Cytotoxicity of Doxorubicin-Curcumin Nanoparticles Conjugated with Two Different Peptides (CKR and EVQ) against FLT3 Protein in Leukemic Stem Cells.

A targeted micellar formation of doxorubicin (Dox) and curcumin (Cur) was evaluated to enhance the efficacy and reduce the toxicity of these drugs in KG1a leukemic stem cells (LSCs) compared to EoL-1 leukemic cells. Dox-Cur-micelle (DCM) was developed to improve the cell uptake of both compounds in LSCs. Cur-micelle (CM) was produced to compare with DCM. DCM and CM were conjugated with two FLT3 (FMS-like tyrosine kinase)-specific peptides (CKR; C and EVQ; E) to increase drug delivery to KG1a via the FLT3 receptor (AML marker). They were formulated using a film-hydration technique together with a pH-induced self-assembly method. The optimal drug-to-polymer weight ratios for the DCM and CM formulations were 1:40. The weight ratio of Dox and Cur in DCM was 1:9. DCM and CM exhibited a particle size of 20-25 nm with neutral charge and a high %EE. Each micelle exhibited colloidal stability and prolonged drug release. Poloxamer 407 (P407) was modified with terminal azides and conjugated to FLT3-targeting peptides with terminal alkynes. DCM and CM coupled with peptides C, E, and C + E exhibited a higher particle size. Moreover, DCM-C + E and CM-C + E showed the highest toxicity in KG-1a and EoL-1 cells. Using two peptides likely improves the probability of micelles binding to the FLT3 receptor and induces cytotoxicity in leukemic stem cells.

CCL2 signaling promotes skeletal muscle wasting in non-tumor and breast tumor models.

Despite advancements in treatment, approximately 25% of patients with breast cancer experience long-term skeletal muscle wasting (SMW), which limits mobility, reduces drug tolerance and adversely impacts survival. By understanding the underlying molecular mechanisms of SMW, we may be able to develop new strategies to alleviate this condition and improve the lives of patients with breast cancer. Chemokines are small soluble factors that regulate homing of immune cells to tissues during inflammation. In breast cancers, overexpression of C-C chemokine ligand 2 (CCL2) correlates with unfavorable prognosis. Elevated levels of CCL2 in peripheral blood indicate possible systemic effects of this chemokine in patients with breast cancer. Here, we investigated the role of CCL2 signaling on SMW in tumor and non-tumor contexts. In vitro, increasing concentrations of CCL2 inhibited myoblast and myotube function through C-C chemokine receptor 2 (CCR2)-dependent mechanisms involving JNK, SMAD3 and AMPK signaling. In healthy mice, delivery of recombinant CCL2 protein promoted SMW in a dose-dependent manner. In vivo knockdown of breast tumor-derived CCL2 partially protected against SMW. Overall, chronic, upregulated CCL2-CCR2 signaling positively regulates SMW, with implications for therapeutic targeting.

Soluble antigen arrays provide increased efficacy and safety over free peptides for tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but clinical implementation has been challenging. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA used (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Over time, the peptides induced an IgE-independent anaphylactic reaction, the incidence of which was significantly delayed when peptides were in SAgA form rather than in free form. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Immunologic anaphylaxis occurred in NOD mice in a dose-dependent manner but not in C57BL/6 or BALB/c mice; however, its incidence did not correlate with the level of anti-peptide antibodies. We provide evidence that SAgAs significantly improve the efficacy of peptides to induce tolerance and prevent autoimmune diabetes while at the same time reducing their anaphylactogenic potential.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration.

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air-liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

Dynamic control of contractile resistance to iPSC-derived micro-heart muscle arrays.

Many types of cardiovascular disease are linked to the mechanical forces placed on the heart. However, our understanding of how mechanical forces exactly affect the cellular biology of the heart remains incomplete. In vitro models based on cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CM) enable researchers to develop medium to high-throughput systems to study cardiac mechanobiology at the cellular level. Previous models have been developed to enable the study of mechanical forces, such as cardiac afterload. However, most of these models require exogenous extracellular matrix (ECM) to form cardiac tissues. Recently, a system was developed to simulate changes in afterload by grafting ECM-free micro-heart muscle arrays to elastomeric substrates of discrete stiffnesses. In the present study, we extended this system by combining the elastomer-grafted tissue arrays with a magnetorheological elastomeric substrate. This system allows iPSC-CM based micro-heart muscle arrays to experience dynamic changes in contractile resistance to mimic dynamically altered afterload. Acute changes in substrate stiffness led to acute changes in the calcium dynamics and contractile forces, illustrating the system's ability to dynamically elicit changes in tissue mechanics by dynamically changing contractile resistance.

CD22L Conjugation to Insulin Attenuates Insulin-Specific B Cell Activation.

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 (125TgSD) 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.

Multivalent Scaffolds to Promote B cell Tolerance.

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.

Novel 3D Force Sensors for a Cost-Effective 3D Force Plate for Biomechanical Analysis.

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.

Soluble antigen arrays improve the efficacy and safety of peptide-based tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but the limited in vivo stability and uptake of peptides impedes clinical implementation. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Both SAgA variants significantly delayed anaphylaxis compared to their respective free peptides. The anaphylaxis, which occurred in NOD mice but not in C57BL/6 mice, was dose-dependent but did not correlate with the production of IgG1 or IgE against the peptides. We provide evidence that SAgAs significantly improve the efficacy and safety of peptide-based immunotherapy.

Design of a Tumor Binding GMCSF as Intratumoral Immunotherapy of Solid Tumors.

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".

Disrupting N-Glycosylation Using Type I Mannosidase Inhibitors Alters B-Cell Receptor Signaling.

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.

Doxorubicin-Loaded Polymeric Micelles Conjugated with CKR- and EVQ-FLT3 Peptides for Cytotoxicity in Leukemic Stem Cells.

Doxorubicin (Dox) is the standard chemotherapeutic agent for acute myeloblastic leukemia (AML) treatment. However, 40% of Dox-treated AML cases relapsed due to the presence of leukemic stem cells (LSCs). Thus, poloxamer 407 and CKR- and EVQ-FLT3 peptides were used to formulate Dox-micelles (DMs) and DM conjugated with peptides (CKR and EVQ) for improving AML-LSC treatment. Results indicated that DMs with a weight ratio of Dox to P407 of 1:200 had a particle size of 23.3 ± 1.3 nm with a high percentage of Dox entrapment. They were able to prolong drug release and maintain physicochemical stability. Following effective DM preparation, P407 was modified and conjugated with FLT3 peptides, CKR and EVQ to formulate DM-CKR, DM-EVQ, and DM-CKR+DM-EVQ. Freshly synthesized DMs displaying FLT3 peptides showed particle sizes smaller than 50 nm and a high drug entrapment level, comparable with DMs. DM-CKR+DM-EVQ was considerably more toxic to KG-1a (AML LSC-like cell model) than Dox-HCl. These FLT3-targeted DMs could increase drug uptake and induce apoptosis induction. Due to an increase in micelle-LSC binding and uptake, DMs displaying both peptides tended to improve the potency of Dox compared to a single peptide-coupled micelle.

Glatiramer Acetate Complexed with CpG as Intratumoral Immunotherapy in Combination with Anti-PD-1.

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.

Strategies to Screen Anti-AQP4 Antibodies from Yeast Surface Display Libraries.

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.

Co-Treatments of Edible Curcumin from Turmeric Rhizomes and Chemotherapeutic Drugs on Cytotoxicity and FLT3 Protein Expression in Leukemic Stem Cells.

This study aims to enhance efficacy and reduce toxicity of the combination treatment of a drug and curcumin (Cur) on leukemic stem cell and leukemic cell lines, including KG-1a and KG-1 (FLT3+ LSCs), EoL-1 (FLT3+ LCs), and U937 (FLT3- LCs). The cytotoxicity of co-treatments of doxorubicin (Dox) or idarubicin (Ida) at concentrations of the IC10-IC80 values and each concentration of Cur at the IC20, IC30, IC40, and IC50 values (conditions 1, 2, 3, and 4) was determined by MTT assays. Dox-Cur increased cytotoxicity in leukemic cells. Dox-Cur co-treatment showed additive and synergistic effects in several conditions. The effect of this co-treatment on FLT3 expression in KG-1a, KG-1, and EoL-1 cells was examined by Western blotting. Dox-Cur decreased FLT3 protein levels and total cell numbers in all the cell lines in a dose-dependent manner. In summary, this study exhibits a novel report of Dox-Cur co-treatment in both enhancing cytotoxicity of Dox and inhibiting cell proliferation via FLT3 protein expression in leukemia stem cells and leukemic cells. This is the option of leukemia treatment with reducing side effects of chemotherapeutic drugs to leukemia patients.

Remote-Controlled 3D Porous Magnetic Interface toward High-Throughput Dynamic 3D Cell Culture.

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.

Glatiramer acetate enhances tumor retention and innate activation of immunostimulants.

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.

Simulating particle movement inside subcutaneous injection site simulator (SCISSOR) using Monte-Carlo method.

This study used Monte Carlo method to simulate particle movement inside a commercialized instrument called Subcutaneous Injection Site Simulator (SCISSOR). A series of parameters related to instrument, injection device, operation, formulation, as well as medium were thoroughly investigated. Also, several events that particles may occur in the subcutaneous (SC) space, including diffusion, binding, and aggregation, were implemented in our Monte Carlo based algorithms. The simulation result revealed that membrane area and position, diffusivity in the simulated SC medium, as well as injection position significantly affected release profile. Diffusivity in the injection volume could only influence release profile when this diffusivity was extremely low. Other factors, including initial injection shape, injection volume, and formulation concentration, had only minor impact on release profile. In addition, binding slowed down release, whereas aggregation reduced both total percentage of release and release rate. This study presented Monte Carlo method would potentially become a powerful tool to support multiple development activities related to experimental design, parameter sensitivity analysis, and control strategy development.

Drug Release Kinetics from Nondegradable Hydrophobic Polymers Can Be Modulated and Predicted by the Glass Transition Temperature.

Controlling drug release kinetics within a desired therapeutic window is the central task when designing polymeric drug delivery systems. Complex polymer chemistries have often been explored to control water penetration, polymer degradation rate, or the mesh network size of delivery systems. Here, a simple parameter for controlling the release rate and duration of nondegradable hydrophobic polymers is discovered. A systematic study involving 59 polymers and multiple drugs demonstrates that the glass transition temperature, Tg , is a critical factor that dictates drug release kinetics from nondegradable hydrophobic polymers. Drug release rate exhibits a unique and simple linear correlation of (T - Tg )0.5 despite variability of polymer structure and type. An empirical model established based on the special correlation can accurately simulate and predict drug release kinetics from polymers saving substantial time typically required to test long-acting drug delivery systems.

Polymer-coated microparticle scaffolds engineered for potential use in musculoskeletal tissue regeneration.

Biomaterials constructed exclusively of sintered microspheres have great potential in tissue engineering scaffold applications, offering the ability to create shape-specific scaffolds with precise controlled release yet to be matched by traditional additive manufacturing methods. The problem is that these microsphere-based scaffolds are limited in their stiffness for applications such as bone regeneration. Our vision to solve this problem was borne from a hierarchical structure perspective, focusing on the individual unit of the structure: the microsphere itself. In a core-shell approach, we envisioned a stiff core to create a stiff microsphere unit, with a polymeric shell that would enable sintering to the other microsphere units. Therefore, the current study provided a comparison of macroscopic biomaterials built on either polymer microspheres or polymer-coated hard glass microspheres. Identical polycaprolactone (PCL) polymer solutions were used to fabricate microspheres and as a thin coating on soda lime glass microspheres (hard phase). The materials were characterized as loose particles and as scaffolds via scanning electron microscopy, thermogravimetry, differential scanning calorimetry, Raman spectroscopy, mechanical testing, and a live/dead analysis with human umbilical cord-derived Wharton's jelly cells. The elastic modulus of the scaffolds with the thinly coated hard phase was about five times higher with glass microspheres (up to about 25 MPa) than pure polymer microspheres, while retaining the structure, cell adhesion, and chemical properties of the PCL polymer. This proof-of-concept study demonstrated the ability to achieve at least a five-fold increase in macroscopic stiffness via altering the core microsphere units with a core-shell approach.