Parkinson's disease, and arthritis, represents one of the major challenges in drug delivery. Monocytes possess a unique ability to target and penetrate into sites of inflammation. Here, we describe a broad approach to take advantage of the natural ability of monocytes to target and deliver flat polymeric particles ("Cellular Backpacks") to inflamed tissues. Cellular backpacks attach strongly to the surface of monocytes but do not undergo phagocytosis due to backpack's size, disk-like shape and flexibility. Following attachment of backpacks, monocytes retain important cellular functions including transmigration through an endothelial monolayer and differentiation into macrophages. In two separate in vivo inflammation models, backpack-laden monocytes exhibit increased targeting to inflamed tissues. Cellular backpacks, and their abilities to attach to monocytes without impairing monocyte functions and 'hitchhike' to a variety of inflamed tissues, offer a new platform for both cell-mediated therapies and broad targeting of inflamed tissues.

Determination of lithium-ion distributions in nanostructured block polymer electrolyte thin films by X-ray photoelectron spectroscopy depth profiling.

X-ray photoelectron spectroscopy (XPS) depth profiling with C60(+) sputtering was used to resolve the lithium-ion distribution in the nanometer-scale domain structures of block polymer electrolyte thin films. The electrolytes of interest are mixtures of lithium trifluoromethanesulfonate and lamellar-forming polystyrene-poly(oligo(oxyethylene)methacrylate) (PS-POEM) copolymer. XPS depth profiling results showed that the lithium-ion concentration was directly correlated with the POEM concentration. Furthermore, chemical state and atomic composition of the film were analyzed through the deconvolution of the C1s signal, indicating that the lithium ions appear to be uniformly distributed in the POEM domains. Overall, the unique capabilities of C60(+) depth profiling XPS provide a powerful tool for the analysis of nanostructured polymer thin films in applications ranging from energy storage and generation to surface coatings and nanoscale templates.

Design and fabrication of zwitter-wettable nanostructured films.

Manipulating surface properties using chemistry and roughness has led to the development of advanced multifunctional surfaces. Here, in a nanostructured polymer film consisting of a hydrophilic reservoir of chitosan/carboxymethyl cellulose capped with various hydrophobic layers, we demonstrate the role of a third design factor, water permeation rate. We use this additional design criterion to produce antifogging coatings that readily absorb water vapor while simultaneously exhibiting hydrophobic character to liquid water. These zwitter-wettable films, produced via aqueous layer-by-layer assembly, consist of a nanoscale thin hydrophobic capping layer (chitosan/Nafion) that enables water vapor to diffuse rapidly into the underlying hydrophilic reservoir rather than nucleating drops of liquid water on the surface. We characterize these novel films using a quartz crystal microbalance with dissipation monitoring (QCM-D) and via depth-profiling X-ray photoelectron spectroscopy (XPS) in addition to extensive testing for fogging/antifogging performance.

Orientation-specific attachment of polymeric microtubes on cell surfaces.

Tubular particles presenting heterogeneous regions of chemistry on the tube-ends versus the side are fabricated and are shown to control the particle orientation on the surface of live lymphocytes. Controlling the orientation of anisotropic microparticles on cell surfaces is of interest for biomedical applications and drug delivery in particular, since it can be used to promote or resist particle internalization.

Supramolecular ion-pair interactions to control monolayer assembly.

We demonstrate that noncovalent ion-pair interactions in solution can be employed to control the molecular spacing of thiols in a self-assembled monolayer (SAM) on gold. Ion-pairs formed between the carboxylate tail-group of 16-mercaptohexadecanoic acid (MHA) and tetraalkylammonium (TAA+) hydroxide salts of various alkyl side-chain lengths remain intact during chemisorption of the thiol on gold. The resulting ion-pair SAMs exhibit a 1:1 molar ratio of MHA:TAA+ on the surface and are covalently bound to the gold surface through the thiol headgroup of MHA. We hypothesize that the incorporation of the bulky TAA+ group competes with the strong tendency of the thiols to organize into an ordered monolayer, which highlights the strength of the ion-pair complexes. The ion-pair films can be converted into a loosely packed MHA monolayer by rinsing the SAM with a solution of potassium perchlorate, which releases the TAA+ from the surface. Contact angle measurements and X-ray spectroscopy (XPS) confirm the stoichiometry and covalent attachment of the monolayers. XPS analysis and contact angle measurements indicate that the surface density of bound MHA decreases with increasing size of the TAA+ cation. These results suggest that steric hindrance created by the bulky side-chains of the TAA+ cation dictates the lateral spacing of MHA chains on the surface.