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Unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs) have a storied history as agonists for Toll-like receptor 9 (TLR9). CpG ODNs have shown promising antitumor effects in preclinical studies by inducing potent proinflammatory immune responses. However, clinical success has been hindered by inconsistent efficacy and immune-related toxicities caused by systemic exposure to CpG ODNs. We previously identified that glatiramer acetate (GA), an FDA-approved, lysine-rich polypeptide, could complex class B CpG into cationic nanoparticles which persist at the intratumoral injection site while mitigating the induction of systemic proinflammatory cytokines in mouse tumor models. To extend GA applications across subtypes of CpG ODN (class A, B, and C), we evaluated physiochemical properties and identified the immunological signaling of GA and its complexes with different classes of CpG ODNs. We compared the physiochemical characteristics of three types of GA-CpG nanoparticles, followed by assessments of cell uptake efficiency and endolysosomal trafficking. We then performed successive in vitro and in vivo assays to evaluate immunological discrepancies. Complexation with GA preserved the immunological activity of CpG ODN subtypes while encapsulating them into cationic spherical nanoparticles. GA improved the cellular uptake of CpG ODNs, generally increased retention in early endosomes, and amplified immunological responses. A subsequent in vivo experiment confirmed the achievement of potent tumor suppression while mitigating systemic immune-related toxicities. Together, these data help elucidate the noncanonical role of GA to serve as a nucleic acid delivery scaffold that can improve the efficacy and safety of CpG adjuvant for clinical cancer immunotherapy.

Pancreatic islet-reactive B lymphocytes promote Type 1 diabetes (T1D) by presenting an antigen to islet-destructive T cells. Teplizumab, an anti-CD3 monoclonal, delays T1D onset in patients at risk, but additional therapies are needed to prevent the disease entirely. Therefore, bifunctional molecules were designed to selectively inhibit T1D-promoting anti-insulin B cells by conjugating a ligand for the B cell inhibitory receptor CD22 (i.e., CD22L) to insulin, which permit these molecules to concomitantly bind to anti-insulin B cell receptors (BCRs) and CD22. Two prototypes were synthesized: 2:2 insulin-CD22L conjugate on a 4-arm PEG backbone, and 1:1 insulin-CD22L direct conjugate. Transgenic mice (125Tg SD ) expressing anti-insulin BCRs provided cells for in vitro testing. Cells were cultured with constructs for 3 days, then assessed by flow cytometry. Duplicate wells with anti-CD40 simulated T cell help. A 2-insulin 4-arm PEG control caused robust proliferation and activation-induced CD86 upregulation. Anti-CD40 further boosted these effects. This may indicate that BCR-cross-linking occurs when antigens are tethered by the PEG backbone as soluble insulin alone has no effect. Addition of CD22L via the 2:2 insulin-CD22L conjugate restored B cell properties to that of controls without an additional beneficial effect. In contrast, the 1:1 insulin-CD22L direct conjugate significantly reduced anti-insulin B cell proliferation in the presence of anti-CD40. CD22L alone had no effect, and the constructs did not affect the WT B cells. Thus, multivalent antigen constructs tend to activate anti-insulin B cells, while monomeric antigen-CD22L conjugates reduce B cell activation in response to simulated T cell help and reduce pathogenic B cell numbers without harming normal cells. Therefore, monomeric antigen-CD22L conjugates warrant futher study and may be promising candidates for preclinical trials to prevent T1D without inducing immunodeficiency.

Autoimmune diseases are characterized by aberrant immune responses toward self-antigens. Current treatments lack specificity, promoting adverse effects by broadly suppressing the immune system. Therapies that specifically target the immune cells responsible for disease are a compelling strategy to mitigate adverse effects. Multivalent formats that display numerous binding epitopes off a single scaffold may enable selective immunomodulation by eliciting signals through pathways unique to the targeted immune cells. However, the architecture of multivalent immunotherapies can vary widely, and there is limited clinical data with which to evaluate their efficacy. Here, we set forth to review the architectural properties and functional mechanisms afforded by multivalent ligands and evaluate four multivalent scaffolds that address autoimmunity by altering B cell signaling pathways. First, we address both synthetic and natural polymer backbones functionalized with a variety of small molecule, peptide, and protein ligands for probing the effects of valency and costimulation. Then, we review nanoparticles composed entirely from immune signals which have been shown to be efficacious. Lastly, we outline multivalent liposomal nanoparticles capable of displaying high numbers of protein antigens. Taken together, these examples highlight the versatility and desirability of multivalent ligands for immunomodulation and illuminate strengths and weaknesses of multivalent scaffolds for treating autoimmunity.

Three-dimensional force plates are important tools for biomechanics discovery and sports performance practice. However, currently, available 3D force plates lack portability and are often cost-prohibitive. To address this, a recently discovered 3D force sensor technology was used in the fabrication of a prototype force plate. Thirteen participants performed bodyweight and weighted lunges and squats on the prototype force plate and a standard 3D force plate positioned in series to compare forces measured by both force plates and validate the technology. For the lunges, there was excellent agreement between the experimental force plate and the standard force plate in the X-, Y-, and Z-axes (r = 0.950-0.999, p < 0.001). For the squats, there was excellent agreement between the force plates in the Z-axis (r = 0.996, p < 0.001). Across axes and movements, root mean square error (RMSE) ranged from 1.17% to 5.36% between force plates. Although the current prototype force plate is limited in sampling rate, the low RMSEs and extremely high agreement in peak forces provide confidence the novel force sensors have utility in constructing cost-effective and versatile use-case 3D force plates.

Next-generation cancer immunotherapies may utilize immunostimulants to selectively activate the host immune system against tumor cells. Checkpoint inhibitors (CPIs) like anti-PD1/PDL-1 that inhibit immunosuppression have shown unprecedented success but are only effective in the 20-30% of patients that possess an already "hot" (immunogenic) tumor. In this regard, intratumoral (IT) injection of immunostimulants is a promising approach since they can work synergistically with CPIs to overcome the resistance to immunotherapies by inducing immune stimulation in the tumor. One such immunostimulant is granulocyte macrophage-colony-stimulating factor (GMCSF) that functions by recruiting and activating antigen-presenting cells (dendritic cells) in the tumor, thereby initiating anti-tumor immune responses. However, key problems with GMCSF are lack of efficacy and the risk of systemic toxicity caused by the leakage of GMCSF from the tumor tissue. We have designed tumor-retentive versions of GMCSF that are safe yet potent immunostimulants for the local treatment of solid tumors. The engineered GMCSFs (eGMCSF) were synthesized by recombinantly fusing tumor-ECM (extracellular matrix) binding peptides to GMCSF. The eGMCSFs exhibited enhanced tumor binding and potent immunological activity in vitro and in vivo. Upon IT administration, the tumor-retentive eGMCSFs persisted in the tumor, thereby alleviating systemic toxicity, and elicited localized immune activation to effectively turn an unresponsive immunologically "cold" tumor "hot".

CpG oligodeoxynucleotides are toll-like receptor 9 agonists capable of inducing potent pro-inflammatory immune responses. Although CpG oligodeoxynucleotides have shown promising antitumor effects, their systemic activity can trigger immune-related toxicity, limiting therapeutic application. We previously identified glatiramer acetate (GA), a cationic polypeptide approved for the treatment of relapsing-remitting multiple sclerosis, as an intratumoral delivery agent capable of complexing with CpG, thereby pinning it to the injection site and limiting systemic exposure. Here, we investigated whether the combination of CpG or GA-CpG polyplexes and intraperitoneal anti-PD-1 therapy would result in synergistic efficacy in AT84 and CT26 murine syngeneic models of head and neck and colon cancers, respectively. In both AT84 and CT26 tumor models, intratumoral CpG or GA-CpG treatment similarly suppressed tumor growth, but the efficacy was not amplified with anti-PD-1. Nevertheless, combination treatment increased cytotoxic T cell, helper T cell, and natural killer cell infiltration into AT84 tumors. Surprisingly, the combination of intratumoral GA and intraperitoneal anti-PD-1 treatment resulted in elevated systemic GM-CSF and IL-2 cytokine levels and demonstrated synergistic antitumor effects in the CT26 mouse tumor model. Moreover, tumors that responded most significantly to anti-PD-1 plus GA treatment showed increased markers of infiltration of CD4 + T cells and natural killer cells. Combinations of intratumoral GA or GA-CpG polyplexes with anti-PD-1 treatment warrant further investigation as combination cancer immunotherapy strategies.

Kifunensine is a known inhibitor of type I α-mannosidase enzymes and has been shown to have therapeutic potential for a variety of diseases and application in the expression of high-mannose N-glycan bearing glycoproteins; however, the compound's hydrophilic nature limits its efficacy. We previously synthesized two hydrophobic acylated derivatives of kifunensine, namely, JDW-II-004 and JDW-II-010, and found that these compounds were over 75-fold more potent than kifunensine. Here we explored the effects of these compounds on different mice and human B cells, and we demonstrate that they affected the cells in a similar fashion to kifunensine, further demonstrating their functional equivalence to kifunensine in assays utilizing primary cells. Specifically, a dose-dependent increase in the formation of high-mannose N-glycans decorated glycoproteins were observed upon treatment with kifunensine, JDW-II-004, and JDW-II-010, but greater potency was observed with the acylated derivatives. Treatment with kifunensine or the acylated derivatives also resulted in impaired B-cell receptor (BCR) signaling of the primary mouse B cells; however, primary human B cells treated with kifunensine or JDW-II-004 did not affect BCR signaling, while a modest increase in BCR signaling was observed upon treatment with JDW-010. Nevertheless, these findings demonstrate that the hydrophobic acylated derivatives of kifunensine can help overcome the mass-transfer limitations of the parent compound, and they may have applications for the treatment of ERAD-related diseases or prove to be more cost-effective alternatives for the generation and production of high-mannose N-glycan bearing glycoproteins., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH 6 ). The synthesis of Fc-insulin 2 from Fc-LPETGGH 6 was evaluated with direct sortase-mediated ligation (SML) and two chemoenzymatic strategies. Direct SML was performed with triglycine-insulin, and chemoenzymatic strategies used to SML fuse either triglycine-azide or triglycine-DBCO prior to linking insulin with copper-catalyzed or strain-promoted azidealkyne cycloaddition. Reaction conditions were optimized by evaluating reagent concentrations, relative equivalents, temperature, and time. Direct SML provided the most effective reaction yields, converting 60-70% of Fc-LPETGGH 6 to Fc-insulin 2 , whereas our optimized chemoenzymatic synthesis converted 30-40% of Fc-LPETGGH 6 to Fc-insulin 2 . Here we show that SML is a practical and efficient method to synthesize Fc fusions and provide an optimized pathway for fusion drug synthesis., Competing Interests: The authors declare no competing financial interest.

Mechanical stimuli have been shown to play a large role in cellular behavior, including cellular growth, differentiation, morphology, homeostasis, and disease. Therefore, developing bioreactor systems that can create complex mechanical environments for both tissue engineering and disease modeling drug screening is appealing. However, many of existing systems are restricted because of their bulky size with external force generators, destructive microenvironment control, and low throughput. These shortcomings have preceded to the utilization of magnetic stimuli responsive materials, given their untethered, fast, and tunable actuation potential at both the microscale and macroscale level, for seamless integration into cell culture wells and microfluidic systems. Nevertheless, magnetic soft materials for cell culture have been limited due to the inability to develop well-defined 3D structures for more complex and physiological relevant mechanical actuation. Herein, we introduce a facile fabrication process to develop magnetic-PDMS (polydimethylsiloxane) porous composite designs with both well-defined and controllable microlevel and macrolevel features to dynamically manipulate 3D cell-laden gel at the scale. The intrinsic stiffness of the magnetic-PDMS porous composites is also modulated to control the deformation potential to mimic physiological relevant strain levels, with 2.89-11% observed in magnetic actuation studies. High cell viability was achieved with the culturing of both human adipose stem cells (hADMSCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in 3D cell-laden gel interfaced with the magnetic-PDMS porous composite. Also, the highly interconnected porous network of the magnetic-PDMS composites facilitated free diffusion throughout the porous structure showcasing the potential of a multisurface contact 3D porous magnetic structure in both reservoir and 96-well plate insert designs for more complex dynamic mechanical actuation. In conclusion, these studies provide a means for establishing a biocompatible, tunable magnetic-PDMS porous composite with fast and programmable dynamic strain potential making it a suitable platform for high-throughput, dynamic 3D cell culture.

Cancer immunotherapy aims to stimulate immune cells to recognize and attack tumor tissue. The immunostimulatory polyanions polyI:C and CpG induce potent pro-inflammatory immune responses as TLR3 and TLR9 agonists, respectively. Clinical trials of TLR agonists, however, have been fraught with immune-related adverse events, even when injecting intratumorally in an effort to minimize systemic exposure. We identified Glatiramer Acetate (GA), a positively-charged polypeptide approved for multiple sclerosis, as a delivery agent capable of complexing with polyI:C or CpG and reducing the mobility of these actives. Small nanoparticles termed polyplexes form when mixing positively-charged GA and negatively-charged immunostimulant (polyI:C or CpG). The ratio of GA to immunostimulant directly affected the potency of TLR activation and the mobility of these actives in simulated tumor tissue. Polyplexes of GA and CpG were injected intratumorally in a tumor model of head and neck cancer (HNC) and significantly mitigated tumor growth as compared to the vehicle controls. Intratumoral injections of CpG showed the slowest tumor growth but exhibited dramatically higher systemic proinflammatory cytokine levels compared to polyplexes of GA with CpG. Sequencing of RNA from resected tumors revealed a similar pattern of upregulated proinflammatory cytokines for CpG and polyplexes, a finding supported by histological tumor staining showing similar infiltration of immune cells induced by these treatments. Intratumoral administration of polyplexes of GA with immunostimulant represents a translational approach to enhance local immune responses while mitigating systemic immune-related adverse events., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Three decades of promise have culminated in the development of gene therapies that can be applied to a broad range of human diseases. After a brief history, we provide an overview of gene therapy types and delivery methods, gene editing technologies, regulatory affairs, clinical trials, approved products, ongoing challenges, and future goals. Information on clinical trials of candidates and on approved products for gene therapy developed between 1988 and 2020 is systematically collated. To obtain this global information, we scanned and reviewed more than 46,000 records of clinical trials from 17 clinical trial database providers. The medical benefits of transformative gene therapies are gradually being accepted by payors, and a significant increase in the number of gene therapy clinical trials and approved gene therapy products has resulted., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The capacity for a soft material to combine remote sensing and remote actuation is highly desirable for many applications in soft robotics and wearable technologies. This work presents a silicone elastomer with a suspension of a small weight fraction of ferromagnetic nickel nanorods, which is capable of both sensing deformation and altering stiffness in the presence of an external magnetic field. Cylinders composed of silicone elastomer and 1% by weight nickel nanorods experience large increases in compressive modulus when exposed to an external magnetic field. Incremental compressions totaling 600 g of force applied to the same silicone-nanorod composites increase the magnetic field strength measured by a Hall effect sensor enabling the material to be used as a soft load cell capable of detecting the rate, duration, and magnitude of force applied. In addition, lattice structures are 3D printed using an ink composed of silicone elastomer and 1% by weight nickel nanorods, which possess the same sensing capacity., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

A synthetic peptide, K-PLP, consisting of 11-unit poly-lysine (K11) linked via polyethylene glycol (PEG) to proteolipid protein epitope (PLP) was synthesized, characterized, and evaluated for efficacy in ameliorating experimental autoimmune encephalomyelitis (EAE) induced by PLP. K-PLP was designed to mimic the cationic nature of the relapsing-remitting multiple sclerosis treatment, glatiramer acetate (GA). With a pI of ~10, GA is able to form visible aggregates at the site of injection via electrostatic interactions with the anionic extracellular matrix. Aggregation further facilitates the retention of GA at the site of injection and draining lymph nodes, which may contribute to its mechanism of action. K-PLP with a pI of ~11, was found to form visible aggregates in the presence of glycosaminoglycans and persist at the injection site and draining lymph nodes in vivo , similar to GA. Additionally, EAE mice treated with K-PLP showed significant inhibition of clinical symptoms compared to free poly-lysine and to PLP, which are the components of K-PLP. The ability of the poly-lysine motif to retain PLP at the injection site, which increased the local exposure of PLP to immune cells may be an important factor affecting drug efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Song, Griffin, Larson, Christopher, Middaugh and Berkland.)

Multiple sclerosis is complex and heterogeneous. Better tools are needed to be able to monitor this disease among individuals, but blood-based biomarkers are often too rare to profile. In this work, we developed antigen-specific biomaterials to replicate the central nervous system niche where multiple sclerosis biomarkers are amplified. We incorporated mouse brain homogenate into a microporous gelatin methacrylate network. Homogenate-containing biomaterials differentially stimulated cells and led to the marked amplification of disease-relevant, antigen-specific B cells. These results demonstrate that biomaterials containing primary tissue homogenate retain antigen specificity and may be a useful tool for decoding human autoimmunity.

Designing an in vitro model of the tumor extracellular microenvironment to screen intratumoral drugs is an active challenge. As recent clinical successes of human intratumoral therapies stimulate research on intratumoral delivery, a need for a 3D tumor model to screen intratumoral therapies arises. When injecting the drug formulation directly into the tumor, the biophysics affecting intratumoral retention must be considered; especially for biologic therapies, which may be dominated by extracellular transport mechanisms. Fibrotic regions in solid tumors are typically rich in collagen I fibers. Using shear rheology, head and neck tumors with higher collagen density show a higher stiffness. Similarly, the stiffness of the hyaluronic acid (HA) hydrogel models is increased by adding collagen fibers to model the bulk biomechanical properties of solid tumors. HA hydrogels are then used as intratumoral injection site simulators to model in vitro the retention of glatiramer acetate (GA) and polyethylene glycol (PEG) administered intratumorally. Both compounds are also injected in murine tumors and retention is studied ex vivo for comparison. Retention of GA in the hydrogels is significantly longer than PEG, analogous to the solid tumors, suggesting the utility of HA hydrogels with collagen I fibers for screening extracellular drug transport after intratumoral administration., (© 2020 Wiley-VCH GmbH.)

Many autoimmune therapies focus on immune suppression to reduce symptom severity and halt disease progression; however, currently approved treatments lack specificity for the autoantigen and rely on more global immune suppression. Multivalent antigen arrays can disarm pathogenic autoimmune B cell populations that specifically recognize the antigen of interest via their B cell receptor (BCR). Disarmament may be achieved by BCR engagement, cross-linking, and sustained receptor occupancy as a result of multivalent, high avidity BCR binding. To engage and explore this mechanism, a tetramer display of the encephalogenic proteolipid peptide (PLP 139-151 ), referred to as 4-arm PLP 139-151 , was synthesized by copper-catalyzed azide-alkyne cycloaddition chemistry. Subcutaneous administration of 4-arm PLP 139-151 completely ameliorated symptoms of paralysis in a mouse model of multiple sclerosis known as experimental autoimmune encephalomyelitis. Competitive binding of 4-arm PLP 139-151 to PLP 139-151 -specific IgG in the mouse serum demonstrated the enhanced avidity associated with the multivalent array compared to the free peptide. Furthermore, key PLP 139-151 -reactive B cells were depleted following 4-arm PLP 139-151 treatment, resulting in significant reduction of proinflammatory cytokines. Together, these data demonstrate the potential of 4-arm PLP 139-151 to silence autoreactive B cell populations and limit the downstream activation of effector cells.

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based immunotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancer therapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemic exposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery. Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also review approved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influences intratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumor retention and limit systemic exposure of potent intratumoral therapies., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Adaptive immunity plays a central role in the pathogenesis of type 1 diabetes. Among past treatment approaches, B cell ablation has yielded unmistakable therapeutic potency; however, global immunosuppression imposes unacceptable risks to a patient population consisting of children. Multivalent antigen arrays represent a compelling strategy for targeted immunosuppression by selectively engaging and inactivating autoreactive B cells. Here, we report the design and characterization of 4-arm polyethylene glycol-insulin (PEG-Ins) conjugates as multivalent arrays for autoreactive B cell engagement. First, we selectively modified human insulin at the B29 residue to retain antigenicity. Next, we conjugated the modified proteins to 20 kDa, 4-arm polyethylene glycol backbones to produce multivalent PEG-Ins constructs. Mass spectrometry, circular dichroism, and dynamic light scattering indicated that the structure of insulin was maintained in the much larger, multivalent construct. PEG-Ins conjugates demonstrated an ex vivo immunological effect in splenocytes harboring an anti-insulin B cell receptor (VH125 SD ) by inactivating B cells and promoting an anergic phenotype that was downregulated in B cell receptor expression (CD79b), and PEG-Ins conjugates did not mobilize calcium upon B cell receptor stimulation. These data support the further study of PEG-Ins conjugates in animal models of type 1 diabetes.

Activation of the immune system to treat cancer has emerged as a powerful therapy tool, however, treatments must overcome the immunosuppressive microenvironment established by tumors. Toll-like receptor (TLR) agonists like CpG and polyI:C are potent stimulators of non-specific, pro-inflammatory immune responses, targeting TLR9 and TLR3, respectively. While these immunostimulants seem promising, systemic exposure can eventually induce severe side effects. Adverse inflammatory reactions in healthy tissues may be avoided by delivering and retaining immunostimulants in proximity to tumors or to primary sites of tumor metastases. Immunostimulants such as CpG and polyI:C cannot be completely immobilized, however, since the target TLR9 and TLR3 are located intracellularly. Previously, polycations like poly-l-lysine (PLL) have been complexed to the anionic CpG or polyI:C with the purpose of improving intracellular delivery and potency. Here, the relationship between PLL molecular weight and immunostimulant complexation, TLR activation, and transport in a simple, model tumor microenvironment was investigated. The polyplexes could be formulated to dramatically limit immunostimulant transport suggesting the potential for injection site retention and minimized systemic exposure of immunostimulants. The molecular weight of PLL and ratio of PLL to immunostimulant affected the accessibility of the immunostimulant within the polyplex and polyplex interaction strength., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Successful gene therapy requires the development of vectors that enable efficient delivery of genetic materials (e.g., pDNA or siRNA) to targeted cells, without degradation of the genetic materials. We have shown that nanoparticles formed by combining cell-penetrating peptide and pDNA (CPP-pDNA) into complexes and condensing them with calcium chloride can provide gene nanoparticles with high transfection efficiency and low cytotoxicity. In this work, we compare in situ measurements of the membrane insertion potential of three arginine-based gene nanoparticles (RW9-NPs, R9-NPs, and RH9-NPs) using four lipid compositions and two types of model membrane (Langmuir monolayers vs. supported bilayers) with their transfection efficiency in two human cancer cell lines. Using a Langmuir trough, we measured the membrane insertion potential of our gene nanoparticles to model membrane monolayers. A Quartz Crystal Microbalance with Dissipation (QCM-D) technique was used to monitor the adsorption of these nanoparticles to lipid bilayers of various compositions. Finally, gene expression using these nanoparticles was measured in breast cancer and cervical cancer cell lines. Our cell culture studies indicate that although R9-NPs and RW9-NPs show a significant increase in transfection efficiency compared to free pDNA, RH9-NPs do not show any significant difference. Both the Langmuir monolayer and QCM-D bilayer studies show that these results are best reflected in the in situ measurement assays when lipid systems containing a mixture of phospholipids, cholesterol, and sphingolipids are used. It is important to note that the mechanism of penetration is expected to differ for RW9 vs. R9; however, gene nanoparticles containing either of these CPPs show similar transfection efficiency. Our results therefore demonstrate that the design of predictive assays for gene therapy using CPPs must involve carefully chosen model lipid membrane systems that accurately represent the varying compositions of cell membranes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antigen-specific immunotherapies (ASIT) present compelling potential for introducing precision to the treatment of autoimmune diseases where nonspecific, global immunosuppression is currently the only treatment option. Central to ASIT design is the delivery of autoantigen, which parallels allergy desensitization approaches. Clinical success in tolerizing allergen-specific responses spans longer than a century, but autoimmune ASITs have yet to see an FDA-approved breakthrough. Allergens and autoantigens differ substantially in physicochemical properties, and these discrepancies influence the nature of their interactions with the immune system. Approved allergen-specific immunotherapies are typically administered as water soluble, neutrally charged protein fractions from 10 to 70 kDa. Conversely, autoantigens are native proteins that exhibit wide-ranging sizes, solubilities, and charges that render them susceptible to immunogenicity. To translate the success of allergen hyposensitization to ASIT, delivery strategies may be necessary to effectively format autoantigens, guide biodistribution, and engage appropriate immune mechanisms., Competing Interests: Declaration of Competing Interest Authors JOS and CJB are co-founders of Orion BioScience, Inc., a company developing technologies for the treatment of autoimmune disease. The other authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Combining autoantigens with immune-modulating drugs has emerged as an attractive approach to selectively reinstate tolerance in autoimmune diseases. The disparate properties of autoantigens and small-molecule immunosuppressants commonly used to treat autoimmune diseases can confound efforts to co-deliver these therapies. However, both components may be co-delivered with adjuvants which have been successful in delivering antigens to immune cells. We evaluated several common adjuvants as vehicles to co-deliver a model antigen and immunosuppressant, ovalbumin (OVA) and dexamethasone (DEX), respectively. Formulations were developed, and the release of DEX from adjuvants was investigated. Next, the effect of adjuvant, DEX, and OVA was tested in vitro using a DC line. A MF59-analog (MF59a) formulation was advanced to more sophisticated co-culture studies using OVA-primed bone marrow-derived dendritic cells and splenocytes or T-cells from OT-II mice. Most of these studies indicated MF59a-based antigen-specific immunotherapies could diminish the markers of inflammation associated with OVA recognition. We rationalized MF59a co-delivery of antigen and drug could reduce the risk of side effects typically associated with these drugs and reinstate immune tolerance, thus prompting continued investigation of emulsion adjuvants as delivery vehicles for antigen-specific immunotherapy of autoimmune diseases., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Relapsing-remitting patterns of many autoimmune diseases such as multiple sclerosis (MS) are perpetuated by a recurring circuit of adaptive immune cells that amplify in secondary lymphoid organs (SLOs) and traffic to compartments where antigen is abundant to elicit damage. Some of the most effective immunotherapies impede the migration of immune cells through this circuit, however, broadly suppressing immune cell migration can introduce life-threatening risks for patients. We developed antigen-specific immune decoys (ASIDs) to mimic tissues targeted in autoimmunity and selectively intercept autoimmune cells to preserve host tissue. Using Experimental Autoimmune Encephalomyelitis (EAE) as a model, we conjugated autoantigen PLP 139-151 to a microporous collagen scaffold. By subcutaneously implanting ASIDs after induction but prior to the onset of symptoms, mice were protected from paralysis. ASID implants were rich with autoimmune cells, however, reactivity to cognate antigen was substantially diminished and apoptosis was prevalent. ASID-implanted mice consistently exhibited engorged spleens when disease normally peaked. In addition, splenocyte antigen-presenting cells were highly activated in response to PLP rechallenge, but CD3 + and CD19  +  effector subsets were significantly decreased, suggesting exhaustion. ASID-implanted mice never developed EAE relapse symptoms even though the ASID material had long since degraded, suggesting exhausted autoimmune cells did not recover functionality. Together, data suggested ASIDs were able to sequester and exhaust immune cells in an antigen-specific fashion, thus offering a compelling approach to inhibit the migration circuit underlying autoimmunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glatiramer acetate (GA) is the active substance of Teva's Copaxone drug, which contains random polypeptides used to treat multiple sclerosis. Glatiramer acetate was originally developed to emulate human myelin basic protein, which contains four different residues [alanine (A), glutamic acid (E), tyrosine (T), and lysine (K)]. We found that GA can complex, condense, and transfect plasmid DNA. Mixing the positively charged GA and the negatively charged genetic material in correct proportions produced small, stable, and highly positively charged nanoparticles. This simple GA-pDNA formulation produced high levels of transfection efficiency with low toxicity in HeLa and A549 cells (lung and cervical cancer cells). Additionally, we studied and compared the nanoparticle properties, gene expression, and cytotoxicity of K 100 -pDNA (high-molecular-weight polylysine) and K 9 -pDNA (low-molecular-weight polylysine) nanoparticles to those of GA-pDNA nanoparticles. We also studied the effect of calcium, which was previously reported to reduce the size and enhance gene expression resulting from similar polyelectrolyte complexes. Adding calcium did not reduce particle size, nor improve the transfection efficiency of GA-pDNA nanoparticles as it did for polylysine-pDNA nanoparticles. GA-pDNA nanoparticles may be prepared by mixing a genetic payload with approved GA therapeutics (e.g., Copaxone), thus offering intriguing possibilities for translational gene therapy studies.

Hydrogels - water swollen cross-linked networks - have demonstrated considerable promise in tissue engineering and regenerative medicine applications. However, ambiguity over which rheological properties are needed to characterize these gels before crosslinking still exists. Most hydrogel research focuses on the performance of the hydrogel construct after implantation, but for clinical practice, and for related applications such as bioinks for 3D bioprinting, the behavior of the pre-gelled state is also critical. Therefore, the goal of this review is to emphasize the need for better rheological characterization of hydrogel precursor formulations, and standardized testing for surgical placement or 3D bioprinting. In particular, we consider engineering paste or putty precursor solutions (i.e., suspensions with a yield stress), and distinguish between these differences to ease the path to clinical translation. The connection between rheology and surgical application as well as how the use of paste and putty nomenclature can help to qualitatively identify material properties are explained. Quantitative rheological properties for defining materials as either pastes or putties are proposed to enable easier adoption to current methods. Specifically, the three-parameter Herschel-Bulkley model is proposed as a suitable model to correlate experimental data and provide a basis for meaningful comparison between different materials. This model combines a yield stress, the critical parameter distinguishing solutions from pastes (100-2000 Pa) and from putties (>2000 Pa), with power law fluid behavior once the yield stress is exceeded. Overall, successful implementation of paste or putty handling properties to the hydrogel precursor may minimize the surgeon-technology learning time and ultimately ease incorporation into current practice. Furthermore, improved understanding and reporting of rheological properties will lead to better theoretical explanations of how materials affect rheological performances, to better predict and design the next generation of biomaterials.

Contemporary approaches to treating autoimmune diseases like multiple sclerosis broadly modulate the immune system and leave patients susceptible to severe adverse effects. Antigen-specific immunotherapies (ASIT) offer a unique opportunity to selectively suppress autoreactive cell populations but have suffered from marginal efficacy even when employing traditional adjuvants to improve delivery. The development of immunologically active antigen delivery vehicles could potentially increase the clinical success of antigen-specific immunotherapies. An emulsion of the antioxidant tocopherol delivering an epitope of proteolipid protein autoantigen (PLP 139-151 ) yielded significant efficacy in mice with experimental autoimmune encephalomyelitis (EAE). In vitro studies indicated tocopherol emulsions reduced oxidative stress in antigen-presenting cells. Ex vivo analysis revealed that tocopherol emulsions shifted cytokine responses in EAE splenocytes. In addition, IgG responses against PLP 139-151 were increased in mice treated with tocopherol emulsions delivering the antigen, suggesting a possible skew in immunity. Overall, tocopherol emulsions provide a functional delivery vehicle for ASIT capable of ameliorating autoimmunity in a murine model.

Glatiramer acetate (GA) is widely prescribed for the treatment of relapsing-remitting multiple sclerosis, however, the mechanism of action is still not fully understood. We investigated the structural properties of GA and examined alterations to the drug upon injection into the subcutaneous space. First, a variety of biophysical characterization techniques were employed to characterize GA in solution. GA was found to exist as alpha helices in solution with a hydrodynamic radius of ~3 nm in size. To simulate GA behavior at the site of injection, GA was injected into a solution of 1.5 MDa hyaluronic acid (HA). Visible aggregates were observed immediately upon injection and subsequent testing indicated aggregation was driven by electrostatic interactions between the positively-charged GA and negatively-charged HA. In vivo testing confirmed GA formed spherical particles in the nano- to micrometer size range, suggesting this mechanism contributes to persistence at the injection site and in draining lymph nodes. The aggregates were found to associate with glycosaminoglycans, suggesting an electrostatic mechanism of induced aggregation like the simulated injection. These novel observations may help explain the complex immunomodulatory mechanisms of GA and adverse injection site reactions seen in patients., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: The current investigation evaluated a novel extended release delivery system for treating inner ear diseases. The platform technology consists of a film forming agent (FFA) and microsphere component to localize and extend drug delivery within the ear., Study Design: Studies evaluated dissolution kinetics of microspheres with multiple encapsulates, testing of a variety of FFAs, and ability to localize to the round window membrane in mice in vivo., Setting: Studies were completed at Orbis Biosciences and The University of Kansas Medical Center., Subjects: In conjunction with in vitro characterization, an infrared dye-containing microsphere formulation was evaluated for round window membrane (RWM) localization and general tolerability in C57/BL6 Mus musculus for 35 days., Methods: In vitro characterization was performed using upright diffusion cells on cellulose acetate membranes, with drug content quantified by high performance liquid chromatography. Mus musculus dosing of infrared dye-containing microspheres was performed under anesthesia with a 27 GA needle and 2.0 μL injection volume RESULTS: In vitro dissolution demonstrates the ability of the FFA with microsphere platform to release steroids, proteins, peptides, and nucleic acids for at least one month, while necroscopy shows the ability of the FFA with dye-loaded microspheres to remain localized to Mus musculus RWM for the same period of time, with favorable tolerability., Conclusions: Combining FFA and microsphere for localized drug delivery may enable cost-effective, extended release local delivery to the inner ear of new and existing small molecules, proteins, peptides, and nucleic acids., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Four molecular weights of poly-l-lysine (PLL) [1000-5000 Da, 1500-8000 Da, 4000-15,000 Da and 15,000-30,000 Da] and three molecular weights of polyethyleneimine (PEI) [800 Da, 2,000 Da, and 25,000 Da] were used to systematically study the effect of calcium (Ca 2+ ) to improve transfection efficiency of polyelectrolyte complexes. Complexes made using different molecular weights of PLL or PEI polymer showed clear differences in the levels of gene expression in the presence and absence of calcium chloride when tested using A549 human lung carcinoma cells. Complexes formed from PLL or PEI 800 Da were exhibited negligible expression of pDNA according to a luciferase reporter assay. Low molecular weight PLL and PEI 800 Da, however, became highly efficient gene delivery vehicles when calcium was added to the nascent complexes while maintaining the low cytotoxicity of low molecular weight polyamines. Additional analyses indicated that the most effective formulations utilized a threshold level of calcium, which created small, stable particles, but also facilitated unpackaging of the gene complexes and release of pDNA., (Published by Elsevier B.V.)

Traumatic brain injury (TBI) is a life-threatening condition defined by internal brain herniation. Severe TBI is commonly treated by a two-stage surgical intervention, where decompressive craniectomy is first conducted to remove a large portion of calvarial bone and allow unimpeded brain swelling. In the second surgery, spaced weeks to months after the first, cranioplasty is performed to restore the cranial bone. Hydrogels with paste-like precursor solutions for surgical placement may potentially revolutionize TBI treatment by permitting a single-stage surgical intervention, capable of being implanted with the initial surgery, remaining pliable during brain swelling, and tuned to regenerate calvarial bone after brain swelling has subsided. The current study evaluated the use of photocrosslinkable pentenoate-functionalized hyaluronic acid (PHA) and non-crosslinking hyaluronic acid (HA) hydrogels encapsulating naturally derived tissue particles of demineralized bone matrix (DBM), devitalized cartilage (DVC), devitalized meniscus (DVM), or devitalized tendon (DVT) for bone regeneration in critical-size rat calvarial defects. All hydrogel precursors exhibited a yield stress for placement and addition of particles increased the average material compressive modulus. The HA-DBM (4-30%), PHA (4%), and PHA-DVT (4-30%) groups had 5 (p < 0.0001), 3.1, and 3.2 (p < 0.05) times greater regenerated bone volume compared to the sham (untreated defect) group, respectively. In vitro cell studies suggested that the PHA-DVT (4-10%) group would have the most desirable performance. Overall, hydrogels containing DVT particles outperformed other materials in terms of bone regeneration in vivo and calcium deposition in vitro. Hydrogels containing DVT will be further evaluated in future rat TBI studies., Statement of Significance: Traumatic brain injury (TBI) is a life-threatening condition characterized by severe brain swelling and is currently treated by a two-stage surgical procedure. Complications associated with the two-stage surgical intervention include the occurrence of the condition termed syndrome of the trephined; however, the condition is completely reversible once the secondary surgery is performed. A desirable TBI treatment would include a single surgical intervention to avoid syndrome of the trephined altogether. The first hurdle in reaching the overall goal is to develop a pliable hydrogel material that can regenerate the patient's bone. The development of a pliable hydrogel technology would greatly impact the field of bone regeneration for TBI application and other areas of bone regeneration., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Interrogating biological systems is often limited by access to biological probes. The emergence of "click chemistry" has revolutionized bioconjugate chemistry by providing facile reaction conditions amenable to both biologic molecules and small molecule probes such as fluorophores, toxins, or therapeutics. One particularly popular version is the copper-catalyzed azide-alkyne cycloaddition (AAC) reaction, which has spawned new alternatives such as the strain-promoted azide-alkyne cycloaddition reaction, among others. This focused review highlights practical approaches to AAC reactions for the synthesis of peptide or protein bioconjugates and contrasts current challenges and limitations in light of recent advances in the field. The conical success of antibody drug conjugates has expanded the toolbox of linkers and payloads to facilitate practical applications of bioconjugation to create novel therapeutics and biologic probes. The AAC reaction in particular is poised to enable a large set of functionalized molecules as a combinatorial approach to high-throughput bioconjugate generation, screening, and honing of lead compounds.

In the treatment of severe traumatic brain injury (TBI), decompressive craniectomy is commonly used to remove a large portion of calvarial bone to allow unimpeded brain swelling. Hydrogels have the potential to revolutionize TBI treatment by permitting a single-surgical intervention, remaining pliable during brain swelling, and tuned to regenerate bone after swelling has subsided. With this motivation, our goal is to present a pliable material capable of regenerating calvarial bone across a critical size defect. We therefore proposed the use of a methacrylated solubilized decellularized cartilage (MeSDCC) hydrogel encapsulating synthetic osteogenic particles of hydroxyapatite nanofibers, bioglass microparticles, or added rat bone marrow-derived mesenchymal stem cells (rMSCs) for bone regeneration in critical-size rat calvarial defects. Fibrin hydrogels were employed as a control material for the study. MeSDCC hydrogels exhibited sufficient rheological performance for material placement before crosslinking ([Formula: see text] > 500 Pa), and sufficient compressive moduli post-crosslinking (E > 150 kPa). In vitro experiments suggested increased calcium deposition for cells seeded on the MeSDCC material; however, in vivo bone regeneration was minimal in both MeSDCC and fibrin groups, even with colloidal materials or added rMSCs. Minimal bone regeneration in the MeSDCC test groups may potentially be attributed to cartilage solubilization after decellularization, in which material signals may have degraded from enzymatic treatment. Looking to the future, an improvement in the bioactivity of the material will be crucial to the success of bone regeneration strategies for TBI treatment.

Microspheres have long been used in drug delivery applications because of their controlled release capabilities. They have increasingly served as the fundamental building block for fabricating scaffolds for regenerative engineering because of their ability to provide a porous network, offer high-resolution control over spatial organization, and deliver growth factors/drugs and/or nanophase materials. Because they provide physicochemical gradients via spatiotemporal release of bioactive factors and nanophase ceramics, microspheres are a desirable tool for engineering complex tissues and biological interfaces. In this review we describe various methods for microsphere fabrication and sintering, and elucidate how these methods influence both micro- and macroscopic scaffold properties, with a special focus on the nature of sintering. Furthermore, we review key applications of microsphere-based scaffolds in regenerating various tissues. We hope to inspire researchers to join a growing community of investigators using microspheres as tissue engineering scaffolds so that their full potential in regenerative engineering may be realized.

Colloidal gels encapsulating natural materials and exhibiting paste-like properties for placement are promising for filling complex geometries in craniofacial bone regeneration applications. Colloidal materials have demonstrated modest clinical outcomes as bone substitutes in orthopedic applications, but limited success in craniofacial applications. As such, development of a novel colloidal gel will fill a void in commercially available products for use in craniofacial reconstruction. One likely application for this technology is cranial reconstruction. Currently, traumatic brain injury (TBI) is often treated with a hemi-craniectomy, a procedure in which half the cranium is removed to allow the injured brain to swell and herniate beyond the enclosed cranial vault. The use of colloidal gels would allow for the design of a pliable material capable of expansion during brain swelling and facilitate cranial bone regeneration alleviating the need for a second surgery to replace the previously removed hemi-cranium. In the current study, colloidal nanoparticles of hydroxyapatite (HAp), demineralized bone matrix (DBM), and decellularized cartilage (DCC) were combined with hyaluronic acid (HA) to form colloidal gels with desirable rheological properties ([Formula: see text] ≥ 100 Pa). BMP-2 and VEGF growth factors were included to assess extracellular matrix (ECM) contribution of DBM and DCC. The HA-HAp (BMP-2) and HA-HAp-DCC group had 89 and 82% higher bone regeneration compared to the sham group, respectively (p < 0.01). Material retention issues observed may be alleviated by implementing chemical crosslinking. Overall, DCC may be a promising material for bone regeneration in general, and colloidal gels may hold significant potential in craniofacial applications.

One of the grand challenges in translational regenerative medicine is the surgical placement of biomaterials. For bone regeneration in particular, malleable and injectable colloidal gelsare frequently designed to exhibit self-assembling and shear-response behavior which facilitates biomaterial placement in tissue defects. The current study demonstrated that by combining native extracellular matrix (ECM) microparticles, i.e., demineralized bone matrix (DBM) and decellularized cartilage (DCC), with hyaluronic acid (HA) and hydroxyapatite (HAP) nanoparticles, a viscoelastic colloidal gel consisting exclusively of natural materials was achieved. Rheological testing of HA-ECM suspensions and HA-HAP-ECM colloidal gels concluded either equivalent or substantially higher storage moduli (G' ≈ 100-10 000 Pa), yield stresses (τ y ≈ 100-1000 Pa), and viscoelastic recoveries (G' recovery ≥ 87%) in comparison with controls formulated without ECM, which indicated a previously unexplored synergy in fluid properties between ECM microparticles and HA-HAP colloidal networks. Notable rheological differences were observed between respective DBM and DCC formulations, specifically in HA-HAP-DBM mixtures, which displayed a mean 3-fold increase in G' and a mean 4-fold increase in τ y from corresponding DCC mixtures. An initial in vitro assessment of these potential tissue fillers as substrates for cell growth revealed that all formulations of HA-ECM and HA-HAP-ECM showed no signs of cytotoxicity and appeared to promote cell viability. Both DBM and DCC colloidal gels represent promising platforms for future studies in bone and cartilage tissue engineering. Overall, the current study identified colloidal gels constructed exclusively of natural materials, with viscoelastic properties that may facilitate surgical placement for a wide variety of therapeutic applications.

To evaluate the potential of cell-penetrating peptide-based delivery of apoptosis-inducer gene in cancer therapy, a modified HIV-1 TAT peptide (dimerized TAT peptide, dTAT) was studied. The dTAT and plasmid DNA (pDNA) complexes (dTAT-pDNA) were condensed using calcium chloride (dTAT-pDNA-Ca 2+ ). This simple nonviral formulation approach showed high levels of gene expression in vitro without any cytotoxicity. In mouse studies, a single intratracheal (IT) aerosol spray or 2 intravenous (IV) injections of the dTAT, apoptosis-inducer gene, angiotensin II type 2 receptor (AT2R), and Ca 2+ complexes (dTAT-pAT2R-Ca 2+ ) significantly attenuated the acutely growing mouse Lewis lung carcinoma allografts in mouse lungs. Furthermore, single IT (p = 0.054) and the combination of IT and IV (p < 0.05) administrations of dTAT-pAT2R-Ca 2+ markedly attenuated slowly growing and relatively large-sized H358 human bronchioloalveolar carcinoma xenografts in mouse lungs. These results indicate that the dTAT-pDNA-Ca 2+ effectively delivered the gene to cancer cells by either IT or IV administration although the local pulmonary delivery of the dTAT-pAT2R-Ca 2+ showed more effective growth inhibition of orthotopic lung cancer grafts. Thus, the present study offers preclinical proof of concept that a dTAT-based nonviral gene delivery method via IT administration may be an effective lung cancer gene therapy., Competing Interests: The authors disclose no potential conflicts of interest., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Extracellular matrix materials such as decellularized cartilage (DCC) and chondroitin sulfate (CS) may be attractive chondrogenic materials for cartilage regeneration. The goal of the current study was to investigate the effects of encapsulation of DCC and CS in homogeneous microsphere-based scaffolds, and to test the hypothesis that encapsulation of these extracellular matrix materials would induce chondrogenesis of rat bone marrow stromal cells. Four different types of homogeneous scaffolds were fabricated from microspheres of poly(D,L-lactic-co-glycolic acid): Blank (poly(D,L-lactic-co-glycolic acid) only; negative control), transforming growth factor-β3 encapsulated (positive control), DCC encapsulated, and CS encapsulated. These scaffolds were then seeded with rat bone marrow stromal cells and cultured for 6 weeks. The DCC and CS encapsulation altered the morphological features of the microspheres, resulting in higher porosities in these groups. Moreover, the mechanical properties of the scaffolds were impacted due to differences in the degree of sintering, with the CS group exhibiting the highest compressive modulus. Biochemical evidence suggested a mitogenic effect of DCC and CS encapsulation on rat bone marrow stromal cells with the matrix synthesis boosted primarily by the inherently present extracellular matrix components. An important finding was that the cell seeded CS and DCC groups at week 6 had up to an order of magnitude higher glycosaminoglycan contents than their acellular counterparts. Gene expression results indicated a suppressive effect of DCC and CS encapsulation on rat bone marrow stromal cell chondrogenesis with differences in gene expression patterns existing between the DCC and CS groups. Overall, DCC and CS were easily included in microsphere-based scaffolds; however, there is a requirement to further refine their concentrations to achieve the differentiation profiles we seek in vitro., Competing Interests: Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2016.)

Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(D,L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the "blank" (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration.

"Raw materials," or materials capable of serving both as building blocks and as signals, which are often but not always natural materials, are taking center stage in biomaterials for contemporary regenerative medicine. In osteochondral tissue engineering, a field leveraging the underlying bone to facilitate cartilage regeneration, common raw materials include chondroitin sulfate (CS) for cartilage and β-tricalcium phosphate (TCP) for bone. Building on our previous work with gradient scaffolds based on microspheres, here we delved deeper into the characterization of individual components. In the current study, the release of CS and TCP from poly(D, L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds was evaluated over a time period of 4 weeks. Raw material encapsulated groups were compared to 'blank' groups and evaluated for surface topology, molecular weight, and mechanical performance as a function of time. The CS group may have led to increased surface porosity, and the addition of CS improved the mechanical performance of the scaffold. The finding that CS was completely released into the surrounding media by 4 weeks has a significant impact on future in vivo studies, given rapid bioavailability. The addition of TCP seemed to contribute to the rough external appearance of the scaffold. The current study provides an introduction to degradation patterns of homogenous raw material encapsulated scaffolds, providing characterization data to advance the field of microsphere-based scaffolds in tissue engineering., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-β3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-β3. DVC consistently outperformed all TGF-β3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.

Noncovalent complexation of plasmid DNA (pDNA) with cell-penetrating peptides (CPPs) forms relatively large complexes with poor gene expression. Yet, condensing these CPP-pDNA complexes via addition of calcium chloride produces small and stable nanoparticles with high levels of gene expression. This simple formulation offered high transfection efficiency and negligible cytotoxicity in HEK-293 (a virus-immortalized kidney cell) and A549 (a human lung cancer cell line). Small changes in CPP charge type, charge spacing, and hydrophobicity were studied by using five arginine-rich CPPs: the well-known hydrophilic polyarginine R9 peptide, a hydrophilic RH9 peptide, and three amphiphilic peptides (RA9, RL9, and RW9) with charge distributions that favor membrane penetration. R9 and RW9 nanoparticles were significantly more effective than the other CPPs under most formulation conditions. However, these CPPs exhibit large differences in membrane penetration potential. Maximum transfection resulted from an appropriate balance of complexing with pDNA, releasing DNA, and membrane penetration potential.

Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Cell-penetrating peptides (CPP) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Here, a synthetic CPP (polylysine, K9 peptide) was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R) and complexes were condensed using calcium chloride. The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro and in vivo. This simple nonviral formulation approach showed negligible cytotoxicity in four different human cell lines (cervix, breast, kidney, and lung cell lines) and one mouse cell line (a lung cancer cell line). In addition, this K9-pDNA-Ca(2+) complex demonstrated cancer-targeted gene delivery when administered via intravenous injection or intratracheal spray. The transfection efficiency was evaluated in Lewis lung carcinoma (LLC) cell lines cultured in vitro and in orthotopic cancer grafts in syngeneic mice. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth, offering preclinical proof-of-concept for a novel nonviral gene delivery method exhibiting effective lung tumor gene therapy via either intravenous or intratracheal administration., (©2015 American Association for Cancer Research.)

Hydrogels are a promising class of materials for tissue regeneration, but they lack the ability to be molded into a defect site by a surgeon because hydrogel precursors are liquid solutions that are prone to leaking during placement. Therefore, although the main focus of hydrogel technology and developments are on hydrogels in their crosslinked form, our primary focus is on improving the fluid behavior of hydrogel precursor solutions. In this work, we introduce a method to achieve paste-like hydrogel precursor solutions by combining hyaluronic acid nanoparticles with traditional crosslinked hyaluronic acid hydrogels. Prior to crosslinking, the samples underwent rheological testing to assess yield stress and recovery using linear hyaluronic acid as a control. The experimental groups containing nanoparticles were the only solutions that exhibited a yield stress, demonstrating that the nanoparticulate rather than the linear form of hyaluronic acid was necessary to achieve paste-like behavior. The gels were also photocrosslinked and further characterized as solids, where it was demonstrated that the inclusion of nanoparticles did not adversely affect the compressive modulus and that encapsulated bone marrow-derived mesenchymal stem cells remained viable. Overall, this nanoparticle-based approach provides a platform hydrogel system that exhibits a yield stress prior to crosslinking, and can then be crosslinked into a hydrogel that is capable of encapsulating cells that remain viable. This behavior may hold significant impact for hydrogel applications where a paste-like behavior is desired in the hydrogel precursor solution.

Extracellular matrix (ECM) components, such as chondroitin sulfate (CS) and tricalcium phosphate, serve as raw materials, and thus spatial patterning of these raw materials may be leveraged to mimic the smooth transition of physical, chemical, and mechanical properties at the bone-cartilage interface. We hypothesized that encapsulation of opposing gradients of these raw materials in high molecular weight poly(d,l-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds would enhance differentiation of rat bone marrow-derived stromal cells. The raw material encapsulation altered the microstructure of the microspheres and also influenced the cellular morphology that depended on the type of material encapsulated. Moreover, the mechanical properties of the raw material encapsulating microsphere-based scaffolds initially relied on the composition of the scaffolds and later on were primarily governed by the degradation of the polymer phase and newly synthesized ECM by the seeded cells. Furthermore, raw materials had a mitogenic effect on the seeded cells and led to increased glycosaminoglycan (GAG), collagen, and calcium content. Interestingly, the initial effects of raw material encapsulation on a per-cell basis might have been overshadowed by medium-regulated environment that appeared to favor osteogenesis. However, it is to be noted that in vivo, differentiation of the cells would be governed by the surrounding native environment. Thus, the results of this study demonstrated the potential of the raw materials in facilitating neo-tissue synthesis in microsphere-based scaffolds and perhaps in combination with bioactive signals, these raw materials may be able to achieve intricate cell differentiation profiles required for regenerating the osteochondral interface.

Autologous bone grafting (ABG) remains entrenched as the gold standard of treatment in bone regenerative surgery. Consequently, many marginally successful bone tissue engineering strategies have focused on mimicking portions of ABG's "ideal" osteoconductive, osteoinductive, and osteogenic composition resembling the late reparative stage extracellular matrix (ECM) in bone fracture repair, also known as the "hard" or "bony" callus. An alternative, less common approach that has emerged in the last decade harnesses endochondral (EC) ossification through developmental engineering principles, which acknowledges that the molecular and cellular mechanisms involved in developmental skeletogenesis, specifically EC ossification, are closely paralleled during native bone healing. EC ossification naturally occurs during the majority of bone fractures and, thus, can potentially be utilized to enhance bone regeneration for nearly any orthopedic indication, especially in avascular critical-sized defects where hypoxic conditions favor initial chondrogenesis instead of direct intramembranous ossification. The body's native EC ossification response, however, is not capable of regenerating critical-sized defects without intervention. We propose that an underexplored potential exists to regenerate bone through the native EC ossification response by utilizing strategies which mimic the initial inflammatory or fibrocartilaginous ECM (i.e., "pro-" or "soft" callus) observed in the early reparative stage of bone fracture repair. To date, the majority of strategies utilizing this approach rely on clinically burdensome in vitro cell expansion protocols. This review will focus on the confluence of two evolving areas, (1) native ECM biomaterials and (2) developmental engineering, which will attempt to overcome the technical, business, and regulatory challenges that persist in the area of bone regeneration. Significant attention will be given to native "raw" materials and ECM-based designs that provide necessary osteo- and chondro-conductive and inductive features for enhancing EC ossification. In addition, critical perspectives on existing stem cell-based therapeutic strategies will be discussed with a focus on their use as an extension of the acellular ECM-based designs for specific clinical indications. Within this framework, a novel realm of unexplored design strategies for bone tissue engineering will be introduced into the collective consciousness of the regenerative medicine field.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the 'raw material' building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca(2+) complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest that in the neutral environment the complexes are unable to penetrate the zwitterionic lipid membranes but can penetrate through the anionic lipid membranes. However, the acidic pH mimicking the local environment in the late endosomes leads to a significant increase in adsorption of the complexes to zwitterionic lipid headgroups and decreases for anionic headgroups. These membrane-gene complex interactions may be responsible for the ability of the complexes to efficiently enter the intracellular environment through endocytosis and escape from the endosomes to effectively deliver their genetic payload.

Background: The microfracture technique for cartilage repair has limited ability to regenerate hyaline cartilage., Aim: The current study made a direct comparison between microfracture and an osteochondral approach with microsphere-based gradient plugs., Materials & Methods: The PLGA-based scaffolds had opposing gradients of chondroitin sulfate and β-tricalcium phosphate. A 1-year repair study in sheep was conducted., Results: The repair tissues in the microfracture were mostly fibrous and had scattered fissures with degenerative changes. Cartilage regenerated with the gradient plugs had equal or superior mechanical properties; had lacunated cells and stable matrix as in hyaline cartilage., Conclusion: This first report of gradient scaffolds in a long-term, large animal, osteochondral defect demonstrated potential for equal or better cartilage repair than microfracture., Competing Interests: Financial & competing interests disclosure The authors gratefully acknowledge funding by the Coulter Foundation. In addition, this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under Award Number R01 AR056347. The authors would also like to recognize support from the Kansas Bioscience Authority Rising Star Award. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.