Ashutosh Chilkoti

Physically cross-linked hydrogels formed through noncovalent interactions offer reversible gelation, injectability, and shear-thinning behavior, making them ideal for biomedical applications. Temperature-responsive systems are particularly attractive, as they enable injection as a solution, and at physiological temperature for minimally invasive delivery. Poly(oligo(ethylene glycol) methacrylate) (POEGMA) is a promising nonimmunogenic polymer that combines PEG-like hydration and protein resistance with tunable thermal responsiveness that does not bind pre-existing anti-PEG antibodies that are ubiquitous and elicits a minimal antibody response against itself. Here, we show that diblock POEGMA copolymerscomposed solely of short ethylene glycol side chainsspontaneously self-assemble into nanoparticles and form physically cross-linked hydrogels above a controllable transition temperature, without chemical modification. Using fluorescence recovery after photobleaching and video particle tracking nanorheology, we identify a thermally induced transition from dynamically arrested viscoelastic hydrogels to dynamically arrested liquid coacervates. These results establish a new class of single-component, nonimmunogenic POEGMA hydrogels with tunable phase behavior and mechanical properties, offering a versatile platform for localized drug delivery, sustained release, and regenerative medicine.

Abstract Excessive alcohol consumption remains a major public health challenge with limited therapeutic options. Both glucagon-like peptide-1 (GLP-1) and fibroblast growth factor-21 (FGF21) independently regulate alcohol intake through complementary metabolic and reward pathways, but their combined potential has not been explored. Here, we report that a long-acting dual agonist, GLP1-ELP-FGF21 modulates behavioural, neurophysiological, and cognitive components of alcohol seeking in mice. A single GLP1-ELP-FGF21 dose reversibly reduces voluntary alcohol intake for at least 72 hours in male mice, has sustained effects in female mice, and markedly blunts nucleus accumbens dopamine transients aligned to the initiation and termination of lick bouts during alcohol consumption. To assess its effects on decision-making, we used a novel two-choice (alcohol versus food) decision task modelled with evidence-accumulation frameworks. Alcohol choice behaviour conformed to evidence accumulation decision models: Linear Ballistic Accumulator (LBM) and Racing diffusion models (RDM). Critically, GLP1-ELP-FGF21 selectively reduces choices for alcohol and slows the latent accumulation rate for alcohol options, without affecting food-directed choice or non-decision processes. Sensory-specific satiety devaluation confirms that reductions in reward value are explained by reductions in accumulation rates. Together, these results highlight GLP1-ELP-FGF21 as a therapeutic strategy for alcohol use disorder via modulation of central reward pathways and decision-making when confronted with alcohol reward

Post translational modifications (PTMs) of proteins are used by natural systems to expand beyond the 20 canonical amino acids. The variation introduced at the sequence level by PTMs after expression leads to changes in both the structure and function of proteins. PTMs expand the chemical repertoire from which new biomaterials can be constructed. Inspired by the post-translational addition of cholesterol to proteins, we have synthesized five new hybrid lipid-protein biomaterials called Sterol modified polypeptides (STaMPs). These STaMPs consist of an elastin like polypeptide (ELP) conjugated to a sterol, namely coprostanol, epicoprostanol, androstanol, galeterone, or dehydroepiandrosterone. We show that the STaMPs form self-assembled nanostructures such as spherical and cylindrical micelles, and vesicles. Furthermore, the sterols modify the typical LCST behavior of ELPs in a predictable fashion depending on the hydrophobicity of the sterol appended. STaMPs could expand the possibilities for drug delivery by enabling the physical encapsulation of hydrophobic drugs, increasing the solubility and half-life of sterol-based chemotherapeutics, and forming thermosensitive drug depots.

A combined theoretical and experimental investigation presents a consistent parabolic potential model for the prediction and optimization of mammalian cell adhesion and detachment from genetically engineered thermoresponsive elastin-like protein (ELP) modified surfaces. Linear ELP chains concatenated with both thiol-gold surface-binding and RGD cell-binding domains serve as thermally responsive cell harvesting surfaces. This architecture of a 1:1 ratio of cell binding domain to linear polymer chain provides precise control of the chemical representation of the cell binding and thermoresponsive properties. The parabolic potential model of surface-grafted phase-separating polymers describing the ELP brush films, combined with surface-bound cell culture measurements, is used to analyze the effects of protein chain length N and surface area per chain σ. The cell binding fractions allow the calculation of system free energies, which are consistent with the parabolic potential model through identification of the underlying polymer lengths. This offers the ability for the model to identify optimal conditions that promote cell attachment and detachment. This model represents a quantitative framework for optimizing surface grafted protein layer thickness and cell displacement energy, which is a crucial technical step forward for programming of thermoresponsive biopolymer substrates for nonenzymatic cell harvesting.

Abstract Despite renewed interest in IL‐12 as a cancer immunotherapy due to its ability to stimulate the adaptive immune system, its short half‐life and narrow therapeutic window continues to present challenges for effective delivery. Previous studies with IL‐12 have investigated the effects of route of delivery or sustained delivery of the cytokine on its efficacy but are unable to simultaneously investigate the effects of both within the same system. This work seeks to address this gap by utilizing an elastin‐like polypeptide (ELP) carrier, which can undergo a thermally triggered phase transition to a gel‐like depot, to probe the effects of both sustained release and spatial delivery of IL‐12. By conjugating IL‐12 with an ELP, this work creates an IL‐12‐ELP fusion that can be injected intratumorally or subcutaneously to form a sustained‐release depot. In a B16F10 murine model, intratumoral injection of a depot‐forming IL‐12‐ELP fusion significantly improved survival compared to free IL‐12. IL‐12‐ELP is retained within the tumor approximately fourfold longer than free IL‐12, resulting in higher CD8+ T cell recruitment at the tumor and local concentrations of inflammatory cytokines at Day 2. Taken together, this work provides insights into rational cytokine delivery, the importance of tumor localization, and the benefits of sustained release.

Abstract Biomaterials can improve cancer immunotherapies by controlling their release and thereby optimizing their time‐dependent engagement of the immune system. In this study, an approach is described to control the release of a potent immunostimulant—CpG oligodeoxynucleotide—from a genetically‐encoded elastin‐like polypeptide (ELP) depot. A CpG‐binding ELP containing an oligolysine domain (ELP‐Lys 12 ) is synthesized that electrostatically complexes CpG and formulate it with an excipient ELP. The ELP‐CpG complex retains the thermally responsive phase behavior of the parent ELP, transitioning into a viscous depot at body temperature. Stepwise addition of excipient ELP predictably changes ELP‐CpG transition temperature, depot dissolution kinetics, and retention of CpG within the depot. Mixtures of ELP‐Lys 12 , excipient ELP, and CpG undergo microphase separation, forming a porous, sponge‐like depot that contains tunable amounts of soluble CpG in the pores. In vivo, the modified formulations exhibit varying degrees of CpG retention over multiple weeks following a single intratumoral injection. Finally, by modifying the release kinetics of CpG, optimized ELP‐CpG achieves greater reduction of metastatic disease in a murine metastatic breast cancer model than soluble CpG. These results demonstrate that ELPs can be used to precisely tune the release kinetics of immunotherapies for better outcomes in the treatment of metastatic cancer.

Abstract The antifouling performance of a previously developed triblock protein, B‐M‐E is optimized, that self‐assembles on gold surfaces to form a nonfouling layer by modifying the sequence of its E block. In this protein, B is a gold‐binding domain, M is a trimerization domain, and E is a synthetic intrinsically disordered protein (IDP) that confers nonfouling behavior. To identify optimal sequences for the nonfouling E block, a few candidate IDPs are screened that provide a proxy for nonfouling behavior, such as extending the half‐life of their fusion partners in systemic circulation and sequences that promote soluble expression of their fusion partners in E coli . One IDP is identified with the sequence [(GAGAIP) 3 ‐(GAGEIP)] 4 as the E block in B‐M‐E brush on gold, forming a nonfouling coating with performance comparable to a self‐assembled monolayer (SAM) of a tetraethylene glycol‐terminated alkanethiol on gold. These B‐M‐E brushes also render gold surfaces resistant to E. coli attachment for at least seven days. The B‐M‐E protein can be synthesized at scale in bacterial expression systems using the upstream fermentation and downstream purification capabilities of the biotechnology industry. It may provide a useful and robust alternative to existing nonfouling coatings based on small molecules or synthetic polymers.

Peripheral nerve injuries are a common and potentially devastating condition affecting over 20 million people in the United States alone, resulting in significant functional disability and chronic pain for patients. Unfortunately, even when repaired under optimal conditions with cutting-edge techniques, current approaches to peripheral nerve repair result in incomplete functional recovery and chronic pain in over half of patients, highlighting the pressing need for the development of new strategies for peripheral nerve repair. Biomaterials, due to their tunable properties, can be rationally designed to address many aspects of peripheral nerve repair, making them a promising solution for improving functional outcomes following nerve repair. This review discusses the current lack of efficacious treatments for peripheral nerve repair and how biomaterials can fill this crucial void, as well as what properties those materials should have from a material, biological, and practical concerns perspective. The review is divided into three main sections, the first of which outlines the complex process of peripheral nerve repair, providing an understandable and clinically germane overview of peripheral nerve repair. Part two of this review discusses biological design principles to engineer biomaterials that favor nerve regeneration. Part three discusses practical considerations for adapting biomaterials for clinical use.

Dry, transduction biomaterial scaffolds (Drydux) represent a novel platform for enhancing viral transduction, achieving drastic improvements in transduction efficiency (from ∼10% to >80%) while simplifying production of potent genetically engineered cells. This technology addresses a critical bottleneck in cell therapy manufacturing, where conventional methods require complex protocols and often yield suboptimal results. However, the underlying material science driving Drydux-enhanced transduction remains unclear. Here, we comprehensively assess biomaterial properties that influence viral transduction enhancement through systematic testing of polysaccharides, proteins, elastin-like polypeptides (ELPs), and synthetic polymers. Our findings reveal that surface porosity and liquid absorption are primary drivers of transduction enhancement, while polymer charge and flexibility play secondary roles. Negatively charged and flexible materials-particularly gelatin, hyaluronan, and alginate-demonstrated superior performance. Notably, despite promising material characteristics, synthetic polymers failed to enhance transduction, highlighting the unique advantages of specific biomaterial compositions. By elucidating these structure-function relationships, this work establishes design principles for optimizing biomaterial-enhanced transduction and expands the Drydux platform's potential for transforming cell therapy manufacturing, regenerative medicine, and beyond.

The efficacy of tumor-targeted therapeutics, engineered to engage specific cellular receptors to promote accumulation and penetration, is strongly influenced by the carrier's affinity for its target and the valency of binding molecules incorporated into the carrier. Previous research has primarily focused on improving targeting by augmenting the number of binding proteins on the carrier, inadvertently raising avidity without isolating the individual effects of binding strength and valency. Herein, we precisely evaluate the impact of multivalency on tumor targeting with a recombinant approach to independently control valency, avidity, and size. Our findings reveal that constructs with equivalent binding strength exhibit comparable receptor engagement and tumor extravasation, regardless of valency. Moreover, excessive avidity adversely affected tumor accumulation and penetration, with the highest-avidity construct showing diminished exposure. These results indicate that overall binding strength, not valency, is the primary determinant of tumor targeting, providing valuable insights for designing effective macromolecular drug carriers.

Nab-paclitaxel (Abraxane), an albumin-bound solvent-free paclitaxel (PTX) formulation that takes advantage of the endogenous albumin transport pathway, is the current gold standard for treatment of solid tumors with PTX. However, nab-paclitaxel has several limitations, including complex manufacturing, immunogenicity, slow drug-release, and a narrow therapeutic window. Nevertheless, no other PTX formulation has gained the Food and Drug Administration approval since Abraxane's 18-year reign. Addressing these concerns, herein, a PTX-loaded nanoparticle of a recombinant polypeptide that-like nab-paclitaxel-capitalizes on the long in vivo half-life of albumin is reported. This genetically engineered nanoparticle packages PTX in the core of the nanoparticle and displays an albumin-binding domain on the exterior of the nanoparticle. Upon in vivo administration, the drug-loaded nanoparticle binds albumin with nanomolar affinity, and acquires an albumin-corona, which eliminates the need to use exogenous albumin. The nanoparticles can be stored at subzero temperature as lyophilized powder without any cryoprotectants for upto a year and can be reconstituted on-demand in aqueous buffer at high concentration, thus greatly simplifying formulation processes. These albumin-binding nanoparticles improve the therapeutic window by at least twofold compared to nonalbumin-binding counterpart and outperform nab-paclitaxel in multiple murine tumor models, results that have been independently replicated by a contract research organization.

Abstract Prostate cancer is the second leading cause of cancer-related death in U.S. men. Genomic and clinical associations have identified loss of RB function as a dominant mechanism driving prostate cancer lethality. We recently demonstrated that ferroptosis induction in vivo by ferroptosis inducers blocks RB1-deficient prostate tumor growth and metastasis and leads to improved survival of the mice. As all ferroptosis inducers are newly discovered, there is a great need to improve their therapeutic potential through different avenues, including innovative drug delivery systems. Poly Lactic-co-glycolic acid (PLGA) is an FDA-approved biodegradable polymer. Due to their strong biocompatibility and consistent release, PLGA-based nanotherapeutics are an attractive carrier for small molecule drugs. This study aims to further improve the efficacy of ferroptosis induction against lethal RB1-deficient prostate cancer through PLGA-based ferroptosis-induced nanoparticles (NPs). The PLGA NPs encapsulating JKE-1674 were synthesized using the solvent evaporation method. The NPs were characterized using dynamic light scattering (DLS) and scanning electron microscopy (SEM) to assess the physicochemical properties of the NPs. The cell-killing efficacy of free-form or PLGA NP encapsulated JKE-1674 was then evaluated in vitro. Our analyses showed that the particle size and polydispersity index (PDI) of JKE-1674 PLGA NPs were 160.8 nm and 0.049, respectively. SEM images of JKE-1674 PLGA NPs further validated the DLS data. These findings suggest that encapsulation of JKE-1674 within the PLGA NPs results in a stable and biocompatible formation. Critically, JKE-1674 encapsulated by PLGA NPs is as potent as JKE-1674 in its free form to induce ferroptosis and associated lipid peroxidation in various human prostate cancer cell lines. This study presents the first effort to develop and validate PLGA-based ferroptosis-induced nanotherapeutics. Based on our in vitro data, we will test the in vivo efficacy of JKE-1674 PLGA NPs against lethal RB1 deficient prostate cancer. Citation Format: Eden M. Jacob, Sarah Y. Kim, Parul Sirohi, Mu-En Wang, Meredith L. Davis, Alyssa R. Bawcom, Ashutosh Chilkoti, Mark R. Wiesner, Jiaoti Huang, Ming Chen. Investigating the induction of ferroptosis by nano drug delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3202.

I will describe a point-of-care diagnostic that we have developed, in which all reagents are printed and stored on a “non-fouling”—protein and cell resistant— polymer brush. The D4 assay involves four sequential events: (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse (across surface); and (4) Detect (binding event). The D4 assay consists of microarrays of printed on the polymer polymer brush yields quantitative results, with picomolar sensitivity within an hour. All reagents are inkjet-printed and stored on D4 chips, which do not require refrigeration. Upon application of a drop of blood to the D4 chip, analyte capture and detection on the D4 chip occurs automatically, generating an image of fluorescent spots that is imaged by a hand-held detector and quantified by an on-board App. Examples of quantitative dose-response from whole blood will be presented and from patient samples. A plasmonically enhanced version of this assay will also be described.

Electrical biosensors, including transistor-based devices (i.e., BioFETs), have the potential to offer versatile biomarker detection in a simple, low-cost, scalable, and point-of-care manner. Semiconducting carbon nanotubes (CNTs) are among the most explored nanomaterial candidates for BioFETs due to their high electrical sensitivity and compatibility with diverse fabrication approaches. However, when operating in solutions at biologically relevant ionic strengths, CNT-based BioFETs suffer from debilitating levels of signal drift and charge screening, which are often unaccounted for or sidestepped (but not addressed) by testing in diluted solutions. In this work, we present an ultrasensitive CNT-based BioFET called the D4-TFT, an immunoassay with an electrical readout, which overcomes charge screening and drift-related limitations of BioFETs. In high ionic strength solution (1X PBS), the D4-TFT repeatedly and stably detects subfemtomolar biomarker concentrations in a point-of-care form factor by increasing the sensing distance in solution (Debye length) and mitigating signal drift effects. Debye length screening and biofouling effects are overcome using a poly(ethylene glycol)-like polymer brush interface (POEGMA) above the device into which antibodies are printed. Simultaneous testing of a control device having no antibodies printed over the CNT channel confirms successful detection of the target biomarker via an on-current shift caused by antibody sandwich formation. Drift in the target signal is mitigated by a combination of: (1) maximizing sensitivity by appropriate passivation alongside the polymer brush coating; (2) using a stable electrical testing configuration; and (3) enforcing a rigorous testing methodology that relies on infrequent DC sweeps rather than static or AC measurements. These improvements are realized in a relatively simple device using printed CNTs and antibodies for a low-cost, versatile platform for the ongoing pursuit of point-of-care BioFETs.

In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs. Nature also diversifies the structure of proteins and thereby their functions by hybridization of the proteins with other moieties such as glycans and lipids; for instance, post-translationally glycosylated and lipidated proteins are important cell membrane components. Additionally, diversity in protein structure and function is achieved in nature through cross-linking proteins within themselves or with other domains to create various topologies. For example, an essential characteristic of the extracellular matrix (ECM) is the cross-linking of its network components, including proteins such as collagen and elastin, as well as polysaccharides such as hyaluronic acid (HA). Inspired by nature, synthetic IDP (SynIDP)-based biomaterials can be designed by employing similar strategies with the goal of introducing structural diversity and hence unique physiochemical properties. This Account describes such materials produced over the past decade and following one or more of the following approaches: (1) incorporating highly ordered domains into SynIDPs, (2) conjugating SynIDPs to other moieties through either genetically encoded post-translational modification or chemical conjugation, and (3) engineering the topology of SynIDPs via chemical modification. These approaches introduce modifications to the primary structure of SynIDPs, which are then translated to unique three-dimensional secondary and tertiary structures. Beginning with completely disordered SynIDPs as the point of origin, structure may be introduced into SynIDPs by each of these three unique approaches individually along orthogonal axes or by combinations of the three, enabling bioinspired designs to theoretically span the entire range of three-dimensional structural possibilities. Furthermore, the resultant structures span a wide range of length scales, from nano- to meso- to micro- and even macrostructures. In this Account, emphasis is placed on the physiochemical properties and structural features of the described materials. Conjugates of SynIDPs to synthetic polymers and materials achieved by simple mixing of components are outside the scope of this Account. Related biomedical applications are described briefly. Finally, we note future directions for the design of functional SynIDP-based biomaterials.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

We report a targeted prodrug delivery platform that can deliver a cytostatic nucleobase analog with high drug loading. We chose fluorouracil (5FU), a drug used to treat various cancers, whose active metabolite 5-fluorodeoxyuridine monophosphate (5-FdUMP) is the antineoplastic agent. We use terminal deoxynucleotidyl transferase (TdT) to polymerize 5-fluorodeoxyuridine triphosphate (5-FdUTP) onto the 3'-end of an aptamer. We find that (i) addition of hydrophobic, unnatural nucleotides at the 3'-end of the 5-FdU polynucleotide by TdT leads to their spontaneous self-assembly into nuclease resistant micelles, (ii) aptamers presented on the micelle corona retain specificity for their cognate receptor on tumor cells, and (iii) the micelles deliver 5FU to tumor cells and exhibit greater cytotoxicity than the free drug. The modular design of our platform, consisting of a targeting moiety, a polynucleotide drug, and a self-assembly domain, can be adapted to encompass a range of polymerizable therapeutic nucleotides and targeting units.

Control of the electrochemical environment in living cells is typically attributed to ion channels. Here we show that the formation of biomolecular condensates can modulate the electrochemical environment in cells, which affects processes globally within the cell and interactions of the cell with its environment. Condensate formation results in the depletion or enrichment of certain ions, generating intracellular ion gradients. These gradients directly affect the electrochemical properties of a cell, including the cytoplasmic pH and hyperpolarization of the membrane potential. The modulation of the electrochemical equilibria between the intra- and extra-cellular environments by biomolecular condensates governs charge-dependent uptake of small molecules by cells, and thereby directly influences bacterial survival under antibiotic stress. The shift of the intracellular electrochemical equilibria by condensate formation also drives a global change of the gene expression profile. The control of the cytoplasmic environment by condensates is correlated with their volume fraction, which can be highly variable between cells due to the stochastic nature of gene expression at the single cell level. Thus, condensate formation can amplify cell-cell variability of the environmental effects induced by the shift of cellular electrochemical equilibria. Our work reveals new biochemical functions of condensates, which extend beyond the biomolecules driving and participating in condensate formation, and uncovers a new role of biomolecular condensates in cellular regulation.

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We can correlate the lamellar structures to \b{eta}-sheet formation and demonstrate similarities between the observed nanoscopic structural arrangement and spider silk. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins.

The ongoing Coronavirus disease 2019 (COVID-19) pandemic illustrates the need for sensitive and reliable tools to diagnose and monitor diseases. Traditional diagnostic approaches rely on centralized laboratory tests that result in long wait times to results and reduce the number of tests that can be given. Point-of-care tests (POCTs) are a group of technologies that miniaturize clinical assays into portable form factors that can be run both in clinical areas --in place of traditional tests-- and outside of traditional clinical settings --to enable new testing paradigms. Hallmark examples of POCTs are the pregnancy test lateral flow assay and the blood glucose meter. Other uses for POCTs include diagnostic assays for diseases like COVID-19, HIV, and malaria but despite some successes, there are still unsolved challenges for fully translating these lower cost and more versatile solutions. To overcome these challenges, researchers have exploited innovations in colloid and interface science to develop various designs of POCTs for clinical applications. Herein, we provide a review of recent advancements in lateral flow assays, other paper based POCTs, protein microarray assays, microbead flow assays, and nucleic acid amplification assays. Features that are desirable to integrate into future POCTs, including simplified sample collection, end-to-end connectivity, and machine learning, are also discussed in this review.

Point-of-care tests (POCTs) are increasingly being used in field settings, particularly outdoors. The performance of current POCTs─most commonly the lateral flow immunoassay─can be adversely affected by ambient temperature and humidity. We developed a self-contained immunoassay platform─the D4 POCT─that can be conducted at the POC by integrating all reagents in a capillary-driven passive microfluidic cassette that minimizes user intervention. The assay can be imaged and analyzed on a portable fluorescence reader─the D4Scope─and provide quantitative outputs. Here, we systematically investigated the resilience of our D4 POCT to varied temperature and humidity and to physiologically diverse human whole blood samples that span a wide range of physiological hematocrit (30-65%). For all conditions, we showed that the platform maintained high sensitivity (0.05-0.41 ng/mL limits of detection). The platform also demonstrated good accuracy in reporting true analyte concentration across environmental extremes when compared to the manually operated format of the same test to detect a model analyte─ovalbumin. Additionally, we engineered an improved version of the microfluidic cassette that improved the ease-of-use of the device and shortened the time-to-result. We implemented this new cassette to create a rapid diagnostic test to detect talaromycosis infection in patients with advanced HIV disease at the POC, demonstrating comparable sensitivity and specificity to the laboratory test for the disease.

ABSTRACT Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy, and the recent engineering of GVs has led to multiple applications including reporter gene imaging, acoustic control, and payload delivery. GVs often cluster into a honeycomb pattern to minimize their occupancy of cytosolic space; however, the molecular mechanism behind this process and its influence on cellular physiology remain unknown. Here, we identified GvpU as the protein governing this process. GvpU-mediated clustering is selective to the genotype of GVs, allowing the design of GV variants with genetically encodable clustering states. Furthermore, we uncovered that the clustering is modulated by phase transition behaviors encoded in the intrinsically disordered region of GvpU through a balanced contribution of acidic and aromatic residues, and such phase transition can directly modulate cellular fitness. Collectively, our findings elucidate the protein player, molecular mechanism, and functional roles of GV clustering, and its programmability for biomedical applications.

<div>AbstractPurpose:<p>Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations.</p>Experimental Design:<p>New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of <i>Pten</i>-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin.</p>Results:<p>The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8<sup>+</sup> T cells was observed exclusively in CP-Dox–treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle–based chemotherapy.</p>Conclusions:<p>These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype–dependent nature of therapeutic response.</p></div>

<div>AbstractPurpose:<p>Nanoparticle-encapsulated drug formulations can improve responses to conventional chemotherapy by increasing drug retention within the tumor and by promoting a more effective antitumor immune response than free drug. New drug delivery modalities are needed in sarcomas because they are often chemoresistant cancers, but the rarity of sarcomas and the complexity of diverse subtypes makes it challenging to investigate novel drug formulations.</p>Experimental Design:<p>New drug formulations can be tested in animal models of sarcomas where the therapeutic response of different formulations can be compared using mice with identical tumor-initiating mutations. Here, using Cre/loxP and CRISPR/Cas9 techniques, we generated two distinct mouse models of <i>Pten</i>-deleted soft-tissue sarcoma: malignant peripheral nerve sheath tumor (MPNST) and undifferentiated pleomorphic sarcoma (UPS). We used these models to test the efficacy of chimeric polypeptide doxorubicin (CP-Dox), a nanoscale micelle formulation, in comparison with free doxorubicin.</p>Results:<p>The CP-Dox formulation was superior to free doxorubicin in MPNST models. However, in UPS tumors, CP-Dox did not improve survival in comparison with free doxorubicin. While CP-Dox treatment resulted in elevated intratumoral doxorubicin concentrations in MPNSTs, this increase was absent in UPS tumors. In addition, elevation of CD8<sup>+</sup> T cells was observed exclusively in CP-Dox–treated MPNSTs, although these cells were not required for full efficacy of the CP nanoparticle–based chemotherapy.</p>Conclusions:<p>These results have important implications for treating sarcomas with nanoparticle-encapsulated chemotherapy by highlighting the tumor subtype–dependent nature of therapeutic response.</p></div>

<p>Supplementary Figures</p>

<p>Supplementary Figures</p>

<div>Abstract<p>The delivery of anticancer therapeutics to solid tumors remains a critical problem in the treatment of cancer. This study reports a new methodology to target a temperature-responsive macromolecular drug carrier, an elastin-like polypeptide (ELP) to solid tumors. Using a dorsal skin fold window chamber model and intravital laser scanning confocal microscopy, we show that the ELP forms micron-sized aggregates that adhere to the tumor vasculature only when tumors are heated to 41.5°C. Upon return to normothermia, the vascular particles dissolve into the plasma, increasing the vascular concentration, which drives more ELPs across the tumor blood vessel and significantly increases its extravascular accumulation. These observations suggested that thermal cycling of tumors would increase the exposure of tumor cells to ELP drug carriers. We investigated this hypothesis in this study by thermally cycling an implanted tumor in nude mice from body temperature to 41.5°C thrice within 1.5 h, and showed the repeated formation of adherent microparticles of ELP in the heated tumor vasculature in each thermal cycle. These results suggest that thermal cycling of tumors can be repeated multiple times to further increase the accumulation of a thermally responsive polymeric drug carrier in solid tumors over a single heat-cool cycle. More broadly, this study shows a new approach—tumor thermal cycling—to exploit stimuli-responsive polymers <i>in vivo</i> to target the tumor vasculature or extravascular compartment with high specificity. [Cancer Res 2007;67(9):4418–24]</p></div>

Supplementary Figure 1 from Thermal Cycling Enhances the Accumulation of a Temperature-Sensitive Biopolymer in Solid Tumors

Supplementary Figure 1 from Thermal Cycling Enhances the Accumulation of a Temperature-Sensitive Biopolymer in Solid Tumors