Nishit Doshi

Red blood cell-mimicking synthetic biomaterial particles

Biomaterials form the basis of current and future biomedical technologies. They are routinely used to design therapeutic carriers, such as nanoparticles, for applications in drug delivery. Current strategies for synthesizing drug delivery carriers are based either on discovery of materials or development of fabrication methods. While synthetic carriers have brought upon numerous advances in drug delivery, they fail to match the sophistication exhibited by innate biological entities. In particular, red blood cells (RBCs), the most ubiquitous cell type in the human blood, constitute highly specialized entities with unique shape, size, mechanical flexibility, and material composition, all of which are optimized for extraordinary biological performance. Inspired by this natural example, we synthesized particles that mimic the key structural and functional features of RBCs. Similar to their natural counterparts, RBC-mimicking particles described here possess the ability to carry oxygen and flow through capillaries smaller than their own diameter. Further, they can also encapsulate drugs and imaging agents. These particles provide a paradigm for the design of drug delivery and imaging carriers, because they combine the functionality of natural RBCs with the broad applicability and versatility of synthetic drug delivery particles.

Flow and adhesion of drug carriers in blood vessels depend on their shape: A study using model synthetic microvascular networks

Development of novel carriers and optimization of their design parameters has led to significant advances in the field of targeted drug delivery. Since carrier shape has recently been recognized as an important design parameter for drug delivery, we sought to investigate how carrier shape influences their flow in the vasculature and their ability to target the diseased site. Idealized synthetic microvascular networks (SMNs) were used for this purpose since they closely mimic key physical aspects of real vasculature and at the same time offer practical advantages in terms of ease of use and direct observation of particle flow. The attachment propensities of surface functionalized spheres, elliptical/circular disks and rods with dimensions ranging from 1microm to 20microm were compared by flowing them through bifurcating SMNs. Particles of different geometries exhibited remarkably different adhesion propensities. Moreover, introduction of a bifurcation as opposed to the commonly used linear channel resulted in significantly different flow and adhesion behaviors, which may have important implications in correlating these results to in vivo behavior. This study provides valuable information for design of carriers for targeted drug delivery.

Macrophages Recognize Size and Shape of Their Targets

Recognition by macrophages is a key process in generating immune response against invading pathogens. Previous studies have focused on recognition of pathogens through surface receptors present on the macrophages surface. Here, using polymeric particles of different geometries that represent the size and shape range of a variety of bacteria, the importance of target geometry in recognition was investigated. The studies reported here reveal that attachment of particles of different geometries to macrophages exhibits a strong dependence on size and shape. For all sizes and shapes studied, particles possessing the longest dimension in the range of 2–3 µm exhibited highest attachment. This also happens to be the size range of most commonly found bacteria in nature. The surface features of macrophages, in particular the membrane ruffles, might play an important role in this geometry-based target recognition by macrophages. These findings have significant implications in understanding the pathogenicity of bacteria and in designing drug delivery carriers.

Endocytosis and Intracellular Distribution of PLGA Particles in Endothelial Cells: Effect of Particle Geometry.

Targeting, internalization, and intracellular trafficking of carriers are key processes in drug delivery to endothelial cells. We synthesized PLGA particles with spherical and elliptical disk geometries and investigated the effect of particle shape on rate of particle endocytosis and their intracellular distribution in endothelial cells. Elliptical disks (aspect ratio of 5) were endocytosed at a slower rate compared to spheres (1.8 µm diameter) of the same volume. However, both particles were eventually internalized and accumulated around the nucleus. We quantified the orientation of elliptical disks and found that disks, on average, oriented tangentially with the nuclear membrane. The non-spherical geometry of elliptical disks brings unique aspects to the kinetics and equilibrium distribution of these particles in cells.

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 =

Platelet mimetic particles for targeting thrombi in flowing blood.

Mammalian platelets are anucleated cell fragments derived from megakaryocytes[1] that play a vital role in several physiologic and pathologic processes such as hemostasis and thrombosis[2], release of growth factors and modulation of inflammatory and immune responses[3]. The contribution to hemostasis, a process during which platelets have a key role in forming the plugs that seal injured vessels and arrest bleeding, is essential for the integrity of blood circulation [4, 5]. This complex function involves the ability to adhere to reactive subendothelial structures exposed at sites of injury and aggregate with one another in flowing blood[6]. To be efficient, this process must take place on both venular and arteriolar side of the circulation, the latter being where greatest is the physical challenge of overcoming the fluid dynamic forces that oppose adhesion and aggregation[7].

Needle-shaped polymeric particles induce transient disruption of cell membranes

Nano- and microparticles of various shapes have recently been introduced for various drug-delivery applications. Shape of particles has been shown to have an impact on various processes including circulation, vascular adhesion and phagocytosis. Here, we assess the role of particle geometry and surface chemistry in their interactions with cell membranes. Using representative particles of different shape (spheres, elongated and flat particles), size (500 nm–1 µm) and surface chemistry (positively and negatively charged), we evaluated the response of endothelial cells to particles. While spherical and elliptical disc-shaped particles did not have an impact on cell spreading and motility, needle-shaped particles induced significant changes in the same. Further studies revealed that needle-shaped particles induced disruption of cell membranes as indicated by the release of lactate dehydrogenase and uptake of extracellular calcein. The effect of needle-shaped particles on cells was transient and was reversed over a time period of 1–48 h depending on particle parameters.

Polymer Nanoneedle‐Mediated Intracellular Drug Delivery

Delivery of drugs into the cellular cytoplasm of target cells represents a major hurdle in treating various diseases. This challenge can be addressed by encapsulation of drugs onto or within nanoparticles, which can then be targeted to diseased cells. Here, needle-shaped particles are shown to exhibit substantially higher cytoplasmic delivery of drugs such as siRNA compared to their spherical counterparts. Furthermore, these needles are designed to lose their sharp tips over time and can render themselves ineffective over time, thereby offering control over their duration of activity and toxicity. Such polymer nanoneedles open new avenues for delivering drug molecules directly into the cytoplasm with low toxicity.

A permeation enhancer for increasing transport of therapeutic macromolecules across the intestine

Delivery of therapeutic macromolecules is limited by the physiological limitations of the gastrointestinal tract including poor intestinal permeability, low pH and enzymatic activity. Several permeation enhancers have been proposed to enhance intestinal permeability of macromolecules; however their utility is often hindered by toxicity and limited potency. Here, we report on a novel permeation enhancer, Dimethyl palmitoyl ammonio propanesulfonate (PPS), with excellent enhancement potential and minimal toxicity. PPS was tested for dose- and time-dependent cytotoxicity, delivery of two model fluorescent molecules, sulforhodamine-B and FITC-insulin in vitro, and absorption enhancement of salmon calcitonin (sCT) in vivo. Caco-2 studies revealed that PPS is an effective enhancer of macromolecular transport while being minimally toxic. TEER measurements in Caco-2 monolayers confirmed the reversibility of the effect of PPS. Confocal microscopy studies revealed that molecules permeate via both paracellular and transcellular pathways in the presence of PPS. In vivo studies in rats showed that PPS enhanced relative bioavailability of sCT by 45-fold after intestinal administration. Histological studies showed that PPS does not induce damage to the intestine. PPS is an excellent permeation enhancer which provides new opportunities for developing efficacious oral/intestinal delivery systems for therapeutic macromolecules.

Mucociliary clearance of micro- and nanoparticles is independent of size, shape and charge--an ex vivo and in silico approach.

The fate of inhaled particles after deposition onto the pulmonary mucosa is far from being solved, in particular with respect to mucociliary clearance and mucus penetration. Due to the fact that these phenomena govern pulmonary residence time and thus bioavailability, they are highly relevant for any kind of controlled release formulation delivered via that route. This study applies ex vivo and in silico approaches to investigate the dependency of muciliary clearance of micro-, submicrometer and nanoparticles on size, shape, charge and surface chemistry of such particles. In addition, measurement of mucociliary clearance of different particles also provided information about their penetration into mucus. Surprisingly, no significant differences in mucociliary clearance could be found for any type of particle under investigation. As revealed by computational modeling, particle penetration into the mucus gel layer was negligible at least within the time frame allowed by horizontal mucus transport. These data suggest that the observed lack of difference in mucociliary clearance is caused by the lack of immediate penetration of deposited aerosol particles through the mucus blanket.