CRISPR-Cas9 knockin mice for genome editing and cancer modeling.

CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three significantly mutated genes in lung adenocarcinoma. Delivery of a single AAV vector in the lung generated loss-of-function mutations in p53 and Lkb1, as well as homology-directed repair-mediated Kras(G12D) mutations, leading to macroscopic tumors of adenocarcinoma pathology. Together, these results suggest that Cas9 mice empower a wide range of biological and disease modeling applications.

Phagocytic Uptake of Polymeric Particles by Immune Cells under Flow Conditions.

Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be established if the circulating cells can take up particles while flowing and if the uptake capacity is similar under static and flow conditions. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to determine if immune cells take up particles under constant fluidic flow. We use polystyrene particles of varying sizes as the model of a polymeric particle for these studies. Our results show that the immune cells do phagocytose under flow conditions. We demonstrate that cell lines representing myeloid cells, primary human neutrophils, and monocytes take up submicrometer-sized particles at similar or better rates under flow compared to static conditions. Experiments with whole human blood show that, even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. Together, these data suggest that circulating immune cells are likely to phagocytose intravenously injected particulates, which has implications for the design of particles to evade or target these cells.

Association of mitochondria with microtubules inhibits mitochondrial fission by precluding assembly of the fission protein Dnm1.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Insights into coumarin-mediated inhibition of biofilm formation in Salmonella Typhimurium.

Coumarins have been shown to possess antimicrobial, anti-quorum sensing and anti-biofilm properties against a wide range of pathogenic bacteria. This study aimed to shed light on the effects of non-substituted coumarin on biofilm formation by the foodborne pathogen Salmonella Typhimurium. Additionally, its efficacy was tested in combination with another potent anti-biofilm agent, resveratrol. Coumarin inhibited biofilm formation for prolonged periods in millimolar concentrations with marginal effects on planktonic growth. It attenuated curli and cellulose production, likely by downregulating the transcript levels of major biofilm formation genes csgD, csgA and adrA. Coumarin further restricted motility in a dose-dependent manner. In addition, coumarin with resveratrol exhibited improved anti-biofilm properties compared with the individual compounds alone. Thus, coumarin alone or with resveratrol can be employed for inhibiting biofilms in food storage and processing units.

Injectable Polymer-Nanoparticle Hydrogels for Local Immune Cell Recruitment.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)-block-polylactide (PEG-b-PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG-b-PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.

Supramolecular PEGylation of biopharmaceuticals.

The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host-guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit[7]uril (CB[7])-PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB[7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy.

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates.

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.

Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes.

The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.

A vector-free microfluidic platform for intracellular delivery.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.

Dendrimer-Inspired Nanomaterials for the in Vivo Delivery of siRNA to Lung Vasculature.

Targeted RNA delivery to lung endothelial cells has the potential to treat conditions that involve inflammation, such as chronic asthma and obstructive pulmonary disease. To this end, chemically modified dendrimer nanomaterials were synthesized and optimized for targeted small interfering RNA (siRNA) delivery to lung vasculature. Using a combinatorial approach, the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length. The top performing materials from in vivo screens were found to primarily target Tie2-expressing lung endothelial cells. At high doses, the dendrimer-lipid derivatives did not cause chronic increases in proinflammatory cytokines, and animals did not suffer weight loss due to toxicity. We believe these materials have potential as agents for the pulmonary delivery of RNA therapeutics.

Electrical Stimulation for Immunomodulation.

The immune system plays a key role in the development and progression of numerous diseases such as chronic wounds, autoimmune diseases, and various forms of cancer. Hence, controlling the behavior of immune cells has emerged as a promising approach for treating these diseases. Current modalities for immunomodulation focus on chemical based approaches, which while effective have the limitations of nonspecific systemic side effects or requiring invasive delivery approaches to reduce the systemic side effects. Recent advances have unraveled the significance of electrical stimulation as an attractive noninvasive approach to modulate immune cell phenotype and activity. This review provides insights on electrical stimulation strategies employed for regulating the behavior of macrophages, T and B cells, and neutrophils. For obtaining a better understanding, two major types of electrical stimulation sources, conventional and self-powered sources, that have been used for immunomodulation are extensively discussed. Next, the strategies of electrical stimulation that may be applied to cells in vitro and in vivo are discussed, with a focus on conventional and stimuli-responsive self-powered sources. A description of how these strategies influence the polarization, phagocytosis, migration, and differentiation of immune cells is also provided. Finally, recent developments in the use of highly localized and efficient platforms for electrical stimulation based immunomodulation are also highlighted.

Cholesterol Levels and Monocyte Phenotype Are Predictors of Nonhealing in Individuals with Low-Grade Diabetic Foot Ulcers: A Prospective Cohort Study.

Objective: Inflammation has been linked to progression of diabetic foot ulcers (DFU); however, specific predictive markers of nonhealing are scarce. The goal of this study was to identify biochemical and immunological parameters from the blood as predictors of nonhealing in grade 1 and grade 2 DFU. Approach: Individuals with low-grade foot ulcers were enrolled in the study to determine if histopathological, biochemical, and immunological parameters could be used to predict individuals whose ulcers would not heal. Data analysis was performed using traditional univariate analyses as well as univariate and multivariable logistic regression, and STROBE guidelines were used for reporting data. Results: Among the 52 individuals who completed the study, we observe that no single histopathological and biochemical parameter was predictive. Conventional univariate analysis and univariate logistic regression analysis showed that the expression of the cell surface proteins CD63, HLA-DR, and CD11b on monocytes was significantly lower in nonhealed individuals, but with moderate discriminative ability. In comparison, a multivariable logistic regression model identified four of the 31 parameters to be salient predictors with low density lipoprotein (LDL) cholesterol (odds ratio [OR] 18.83, confidence interval [CI] 18.83-342) and cell-surface expression of CD63 on monocytes (OR 0.12, CI 0.12-0.45) showing significance and demonstrating high discrimination ability. Innovation: The approach of using a combination of biochemical and immunological parameters to predict ulcer healing is new. Conclusion: Through this study we conclude that LDL cholesterol and cell-surface expression of CD63 on monocytes strongly correlate with nonhealing in individuals with grade 1 and grade 2 DFU.

The consequences of particle uptake on immune cells.

Particle-based systems are becoming ubiquitous in the clinic. When administered in the body, phagocytic immune cells recognize these particles as foreign substances, resulting in their cellular engulfment for degradation and elimination. However, the internalization of particles may induce unintended effects on the functions of these immune cells. Understanding the consequences of particle uptake on immune cells is imperative to design biocompatible and efficient particulate formulations for diagnostic and therapeutic use. Here, we review the recent literature that describes the changes induced in immune cells following internalization of cargo-free particles. We also discuss how the inherent immunomodulatory properties of particles may be leveraged for therapeutic applications. We conclude with suggestions on newer methods to evaluate the effects of particles on immune cells.

Sterile inflammation alters neutrophil kinetics in mice.

Neutrophils play a crucial role in establishing inflammation in response to an infection or injury, but their production rates, as well as blood and tissue residence times, remain poorly characterized under these conditions. Herein, using a biomaterial implant model to establish inflammation followed by in vivo tracking of newly formed neutrophils, we determine neutrophil kinetics under inflammatory conditions. To obtain quantifiable information from our experimental observations, we develop an ordinary differential equation-based mathematical model to extract kinetic parameters. Our data show that in the presence of inflammation resulting in emergency granulopoiesis-like conditions, neutrophil maturation time in the bone marrow reduces by around 60% and reduced half-life in the blood, compared with noninflammatory conditions. Additionally, neutrophil residence time at the inflammatory site increases by 2-fold. Together, these data improve our understanding of neutrophil kinetics under inflammatory conditions, which could pave the way for therapies that focus on modulating in vivo neutrophil dynamics.

Emergent dynamics of a three-node regulatory network explain phenotypic switching and heterogeneity: a case study of Th1/Th2/Th17 cell differentiation.

Naïve helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a "master regulator"-T-bet (Th1), GATA3 (Th2), and RORγT (Th17)-that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotype, Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17, and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remain unclear. Here through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any master regulator can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/RORγT.

Immunomodulatory Bandage for Accelerated Healing of Diabetic Wounds.

Diabetic foot ulcers are challenging to treat. Current strategies to treat these wounds focus on preventing infection and promoting tissue regrowth but are ineffective in many individuals. Low-grade chronic inflammation is present in individuals with diabetes, and altering the inflammatory responses at the wound site could be an alternate approach to promote healing. We hypothesized that immunomodulation of the wound microenvironment would result in accelerated healing. To test this hypothesis, we began by characterizing the changes in the myeloid cell phenotype in a mouse model [leptin receptor knockout (KO) mouse] that closely mimics the type 2 diabetes condition observed in humans. We observed increased numbers of monocytes and neutrophils in the circulation of the KO mice compared to that in wild-type control mice. We also observed several phenotypic changes in neutrophils from the KO diabetic mice, suggesting low-grade systemic inflammation. Hence, we developed a rapamycin-loaded chitosan scaffold that may be used to modulate immune responses. The use of these immunomodulatory scaffolds at a wound site resulted in accelerated healing compared to the healing using blank scaffolds. In summary, our data suggest that immunomodulation may be a viable strategy to promote the healing of wounds in individuals with diabetes.

Surface-Coated Cerium Nanoparticles to Improve Chemotherapeutic Delivery to Tumor Cells.

The antioxidant property of cerium oxide nanoparticles has increased their demand as a nanocarrier to improve the delivery and therapeutic efficacy of anticancer drugs. Here, we report the synthesis of alginate-coated ceria nanoformulations (ceria NPs) and characterization using FTIR spectroscopy, Raman microscopy, and X-ray diffraction. The synthesized ceria NPs show negligible inherent in vitro toxicity when tested on a MDA-MB-231 breast cancer cell line at higher particle concentrations. Upon loading these particles with doxorubicin (Dox) and paclitaxel (PTX) drugs, we observe a potential synergistic cytotoxic effect mediated by the drug and the ceria NPs, resulting in the better killing capacity as well as suppression of cell migration against the MDA-MB-231 cell line. Further, to verify the immune-escaping capacity before targeting cancer cells, we coated the drug-loaded ceria NPs with the membrane of MDA-MB-231 cells using an extrusion method. The resultant delivery system exhibited in vitro preferential uptake by the MDA-MB-231 cell line and showed reduced uptake by the murine macrophage cell line (RAW 264.7), assigning its potential application as non-immunogenic personalized therapy in targeting and killing of cancer cells.

Particle uptake driven phagocytosis in macrophages and neutrophils enhances bacterial clearance.

Humans are exposed to numerous synthetic foreign particles in the form of drug delivery systems and diagnostic agents. Specialized immune cells (phagocytes) clear these particles by phagocytosing and attempting to degrade them. The process of recognition and internalization of the particles may trigger changes in the function of phagocytes. Some of these changes, especially the ability of a particle-loaded phagocyte to take up and neutralize pathogens, remains poorly studied. Herein, we demonstrate that the uptake of non-stimulatory cargo-free particles enhances the phagocytic ability of monocytes, macrophages and neutrophils. The enhancement in phagocytic ability was independent of particle properties, such as size or the base material constituting the particle. Additionally, we show that the increased phagocytosis was not a result of cellular activation or cellular heterogeneity but was driven by changes in cell membrane fluidity and cellular compliance. A consequence of the enhanced phagocytic activity was that particulate-laden immune cells neutralize Escherichia coli (E. coli) faster in culture. Moreover, when administered in mice as a prophylactic, particulates enable faster clearance of E. coli and Staphylococcus epidermidis. Together, we demonstrate that the process of uptake induces cellular changes that favor additional phagocytic events. This study provides insights into using non-stimulatory cargo-free particles to engineer immune cell functions for applications involving faster clearance of phagocytosable abiotic and biotic material.