Electrokinetic Active Particles for Motion-Based Biomolecule Detection.

Detection of biomolecules is essential for patient diagnosis, disease management, and numerous other applications. Recently, nano- and microparticle-based detection has been explored for improving traditional assays by reducing required sample volumes and assay times as well as enhancing tunability. Among these approaches, active particle-based assays that couple particle motion to biomolecule concentration expand assay accessibility through simplified signal outputs. However, most of these approaches require secondary labeling, which complicates workflows and introduces additional points of error. Here, we show a proof-of-concept for a label-free, motion-based biomolecule detection system using electrokinetic active particles. We prepare induced-charge electrophoretic microsensors (ICEMs) for the capture of two model biomolecules, streptavidin and ovalbumin, and show that the specific capture of the biomolecules leads to direct signal transduction through ICEM speed suppression at concentrations as low as 0.1 nM. This work lays the foundation for a new paradigm of rapid, simple, and label-free biomolecule detection using active particles.

Two-dimensional diffusiophoretic colloidal banding: optimizing the spatial and temporal design of solute sinks and sources.

Diffusiophoresis refers to the phenomenon where colloidal particles move in response to solute concentration gradients. Existing studies on diffusiophoresis, both experimental and theoretical, primarily focus on the movement of colloidal particles in response to one-dimensional solute gradients. In this work, we numerically investigate the impact of two-dimensional solute gradients on the distribution of colloidal particles, i.e., colloidal banding, induced via diffusiophoresis. The solute gradients are generated by spatially arranged sources and sinks that emit/absorb a time-dependent solute molar rate. First we study a dipole system, i.e., one source and one sink, and discover that interdipole diffusion and molar rate decay timescales dictate colloidal banding. At timescales shorter than the interdipole diffusion timescale, we observe a rapid enhancement in particle enrichment around the source due to repulsion from the sink. However, at timescales longer than the interdipole diffusion timescale, the source and sink screen each other, leading to a slower enhancement. If the solute molar rate decays at the timescale of interdipole diffusion, an optimal separation distance is obtained such that particle enrichment is maximized. We find that the partition coefficient of solute at the interface between the source and bulk strongly impacts the optimal separation distance. Surprisingly, the diffusivity ratio of solute in the source and bulk has a much weaker impact on the optimal dipole separation distance. We also examine an octupole configuration, i.e., four sinks and four sources, arranged in a circle, and demonstrate that the geometric arrangement that maximizes enrichment depends on the radius of the circle. If the radius of the circle is small, it is preferred to have sources and sinks arranged in an alternating fashion. However, if the radius of the circle is large, a consecutive arrangement of sources and sinks is optimal. Our numerical framework introduces a novel method for spatially and temporally designing the banded structure of colloidal particles in two dimensions using diffusiophoresis and opens up new avenues in a field that has primarily focused on one-dimensional solute gradients.

Rapid capture of biomolecules from blood via stimuli-responsive elastomeric particles for acoustofluidic separation.

The detection of biomarkers in blood often requires extensive and time-consuming sample preparation to remove blood cells and concentrate the biomarker(s) of interest. We demonstrate proof-of-concept for a chip-based, acoustofluidic method that enables the rapid capture and isolation of a model protein biomarker (i.e., streptavidin) from blood for off-chip quantification. Our approach makes use of two key components - namely, soluble, thermally responsive polypeptides fused to ligands for the homogeneous capture of biomarkers from whole blood and silicone microparticles functionalized with similar, tethered, thermally responsive polypeptides. When the two components are mixed together and subjected to a mild thermal trigger, the thermally responsive moieties undergo a phase transition, causing the untethered (soluble) polypeptides to co-aggregate with the particle-bound polypeptides. The mixture is then diluted with warm buffer and injected into a microfluidic channel supporting a bulk acoustic standing wave. The biomarker-bearing particles migrate to the pressure antinodes, whereas blood cells migrate to the pressure node, leading to rapid separation with efficiencies exceeding 90% in a single pass. The biomarker-bearing particles can then be analyzed via flow cytometry, with a limit of detection of 0.75 nM for streptavidin spiked in blood plasma. Finally, by cooling the solution below the solubility temperature of the polypeptides, greater than 75% of the streptavidin is released from the microparticles, offering a unique approach for downstream analysis (e.g., sequencing or structural analysis). Overall, this methodology has promise for the detection, enrichment and analysis of some biomarkers from blood and other complex biological samples.

Assembly of hard spheres in a cylinder: a computational and experimental study.

Hard spheres are an important benchmark of our understanding of natural and synthetic systems. In this work, colloidal experiments and Monte Carlo simulations examine the equilibrium and out-of-equilibrium assembly of hard spheres of diameter σ within cylinders of diameter σ≤D≤ 2.82σ. Although phase transitions formally do not exist in such systems, marked structural crossovers can nonetheless be observed. Over this range of D, we find in simulations that structural crossovers echo the structural changes in the sequence of densest packings. We also observe that the out-of-equilibrium self-assembly depends on the compression rate. Slow compression approximates equilibrium results, while fast compression can skip intermediate structures. Crossovers for which no continuous line-slip exists are found to be dynamically unfavorable, which is the main source of this difference. Results from colloidal sedimentation experiments at low diffusion rate are found to be consistent with the results of fast compressions, as long as appropriate boundary conditions are used.

Highly parallel acoustic assembly of microparticles into well-ordered colloidal crystallites.

The precise arrangement of microscopic objects is critical to the development of functional materials and ornately patterned surfaces. Here, we present an acoustics-based method for the rapid arrangement of microscopic particles into organized and programmable architectures, which are periodically spaced within a square assembly chamber. This macroscale device employs two-dimensional bulk acoustic standing waves to propel particles along the base of the chamber toward pressure nodes or antinodes, depending on the acoustic contrast factor of the particle, and is capable of simultaneously creating thousands of size-limited, isotropic and anisotropic assemblies within minutes. We pair experiments with Brownian dynamics simulations to model the migration kinetics and assembly patterns of spherical microparticles. We use these insights to predict and subsequently validate the onset of buckling of the assemblies into three-dimensional clusters by experiments upon increasing the acoustic pressure amplitude and the particle concentration. The simulations are also used to inform our experiments for the assembly of non-spherical particles, which are then recovered via fluid evaporation and directly inspected by electron microscopy. This method for assembly of particles offers several notable advantages over other approaches (e.g., magnetics, electrokinetics and optical tweezing) including simplicity, speed and scalability and can also be used in concert with other such approaches for enhancing the types of assemblies achievable.

Elastomeric negative acoustic contrast particles for capture, acoustophoretic transport, and confinement of cells in microfluidic systems.

We present a particle-based method for the immunospecific capture and confinement of cells using acoustic radiation forces. Ultrasonic standing waves in microfluidic systems have previously been used for the continuous focusing of cells in rapid screening and sorting applications. In aqueous fluids, cells typically exhibit positive acoustic contrast and are thus forced toward the pressure nodes of a standing wave. Conversely, elastomeric particles exhibit negative acoustic contrast and travel toward the pressure antinodes. We have developed a class of elastomeric particles that are synthesized in bulk using a simple nucleation and growth process, providing precise control over their size and functional properties. We demonstrate that the biofunctionalization of these particles can allow the capture and transport of cells to the pressure antinodes solely via acoustic radiation forces, which may enable new acoustics-based cell handling techniques such as the washing, labeling, and sorting of cells with minimal preparatory steps.

Nucleation and growth synthesis of siloxane gels to form functional, monodisperse, and acoustically programmable particles.

Nucleation and growth methods offer scalable means of synthesizing colloidal particles with precisely specified size for applications in chemical research, industry, and medicine. These methods have been used to prepare a class of silicone gel particles that display a range of programmable properties and narrow size distributions. The acoustic contrast factor of these particles in water is estimated and can be tuned such that the particles undergo acoustophoresis to either the pressure nodes or antinodes of acoustic standing waves. These particles can be synthesized to display surface functional groups that can be covalently modified for a range of bioanalytical and acoustophoretic sorting applications.

Field-directed of patchy anisotropic microparticles with defined shape.

Electromagnetic fields can generate orientation-dependent, long range interactions between colloidal components that direct their into highly ordered structures, such as small ordered clusters, chains, and large crystalline lattices. While much effort has been devoted to exploring the assembly of spherical colloids, few reports have investigated the directed assembly of non-spherical particles with Janus or patchy morphologies. Here, we use photolithographic techniques to fabricate a wide range of anisotropically shaped patchy particles and follow their in liquid suspensions under the influence of electric and magnetic fields. We analyze the assembly of several types of patchy particles across a range of field parameters and fluid compositions, and report a number of distinct, well-ordered, architectures including cylindrical, prismatic, and staggered chains. The structures assembled from anisotropic patchy components provide a glimpse into the range of architectures that can be created by combining field directed with rationally designed particles. By using numerical simulations to model the electric and magnetic field interactions between these particles, we interpret the results of the assembly process and explain how they can be controlled by the position of the metal facet, the frequency (for AC fields), or magnetic susceptibility of the medium. The resulting structures, and similar ones produced through the field-directed assembly of patchy anisotropic particles, can possess unique electrical and optical properties and may have potential applications in a number of future technology applications such as microactuators, metamaterials and multiferroic materials.

Dissolved gases from pressure changes in the lungs elicit an immune response in human peripheral blood.

Conventional dogma suggests that decompression sickness (DCS) is caused by nitrogen bubble nucleation in the blood vessels and/or tissues; however, the abundance of bubbles does not correlate with DCS severity. Since immune cells respond to chemical and environmental cues, we hypothesized that the elevated partial pressures of dissolved gases drive aberrant immune cell phenotypes in the alveolar vasculature. To test this hypothesis, we measured immune responses within human lung-on-a-chip devices established with primary alveolar cells and microvascular cells. Devices were pressurized to 1.0 or 3.5 atm and surrounded by normal alveolar air or oxygen-reduced air. Phenotyping of neutrophils, monocytes, and dendritic cells as well as multiplexed ELISA revealed that immune responses occur within 1 hour and that normal alveolar air (i.e., hyperbaric oxygen and nitrogen) confer greater immune activation. This work strongly suggests innate immune cell reactions initiated at elevated partial pressures contribute to the etiology of DCS.

Microrobots for Biomedicine: Unsolved Challenges and Opportunities for Translation.

Microrobots are being explored for biomedical applications, such as drug delivery, biological cargo transport, and minimally invasive surgery. However, current efforts largely focus on proof-of-concept studies with nontranslatable materials through a "design-and-apply" approach, limiting the potential for clinical adaptation. While these proof-of-concept studies have been key to advancing microrobot technologies, we believe that the distinguishing capabilities of microrobots will be most readily brought to patient bedsides through a "design-by-problem" approach, which involves focusing on unsolved problems to inform the design of microrobots with practical capabilities. As outlined below, we propose that the clinical translation of microrobots will be accelerated by a judicious choice of target applications, improved delivery considerations, and the rational selection of translation-ready biomaterials, ultimately reducing patient burden and enhancing the efficacy of therapeutic drugs for difficult-to-treat diseases.

Magnetically locked Janus particle clusters with orientation-dependent motion in AC electric fields.

Active particles, or micromotors, locally dissipate energy to drive locomotion at small length scales. The type of trajectory is generally fixed and dictated by the geometry and composition of the particle, which can be challenging to tune using conventional fabrication procedures. Here, we report a simple, bottom-up method to magnetically assemble gold-coated polystyrene Janus particles into "locked" clusters that display diverse trajectories when stimulated by AC electric fields. The orientation of particles within each cluster gives rise to distinct modes of locomotion, including translational, rotational, trochoidal, helical, and orbital. We model this system using a simplified rigid beads model and demonstrate qualitative agreement between the predicted and experimentally observed cluster trajectories. Overall, this system provides a facile means to scalably create micromotors with a range of well-defined motions from discrete building blocks.

Systemic Tumor Suppression via Macrophage-Driven Automated Homing of Metal-Phenolic-Gated Nanosponges for Metastatic Melanoma.

Cell-based therapies comprising the administration of living cells to patients for direct therapeutic activities have experienced remarkable success in the clinic, of which macrophages hold great potential for targeted drug delivery due to their inherent chemotactic mobility and homing ability to tumors with high efficiency. However, such targeted delivery of drugs through cellular systems remains a significant challenge due to the complexity of balancing high drug-loading with high accumulations in solid tumors. Herein, a tumor-targeting cellular drug delivery system (MAGN) by surface engineering of tumor-homing macrophages (Mφs) with biologically responsive nanosponges is reported. The pores of the nanosponges are blocked with iron-tannic acid complexes that serve as gatekeepers by holding encapsulated drugs until reaching the acidic tumor microenvironment. Molecular dynamics simulations and interfacial force studies are performed to provide mechanistic insights into the "ON-OFF" gating effect of the polyphenol-based supramolecular gatekeepers on the nanosponge channels. The cellular chemotaxis of the Mφ carriers enabled efficient tumor-targeted delivery of drugs and systemic suppression of tumor burden and lung metastases in vivo. The findings suggest that the MAGN platform offers a versatile strategy to efficiently load therapeutic drugs to treat advanced metastatic cancers with a high loading capacity of various therapeutic drugs.

Magnetic systems for cancer immunotherapy.

Immunotherapy is a rapidly developing area of cancer treatment due to its higher specificity and potential for greater efficacy than traditional therapies. Immune cell modulation through the administration of drugs, proteins, and cells can enhance antitumoral responses through pathways that may be otherwise inhibited in the presence of immunosuppressive tumors. Magnetic systems offer several advantages for improving the performance of immunotherapies, including increased spatiotemporal control over transport, release, and dosing of immunomodulatory drugs within the body, resulting in reduced off-target effects and improved efficacy. Compared to alternative methods for stimulating drug release such as light and pH, magnetic systems enable several distinct methods for programming immune responses. First, we discuss how magnetic hyperthermia can stimulate immune cells and trigger thermoresponsive drug release. Second, we summarize how magnetically targeted delivery of drug carriers can increase the accumulation of drugs in target sites. Third, we review how biomaterials can undergo magnetically driven structural changes to enable remote release of encapsulated drugs. Fourth, we describe the use of magnetic particles for targeted interactions with cellular receptors for promoting antitumor activity. Finally, we discuss translational considerations of these systems, such as toxicity, clinical compatibility, and future opportunities for improving cancer treatment.

Drug delivery to macrophages: A review of targeting drugs and drug carriers to macrophages for inflammatory diseases.

Macrophages play a key role in defending against foreign pathogens, healing wounds, and regulating tissue homeostasis. Driving this versatility is their phenotypic plasticity, which enables macrophages to respond to subtle cues in tightly coordinated ways. However, when this coordination is disrupted, macrophages can aid the progression of numerous diseases, including cancer, cardiovascular disease, and autoimmune disease. The central link between these disorders is aberrant macrophage polarization, which misguides their functional programs, secretory products, and regulation of the surrounding tissue microenvironment. As a result of their important and deterministic roles in both health and disease, macrophages have gained considerable attention as targets for drug delivery. Here, we discuss the role of macrophages in the initiation and progression of various inflammatory diseases, summarize the leading drugs used to regulate macrophages, and review drug delivery systems designed to target macrophages. We emphasize strategies that are approved for clinical use or are poised for clinical investigation. Finally, we provide a prospectus of the future of macrophage-targeted drug delivery systems.

Materials for Immunotherapy.

Breakthroughs in materials engineering have accelerated the progress of immunotherapy in preclinical studies. The interplay of chemistry and materials has resulted in improved loading, targeting, and release of immunomodulatory agents. An overview of the materials that are used to enable or improve the success of immunotherapies in preclinical studies is presented, from immunosuppressive to proinflammatory strategies, with particular emphasis on technologies poised for clinical translation. The materials are organized based on their characteristic length scale, whereby the enabling feature of each technology is organized by the structure of that material. For example, the mechanisms by which i) nanoscale materials can improve targeting and infiltration of immunomodulatory payloads into tissues and cells, ii) microscale materials can facilitate cell-mediated transport and serve as artificial antigen-presenting cells, and iii) macroscale materials can form the basis of artificial microenvironments to promote cell infiltration and reprogramming are discussed. As a step toward establishing a set of design rules for future immunotherapies, materials that intrinsically activate or suppress the immune system are reviewed. Finally, a brief outlook on the trajectory of these systems and how they may be improved to address unsolved challenges in cancer, infectious diseases, and autoimmunity is presented.

Design principles of drug combinations for chemotherapy.

Combination chemotherapy is the leading clinical option for cancer treatment. The current approach to designing drug combinations includes in vitro optimization to maximize drug cytotoxicity and/or synergistic drug interactions. However, in vivo translatability of drug combinations is complicated by the disparities in drug pharmacokinetics and activity. In vitro cellular assays also fail to represent the immune response that can be amplified by chemotherapy when dosed appropriately. Using three common chemotherapeutic drugs, gemcitabine (GEM), irinotecan (IRIN), and a prodrug form of 5-flurouracil (5FURW), paired with another common drug and immunogenic cell death inducing agent, doxorubicin (DOX), we sought to determine the in vitro parameters that predict the in vivo outcomes of drug combinations in the highly aggressive orthotopic 4T1 murine breast cancer model. With liposomal encapsulation of each drug pair, we enabled uniform drug pharmacokinetics across the drug combinations, thus allowing us to study the inherent benefits of the drug pairs and compare them to DOX liposomes representative of DOXIL®. Surprisingly, the Hill coefficient (HC) of the in vitro dose-response Hill equation provided a better prediction of in vivo efficacy than drug IC50 or combination index. GEM/DOX liposomes exhibited a high HC in vitro and an increase in M1/M2 macrophage ratio in vivo. Hence, GEM/DOX liposomes were further investigated in a long-term survival study and compared against doxorubicin liposomes and gemcitabine liposomes. The GEM/DOX liposome-treated group had the longest median survival time, double that of the DOX liposome-treated group and 3.4-fold greater than that of the untreated controls. Our studies outline the development of a more efficacious formulation than clinically representative liposomal doxorubicin for breast cancer treatment and presents a novel strategy for designing cancer drug combinations.

Migration of Microparticle-Containing Amoeba through Constricted Environments.

In many situations, cells migrate through tiny orifices. Examples include the extravasation of immune cells from the bloodstream for fighting infections, the infiltration of cancer cells during metastasis, and the migration of human pathogens. An extremely motile and medically relevant type of human pathogen is Acanthamoeba castellanii. In the study presented here, we investigated how a combination of microparticles and microstructured interfaces controls the migration of A. castellanii trophozoites. The microinterfaces comprised well-defined micropillar arrays, and the trophozoites easily migrated through the given constrictions by adapting the shape and size of their intracellular vacuoles and by adapting intracellular motion. After feeding the trophozoite cells in microinterfaces with synthetic, stiff microparticles of various sizes and shapes, their behavior changed drastically: if the particles were smaller than the micropillar gap, migration was still possible. If the cells incorporated particles larger than the pillar gap, they could become immobilized but could also display remarkable problem-solving capabilities. For example, they turned rod-shaped microparticles such that their short axis fit through the pillar gap or they transported the particles above the structure. As migration is a crucial contribution to A. castellanii pathogenicity and is also relevant to other biological processes in microenvironments, such as cancer metastasis, our results provide an interesting strategy for controlling the migration of cells containing intracellular particles by microstructured interfaces that serve as migration-limiting environments.

Engineering of Living Cells with Polyphenol-Functionalized Biologically Active Nanocomplexes.

Approaches to safely and effectively augment cellular functions without compromising the inherent biological properties of the cells, especially through the integration of biologically labile domains, remain of great interest. Here, a versatile strategy to assemble biologically active nanocomplexes, including proteins, DNA, mRNA, and even viral carriers, on cellular surfaces to generate a cell-based hybrid system referred to as "Cellnex" is established. This strategy can be used to engineer a wide range of cell types used in adoptive cell transfers, including erythrocytes, macrophages, NK cells, T cells, etc. Erythrocytenex can enhance the delivery of cargo proteins to the lungs in vivo by 11-fold as compared to the free cargo counterpart. Biomimetic microfluidic experiments and modeling provided detailed insights into the targeting mechanism. In addition, Macrophagenex is capable of enhancing the therapeutic efficiency of anti-PD-L1 checkpoint inhibitors in vivo. This simple and adaptable approach may offer a platform for the rapid generation of complex cellular systems.

Cellular backpacks for macrophage immunotherapy.

Adoptive cell transfers have emerged as a disruptive approach to treat disease in a manner that is more specific than using small-molecule drugs; however, unlike traditional drugs, cells are living entities that can alter their function in response to environmental cues. In the present study, we report an engineered particle referred to as a "backpack" that can robustly adhere to macrophage surfaces and regulate cellular phenotypes in vivo. Backpacks evade phagocytosis for several days and release cytokines to continuously guide the polarization of macrophages toward antitumor phenotypes. We demonstrate that these antitumor phenotypes are durable, even in the strongly immunosuppressive environment of a murine breast cancer model. Conserved phenotypes led to reduced metastatic burdens and slowed tumor growths compared with those of mice treated with an equal dose of macrophages with free cytokine. Overall, these studies highlight a new pathway to control and maintain phenotypes of adoptive cellular immunotherapies.

Role of synergy and immunostimulation in design of chemotherapy combinations: An analysis of doxorubicin and camptothecin.

Combination chemotherapy is often employed to improve therapeutic efficacies of drugs. However, traditional combination regimens often utilize drugs at or near-their maximum tolerated doses (MTDs), elevating the risk of dose-related toxicity and impeding their clinical success. Further, high doses of adjuvant or neoadjuvant chemotherapies can cause myeloablation, which compromises the immune response and hinders the efficacy of chemotherapy as well as accompanying treatments such as immunotherapy. Clinical outcomes can be improved if chemotherapy combinations are designed to reduce the overall doses without compromising their therapeutic efficacy. To this end, we investigated a combination of camptothecin (CPT) with doxorubicin (DOX) as a low-dose treatment option for breast cancer. DOX-CPT combinations were synergistic in several breast cancer cell lines in vitro and one particular ratio displayed extremely high synergy on human triple negative breast cancer cells (MDA-MB-231). This combination led to excellent long-term survival of mice bearing MDA-MB-231 tumors at doses roughly five-fold lower than the reported MTD values of its constituent drugs. Impact of low dose DOX-CPT treatment on local tumor immune environment was assessed in immunocompetent mice bearing breast cancer (4T1) tumors. The combination was not only superior in inhibiting the disease progression compared to individual drugs, but it also generated a more favorable antitumor immunogenic response. Engineering DOX and CPT ratios to manifest synergy enables treatment at doses much lower than their MTDs, which could ultimately facilitate their translation into the clinic as a promising combination for breast cancer treatment.