Jonathan B. Gilbert

Cell-Based Drug Delivery Devices Using Phagocytosis- Resistant Backpacks

Macrophages, ubiquitous phagocytic cells in the human immune system, play a key role in homeostatic, immunological, and infl ammatory processes. [ 1‐2 ] Macrophages are widely distributed in various tissues and play a central role in clearing invading pathogens, dead cells, and foreign entities through phagocytosis. [ 3 ] Their wide presence in various organs and tissues makes them particularly suited to provide an immediate defense against invading threats. Moreover, macrophages are rapidly recruited to the diseased site by signaling molecules such as cytokines. Hence, macrophages are involved in a wide range of pathological conditions including cancer, atherosclerosis, various infl ammatory diseases such as vasculitis and asthma, and many others. Since macrophages play an indispensable role in most pathological conditions, they represent an ideal target for therapeutic applications. Several approaches seeking to use macrophages for targeted therapies involve feeding therapeutic nanoparticles to macrophages ex vivo, followed by re-injection of the macrophages to target the diseased site. This approach has shown promising results for treating HIV infections [ 4 ] , brain disorders, [ 5 ] and solid tumors. [ 6 ] While this strategy is effective for certain conditions, its applications are limited by the fact that the drug carriers are sequestered within the phagosome of macrophages, which reduces the release rates, and in certain cases, degrades the drug. This limitation can be potentially addressed by designing particles that: i) attach to the macrophage surface, ii) avoid internalization, iii) do not interfere with macrophage function, and iv) release the encapsulated drugs in a controlled manner. However, development of materials that simultaneously fulfi ll these requirements is a signifi cant challenge. Herein, we report the ability of cellular backpacks to successfully encapsulate and controllably release drugs and avoid phagocytic internalization while remaining on the macrophage’s surface. These characteristics point to new possibilities in creating cell-based bio-hybrid devices that leverage both the functions of the encapsulated cargo (drugs, nanoparticles, etc.) and the native functions of the cell. Cellular backpacks are fabricated using a standard photolithography lift-off technique of layer-by-layer and spray deposited fi lm. [ 7 ] Briefl y, a positive photoresist is patterned with regularly spaced 7- μ m-diameter holes that extend down to the substrate. Next, a layer-by-layer deposited fi lm consisting of alternating hydrogen bond donor‐acceptor pairs is deposited, and this layer comprises the release region that tethers the rest of the backpack to the substrate. Two hydrogen-bonded regions were used, and details can be found elsewhere. [ 7 ] Next, a Polyelectrolyte multilayer (PEM) of either (FITC-PAH/MNP) or (PDAC/SPS) is deposited to provide suffi cient mechanical rigidity for the backpack to survive the fi nal acetone sonication step [FITC =

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 cm2/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.

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.

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.

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.

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.

Microfluidic-Enabled Intracellular Delivery of Membrane Impermeable Inhibitors to Study Target Engagement in Human Primary Cells

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.

Abstract 2293: Vector-free engineering of immune cells for adoptive cell therapy

Ex vivo manipulation of the immune system for therapeutic purposes has shown incredible clinical promise with the advent of cell therapies such as Chimeric Antigen Receptor modified T-Cell Therapies (CAR-T). However expanding methods to manipulate cells beyond using plasmids and viruses requires a new delivery paradigm. Here we describe a microfluidic approach discovered at MIT where cells are mechanically deformed as they pass through a constriction smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer directly into the cytosol. The method was recognized as one of the World Changing Ideas of 2014 by Scientific American since it has demonstrated the ability to deliver a range of material, such as nanoparticles and proteins to primary cells including embryonic stem cells and immune cells. This is in contrast to existing vector-based and physical methods that have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or oncogenic off-target effects. Supporting the enabling potential of the new deformation based method, we previously reported that delivering protein transcription factors to primary fibroblasts produces a 10-fold improvement in induced pluripotent stem cell colony formation relative to electroporation and cell-penetrating peptides. In this work we describe the use of the vector-free technology to deliver antigen protein directly to the cytoplasm of antigen presenting cells to drive a powerful antigen specific T-cell response. Current efforts to use antigen presenting cells to drive T-cell responses rely on an inefficient process called cross-presentation that relies on material escaping the endosome and entering the cytoplasm. We believe that by delivering antigen directly to the cytoplasm of antigen presenting cells we can overcome this long standing barrier and drive powerful and specific T-cell responses. Our results show that by adoptively transferring antigen presenting cells that have antigen delivered into them we can drive a significant T-cell response. Specifically, we found that this results in a ∼50x increase in antigen specific T-cells in vivo when compared to endocytosis. This advance has the potential to dramatically enhance the therapeutic potential of therapeutic vaccination with antigenic material for the treatment of a wide variety of cancers. Indeed, the ability to deliver structurally diverse materials to difficult-to-transfect primary cells indicate that this method could potentially enable many novel clinical applications. Citation Format: Armon Sharei, Jonathan Gilbert, Darrell Irvine, Klavs Jensen, Robert Langer. Vector-free engineering of immune cells for adoptive cell therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2293.