Cortical spheroid on perfusable microvascular network in a microfluidic device.

Human induced pluripotent stem cell (hiPSC)-derived brain spheroids can recapitulate the complex cytoarchitecture of the brain, as well as the genetic/epigenetic footprint of human brain development. However, hiPSC-derived 3D models such as spheroid and organoids does not have a perfusable microvascular network, which plays a vital role in maintaining homeostasis in vivo. With the critical balance of positive and negative angiogenic modulators, 3D microvascular network can be achieved by angiogenesis. This paper reports on a microfluidic-based three-dimensional, cortical spheroid grafted on the vascular-network. Vascular network was formed by inducing angiogenic sprouting using concentration gradient-driven angiogenic factors in the microfluidic device. We investigate critical factors for angiogenic vascular network formation with spheroid placement, including 1) a PKCα activator, phorbol-12-myristate-13-acetate (PMA); 2) orientation of endothelial cells under perfusion and permeability of vascular network; 3) effect of extracellular matrix (ECM) types and their densities on angiogenesis; and 4) integration with cortical spheroid on vascular network. This paper demonstrates proof of concept for the potential utility of a membrane-free in vitro cortical spheroid tissue construct with perfusable microvascular network that can be scaled up to a high throughput platform. It can provide a cost-effective alternative platform to animal testing by modeling brain diseases and disorders, and screening drugs.

Effects of Altering Magnesium Metal Surfaces on Degradation In Vitro and In Vivo during Peripheral Nerve Regeneration.

In vivo use of biodegradable magnesium (Mg) metal can be plagued by too rapid a degradation rate that removes metal support before physiological function is repaired. To advance the use of Mg biomedical implants, the degradation rate may need to be adjusted. We previously demonstrated that pure Mg filaments used in a nerve repair scaffold were compatible with regenerating peripheral nerve tissues, reduced inflammation, and improved axonal numbers across a short-but not long-gap in sciatic nerves in rats. To determine if the repair of longer gaps would be improved by a slower Mg degradation rate, we tested, in vitro and in vivo, the effects of Mg filament polishing followed by anodization using plasma electrolytic oxidation (PEO) with non-toxic electrolytes. Polishing removed oxidation products from the surface of as-received (unpolished) filaments, exposed more Mg on the surface, produced a smoother surface, slowed in vitro Mg degradation over four weeks after immersion in a physiological solution, and improved attachment of cultured epithelial cells. In vivo, treated Mg filaments were used to repair longer (15 mm) injury gaps in adult rat sciatic nerves after placement inside hollow poly (caprolactone) nerve conduits. The addition of single Mg or control titanium filaments was compared to empty conduits (negative control) and isografts (nerves from donor rats, positive control). After six weeks in vivo, live animal imaging with micro computed tomography (micro-CT) showed that Mg metal degradation rates were slowed by polishing vs. as-received Mg, but not by anodization, which introduced greater variability. After 14 weeks in vivo, functional return was seen only with isograft controls. However, within Mg filament groups, the amount of axonal growth across the injury site was improved with slower Mg degradation rates. Thus, anodization slowed degradation in vitro but not in vivo, and degradation rates do affect nerve regeneration.

Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-culture from Human Stem Cells.

Background: Recently, the in vitro blood brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications it has with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for the iPCs and iECs in transwell system and 3-D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, as well as brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, as well as BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid ß42 oligomers by the upregulation of expression of TNFa, IL6, NFKB, Casp3, SOD2 and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof-of-concept vascularization strategy was demonstrated in a 3-D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.

A Three-Dimensional Brain-on-a-Chip Using Human iPSC-Derived GABAergic Neurons and Astrocytes.

Brain-on-a-chip is a miniaturized engineering platform to mimic the structural and functional aspects of brain tissue. We describe a method to construct a three-dimensional (3D) brain-on-a-chip in this chapter. We firstly portray the method of a brain-on-a-chip model with cocultured mice neurons, microglia, and astrocytes to mimic brain tissue and membrane-free perfusion with endothelial cells, in which we successfully build the blood-brain barrier to screen neurotoxicity. Then we describe a method to construct a brain-on-a-chip with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded GABAergic neurons and astrocytes and a perfusion channel with dynamic flow. We also include the broader applicability test of this model using an organophosphate (OP), malathion, to induce acute and chronic neurotoxicity, and then using butyrylcholinesterase (BuChE) as an exogenous bioscavenger of OP. Following the methods listed in this chapter, we are able to measure the neurotoxic effects on construct integrity, viability, and total AChE and BuChE activity.

Mechanically Tunable Extracellular Matrix of Genipin Crosslinked Collagen and Its Effect on Endothelial Function.

Mechanical rigidity of a matrix, to which cells adhere, plays a significant role in regulating phenotypic cellular behaviors such as spreading and junction formation because vascular cells sense and respond to changes in their mechanical environment. Controlling mechanical properties of extracellular matrix by using a crosslinker is important for cell and tissue mechanobiology. In this paper, we explored genipin, a natural plant extract, to crosslink collagen-I in order to enhance mechanical properties with low cytotoxicity. We characterized the effects of genipin concentration on the mechanical properties, color change, degradation, structure, cell viability, and endothelial function such as transendothelial electrical resistance (TEER). Through the analysis of both material properties and endothelial response, it was found that genipin-based glycation caused an increase in viscoelastic moduli in collagen hydrogels, as well as increased fiber density in their structural morphology. Endothelial cells were found to form better barriers, express higher levels of tight junction proteins, and exhibit better adhesion on stiffer matrices.

Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces.

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.

Three-dimensional brain-on-chip model using human iPSC-derived GABAergic neurons and astrocytes: Butyrylcholinesterase post-treatment for acute malathion exposure.

Organophosphates (OPs) induce acute and chronic neurotoxicity, primarily by inhibiting acetylcholinesterase (AChE) activity as well as by necrosis, and apoptosis. Butyrylcholinesterase (BuChE), an exogenous bioscavenger of OPs, can be used as a treatment for OP exposure. It is prerequisite to develop in vitro brain models that can study BuChE post-treatment for acute OP exposure. In this study, we developed a three-dimensional (3D) brain-on-chip platform with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. The platform consists of two compartments: 1) a hydrogel embedded with human iPSC-derived GABAergic neurons and astrocytes and 2) a perfusion channel with dynamic medium flow. The brain tissue constructs were exposed to Malathion (MT) at various concentrations and then treated with BuChE after 20 minutes of MT exposure. Results show that the iPSC-derived neurons and astrocytes directly interacted and formed synapses in the 3D matrix, and that treatment with BuChE improved viability after MT exposure up to a concentration of 10-3 M. We conclude that the 3D brain-on-chip platform with human iPSC-derived brain cells is a suitable model to study the neurotoxicity of OP exposure and evaluate therapeutic compounds for treatment.

Three-dimensional (3D) brain microphysiological system for organophosphates and neurochemical agent toxicity screening.

We investigated a potential use of a 3D tetraculture brain microphysiological system (BMPS) for neurotoxic chemical agent screening. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded neuroblastoma cells, microglia, and astrocytes, and vascular tissue with dynamic flow and membrane-free culture of the endothelial layer. We tested the broader applicability of this model, focusing on organophosphates (OPs) Malathion (MT), Parathion (PT), and Chlorpyrifos (CPF), and chemicals that interact with GABA and/or opioid receptor systems, including Muscimol (MUS), Dextromethorphan (DXM), and Ethanol (EtOH). We validated the BMPS platform by measuring the neurotoxic effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability, and residual OP concentration. The results show that OPs penetrated the model blood brain barrier (BBB) and inhibited AChE activity. DXM, MUS, and EtOH also penetrated the BBB and induced moderate toxicity. The results correlate well with available in vivo data. In addition, simulation results from an in silico physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model that we generated show good agreement with in vivo and in vitro data. In conclusion, this paper demonstrates the potential utility of a membrane-free tetraculture BMPS that can recapitulate brain complexity as a cost-effective alternative to animal models.

Comparison of endothelial cell attachment on surfaces of biodegradable polymer-coated magnesium alloys in a microfluidic environment.

Polymeric coatings can provide temporary stability to bioresorbable metallic stents at the initial stage of deployment by alleviating rapid degradation and providing better interaction with surrounding vasculature. To understand this interfacing biocompatibility, this study explored the endothelial-cytocompatibility of polymer-coated magnesium (Mg) alloys under static and dynamic conditions compared to that of non-coated Mg alloy surfaces. Poly (carbonate urethane) urea (PCUU) and poly (lactic-co-glycolic acid) (PLGA) were coated on Mg alloys (WE43, AZ31, ZWEKL, ZWEKC) and 316L stainless steel (316L SS, control sample), which were embedded into a microfluidic device to simulate a vascular environment with dynamic flow. The results from attachment and viability tests showed that more cells were attached on the polymer-coated Mg alloys than on non-coated Mg alloys in both static and dynamic conditions. In particular, the attachment and viability on PCUU-coated surfaces were significantly higher than that of PLGA-coated surfaces of WE43 and ZWEKC in both static and dynamic conditions, and of AZ31 in dynamic conditions (P<0.05). The elementary distribution map showed that there were relatively higher Carbon weight percentages and lower Mg weight percentages on PCUU-coated alloys than PLGA-coated alloys. Various levels of pittings were observed underneath the polymer coatings, and the pittings were more severe on the surface of Mg alloys that corroded rapidly. Polymer coatings are recommended to be applied on Mg alloys with relatively low corrosion rates, or after pre-stabilizing the substrate. PCUU-coating has more selective potential to enhance the biocompatibility and mitigate the endothelium damage of Mg alloy stenting.

Three-dimensional (3D) tetra-culture brain on chip platform for organophosphate toxicity screening.

Organophosphate-based compounds (OPs) represent a significant threat to warfighters (nerve agents) and civilian populations (pesticides). There is a pressing need to develop in vitro brain models that correlate to the in vivo brain to rapidly study OPs for neurotoxicity. Here we report on a microfluidic-based three-dimensional, four-cell tissue construct consisting of 1) a blood-brain barrier that has dynamic flow and membrane-free culture of the endothelial layer, and 2) an extracellular matrix (ECM)-embedded tissue construct with neuroblastoma, microglia, and astrocytes. We demonstrated this platform's utility by measuring OP effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability and residual OP concentration with four model OPs. The results show that the OPs penetrate the blood brain barrier (BBB) and rapidly inhibit AChE activity, and that in vitro toxicity was correlated with available in vivo data. This paper demonstrates the potential utility of a membrane-free tetra-cultured brain on chip that can be scaled to high throughput as a cost-effective alternative method to animal testing.

The Effects of Static and Dynamic Loading on Biodegradable Magnesium Pins In Vitro and In Vivo.

Here we systematically assess the degradation of biodegradable magnesium pins (as-drawn pure Mg, as-cast Mg-Zn-Mn, and extruded Mg-Zn-Mn) in a bioreactor applying cyclical loading and simulated body fluid (SBF) perfusion. Cyclical mechanical loading and interstitial flow accelerated the overall corrosion rate, leading to loss of mechanical strength. When compared to the in vivo degradation (degradation rate, product formation, uniform or localized pitting, and stress distribution) of the same materials in mouse subcutaneous and dog tibia implant models, we demonstrate that the in vitro model facilitates the analysis of the complex degradation behavior of Mg-based alloys in vivo. This study progresses the development of a suitable in vitro model to examine the effects of mechanical stress and interstitial flow on biodegradable implant materials.

Biodegradability and platelets adhesion assessment of magnesium-based alloys using a microfluidic system.

Magnesium (Mg)-based stents are extensively explored to alleviate atherosclerosis due to their biodegradability and relative hemocompatibility. To ensure the quality, safety and cost-efficacy of bioresorbable scaffolds and full utilization of the material tunability afforded by alloying, it is critical to access degradability and thrombosis potential of Mg-based alloys using improved in vitro models that mimic as closely as possible the in vivo microenvironment. In this study, we investigated biodegradation and initial thrombogenic behavior of Mg-based alloys at the interface between Mg alloys' surface and simulated physiological environment using a microfluidic system. The degradation properties of Mg-based alloys WE43, AZ31, ZWEK-L, and ZWEK-C were evaluated in complete culture medium and their thrombosis potentials in platelet rich plasma, respectively. The results show that 1) physiological shear stress increased the corrosion rate and decreased platelets adhesion rate as compared to static immersion; 2) secondary phases and impurities in material composition induced galvanic corrosion, resulting in higher corrosion resistance and platelet adhesion rate; 3) Mg-based alloys with higher corrosion rate showed higher platelets adhesion rate. We conclude that a microfluidic-based in vitro system allows evaluation of biodegradation behaviors and platelets responses of Mg-based alloys under specific shear stress, and degradability is related to platelets adhesion.

A Smartphone-Based Automatic Measurement Method for Colorimetric pH Detection Using a Color Adaptation Algorithm.

This paper proposes a smartphone-based colorimetric pH detection method using a color adaptation algorithm for point-of-care applications. Although a smartphone camera can be utilized to measure the color information of colorimetric sensors, ambient light changes and unknown built-in automatic image correction operations make it difficult to obtain stable color information. This paper utilizes a 3D printed mini light box and performs a calibration procedure with a paper-printed comparison chart and a reference image which overcomes the drawbacks of smartphone cameras and the difficulty in preparing for the calibration procedure. The color adaptation is performed in the CIE 1976 u'v' color space by using the reference paper in order to stabilize the color variations. Non-rigid u'v' curve interpolation is used to produce the high-resolution pH estimate. The final pH value is estimated by using the best-matching method to handle the nonlinear curve properties of multiple color patches. The experimental results obtained using a pH indicator paper show that the proposed algorithm provides reasonably good estimation of pH detection. With paper-printed accurate color comparison charts and smart color adaptation techniques, superior estimation is achieved in the smartphone-based colorimetric pH detection system for point-of-care application.

Expandable Mg-based Helical Stent Assessment using Static, Dynamic, and Porcine Ex Vivo Models.

A bioresorbable metallic helical stent was explored as a new device opportunity (magnesium scaffold), which can be absorbed by the body without leaving a trace and simultaneously allowing restoration of vasoreactivity with the potential for vessel remodeling. In this study, developed Mg-based helical stent was inserted and expanded in vessels with subsequent degradation in various environments including static, dynamic, and porcine ex vivo models. By assessing stent degradation in three different environments, we observed: (1) stress- and flow-induced degradation; (2) a high degradation rate in the dynamic reactor; (3) production of intermediate products (MgO/Mg(OH)2 and Ca/P) during degradation; and (4) intermediate micro-gas pocket formation in the neighboring tissue ex vivo model. Overall, the expandable Mg-based helical stent employed as a scaffold performed well, with expansion rate (>100%) in porcine ex vivo model.

An evaluation of the sonoporation potential of low-boiling point phase-change ultrasound contrast agents in vitro.

BACKGROUND: Phase-change ultrasound contrast agents (PCCAs) offer a solution to the inherent limitations associated with using microbubbles for sonoporation; they are characterized by prolonged circulation lifetimes, and their nanometer-scale sizes may allow for passive accumulation in solid tumors. As a first step towards the goal of extravascular cell permeabilization, we aim to characterize the sonoporation potential of a low-boiling point formulation of PCCAs in vitro. METHODS: Parameters to induce acoustic droplet vaporization and subsequent microbubble cavitation were optimized in vitro using high-speed optical microscopy. Sonoporation of pancreatic cancer cells in suspension was then characterized at a range of pressures (125-600 kPa) and pulse lengths (5-50 cycles) using propidium iodide as an indicator molecule. RESULTS: We achieved sonoporation efficiencies ranging from 8 ± 1% to 36 ± 4% (percent of viable cells), as evidenced by flow cytometry. Increasing sonoporation efficiency trended with increasing pulse length and peak negative pressure. CONCLUSIONS: We conclude that PCCAs can be used to induce the sonoporation of cells in vitro, and our results warrant further investigation into the use of PCCAs as extravascular sonoporation agents in vivo.

A study of long-term static load on degradation and mechanical integrity of Mg alloys-based biodegradable metals.

Predicting degradation behavior of biodegradable metals in vivo is crucial for the clinical success of medical devices. This paper reports on the effect of long-term static stress on degradation of magnesium alloys and further changes in mechanical integrity. AZ31B (H24) and ZE41A (T5) alloys were tested to evaluate stress corrosion cracking (SCC) in a physiological solution for 30 days and 90 days (ASTM G39 testing standard). Scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX) and micro-computed tomography (micro-CT) were used to characterize surface morphology and micro-structure of degraded alloys. The results show the different mechanisms of stress corrosion cracking for AZ31B (transgranular stress corrosion cracking, TGSCC) and ZE41A (intergranular stress corrosion cracking, IGSCC). AZ31B was more susceptible to stress corrosion cracking under a long term static load than ZE41A. In conclusion, we observed that long-term static loading accelerated crack propagation, leading to the loss of mechanical integrity.

Ex vivo blood vessel bioreactor for analysis of the biodegradation of magnesium stent models with and without vessel wall integration.

Current in vitro models fail in predicting the degradation rate and mode of magnesium (Mg) stents in vivo. To overcome this, the microenvironment of the stent is simulated here in an ex vivo bioreactor with porcine aorta and circulating medium, and compared with standard static in vitro immersion and with in vivo rat aorta models. In ex vivo and in vivo conditions, pure Mg wires were exposed to the aortic lumen and inserted into the aortic wall to mimic early- and long-term implantation, respectively. Results showed that: 1) Degradation rates of Mg were similar for all the fluid diffusion conditions (in vitro static, aortic wall ex vivo and in vivo); however, Mg degradation under flow condition (i.e. in the lumen) in vivo was slower than ex vivo; 2) The corrosion mode in the samples can be mainly described as localized (in vitro), mixed localized and uniform (ex vivo), and uniform (in vivo); 3) Abundant degradation products (MgO/Mg(OH)2 and Ca/P) with gas bubbles accumulated around the localized degradation regions ex vivo, but a uniform and thin degradation product layer was found in vivo. It is concluded that the ex vivo vascular bioreactor provides an improved test setting for magnesium degradation between static immersion and animal experiments and highlights its promising role in bridging degradation behavior and biological response for vascular stent research. STATEMENT OF SIGNIFICANCE: Magnesium and its alloys are candidates for a new generation of biodegradable stent materials. However, the in vitro degradation of magnesium stents does not match the clinical degradation rates, corrupting the validity of conventional degradation tests. Here we report an ex vivo vascular bioreactor, which allows simulation of the microenvironment with and without blood vessel integration to study the biodegradation of magnesium implants in comparison with standard in vitro test conditions and with in vivo implantations. The bioreactor did simulate the corrosion of an intramural implant very well, but showed too high degradation for non-covered implants. It is concluded that this system is in between static incubation and animal experiments concerning the predictivity of the degradation.

Effects of polydeoxyribonucleotides (PDRN) on wound healing: Electric cell-substrate impedance sensing (ECIS).

Polydeoxyribonucleotides (PDRN) have been explored as an effective treatment for tissue repair in peripheral artery occlusive disease, diabetic foot ulcers, and eye lotion. We report on the effect of polydeoxyribonucleotides (PDRN) on wound healing by using the electric cell-substrate impedance sensing (ECIS) system and viability testing. Human osteoblasts (U2OS) and primary human dermal fibroblasts (HDF) were used to study the effect of PDRN on migration and proliferation. ECIS allowed the creation of a wound by applying high current, and then monitoring the healing process by measuring impedance in real time. The traditional culture-insert gap-closure migration assay was performed and compared with the ECIS wound assay. PDRN-treated U2OS and HDF cells affected cell motilities to wounding site. Viability test results show that HDF and U2OS proliferation depended on PDRN concentration. Based on the results, a PDRN compound can be useful in wound healing associated with bone and skin.

Biodegradable, elastomeric coatings with controlled anti-proliferative agent release for magnesium-based cardiovascular stents.

Vascular stent design continues to evolve to further improve the efficacy and minimize the risks associated with these devices. Drug-eluting coatings have been widely adopted and, more recently, biodegradable stents have been the focus of extensive evaluation. In this report, biodegradable elastomeric polyurethanes were synthesized and applied as drug-eluting coatings for a relatively new class of degradable vascular stents based on Mg. The dynamic degradation behavior, hemocompatibility and drug release were investigated for poly(carbonate urethane) urea (PCUU) and poly(ester urethane) urea (PEUU) coated magnesium alloy (AZ31) stents. Poly(lactic-co-glycolic acid) (PLGA) coated and bare stents were employed as control groups. The PCUU coating effectively slowed the Mg alloy corrosion in dynamic degradation testing compared to PEUU-coated, PLGA-coated and bare Mg alloy stents. This was confirmed by electron microscopy, energy-dispersive x-ray spectroscopy and magnesium ion release experiments. PCUU-coating of AZ31 was also associated with significantly reduced platelet adhesion in acute blood contact testing. Rat vascular smooth muscle cell (rSMC) proliferation was successfully inhibited when paclitaxel was released from pre-loaded PCUU coatings. The corrosion retardation, low thrombogenicity, drug loading capacity, and high elasticity make PCUU an attractive option for drug eluting coating on biodegradable metallic cardiovascular stents.

Nanovesicle-targeted Kv1.3 knockdown in memory T cells suppresses CD40L expression and memory phenotype.

Ca(2+) signaling controls activation and effector functions of T lymphocytes. Ca(2+) levels also regulate NFAT activation and CD40 ligand (CD40L) expression in T cells. CD40L in activated memory T cells binds to its cognate receptor, CD40, on other cell types resulting in the production of antibodies and pro-inflammatory mediators. The CD40L/CD40 interaction is implicated in the pathogenesis of autoimmune disorders and CD40L is widely recognized as a therapeutic target. Ca(2+) signaling in T cells is regulated by Kv1.3 channels. We have developed lipid nanoparticles that deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to CD45RO(+) memory T cells and reduce the activation-induced Ca(2+) influx. Herein we report that Kv1.3-NPs reduced NFAT activation and CD40L expression exclusively in CD45RO(+) T cells. Furthermore, Kv1.3-NPs suppressed cytokine release and induced a phenotype switch of T cells from predominantly memory to naïve. These findings indicate that Kv1.3-NPs operate as targeted immune suppressive agents with promising therapeutic potentials.