A number of potent chemopreventives like curcumin and niclosamide have shown promising activities in cancer, although having the drawback of poor bioavailability. To improve the utility of these ...drugs in cancer therapy, we have encapsulated curcumin and niclosamide in poly(lactic-co-glycolide) (PLGA) nanoparticles, in the presence of poly(vinyl alcohol), using the nanoprecipitation method. We successfully synthesised spherical PLGA nanoparticles, either with each of the single drug individually or with both drugs together, for dual drug loaded samples. FTIR spectroscopy confirmed the successful loading of curcumin and niclosamide in PLGA nanoparticles. The encapsulation efficiency was 48.15% and 70.27% for curcumin and niclosamide, respectively, when loaded individually; which increased to 58.09% for curcumin and 85.36% for niclosamide, with simultaneous dual drug encapsulation
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In vitro drug release showed that, as required, a much higher amount of drug released at the acidic pH 6.0 of cancer cells (86.01%, 95.04%, for curcumin and niclosamide, respectively), compared to normal, healthy cell’s pH of 7.4 (25.70% and 60.92% for these two same drugs). MTT assay revealed that dual-drug loaded PLGA nanoparticles exhibited a higher anticancer effect, compared to a bare mixture of two drugs in DMSO (having no PLGA). Therefore, PLGA nanoparticles can be used as an effective carrier to deliver the two hydrophobic drugs to MDA-MB-231 breast cancer cells, for a superior anticancer effect.
Macrophages are phagocytic innate immune cells capable of phenotypical switching in response to the local microenvironment. Studies often use either primary macrophages or immortalized cell lines for ...hypothesis testing, therapeutic assessment, and biomaterial evaluation without carefully considering the potential effects of cell source and tissue of origin, which strongly influence macrophage response. Surprisingly, limited information is available about how, under similar stimuli, immortalized cell lines and primary cells respond in both phenotypical and functional changes. To address this need, in this work, we cultured immortalized macrophage cell lines derived from different origins ( i.e., blood, lung, peritoneal) to understand and compare macrophage phenotypical responses, including polarization and plasticity, morphological changes, and phagocytic functionalities, as well as compared primary macrophages extracted from peritoneal and bone marrow to their immortalized cell line counterparts. We found significant differences in baseline expression of different markers (e.g., CD86, MHCII, CD206, and EGR2) amongst different cell lines, which further influence both polarization and repolarization of the cells, in addition to their phagocytic functionality. Additionally, we observed that, while RAW 264.7 cells behave similarly to the primary bone marrow-derived macrophages, there are noticeable phenotypical and functional differences in cell line (IC-21) and primary peritoneal macrophages, highlighting tissue-specific differences in macrophage response amongst cell lines and primary cells. Moving to three-dimensional (3D) culture in well-defined biomaterials, blood-derived primary and cell line macrophages were encapsulated within hydrogel-based synthetic extracellular matrices and their polarization profiles and cell morphologies were compared. Macrophages exhibited less pronounced polarization during 3D culture in these compliant, soft materials compared to two-dimensional (2D) culture on rigid, tissue culture plastic plates. Overall, our findings highlight origin-specific differences in macrophage response, and therefore, careful considerations must be made to identify the appropriate cell source for the application of interest.
The immune system, consisting of innate and adaptive immunity, acts as the body's defense mechanism to protect against disease-causing pathogens. Innate immune cells, such as macrophages and ...dendritic cells, act as an initial line of defense in the human body to protect against pathogens non-specifically. Adaptive immune cells, such as B and T-cells, are a part of acquired immunity that specifically targets the disease-causing agent. In a healthy state, innate and adaptive immune cells work together to protect the body against pathogens. However, in a diseased state, innate and adaptive immune cells execute a complex cascade of events that can reinforce or exacerbate disease progression. Engineered systems are needed to understand these complex processes and direct them for improved treatments. Synthetic culture platforms, such as hydrogels, present opportunities to dissect this complexity and mimic key aspects of tissue microenvironments in both i) fundamental studies of immune cell responses to specific extracellular stimuli (e.g., microenvironment changes during disease progression) and ii) applied studies that utilize these extracellular cues to direct immune cell phenotype and enable their engineering and expansion. In this thesis, I designed and applied hydrogel-based platforms with tunable biophysical and biochemical properties to investigate and modulate responses of both innate and adaptive immune cells with the goal of developing new therapeutic strategies and cell manufacturing approaches.Focusing first on innate immunity, I studied the role of two individual components that are critical in developing the hydrogel-based in vitro culture platform: cells and biomaterials. For innate immune cells, I studied how tissue of origin can impact cell response, particularly for macrophages. By culturing cells from different origins, including lungs, peritoneal cavity, and monocyte-derived, I documented aspects of both phenotype and function that are heavily influenced by macrophage tissue origin. Next, I studied how to tune the properties of our hydrogel platform based on the application of interest, with a focus on probing macrophage responses to microenvironment cues. To develop a 2D hydrogel culture platform with tunable degradation or stability for macrophage culture, I investigated how the chirality of the amino acids plays a role in the degradability and cytocompatibility of both peptides and peptide-linked hydrogels. By systematically substituting D-amino acids for L-amino acids within a commonly used linker peptide sequence (VPMS↓MRGG), I successfully created a library of peptide sequences with increased resistance to enzymatic degradation. However, this trend was accompanied by increased cytotoxicity for immune cells. This work established strategies that could be easily employed to design and evaluate other enzymatically responsive linker peptides with tunable degradation properties and the important interplay between peptide D-AA content, degradability, and cytotoxicity.Based on the insights gained from these studies, I created a culture platform with tunable biophysical and biochemical properties inspired by the pulmonary microenvironment to study the role of macrophages in the initiation and progression of fibrosis. I utilized a 2-factorial design of experiment (DOE) approach to study how macrophages respond to pro-fibrotic stimuli: increased matrix stiffness and IL-13, a profibrotic soluble factor linked with disease severity. This approach allowed us to study the individual and combinatorial effects of profibrotic stimuli on macrophage phenotype and function. I found that macrophage morphology, phenotype, and particle uptake were influenced by substrate stiffness and IL13 independently. In addition, both stiffness and IL13 worked synergistically to further influence macrophage phenotype and diminish efferocytosis. These results demonstrate how bioinspired hydrogel platforms can be effectively utilized for investigating immune cell responses in diseased states.Focusing next on adaptive immunity, I established a bioinspired hydrogel platform that can be integrated within a flow-based device for T cell activation and engineering for the production of cell therapies. I first collaboratively created a soft hydrogel platform with bioactive antibodies (anti-CD3 and anti-CD28) to stimulate T cells and found that cells interacting with these platforms had increased proliferation, similar activation, and a more desirable memory phenotype with lower exhaustion compared to cells activated on stiff plastic. I then showed how these soft hydrogels can be incorporated with existing membrane-based flow devices to increase the transduction efficiency of the cells. Overall, I showed how a soft bioinspired material system can effectively tune immune cell responses for cell therapy applications.In conclusion, my dissertation delves into the use of bioinspired hydrogel platforms to understand and manipulate both innate and adaptive immune cells. I demonstrated new opportunities for tailoring the hydrogels to the application of interest, studying complex immune responses, and manipulating the microenvironment for improved cell therapy manufacturing.
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Aerosolization of immunotherapies poses incredible potential for manipulating the local mucosal-specific microenvironment, engaging specialized pulmonary cellular defenders, and ...accessing mucosal associated lymphoid tissue to redirect systemic adaptive and memory responses. In this review, we breakdown key inhalable immunoengineering strategies for chronic, genetic, and infection-based inflammatory pulmonary disorders, encompassing the historic use of immunomodulatory agents, the transition to biological inspired or derived treatments, and novel approaches of complexing these materials into drug delivery vehicles for enhanced release outcomes. Alongside a brief description of key immune targets, fundamentals of aerosol drug delivery, and preclinical pulmonary models for immune response, we survey recent advances of inhaled immunotherapy platforms, ranging from small molecules and biologics to particulates and cell therapies, as well as prophylactic vaccines. In each section, we address the formulation design constraints for aerosol delivery as well as advantages for each platform in driving desirable immune modifications. Finally, prospects of clinical translation and outlook for inhaled immune engineering are discussed.
Many chemotherapeutic drugs are unable to achieve their potency because of their hydrophobic nature e. g. curcumin (Cur), coupled to its inability to reach the target site, lower circulation time and ...low half-lifetime. To overcome these, PLGA nanoparticles were synthesized, loaded with curcumin, coated with polyethylene glycol (PEG) and conjugated with different targeting moieties - either folic acid (FA), or hyaluronic acid (HA), or transferrin (Tf) - in the quest to find the best possible targeting agent. TEM images confirm uniform coating of PEG over the PLGA nanoparticle surface, with a size increment from 85 nm (for only PLGA) to ~124 nm (for PLGA-Cur-PEG-ligand nanoparticles), subsequent to PEG coating and attachment of targeting moieties. Cell viability experiments at 80 μM drug concentration on MDA-MB-231 cells confirmed that, viability gradually decreased from 92% to 41%, 32%, 39%, 19% and 8% for free Cur in PBS, to PLGA-Cur, PLGA-Cur-PEG, PLGA-Cur-PEG-Tf, PLGA-Cur-PEG-HA and PLGA-Cur-PEG-FA, respectively. Confocal microscopy and flow cytometry established a higher percentage of curcumin internalization in cells, leading to enhanced cancer cell killing with targeted nanoparticles. Therefore, HA and FA ligands are better targeting moieties, to treat highly aggressive and metastatic MDA-MB-231 breast cancer cells using PLGA nanoparticles.
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Despite recent clinical successes of chimeric antigen receptor T cell therapies in treating liquid cancers, many lingering challenges stand in the way of therapeutic translation to broader types of ...malignancies. Macrophages have been proposed as alternatives to T cells given macrophages’ advantages in promoting tumor infiltration, acquiring diverse antigens, and possessing the ability to continuously stimulate adaptive responses. However, the poor survival of macrophages upon transplantation in addition to transient anti-tumor phenotypical states have been major obstacles standing in the way of macrophage-based cell therapies. Given recent discoveries of nanoparticle strategies in improving macrophage survival and promoting phenotype retention, we herein report the ability to extend the survival and phenotype of macrophage transplants in murine lungs via pre-treatment with nanoparticles of varying degradation rates. Macrophages pre-treated with 100 µg/ml dose of poly(ethylene glycol) diacrylate nanoparticle formulations improve pulmonary macrophage transplant survival over untreated cells beyond 7 days, where degradable nanoparticle formulations result in over a 50% increase in retention of transplanted cell counts relative to untreated cells. Furthermore, pre-treated macrophages more efficiently retain an imposed pro-inflammatory-like polarization state following transplantation out to 7 days compared to macrophages pre-treated with a classical pro-inflammatory stimulus, interferon-gamma, where CD86 costimulatory molecule expression is greater than 150% higher in pre-treated macrophage transplants compared to untreated counterparts. These findings provide an avenue for a major improvement in the lifespan and efficacy of macrophage-based cell therapies and have broader implications to other phagocyte-based cellular therapeutics and administration routes.
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Enzymatically degradable peptides are commonly used as linkers within hydrogels for biological applications; however, controlling the degradation of these engineered peptides with different contexts ...and cell types can prove challenging. In this work, we systematically examined the substitution of d-amino acids (D-AAs) for different l-amino acids in a peptide sequence commonly utilized in enzymatically degradable hydrogels (VPMS↓MRGG) to create peptide linkers with a range of different degradation times, in solution and in hydrogels, and investigated the cytocompatibility of these materials. We found that increasing the number of D-AA substitutions increased the resistance to enzymatic degradation both for free peptide and peptide-linked hydrogels; yet, this trend also was accompanied by increased cytotoxicity in cell culture. This work demonstrates the utility of D-AA-modified peptide sequences to create tunable biomaterials platforms tempered by considerations of cytotoxicity, where careful selection and optimization of different peptide designs is needed for specific biological applications.
The COVID-19 pandemic caused by the global spread of the SARS-CoV-2 virus has led to a staggering number of deaths worldwide and significantly increased burden on healthcare as nations scramble to ...find mitigation strategies. While significant progress has been made in COVID-19 diagnostics and therapeutics, effective prevention and treatment options remain scarce. Because of the potential for the SARS-CoV-2 infections to cause systemic inflammation and multiple organ failure, it is imperative for the scientific community to evaluate therapeutic options aimed at modulating the causative host immune responses to prevent subsequent systemic complications. Harnessing decades of expertise in the use of natural and synthetic materials for biomedical applications, the biomaterials community has the potential to play an especially instrumental role in developing new strategies or repurposing existing tools to prevent or treat complications resulting from the COVID-19 pathology. Leveraging microparticle- and nanoparticle-based technology, especially in pulmonary delivery, biomaterials have demonstrated the ability to effectively modulate inflammation and may be well-suited for resolving SARS-CoV-2-induced effects. Here, we provide an overview of the SARS-CoV-2 virus infection and highlight current understanding of the host’s pulmonary immune response and its contributions to disease severity and systemic inflammation. Comparing to frontline COVID-19 therapeutic options, we highlight the most significant untapped opportunities in immune engineering of the host response using biomaterials and particle technology, which have the potential to improve outcomes for COVID-19 patients, and identify areas needed for future investigations. We hope that this work will prompt preclinical and clinical investigations of promising biomaterials-based treatments to introduce new options for COVID-19 patients.
The pulmonary fibrotic microenvironment is characterized by increased stiffness of lung tissue and enhanced secretion of profibrotic soluble cues contributing to a feedback loop that leads to ...dysregulated wound healing and lung failure. Pinpointing the individual and tandem effects of profibrotic stimuli in impairing immune cell response remains difficult and is needed for improved therapeutic strategies. We utilized a statistical design of experiment (DOE) to investigate how microenvironment stiffness and interleukin 13 (IL13), a profibrotic soluble factor linked with disease severity, contribute to the impaired macrophage response commonly observed in pulmonary fibrosis. We used engineered bioinspired hydrogels of different stiffness, ranging from healthy to fibrotic lung tissue, and cultured murine alveolar macrophages (MH-S cells) with or without IL13 to quantify cell response and analyze independent and synergistic effects. We found that, while both stiffness and IL13 independently influence macrophage morphology, phenotype, phagocytosis and efferocytosis, these factors work synergistically to exacerbate impaired macrophage phenotype and efferocytosis. These unique findings provide insights into how macrophages in fibrotic conditions are not as effective in clearing debris, contributing to fibrosis initiation/progression, and more broadly inform how underlying drivers of fibrosis modulate immune cell response to facilitate therapeutic strategies.
Alternative activation (M2 phenotype) of macrophages and reduced phagocytosis is regulated synergistically by increased substrate stiffness and presence of profibrotic soluble cues.
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