A microphysiological assay for studying T-cell chemotaxis, trafficking and tumor killing.

Tumors in patients non-responsive to immunotherapy harbor a series of barriers that impede the efficacy of effector T-cells. Consequently, therapeutically modulating the chemotaxis machinery to enable effector T cell infiltration and function in the tumor could result in more successful therapeutic outcomes. Complex in-vitro models allow re-creation of in-vivo tumor complexities in an in-vitro setting, allowing improved translatability to patient biology at the laboratory scale. We identified a gap in available industrial scale microphysiological (MPS) assays for faster validation of targets and strategies that enable T-cell chemotaxis and effector function within tumor microenvironments. Using a commercially available, 96 -chip 2-lane microfluidic assay system, we present a novel, scalable, complex in vitro microphysiological assay to study 3D T-cell chemotaxis and function within native, extracellular matrix (ECM)-rich multicellular tumor environments. Activated or naïve CD3+ T-cells stained with far-red nuclear stain responded to the chemokine gradients generated within the matrigel-collagen ECM by migrating into the microfluidic channel (~5 mm horizontal window), in a concentration- and cell type-dependent manner. Furthermore, we observed and tracked chemotaxis and cancer cell killing function of antigen-specific CD4.CD8.CAR-T cells (chimeric antigen receptor (CAR)-T cells) that responded to CXCR3 agonist gradient built through the expansive 5 mm of cancer cell colony containing stroma. The 2-lane assay system yielded useful information regarding donor and dose-dependent differences in CAR-T cell chemotaxis and tumor killing. The scalable assay system allows a granular window into immune cell migration and function in tissue spaces beyond endothelium, addressing a missing gap in studying tissue-specific immune cell chemotaxis and function to bring forward advancements in cancer immunotherapy. .

The Current Status and Use of Microphysiological Systems by the Pharmaceutical Industry: The International Consortium for Innovation and Quality Microphysiological Systems Affiliate Survey and Commentary.

Microphysiological systems (MPS) are comprised of one or multiple cell types of human or animal origins that mimic the biochemical/electrical/mechanical responses and blood-tissue barrier properties of the cells observed within a complex organ. The goal of incorporating these in vitro systems is to expedite and advance the drug discovery and development paradigm with improved predictive and translational capabilities. Considering the industry need for improved efficiency and the broad challenges of model qualification and acceptance, the International Consortium for Innovation and Quality (IQ) founded an IQ MPS working group in 2014 and Affiliate in 2018. This group connects thought leaders and end users, provides a forum for crosspharma collaboration, and engages with regulators to qualify translationally relevant MPS models. To understand how pharmaceutical companies are using MPS, the IQ MPS Affiliate conducted two surveys in 2019, survey 1, and 2021, survey 2, which differed slightly in the scope of definition of the complex in vitro models under question. The surveys captured demographics, resourcing, rank order for organs of interest, compound modalities tested, and MPS organ-specific questions, including nonclinical species needs and cell types. The major focus of this manuscript is on results from survey 2, where we specifically highlight the context of use for MPS within safety, pharmacology, or absorption, disposition, metabolism, and excretion and discuss considerations for including MPS data in regulatory submissions. In summary, these data provide valuable insights for developers, regulators, and pharma, offering a view into current industry practices and future considerations while highlighting key challenges impacting MPS adoption. SIGNIFICANCE STATEMENT: The application of microphysiological systems (MPS) represents a growing area of interest in the drug discovery and development framework. This study surveyed 20+ pharma companies to understand resourcing, current areas of application, and the key challenges and barriers to internal MPS adoption. These results will provide regulators, tech providers, and pharma industry leaders a starting point to assess the current state of MPS applications along with key learnings to effectively realize the potential of MPS as an emerging technology.

Lung tumor microphysiological system with 3D endothelium to evaluate modulators of T-cell migration.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor-on-chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a HUVEC-derived vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells migrate; and a chemoattractant/tumor (HCC0827 or NCI-H520) compartment. Here, activated T-cells extravasate and migrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose translational strategies by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

A pharmaceutical industry perspective on microphysiological kidney systems for evaluation of safety for new therapies.

The human kidney contains approximately one million nephrons. As the functional unit of the kidney, the nephron affords an opportunity to approximate the kidney at a microphysiological scale. Recent emergence of physiologically accurate human tissue models has radically advanced the possibilities of mimicking organ biology and multi-organ combinations in vitro. Anatomically, the nephron is one of the most complex, sequentially integrated microfluidic units in the body making the miniaturized microfluidic systems excellent candidates for capturing the kidney biology in vitro. While these models are promising, there are a number of considerations for practical implementation into a drug development paradigm. Opportunities for pharmaceutical industry applications of new MPS models often start with drug safety testing. As such, the intent of this article is to focus on safety and ADME applications. This article reviews biological functions of the kidney and options for characterizing known roles in nephrotoxicity. The concept of "context-of-use" is introduced as a framework for describing and verifying the specific features of an MPS platform for use in drug development. Overall, we present a perspective on key attributes of microphysiological kidney models, which the pharmaceutical industry could leverage to improve confident safety and ADME evaluations of experimental therapies.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering.

Chemotherapeutic Drug-Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA.

Plasmid DNA (pDNA) is an attractive therapeutic biomolecule in several diseases including cancer, AIDS, cystic fibrosis, Parkinson's disease, and Alzheimer's disease. Increasing demand for plasmid DNA as a therapeutic biomolecule for transgene expression or vaccine applications necessitate novel approaches to bioprocessing. The synthesis, characterization and evaluation of aminoglycoside-derived hydrogel microbeads (Amikabeads) for pDNA binding is described previously. Here, the generation and evaluation of novel chemotherapeutic drug-conjugated microbeads for application in pDNA binding and recovery is described. Chemotherapeutic drug-conjugated Amikabeads demonstrate higher binding of methylated pDNA compared to unmethylated pDNA in presence of high salt concentrations. Desorption of plasmids from drug-conjugated microbeads is facilitated by the use of organic modifiers. The observed differences in binding methylated versus unmethylated DNA can make drug-conjugated microbeads useful in diagnostic as well as therapeutic applications. These results demonstrate that anti-cancer drugs represent a diverse set of ligands that may be exploited for molecular engineering of novel DNA binding materials for applications in delivery, diagnostics, and biomanufacturing.

Parallel fabrication of macroporous scaffolds.

Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques.

Investigation into Pseudo-Capacitance Behavior of Glycoside-Containing Hydrogels.

Electrochemical pseudocapacitors are an attractive choice for energy storage applications because they offer higher energy densities than electrostatic or electric double layer capacitors. They also offer higher power densities in shorter durations of time, as compared to batteries. Recent efforts on pseudocapacitors include biocompatible hydrogel electrolytes and transition metal electrodes for implantable energy storage applications. Pseudocapacitive behavior in these devices has been attributed to the redox reactions that occur within the electric double layer, which is formed at the electrode-electrolyte interface. In the present study, we describe a detailed investigation on redox reactions responsible for pseudocapacitive behavior in glycoside-containing hydrogel formulations. Pseudocapacitive behavior was compared among various combinations of biocompatible hydrogel electrolytes, using carbon tape electrodes and transition metal electrodes based on fluorine-doped tin oxide. The hydrogels demonstrated a pseudocapacitive response only in the presence of transition metal electrodes but not in the presence of carbon electrodes. Hydrogels containing amine moieties showed greater energy storage than gels based purely on hydroxyl functional groups. Furthermore, energy storage increased with greater amine content in these hydrogels. We claim that the redox reactions in hydrogels are largely based on Lewis acid-base interactions, facilitated by amine and hydroxyl side groups along the electrolyte chain backbones, as well as hydroxylation of electrode surfaces. Water plays an important role in these reactions, not only in terms of providing ionic radicals but also in assisting ion transport. This understanding of redox reactions will help determine the choice of transition metal electrodes, Lewis acid-base pairs in electrolytes, and medium for ionic transport in future biocompatible pseudocapacitors.

Aminoglycoside-derived amphiphilic nanoparticles for molecular delivery.

The development of effective drug carriers can lead to improved outcomes in a variety of disease conditions. Aminoglycosides have been used as antibacterial therapeutics, and are attractive as monomers for the development of polymeric materials in various applications. Here, we describe the development of novel aminoglycoside-derived amphiphilic nanoparticles for drug delivery, with an eye towards ablation of cancer cells. The aminoglycoside paromomycin was first cross-linked with resorcinol diglycidyl ether leading to the formation of a poly (amino ether), PAE. PAE molecules were further derivatized with methoxy-terminated poly(ethylene glycol) or mPEG resulting in the formation of mPEG-PAE polymer, which self-assembled to form nanoparticles. Formation of the mPEG-PAE amphiphile was characterized using (1)H NMR, (13)C NMR, gel permeation chromatography (GPC) and FTIR spectroscopy. Self-assembly of the polymer into nanoparticles was characterized using dynamic light scattering, zeta potential analyses, atomic force microscopy (AFM) and the pyrene fluorescence assay. mPEG-PAE nanoparticles were able to carry significant amounts of doxorubicin (DOX), presumably by means of hydrophobic interactions between the drug and the core. Cell-based studies indicated that mPEG-PAE nanoparticles, loaded with doxorubicin, were able to induce significant loss in viabilities of PC3 human prostate cancer, MDA-MB-231 human breast cancer, and MB49 murine bladder cancer cells; empty nanoparticles resulted in negligible losses of cell viability under the conditions investigated. Taken together, our results indicate that the mPEG-PAE nanoparticle platform is attractive for drug delivery in different applications, including cancer.

Aminoglycoside antibiotic-derived anion-exchange microbeads for plasmid DNA binding and in situ DNA capture.

Plasmid DNA (pDNA) therapeutics are being investigated for gene therapy and DNA vaccines against diseases including cancer, cystic fibrosis and AIDS. In addition, several applications in modern biotechnology require pDNA for transient protein production. Here, we describe the synthesis, characterization, and evaluation of microbeads ("Amikabeads") derived from the aminoglycoside antibiotic amikacin for pDNA binding and in situ DNA capture from mammalian cells. The parental aminoglycoside-derived microbeads (Amikabeads-P) acted as anion-exchange materials, and demonstrated high capacities for binding pDNA. Binding of pDNA was significantly enhanced following quaternization of the amines on the microbeads (Amikabeads-Q). Amikabeads were further employed for the disruption and extraction of DNA from mammalian cells, indicating their utility for in situ DNA capture. Our results indicate that Amikabeads are a novel material, with multiple reactive groups for further conjugation, and can have several applications in plasmid DNA biotechnology.

Design, synthesis, and functionalization of nanomaterials for therapeutic drug delivery.

Nanomaterials have the potential to solve some of the toughest challenges facing modern medicine. Their unique optical, magnetic and chemical properties at the nanoscale make them different from their macroscale counterparts. Successful application of nanomaterials can revolutionize therapeutics, diagnostics and imaging in several biomedical applications. Self-assembled amphiphilic polymeric nanoparticles have been employed to carry poorly soluble chemotherapeutic drugs. Loading of anticancer chemotherapeutic drugs into self assembled polymeric nanoparticles have shown to increase their circulation time, tumor localization and therapeutic potential. This book chapter provides an introductory discussion to organic nanotechnologies for drug delivery. Promising advances in the field of nanomedicine will be discussed and an outlook to the future will be provided.

Sensitizing cancer cells to TRAIL-induced death by micellar delivery of mitoxantrone.

TNFα-related apoptosis-inducing ligand (TRAIL) induces death selectively in cancer cells. However, subpopulations of cancer cells are either resistant to or can develop resistance to TRAIL-induced death. As a result, strategies that overcome this resistance are currently under investigation. We have recently identified several US FDA-approved drugs with TRAIL-sensitization activity against prostate, breast and pancreatic cancer cells. Mitoxantrone, a previously unknown TRAIL sensitizer identified in the screen, was successfully encapsulated in methoxy-, amine- and carboxyl-terminated PEG-DSPE micelles in order to facilitate delivery of the drug to cancer cells. All three micelle types were extensively characterized for their physicochemical properties and evaluated for their ability to sensitize cancer cells to TRAIL-induced death. Our results indicate that micelle-encapsulated mitoxantrone can be advantageously employed in synergistic treatments with TRAIL, leading to a biocompatible delivery system and amplified cell killing activity for combination chemotherapeutic cancer treatments.

Discovery of antibiotics-derived polymers for gene delivery using combinatorial synthesis and cheminformatics modeling.

We describe the combinatorial synthesis and cheminformatics modeling of aminoglycoside antibiotics-derived polymers for transgene delivery and expression. Fifty-six polymers were synthesized by polymerizing aminoglycosides with diglycidyl ether cross-linkers. Parallel screening resulted in identification of several lead polymers that resulted in high transgene expression levels in cells. The role of polymer physicochemical properties in determining efficacy of transgene expression was investigated using Quantitative Structure-Activity Relationship (QSAR) cheminformatics models based on Support Vector Regression (SVR) and 'building block' polymer structures. The QSAR model exhibited high predictive ability, and investigation of descriptors in the model, using molecular visualization and correlation plots, indicated that physicochemical attributes related to both, aminoglycosides and diglycidyl ethers facilitated transgene expression. This work synergistically combines combinatorial synthesis and parallel screening with cheminformatics-based QSAR models for discovery and physicochemical elucidation of effective antibiotics-derived polymers for transgene delivery in medicine and biotechnology.