As a model of the intestinal epithelium, intestinal stem cells (ISCs) have been grown and differentiated as monolayers on materials where stochastic organization of the crypt and villi cells occurs. We developed an allyl sulfide crosslinked photoresponsive hydrogel with a shear modulus of 1.6 kPa and functionalized with GFOGER, Bm-binder peptide ligands for monolayer growth of ISCs. The allyl sulfide chemistry allowed control of mechanics in the presence of growing ISC monolayers, and structured irradiation afforded spatial regulation of the hydrogel properties. Specifically, ISC monolayers grown on 1.6 kPa substrates were i softened to 0.29 kPa, using circular patterns 50, 75, and 100 μm in diameter, during differentiation, resulting in control over the size and arrangement of crypts and monolayer cellularity. These photoresponsive materials should prove useful in applications ranging from studying crypt evolution to drug screening and transport across tissues of changing cellular composition.

Magnetic particle imaging (MPI) is an emerging modality that can address longstanding technological challenges encountered with magnetic particle hyperthermia (MPH) cancer therapy. MPI is a tracer technology compatible with MPH for which magnetic nanoparticles (MNPs) provide signal for MPI and heat for MPH. Identifying whether a specific MNP formulation is suitable for both modalities is essential for clinical implementation. Current models predict that functional requirements of each modality impose conflicting demands on nanoparticle magnetic properties. This objective here is to develop a measurement and ranking scheme based on end-use performance to streamline evaluation of candidate MNP formulations. The measured MPI point-spread function (PSF) and specific loss power (SLP) is combined to generate a single numerical value for comparison on a relative ranking scale, or figure of merit (FoM). 12 aqueous iron-containing formulations are evaluated, including FDA-approved (parenteral) iron-containing colloids. MNPs with high (Synomag-D70: 123.4), medium (Synomag-D50: 63.2), and low (NanoXact: 0.147) FoM values are selected for in vivo validation of the selection scheme in subcutaneous 4T1 tumors. Results demonstrate that the proposed ranking accurately assessed the relative performance of MNPs for MPI and MPH. Data demonstrated that image quality and tumor temperature rise increased with FoM ranking, validating predictions. It isshown that the MPI signal correlated with MNP concentration in tissue. Computational heat transfer models anchored on tumor MPI data harmonized with experimental results to within an average of 2 °C when MNP content estimated from MPI data is included. Computational studies emphasized the importance of post-injection MNP quantitation and MPI spatial resolution.

N-dihydrogalactochitosan (GC) is an immune stimulant/adjuvant. Synthesized from chitosan and galactose, GC is a new chemical entity that significantly enhances the immune-stimulating properties of its parental material, chitosan, making it a promising therapeutic agent. When used in combination with antigenic material, GC stimulates innate and adaptive antitumor and antiviral immunities. However, its mechanism has not been fully investigated. Herein we demonstrate that GC drives type I IFN activation in antigen-presenting cells (APCs). More importantly, GC drives alternative STING pathways, leading to inflammatory cell death that enhances dendritic cell (DC) activation. GC-activated DCs trigger a variety of nucleic acid sensing pattern recognition receptors (PRRs) pathways and IL-1β production via the activation of the inflammasome. GC induces a potent response of type I IFNs and upregulates genes associated with STING signaling within the tumor microenvironment (TME). Moreover, intratumoral delivery of GC reduces the numbers of M2-like macrophages and increases M1-like macrophages residing within the TME, while subsequently increasing the number of activated DCs. Our findings demonstrate that GC acts as a multimodal immune stimulant via STING to generate a broad type I IFN response. This uniquely broad response holds therapeutic promise in generating enhanced antitumor and antiviral immunities.

The transport of fluids and solids is a vital process inside the human body, facilitated by the wave-like motion in the lumen called peristalsis. However, peristalsis may be compromised due to tumor growth, resulting in difficulties in lumen motility. The dysmotility of the human lumen can result in blockages and pose numerous challenges, including aspiration in the lungs and reproductive issues in the female oviduct. Restoring peristalsis in medical devices, such as medical stents, can prevent device blockage and promote effective transport. Here, a wirelessly actuated soft robotic undulating pump designed to efficiently transport both viscous fluidic and solid cargos is proposed. The kinematics of the single sheet and the coordination between pairs are systematically designed to generate undulation and peristalsis, enabling the pumping of both liquids and solids. The integration of the undulating pump is demonstrated onto an esophageal stent. The same undulating motion-based pumping mechanism can be adapted for usage in other organs, such as the female oviduct, thereby offering potential applications for treating lumen dysmotility in various diseases. The proposed wirelessly actuated robotic pumping mechanism holds promise in facilitating diverse implantable medical devices aimed at treating diseases characterized by impaired peristalsis and dysmotility.

3D printed biomaterial implants are revolutionizing personalized medicine for tissue repair, especially in orthopedics. In this study, a radiopaque bismuth oxide (BiO) doped polycaprolactone (PCL) composite is developed and implemented to enable the use of diagnostic X-ray technologies, especially spectral photon counting X-ray computed tomography (SPCCT), for comprehensive tissue engineering scaffold (TES) monitoring. PCL filament with homogeneous BiO nanoparticle (NP) dispersion (0.8 to 11.7 wt%) are first fabricated. TES are then 3D printed with the composite filament, optimizing printing parameters for small features and severely overhung geometries. These composite TES are characterized via micro-computed tomography (μCT), tensile testing, and a cytocompatibility study, with 2 wt% BiO NPs providing improved tensile properties, equivalent cytocompatibility to neat PCL, and excellent radiographic distinguishability. Radiographic performance is validated in situ by imaging 4 and 7 wt% BiO doped PCL TES in a mouse model with μCT, showing excellent agreement with in vitro measurements. Subsequently, CT image-derived swine menisci are 3D printed with composite filament and re-implanted in corresponding swine legs ex vivo. Re-imaging the swine legs via clinical CT allows facile identification of device location and alignment. Finally, the emergent technology of SPCCT unambiguously distinguishes the implanted meniscus in situ via means of color K-edge imaging.

Self-assembly of siRNA with a block copolymer featuring guanidinium and zwitterion functionalized blocks generates core-shell-like nanovectors that provide cytosolic access to siRNA and efficiently evade phagocytic clearance. The guanidinium-functionalized inner block complexes siRNA in the nanovector interior and enables cytosolic delivery. The zwitterionic outer block provides a non-interacting shell on the nanovectors that reduces macrophage uptake and phagocytic clearance and enhances tumor localization . These nanovectors were used to treat a 4T1 (murine) model of triple-negative breast cancer (TNBC). The nanovectors deliver siRNA efficiently to 4T1 triple-negative breast cancer cells , with high selectivity relative to macrophages. This efficiency and selectivity translate into efficacy: diblock nanovectors evaded phagocytic clearance and efficiently localized in an aggressive murine 4T1 orthotopic model, with a ~3-fold increase of vector residing in the tumor compared to the homopolymer nanovectors. This increased localization efficiently knocked down STAT3 (~80%) and provided tumorostasis (100% growth inhibition) at a low dose of 0.14 mg/kg. The and efficacy of these nanovectors demonstrate the potential of engineered polymer architectures to generate effective self-assembled siRNA therapeutics that avoid phagocytic clearance for the treatment of diseases requiring systemic administration.

Traditional deep fluorescence imaging has primarily focused on red-shifting imaging wavelengths into the near-infrared (NIR) windows or implementation of multi-photon excitation approaches. Here, we combine the advantages of NIR and multiphoton imaging by developing a dual-infrared two-photon microscope to enable high-resolution deep imaging in biological tissues. We first computationally identify that photon absorption, as opposed to scattering, is the primary contributor to signal attenuation. We next construct a NIR two-photon microscope with a 1640 nm femtosecond pulsed laser and a NIR PMT detector to image biological tissues labeled with fluorescent single-walled carbon nanotubes (SWNTs). We achieve spatial imaging resolutions close to the Abbe resolution limit and eliminate blur and background autofluorescence of biomolecules, 300 μm deep into brain slices and through the full 120 μm thickness of a leaf. We also demonstrate that NIR-II two-photon microscopy can measure tissue heterogeneity by quantifying how much the fluorescence power law function varies across tissues, a feature we exploit to distinguish Huntington's Disease afflicted mouse brain tissues from wildtype. Our results suggest dual-infrared two-photon microscopy could accomplish in-tissue structural imaging and biochemical sensing with a minimal background, and with high spatial resolution, in optically opaque or highly autofluorescent biological tissues.

Flexible and stretchable strain sensors are in high demand in sports performance monitoring, structural health monitoring, and biomedical applications. However, existing stretchable soft sensors, primarily based on soft polymer materials, often suffer from drawbacks, including high hysteresis, low durability, and delayed response. To overcome these limitations, we introduced a stretchable miniature fiber sensor comprised of a stretchable core tightly coiled with parallel conductive wires. This fiber sensor is flexible and stretchable while exhibiting low hysteresis, a remarkable theoretical resolution of 0.015%, a response time of less than 30 milliseconds, and excellent stability after extensive cycling tests of over 16,000 cycles. To understand and predict the capacitive sensor response of the proposed sensor, an analytical expression was derived and proved to have good agreements with both experimental results and numerical simulation. The potential of the strain sensor as a wearable device is demonstrated by embedding it into belts, gloves, and knee protectors. Additionally, the sensor could extend its applications beyond wearable devices, as demonstrated by its integration into bladder and life safety rope monitoring systems. We envision our sensor can find applications in the field of sports performance evaluations, health care monitoring, and structural safety assessments.

Peripherally injected local anesthetics exhibit limited ability to penetrate peripheral nerve barriers (PNBs), which limits their effectiveness in peripheral nerve block and increases the risk of adverse effects. In this work, we demonstrated that exosomes derived from Human Embryo Kidney (HEK) 293 cells can effectively traverse the perineurium, which is the rate-limiting barrier within PNBs that local anesthetics need to cross before acting on axons. Based on this finding, we use these exosomes as a carrier for bupivacaine (BUP), a local anesthetic commonly used in clinical settings. The assessments revealed that the prepared exosomal bupivacaine (BUP@EXO) achieves a BUP loading capacity of up to 82.33% and sustained release of BUP for over 30 days. In rats, a single peripheral injection of BUP@EXO, containing 0.75 mg of BUP, which is ineffective for BUP alone, induced a 2-hour sensory nerve blockade without significant motor impairments. Increasing the BUP dose in BUP@EXO to 2.5 mg, a highly toxic dose for BUP alone, extended the sensory nerve blockade to 12 hours without causing systemic cardiotoxicity and local neurotoxicity and myotoxicity.

Exosomes derived from mesenchymal stem cells are an active area of research due to their therapeutic potential in treating osteoporosis. To further harness their therapeutic performance in modulating bone resorption, we have equipped exosomes with osteoclast-targeting moieties on their surface as well as chemokine receptor antagonists blocking osteoclast recruitment. Phosphatidylserine (PS), a membrane lipid exerting immunosuppressive and phagocytic signals, was incorporated in the membrane of exosome mimetics (EMs) to achieve a marked affinity for osteoclast precursors and potential anti-resorptive effects. We also aimed to tackle a CXCL9-CXCR3 ligand-receptor axis, a critical signaling axis in regulating osteoclast precursor recruitment and differentiation at bone resorption sites, by encapsulating a chemical antagonist of CXCR3, AMG487, in the PS-incorporated EMs (PS-EMs). The osteoclast-targeting PS-EMs loaded with AMG487 effectively protected against bone loss in an ovariectomized mouse model. Our findings demonstrate the great promise of PS-EMs as anti-resorptive nanotherapies for alleviating osteoporosis.

Covalent and defect-free surface-grafted solid lubricating chains that can impart slippery behavior have proven advantageous over lubricant infused and textured anti-wetting surfaces. Herein, the co-hydrolysis and co-condensation of a mixture of organosilanes followed by the epoxy-amine ring opening reaction at the interface results in a highly robust, transparent and solid slippery omniphobic coating (LL-OSC). The presence of the epoxy-terminated organosilane a) acts as a molecular spacer in between the low-surface energy, rigid fluorine terminated silane and b) provides 'reactive' epoxy groups for covalent binding to a pre-functionalized amine surface for potential applicability in droplet transport and manipulation, diagnostics etc. LL-OSC exhibits resistance to both solid and liquid abrasions such as sandpaper abrasions, prolonged UV irradiation, DI water and high temperature (30 days), submersion in chemically contaminated aqueous solutions. This is the first report of a hemocompatible solid slippery coating for inhibiting platelet adhesion, thus, paving way for blood-contacting medical device applications. Our LL-OSC exhibits remarkable cytocompatibility, repellence to plasma protein, cells and prevents biofilm formation. Additionally, the substrate independent LL-OSC can be applied onto metals and polymers. We envision that the reported durable, solid slippery coating will find widespread applicability in hospital settings, electronic devices etc.

Decellularized small intestine submucosa (dSIS) is a promising biomaterial for promoting tissue regeneration. Isolated from the submucosal layer of animal jejunum, SIS is rich in extracellular matrix (ECM) proteins, including collagen, laminin, and fibronectin. Following mild decellularization, dSIS becomes an acellular matrix that supports cell adhesion, proliferation, and differentiation. Conventional dSIS matrix is usually obtained by thermal crosslinking, which yields a soft scaffold with low stability. To address these challenges, dSIS has been modified with methacrylate groups for photocrosslinking into stable hydrogels. However, dSIS has not been modified with clickable handles for orthogonal crosslinking. Here, we report the development of norbornene-modified dSIS, named dSIS-NB, via reacting amine groups of dSIS with carbic anhydride in acidic aqueous reaction conditions. Using triethylamine (TEA) as a mild base catalyst, we obtained high degrees of NB substitution on dSIS. In addition to describing the synthesis of dSIS-NB, we explored its adaptability in orthogonal hydrogel crosslinking and used dSIS-NB hydrogels for cancer and vascular tissue engineering. Impressively, compared with physically crosslinked dSIS and collagen matrices, orthogonally crosslinked dSIS-NB hydrogels supported rapid dissemination of cancer cells and superior vasculogenic and angiogenic properties. dSIS-NB was also exploited as a versatile bioink for 3D bioprinting applications.

Tissue self-assembly relies on the interplay between structural cues imparted by the extracellular matrix and instructive chemical factors that guide cellular signaling pathways. Here, we report that endothelial cell-laden gelatin-based hydrogels with optimized mechanical and chemical properties facilitate vasculogenesis and recruitment of endogenous blood vessels . We demonstrate that these engineered matrices, with tailored viscoelastic features and stiffness, drive vascular self-assembly in a yes-associated protein mechanosensing-dependent manner through αvβ3 integrin and matrix metalloproteinase 2 activity. Our research highlights how the extracellular matrix, in the form of gelatin-based hydrogels with adjustable stress relaxation rates, drive vascular morphogenesis in the absence of growth factor supplementation, lending to a minimalistic platform for discretizing features of the microenvironment niche. Collectively, these results demonstrate a testbed that enables mechanistic evaluation of morphogenetic processes. Specifically, our results show how mechanical cues impact signaling pathways that modulate vascular remodeling, a critical tissue engineering paradigm needed for the translational application of vascularized grafts for regenerative medicine applications.

Three-dimensional (3D) bioprinting using photocrosslinkable hydrogels has gained considerable attention due to its versatility in various applications, including tissue engineering and drug delivery. Egg White (EW) is an organic biomaterial with excellent potential in tissue engineering. It provides abundant proteins, along with biocompatibility, bioactivity, adjustable mechanical properties, and intrinsic antiviral and antibacterial features. Here, we have developed a photocrosslinkable hydrogel derived from EW through methacryloyl modification, resulting in Egg White methacryloyl (EWMA). Upon exposure to UV light, synthesized EWMA becomes crosslinked, creating hydrogels with remarkable bioactivity. These hydrogels offer adjustable mechanical and physical properties compatible with most current bioprinters. The EWMA hydrogels closely resemble the native extracellular matrix (ECM) due to cell-binding and matrix metalloproteinase-responsive motifs inherent in EW. In addition, EWMA promotes cell growth and proliferation in 3D cultures. It facilitates vascularization when investigated with human umbilical vein endothelial cells (HUVECs), making it an attractive replacement for engineering hemocompatible vascular grafts and biomedical implants. In summary, the EWMA matrix enables the biofabrication of various living constructs. This breakthrough enhances the development of physiologically relevant 3D models and opens many opportunities in regenerative medicine.

Single-walled carbon nanotubes (SWCNTs) are desirable nanoparticles for sensing biological analytes due to their photostability and intrinsic near-infrared fluorescence. Previous strategies for generating SWCNT nanosensors have leveraged nonspecific adsorption of sensing modalities to the hydrophobic SWCNT surface that often require engineering new molecular recognition elements. An attractive alternate strategy is to leverage pre-existing molecular recognition of proteins for analyte specificity, yet attaching proteins to SWCNT for nanosensor generation remains challenging. Towards this end, we introduce a generalizable platform to generate protein-SWCNT-based optical sensors and use this strategy to synthesize a hydrogen peroxide (HO) nanosensor by covalently attaching horseradish peroxidase (HRP) to the SWCNT surface. We demonstrate a concentration-dependent response to HO, confirm the nanosensor can image HO in real-time, and assess the nanosensor's selectivity for HO against a panel of biologically relevant analytes. Taken together, these results demonstrate successful covalent attachment of enzymes to SWCNTs while preserving both intrinsic SWCNT fluorescence and enzyme function. We anticipate this platform can be adapted to covalently attach other proteins of interest including other enzymes for sensing or antibodies for targeted imaging and cargo delivery.

Stretchable electrodes are an essential component in soft actuator systems. In particular, Joule heating electrodes (JHEs) are required for thermal actuation systems. A highly stretchable, patternable, and low-voltage operating JHE based on hybrid layers of silver nanowires (AgNWs) and carbon nanotubes (CNTs) is reported. The conductive layers were applied on a locally pre-strained bistable electroactive polymer (BSEP) membrane to form a wrinkled conductive surface with a low resistance of 300 Ω/sq, and subsequently patterned to a serpentine trace by laser engraving. The resistance of the resulting electrode remains nearly unchanged up to ~80-90% area strain. By applying a voltage of 7 - 9 V to the electrode, the temperature of the BSEP membrane increased to more than 60 °C, well above the polymer's phase transition temperature of 46 °C, thereby lowering its modulus by a factor of 10. An electronic Braille device based on the JHEs on a BSEP membrane was assembled with a diaphragm chamber. The electrode was patterned into 3 × 2 individually addressable pixels according to the standard U.S. Braille cell format. Through Joule heating of the pixels and local expansion of the BSEP membrane using a small pneumatic pressure, the pixels deformed out of the plane by over 0.5 mm to display specific Braille letters. The Braille content can be refreshed for 20,000 cycles at the same operating voltage.

Microfluidic valves play a key role within microfluidic systems by regulating fluid flow through distinct microchannels, enabling many advanced applications in medical diagnostics, lab-on-chips, and laboratory automation. While microfluidic systems are often limited to planar structures, 3D printing enables new capabilities to generate complex designs for fluidic circuits with higher densities and integrated components. However, the control of fluids within 3D structures presents several difficulties, making it challenging to scale effectively and many fluidic devices are still often restricted to quasi-planar structures. Incorporating mechanical metamaterials that exhibit spatially adjustable mechanical properties into microfluidic systems provides an opportunity to address these challenges. Here, we have performed systematic computational and experimental characterization of a modified re-entrant honeycomb structure to generate a modular metamaterial for an active device that allows us to directly regulate flow through integrated, multiplexed fluidic channels "one-at-a-time," in a manner that is highly scalable. We present a design algorithm so that this architecture can be extended to arbitrary geometries, and we expect that by incorporation of mechanical metamaterial designs into 3D printed fluidic systems, which themselves are readily expandable to any complex geometries, will enable new biotechnological and biomedical applications of 3D printed devices.

3-D bioprinting is a promising technology to fabricate custom geometries for tissue engineering. However, most bioprintable hydrogels are weak and fragile, difficult to handle and cannot mimetic the mechanical behaviors of the native soft elastic tissues. We have developed a visible light crosslinked, single-network, elastic and biocompatible hydrogel system based on an acrylated triblock copolymer of poly(ethylene glycol) PEG and polycaprolactone (PCL) (PEG-PCL-DA). To enable its application in bioprinting of soft tissues, we have modified the hydrogel system on its printability and biodegradability. Furthermore, we hypothesize that this elastic material can better transmit pulsatile forces to cells, leading to enhanced cellular response under mechanical stimulation. This central hypothesis was tested using vascular conduits with smooth muscle cells (SMCs) cultured under pulsatile forces in a custom-made bioreactor. The results showed that vascular conduits made of PEG-PCL-DA hydrogel faithfully recapitulate the rapid stretch and recoil under the pulsatile pressure from 1 to 3 Hz frequency, which induced a contractile SMC phenotype, consistently upregulated the core contractile transcription factors. In summary, our work demonstrates the potential of elastic hydrogel for 3D bioprinting of soft tissues by fine tuning the printability, biodegradability, while possess robust elastic property suitable for manual handling and biomechanical stimulation.

Blood scarcity is one of the main causes of healthcare disruptions worldwide, with blood shortages occurring at an alarming rate. Over the last decades, blood substitutes has aimed at reinforcing the supply of blood, with several products (e.g., hemoglobin-based oxygen carriers, perfluorocarbons) achieving a limited degree of success. Regardless, there is still no widespread solution to this problem due to persistent challenges in product safety and scalability. In this Review, we describe different advances in the field of blood substitution, particularly in the development of artificial red blood cells, otherwise known as engineered erythrocytes. We categorize the different strategies into natural, synthetic, or hybrid approaches, and discuss their potential in terms of safety and scalability. We identify synthetic engineered erythrocytes as the most powerful approach, and describe erythrocytes from a materials engineering perspective. We review their biological structure and function, as well as explore different methods of assembling a material-based cell. Specifically, we discuss how to recreate size, shape, and deformability through particle fabrication, and how to recreate the functional machinery through synthetic biology and nanotechnology. We conclude by describing the versatile nature of synthetic erythrocytes in medicine and pharmaceuticals and propose specific directions for the field of erythrocyte engineering.

Chimeric antigen receptor (CAR) monocyte and macrophage therapies are promising solid tumor immunotherapies that can overcome the challenges facing conventional CAR T cell therapy. mRNA lipid nanoparticles (mRNA-LNPs) offer a viable platform for engineering of CAR monocytes with transient and tunable CAR expression to reduce off-tumor toxicity and streamline cell manufacturing. However, identifying LNPs with monocyte tropism and intracellular delivery potency is difficult using traditional screening techniques. Here, ionizable lipid design and high-throughput screening are utilized to identify a new class of oxidized LNPs with innate tropism and mRNA delivery to monocytes. A library of oxidized (oLNPs) and unoxidized LNPs (uLNPs) is synthesized to evaluate mRNA delivery to immune cells. oLNPs demonstrate notable differences in morphology, ionization energy, and p, therefore enhancing delivery to human macrophages, but not T cells. Subsequently, library screening with DNA barcodes identifies an oLNP formulation, C14-O2, with innate tropism to monocytes. In a proof-of-concept study, the C14-O2 LNP is used to engineer functional CD19-CAR monocytes for robust B cell aplasia (45%) in healthy mice. This work highlights the utility of oxidized LNPs as a promising platform for engineering CAR macrophages/monocytes for solid tumor CAR monocyte therapy.

The physiological property of mucus is an important biomarker for monitoring the human health conditions and helping understand disease development, as mucus property such as viscosity is highly correlated with inflammation and other diseases. However, it remains challenging to sense mucus viscosity using pure medical imaging. Collecting and analyzing mucus sample in vitro using flexible endoscopes and capsule endoscope robots is also challenging due to their difficulty of accessing very confined, tortuous, and small spaces, and the sample may not reflect the real mucus property. Here a novel method is proposed to enable sensing mucus viscosity in situ by wireless miniature sensors actuated by magnetic fields and tracked by medical imaging. These miniature viscosity sensors can be delivered with minimal invasion using a novel sensor delivery mechanism by controlling a magnetically actuated millimeter-scale soft climbing robot. As the soft robot can access confined and narrow spaces, and reliably deploy the sensor on soft tissue surfaces, multiple sensors can be delivered on soft biological tissues to sense biofluid viscosity spatiotemporally. The proposed minimally invasive robotic delivery and viscosity sensing method thus paves the way toward sensing biofluid properties deep inside the body for future disease monitoring and early diagnosis functions.