Tumor biomechanics as a novel imaging biomarker to assess response to immunotherapy in a murine glioma model.

Glioblastoma is the most common and aggressive primary malignant brain tumor with poor prognosis. Novel immunotherapeutic approaches are currently under investigation. Even though magnetic resonance imaging (MRI) is the most important imaging tool for treatment monitoring, response assessment is often hampered by therapy-related tissue changes. As tumor and therapy-associated tissue reactions differ structurally, we hypothesize that biomechanics could be a pertinent imaging proxy for differentiation. Longitudinal MRI and magnetic resonance elastography (MRE) were performed to monitor response to immunotherapy with a toll-like receptor 7/8 agonist in orthotopic syngeneic experimental glioma. Imaging results were correlated to histology and light sheet microscopy data. Here, we identify MRE as a promising non-invasive imaging method for immunotherapy-monitoring by quantifying changes in response-related tumor mechanics. Specifically, we show that a relative softening of treated compared to untreated tumors is linked to the inflammatory processes following therapy-induced re-education of tumor-associated myeloid cells. Mechanistically, combined effects of myeloid influx and inflammation including extracellular matrix degradation following immunotherapy form the basis of treated tumors being softer than untreated glioma. This is a very early indicator of therapy response outperforming established imaging metrics such as tumor volume. The overall anti-tumor inflammatory processes likely have similar effects on human brain tissue biomechanics, making MRE a promising tool for gauging response to immunotherapy in glioma patients early, thereby strongly impacting patient pathway.

Host-functionalization of macrin nanoparticles to enable drug loading and control tumor-associated macrophage phenotype.

Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified ß-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of ß-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug's effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24 h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages targeted with R848-loaded macrins, relative to vehicle controls, validating the desired pro-inflammatory reprogramming of TAMs in vivo by cell-targeted drug delivery. Finally, in an intradermal MC38 tumor model, cyclodextrin-modified macrin NPs loaded with immunostimulatory drugs significantly reduced tumor growth. Therefore, efficient and effective repolarization of tumor-associated macrophages to an M1-like phenotype-via drug-loaded macrins-inhibits tumor growth and may be useful as an adjuvant to existing immune checkpoint therapies.

Injectable Granular Hydrogels Enable Avidity-Controlled Biotherapeutic Delivery.

Protein therapeutics represent a rapidly growing class of pharmaceutical agents that hold great promise for the treatment of various diseases such as cancer and autoimmune dysfunction. Conventional systemic delivery approaches, however, result in off-target drug exposure and a short therapeutic half-life, highlighting the need for more localized and controlled delivery. We have developed an affinity-based protein delivery system that uses guest-host complexation between ß-cyclodextrin (CD, host) and adamantane (Ad, guest) to enable sustained localized biomolecule presentation. Hydrogels were formed by the copolymerization of methacrylated CD and methacrylated dextran. Extrusion fragmentation of bulk hydrogels yielded shear-thinning and self-healing granular hydrogels (particle diameter = 32.4 ± 16.4 µm) suitable for minimally invasive delivery and with a high host capacity for the retention of guest-modified proteins. Bovine serum albumin (BSA) was controllably conjugated to Ad via EDC chemistry without affecting the affinity of the Ad moiety for CD (KD = 12.0 ± 1.81 µM; isothermal titration calorimetry). The avidity of Ad-BSA conjugates was directly tunable through the number of guest groups attached, resulting in a fourfold increase in the complex half-life (t1/2 = 5.07 ± 1.23 h, surface plasmon resonance) that enabled a fivefold reduction in protein release at 28 days. Furthermore, we demonstrated that the conjugation of Ad to immunomodulatory cytokines (IL-4, IL-10, and IFNγ) did not detrimentally affect cytokine bioactivity and enabled their sustained release. Our strategy of avidity-controlled delivery of protein-based therapeutics is a promising approach for the sustained local presentation of protein therapeutics and can be applied to numerous biomedical applications.

Mesenchymal Stem Cells Delivered Locally to Ischemia-Reperfused Kidneys via Injectable Hyaluronic Acid Hydrogels Decrease Extracellular Matrix Remodeling 1 Month after Injury in Male Mice.

The translation of stem cell therapies has been hindered by low cell survival and retention rates. Injectable hydrogels enable the site-specific delivery of therapeutic cargo, including cells, to overcome these challenges. We hypothesized that delivery of mesenchymal stem cells (MSC) via shear-thinning and injectable hyaluronic acid (HA) hydrogels would mitigate renal damage following ischemia-reperfusion acute kidney injury. Acute kidney injury (AKI) was induced in mice by bilateral or unilateral ischemia-reperfusion kidney injury. Three days later, mice were treated with MSCs either suspended in media injected intravenously via the tail vein, or injected under the capsule of the left kidney, or MSCs suspended in HA injected under the capsule of the left kidney. Serial measurements of serum and urine biomarkers of renal function and injury, as well as transcutaneous glomerular filtration rate (tGFR) were performed. In vivo optical imaging showed that MSCs localized to both kidneys in a sustained manner after bilateral ischemia and remained within the ipsilateral treated kidney after unilateral ischemic AKI. One month after injury, MSC/HA treatment significantly reduced urinary NGAL compared to controls; it did not significantly reduce markers of fibrosis compared to untreated controls. An analysis of kidney proteomes revealed decreased extracellular matrix remodeling and high overlap with sham proteomes in MSC/HA-treated animals. Hydrogel-assisted MSC delivery shows promise as a therapeutic treatment following acute kidney injury.

T cell-independent eradication of experimental glioma by intravenous TLR7/8-agonist-loaded nanoparticles.

Glioblastoma, the most common and aggressive primary brain tumor type, is considered an immunologically "cold" tumor with sparse infiltration by adaptive immune cells. Immunosuppressive tumor-associated myeloid cells are drivers of tumor progression. Therefore, targeting and reprogramming intratumoral myeloid cells is an appealing therapeutic strategy. Here, we investigate a ß-cyclodextrin nanoparticle (CDNP) formulation encapsulating the Toll-like receptor 7 and 8 (TLR7/8) agonist R848 (CDNP-R848) to reprogram myeloid cells in the glioma microenvironment. We show that intravenous monotherapy with CDNP-R848 induces regression of established syngeneic experimental glioma, resulting in increased survival rates compared with unloaded CDNP controls. Mechanistically, CDNP-R848 treatment reshapes the immunosuppressive tumor microenvironment and orchestrates tumor clearing by pro-inflammatory tumor-associated myeloid cells, independently of T cells and NK cells. Using serial magnetic resonance imaging, we identify a radiomic signature in response to CDNP-R848 treatment and ultrasmall superparamagnetic iron oxide (USPIO) imaging reveals that immunosuppressive macrophage recruitment is reduced by CDNP-R848. In conclusion, CDNP-R848 induces tumor regression in experimental glioma by targeting blood-borne macrophages without requiring adaptive immunity.

Sustained release of drug-loaded nanoparticles from injectable hydrogels enables long-term control of macrophage phenotype.

Injectable hydrogels may be pre-formed through dynamic crosslinks, allowing for injection and subsequent retention in the tissue by shear-thinning and self-healing processes, respectively. These properties enable the site-specific delivery of encapsulated therapeutics; yet, the sustained release of small-molecule drugs and their cell-targeted delivery remains challenging due to their rapid diffusive release and non-specific cellular biodistribution. Herein, we develop an injectable hydrogel system composed of a macrophage-targeted nanoparticle (cyclodextrin nanoparticles, CDNPs) crosslinked by adamantane-modified hyaluronic acid (Ad-HA). The polymer-nanoparticle hydrogel uniquely leverages cyclodextrin's interaction with small molecule drugs to create a spatially discrete drug reservoir and with adamantane to yield dynamic, injectable hydrogels. Through an innovative two-step drug screening approach and examination of 45 immunomodulatory drugs with subsequent in-depth transcriptional profiling of both murine and human macrophages, we identify celastrol as a potent inhibitor of pro-inflammatory (M1-like) behavior that furthermore promotes a reparatory (M2-like) phenotype. Celastrol encapsulation within the polymer-nanoparticle hydrogels permitted shear-thinning injection and sustained release of drug-laden nanoparticles that targeted macrophages to modulate cell behavior for greater than two weeks in vitro. The modular hydrogel system is a promising approach to locally modulate cell-specific phenotype in a range of applications for immunoregenerative medicine.

Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells.

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R- cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor-mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell-targeted immunotherapies and promote antitumor immunity.See related Spotlight by Burrello and de Visser, p. 4.

Therapeutically reprogrammed nutrient signalling enhances nanoparticulate albumin bound drug uptake and efficacy in KRAS-mutant cancer.

Nanoparticulate albumin bound paclitaxel (nab-paclitaxel, nab-PTX) is among the most widely prescribed nanomedicines in clinical use, yet it remains unclear how nanoformulation affects nab-PTX behaviour in the tumour microenvironment. Here, we quantified the biodistribution of the albumin carrier and its chemotherapeutic payload in optically cleared tumours of genetically engineered mouse models, and compared the behaviour of nab-PTX with other clinically relevant nanoparticles. We found that nab-PTX uptake is profoundly and distinctly affected by cancer-cell autonomous RAS signalling, and RAS/RAF/MEK/ERK inhibition blocked its selective delivery and efficacy. In contrast, a targeted screen revealed that IGF1R kinase inhibitors enhance uptake and efficacy of nab-PTX by mimicking glucose deprivation and promoting macropinocytosis via AMPK, a nutrient sensor in cells. This study thus shows how nanoparticulate albumin bound drug efficacy can be therapeutically improved by reprogramming nutrient signalling and enhancing macropinocytosis in cancer cells.

Quantification of Cellular Drug Biodistribution Addresses Challenges in Evaluating in vitro and in vivo Encapsulated Drug Delivery.

Nanoencapsulated drug delivery to solid tumors is a promising approach to overcome pharmacokinetic limitations of therapeutic drugs. However, encapsulation leads to complex drug biodistribution and delivery making analysis of delivery efficacy challenging. As proxies, nanocarrier accumulation or total tumor drug uptake in the tumor are used to evaluate delivery. Yet, these measurements fail to assess delivery of active, released drug to the target, and thus it commonly remains unknown if drug-target occupancy has been achieved. Here, we develop an approach to evaluate the delivery of encapsulated drug to the target, where residual drug target vacancy is measured using a fluorescent drug analog. In vitro measurements reveal that burst release governs drug delivery independent of nanoparticle uptake, and highlight limitations of evaluating nanoencapsulated drug delivery in these models. In vivo, however, our approach captures successful nanoencapsulated delivery, finding that tumor stromal cells drive nanoparticle accumulation and mediate drug delivery to adjacent cancer cells. These results, and generalizable approach, provide a critical advance to evaluate delivery of encapsulated drug to the drug target - the central objective of nanotherapeutics.

Applications of Macrocyclic Host Molecules in Immune Modulation and Therapeutic Delivery.

The immune system plays a central role in the development and progression of human disease. Modulation of the immune response is therefore a critical therapeutic target that enables us to approach some of the most vexing problems in medicine today such as obesity, cancer, viral infection, and autoimmunity. Methods of manipulating the immune system through therapeutic delivery centralize around two common themes: the local delivery of biomaterials to affect the surrounding tissue or the systemic delivery of soluble material systems, often aided by context-specific cell or tissue targeting strategies. In either case, supramolecular interactions enable control of biomaterial composition, structure, and behavior at the molecular-scale; through rational biomaterial design, the realization of next-generation immunotherapeutics and immunotheranostics is therefore made possible. This brief review highlights methods of harnessing macromolecular interaction for immunotherapeutic applications, with an emphasis on modes of drug delivery.

Myeloid Cell-Targeted Nanocarriers Efficiently Inhibit Cellular Inhibitor of Apoptosis for Cancer Immunotherapy.

Immune-checkpoint blockers can promote sustained clinical responses in a subset of cancer patients. Recent research has shown that a subpopulation of tumor-infiltrating dendritic cells functions as gatekeepers, sensitizing tumors to anti-PD-1 treatment via production of interleukin-12 (IL-12). Hypothesizing that myeloid cell-targeted nanomaterials could be used to deliver small-molecule IL-12 inducers, we performed high-content image-based screening to identify the most efficacious small-molecule compounds. Using one lead candidate, LCL161, we created a myeloid-targeted nanoformulation that induced IL-12 production in intratumoral myeloid cells in vivo, slowed tumor growth as a monotherapy, and had no significant systemic toxicity. These results pave the way for developing combination immunotherapeutics by harnessing IL-12 production for immunostimulation.

Injectable Shear-Thinning Hydrogels Prevent Ischemic Mitral Regurgitation and Normalize Ventricular Flow Dynamics.

Injectable hydrogels are known to attenuate left-ventricular (LV) remodeling following myocardial infarction (MI), dependent on material mechanical properties. The effect of hydrogel injection on ischemic mitral regurgitation (IMR) resultant from LV remodeling remains relatively unexplored. This study uses multiple imaging methods to evaluate the efficacy of injectable hydrogels with tunable modulus to prevent post-MI development of IMR. Posterolateral MI was induced in 20 sheep with subsequent epicardial injection of saline (control (MI); n = 7), soft hydrogel (guest-host crosslinking, modulus <1 kPa, n = 7), or stiff hydrogel (dual-crosslinking, modulus = 41.4 ± 4.3 kPa, n = 6) within the infarct region and 8-week follow-up. IMR and valve geometry were assessed by echocardiography. LV geometry (long-axis dimension, posterior chordae length) and ventricular flow dynamics were assessed by magnetic resonance imaging. IMR developed in MI controls at 8 weeks and was attenuated with hydrogel treatment (IMR grade for MI: 1.86 ± 0.69; guest-host crosslinking: 1.29 ± 1.11; dual-crosslinking: 0.50 ± 0.55, P = 0.02 vs MI). Tethering of the posterior leaflet increased in MI controls, but not with stiff hydrogel treatment. Across cohorts, IMR was correlated with changes in the long-axis dimension (Spearman R = 0.77) and posterior chordae length (Spearman R = 0.64). Intraventricular flow dynamics were highly disturbed in MI controls, but stiff hydrogel treatment normalized flow patterns and reduced the prevalence of large (≥2+ MR, >5 mL) regurgitant volumes. Injectable hydrogels attenuated subvalvular remodeling and leaflet tethering, preventing IMR development and normalizing LV flow dynamics. Hydrogels with a supraphysiological modulus yielded best outcomes.

Development of Adamantane-Conjugated TLR7/8 Agonists for Supramolecular Delivery and Cancer Immunotherapy.

Tumor-associated macrophages (TAMs) are often abundant in solid cancers, assuming an immunosuppressive (M2-like) phenotype which supports tumor growth and immune escape. Recent methods have focused on identification of means (e.g., drugs, nanomaterials) that polarize TAMs to a tumor suppressive (M1-like) phenotype; however, reducing the systemic side effects of these therapies and enabling their delivery to TAMs has remained a challenge. Methods: Here, we develop R848-Ad, an adamantane-modified derivative of the toll-like receptor (TLR) 7/8 agonist resiquimod (R848) through iterative drug screening against reporter cell lines. The adamantane undergoes guest-host interaction with cyclodextrin nanoparticles (CDNPs), enabling drug loading under aqueous conditions and TAM-targeted drug delivery. Therapeutic efficacy and systemic side effects were examined in a murine MC38 cancer model. Results: R848-Ad retained macrophage polarizing activity through agonization of TLR7/8, and the adamantane moiety improved drug affinity for the CDNP. In preclinical studies, nanoformulated R848-Ad resulted in a drastic reduction in measurable systemic effects (loss of body weight) relative to similarly formulated R848 alone while arresting tumor growth. Conclusions: The findings demonstrate the ability of strong nanoparticle-drug interactions to limit systemic toxicity of TLR agonists while simultaneously maintaining therapeutic efficacy.

Screening for new macrophage therapeutics.

Myeloid derived macrophages play a key role in many human diseases, and their therapeutic modulation via pharmacological means is receiving considerable attention. Of particular interest is the fact that these cells are i) dynamic phenotypes well suited to therapeutic manipulation and ii) phagocytic, allowing them to be efficiently targeted with nanoformulations. However, it is important to consider that macrophages represent heterogeneous populations of subtypes with often competing biological behaviors and functions. In order to develop next generation therapeutics, it is therefore essential to screen for biological effects through a combination of in vitro and in vivo assays. Here, we review the state-of-the-art techniques, including both cell based screens and in vivo imaging tools that have been developed for assessment of macrophage phenotype. We conclude with a forward-looking perspective on the growing need for noninvasive macrophage assessment and laboratory assays to be put into clinical practice and the potential broader impact of myeloid-targeted therapeutics.

Cathelicidin Related Antimicrobial Peptide (CRAMP) Enhances Bone Marrow Cell Retention and Attenuates Cardiac Dysfunction in a Mouse Model of Myocardial Infarction.

BACKGROUND: Acute myocardial infarction (MI) and the ensuing ischemic heart disease are approaching epidemic state. Unfortunately, no definitive therapies are available and human regenerative therapies have conflicting results. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Cathelicidin related antimicrobial peptides (CRAMPs) enhance chemotactic responsiveness of BMSPCs to low SDF-1 gradients, suggesting a potential role in BMSPCs engraftment. Here, we assessed the therapeutic efficacy of CRAMPs in the context of BMSPCs recruitment and retention via intracardiac delivery of CRAMP-treated BMSPCs or CRAMP-releasing hydrogels (HG) post-AMI. METHODS: For cell transplantation experiments, mice were randomized into 3 groups: MI followed by injection of PBS, BMMNCs alone, and BMMNCs pre-incubated with CRAMP. During the in vivo HG studies, BM GFP chimera mice were randomized into 4 groups: MI followed by injection of HG alone, HG + SDF-1, HG + CRAMP, HG + SDF-1 + CRAMP. Changes in cardiac function at 5 weeks after MI were assessed using echocardiography. Angiogenesis was assessed using isolectin staining for capillary density. RESULTS: Mice treated with BMMNCs pre-incubated with CRAMP had smaller scars, enhanced cardiac recovery and less adverse remodeling. Histologically, this group had higher capillary density. Similarly, sustained CRAMP release from hydrogels enhanced the therapeutic effect of SDF-1, leading to enhanced functional recovery, smaller scar size and higher capillary density. CONCLUSION: Cathelicidins enhance BMMNC retention and recruitment after intramyocardial administration post-AMI resulting in improvements in heart physiology and recovery. Therapies employing these strategies may represent an attractive method for improving outcomes of regenerative therapies in human studies.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.

TLR7/8-agonist-loaded nanoparticles promote the polarization of tumour-associated macrophages to enhance cancer immunotherapy.

Tumour-associated macrophages are abundant in many cancers, and often display an immune-suppressive M2-like phenotype that fosters tumour growth and promotes resistance to therapy. Yet, macrophages are highly plastic and can also acquire an anti-tumorigenic M1-like phenotype. Here, we show that R848, an agonist of the toll-like receptors TLR7 and TLR8 identified in a morphometric-based screen, is a potent driver of the M1 phenotype in vitro and that R848-loaded ß-cyclodextrin nanoparticles (CDNP-R848) lead to efficient drug delivery to tumour-associated macrophages in vivo. As a monotherapy, the administration of CDNP-R848 in multiple tumour models in mice altered the functional orientation of the tumour immune microenvironment towards an M1 phenotype, leading to controlled tumour growth and protecting the animals against tumour rechallenge. When used in combination with the immune checkpoint inhibitor anti-PD-1, we observed improved immunotherapy response rates, including in a tumour model resistant to anti-PD-1 therapy alone. Our findings demonstrate the ability of rationally engineered drug-nanoparticle combinations to efficiently modulate tumour-associated macrophages for cancer immunotherapy.

Injectable Shear-Thinning Hydrogels for Minimally Invasive Delivery to Infarcted Myocardium to Limit Left Ventricular Remodeling.

BACKGROUND: Injectable, acellular biomaterials hold promise to limit left ventricular remodeling and heart failure precipitated by infarction through bulking or stiffening the infarct region. A material with tunable properties (eg, mechanics, degradation) that can be delivered percutaneously has not yet been demonstrated. Catheter-deliverable soft hydrogels with in vivo stiffening to enhance therapeutic efficacy achieve these requirements. METHODS AND RESULTS: We developed a hyaluronic acid hydrogel that uses a tandem crosslinking approach, where the first crosslinking (guest-host) enabled injection and localized retention of a soft (<1 kPa) hydrogel. A second crosslinking reaction (dual-crosslinking) stiffened the hydrogel (41.4±4.3 kPa) after injection. Posterolateral infarcts were investigated in an ovine model (n≥6 per group), with injection of saline (myocardial infarction control), guest-host hydrogels, or dual-crosslinking hydrogels. Computational (day 1), histological (1 day, 8 weeks), morphological, and functional (0, 2, and 8 weeks) outcomes were evaluated. Finite-element modeling projected myofiber stress reduction (>50%; P<0.001) with dual-crosslinking but not guest-host injection. Remodeling, assessed by infarct thickness and left ventricular volume, was mitigated by hydrogel treatment. Ejection fraction was improved, relative to myocardial infarction at 8 weeks, with dual-crosslinking (37% improvement; P=0.014) and guest-host (15% improvement; P=0.058) treatments. Percutaneous delivery via endocardial injection was investigated with fluoroscopic and echocardiographic guidance, with delivery visualized by magnetic resonance imaging. CONCLUSIONS: A percutaneous delivered hydrogel system was developed, and hydrogels with increased stiffness were found to be most effective in ameliorating left ventricular remodeling and preserving function. Ultimately, engineered systems such as these have the potential to provide effective clinical options to limit remodeling in patients after infarction.

Delivery of interleukin-10 via injectable hydrogels improves renal outcomes and reduces systemic inflammation following ischemic acute kidney injury in mice.

Injectable hydrogels can be used to deliver drugs in situ over a sustained period of time. We hypothesized that sustained delivery of interleukin-10 (IL-10) following acute kidney injury (AKI) would mitigate the local and systemic proinflammatory cascade induced by AKI and reduce subsequent fibrosis. Wild-type C57BL/6 mice underwent ischemia-reperfusion AKI with avertin anesthesia. Three days later, mice were treated with either hyaluronic acid injectable hydrogel with or without IL-10, or IL-10 suspended in saline, injected under the capsule of the left kidney, or hydrogel with IL-10 injected subcutaneously. Untreated AKI served as controls. Serial in vivo optical imaging tracked the location and degradation of the hydrogel over time. Kidney function was assessed serially. Animals were killed 28 days following AKI and the following were evaluated: serum IL-6, lung inflammation, urine neutrophil gelatinase-associated lipocalin, and renal histology for fibroblast activity, collagen type III deposition and fibrosis via Picrosirius Red staining and second harmonic imaging. Our model shows persistent systemic inflammation, and renal inflammation and fibrosis 28 days following AKI. The hydrogels are biocompatible and reduced serum IL-6 and renal collagen type III 28 days following AKI even when delivered without IL-10. Treatment with IL-10 reduced renal and systemic inflammation, regardless of whether the IL-10 was delivered in a sustained manner via the injectable hydrogel under the left kidney capsule, as a bolus injection via saline under the left kidney capsule, or via the injectable hydrogel subcutaneously. Injectable hydrogels are suitable for local drug delivery following renal injury, are biocompatible, and help mitigate local and systemic inflammation.

Injectable shear-thinning hydrogels used to deliver endothelial progenitor cells, enhance cell engraftment, and improve ischemic myocardium.

OBJECTIVES: The clinical translation of cell-based therapies for ischemic heart disease has been limited because of low cell retention (<1%) within, and poor targeting to, ischemic myocardium. To address these issues, we developed an injectable hyaluronic acid (HA) shear-thinning hydrogel (STG) and endothelial progenitor cell (EPC) construct (STG-EPC). The STG assembles as a result of interactions of adamantine- and ß-cyclodextrin-modified HA. It is shear-thinning to permit delivery via a syringe, and self-heals upon injection within the ischemic myocardium. This directed therapy to the ischemic myocardial border zone enables direct cell delivery to address adverse remodeling after myocardial infarction. We hypothesize that this system will enhance vasculogenesis to improve myocardial stabilization in the context of a clinically translatable therapy. METHODS: Endothelial progenitor cells (DiLDL(+) VEGFR2(+) CD34(+)) were harvested from adult male rats, cultured, and suspended in the STG. In vitro viability was quantified using a live-dead stain of EPCs. The STG-EPC constructs were injected at the border zone of ischemic rat myocardium after acute myocardial infarction (left anterior descending coronary artery ligation). The migration of the enhanced green fluorescent proteins from the construct to ischemic myocardium was analyzed using fluorescent microscopy. Vasculogenesis, myocardial remodeling, and hemodynamic function were analyzed in 4 groups: control (phosphate buffered saline injection); intramyocardial injection of EPCs alone; injection of the STG alone; and treatment with the STG-EPC construct. Hemodynamics and ventricular geometry were quantified using echocardiography and Doppler flow analysis. RESULTS: Endothelial progenitor cells demonstrated viability within the STG. A marked increase in EPC engraftment was observed 1-week postinjection within the treated myocardium with gel delivery, compared with EPC injection alone (17.2 ± 0.8 cells per high power field (HPF) vs 3.5 cells ± 1.3 cells per HPF, P = .0002). A statistically significant increase in vasculogenesis was noted with the STG-EPC construct (15.3 ± 5.8 vessels per HPF), compared with the control (P < .0001), EPC (P < .0001), and STG (P < .0001) groups. Statistically significant improvements in ventricular function, scar fraction, and geometry were noted after STG-EPC treatment compared with the control. CONCLUSIONS: A novel injectable shear-thinning HA hydrogel seeded with EPCs enhanced cell retention and vasculogenesis after delivery to ischemic myocardium. This therapy limited adverse myocardial remodeling while preserving contractility.