PEGylation enables subcutaneously administered nanoparticles to induce antigen-specific immune tolerance.

The development of nanomaterials to induce antigen-specific immune tolerance has shown promise for treating autoimmune diseases. While PEGylation has been widely used to reduce host immune responses to nanomaterials, its tolerogenic potential has not been reported. Here, we report for the first time that a subcutaneous injection of PEGylated poly(lactide-co-glycolide) (PLGA) nanoparticles containing auto-antigen peptide MOG35-55 without any tolerogenic drugs is sufficient to dramatically ameliorate symptoms after disease onset in an antigen-specific manner in a mouse model of multiple sclerosis. Neither free MOG35-55 nor particles without PEG exhibit this efficacy. Interestingly, mechanistic studies indicate that PEGylation of nanoparticles does not reduce dendritic cell activation through direct nanoparticle-cell interactions. Instead, PEGylated nanoparticles induce lower complement activation, neutrophil recruitment, and co-stimulatory molecule expression on dendritic cells around the injection sitecompared to non-PEGylated PLGA nanoparticles, creating a more tolerogenic microenvironment in vivo. We further demonstrate that the locally recruited dendritic cells traffic to lymphoid organs to induce T cell tolerance. These results highlight the critical role of surface properties of nanomaterials in inducing immune tolerance via subcutaneous administration.

Engineering Protein Delivery Depots for Cancer Immunotherapy.

Cancer immunotherapy that induces a tumor-specific immune response for cancer eradication has received tremendous attention. To enhance the immunotherapeutic effects, many drug delivery strategies have been developed to overcome the physiological barriers as well as to reduce toxicity. For example, intratumoral or peritumoral administration of injectable depot formulations can directly target tumor sites for immunotherapy. Compared with systemic administration of therapeutics, this strategy has minimal side effects. Such local treatment can also trigger a systemic immune response for inhibiting tumor metastasis. This Topical Review highlights the recent studies on depot-mediated delivery of protein/peptide therapeutics for cancer immunotherapy. Further opportunities and challenges in this field are also discussed.

A new class of biological materials: Cell membrane-derived hydrogel scaffolds.

Biological materials are superior to synthetic biomaterials in biocompatibility and active interactions with cells. Here, a new class of biological materials, cell membrane-derived hydrogel scaffolds are reported for harnessing these advantages. To form macroporous scaffolds, vesicles derived from red blood cell membranes (RBCMs) are chemically crosslinked via cryogelation. The RBCM scaffolds with a pore size of around 70 µm are soft and injectable. Highly biocompatible scaffolds are typically made of superhydrophilic polymers and lack the ability to encapsulate and release hydrophobic drugs in a controlled manner. However, hydrophobic molecules can be efficiently encapsulated inside RBCM scaffolds and be sustainedly released. RBCM scaffolds show low neutrophil infiltration after subcutaneous injection in mice, and a significantly higher number of infiltrated macrophages than methacrylate alginate (MA-alginate) scaffolds. According to gene expression and surface markers, these macrophages have an M2-like phenotype, which is anti-inflammatory and immune suppressive. There are also higher percentages of macrophages presenting immunosuppressive PD-L1 in RBCM-scaffolds than in MA-alginate scaffolds. Interestingly, the concentrations of anti-inflammatory cytokine, IL-10 in both types of scaffolds are higher than those in normal organ tissues. This study sheds light on cell membrane-derived hydrogels, which can actively modulate cells in unique ways unavailable to existing hydrogel scaffolds.

Dense and Dynamic Polyethylene Glycol Shells Cloak Nanoparticles from Uptake by Liver Endothelial Cells for Long Blood Circulation.

Research into long-circulating nanoparticles has in the past focused on reducing their clearance by macrophages. By engineering a hierarchical polyethylene glycol (PEG) structure on nanoparticle surfaces, we revealed an alternative mechanism to enhance nanoparticle blood circulation. The conjugation of a second PEG layer at a density close to but lower than the mushroom-to-brush transition regime on conventional PEGylated nanoparticles dramatically prolongs their blood circulation via reduced nanoparticle uptake by non-Kupffer cells in the liver, especially liver sinusoidal endothelial cells. Our study also disclosed that the dynamic outer PEG layer reduces protein binding affinity to nanoparticles, although not the total number of adsorbed proteins. These effects of the outer PEG layer diminish in the higher density regime. Therefore, our results suggest that the dynamic topographical structure of nanoparticles is an important factor in governing their fate in vivo. Taken together, this study advances our understanding of nanoparticle blood circulation and provides a facile approach for generating long circulating nanoparticles.

Cell Membrane Bioconjugation and Membrane-Derived Nanomaterials for Immunotherapy.

Cell membrane engineering, including live cell membrane bioconjugation and cell membrane-derived nanomaterials is a highly promising strategy to modulate immune responses for treating diseases. Many cell membrane engineering methods have potential for translation for human clinical use in the near future. In this Topical Review, we summarize the cell membrane conjugation strategies that have been investigated for cancer immunotherapy, the prevention of immune rejection to donor cells and tissues, and the induction of antigen-specific tolerance in autoimmune diseases. Additionally, cell membrane-derived or membrane-coated nanomaterials are an emerging class of nanomaterials that is attracting significant attention in the field of nanomedicine. Some of these nanomaterials have been employed to elicit immune responses against cancer, toxins, and bacteria, although their application in establishing immune tolerance has not been explored. In addition to discussing potential problems, we provide our perspectives for promising future directions.

Targeting Chondroitin Sulfate Glycosaminoglycans to Treat Cardiac Fibrosis in Pathological Remodeling.

BACKGROUND: Heart failure is a leading cause of mortality and morbidity, and the search for novel therapeutic approaches continues. In the monogenic disease mucopolysaccharidosis VI, loss-of-function mutations in arylsulfatase B lead to myocardial accumulation of chondroitin sulfate (CS) glycosaminoglycans, manifesting as myriad cardiac symptoms. Here, we studied changes in myocardial CS in nonmucopolysaccharidosis failing hearts and assessed its generic role in pathological cardiac remodeling. METHODS: Healthy and diseased human and rat left ventricles were subjected to histological and immunostaining methods to analyze glycosaminoglycan distribution. Glycosaminoglycans were extracted and analyzed for quantitative and compositional changes with Alcian blue assay and liquid chromatography-mass spectrometry. Expression changes in 20 CS-related genes were studied in 3 primary human cardiac cell types and THP-1-derived macrophages under each of 9 in vitro stimulatory conditions. In 2 rat models of pathological remodeling induced by transverse aortic constriction or isoprenaline infusion, recombinant human arylsulfatase B (rhASB), clinically used as enzyme replacement therapy in mucopolysaccharidosis VI, was administered intravenously for 7 or 5 weeks, respectively. Cardiac function, myocardial fibrosis, and inflammation were assessed by echocardiography and histology. CS-interacting molecules were assessed with surface plasmon resonance, and a mechanism of action was verified in vitro. RESULTS: Failing human hearts displayed significant perivascular and interstitial CS accumulation, particularly in regions of intense fibrosis. Relative composition of CS disaccharides remained unchanged. Transforming growth factor-ß induced CS upregulation in cardiac fibroblasts. CS accumulation was also observed in both the pressure-overload and the isoprenaline models of pathological remodeling in rats. Early treatment with rhASB in the transverse aortic constriction model and delayed treatment in the isoprenaline model proved rhASB to be effective at preventing cardiac deterioration and augmenting functional recovery. Functional improvement was accompanied by reduced myocardial inflammation and overall fibrosis. Tumor necrosis factor-α was identified as a direct binding partner of CS glycosaminoglycan chains, and rhASB reduced tumor necrosis factor-α-induced inflammatory gene activation in vitro in endothelial cells and macrophages. CONCLUSIONS: CS glycosaminoglycans accumulate during cardiac pathological remodeling and mediate myocardial inflammation and fibrosis. rhASB targets CS effectively as a novel therapeutic approach for the treatment of heart failure.

Cell membrane coating for reducing nanoparticle-induced inflammatory responses to scaffold constructs.

The controlled release of therapeutics from micro or nanoparticles has been well-studied. Incorporation of these particles inside biomaterial scaffolds is promising for tissue regeneration and immune modulation. However, these particles may induce inflammatory and foreign body responses to scaffold constructs, limiting their applications. Here we show that widely used poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) formed by double emulsion dramatically increased neutrophil infiltration and pro-inflammatory cytokines in alginate scaffolds 1 day after the subcutaneous injection of the scaffolds into mice. The coating of red blood cell (RBC) membranes on PLGA NPs completely eliminated these short-term inflammatory responses. For a longer term of 10 days, neither PLGA NPs nor RBC membrane-coated nanoparticles exerted a significant effect on the infiltration of neutrophils or macrophages in alginate scaffolds possibly due to the degradation and/or clearance of nanoparticles by infiltrating cells by that time. Despite the extensive exploration of cell membrane-coated nanoparticles, our study is the first to investigate the effects of cell membrane coating on foreign body reaction to nanoparticles. Harnessing the natural biocompatibility of cell membranes, our strategy of anti-inflammatory protection for scaffolds may be pivotal for many applications, such as those relying on the recruitment of stem cells and/or progenitor cells to scaffolds.

Single cardiomyocyte nuclear transcriptomes reveal a lincRNA-regulated de-differentiation and cell cycle stress-response in vivo.

Cardiac regeneration may revolutionize treatment for heart failure but endogenous progenitor-derived cardiomyocytes in the adult mammalian heart are few and pre-existing adult cardiomyocytes divide only at very low rates. Although candidate genes that control cardiomyocyte cell cycle re-entry have been implicated, expression heterogeneity in the cardiomyocyte stress-response has never been explored. Here, we show by single nuclear RNA-sequencing of cardiomyocytes from both mouse and human failing, and non-failing adult hearts that sub-populations of cardiomyocytes upregulate cell cycle activators and inhibitors consequent to the stress-response in vivo. We characterize these subgroups by weighted gene co-expression network analysis and discover long intergenic non-coding RNAs (lincRNA) as key nodal regulators. KD of nodal lincRNAs affects expression levels of genes related to dedifferentiation and cell cycle, within the same gene regulatory network. Our study reveals that sub-populations of adult cardiomyocytes may have a unique endogenous potential for cardiac regeneration in vivo.Adult mammalian cardiomyocytes are predominantly binucleated and unable to divide. Using single nuclear RNA-sequencing of cardiomyocytes from mouse and human failing and non-failing adult hearts, See et al. show that some cardiomyocytes respond to stress by dedifferentiation and cell cycle re-entry regulated by lncRNAs.

A landscape of circular RNA expression in the human heart.

Aims: Circular RNA (circRNA) is a newly validated class of single-stranded RNA, ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types, genome-wide circRNA expression in the heart has yet to be described in detail. Methods and results: We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts, 25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools, we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse, respectively. Their abundance generally correlates with the abundance of their cognate linear RNA, but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN), RYR2, and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms, the majority (83%) of which originates from the I-band domain. Finally, we confirmed the expression of selected cardiac circRNA by RT-PCR, Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. Conclusions: Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA.

Non-genetic engineering of cells for drug delivery and cell-based therapy.

Cell-based therapy is a promising modality to address many unmet medical needs. In addition to genetic engineering, material-based, biochemical, and physical science-based approaches have emerged as novel approaches to modify cells. Non-genetic engineering of cells has been applied in delivering therapeutics to tissues, homing of cells to the bone marrow or inflammatory tissues, cancer imaging, immunotherapy, and remotely controlling cellular functions. This new strategy has unique advantages in disease therapy and is complementary to existing gene-based cell engineering approaches. A better understanding of cellular systems and different engineering methods will allow us to better exploit engineered cells in biomedicine. Here, we review non-genetic cell engineering techniques and applications of engineered cells, discuss the pros and cons of different methods, and provide our perspectives on future research directions.

Structural elucidation of cell membrane-derived nanoparticles using molecular probes.

Cell membrane-derived nanoparticles (CNPs) are a novel class of materials and are superior to synthetic nanomaterials in certain aspects due to their biological origin. Although their medical applications have been actively explored, the fundamental structure of CNPs is rarely studied. For example, the membrane orientation of CNPs is critical for their pharmacokinetics, but the previous characterizations were mostly qualitative. Herein, we report a method to quantitatively study membrane orientation of CNPs by using a 6-FAM ssDNA probe and a BHQ1 ssDNA quencher with a complementary sequence. This method utilizes specific DNA hybridization and fluorescence resonance energy transfer between 6-FAM and BHQ1. When ssDNA probes are conjugated on cell membranes, the probe marks the outer leaflet of cell membranes. The fluorescence intensities of particle solutions before and after the addition of the ssDNA quencher can be measured to quantitatively determine the fraction of CNPs with a correct outside-out (also called right-side-out) membrane orientation. Red blood cell membrane-derived nanoparticles (RBC-NPs) were fabricated and determined to have an 84% correct orientation. The quenching of membrane-bound nitrobenzoxadiazole (NBD) was used to study the permeability of RBC-NPs. It was found that RBC-NPs have a significantly higher permeability to the NBD quencher, dithionite ions, compared to live cells and egg PC/cholesterol liposomes. The ubiquitous methods using molecular probes can elucidate some structural properties of CNPs in general, enabling direct comparisons among CNPs that are derived from different cells and convenient optimization of particle fabrication.

Cellometer vision as an alternative to flow cytometry for cell cycle analysis, mitochondrial potential, and immunophenotyping.

Cell phenotyping and cell cycle analysis are two commonly used assays in both clinical diagnosis and biomedical research. Cell phenotyping by identifying different biomarkers is essential for the diagnosis of hematologic malignancy, sub-classifying diseases, monitoring response to treatment, predicting prognosis, detecting rare cell populations and residual malignant cells. Cell cycle analysis distinguishes cells in different phases of cell cycle and is often used to determine the cellular response to drugs and biological stimulations. These assays have been traditionally carried out by sensitive fluorescence detection methods such as flow cytometry and laser scanning cytometry for fluorescence-based cell population analysis. However, these instruments remain relatively expensive, large in size, and require a considerable amount of maintenance, which may not be feasible for smaller research groups that do not have access to these equipments or field clinics that require quick diagnostic results on site. Recently, a small portable imaging cytometry system (Cellometer Vision) has been developed by Nexcelom Bioscience LLC (Lawrence, MA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Here we report new applications of the Cellometer imaging cytometry for fluorescence-based cell population analysis and compared them with conventional flow cytometry. Cell population analysis assays such as immunophenotyping, cell cycle, and mitochondrial membrane potential detection methods have not yet been reported for the Cellometer Vision system. Using this imaging cytometry method for fluorescence-based assays that are typically done by flow cytometry offers a quick, simple, and inexpensive alternative method for biomedical research, which may be beneficial for smaller research laboratories and clinics.