Modeling of spatiotemporal dynamics of ligand-coated particle flow in targeted drug delivery processes.

Nanoparticles tethered with vasculature-binding epitopes have been used to deliver the drug into injured or diseased tissues via the bloodstream. However, the extent that blood flow dynamics affects nanoparticle retention at the target site after adhesion needs to be better understood. This knowledge gap potentially underlies significantly different therapeutic efficacies between animal models and humans. An experimentally validated mathematical model that accurately simulates the effects of blood flow on nanoparticle adhesion and retention, thus circumventing the limitations of conventional trial-and-error-based drug design in animal models, is lacking. This paper addresses this technical bottleneck and presents an integrated mathematical method that derives heavily from a unique combination of a mechanics-based dispersion model for nanoparticle transport and diffusion in the boundary layers, an asperity model to account for surface roughness of endothelium, and an experimentally calibrated stochastic nanoparticle-cell adhesion model to describe nanoparticle adhesion and subsequent retention at the target site under external flow. PLGA-b-HA nanoparticles tethered with VHSPNKK peptides that specifically bind to vascular cell adhesion molecules on the inflamed vascular wall were investigated. The computational model revealed that larger particles perform better in adhesion and retention at the endothelium for the particle sizes suitable for drug delivery applications and within physiologically relevant shear rates. The computational model corresponded closely to the in vitro experiments which demonstrates the impact that model-based simulations can have on optimizing nanocarriers in vascular microenvironments, thereby substantially reducing in vivo experimentation as well as the development costs.

Neuronal innervation regulates the secretion of neurotrophic myokines and exosomes from skeletal muscle.

Myokines and exosomes, originating from skeletal muscle, are shown to play a significant role in maintaining brain homeostasis. While exercise has been reported to promote muscle secretion, little is known about the effects of neuronal innervation and activity on the yield and molecular composition of biologically active molecules from muscle. As neuromuscular diseases and disabilities associated with denervation impact muscle metabolism, we hypothesize that neuronal innervation and firing may play a pivotal role in regulating secretion activities of skeletal muscles. We examined this hypothesis using an engineered neuromuscular tissue model consisting of skeletal muscles innervated by motor neurons. The innervated muscles displayed elevated expression of mRNAs encoding neurotrophic myokines, such as interleukin-6, brain-derived neurotrophic factor, and FDNC5, as well as the mRNA of peroxisome-proliferator-activated receptor γ coactivator 1α, a key regulator of muscle metabolism. Upon glutamate stimulation, the innervated muscles secreted higher levels of irisin and exosomes containing more diverse neurotrophic microRNAs than neuron-free muscles. Consequently, biological factors secreted by innervated muscles enhanced branching, axonal transport, and, ultimately, spontaneous network activities of primary hippocampal neurons in vitro. Overall, these results reveal the importance of neuronal innervation in modulating muscle-derived factors that promote neuronal function and suggest that the engineered neuromuscular tissue model holds significant promise as a platform for producing neurotrophic molecules.

Graphene Hybrid Tough Hydrogels with Nanostructures for Tissue Regeneration.

Over the past few decades, hydrogels have attracted considerable attention as promising biomedical materials. However, conventional hydrogels require improved mechanical properties, such as brittleness, which significantly limits their widespread use. Recently, hydrogels with remarkably improved toughness have been developed; however, their low biocompatibility must be addressed. In this study, we developed a tough graphene hybrid hydrogel with nanostructures. The resultant hydrogel exhibited remarkable mechanical properties while representing an aligned nanostructure that resembled the extracellular matrix of soft tissue. Owing to the synergistic effect of the topographical properties, and the enhanced biochemical properties, the graphene hybrid hydrogel had excellent stretchability, resilience, toughness, and biocompatibility. Furthermore, the hydrogel displayed outstanding tissue regeneration capabilities (e.g., skin and tendons). Overall, the proposed graphene hybrid tough hydrogel may provide significant insights into the application of tough hydrogels in tissue regeneration.

Intracerebral Nanoparticle Transport Facilitated by Alzheimer Pathology and Age.

Nanoparticles have emerged as potential transporters of drugs targeting Alzheimer's disease (AD), but their design should consider the blood-brain barrier (BBB) integrity and neuroinflammation of the AD brain. This study presents that aging is a significant factor for the brain localization and retention of nanoparticles, which we engineered to bind with reactive astrocytes and activated microglia. We assembled 200 nm-diameter particles using a block copolymer of poly(lactic-co-glycolic acid) (PLGA) and CD44-binding hyaluronic acid (HA). The resulting PLGA-b-HA nanoparticles displayed increased binding to CD44-expressing reactive astrocytes and activated microglia. Upon intravascular injection, nanoparticles were localized to the hippocampi of both APP/PS1 AD model mice and their control littermates at 13-16 months of age due to enhanced transvascular transport through the leaky BBB. No particles were found in the hippocampi of young adult mice. These findings demonstrate the brain localization of nanoparticles due to aging-induced BBB breakdown regardless of AD pathology.

In-drop thermal cycling of microcrystal assembly for senescence control (MASC) with minimal variation in efficacy.

The secretome from mesenchymal stem cells (MSCs) has recently gained attention for new therapeutics. However, clinical application requires in vitro cell manufacturing to attain enough cells. Unfortunately, this process often drives MSCs into a senescent state that drastically changes cellular secretion activities. Antioxidants are used to reverse and prevent the propagation of senescence; however, their activity is short-lived. Polymer-stabilized crystallization of antioxidants has been shown to improve bioactivity, but the broad crystal size distribution (CSD) significantly increases the efficacy variation. Efforts were made to crystalize drugs in microdroplets to narrow the CSD, but the fraction of drops containing at least one crystal can be as low as 20%. To this end, this study demonstrates that in-drop thermal cycling of hyaluronic acid-modified antioxidant crystals, named microcrystal assembly for senescence control (MASC), can drive the fraction of microdrops containing crystals to >86% while achieving significantly narrower CSDs (13±3µm) than in bulk (35±11µm). Therefore, this approach considerably improves the practicality of CSD-control in drops. In addition to exhibiting uniform release, MASC made with antioxidizing N-acetylcysteine extended the release time by 40%. MASC further improves the restoration of reactive oxygen species homeostasis in MSCs, thus minimizing cellular senescence and preserving desired secretion activities. We propose that MASC is broadly useful to controlling senescence of a wide array of therapeutic cells during biomanufacturing.

Antimicrobial efficacy of self-locomotive manganese oxide nanozyme-doped diatom microbubbler on orthodontic brackets in vitro.

BACKGROUND: Orthodontic brackets provide a favorable environment for Streptococcus mutans biofilm formation, increasing the risk of white spots and dental caries. Manganese oxide (MnO2) nanozyme-doped diatom microbubbler (DM) is a recently developed material for biofilm removal. DM can generate oxygen by catalase-mimicking activity in Hydrogen peroxide (H2O2) solution and move with ejecting oxygen microbubbles to produce a mechanical self-cleansing effect. This study aimed to evaluate the feasibility of DM as a novel bracket cleaner. METHODS: DM was prepared according to the protocol and analyzed using a scanning electron microscope (SEM). We treated S. mutans biofilms grown over bracket with phosphate-buffered saline (PBS group), 0.12% chlorhexidine (CHX group), 3% H2O2 (H2O2 group), and co-treatment with 3 mg/mL of DM and 3% H2O2 (DM group). The biofilm removal effect was analyzed using crystal violet assay, and the results were observed using SEM. The viability of S. mutans in remaining biofilms was evaluated using confocal laser scanning microscopy (CLSM). Finally, we examined the effect of all materials on mature multispecies biofilms formed on debonded brackets. RESULTS: Crystal violet assay results revealed that the CHX group removed more biofilms than the control group, and the DM group removed biofilms more effectively than the CHX group (p < 0.0001). SEM and CLSM images showed that CHX killed S. mutans but failed to remove most biofilms on brackets. However, DM effectively removed biofilms and mature multispecies biofilms on debonded brackets (p < 0.0001). CONCLUSIONS: Co-treatment with DM and H2O2 is effective in removing biofilms on orthodontic brackets compared to conventional antibacterial agents.

Biohybrid valveless pump-bot powered by engineered skeletal muscle.

Pumps are critical life-sustaining components for all animals. At the earliest stages of life, the tubular embryonic heart works as a valveless pump capable of generating unidirectional blood flow. Inspired by this elementary pump, we developed an example of a biohybrid valveless pump-bot powered by engineered skeletal muscle. Our pump-bot consists of a soft hydrogel tube connected at both ends to a stiffer polydimethylsiloxane (PDMS) scaffold, creating an impedance mismatch. A contractile muscle ring wraps around the hydrogel tube at an off-center location, squeezing the tube with or without buckling it locally. Cyclic muscle contractions, spontaneous or electrically stimulated, further squeeze the tube, resulting in elastic waves that propagate along the soft tube and get reflected back at the soft/stiff tube boundaries. Asymmetric placement of muscle ring results in a time delay between the wave arrivals, thus establishing a net unidirectional fluid flow irrespective of whether the tube is buckled or not. Flow rates of up to 22.5 µL/min are achieved by the present pump-bot, which are at least three orders of magnitude higher than those from cardiomyocyte-powered valve pumps of similar size. Owning to its simple geometry, robustness, ease of fabrication, and high pumping performance, our pump-bot is particularly well-suited for a wide range of biomedical applications in microfluidics, drug delivery, biomedical devices, cardiovascular pumping system, and more.

1α,25-Dihydroxyvitamin D3 Encapsulated in Nanoparticles Prevents Venous Neointimal Hyperplasia and Stenosis in Porcine Arteriovenous Fistulas.

BACKGROUND: Few therapies prevent venous neointimal hyperplasia (VNH) and venous stenosis (VS) formation in arteriovenous fistulas (AVF). Expression of the immediate early response gene X-1 (Iex-1), also known as Ier3, is associated with VNH and stenosis in murine AVFs. The study aimed to determine if local release of Ier3 long-acting inhibitor 1α,25(OH)2D3 from poly(lactic-co-glycolic acid) (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (1,25 NP) could affect VNH/VS formation in a large animal model. METHODS: Immediately after AVF creation in a porcine model of renal failure, 1,25 NP or vehicle control was injected into the adventitia space of AVF outflow veins. Scanning electron microscopy and dynamic light scattering characterized drug and control nanoparticles. Animals were sacrificed 3 and 28 days later for gene expression, immunohistologic, magnetic resonance imaging and angiography, and ultrasound analyses. Whole transcriptome RNA sequencing with differential gene expression analysis was performed on outflow veins of AVF. RESULTS: Encapsulation of 1α,25(OH)2D3 in PLGA nanoparticles formed nanoparticles of uniform size that were similar to nanoparticles without 1α,25(OH)2D3. The 1,25 NP-treated AVFs exhibited lower VNH/VS, Ier3 gene expression, and IER-3, MCP-1, CD68, HIF-1α, and VEGF-A immunostaining, fibrosis, and proliferation. Blood flow and lumen area increased significantly, whereas peak systolic velocity and wall shear stress decreased. Treatment increased Young's modulus and correlated with histologic assessment of fibrosis and with no evidence of vascular calcification. RNA sequencing analysis showed changes in the expression of genes associated with inflammatory, TGFß1, and apoptotic pathways. CONCLUSIONS: Local release of 1,25 NP improves AVF flow and hemodynamics, and reduces stenosis in association with reduction in inflammation, apoptosis, and fibrosis in a porcine model of arteriovenous fistula.

Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Conditions of extended bed rest and limb immobilization can initiate rapid and significant loss of skeletal muscle mass and function. Physical rehabilitation is standard practice following a period of disuse, yet mobility may be severely compromised, and recovery is commonly delayed or incomplete in special populations. Thus, a novel approach toward recovery of muscle mass is highly desired. Pericytes [neuron-glial antigen 2 (NG2)+CD31-CD45- (Lineage- [Lin-]) and CD146+Lin-] demonstrate capacity to facilitate muscle repair, yet the ability to enhance myofiber growth following disuse is unknown. In the current study, 3-4-mo-old mice were unilaterally immobilized for 14 d (IM) or immobilized for 14 d followed by 14 d of remobilization (RE). Flow cytometry and targeted gene expression analyses were completed to assess pericyte quantity and function following IM and RE. In addition, a transplantation study was conducted to assess the impact of pericytes on recovery. Results from targeted analyses suggest minimal impact of disuse on pericyte gene expression, yet NG2+Lin- pericyte quantity is reduced following IM (P < 0.05). Remarkably, pericyte transplantation recovered losses in myofiber cross-sectional area and the capillary-to-fiber ratio following RE, whereas deficits remained with vehicle alone (P = 0.01). These findings provide the first evidence that pericytes effectively rehabilitate skeletal muscle mass following disuse atrophy.-Munroe, M., Dvoretskiy, S., Lopez, A., Leong, J., Dyle, M. C., Kong, H., Adams, C. M., Boppart, M. D. Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Preoperative vascular surgery model using a single polymer tough hydrogel with controllable elastic moduli.

Materials used in organ mimics for medial simulation and education require tissue-like softness, toughness, and hydration to give clinicians and students accurate tactile feedback. However, there is a lack of materials that satisfy these requirements. Herein, we demonstrate that a stretchable and tough polyacrylamide hydrogel is useful to build organ mimics that match softness, crack growth resistance, and interstitial water of real organs. Varying the acrylamide concentration between 29 or 62% w/w with a molar ratio between cross-linker and acrylamide of 1 : 10 800 resulted in a fracture energy around ∼2000 J m-2. More interestingly, this tough gel permitted variation of the elastic modulus from 8 to 62 kPa, which matches the softness of brain to vascular and muscle tissue. According to the rheological frequency sweep, the tough polyacrylamide hydrogels had a greatly decreased number of flow units, indicating that when deformed, stress was dispersed over a greater area. We propose that such molecular dissipation results from the increased number of entangled polymers between distant covalent cross-links. The gel was able to undergo various manipulations including stretching, puncture, delivery through a syringe tip, and suturing, thus enabling the use of the gel as a blood vessel model for microsurgery simulation.

Engineering the Surface of Therapeutic "Living" Cells.

Biological cells are complex living machines that have garnered significant attention for their potential to serve as a new generation of therapeutic and delivery agents. Because of their secretion, differentiation, and homing activities, therapeutic cells have tremendous potential to treat or even cure various diseases and injuries that have defied conventional therapeutic strategies. Therapeutic cells can be systemically or locally transplanted. In addition, with their ability to express receptors that bind specific tissue markers, cells are being studied as nano- or microsized drug carriers capable of targeted transport. Depending on the therapeutic targets, these cells may be clustered to promote intercellular adhesion. Despite some impressive results with preclinical studies, there remain several obstacles to their broader development, such as a limited ability to control their transport, engraftment, secretion and to track them in vivo. Additionally, creating a particular spatial organization of therapeutic cells remains difficult. Efforts have recently emerged to resolve these challenges by engineering cell surfaces with a myriad of bioactive molecules, nanoparticles, and microparticles that, in turn, improve the therapeutic efficacy of cells. This review article assesses the various technologies developed to engineer the cell surfaces. The review ends with future considerations that should be taken into account to further advance the quality of cell surface engineering.

Matrix Softness-Mediated 3D Zebrafish Hepatocyte Modulates Response to Endocrine Disrupting Chemicals.

Endocrine disrupting chemicals (EDC) include synthetic compounds that mimic the structure or function of natural hormones. While most studies utilize live embryos or primary cells from adult fish, these cells rapidly lose functionality when cultured on plastic or glass substrates coated with extracellular matrix proteins. This study hypothesizes that the softness of a matrix with adhered fish cells can regulate the intercellular organization and physiological function of engineered hepatoids during EDC exposure. We scrutinized this hypothesis by culturing zebrafish hepatocytes (ZF-L) on collagen-based hydrogels with controlled elastic moduli by examining morphology, urea production, and intracellular oxidative stress of hepatoids exposed to 17ß-estradiol. Interestingly, the softer gel drove cells to form a cell sheet with a canaliculi-like structure compared to its stiffer gel counterpart. The hepatoids cultured on the softer gel exhibited more active urea production upon exposure to 17ß-estradiol and displayed faster recovery of intracellular reactive oxygen species level confirmed by gradient light interference microscopy (GLIM), a live-cell imaging technique. These results are broadly useful to improve screening and understanding of potential EDC impacts on aquatic organisms and human health.

Surface tethering of stromal cell-derived factor-1α carriers to stem cells enhances cell homing to ischemic muscle.

Mesenchymal stem cells are promising medicine for treating diseases and tissue defects because of their innate ability to secrete therapeutic factors. Intravenous delivery of stem cells, although favored for its minimal invasiveness, is often plagued by low cellular engraftment in the target tissue. To this end, this study hypothesizes that in situ activation of cellular expression of CXC chemokine 4 (CXCR4) would significantly improve cellular migration to injured tissue. This hypothesis was examined by tethering the surface of stem cells with poly(D,L-lactide-co-glycolide)-block-hyaluronic acid (HA) particles containing stromal cell-derived factor-1α, a model chemokine to sensitize CXCR4. The HA blocks in the particles enhanced the association rate constant to stem cells by 3.3-fold, and in turn, increased the number of cells expressing CXCR4 receptors. Consequently, these cells displayed 1.2-fold higher transendothelial migration in vitro and 1.7-fold greater trafficking to the ischemic hindlimb of a mouse than that of the untethered cells.

Stimulus-Responsive Anti-Oxidizing Drug Crystals and their Ecological Implication.

Various antioxidants are being used to neutralize the harmful effects of reactive oxygen species (ROS) overproduced in diseased tissues and contaminated environments. Polymer-directed crystallization of antioxidants has attracted attention as a way to control drug efficacy through molecular dissolution. However, most recrystallized antioxidants undertake continuous dissolution independent of the ROS level, thus causing side-effects. This study demonstrates a unique method to assemble antioxidant crystals that modulate their dissolution rate in response to the ROS level. We hypothesized that antioxidants recrystallized using a ROS-labile polymer would be triggered to dissolve when the ROS level increases. We examined this hypothesis by using catechin as a model antioxidant. Catechin was recrystallized using polyethylenimine cross-linked with ROS-labile diselanediylbis-(ethane-2,1-diyl)-diacrylate. Catechin crystallized with the ROS-labile polymer displays accelerated dissolution proportional to the H2 O2 concentration. The ROS-responsive catechin crystals protect vascular cells from oxidative insults by activating intracellular glutathione peroxidase expression and, in turn, inhibiting an increase in the intracellular oxidative stress. In addition, ROS-responsive catechin crystals alleviate changes in the heart rate of Daphnia magna in oxidative media. We propose that the results of this study would be broadly useful for improving the therapeutic efficacy of a broad array of drug compounds.

Potential lymphangiogenesis therapies: Learning from current antiangiogenesis therapies-A review.

In recent years, lymphangiogenesis, the process of lymphatic vessel formation from existing lymph vessels, has been demonstrated to have a significant role in diverse pathologies, including cancer metastasis, organ graft rejection, and lymphedema. Our understanding of the mechanisms of lymphangiogenesis has advanced on the heels of studies demonstrating vascular endothelial growth factor C as a central pro-lymphangiogenic regulator and others identifying multiple lymphatic endothelial biomarkers. Despite these breakthroughs and a growing appreciation of the signaling events that govern the lymphangiogenic process, there are no FDA-approved drugs that target lymphangiogenesis. In this review, we reflect on the lessons available from the development of antiangiogenic therapies (26 FDA-approved drugs to date), review current lymphangiogenesis research including nanotechnology in therapeutic drug delivery and imaging, and discuss molecules in the lymphangiogenic pathway that are promising therapeutic targets.

Pore Diameter of Mesoporous Silica Modulates Oxidation of H2O2-Sensing Chromophore in a Porous Matrix.

Hydrogen peroxide (H2O2) is an attractive chemical because of its bleaching properties in paper and pulp industry and as a disinfectant in the food, water, and medical industries. However, it is important to monitor the residual H2O2 level after its usage and prevent any unintended health problems or chemical reactions. Most H2O2 sensors often utilize fluorophores or electrical circuitry that requires an additional irradiation or a digital display. To this end, this study presents a 3,3',5,5'-tetramethylbenzidine (TMB)/horseradish peroxidase (HRP)-loaded patch that alerts the presence of high H2O2 levels by generating a visible blue color. We hypothesized that water-insoluble TMB immobilized within mesoporous silica particles of proper pore diameter and structure would act as a colorimetric indicator through the H2O2-mediated oxidation within a cross-linked patch. We examined this hypothesis by immobilizing TMB molecules in mesoporous silica particles with 2 and 7 nm diameter cylindrical pores as well as on nonporous silica particles. Then, we loaded these TMB-silica particles and HRP in a porous alginate patch via sequential in situ cross-linking reaction and lyophilization. In the presence of 25-5000 µM H2O2, which simulate H2O2 concentrations found in residual disinfecting fluids, the patch loaded with TMB-mesoporous silica particles with 7 nm diameter pores generated a distinct blue color with varying intensities depending on the H2O2 concentration. The design principles demonstrated in this study should be applicable to a broad array of sensors to be integrated into a moldable, three-dimensional matrix.

Spatial Organization of Superparamagnetic Iron Oxide Nanoparticles in/on Nano/Microsized Carriers Modulates the Magnetic Resonance Signal.

Superparamagnetic iron oxide nanoparticles (SPIONs) are often encapsulated into drug-carrying nano/microsized particles for simultaneous magnetic resonance (MR) imaging and treatment of diseased tissues. Unfortunately, encapsulated SPIONs may have a limited ability to modulate the T2-weighted relaxation of water protons, but this insight has not been examined systematically. This study demonstrates that SPIONs immobilized on 200 nm diameter poly(lactic- co-glycolic acid) (PLGA) nanoparticles using Pickering emulsification present 18-fold higher relaxivity than encapsulated SPIONs and 1.5-fold higher relaxivity than free SPIONs. In contrast, the SPIONs immobilized on 10 µm diameter PLGA particles exhibit a minor increase in MR relaxivity. This interesting finding will significantly impact current efforts to synthesize and assemble advanced MR contrast agents.

Disease-directed design of biodegradable polymers: Reactive oxygen species and pH-responsive micellar nanoparticles for anticancer drug delivery.

Herein, we report reactive oxygen species (ROS)- and pH-responsive biodegradable polyethylene glycol (PEG)-block-polycarbonate by installing thioether groups onto the polycarbonate and its self-assembled core/shell structured micelles for anticancer drug delivery. Oxidation of thioethers to sulfoxide and subsequently sulfone induces an increase in hydrophilicity, resulting in more hydrophilic micellar core. This phase-change caused the micelles to swell and enhance cargo release. Carboxylic acid groups have also been installed onto thioether-containing polycarbonate to promote loading of amine-containing anticancer doxorubicin through electrostatic interaction. Urea-functionalized thioether-containing PEG-block-polycarbonates were synthesized to mix with the acid-functionalized PEG-block-polycarbonate for stabilizing micelle structure through hydrogen-bonding interaction. The mixed micelles were 50 nm in diameter and had a 25 wt% loading capacity for doxorubicin. Enhanced drug release from the micelles was triggered by low pH and high content of ROS. Drug-encapsulated micelles accumulated in tumors through leaky tumor vasculature in PC-3 human prostate cancer xenograft mouse model.

Poly(ethylene glycol)-Mediated Collagen Gel Mechanics Regulates Cellular Phenotypes in a Microchanneled Matrix.

For the past few decades, efforts have been extensively made to reproduce tissue of interests for various uses including fundamental bioscience studies, clinical treatments, and even soft robotic systems. In these studies, cells are often cultured in micropores introduced in a provisional matrix despite that bulk rigidity may negatively affect cellular differentiation involved in tissue formation. To this end, we hypothesized that suspending cells within a soft fibrous matrix that is encapsulated within the microchannels of a provisional matrix would allow us to mediate effects of the matrix rigidity on cells and, in turn, to increase the cell differentiation level. We examined this hypothesis by filling microchannels interpenetrating alginate matrices with collagen gels of controlled elastic moduli (i.e., 125 to 1 Pa). Myoblasts used as a model predifferentiated cell were suspended within the collagen gels. The elastic modulus of the collagen gels was decreased through the addition of poly(ethylene glycol) during the gel preparation. Myoblasts loaded in the collagen gel exhibited a higher myogenic differentiation level than those adhered to the collagen-coated microchannel wall. Furthermore, the collagen gel softened by poly(ethylene glycol) further increased the volume of the multinucleated myofibers. The role of collagen gel softness on cell differentiation became more significant when the bulk elastic modulus of the alginate matrix was tuned to be close to that of muscle tissue (i.e., 11 kPa). We believe that the results of this study would be useful to understanding phenotypic activities of a wide array of cells involved in tissue development and regeneration.

3D printing enables separation of orthogonal functions within a hydrogel particle.

Multifunctional particles with distinct physiochemical phases are required by a variety of applications in biomedical engineering, such as diagnostic imaging and targeted drug delivery. This motivates the development of a repeatable, efficient, and customizable approach to manufacturing particles with spatially segregated bioactive moieties. This study demonstrates a stereolithographic 3D printing approach for designing and fabricating large arrays of biphasic poly (ethylene glycol) diacrylate (PEGDA) gel particles. The fabrication parameters governing the physical and biochemical properties of multi-layered particles are thoroughly investigated, yielding a readily tunable approach to manufacturing customizable arrays of multifunctional particles. The advantage in spatially organizing functional epitopes is examined by loading superparamagnetic iron oxide nanoparticles (SPIONs) and bovine serum albumin (BSA) in separate layers of biphasic PEGDA gel particles and examining SPION-induced magnetic resonance (MR) contrast and BSA-release kinetics. Particles with spatial segregation of functional moieties have demonstrably higher MR contrast and BSA release. Overall, this study will contribute significant knowledge to the preparation of multifunctional particles for use as biomedical tools.