Protein-Functionalized Gold Nanospheres with Tunable Photothermal Efficiency for the Near-Infrared Photothermal Ablation of Biofilms.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multidrug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). The temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34 to 42 °C. Then, by functionalizing the AuNP with an additional small globular protein, we could extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed an enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68 ± 6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for the photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced antibiofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel antibiofilm nanomaterial with low cytotoxicity.

Correction Notice: Microscopy and Plate Reader-based Methods for Monitoring the Interaction of Platelets and Tumor Cells in vitro.

Platelets and their activation status play an essential role in cancer metastasis. Therefore, the anti-metastatic potential of antiplatelet drugs has been investigated for many years. However, the initial screening of these antiplatelet drugs to determine which agents can inhibit the interactions of platelets and tumor cells is very limited due to reliance upon expensive, time-consuming, and low-throughput animal experiments for screening. In vitro models of the platelet-tumor cell interaction can be a useful tool to rapidly screen multiple antiplatelet drugs and compare their ability to disrupt platelet-tumor cell interactions, while also identifying optimal concentrations to move forward for in vivo validation. Hence, we adopted methods used in platelet activation research to isolate and label platelets before mixing them with tumor cells (MDA-MB-231-RFP cells) in vitro in a static co-culture model. Platelets were isolated from other blood components by centrifugation, followed by fluorescent labeling using the dye CMFDA (CellTrackerTM Green). Labeling platelets allows microscopic observation of the introduced platelets with tumor cells grown in cell culture dishes. These methods have facilitated the study of platelet-tumor cell interactions in tissue culture. Here, we provide details of the methods we have used for platelet isolation from humans and mice and their staining for further interaction with tumor cells by microscopy and plate reader-based quantification. Moreover, we show the utility of this assay by demonstrating decreased platelet-tumor cell interactions in the presence of the T-Prostanoid receptor (TPr) inhibitor ifetroban. The methods described here will aid in the rapid discovery of antiplatelet agents, which have potential as anti-metastatic agents as well. Key features • Analysis of platelet-tumor cell binding dynamics. • In vitro methods developed for measuring platelet-tumor cell binding to enable rapid testing of antiplatelet and other compounds. • Complementary analysis of platelet-tumor cell binding by imaging and fluorimetry-based readings. • Representative results screening the effect of the antiplatelet drug, ifetroban, on platelet-tumor cell binding using the protocol. • Validation results were presented with both a TPr agonist and ifetroban (antagonist).

Near-Infrared Photothermal Ablation of Biofilms using ProteinFunctionalized Gold Nanospheres with a Tunable Temperature Response.

Temperature-responsive nanostructures with high antimicrobial efficacy are attractive for therapeutic applications against multi-drug-resistant bacteria. Here, we report temperature-responsive nanospheres (TRNs) that are engineered to undergo self-association and agglomeration above a tunable transition temperature (Tt). Temperature-responsive behavior of the nanoparticles is obtained by functionalizing citrate-capped, spherical gold nanoparticles (AuNPs) with elastin-like polypeptides (ELPs). Using protein design principles, we achieve a broad range of attainable Tt values and photothermal conversion efficiencies (η). Two approaches were used to adjust this range: First, by altering the position of the cysteine residue used to attach ELP to the AuNP, we attained a Tt range from 34-42 °C. Then, functionalizing the AuNP with an additional small globular protein, we were able to extend this range to 34-50 °C. Under near-infrared (NIR) light exposure, all TRNs exhibited reversible agglomeration. Moreover, they showed enhanced photothermal conversion efficiency in their agglomerated state relative to the dispersed state. Despite their spherical shape, TRNs have a photothermal conversion efficiency approaching that of gold nanorods (η = 68±6%), yet unlike nanorods, the synthesis of TRNs requires no cytotoxic compounds. Finally, we tested TRNs for photothermal ablation of biofilms. Above Tt, NIR irradiation of TRNs resulted in a 10,000-fold improvement in killing efficiency compared to untreated controls (p < 0.0001). Below Tt, no enhanced anti-biofilm effect was observed. In conclusion, engineering the interactions between proteins and nanoparticles enables the tunable control of TRNs, resulting in a novel, anti-biofilm nanomaterial with low cytotoxicity.

Selective Blood Cell Hitchhiking in Whole Blood with Ionic Liquid-Coated PLGA Nanoparticles to Redirect Biodistribution After Intravenous Injection.

Less than 5% of intravenously-injected nanoparticles (NPs) reach destined sites in the body due to opsonization and immune-based clearance in vascular circulation. By hitchhiking in situ onto specific blood components post-injection, NPs can selectively target tissue sites for unprecedentedly high drug delivery rates. Choline carboxylate ionic liquids (ILs) are biocompatible liquid salts <100X composed of bulky asymmetric cations and anions. This class of ILs has been previously shown to significantly extend circulation time and redirect biodistribution in BALB/c mice post-IV injection via hitchhiking on red blood cell (RBC) membranes. Herein, we synthesized & screened 60 choline carboxylic acid-based ILs to coat PLGA NPs and present the impact of structurally engineering the coordinated anion identity to selectively interface and hitchhike lymphocytes, monocytes, granulocytes, platelets, and RBCs in whole mouse blood for in situ targeted drug delivery. Furthermore, we find this nanoparticle platform to be biocompatible (non-cytotoxic), translate to human whole blood by resisting serum uptake and maintaining modest hitchhiking, and also significantly extend circulation retention over 24 hours in BALB/c healthy adult mice after IV injection. Because of their altered circulation profiles, we additionally observe dramatically different organ accumulation profiles compared to bare PLGA NPs. This study establishes an initial breakthrough platform for a modular and transformative targeting technology to hitchhike onto blood components with high efficacy and safety in the bloodstream post-IV administration.

Current and future directions of drug delivery for the treatment of mental illnesses.

Mental illnesses including anxiety disorders, autism spectrum disorder, post-traumatic stress disorder, schizophrenia, depression, and others exact an immense toll on the healthcare system and society at large. Depression alone impacts 21 million adults and costs over $200 billion annually in the United States. However, pharmaceutical strategies to treat mental illnesses are lagging behind drug development in many other disease areas. Because many of the shortcomings of therapeutics for mental illness relate to delivery problems, drug delivery technologies have the potential to radically improve the effectiveness of therapeutics for these diseases. This review describes the current pharmacotherapeutic approaches to treating mental illnesses as well as drug delivery approaches that have improved existing therapies. Approaches to improve drug bioavailability, provide controlled release of therapeutics, and enable drug targeting to the central nervous system (CNS) will be highlighted. Moreover, next-generation delivery approaches such as environmentally-controlled release and interval/sequential drug release will be addressed. Based on the evolving landscape of the treatment of mental illnesses, the nascent field of drug delivery in mental health has tremendous potential for growth in terms of both economic and patient impact.

Interval delivery of 5HT2A agonists using multilayered polymer films.

There is an urgent unmet medical need to develop therapeutic options for the ~50% of depression patients suffering from treatment-resistant depression, which is difficult to treat with existing psycho- and pharmaco-therapeutic options. Classical psychedelics, such as the 5HT2A agonists, have re-emerged as a treatment paradigm for depression. Recent clinical trials highlight the potential effectiveness of 5HT2A agonists to improve mood and psychotherapeutic growth in treatment-resistant depression patients, even in those who have failed a median of four previous medications in their lifetime. Moreover, microdosing could be a promising way to achieve long-term alleviation of depression symptoms without a hallucinogenic experience. However, there are a gamut of practical barriers that stymie further investigation of microdosing 5HT2A agonists, including: low compliance with the complicated dosing regimen, high risk of diversion of controlled substances, and difficulty and cost administering the long-term treatment regimens in controlled settings. Here, we developed a drug delivery system composed of multilayered cellulose acetate phthalate (CAP)/Pluronic F-127 (P) films for the encapsulation and interval delivery of 5HT2A agonists from a fully biodegradable and biocompatible implant. CAPP film composition, thickness, and layering strategies were optimized, and we demonstrated three distinct pulses from the multilayered CAPP films in vitro. Additionally, the pharmacokinetics and biodistribution of the 5HT2A agonist 2,5-Dimethoxy-4-iodoamphetamine (DOI) were quantified following the subcutaneous implantation of DOI-loaded single and multilayered CAPP films. Our results demonstrate, for the first time, the interval delivery of psychedelics from an implantable drug delivery system and open the door to future studies into the therapeutic potential of psychedelic delivery.

Dual-Responsive Glycopolymers for Intracellular Codelivery of Antigen and Lipophilic Adjuvants.

Traditional approaches to vaccines use whole organisms to trigger an immune response, but they do not typically generate robust cellular-mediated immunity and have various safety risks. Subunit vaccines composed of proteins and/or peptides represent an attractive and safe alternative to whole organism vaccines, but they are poorly immunogenic. Though there are biological reasons for the poor immunogenicity of proteins and peptides, one other key to their relative lack of immunogenicity could be attributed to the poor pharmacokinetic properties of exogenously delivered proteins and peptides. For instance, peptides often aggregate at the site of injection and are not stable in biological fluids, proteins and peptides are rapidly cleared from circulation, and both have poor cellular internalization and endosomal escape. Herein, we developed a delivery system to address the lack of protein immunogenicity by overcoming delivery barriers as well as codelivering immune-stimulating adjuvants. The glycopolymeric nanoparticles (glycoNPs) are composed of a dual-stimuli-responsive block glycopolymer, poly[2-(diisopropylamino)ethyl methacrylate]-b-poly[(pyridyl disulfide ethyl methacrylate)-co-(methacrylamidoglucopyranose)] (p[DPA-b-(PDSMA-co-MAG)]). This polymer facilitates protein conjugation and cytosolic release, the pH-responsive release of lipophilic adjuvants, and pH-dependent membrane disruption to ensure cytosolic delivery of antigens. We synthesized p[DPA-b-(PDSMA-co-MAG)] by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by the formation and physicochemical characterization of glycoNPs using the p[DPA-b-(PDSMA-co-MAG)] building blocks. These glycoNPs conjugated the model antigen ovalbumin (OVA) and released OVA in response to elevated glutathione levels. Moreover, the glycoNPs displayed pH-dependent drug release of the model hydrophobic drug Nile Red while also exhibiting pH-responsive endosomolytic behavior as indicated by a red blood cell hemolysis assay. GlycoNPs coloaded with OVA and the toll-like receptor 7/8 (TLR-7/8) agonist Resiquimod (R848) activated DC 2.4 dendritic cells (DCs) significantly more than free OVA and R848 and led to robust antigen presentation of the OVA epitope SIINFEKL on major histocompatibility complex I (MHC-I). In sum, the dual-stimuli-responsive glycopolymer introduced here overcomes major protein and peptide delivery barriers and could vastly improve the immunogenicity of protein-based vaccines.

Assessment of the Immune Response to Tumor Cell Apoptosis and Efferocytosis.

Apoptotic cells are cleared from the body principally through recognition and engulfment by neighboring phagocytes, a process known as efferocytosis. During efferocytosis, phagocytes are recruited to the site/activated by "find me" signals released from apoptotic cells, precisely identify apoptotic cells by the recognition of "eat me" signals on the apoptotic cell surface, and engulf the apoptotic cells to prevent secondary necrosis and inflammation. Thus, efferocytosis is critical for tissue homeostasis in normal physiology. However, efferocytosis of apoptotic tumor cells-performed by tumor-associated macrophages-suppresses immunity within the tumor microenvironment and limits the antitumor response. This phenomenon is further exacerbated in tumor residual disease because of the high apoptotic cell burden generated by cytotoxic therapies. Blocking efferocytosis could be a powerful approach to boost tumor immunogenicity, particularly as a combination approach with cytotoxic therapies that produce many apoptotic cells, but little is currently known about the immune response to efferocytosis. Moreover, there is a dearth of in vivo models available to study the immunologic and therapeutic consequences of blocking efferocytosis in tumor residual disease.Here, we describe a model that enables in vivo studies of tumor immunology in the aftermath of cytotoxic therapy with an emphasis on the impact of efferocytosis. Orthotopic HER2+ mammary tumors are established in immune-competent mice, followed by a single administration of lapatinib, a receptor tyrosine kinase inhibitor of HER2, to the mice that induces widespread, transient apoptosis in the tumor microenvironment. In the days following lapatinib treatment, agents that block efferocytosis such as BMS-777607 are administered. Tissue is collected from cohorts of mice at day 2 (after lapatinib treatment only) to assess apoptosis, day 8 (after lapatinib treatment followed by blockade of efferocytosis) to assess the immune response to apoptosis and efferocytosis, and day 28 (after 4 consecutive weeks of treatment) to assess therapeutic efficacy. This model enables mechanistic studies of tumor immunology in residual disease as well as therapeutic efficacy studies of targeted agents that disrupt efferocytosis.

ROS-Responsive Glycopolymeric Nanoparticles for Enhanced Drug Delivery to Macrophages.

Macrophages play a diverse, key role in many pathologies, including inflammatory diseases, cardiovascular diseases, and cancer. However, many therapeutic strategies targeting macrophages suffer from systemic off-target toxicity resulting in notoriously narrow therapeutic windows. To address this shortcoming, the development of poly(propylene sulfide)-b-poly(methacrylamidoglucopyranose) [PPS-b-PMAG] diblock copolymer-based nanoparticles (PMAG NPs) capable of targeting macrophages and releasing drug in the presence of reactive oxygen species (ROS) is reported. PMAG NPs have desirable physicochemical properties for systemic drug delivery, including slightly negative surface charge, ≈100 nm diameter, and hemo-compatibility. Additionally, due to the presence of PPS in the NP core, PMAG NPs release drug cargo preferentially in the presence of ROS. Importantly, PMAG NPs display high cytocompatibility and are taken up by macrophages in cell culture at a rate ≈18-fold higher than PEGMA NPs-NPs composed of PPS-b-poly(oligoethylene glycol methacrylate). Computational studies indicate that PMAG NPs likely bind with glucose transporters such as GLUT 1/3 on the macrophage cell surface to facilitate high levels of internalization. Collectively, this study introduces glycopolymeric NPs that are uniquely capable of both receptor-ligand targeting to macrophages and ROS-dependent drug release and that can be useful in many immunotherapeutic settings.

Comparative Investigation of the Hydrolysis of Charge-Shifting Polymers Derived from an Azlactone-Based Polymer.

Poly 2-vinyl-4,4-dimethylazlactone (PVDMA) has received much attention as a "reactive platform" to prepare charge-shifting polycations via post-polymerization modification with tertiary amines that possess primary amine or hydroxyl reactive handles. Upon hydrolysis of the resulting amide or ester linkages, the polymers can undergo a gradual transition in net charge from cationic to anionic. Herein, a systematic investigation of the hydrolysis rate of PVDMA-derived charge-shifting polymers is described. PVDMA is modified with tertiary amines bearing either primary amine, hydroxyl, or thiol reactive handles. The resulting polymers possess tertiary amine side chains connected to the backbone via amide, ester, or thioester linkages. The hydrolysis rates of each PVDMA derivative are monitored at 25 and 50 °C at pH values of 5.5, 7.5, and 8.5, respectively. While the hydrolysis rate of the amide-functionalized PVDMA is negligible over the period investigated, the hydrolysis rates of the ester- and thioester-functionalized PVDMA increase with increasing temperature and pH. Interestingly, the hydrolysis rate of the thioester-functionalized PVDMA appears to be more rapid than the ester-functionalized PVDMA at all pH values and temperatures investigated. It is believed that these results can be utilized to inform the future preparation of PVDMA-based charge-shifting polymers for biomedical applications.

Improved nanoformulation and bio-functionalization of linear-dendritic block copolymers with biocompatible ionic liquids.

Linear-dendritic block copolymers (LDBCs) have emerged as promising materials for drug delivery applications, with their hybrid structure exploiting advantageous properties of both linear and dendritic polymers. LDBCs have promising encapsulation efficiencies that can be used to encapsulate both hydrophobic and hydrophilic dyes for bioimaging, cancer therapeutics, and small biomolecules. Additionally, LDBCS can be readily functionalized with varying terminal groups for more efficient targeted delivery. However, depending on structural composition and surface properties, LDBCs also exhibit high dispersities (D), poor shelf-life, and potentially high cytotoxicity to non-target interfacing blood cells during intravenous drug delivery. Here, we show that choline carboxylic acid-based ionic liquids (ILs) electrostatically solvate LDBCs by direct dissolution and form stable and biocompatible IL-integrated LDBC nano-assemblies. These nano-assemblies are endowed with red blood cell-hitchhiking capabilities and show altered cellular uptake behavior ex vivo. When modified with choline and trans-2-hexenoic acid, IL-LDBC dispersity dropped by half compared to bare LDBCs, and showed a significant shift of the cationic surface charge towards neutrality. Proton nuclear magnetic resonance spectroscopy evidenced twice the total amount of IL on the LDBCs relative to an established IL-linear PLGA platform. Transmission electron microscopy suggested the formation of a nanoparticle surface coating, which acted as a protective agent against RBC hemolysis, reducing hemolysis from 73% (LDBC) to 25% (IL-LDBC). However, dramatically different uptake behavior of IL-LDBCs vs. IL-PLGA NPs in RAW 264.7 macrophage cells suggests a different conformational IL-NP surface assembly on the linear versus the linear-dendritic nanoparticles. These results suggest that by controlling the physical chemistry of polymer-IL interactions and assembly on the nanoscale, biological function can be tailored toward the development of more effective and more precisely targeted therapies.

Effect of pore size and spacing on neovascularization of a biodegradble shape memory polymer perivascular wrap.

Neointimal hyperplasia (NH) is a main source of failures in arteriovenous fistulas and vascular grafts. Several studies have demonstrated the promise of perivascular wraps to reduce NH via promotion of adventitial neovascularization and providing mechanical support. Limited clinical success thus far may be due to inappropriate material selection (e.g., nondegradable, too stiff) and geometric design (e.g., pore size and spacing, diameter). The influence of pore size and spacing on implant neovascularization is investigated here for a new biodegradable, thermoresponsive shape memory polymer (SMP) perivascular wrap. Following an initial pilot, 21 mice were each implanted with six scaffolds: four candidate SMP macroporous designs (a-d), a nonporous SMP control (e), and microporous GORETEX (f). Mice were sacrificed after 4 (N = 5), 14 (N = 8), and 28 (N = 8) days. There was a statistically significant increase in neovascularization score between all macroporous groups compared to nonporous SMP (p < .023) and microporous GORETEX (p < .007) controls at Day 28. Wider-spaced, smaller-sized pore designs (223 µm-spaced, 640 µm-diameter Design c) induced the most robust angiogenic response, with greater microvessel number (p < .0114) and area (p < .0055) than nonporous SMPs and GORETEX at Day 28. This design also produced significantly greater microvessel density than nonporous SMPs (p = 0.0028) and a smaller-spaced, larger-sized pore (155 µm-spaced, 1,180 µm-sized Design b) design (p = .0013). Strong neovascularization is expected to reduce NH, motivating further investigation of this SMP wrap with controlled pore spacing and size in more advanced arteriovenous models.

Immunostimulatory biomaterials to boost tumor immunogenicity.

Cancer immunotherapy is exhibiting great promise as a new therapeutic modality for cancer treatment. However, immunotherapies are limited by the inability of some tumors to provoke an immune response. These tumors with a 'cold' immunological phenotype are characterized by low numbers of tumor-infiltrating lymphocytes, high numbers of immunosuppressive leukocytes (e.g. regulatory T cells, tumor-associated macrophages), and high production of immune-dampening signals (e.g. IL-10, TGF-ß, IDO-1). Strategies to boost the aptitude of tumors to initiate an immune response (i.e. boost tumor immunogenicity) will turn 'cold' tumors 'hot' and augment the anti-tumor efficacy of current immunotherapies. Approaches to boost tumor immunogenicity already show promise; however, multifaceted delivery and immunobiology challenges exist. For instance, systemic delivery of many immune-stimulating agents causes off-target toxicity and/or the development of autoimmunity, limiting the administrable dose below the threshold needed to achieve efficacy. Moreover, once administered in vivo, molecules such as the nucleic acid-based agonists for many pattern recognition receptors are either rapidly cleared or degraded, and don't efficiently traffic to the intracellular compartments where the receptors are located. Thus, these nucleic acid-based drugs are ineffective without a delivery system. Biomaterials-based approaches aim to enhance current strategies to boost tumor immunogenicity, enable novel strategies, and spare dose-limiting toxicities. Here, we review recent progress to improve cancer immunotherapies by boosting immunogenicity within tumors using immunostimulatory biomaterials.

Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study.

Although new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2-prostanoid receptor (TPr), a G-protein-coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for CPI211, we performed a phenome-wide association study (PheWAS) of the gene encoding TPr, TBXA2R, using robust deidentified health records and matched genomic data from more than 29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single-nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated 200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered an rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced transendothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as antimetastatic agents and highlighting the use of PheWAS as an approach to accelerate drug repurposing.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously 'undruggable' hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (< pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability.

Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly ( r = 0.95 and p < 10-4) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly[(ethylene glycol)- b-[(2-(dimethylamino)ethyl methacrylate)- co-(butyl methacrylate)]] [PEG-(DMAEMA- co-BMA)] siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization ( r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake ( r = 0.73 and p < 10-3). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z' of 0.6 (whereas assays with Z' > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization ( p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors ( p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.

Treatment-Induced Tumor Cell Apoptosis and Secondary Necrosis Drive Tumor Progression in the Residual Tumor Microenvironment through MerTK and IDO1.

Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor 'homeostasis' and progression. SIGNIFICANCE: These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.

Efferocytosis in the tumor microenvironment.

Within the course of a single minute, millions of cells in the human body will undergo programmed cell death in response to physiological or pathological cues. The diminished energetic capacity of an apoptotic cell renders the cell incapable of sustaining plasma membrane integrity. Under these circumstances, intracellular contents that might leak into the surrounding tissue microenvironment, a process referred to as secondary necrosis, could induce inflammation and tissue damage. Remarkably, in most cases of physiologically rendered apoptotic cell death, inflammation is avoided because a mechanism to swiftly remove apoptotic cells from the tissue prior to their secondary necrosis becomes activated. This mechanism, referred to as efferocytosis, uses phagocytes to precisely identify and engulf neighboring apoptotic cells. In doing so, efferocytosis mantains tissue homeostasis that would otherwise be disrupted by normal cellular turnover and exacerbated further when the burden of apoptotic cells becomes elevated due to disease or insult. Efferocytosis also supports the resolution of inflammation, restoring tissue homesostasis. The importance of efferocytosis in health and disease underlies the increasing research efforts to understand the mechanisms by which efferocytosis occurs, and how a failure in the efferocytic machinery contributes to diseases, or conversely, how cancers effectively use the existing efferocytic machinery to generate a tumor-tolerant, immunosuppressive tumor microenvironment. We discuss herein the molecular mechanisms of efferocytosis, how the process of efferocytosis might support a tumor 'wound healing' phenotype, and efforts to target efferocytosis as an adjunct to existing tumor treatments.

Molecular Regulation of Acute Tie2 Suppression in Sepsis.

OBJECTIVES: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness. DESIGN: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients. SETTING: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen. PATIENTS: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22). INTERVENTIONS: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-α, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor-α-treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p < 0.005). CONCLUSIONS: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript.