Bioprintable hydrogel and bioink

Abstract

Bioprintable hydrogels, methods of synthesizing, and methods of use thereof are provided.

Description

[0001] BIOPRINTABLE HYDROGEL AND BIOINK

[0002] Bin Duan Wen Shi Mitchell Kuss

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 535,197, filed August 29, 2023. The foregoing application is incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] This application relates to the field of hydrogels. More specifically, this invention provides bioprintable hydrogels and methods of use thereof.

[0006] BACKGROUND OF THE INVENTION

[0007] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0008] Hydrogels display soft tissue-like mechanical properties and have emerged as good candidates for wearable electronic devices. These devices can sense external stimuli, such as pressure, strain, temperature, and moisture, and convert them into electronic signals, including current, resistance, and capacitance. This enables real-time monitoring of human motion and health parameters (Ying, et al. (2021) i Science 24(11): 103174). Among different types of sensors grouped by their working mechanisms, the capacitive sensors have gained more and more attention due to their linear response, easy signal acquisition, and low power consumption (Zhang, et al. (2021) Adv. Electronic Mater., 7(7):2100174; Rao, et al. (2020) J. Mater. Chem. C, 8(18):6034-6041). Hydrogels made from various compositions via different fabrication methods have been successfully developed as capacitive wearable sensors (Cheng, et al. (2023) Adv. Mater. Tech., 8(l l):2201959).

[0009] Despite the intriguing stretchability and flexibility of hydrogels, many challenges still exist for hydrogel-based devices. For example, under extremely cold temperature conditions, soft hydrogels become rigid counterparts and are poorly conductive due to the water in them freezing (Liu, et al. (2020) ACS Appl. Mater. Interfaces 12(22):25334- 25344). Meanwhile, hydrogels also suffer from poor water retention in the ambient environment because of water evaporation (Ma, et al. (2021) Chem. Engr. J., 420: 129865). The transparency of hydrogels is greatly deteriorated in both scenarios (Shi, et al., (2020) Research 2020:2505619). These issues have substantially restricted the application of hydrogels as wearable sensors. One simple and effective solution to the issues is to percolate hydrogels in high-concentration salt solutions. This process reduces the glass transition temperature of a hydrogel and prevents the water inside from freezing (Wu, et al. (2021) J. Mater. Chem. C 9(39): 13668-13679). Moreover, the free ions introduced by the salt solution act as electrolytes, enhancing the conductivity of the hydrogel. In addition, specific salts, such as lithium chloride (LiCl) and lithium bromide (LiBr), have a strong binding affinity to the water molecules and can enhance the water retention and contribute to the long-term stability of the hydrogel (Wu, et al. (2021) J. Mater. Chem. C 9(39): 13668-13679; Sui, et al. (2021) Chem. Engr. J. 419: 129478). Likewise, ionic hydrogel-based capacitive sensors have shown robust performance and enhanced sensitivity (Yin, et al. (2019) Nano Energy 58:96-104; Wu, et al. (2021) ACS Appl. Mater. Interfaces 13(18):21854-21864; Wu, et al. (2021) Composites Part A: Appl. Sci. Manuf., 145: 106373).

[0010] The fabrication method is another important aspect to consider. Traditional fabrication methods, such as molding and casting, are difficult and time-consuming into making customized or complex objects that satisfy the users’ needs, thus hindering the development of hydrogel -based sensors (Xiong, et al. (2023) Nature Comm., 14(1): 1331). To tackle the problems, three-dimensional (3D) printing has become a prevailing technique for constructing wearable devices with dedicated structures due to its inherent advantages, such as free design, simple manipulation, and low cost (Puza, et al. (2022) Adv. Funct. Mater., 32(39):2205345; Wang, et al. (2018) ACS Biomater. Sci. Engr., 4(12):4401-4411; Shi, et al. (2021) Adv. Healthcare Mater., 10(12):2100180). However, the most widely used 3D printing method, extrusion-based 3D printing, faces a major challenge in fabricating sophisticated objects, like the gyroid structure (Hou, et al. (2021) Front. Mater., 8:647229). In contrast, vat photopolymerization (VP) is gaining popularity due to its capability to achieve a higher resolution and print more complex 3D architectures (Al Rashid, et al. (2021) Additive Manuf., 47: 102279). Digital light processing (DLP) 3D printing, a typical VP technology, is featured by its high printing resolution and fast printing speed and is considered an ideal technique for preparing hydrogels with refined structures (Zhang, et al. (2020) Inti. J. Bioprinting 6(1):242; Ding, et al. (2022) Mol. Sys. Design Engr., 7(9): 1017-1029). Build materials for DLP are commonly composed of photoreactive polymers, photoinitiators, and photo-absorbers (Benjamin, et al. (2019) Biomed. Physics Engr. Express 5(2):025035). The mechanical properties of the DLP-printed hydrogels can be tailored by adjusting the contents and ratio of the first two components. The photo-absorber is also critical because it impacts the printing resolution in the photo-curing process (Huh, et al. (2021) Biofabrication 13(3) :034103). Ionic hydrogels prepared using DLP printing have generated increasing research interest, but it remains challenging to use DLP to print a hydrogel that can achieve a well-balanced combination of conductivity, transparency, water retention, and extreme temperature tolerance (He, et al. (2021) ACS Appl. Mater. Interfaces 13(30):36286-36294; Ge, et al. (2021) Adv. Funct. Mater., 31(52):2107437; Yin, et al. (2019) Materials Horizons 6(4):767-780; Lopez-Larrea, et al. (2022) ACS Appl. Polymer Mater., 4(9):6749-6759).

[0011] Hydrogels with a shape-memory property can be programmed into temporary shapes and recover to their initial shapes upon external stimuli, such as temperature, light, and other environmental factors (Zhan, et al. (2022) Inti. J. Extreme Manuf., 4(l):015302; Costa, et al. (2022) ACS Mater. Letters 4(4):701-706). Shape-memory hydrogels are useful for developing various flexible electronics (Costa, et al. (2022) ACS Mater. Letters 4(4):701-706; Feng, et al. (2022) Chem. Engr. J., 432: 134406). For example, a poly(ethylene glycol)-poly(E-caprolactone) hydrogel with the shape-memory ability has been designed, and a device that can geometrically adapt to the detection joint of human bodies through shape programming of the hydrogel has been fabricated (Wu, et al. (2023) Adv. Funct. Mater., 33(36):2303292). Four-dimensional (4D) printing is an efficient strategy for printing objects that can be programmed to change their shapes through specific stimuli (Ahmed, et al. (2021) Polymer 228: 123926; Spiegel, et al. (2022) Adv. Funct. Mater., 32(51):2110580). 4D-printed wearable hydrogel sensors with a shape-memory property and programmability would be highly valuable, not only due to the simplified fabrication process but also because of the convenience it achieves in terms of device storage and long-term stability. Unfortunately, there are very few studies focused on the development of novel hydrogels specifically for wearable device application (Peng, et al. (2022) Chem. Engr. J., 439: 135593).

[0012] SUMMARY OF THE INVENTION

[0013] In accordance with the instant invention, hydrogel precursors are provided. In certain embodiments, the hydrogel precursor comprises an amphiphilic block copolymer and a photoinitiator, wherein the amphiphilic block copolymer comprises at least one terminal photoactive group. In certain embodiments, the amphiphilic block copolymer comprises two terminal photoactive groups. In certain embodiments, the photoactive group is an acrylate group. In certain embodiments, the hydrogel precursor further comprises at least one photoactive polymer, particularly a photoactive monomer and / or copolymer. In certain embodiments, the hydrogel precursor further comprises a photoabsorber. In certain embodiments, the amphiphilic block copolymer is a poloxamer such as poloxamer 407. In certain embodiments, the photoinitiator is lithium phenyl- 2,4,6-trimethylbenzoylphosphinate (LAP). In certain embodiments, the photoabsorber is tartazine. In certain embodiments, the hydrogel precursor comprises poloxamer 407 diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartazine. In certain embodiments, the hydrogel precursor further comprises a therapeutic agent, cell, tissue, and / or electrically conductive compound.

[0014] In accordance with another aspect of the instant invention, hydrogels are provided. The hydrogel may be made from the hydrogel precursor of the instant invention. In certain embodiments, the hydrogel precursor is exposed to light for activation of the photoinitiator. In certain embodiments, the hydrogel is three- dimensionally printed such as by light and / or laser-based, 3D printing or digital light processing 3D printing using the hydrogel precursor of the instant invention. In certain embodiments, the hydrogel comprises intermicellarly crosslinked poloxamer 407. In certain embodiments, the hydrogel of the instant invention further comprises an ionic salt or conductive filler. In certain embodiments, the ionic salt is lithium chloride or lithium bromide.

[0015] In accordance with another aspect of the instant invention, methods of synthesizing a hydrogel are provided. In certain embodiments, the method comprises 3D printing the hydrogel using the hydrogel precursor of the instant invention. In certain embodiments the method comprises light and / or laser-based, 3D printing the hydrogel. In certain embodiments the method comprises digital light processing, 3D printing the hydrogel. The method may further comprise washing the hydrogel and / or adding an ionic salt or conductive filler to the hydrogel. In certain embodiments, the method further comprises adding a therapeutic agent, cell, tissue, and / or electrically conductive compound.

[0016] In accordance with another aspect of the instant invention sensors or biosensors comprising the hydrogel of the instant invention are provided. In certain embodiments, the sensor or biosensor is a tactile sensor, pressure sensor, motion sensor, or stain sensor. In accordance with another aspect of the instant invention, methods for delivering a compound such as a therapeutic agent or cell to a subject are provided. The method comprises administering or implanting a hydrogel of the instant invention into a subject. The hydrogel may comprise and / or encapsulate the compound (e.g., therapeutic agent or cell).

[0017] In accordance with another aspect of the instant invention, methods of detecting nitric oxide in a subject are provided. The methods comprise administering or implanting a hydrogel of the instant invention into the subject and detecting fluorescence from the hydrogel, wherein the hydrogel comprises and / or encapsulates a carbon nanotube. In certain embodiments, the carbon nanotube comprises a single-walled carbon nanotube (SWNT) or a DNA-wrapped SWNT.

[0018] In accordance with another aspect of the instant invention, methods for producing a cell spheroid are provided. The methods comprise culturing cells on a hydrogel of the instant invention, thereby producing the cell spheroid. The methods may further comprise isolating the cell spheroid. In certain embodiments, the hydrogel is shaped or printed as a tissue culture well insert.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A provides a schematic illustration for the preparation and application of the poloxamer-based ionic hydrogel as a wearable sensor. DLP printing of Pluronic® F- 127 diacrylate micelles followed by lithium salt percolation. The ionic hydrogel can be customized and applied as either a strain sensor or tactile sensor for real-time motion monitoring, even at extreme temperatures. Figure IB provides an illustration of F-127 serving as both monomer and crosslinker during the DLP process. Figure 1C provides a schematic of the synthesis strategy of the F-127 diacrylate. Figure ID provides the particle size distribution of F-127 diacrylate micelles measured by dynamic light scattering (DLS) nanoparticle analysis. Figure IE provides the zeta potential of F-127 diacrylate micelles with different concentrations. Figure IF provides transmission electron microscopic (TEM) images of F-127 diacrylate micelles. Scale bars: 500 nm. Figures 1G and 1H provide F-127 printing parameter optimization. Circle: printable, x: not printable, Square: printable, but the shape or size does not match the CAD design. Figure 1G shows the printability of an 8x3 mm cylinder for 6.5% and 12.5% F-127 over various printing times per layer. Figure 1H shows the printability of the various channel sizes in the channel testing cuboid for several tartrazine photo-absorber concentrations. Figure 2A provides the storage modulus of hydrogels immediately after DLP printing under different curing times at two respective precursor concentrations (n = 3; for certain points, the error bars were shorter than the height of the symbol; therefore, the error bars were not drawn). Figure 2B provides a comparison of the storage modulus before and after washing with water (n = 3). Figure 2C provides the design of the channel testing cuboid with 0.5, 1, 1.5, and 2 mm diameter channels for photoabsorber (tartrazine) concentration optimization. Figure 2D provides a visualization of the printed channel testing cuboids made from hydrogel precursors with 0.5, 1, and 1.5 mM tartrazine concentrations. Printed channels were filled with food color dye. Figure 2E provides the measurement of the measured diameters of the channels compared to the designed diameters for the same channels (n = 3). Figure 2F provides the slope of the line for each photoabsorber (tartrazine) concentration group (n = 3). Figure 2G provides an image of four printed 5 mm x 5 mm x 5 mm pyramids connected at the base. Figure 2H provides an image of printed 10 mm x 10 mm x 10 mm lattice cube with circular channels in all directions. Figure 21 provides an image of a printed 6 mm outer diameter tube with a 1 mm thick wall with 0.5 mm x 1 mm diamond cutouts. Figure 2J provides an image of printed 15 mm x 15 mm x 15 mm gyroid cube (design modified from thingiverse.eom / thing:757884). Figure 2K provides an image of a manually compressed lattice cube. Figure 2L provides an image of a lattice cube released back to its natural shape. Figure 2M provides an image of a manually compressed gyroid cube. Figure 2N provides an image of a gyroid cube released back to its natural shape. Figure 20 provides cyclic compressive force-strain curves of the lattice cube. Figure 2P provides cyclic compressive force-strain curves of the gyroid cube. Figure 2Q provides the cyclic compressive force-strain curves of the 5% PEDOT: PSS hydrogel (inset: solid cubic 5% PEDOT:PSS hydrogel). Figure 2R provides a distribution plot of the 5% PEDOT: PSS hydrogel conductivity.

[0021] Figures 3 A-3I show the shape-memory and geometry-adaptivity of the DLP 3D printed pol oxamer hydrogels. Figure 3 A: 3D design of the solid object with an open serpentine channel. Top view (Fig. 3B) and side view (Fig. 3C) of the DLP -printed poloxamer hydrogel with the channel. Fig. 3D: Dehydrated poloxamer hydrogel with the channel. Fig. 3E: Rehydrated poloxamer hydrogel with the channel after dehydration. Fig. 3F: Rehydrated poloxamer hydrogel with the channel visualized with dye throughout the channel. Figures 3G-3I: Images showing the shape-adaptivity of the DLP-printed poloxamer hydrogel on a plastic tube that mimics the human finger through dehydration and rehydration mediated shape programming.

[0022] Figures 4A-4I provide in vitro and in vivo biocompatibility studies of the DLP- printed poloxamer hydrogels. Live and dead images of the ADMSCs cocultured with the printed hydrogel on day 3 (Fig. 4A) and day 7 (Fig. 4B). Fig. 4C shows the proliferation of the ADMSCs with or without hydrogel coculture on days 3 and 7. Figure 4D provides an image of the poloxamer hydrogel after 4-week subcutaneous implantation. Figures 4E and 4F provide hematoxylin and eosin (H&E) staining and Masson’s tri chrome staining, respectively, of the skin tissue close to the implanted poloxamer hydrogel. Figure 4G provides an image of the PEG hydrogel after 4-week subcutaneous implantation. Figures 4H and 41 provide H&E staining and Masson’s tri chrome staining, respectively, of the skin tissue close to the PEG hydrogel. Scale bars: 100 pm (Figs. 4A and 4B), 500 pm (Figs. 4E, 4F, 4H, and 41), 8 mm (Figs. 4D and 4G).

[0023] Figure 5A shows the transmittance and optical images of the DLP-printed poloxamer hydrogel and the 30% LiCl percolated ionic hydrogel. Figure 5B provides the swelling ratio of the poloxamer hydrogels and the ionic hydrogels at different temperatures (n = 3). Figure 5C provides images of the poloxamer hydrogel and the ionic hydrogel before and after 24 hour exposure to the ambient environment (22°C and 50% RH). Figure 5D provides the weight ratio change of the poloxamer hydrogels and ionic hydrogels after long-term storage in ambient environments (22°C and 50% RH). Figure 5E provides an image of the dehydrated ionic hydrogel. Scale bar: 5 mm. Figures 5F and 5G provide images of the top view and side view, respectively, of the rehydrated ionic hydrogel with the channel visualized with dye throughout the channel. Figure 5H provides optical images of the ionic hydrogel under 0% and 300% strain. Figure 51 provides a graph of tensile stress-strain curves of the ionic hydrogel and normal hydrogel. Figures 5 J and 5K provide graphs of Young’s modulus and the storage modulus, respectively, of the ionic hydrogels.

[0024] Figure 6A provides a graph of the relative capacitance changes of the ionic hydrogel and normal hydrogel as a function of the applied tensile strain. Figure 6B provides the dynamic response of the capacitive strain sensor based on the ionic hydrogel under cyclic stretching and releasing. Figure 6C shows the durability and stability performance of the capacitive strain sensor with a cycling test of under 50% tensile strain. Figure 6D provides optical images of the capacitive strain sensor attached to the knee during standing and squatting. Figure 6E shows the relative capacitance changes depending on various movements, including standing, squatting, walking, and running. Figure 6F provides optical images of the customized and 4D-printed wearable hydrogel. Figure 6G shows the relative capacitance changes of the wearable capacitive strain sensor depending on the different bending angles of the finger (n = 3). Figure 6H provides a graph of the dynamic response of the capacitive strain sensor during the bending and straightening of the finger. Figure 61 provides a graph of the ionic conductivity of the ionic hydrogel and normal hydrogel at various temperatures (n=3). Figure 6J shows the capacitance change of the ionic hydrogel under continuous stretching-releasing at a 50% tensile strain. Figure 6K shows the relative capacitance changes of the capacitive strain sensor depending on the different bending angles of the finger (n=3). Figure 6L shows the dynamic response of the capacitive strain sensor during bending and straightening of the finger. Figure 6M shows the relative capacitance changes of the capacitive strain sensor depending on the different bending angles of the elbow (n=3). Figure 6N shows the dynamic response of the capacitive strain sensor during bending and straightening of the elbow. Figure 60 provides a design of the finger splint.

[0025] Figure 7A shows the cyclic compressive stress-strain curves of the solid and porous ionic hydrogels. Figure 7B provides the relative capacitance changes of the solid ionic hydrogels depending on the applied pressure under a 50% loading-unloading compressive strain. Figure 7C shows the relative capacitance changes of the porous ionic hydrogels depending on the applied pressure under a 50% loading-unloading compressive strain. Figure 7D provides the pressure sensitivity of the ionic hydrogels in the pressure range of 0-40 kPa. Figure 7E provides a graph of the capacitance of the porous hydrogel under a cyclic 50% compression strain. Figure 7F shows the relative capacitance changes of the porous hydrogel after 200 loading and unloading cycles with 50% compression. Figure 7G provides optical images of the customized ring-shaped solid and porous ionic hydrogels worn on the finger as a capacitive tactile sensor. Scale bar: 5 mm. Figure 7H provides the relative capacitance changes of the capacitive tactile sensor during the typing of “pressure sensor” on the keyboard. Figure 71 provides an image of the designs for the tactile sensors.

[0026] Figure 8A provides images of the ionic hydrogel and the normal hydrogel before and after freezing them at -20°C. Figure 8B provides a DSC thermogram of the ionic hydrogel and normal hydrogel. Figure 8C provides optical images of ionic hydrogelbased capacitive strain sensors mounted on a robotic hand in a freezer. Figure 8D provides graphs of dynamic capacitance response of the strain sensors under the given hand gestures of the sign language letter of “WORLD” under low temperature monitored by a thermocouple. Figure 8E provides a graph of the tensile strength of the ionic hydrogel at -20°C.

[0027] Figure 9A provides graphs of the compressive cycle stability of 1-mm, 1.5-mm, and 2-mm thick hydrogels at a 40% compression strain with a strain rate of 0.2 mm / s. Figure 9B provides a schematic of a 3D-printed liquid-core hydrogel design. Figure 9C provides a graph showing the linear correlation between the concentrations of SWNT loaded into the hydrogel (15, 30, 50, and 100 mg / L) and the fluorescence intensity. Figure 9D provides a graph of the fluorescence stability of 1-, 1.5-, and 2-mm thick hydrogels over a 4-hour duration. Figure 9E shows that he DNA-wrapped SWNT sensors in 1.5-thick hydrogels respond to the addition of 30 pM nitric oxide (NO) at 990 nm (final NO concentration is 10 pM). Figure 9F provides a graph of the fluorescence stability of a 30 mg / L DNA-wrapped SWNT loaded in a 1.5-mm thick hydrogel over 90 days. Figure 9G shows that PVA-wrapped SWNTs in a 1.5-mm thick hydrogel did not respond to the addition of 30 pM NO. Figure 9H provides the fluorescence spectral quenching upon the addition of various NO concentrations (0, 1, 2, 5, 10, 20, 30, and 50 pM) using 1.5-mm thick 30 mg / L SWNT-loaded hydrogels via a near-infrared (nIR) spectrometer. The corresponding fluorescence quenching of 1.5-mm thick 30 mg / L SWNT-loaded hydrogels at 990 nm from 0 pM to 50 pM is provided in Figure 91. Figure 9J shows a linear fitting curve of the fluorescence quenching of the hydrogel at 990 nm towards the concentrations of NO from 1 pM to 50 pM. Figure 9K provides digital images of the 1.5 mm-thick SWNT sensor-loaded liquid-core hydrogels inserted between the skin and muscle of a chicken breast. Figure 9L provides a fluorescence intensity heat map of the implanted hydrogel. Figure 9M provides a graph of the percent fluorescence spectra of the DNA-wrapped SWNT sensor-loaded hydrogels before and after inserting them underneath the chicken skin. Figure 9N shows the implanted hydrogel response to the addition of 30 pM NO at 990 nm (final NO concentration is 10 pM) before (g), during (h), and after (i) the quenching.

[0028] Figure 10A provides images of the plate inserts in 0.5 mm wells (left) and 0.25 mm wells (right). Figure 10B provides images of cells upon seeding on the plate insert in 0.5 mm wells (left) and 0.25 mm wells (right). Figure 10C provides images of cells after overnight culture on the plate insert in 0.5 mm wells (left) and 0.25 mm wells (right). DETAILED DESCRIPTION OF THE INVENTION

[0029] The lack of both digital light processing (DLP) compatible and biocompatible photopolymers, along with inappropriate material properties required for wearable sensor applications, substantially hinder the employment of DLP 3D printing in the fabrication of multifunctional hydrogels. Herein, a photoreactive poloxamer derivative, Pluronic® F-127 diacrylate, is synthesized which overcomes these limitations and is optimized to achieve DLP 3D printed micelle-based hydrogels with high structural complexity, resolution, and precision. The dehydrated hydrogels exhibit a shape-memory effect and are conformally attached to the geometry of the detection point after rehydration, which implies the 4D printing characteristic of the fabrication process and is beneficial for the storage and application of the device. The excellent cytocompatibility and in vivo biocompatibility further promote the use of the poloxamer hydrogels as a platform for multifunctional wearable systems. After processing with lithium chloride solution, multifunctional conductive ionic hydrogels with anti-freezing and anti-swelling properties along with good transparency and water retention are easily prepared. As capacitive flexible sensors, the DLP 3D printed micelle-based hydrogel devices exhibit excellent sensitivity, cycling stability, and durability in detecting multimodal deformations. Moreover, the DLP 3D printed conductive hydrogels are successfully applied as real-time human motion and tactile sensors with satisfactory sensing performances, even at a -20°C low-temperature environment.

[0030] Pluronic® F-127, which consists of a central hydrophobic block of polypropylene glycol) and two terminal blocks of hydrophilic poly(ethylene glycol), belongs to one type of the poloxamers and is generally recognized as safe by the Food and Drug Administration (FDA) and ready for use as an excipient (Pitto-Barry, et al. (2014) Polymer Chem., 5(10):3291-3297). It forms micelles in solution due to its amphiphilic characteristic and has been used to enhance the solubility of hydrophobic drugs (Basak, et al. (2013) Langmuir 29(13):4350-4356; Shi, et al. (2014) Biomaterials 35(22):5760-5770; Wang, et al. (2019) Int. J. Nanomed., 14:963-976). This polymer is thermo-responsive and presents a sol-gel transition at the gelation temperature in an aqueous solution (Shriky, et al. (2020) J. Colloid Interface Sci., 565: 119-130). In addition, Pluronic® F-127 thermo-reversible hydrogels are biocompatible and can be used in 3D cell culture and tissue engineering (Diniz, et al. (2015) Mater. Med., 26(3): 153). Other poloxamers, such as L-121 and P-123, have not been approved by the FDA. Another FDA-approved pol oxamer, F-68, has a much higher critical micelle concentration of 0.4%, which means it is not as stable as F-127 micelles (Pitto-Barry, et al. (2014) Polym. Chem., 5(10):3291-3297). In addition, there are a limited number of studies reporting the physicochemical and biological properties of F-68-based hydrogels (Khaliq, et al. (2023) Pharmaceutics 15(8):2102).

[0031] The terminal hydroxyl groups of the F-127 polymer can be functionalized and a F-127 diacrylate polymer can be used for photo-cured hydrogels (Li, et al. (2020) RSC Adv., 10(72):44494-44502). However, the amphiphilic photo-reactive polymer has never been used in DLP 3D printing. F-127 diacrylate itself can serve as both the monomer and the crosslinker in photopolymerization and be DLP printed (Figure IB). It can copolymerize with other monomers species, making those DLP noncompatible monomers being incorporated into the hydrogel network (Zhang, et al. (2019) Mater. Lett., 243: 112-115). The amphiphilic property of F-127 prevents the F-127 hydrogel from excessive swelling at ambient temperatures, which is crucial for hydrogel-based sensors intended for use in moisture-rich environments, like sweating or swimming scenarios (Chen, et al. (2022) J. Colloid Interface Sci., 628:287-298). In contrast, other 3D printed PEG, acrylamide, and hyaluronic acid-based hydrogels tend to swell substantially after water soaking. In addition, F-127 can also work as a drug carrier to sustainedly release hydrophobic drugs to exert other functions such as antibacterial and anti-inflammation (Fang, et al. (2020) ACS Appl. Mater. Interfaces 12(47):52307-52318; Kong, et al. (2023) J. Controlled Release 357:319-332).

[0032] The cross-linked micelles of the instant hydrogels exhibit dynamic properties, resulting in excellent resilience, recovery of the hydrogel, and potentially even a shapememory effect (Xia, et al. (2019) Chem. Mater., 31(22):9522-9531). Herein, DLP was used to rapidly construct Pluronic® F-127 hydrogels with tunable mechanical properties and tailored structures with both high resolution and precision. This is distinguished from a study showing photopolymerization of F-127 diacrylate in water not leading to hydrogels (Di Biase, et al. (2011) Soft Matter 7(10):4928-4937), which used the low- efficiency photoinitiator eosin Y at a low concentration.

[0033] The hydrogel of the instant invention is generally transparent, particularly after washing away the photoabsorber. After the hydrogel is dehydrated, it can efficiently recover to its original shape after the rehydration. The hydrogel can form a conformal skin-device interface through shape programming. Moreover, the hydrogel is biocompatible and biodegradable. After soaking it in a LiCl solution overnight, the hydrogel undergoes a transformation, becoming conductive and anti-swelling and exhibiting an enhanced stretchability and Young’s modulus while retaining moisture. Furthermore, an ionic hydrogel-based capacitive strain sensor and pressure sensor made from this material can realize real-time human motion monitoring and exhibit extreme temperature tolerance. The hydrogel provides a convenient platform for fabricating customized wearable sensors with multifunctional properties.

[0034] In accordance with the instant invention, compositions which can be used in 3D printing are provided. The composition can be used for light and / or laser-based 3D printing (e.g., digital light processing or stereolithography (SLA)). In certain embodiments, the composition can be used in digital light processing 3D printing. In certain embodiments, the composition can be used in bioprinting. In certain embodiments, the composition is a bioink (e.g., a composition comprising one or more natural and / or synthetic polymers which are biocompatible). In certain embodiments, the composition is a 3D printable hydrogel precursor. In certain embodiments, the composition is a liquid (e.g., at room temperature).

[0035] The composition may comprise at least one amphiphilic block copolymer. In certain embodiments, the amphiphilic block copolymer comprises hydrophilic polyethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO) blocks. In certain embodiments, the amphiphilic block copolymer comprises a poloxamer (e.g., Pluronic®). In certain embodiments, the amphiphilic block copolymer is a triblock copolymer comprising a central hydrophobic PPO block flanked by two hydrophilic PEO blocks (i.e., an A-B-A triblock structure). Examples of poloxamers include, without limitation: Pluronic® L31, L35, F38, L42, L44, L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88, L92, P94, F98, L101, P103, P104, P105, F108, L121, L122, L123, P123, F127, 10R5, 10R8, 12R3, 17R1, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8, 31R1, 31R2, and 31R4. In certain embodiments, the amphiphilic block copolymer is selected from the group consisting of Pluronic® P85, L121, P123, F127, L31, F38, L61, L64, F68, F77, F87, P94, P103, and P105. In certain embodiments, the amphiphilic block copolymer comprises poloxamer 407 (Pluronic® F127). The composition may comprise at least one additional polymer. In certain embodiments, the additional polymer is a copolymer. In certain embodiments, the additional polymer is a monomer. In certain embodiments, the additional polymer is photoactive (e.g., with a photoactive group). In certain embodiments, the additional polymer is a photoactive monomer (e.g., an acrylic acid or acrylamide). In certain embodiments, the additional polymer is a photoactive copolymer (e.g., sulfobetaine methacrylate (SBMA). In certain embodiments, the additional polymer is a photoactive monomer copolymer.

[0036] Generally, the amphiphilic block copolymer of the composition comprises at least one terminal photoactive group. In certain embodiments, the amphiphilic block copolymer has two terminal photoactive groups. Photoactive groups are known in the art. Examples of photoactive groups include, without limitation: acrylate, methacrylate, 2-chloroacrylate, 2-phenylacrylate, acrylamide, methacrylamide, 2 -chloroacrylamide, 2- phenyl acrylamide, N-lower alkyl substituted acrylamide, N-lower alkyl substituted methacrylamide, N-lower alkyl substituted 2- chloroacrylamide, N-lower alkyl substituted 2-phenylacrylamide, vinyl ether, vinyl ester, styrene, diamine, amide, imide, siloxane, amic ester, amic acid, and derivatives thereof. In certain embodiments, the photoactive group is acrylate, methacrylate, 2-chloroacrylate, 2-phenylacrylate, vinyl ether, vinyl ester, styrene, siloxane, or derivatives thereof. In certain embodiments, the photoactive group is acrylate or methacrylate. In certain embodiments, the photoactive group is acrylate (CH2=CH(C0)0-; wherein the terminal oxygen may be part of the amphiphilic block copolymer). In certain embodiments, the amphiphilic block copolymer is a diacrylate. In certain embodiments, the amphiphilic copolymer is poloxamer 407 (Pluronic® F127) diacrylate.

[0037] The amphiphilic block copolymer may be present at a concentration such that the composition remains a liquid. In certain embodiments, about 1% to about 20%, about 1% to about 15%, about 5% to about 15%, about 6.5% to about 12.5%, about 10% to about 15%, about 5% to about 10%, about 11% to about 14%, or about 12.5% (e.g., w / v) of the composition is an amphiphilic block copolymer (e.g., a poloxamer (e.g., poloxamer 407)).

[0038] The compositions of the instant invention may further comprise a photoabsorber. Photoabsorbers are known in the art. Examples of photoabsorbers include, without limitation: methylene blue, coccine, and tartrazine. In certain embodiments, the photoabsorber is tartazine. In certain embodiments, the concentration of the photoabsorber in the composition is at least 1.5 mM. In certain embodiments, the photoabsorber is present at about 0.5 mM to about 10 mM, 1.0 mM to about 5 mM, about 1.5 mM to about 5 mM, about 1.5 mM to about 3 mM, or about 1.5 mM. The compositions of the instant invention may further comprise a photoinitiator. Photoinitiators are known in the art. Generally, a photoinitiator absorbs energy such as visible light or UV light and starts a chemical reaction that solidifies a liquid. In certain embodiments, the photoinitiator is a crosslinker or photocrosslinker. Examples of photoinitiators include, without limitation: lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), benzophenone, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (DPPO), phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO, Irgacure 819), 2-hydroxy-2-methyl-l-phenyl-propan-I-one (Irgacure 1173), 2-hydroxy- 4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), 2,2'-azobis[2-methyl-n- (2-hydroxy ethyl) propionamide) (VA-086), 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651 or DMPA), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (Darocure TPO; Lucirin TPO), and ethyl (2,4,6-trimethylbenzoyl) phenylphosphinate (Lucirin TPO-L). In certain embodiments, the photoinitiator is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP). In certain embodiments, the concentration of the photoinitiator is at least 0.05 g / ml. In certain embodiments, the photoinitiator is present at about 0.01 g / ml to about 0.1 g / ml, about 0.05 g / ml to about 0.3 g / ml, or about 0.1 g / ml.

[0039] In certain embodiments, the composition of the instant invention comprises poloxamer 407 (Pluronic® F127) diacrylate, a photoabsorber, and a photoinitiator. In certain embodiments, the composition comprises poloxamer 407 (Pluronic® Fl 27) diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and a photoabsorber. In certain embodiments, the composition comprises poloxamer 407 (Pluronic® Fl 27) diacrylate, tartazine, and a photoinitiator. In certain embodiments, the composition comprises poloxamer 407 (Pluronic® F127) diacrylate, tartazine, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0040] In accordance with another aspect of the instant invention, hydrogels are provided. The hydrogels of the instant invention are biocompatible, have a low swell ratio at physiologic temperature, are cyto-compatible with modulable stiffness and elasticity, stretchable, and / or shows shape retention ability. The characteristics of the hydrogel facilitates its application, without limitation, as a drug delivery system, coating system, tissue engineering scaffold, as well as in biomedical devices. In certain embodiments, the hydrogel is a solid at 37°C or higher, at 40°C or higher, 42°C or higher, or even higher. In certain embodiments, the hydrogel comprises or is derived from the hydrogel precursor of the instant invention. In certain embodiments, the hydrogel is 3D printed (e.g., laser and / or light 3D printed). In certain embodiments, the hydrogel is digital light processing 3D printed. In certain embodiments, the hydrogel is bioprinted. In certain embodiments the 3D printing comprises exposing the hydrogel precursor to a light and / or laser of the wavelength to activate the photoinitiator (e.g., 405 nm).

[0041] In certain embodiments, the hydrogel comprises crosslinked amphiphilic block copolymers (e.g., amphiphilic block copolymer diacrylates). In certain embodiments, the hydrogel comprises crosslinked poloxamer 407 (e.g., poloxamer 407 diacrylates). In certain embodiments, the hydrogel comprises poloxamer 407 (Pluronic® F127) diacrylate, a photoabsorber, and a photoinitiator. In certain embodiments, the hydrogel comprises poloxamer 407 (Pluronic® Fl 27) diacrylate, lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), and a photoabsorber. In certain embodiments, the hydrogel comprises poloxamer 407 (Pluronic® Fl 27) diacrylate, tartazine, and a photoinitiator. In certain embodiments, the hydrogel comprises poloxamer 407 (Pluronic® Fl 27) diacrylate, tartazine, and lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP). In certain embodiments, the hydrogel comprises crosslinked components (e.g., crosslinked amphiphilic block copolymer (e.g., via acrylate groups)). In certain embodiments, the hydrogel comprises crosslinked amphiphilic block copolymers with any additional polymers. In certain embodiments, the hydrogel comprises crosslinked micelles (e.g., micelles formed by the amphiphilic block copolymer). In certain embodiments, the hydrogel comprises intramicellar crosslinking of the amphiphilic block copolymer (e.g., poloxamer 407). In certain embodiments, the hydrogel comprises intermicellar crosslinking of the amphiphilic block copolymer (e.g., poloxamer 407). In certain embodiments, the hydrogel comprises intramicellar and intermicellar crosslinking of the amphiphilic block copolymer (e.g., poloxamer 407).

[0042] The hydrogels of the instant invention may be of any shape. Indeed, 3D printing allows for the hydrogels to be made into any desired shape. In certain embodiments, the hydrogel is shaped as a medical implant (e.g., meniscus, intervertebral disc, etc.). In certain embodiments, the hydrogel (optionally comprising an antibiotic) is a coating for a medical implant (e.g., to inhibit and / or prevent biofilm formation). The hydrogel of the instant invention does not support cell adhesion on the surface and is soft and biocompatible, thereby improving patient compliance. In certain embodiments, the hydrogel is wearable. In certain embodiments, the hydrogel is a ring. In certain embodiments, the hydrogel is a tactile sensor. In certain embodiments, the hydrogel is a finger splint. In certain embodiments, the hydrogel is a well insert (e.g., a tissue culture well insert). In certain embodiments, the hydrogel is dish shaped. In certain embodiments, the hydrogel comprises a core such as a liquid core. In certain embodiments, the hydrogel is a capsule or pill shaped. In certain embodiments, the hydrogel is cylindrical with closed ends. In certain embodiments, the hydrogel is a microfluidic device (e.g., for use in micro-physiological modeling (e.g., for therapeutic target identification and drug screening purposes of disorders and disease). In certain embodiments, the surface of the hydrogel is derivatized with small molecules, peptides, or proteins containing free thiol group or acrylate (e.g., methacrylate) groups.

[0043] In certain embodiments, the hydrogel of the instant invention further comprises an ionic salt (e.g., an ionic hydrogel). In certain embodiments, the hydrogel of the instant invention further comprises a conductive filler. In certain embodiments, the ionic salt is a sodium, potassium, or lithium salt. In certain embodiments, the ionic salt is a lithium salt. In certain embodiments, the ionic salt is lithium chloride. In certain embodiments, the ionic salt is lithium bromide. Examples of conductive fillers include, without limitation: ionic liquids (e.g., l-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS).

[0044] The hydrogel of the instant invention may further comprise at least one therapeutic agent (e.g., drug) and / or cell. In certain embodiments, the therapeutic agent is hydrophobic. Therapeutic agents of the instant invention may include, without limitation, drugs, pharmaceuticals, biologies, growth factor, cytokines, chemokines, antibodies, antibody fragments, small molecules, peptides, proteins, nucleic acid molecules, DNA, RNA, and other known biologic substances. In certain embodiments, the therapeutic agent is an antimicrobial or antibiotic. In certain embodiments, the therapeutic agent is an analgesic.

[0045] Antimicrobials may include, without limitation, small molecules, peptides, proteins, DNA, RNA, and other known biologic substances. In a particular embodiment, the antimicrobial is a small molecule. In a particular embodiment, the antimicrobial is an antiviral, antifungal, antibiotic or antibacterial, particularly an antibiotic, antibacterial, or antimicrobial peptide (e.g., LL-37 or a fragment or derivative thereof (e.g., those provided in Wang et al. (2019) Adv. Exp. Med. Biol., 1117:215-240, incorporated herein by reference). In a particular embodiment, the antimicrobial is a small molecule antibiotic. Examples of antibiotics include, without limitation, beta-lactams (e.g., penicillin, ampicillin, oxacillin, cioxacillin, methicillin, cephalosporin, etc.), monobactams (e.g., aztreonam, tigemonam, nocardicin A, tabtoxin, etc.), carbapenems (e.g., imipenem, meropenem, ertapenem, doripenem, etc.), cephalosporins (e.g., cefdinir, cefaclor, cephalexin, cefixime, cefepime, etc.), carbacephems, cephamycins, macrolides (e.g., erythromycin, clarithromycin, azithromycin etc.), quinolones or fluoroquinolones (e.g., ciprofloxacin, levofloxacin, ofloxacin, delafloxacin, etc.), tetracyclines (e.g., tetracycline, doxycycline etc.), sulfonamides (e.g., sulfamethoxazole, sulfafuraxole, etc.), aminoglycosides (e.g., gentamicin, neomycin, tobramycin, kanamycin, etc.), oxazolidinones (e.g., linezolid, posizolid, tedizolid, radezolid, contezolid, etc.), lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), moenomycins, ansamycins (e.g., rifamycins, rifampin), aminocoumarins (e.g., novobiocin), co- trimoxazoles (e.g., trimethoprim and sulfamethoxazole), lincosamides (e.g., clindamycin and lincomycin), polypeptides (e.g., colistin), and glycopeptides (e.g., vancomycin).

[0046] In certain embodiments, the hydrogel of the instant invention may comprise and / or encapsulate cells and / or tissue comprising cells. For example, the methods of the instant invention may comprise seeding cells and / or tissue on and / or in the hydrogel. In certain embodiments, the cells are autologous to the subject to receive or be treated with the hydrogel. In certain embodiments, the cells are allogenic. The hydrogel may comprise and / or encapsulate any cell type. Cell types include, without limitation: embryonic stem cells, adult stem cells, bone marrow stem cells, induced pluripotent stem cells, progenitor cells (e.g., neural progenitor cells), embryonic-like stem cells, mesenchymal stem cells, CAR-T cells, immune cells (including but not limited to T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells and modified forms of these cells and various combinations thereof), cell-based vaccines, and cell lines expressing desired therapeutic proteins and / or genes. In a particular embodiment, the cells comprise stem cells. In a particular embodiment, the cells comprise islet cells (e.g., allogenous islet cells). In a particular embodiment, the hydrogel comprises and / or encapsulates cell spheroids. In a particular embodiment, the hydrogel comprises and / or encapsulates tissue samples (e.g., minced tissue), such as skin tissue samples or bone samples. The cells or tissue may be cultured on and / or within the hydrogel (e.g., the cells or tissue may be cultured for sufficient time to allow for growth on and / or within and / or infiltration into the hydrogel). For example, the cells or tissue may be cultured on and / or in the hydrogel for 1 day, 2 days, 3 days, 4 days, 5 days, or more. In certain embodiments, the hydrogel comprises electrically conductive compounds or components (e.g., conductive metals). In certain embodiments, the hydrogel comprises a carbon nanotube. In certain embodiments, the hydrogel comprises a single-walled carbon nanotube (SWNT). In certain embodiments, the hydrogel comprises a DNA-wrapped single-walled carbon nanotube. In certain embodiments, the DNA is single-stranded.

[0047] The hydrogels may comprise a cavity so as to encapsulate one or more compounds or a composition comprising a carrier and one or more compounds. In certain embodiments, the hydrogel encapsulates at least one therapeutic agent and / or cell. In certain embodiments, the hydrogel encapsulates an electrically conductive compound or component (e.g., conductive metal). In certain embodiments, the hydrogel encapsulates a carbon nanotube. In certain embodiments, the hydrogel encapsulates a single-walled carbon nanotube (SWNT). In certain embodiments, the hydrogel encapsulates a DNA-wrapped single-walled carbon nanotube. The compounds may be contained within the core of a hydrogel capsule.

[0048] Compositions comprising at least one hydrogel are also encompassed by the instant invention. In certain embodiments, the composition further comprises a carrier such as a pharmaceutically acceptable carrier. In certain embodiments, the hydrogel of the instant invention is maintained or stored dehydrated.

[0049] In accordance with the instant invention, methods of synthesizing hydrogels are provided. In certain embodiments, the method comprises 3D printing the hydrogel with the composition (e.g., bioink) of the instant invention. In certain embodiments, the method comprises light and / or laser-based 3D printing (e.g., digital light processing or stereolithography (SLA)). In certain embodiments, the method comprises digital light processing 3D printing. In certain embodiments, the method comprises bioprinting.

[0050] The methods of the instant invention may further comprise washing the printed hydrogel. In certain embodiments, the printed hydrogel is washed with water (e.g., deionized water) or a carrier. In certain embodiment, the hydrogel is washed to remove the photoabsorber.

[0051] The methods of the instant invention may further comprise adding an ionic salt to the printed hydrogel. In certain embodiments, the method comprises placing and / or submersing the printed hydrogel into a solution of the ionic salt (e.g., in water). In certain embodiments, the ionic salt solution comprises at least 10%, at least 20%, at least 25%, or at least 30% (w / v) of the ionic salt (e.g., Li Cl or LiBr). The methods of the instant invention may further comprise adding an additional component to the printed hydrogel. For example, components may be added to the composition (e.g., hydrogel precursor) prior to printing and / or added to the hydrogel after printing. As explained hereinabove, the hydrogels of the instant invention may comprise and / or encapsulate a therapeutic agent, cell, tissue, and / or electrically conductive compound (e.g., carbon nanotube or SWNT). The additional components may be added by soaking the hydrogel in a solution comprising the additional component. In certain embodiments, the additional components are added to the hydrogel by insertion or injection into the hydrogel (e.g., into a cavity of the hydrogel). For example, cells may be inserted or injected into the printed hydrogel and / or carbon nanotubes may be inserted or injected into the printed hydrogel.

[0052] In accordance with the instant invention, sensors and biosensors are provided. The sensors and biosensors comprise the hydrogel of the instant invention. In certain embodiments, the hydrogel is an ionic hydrogel. In certain embodiments, the hydrogel is in electronic communication with the sensor or biosensor. In certain embodiments, the sensor or biosensor is a pressure sensor. In certain embodiments, the sensor or biosensor is a strain sensor. In certain embodiments, the sensor or biosensor is a wearable technology or device. In certain embodiments, the sensor or biosensor is for haptics, motion monitoring, robotics, and / or gait monitoring. In certain embodiments, the sensor or biosensor is for real-time motion sensing. In certain embodiments, the sensor or biosensor is for detecting nitric oxide and the hydrogel comprises electrically conductive components such as carbon nanotubes (e.g., SWNTs or DNA-wrapped SWNTs). As explained herein, the hydrogels of the instant invention display different electrical properties such as changes in current, resistance, and / or capacitance due to external stimuli such as pressure and / or force. These changes in electrical properties can be measured for the sensing applications.

[0053] In accordance with another aspect of the instant invention, methods for culturing and / or generating cell spheroids are provided. In certain embodiments, the method comprises seeding or adding cells to the surface of a hydrogel of the instant invention. In certain embodiments, the printed hydrogel is a tissue culture dish. In certain embodiments, the printed hydrogel is a well insert. The method may further comprise culturing the cells on the printed hydrogel until cell spheroids are formed. For example, the cells be cultured on the hydrogel for 1 day, 2 days, 3 days, 4 days, 5 days, or more. The methods may further comprise isolating the formed cell spheroids. In accordance with the instant invention, methods of delivering a compound (e.g., therapeutic agent) or cell to a subject are provided. The method comprises administering to a subject a hydrogel of the instant invention comprising the compound or cell. The methods may be to treat, inhibit, and / or prevent a disease or disorder in a subject in need thereof. For example, the hydrogels of the instant invention can comprise or encapsulate insulin producing cells (e.g., islet cells) for treating diabetes. In certain embodiments, the hydrogel is a drug or therapeutic agent delivery device (e.g., drug eluting stent). In certain embodiments, the compound (e.g., therapeutic agent) or cell is encapsulated by the hydrogel. Encapsulated compounds can be released slowly from the hydrogel, thereby allowing sustained delivery of the compound to the subject. Indeed, loaded drugs will diffuse out sustainably in the target site from the construct to achieve a better therapeutic outcome. Encapsulated cells (e.g., allogenous cells (e.g., islet cells)) are protected from the host immune while functional to secrete agents (e.g., insulin).

[0054] In accordance with another aspect of the instant invention, methods of detecting and / or measuring nitric oxide in a subject are provided. Nitric oxide has been implicated in the progression of several diseases such as, without limitation: cancer, sepsis, multiple sclerosis, and various autoimmune diseases (Meier, et al. (2021) Nanomater., 11 :243). The method comprises administering or implanting a hydrogel comprising and / or encapsulating carbon nanotubes (e.g., SWNT or DNA-wrapped SWNT) to the subject and detecting and / or measuring fluorescence (e.g., at 990 nm) from the hydrogel in the subject. A decrease or quenching of the fluorescence signal or intensity indicates the presence of nitric oxide. In certain embodiments, the fluorescence is measured at more than one timepoint after administration or implantation. In certain embodiments, the fluorescence is compared to a standard curve (e.g., generated on the subject being studied or on control subjects or samples).

[0055] The present invention encompasses administering and / or implanting a hydrogel of the instant invention into a subject. In certain embodiments, the methods comprise administering the hydrogel of the instant invention to the desired site. The hydrogel of the instant invention can be administered to or implanted in an animal, in particular a mammal, more particularly a human. The compositions of the instant invention may also comprise at least one therapeutic agent (e.g., an antimicrobial) and / or cells. The additional therapeutic agent and / or cells may also be administered in a separate pharmaceutical composition from the compositions of the instant invention. The pharmaceutical compositions may be administered at the same time or at different times (e.g., sequentially).

[0056] The dosage ranges for the administration of the compositions of the invention are those large enough to produce the desired effect. The dosage should not be so large as to cause significant adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counter indications.

[0057] The compositions described herein will generally be administered to a patient as a pharmaceutical composition. The term “patient” as used herein refers to human or animal subjects. These compositions may be employed therapeutically, under the guidance of a physician.

[0058] The pharmaceutical compositions of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the complexes may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents, or suitable mixtures thereof, particularly an aqueous solution. The concentration of the components in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical composition. Except insofar as any conventional media or agent is incompatible with the component to be administered, its use in the pharmaceutical composition is contemplated.

[0059] Selection of a suitable pharmaceutical composition will also depend upon the mode of administration chosen. For example, the adhesive of the invention may be administered by direct injection or surgical implantation. In this instance, a pharmaceutical composition comprises the component dispersed in a medium that is compatible with the site of injection or implantation. Pharmaceutical compositions for injection or implantation are known in the art.

[0060] Definitions

[0061] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0062] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.

[0063] As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind.

[0064] “Hydrophobic” designates a preference for apolar environments (e.g., a hydrophobic substance or moiety is more readily dissolved in or wetted by non-polar solvents, such as hydrocarbons, than by water). In a particular embodiment, hydrophobic polymers may have aqueous solubility less than about 1% wt. at 37°C. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point below about 37°C, particularly below about 34°C, may be considered hydrophobic.

[0065] As used herein, the term “hydrophilic” means the ability to dissolve in water. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point above about 37°C, particularly above about 40°C, may be considered hydrophilic.

[0066] As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids / apolar environments. Typically, an amphiphilic compound comprises a hydrophilic portion and a hydrophobic portion.

[0067] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human. As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

[0068] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0069] A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, treat, or lessen the symptoms of a particular disorder or disease. For example, the treatment of an infection herein may refer to reducing, curing, and / or relieving the infection, the symptom(s) of it, or the predisposition towards it.

[0070] The term “crosslink” refers to a bond or chain of atoms attached between and linking two different molecules (e.g., polymer chains). The term “crosslinker” refers to a molecule capable of forming a covalent linkage between compounds. A “photocrosslinker” refers to a molecule capable of forming a covalent linkage between compounds after photoinduction (e.g., exposure to electromagnetic radiation in the visible and near-visible range). Crosslinkers are well known in the art (e.g., formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, etc.). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent.

[0071] The term “conductive” or “electrically conductive” mean a material which is capable of electrical conductivity without the addition of other elements.

[0072] As used herein, the term “analgesic” refers to an agent that lessens, alleviates, reduces, relieves, or extinguishes pain in an area of a subject's body (i.e., an analgesic has the ability to reduce or eliminate pain and / or the perception of pain).

[0073] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids.

[0074] The following examples illustrate certain embodiments of the invention. They are not intended to limit the invention in any way. EXAMPLE 1

[0075] Materials and methods

[0076] Synthesis and Characterization ofPluronic® F-127 diacrylate

[0077] Pluronic® F-127 (0.5 mmole, Sigma) was first dissolved in 12 ml of dichloromethane (Acros Organics) under constant stirring followed by the addition of 460 pL of acrylic anhydride (4 mmole, Combi-blocks) and 0.4 ml of triethylamine (TEA, TCI America). The mixture was stirred at room temperature for 4 days. After the reaction, the F-127 diacrylate was precipitated in ice chilled ethyl ether (Millipore). The precipitate was collected after centrifugation and re-dissolved in a water and methanol mixture (1 / 1, v / v). After dialyzing it against water for 3 days at 4°C, the purified F-127 diacrylate was freeze-dried in a benchtop lyophilizer (Labconco).

[0078] TheJH NMR of the product was measured on a 500 MHz Bruker NMR system and analyzed with the Topspin 4.0 software (Shi, et al. (2020) Carbohydr. Polym., 233 : 115803). The polymer conjugate was dissolved in D2O (Acros Organic) at 6 mg / mL in the NMR tube, and the chemical shifts were referred to the solvent peak of D2O at 4.78 ppm at 25 °C. The substitution degree of F-127 diacrylate was calculated from the ratio of the integral of the alkene protons from the attached acrylate group (between 6.0 and 6.5 ppm, 2*C2H3) to the integral of F-127 methyl proton peak (1.1-1.2 ppm, 65*CH3).

[0079] Pluronic® F-127 diacrylate was dissolved in DI water to achieve concentrations of 6.5% or 12.5% (w / v). The size and size distribution of the Pluronic® F-127 diacrylate micelles along with the surface charge were measured using the Zetasizer Nano instrument (Malvern). The micelle morphology was evaluated by transmission electron microscopy (TEM, Hitachi H7500; Hitachi) as described (Xue, et al. (2022) Acta Biomaterialia 138: 182-192).

[0080] DLP printing of poloxamer micelle-based hydrogels

[0081] To prepare the 3D printable hydrogel precursor for 3D printing, Pluronic® F-127 diacrylate was dissolved in deionized (DI) water, together with a tartrazine solution that is used as a photo-absorber as well as an LAP solution that is used as a photoinitiator. To fulfill this, a corresponding amount of 6.5% or 12.5% (w / v) Pluronic® F-127 diacrylate was weighed out and placed into a sealable tube. Then DI water and the tartrazine stock solution (5, 10, or 15 mM, 1 / 10 of total volume) were added to the tube at their respective necessary volumes. After stirring the mixture to fully soak the solids and start the dissolving process, the tube was placed into an ice bath to cool the solution. The solution was stirred periodically until all the Pluronic® diacrylate was dissolved. In the end, the 0.3 g / mL LAP stock solution (1 / 3 of total volume) was added right before DLP printing.

[0082] The objects to be printed with the Pluronic® F-127 diacrylate precursor were designed for 3D printing using Solidworks 3D modeling software and then converted to STL files for use by the printer. The 3D printing was performed on a LumenX™ (CELLINK) DLP 3D printer. Briefly, the Pluronic® F-127 diacrylate precursor was deposited onto the center of a polydimethylsiloxane-coated dish (CELLINK). A 405 nm UV light projector cured the precursor using a digital photomask, while a 2.5 * 4 cm2print platform moved vertically to allow the object to print in a layer-by-layer manner. The total printing time with this material is dependent on the height of the object to be printed. However, multiple objects can be printed at a time to increase the efficiency. For example, 6 cubes of 5 mm height, which are 50 layers thick, can be printed in about 20 minutes. Once the printing was finished, the object was removed from the print platform and washed in DI water to remove any excess / unreacted agents. The objects were then allowed to soak in DI water at room temperature to remove the yellow-colored photo-absorber.

[0083] Preparation of ionic conductive hydrogels

[0084] The DLP 3D printed poloxamer hydrogels (12.5%) were washed in DI water at room temperature for at least 4 hours with the water changed every 30 minutes. After that, the hydrogels were soaked in 30% (w / v) LiCl (Acros Organics) solution overnight at room temperature to prepare the ionic hydrogels. The volume of the LiCl solution was 20 times more than the volume of the hydrogels.

[0085] Swelling of hydrogels

[0086] DLP 3D printed hydrogel cubes (5 mm side length) were incubated in water at 4, 22, and 37°C for up to 3 days. At each time point, the hydrogels were taken out from the water, and the side length of each hydrogel sample (Ls) was measured. The side length of the hydrogel immediately after 3D printing was set as Lo. The swelling of the ionic conductive hydrogels was determined in ones using the 30% LiCl solution at 37°C. The hydrogel swelling was presented as the side length change and calculated by the equation: side length swelling (%) = Ls / Lox100. Each group had three replicated measurements.

[0087] Transparency evaluation of hydrogels

[0088] The transparency of the hydrogels was evaluated by a microplate reader (Biotek). The DLP 3D printed and water-washed hydrogel samples (disk shape, 8 mm in diameter and 1 mm thickness) before and after LiCl solution soaking were placed into the empty wells of a 96-well plate. A volume of 50 pL of DI water was added into empty wells of the 96-well plate to be utilized as control samples. The absorbance of the hydrogels was measured at wavelengths ranging from 400 to 700 nm and divided by the absorbance of the water at each wavelength. The absorbance ratio was converted into transmittance based on the Beer-Lambert law.

[0089] Water retention test

[0090] The DLP 3D printed and water-washed hydrogel samples (porous disk, 8 mm in diameter and 1 mm thickness) before and after LiCl soaking were placed in an ambient environment (22 °C and 50% RH) for 14 days. The weight of the hydrogels was measured at each predetermined time point as Mt, and the water retention was calculated by the equation: water retention (%) = Mt / Mox100, where Mo was the weight of hydrogels before environmental exposure.

[0091] Rheological characterization

[0092] The rheological properties of the hydrogels were characterized at 37 °C using a Discovery HR-2 rheometer (TA Instruments) (Shi, et al. (2021) Biofabrication 14(1): 014107). The hydrogels were printed as cylindrical disks with diameters of 20 mm and thicknesses of 1 mm. The hydrogels were transferred into the geometry gap between the 20 mm parallel plate and the base plate. The time sweep method was carried out at a fixed angular frequency (2K rad / s) and 10% strain to measure the storage (G’) and loss (G”) moduli.

[0093] Mechanical experiments

[0094] DLP 3D printed dogbone-shaped hydrogels were prepared for mechanical testing. Tensile and compression stress-strain curves of the hydrogels were obtained by a strength tester (Mark-10, ESM303 with Series 5 digital force gauge, Dynamic Measurement Systems) at room temperature. The tensile stress-strain and compression stress-strain tests were conducted at 5 and 10 mm min'1strain rates, respectively. The strain (a) was calculated by dividing the elongation or contraction (Al) by the initial length (lo) and expressing it as a percentage (a = Al / lox100%). The stress (c) was defined as the load force (F) divided by the original cross-sectional area of the sample (Ao). The Young’s modulus of the hydrogels was determined within the area of the stress-strain curve that exhibits a linear region (between 5% and 20% of strain). For the cyclic stretching, a dynamic strain was applied to the hydrogel by an IPC flexural endurance tester (CK-700FET, CKSI).

[0095] Ionic conductivity measurement

[0096] The electrochemical impedance analysis was performed to evaluate the ionic conductivity of the hydrogels by an impedance analyzer (Multi / Autolab M204, FRA32M module, Metrohm) at various temperatures. The impedance measurements were conducted with a frequency range of 0.1 Hz to 1 MHz and an excitation voltage of 10 mV under open-circuit conditions. The ionic conductivity was calculated by the following formula: c = (1 / Rb)(d / S). Here, c is the ionic conductivity, Rb is the bulk resistance, d is the thickness of the hydrogel, and S is the cross-sectional area of the sample. The bulk resistance was determined by the electrochemical circle fit from the Nyquist plot of the sample using NOVA 2.1.5 software.

[0097] DSC analysis

[0098] The water-washed and LiCl-soaked hydrogels were placed in aluminum pans for DSC analysis using the 204 Fl Phoenix® (Netzsch) DSC system. The samples were cooled from 25 to -160 °C at a rate of -5 °C min'1and then warmed back to 25 °C at a rate of 5 °C min'1. The heat flow was monitored during the study’s process.

[0099] Cell culture, live and dead staining, and cell proliferation assay

[0100] Primary human ADMSCs were purchased from Lonza and cultured and expanded in growth medium containing DMEM / F12 medium (Invitrogen), 10% fetal bovine serum (FBS, Invitrogen), and 1% penicillin / streptomycin (P / S, Invitrogen) at 37 °C in a humidified incubator containing 5% CO2. (Shi, et al. (2021) J. Mater. Chem. B 9(35):7182-7195; Jiang, t al. (2018) ACS Macro Letters 7(10): 1192-1197).

[0101] T1 For the cytocompatibility study, ADMSCs (passage 2-5) were seeded in each well of a 24-well plate at 1 * 104cells / well overnight. The F-127 diacrylate polymer solution with LAP was sterile filtered and DLP 3D printed in a biosafety hood to construct small disks (8 mm diameter and 1 mm thickness). After thoroughly washing them in sterile DI water to remove excess photo-absorbers and LAP, a hydrogel disk was added to each well of cells. The control group had no hydrogel. The cell culture medium was changed every other day. After 3 and 7-day incubation, the cells were stained by live / dead fluorescent staining dyes and imaged by a Zeiss 880 confocal microscope to quantify the cellular viabilities (Wang, et al. (2018) ACS Biomater. Sci. Engr., 4(12):4401-4411). Meanwhile, another 4 wells from each group were used for CCK-8 (Abeam) assays to evaluate the cell proliferation. The absorbance was measured at 460 nm using a microplate reader (BioTek), according to the manufacturer’s protocol.

[0102] In vivo stability and biocompatibility study

[0103] All animal procedures have been approved by the UNMC IACUC. The sterile F- 127 diacrylate was DLP 3D printed into small disks (8 mm diameter and 1 mm thickness). After washing them in sterile DI water, the hydrogel disks were subcutaneously implanted into the backs of C57BL / 6 mice (Female, 10 weeks old, weighing 18 - 20 g, Charles River Laboratories). For the comparison study, PEG hydrogels (10%) were DLP 3D printed into the same shape using PEG diacrylate (Mw 4600, Sigma) and subcutaneously implanted into the mice. The mice were euthanized 1 month after implantation. The major organs and the skin tissues with the hydrogel were isolated for histological assessments.

[0104] H&E and Masson's trichrome staining

[0105] The major organs and the skin tissues with the implanted hydrogels were fixed in 4% paraformaldehyde solution (PF A) and then dehydrated in 70% ethanol. The tissue samples were embedded in paraffin by the UNMC Tissue Sciences Facility. 10 pM thick cross sections of the tissue samples were cut using a microtome. The H&E and Masson’s tri chrome staining were carried out by following the standard protocols in the Tissue Sciences Facility. Images of the H&E and Masson’s tri chrome-stained sections were acquired with a Zeiss optical microscope.

[0106] Application of the ionic hydrogel as a capacitive sensor The capacitance of the hydrogels was measured with an LCR meter (E4980AL, Keysight) and a digit multimeter (DM6500 6 ’A, Keithley). It is noted that PDMS layers were used to encapsulate the hydrogels and metal wires during the electrical measurement under tensile and compressive strains. The relative capacitance changes (AC / Co) of the strain and pressure sensors were calculated by the following equation: AC / Co = (C-Co) / Co, where C is the measured capacitance under the tensile and compressive strains and Co is the initial capacitance. The sensitivity (S) of the pressure sensor was defined as: S = 5( AC / Co ) / 5P, where P is the applied pressure. The sensitivities of the pressure sensor at various pressure intervals were determined using segmented linear fitting analysis. In the low temperature experiment, the real-time temperature in the freezer was obtained by using Fluke View Form software coupled with an infrared thermometer (Fluke 566) and a type K thermocouple probe.

[0107] Statistical methods

[0108] Replicate measurements were shown as the mean ± SD. The statistical analysis was performed in GraphPad Prism 7.0 with the Student’s t-test. P-values < 0.05 were considered statistically significant. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0109] Results

[0110] DLP 3D printing of the poloxamer hydrogel

[0111] To evaluate the potential of the F-127 amphiphilic block copolymer for the DLP 3D printing process, F-127 diacrylate was first synthesized using a modified method (Fig. 1C).JH Nuclear Magnetic Resonance (NMR) study indicated an average of a 55% degree of acrylation, which met the need of the photo-reactive polymer. As expected, F- 127 diacrylate can form micellar structures due to its amphiphilic nature. The average nanoparticle sizes were 257.3 ± 39.7 nm and 283.3 ± 26.6 nm for 6.5% and 12.5% F-127 diacrylate, respectively (Figure ID). The surface potential for both concentrations was also measured, revealing comparable negative potentials of -5.8 ± 0.2 for 6.5% F-127 diacrylate and -5.6 ± 0.04 for 12.5% (Figure IE). Transmission electron microscopic (TEM) images also confirmed the nanoparticle structures for both concentrations (Figure IF). Previous studies implied that F-127 diacrylate could form a physical hydrogel at a 15% concentration (Di Biase, et al. (2011) Soft Matter 7(10):4928-4937), which may hamper the DLP printing. It was confirmed that it was still in liquid form at 12.5%, which was good for sample transfer before printing. For comparison purposes and a better understanding of the printability at a lower concentration, the 12.5% was decreased by nearly half and 6.5% was used for the study. These two concentrations were further used for DLP 3D printing. To initiate the DLP -based photopolymerization, two commonly used photoinitiators (lithium phenyl-2,4,6-trimethylbenzoyl -phosphinate (LAP) and Irgacure 2959) were initially tested, and LAP was selected based on its better photo-curing ability at 405 nm (Fig. 1 A), which is the wavelength used by the 3D printer. Under light exposure, LAP generates the free radical and initiate the free radical chain polymerization of the F-127 diacrylate monomer / cross-linker. Tartrazine, a water- soluble food dye with maximum absorbance at around 420 nm, can control the penetration depth of the DLP laser light within the ink and is expected to improve the resolution. The influence of different ink formulations and printing parameters on the rheological properties and structural fidelity of the DLP printed hydrogels was characterized (Figs. 1G, 1H). When the LAP concentration was fixed, the DLP printed hydrogels were rapidly fabricated with 5 seconds of curing time per layer even with a polymer precursor content as low as 6.5% (w / v).

[0112] The storage modulus (G’) was dependent on the initial concentration of the F-127 diacrylate precursor, and it changed from about 550 Pa at 6.5% concentration to 3900 Pa at 12.5% concentration (Fig. 2A). Increasing the curing time per layer to 10 seconds further increased the G’ at both concentrations. However, no further increase in the G’ was found when the curing time was more than 10 seconds. The 6.5% hydrogels showed a maximum G’ of around 1800 Pa, and the 12.5% hydrogels showed a maximum G’ of around 9500 Pa. To remove the photo-absorber content after 3D printing, the hydrogels were washed in DI water. The color of the hydrogels changed from yellow to transparent as the dye quickly diffused from the hydrogels. The washing process led to a slight decrease in the G’, as the 6.5% hydrogels showed an average of 1500 Pa, and the 12.5% hydrogels showed an average of around 6000 Pa (Fig. 2B). Next, the effects of the tartrazine concentration on the fidelity of the printed structures was investigated by printing the multi-channel structure with various channel diameters (D = 0.5, 1, 1.5, and 2 mm) (Fig. 2C) at a fixed printing conditions (12.5% F-127 diacrylate content and 10 seconds per layer) (Huh, et al. (2021) Biofabrication 13(3):034103). In the multichannel scaffold printed in the presence of 0.5 mM tartrazine, the channel was blocked in most diameters except the one with 2 mm diameter, and this channel was smaller than the designed dimension (Fig. 2D). A 1 mM tartrazine concentration resulted in most channels being smooth, except for the 0.5 mm channel, but the actual sizes were still smaller than the designed values (Fig. 2E). The channels printed in the presence of 1.5 mM tartrazine showed the best resolution, and the measured channel diameters matched well with the designed values, with the slope close to 1.0 (Fig. 2F). Photoabsorbers, such as tartrazine, due to their UV light absorption ability, can increase the printing resolution during the DLP process by preventing the overcross-linking of the hydrogels. When tartrazine is absent, projected UV light overcures a region larger than the projected zone. At lower concentrations of tartrazine, the resolution was low because the projected light could also cause undesired curing of the hydrogels. Only when the concentration was at least 1.5 mM, did the channel dimensions match the design. Therefore, 1.5 mM tartrazine was selected as the optimal concentration of the photo-absorber to combine with 12.5% F-127 diacrylate due to the high printability and fidelity. A porous cube design was used, and three constructs were DLP 3D printed to evaluate the printing accuracy. The key dimensions were measured and compared to the design. The overall deviation is less than 0.1%, which demonstrates high accuracy.

[0113] After determining the ink formulation, the next objective was to print objects with various shapes, sizes, and complexities to assess the fidelity of the printing process in accurately reproducing small and varied features. To achieve this, four constructs were printed with increasing structural complexities (Figs. 2G-2J): four 5 x 5 x 5 mm3square pyramids that were connected to each other by a 1 mm thick base (Fig. 2G), a 6 mm outer diameter tube with a wall thickness of 1 mm that had 0.5 x 1 mm2diamonds cut through it (Fig. 21), a 10 x 10 x 10 mm3lattice cube with circular channels in all directions (Fig. 2H), and a l5 x 15 x 15 mm3gyroid cube (Fig. 2J). The successful printing of these objects demonstrated that the fabricated hydrogel exhibits excellent shape fidelity for DLP 3D printing, allowing for the creation of complex structures with precisely designed features. After printing and further washing, the hydrogels became optically clear. The lattice cube and gyroid cube were able to be fully compressed and released to return to their natural shapes without any damage (Figs. 2K-2P). This indicated that the DLP printed hydrogels had excellent physical resilience and recovery properties.

[0114] Interestingly, other components could be incorporated into the ink formulation without impairing the printing resolution and accuracy. For example, conductive polymers, such as PEDOT: PSS, were successfully integrated without interfering with the DLP printing, offering possibilities for the fabrication of conductive hydrogels. Figure 2Q illustrates the compressive force-strain curve of 5% (w / w) PEDOT:PSS hydrogels when subjected to up to 90% compressive strain, showing good recovery to its original shape upon the release of force. Additionally, the 5% PEDOT:PSS hydrogels exhibited an average conductivity of approximately 0.42 S / cm, as demonstrated in Figure 2R.

[0115] Shape-memory and adaptive behavior of the DLP printed hydrogels

[0116] An interesting characteristic of the DLP printed F 127 hydrogels was the water activated shape-memory property. This was depicted by DLP printing the poloxamer into a microfluidic device (Fig. 3 A). The channel was proven to be clear by perfusing a dye-colored solution from one side to the other (Fig. 3B and 3C). The hydrogel was dehydrated through incubation in a 37°C dry oven overnight (Fig. 3D). Then the dehydrated hydrogel quickly recovered to its original shape after immersing it in water for 30 minutes (Fig. 3E). Further perfusion with the dye-colored solution confirmed that the patency of the channel was not compromised after recovery (Fig. 3F). Similarly, a freeze-dried hydrogel also has the water activated shape-memory property. These findings demonstrated the 4D printing characteristic of the F-127 hydrogel.

[0117] A DLP 3D printed polyethylene glycol diacrylate (PEGDA) construct was also generated. After freeze-drying, the printed PEGDA constructs showed significant alteration of morphology and structure. After further rehydration, the constructs were delaminated and broken. When DI water with red food dye was infused into the channel, the red dye diffused throughout the entire structure rather than remaining within the channel, indicating the disruption of the channel structure.

[0118] The shape-memory ability of the 3D printed hydrogels enables them to be stably stored for extended periods of time without compromising their structures and properties. In addition, it allows the hydrogels to adapt their shape to match specific geometries of detection locations on the human body (Wu, et al. (2023) Adv. Funct. Mater., 33(36):2303292). This can be exemplified by the dehydration and rehydration process of a printed hydrogel fitted to a plastic tube, where the tube is used as a mimic of the human finger. As shown in Figs. 3G-3I, the poloxamer hydrogel was printed into a tube shape, which could fit around a plastic tube with smaller diameter. After dehydration, the hydrogel became a film that can be easily folded. After rehydration, the hydrogel was able to fit onto the plastic tube again, which proved its shape-adaptivity. Compression was further applied during both oven and freeze-drying of the 3D printed poloxamer hydrogel tube. Post-rehydration, the tube maintained its original shape. It can be imagined that after the hydrogel is 3D printed to match a certain geometry of a targeted human tissue (such as in the form of the finger splint, wrist band, etc.), the tailored wearable sensors can form a conformal attachment to the desired tissue locations through rehydration-mediated or water-activated shape recovery after dehydration and long-term storage. Importantly, the shape-memory hydrogels were distinguished from previously reported shape-memory hydrogels, which suffer from issues like being non-3D printable (Wu, et al. (2023) Adv. Funct. Mater., 33(36):2303292) or non-transparent (He, et al. (2021) ACS Appl. Mater. Interfaces 13 (30): 36286-36294).

[0119] In vitro and in vivo biocompatibility of the poloxamer hydrogels

[0120] The biocompatibility of hydrogels is critical for biomedical applications. The thermo-responsive Pluronic® F-127 polymer could form physically crosslinked hydrogels at 37°C when the content is above 20%, yielding high viability and proliferation of the encapsulated mesenchymal stem cells (Diniz, et al. (2015) J. Mater. Sci. Mater. Med., 26(3): 153). Due to the excellent cyto-compatibility of the major component F-127, the primary human adipose derived mesenchymal stem cells (ADMSCs) showed over 95% viability on both day 3 and day 7 when incubated with the DLP-printed F-127 hydrogels (Fig. 4A and 4B). The study also showed that the ADMSCs could maintain over 80% viability even after DLP 3D bioprinting with cells encapsulated inside the F-127 hydrogels. A CCK-8 assay was utilized to assess the cell proliferation, and the results showed a comparable number of cells between the control group and the hydrogel incubation group after 3 and 7 days of culture. This indicates that the hydrogels did not have an influence on cell proliferation in vitro. (Fig. 4C). The in vivo stability and biocompatibility of the DLP 3D printed F-127 hydrogels was evaluated by implanting them subcutaneously in mice. The hydrogels degraded by approximately 50% within one month (Fig. 4D). Histological staining results revealed only limited inflammatory cell infiltration and tissue fibrosis (Fig. 4E and 4F), indicating a minimal foreign body reaction towards the printed poloxamer hydrogels. On the contrary, DLP printed polyethylene glycol (PEG) hydrogels showed minimal degradation (Fig. 4G) and substantial immune reactions, including abundant tissue fibrosis and inflammatory cell infiltration (Fig. 4H and 41), even though PEG is generally considered as a biocompatible material. Although both PEG and F-127 hydrogels could prevent protein adsorption at a close level (Li, et al. (2020) RSC Adv., 10(72):44494- 44502), the excellent biocompatibility of the DLP printed F-127 hydrogel may be due to its better in vivo degradability, which could be explained from two perspectives. Firstly, the molar concentration of PEG was higher than that of F- 127 due to its smaller molecular weight, leading to a higher degree of cross-linking of the PEG monomer. Secondly, during photopolymerization, the F-127 diacrylate was crosslinked not only inter-micellarly but also intra-micellarly (Di Biase, et al. (2011) Soft Matter 7(10):4928- 4937). The relatively weaker connection between each micelle could lead to faster degradation of the micelle as a cohesive unit within the hydrogel. Histological staining of major organs collected from the mice implanted with the 3D printed F127 hydrogel also indicated no observed tissue toxicity. The excellent biodegradability and biocompatibility are expected to facilitate the translational application of the hydrogels and make them much more environmentally friendly.

[0121] Generation and characterization of ionic hydrogels

[0122] DLP printed hydrogels were then modified, specifically focusing on the fabrication and characterization of ionic hydrogels. These hydrogels were facilely prepared by soaking the water-washed hydrogels in 30% LiCl solution overnight. The ionic hydrogels were also transparent, with a transmittance value > 85% over a spectrum from 400 to 700 nm (Fig. 5A). Even though the value was slightly lower than the hydrogels after the water wash, the transparency was still better than other ionic hydrogels (Ma, et al. (2021) Chem. Engr. J., 420: 129865; Wu, et al. (2021) J. Mater. Chem. C 9(39): 13668-13679; Sui, et al. (2021) Chem. Engr. J., 419: 129478). The swelling ratio of the water-washed hydrogels was inversely related to the water temperature, with 4°C showing the most swelling and 37 °C showing the least swelling. This agrees with the thermo-responsive behavior of the F-127 polymer and UV cured F- 127 diacrylate hydrogels (Li, et al. (2020) RSC Adv., 10(72):44494-44502). When the temperature is low (4°C), the F-127 polymer is more hydrophilic because of the hydrophilic transition of the polypropylene oxide) block, which facilitated the interaction between water and the polymer network, resulting in substantial swelling. In contrast, at the high temperature (37°C), hydrophobic interactions between the polypropylene oxide) blocks are stronger, leading to decreased water inclusion in the polymer network and thus reduced swelling (Shen, et al. (2019) Lab Chip 19 (23) : 3962- 3973). Nonetheless, the ionic hydrogels exhibited negligible swelling in comparison to the designed dimensions (Fig. 5B), thereby preserving the resolution of the printed structure even after washing. Moreover, the presence of lithium ions facilitated the water retention ability of the ionic hydrogels by efficiently immobilizing water molecules in their vicinity (Fig. 5C). The ionic hydrogels kept 100% of their weights on day 1 and -90% of their original weights even on day 14 at room temperature with 50% relative humidity (RH), which was comparable to many LiCl ionic hydrogels (Ma, et al. (2021) Chem. Engr. J., 420: 129865; Wu, et al. (2021) J. Mater. Chem. C 9(39): 13668-13679; Sui, et al. (2021) Chem. Engr. J., 419: 129478). LiCl salt shows strong interactions with water molecules via the ionic solvation effect (Mahler et al. (2012) Inorg. Chem., 51 (l):425-38). At room temperature, an equilibrium can be achieved between the water evaporation from the hydrogels and the water absorption into the hydrogels because of the LiCl solvation, which results in the net effect of no water loss (Sui, et al. (2021) Chem. Engr. J., 419: 129478). In contrast, without the LiCl soaking, the hydrogels were easily dehydrated, resulting in an approximately 85% loss of their original weights within one day. The dehydration trend correlated well with the polymer concentration (Fig. 5D). The mechanical and rheological properties of the ionic hydrogels were also characterized. The fracture stress and the elongation at the break of the ionic hydrogels with a dogbone structure were significantly increased by over five times and two times (0.1 MPa and -330%), respectively, over the original hydrogels, as shown in Figs. 5H and 51. The enhancement in the mechanical properties of the LiCl-soaked hydrogels may be ascribed to the increased hydrophobic associations between the poly-(propylene glycol) blocks intramicellularly due to salt percolation into the micelle (Zhang, et al. (2020) Soft Matter 16(3):738-746; Chen, et al. (2020) Cellulose 27: 1113-1126). The stress is greater than many other reported DLP -printed hydrogels (Xiong, et al. (2023) Nat. Commun, 14(1): 1331; Wang, et al. (2020) Research 2020: 1426078; Ge, et al. (2021) Sci. Adv., 7(2):eaba4261; Caprioli, et al. (2021) Nat. Commun., 12(1):2462; Guo, et al. (2023) Composites Communications 37: 101454; He, et al. (2022) Bioactive Materials 17:234-247). The Young’s modulus of the ionic hydrogel also showed a 1.8- times increase to 49 kPa (Fig. 5 J), but it was still in the range of those of human soft tissues (Ye, et al. (2022) Adv. Mater., 34(4):2105120). However, no significant difference in the G’ of the hydrogel was found before and after LiCl percolation (Fig. 5K).

[0123] Furthermore, it was found that the ionic hydrogels were not easily dehydrated at 37 °C in the dry oven. Only -25% of the original weight was lost after 24 hours of drying due to the water retention ability of the ionic hydrogels. The drying temperature was thus increased to 60 °C, and the ionic hydrogels were dehydrated, with the salt leaching out of the hydrogels, after 24 hours of drying (Fig. 5E). After immersing the dehydrated ionic hydrogel in a 30% LiCl solution for 30 minutes, it promptly regained its original shape while maintaining a clear channel (Fig. 5F and G).

[0124] Performance of 3D printed ionic hydrogels as strain sensors

[0125] To visualize the conductivity of the ionic hydrogels, a circuit with a lightemitting diode (LED) was designed. The ionic hydrogel achieved a conductivity of 0.373 ± 0.0035 S / m at room temperature, while the original hydrogel showed a value of only -0.001 S / m (Fig. 61). Although the conductivity was moderate, it can be further increased by soaking it in an ionic liquid.

[0126] For the application of the ionic hydrogels as a wearable sensor, the variations of the capacitance of the hydrogels under mechanical deformation were investigated. As shown in Fig. 6A, the change in capacitance of the ionic hydrogels increased as the strain increased. Upon application of stretching, the gauge factor exhibited a value of 4.37 up to a strain of 10%. From 10% to 50% strain, the gauge factor was recorded as 1.34. In contrast, there was no significant capacitance change in the hydrogels under strain. The dynamic response of the capacitive pressure sensor that was based on the ionic hydrogels under cyclic stretching and releasing is demonstrated in Fig. 6B. The capacitance of the ionic hydrogels decreased upon stretching them (Rao, et al. (2020) J. Mater. Chem. C 8(18):6034-6041). The results also indicated that the relative change of the capacitance was repeatable in each cycle, and the baseline signal was very stable within a few thousand cycles (Fig. 6J). Fig. 6C depicts the durability of the capacitive pressure sensor after hundreds of tensile strain cycles, and the response signal remained nearly unchanged at a large strain of 50%. Based on the excellent comprehensive performance, the ionic hydrogels were further evaluated for real-time detection of human motion as a wearable sensor. The ionic hydrogel capacitive pressure sensor was first placed on the knee, as illustrated in Fig. 6D. Fig. 6E represents the relative capacitance changes depending on various movements, which were standing, squatting, walking, and running. A capacitive pressure sensor was mounted onto the elbow and finger and the relative capacitance changes corresponding to the bending angle of each joint was measured (Figs. 6K-6N). The results show a direct correlation between joint bending and the change in capacitance, with higher joint bending resulting in a more significant capacitance change. Specifically, in joints such as the elbow, where larger strains are experienced compared to the finger, the changes in the capacitance were more pronounced (Fig. 6M and 6N). A wearable capacitive pressure sensor was developed that can be worn on the fingers and designed with various dimensions, as shown in Fig. 6F and Fig. 60. This tailored ionic hydrogel-based finger splint is flexible and stretchable, making it simple to wear on the finger and matches perfectly with the user’s size. It is 30 mm long with a 1.5 mm thick wall and tapered from a 20 mm diameter at one end to a 15.7 mm diameter at the other end. It also has cutouts on all four sides of the finger that fit around the knuckle area, to allow for a better fit and bending of the hydrogel. It matches perfectly with the user’s finger size and bends freely with the finger and returns to its original shape when the finger is straightened. Fig. 6G presents the relative capacitance changes depending on the different bending angles of the finger. As shown in Fig. 6H, the dynamic response of the tailored capacitive pressure sensor indicates that the capacitance changed linearly during finger bending, which indicated a good sensing performance as a flexible strain sensor.

[0127] Performance of 3D printed ionic hydrogels as tactile sensors

[0128] The capacitance changes of the ionic hydrogels were investigated under compressive stress in order to explore their potential application as capacitive tactile sensors. The compressive loading-unloading curves of cubic ionic hydrogels are represented in Fig. 7A. Initially, both solid and porous hydrogels require similar forces for initial deformation, determined by their chemical composition and stiffness. As deformation progresses, the structure of the hydrogels plays a crucial role. Porous hydrogels, due to their collapsible pores, can undergo significant deformation with less additional force. In contrast, solid hydrogels, being denser and without pores, need more force for further deformation as they become more compact under compression. Introducing highly interconnected pores into the hydrogel structure can increase the sensitivity and improve the comfort of wearing the hydrogel sensor (Zhang, et al. (2021) Composites Sci. Tech., 213: 108968). DLP 3D printing is a perfect approach for fabricating well-defined porous hydrogels. Therefore, hydrogels were 3D printed and the performance of hydrogel sensors with solid and porous cubic structures were compared. The hysteresis of the solid hydrogels was much smaller than that of the porous hydrogels (Fig.7A). In addition, the compressive stress of the porous hydrogels was two times smaller than the solid hydrogels. Due to the high freedom of structural engineering afforded by 3D printing, it is straightforward to implement the desired mechanical properties in the fabrication of hydrogels as tactile sensors. The relative capacitance changes of the solid and porous hydrogels under various pressures are characterized in Fig. 7B and 7C. The porous hydrogels showed a more sensitive response to the applied pressure due to increased deformation (Fig. 7D) (Zhang, et al. (2021) Adv. Electronic Mater., 7(7):2100174). The sensitivity of the porous hydrogels was nearly three times higher than that of the solid hydrogels in the 3 - 20 kPa pressure region and six times higher in the 25 - 40 kPa pressure region. In contrast, the solid hydrogels exhibited a more linear but relatively lower response at a wide range of pressures (0.036 kPa-1 in 3 - 40 kPa pressure region). The results indicate that 3D printed hydrogels can be used to construct sensors that can detect very narrow pressure regions with high sensitivity or can cover a broad pressure range. Fig. 7E demonstrates the capacitance of the porous hydrogels under cyclic 50% compression. The measured capacitance of the hydrogels increased upon applying pressure due to decreased distance between the top and bottom electrodes (Ha, et al. (2022) ACS Nano 16(3):3442-3448). As shown in Fig. 7F, the relative capacitance change of the porous hydrogels remained constant after 200 loading and unloading cycles with a 50% compression. Ring-shaped ionic hydrogels were used to validate the use of the 3D-printed ionic hydrogel as wearable sensors (Fig. 71). Fig. 7G presents photographs of solid and porous hydrogels printed into wearable ring shapes. The devices are made up of a 10 mm diameter and 2 mm thick cylindrical base and a 2 mm thick ring that is an approximately 7.25 mm x 10 mm oval shape (Fig. 71). The ring stretches to fit snugly over the finger, while the base sits under the fingertip (Fig. 7G). The porous version has 0.75 mm channels horizontally through the ring and 1 mm channels vertically through the base. The change in capacitance of the ionic hydrogels worn on the left index finger was measured during typing 'pressure sensor' on the keyboard with both hands (Fig. 7H). The solid hydrogels exhibited highly repeatable and responding capacitance changes during typing on the keyboard. Although the porous hydrogels were reproducible and were more sensitive in terms of motion sensing, the relative capacitance change was slightly less consistent solid hydrogels. Nevertheless, the overall electronic performances demonstrate the significant potential of the DLP printed poloxamer-based pressure sensor for use in capacitive tactile sensing. In summary, the soft, stretchy, and customized ionic hydrogels make them suitable candidates for wearable electronics designed for curvilinear surfaces and dynamic circumstances.

[0129] Anti-freezing performance of the ionic hydrogel-based sensors One of the major issues with hydrogel -based sensors is their poor freezing tolerance. In freezing conditions under subzero temperatures, conventional hydrogels that employ water as the dispersing medium will undergo solidification, resulting in the loss of their original mechanical and electrical properties (Liu, et al. (2020) ACS Applied Mater. Interfaces 12(22):25334-25344). Ionic hydrogels could tolerate extremely low temperature environmental conditions because of the decreased freezing point of the ionic hydrogels (Ma, et al. (2021) Chem. Engr. J., 420: 129865; Sui, et al. (2021) Chem. Engr. J., 419: 129478). The anti-freezing ability of the ionic hydrogel was first tested by keeping the water-washed hydrogels and LiCl-soaked hydrogels in a -20 °C freezer. After freezing them for 2 hours, the hydrogels transitioned from a transparent and flexible state to a white and rigid state (Fig. 8A). On the contrary, the ionic hydrogels remained transparent, and the stretchability only slightly decreased compared to that at 25°C (from -330 to -300%; Fig. 8E). Differential scanning calorimetry (DSC) was next used to determine the freezing point of the water-washed hydrogels and the ionic hydrogels, as shown in Fig. 8B. A significant crystallization peak was found for the water-washed hydrogels at -20 °C, while no significant crystallization peak was noticeable for the ionic hydrogels, which agreed well with the findings. The ionic hydrogels were mostly amorphous and showed a glass transition temperature (Tg) of - 124°C. In addition, the ionic hydrogels maintained a conductivity of 0.34 S / m even at - 20°C, which only slightly decreased (-10%) when compared to the value at room temperature.

[0130] The well-retained stretchability and conductivity of the ionic hydrogel at subzero temperatures provides convenience for the application of hydrogel sensors in monitoring finger movement at extremely low temperatures. Fig. 8C presents photographs of ionic hydrogel-based capacitive strain sensors mounted on a robotic hand in a freezer and an infrared thermometer (Fluke 566) with a type K thermocouple probe. The capacitive strain sensors can be appropriately affixed to the joints of a curved robotic hand due to their mechanically stretchable and deformable properties. Furthermore, integrating sensors and the robotic hand provides the robots with advanced capabilities, including gesture recognition. The strain sensors convert the gesture into capacitance, an electrical parameter, thereby facilitating sign language comprehension. The robotic hand was programmed to correspond to each letter (W, O, R, L, and D, respectively). Under subzero temperatures (-20°C), the relative capacitance changes of the strain sensors on the robot hand were measured depending on five different gestures (Fig. 8D). As each finger of the robot was bent, the relative capacitance changes of the strain sensor increased. The capacitance change of the five sensors can recognize and respond to the bending of each finger. As illustrated previously, ionic hydrogels demonstrate remarkable potential for use as wearable sensors, even in challenging low-temperature environments. Upon comparing the performance of the DLP -printed poloxamer micellebased sensor with several reported sensors, its overall performance was comparable or surpassed many other DLP-printed sensors (Xiong, et al. (2023) Nat. Commun., 14(1): 1331; He, et al. (2021) ACS Appl. Mater. Interfaces 13(30):36286-36294; Peng, et al. (2022) Chem. Engr. J., 439: 135593; Peng, et al. (2021) Adv. Funct. Mater., 31 (10):2008729).

[0131] Previous work applying Pluronic® F-127 in 3D printing relies on the extrusionbased printing method, which suffers from low resolution and precision. The resultant physically cross-linked hydrogels have very poor mechanical properties and dissemble and degrade easily at low temperature and thus cannot be applied as a motion sensor. Herein, the DLP technique easily fabricated hydrogels made of Pluronic® F-127 diacrylate-based micelle precursors, and 3D printed hydrogels with tunable mechanical properties were fabricated with high resolution and complexity after optimizing the ink formulation and printing parameters. The DLP printed poloxamer hydrogels were biodegradable and biocompatible and equipped with shape-memory property. After LiCl percolation, conductive and stretchable anti-freezing ionic hydrogels with high transparency and moisture retention were facilely prepared. The ionic hydrogels exhibited good sensitivity and cyclic stability as both capacitive strain sensors and tactile sensors, successfully detecting different modes of human motions. Moreover, DLP 3D printing enabled the precise and simple fabrication of user-specific wearable sensors. It harnessed the geometric adaptivity powered by the shape-memory effect, allowing tailored hydrogels to conform to the detection locations (finger, wrist, etc.) through dehydration and rehydration-mediated shape programming. Furthermore, satisfactory functioning of the hydrogel sensor in an extreme environment was demonstrated by the real-time signal reflecting the finger joint movement of a robotic hand kept in the -20 °C conditions. Overall, considering the performances of the electrical and mechanical properties and the convenient and customizable manufacturing capabilities, these hydrogels are versatile material platform towards seamlessly integrated wearable and bioelectronics. EXAMPLE 2

[0132] Due to bioprinting technologies using a comparatively small amount of material, especially those that use print-on-demand techniques, they are considered to be promising production approaches. Though 3D bioprinting technologies are widely used in the creation of artificial tissues and organs, very little research has been done on their biosensor applications. Single-walled carbon nanotube (SWNT)-based optical sensors are highly praised for their remarkable sensitivity and specificity for a wide range of analytes. Nevertheless, due to the complexity of packaging and immobilization, problems continue to arise when incorporating SWNT sensors into both in vitro and in vivo applications.

[0133] Herein, 3D printing technology was used to improve SWNT sensor integration, resulting in adaptable and efficient platform designs. The optimized bioprinted hydrogel, composed of 3D-printable, modified Pluronic® F-127, improves SWNT containment, ensuring stable fluorescence readings and increased fluorescence quenching in the presence of nitric oxide (NO). The hydrogel-encapsulated sensor maintains a ~95% fluorescence intensity over 90 days. Following the hydrogel parameter optimization, NO solutions were introduced to a 1.5-mm thick walled 30 mg / L SWNT -loaded hydrogel, revealing a gradual fluorescence intensity decrease with higher NO concentrations. When inserted under chicken skin, the sensor exhibits stable fluorescence readings, demonstrating its potential as a real-time in vivo sensor for minimally invasive health monitoring.

[0134] Figure 9A provides compressive cycle stability of 1-mm, 1.5-mm, and 2-mm thick hydrogels at a 40% compression strain with a strain rate of 0.2 mm / s. Figure 9B provides a schematic illustration of a 3D printed liquid-core hydrogel design. SWNT- loaded 1.5 mm thick liquid-core hydrogel samples were synthesized and their fluorescence image was measured at 990 nm. As seen in Figure 9C, there is a linear correlation between the concentrations of SWNT loaded into the hydrogel (15, 30, 50, and 100 mg / L) and the fluorescence intensity. Figure 9D shows the fluorescence stability of 1-, 1.5-, and 2-mm thick hydrogels over a 4-hour duration. Figure 9E shows that DNA- wrapped SWNT ((AT)is (SEQ ID NO: l)-wrapped SWNT) sensors in 1.5- thick hydrogels respond to the addition of 30 pM NO at 990 nm (final NO concentration is 10 pM). Fluorescence stability of a 30 mg / L (AT) 15 (SEQ ID NO: l)-wrapped SWNT loaded in a 1.5-mm thick hydrogel over 90 days is shown in Figure 9F. No statistical difference was found for any of the time points. Notably, PVA- wrapped SWNTs in a 1.5-mm thick hydrogel did not respond to the addition of 30 pM NO (Figure 9G). Figure 9H shows the fluorescence spectral quenching upon the addition of various NO concentrations (0 - 50 pM) using 1.5-mm thick 30 mg / L SWNT-loaded hydrogels via an nIR spectrometer. Figure 91 provides the corresponding fluorescence quenching of 1.5- mm thick 30 mg / L SWNT-loaded hydrogels at 990 nm from 0 pM to 50 pM. A linear fitting curve of the fluorescence quenching of the hydrogel at 990 nm towards the concentrations of NO from 1 pM to 50 pM is shown in Figure 9J.

[0135] Lastly, the SWNT sensor-loaded liquid-core hydrogels was inserted between the skin and muscle of a chicken breast (Fig. 9K). The corresponding fluorescence intensity heat map is provided in Figure 9L. Figure 9M provides the percent fluorescence spectra of (AT) 15 (SEQ ID NO: l)-wrapped SWNT sensor-loaded hydrogels before and after inserting them underneath the chicken skin. The response of the inserted 1.5 mm-thick hydrogel response to the addition of 30 pM NO at 990 nm (final NO concentration is 10 pM) is provided in Figure 9N.

[0136] EXAMPLE 3

[0137] The Pluronic® F-127 acrylate was used to print 12-well plate inserts with designed microwells for the quick and large-scale production of cell spheroids. The size and number of the spheroids created can be adjusted by changing the size of the wells in the inserts. Featured here are 0.5 and 0.25 mm diameter wells. The 3D-printed structures allow for cheap, customized, and scalable production of cell spheroids. The Pluronic® F-127 acrylate has low cell attachment, which allows for cellular aggregation into spheroids as well as the release of the spheroids into the wells of the plate for culture.

[0138] Briefly, the process comprises: 1) seed cells on the surface of the inserts; 2) incubating with medium for cell aggregation into spheroids; and 3) washing the inserts with medium to release the spheroids from the inserts. The cell spheroids can then be further cultured (e.g., in the plate).

[0139] Figure 10A provides images of the plate inserts in 0.5 mm wells and 0.25 mm wells. Figure 10B and 10C provide images of cells upon seeding and after overnight culture, respectively. The presence of cell spheroids is visible. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A hydrogel precursor comprising an amphiphilic block copolymer, a photoabsorber, and a photoinitiator, wherein said amphiphilic block copolymer comprises terminal photoactive groups.

2. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer comprises terminal acrylate groups.

3. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer is a poloxamer.

4. The hydrogel precursor of claim 3, wherein said poloxamer is poloxamer 407.

5. The hydrogel precursor of claim 1, further comprising an additional photoactive polymer.

6. The hydrogel of claim 5, wherein said additional photoactive polymer is an acrylic acid, acrylamide, or sulfobetaine methacrylate (SBMA).

7. The hydrogel precursor of any one of claims 1-6, wherein said photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

8. The hydrogel precursor of any one of claims 1-6, wherein said photoabsorber is tartazine.

9. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer is poloxamer 407 diacrylate, said photoinitiator is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), and said photoabsorber is tartazine.

10. The hydrogel precursor of claim 1, further comprising a therapeutic agent, cell, tissue, and / or electrically conductive compound.

11. A hydrogel which is light and / or laser-based, three-dimensionally printed from the hydrogel precursor of claim 1.

12. The hydrogel of claim 11, wherein said hydrogel is digital light processing, three- dimensionally printed.

13. The hydrogel of claim 11, wherein said hydrogel precursor comprises pol oxamer 407 diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartazine.

14. A hydrogel comprising crosslinked poloxamer 407, wherein said hydrogel comprises intermicellar crosslinking of said poloxamer 407.

15. The hydrogel of any one of claims 11-14, further comprising an ionic salt and / or conductive filler.

16. The hydrogel of claim 15, wherein said ionic salt is lithium chloride or lithium bromide.

17. A method of synthesizing a hydrogel, said method comprising light and / or laserbased, three-dimensionally printing the hydrogel precursor of claim 1.

18. The method of claim 17, comprising digital light processing three-dimensionally printing the hydrogel precursor.

19. The method of claim 17, wherein said hydrogel precursor comprises poloxamer 407 diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartazine.

20. The method of claim 17, further comprising washing the printed hydrogel.

21. The method of claim 17, further comprising adding an ionic salt or conductive filler to the printed hydrogel.

22. The method of claim 17, further comprising adding a therapeutic agent, cell, tissue, and / or electrically conductive compound.

23. A sensor or biosensor comprising the hydrogel of any one of claims 11-14, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

24. The sensor or biosensor of claim 23 which is a tactile sensor, pressure sensor, motion sensor, or stain sensor.

25. A method of delivering a therapeutic agent or cell to a subject, said method comprising administering or implanting a hydrogel of any one of claims 11-14 into said subject, wherein said hydrogel comprises and / or encapsulates said therapeutic agent or cell, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

26. A method of detecting nitric oxide in a subject, said method comprising administering or implanting a hydrogel of any one of claims 11-14 into said subject and detecting fluorescence from the hydrogel, wherein said hydrogel comprises and / or encapsulates a carbon nanotube, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

27. The method of claim 26, wherein said carbon nanotube comprises a single-walled carbon nanotube (SWNT) or a DNA-wrapped SWNT.

28. A method for producing a cell spheroid, said method comprising culturing cells on a hydrogel of any one of claims 11-14, thereby producing said cell spheroid, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

29. The method of claim 28, further comprising isolating said cell spheroid.

30. The method of claim 28, wherein said hydrogel is a tissue culture well insert.ABSTRACTBioprintable hydrogels, methods of synthesizing, and methods of use thereof are provided.What is claimed is:

1. A hydrogel precursor comprising an amphiphilic block copolymer, a photoabsorber, and a photoinitiator, wherein said amphiphilic block copolymer comprises terminal photoactive groups.

2. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer comprises terminal acrylate groups.

3. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer is a poloxamer.

4. The hydrogel precursor of claim 3, wherein said poloxamer is poloxamer 407.

5. The hydrogel precursor of claim 1, further comprising an additional photoactive polymer.

6. The hydrogel of claim 5, wherein said additional photoactive polymer is an acrylic acid, acrylamide, or sulfobetaine methacrylate (SBMA).

7. The hydrogel precursor of any one of claims 1-6, wherein said photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

8. The hydrogel precursor of any one of claims 1-6, wherein said photoabsorber is tartazine.

9. The hydrogel precursor of claim 1, wherein said amphiphilic block copolymer is poloxamer 407 diacrylate, said photoinitiator is lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), and said photoabsorber is tartazine.

10. The hydrogel precursor of claim 1, further comprising a therapeutic agent, cell, tissue, and / or electrically conductive compound.

11. A hydrogel which is light and / or laser-based, three-dimensionally printed from the hydrogel precursor of claim 1.

12. The hydrogel of claim 11, wherein said hydrogel is digital light processing, three- dimensionally printed.

13. The hydrogel of claim 11, wherein said hydrogel precursor comprises pol oxamer 407 diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartazine.

14. A hydrogel comprising crosslinked poloxamer 407, wherein said hydrogel comprises intermicellar crosslinking of said poloxamer 407.

15. The hydrogel of any one of claims 11-14, further comprising an ionic salt and / or conductive filler.

16. The hydrogel of claim 15, wherein said ionic salt is lithium chloride or lithium bromide.

17. A method of synthesizing a hydrogel, said method comprising light and / or laserbased, three-dimensionally printing the hydrogel precursor of claim 1.

18. The method of claim 17, comprising digital light processing three-dimensionally printing the hydrogel precursor.

19. The method of claim 17, wherein said hydrogel precursor comprises poloxamer 407 diacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and tartazine.

20. The method of claim 17, further comprising washing the printed hydrogel.

21. The method of claim 17, further comprising adding an ionic salt or conductive filler to the printed hydrogel.

22. The method of claim 17, further comprising adding a therapeutic agent, cell, tissue, and / or electrically conductive compound.

23. A sensor or biosensor comprising the hydrogel of any one of claims 11-14, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

24. The sensor or biosensor of claim 23 which is a tactile sensor, pressure sensor, motion sensor, or stain sensor.

25. A method of delivering a therapeutic agent or cell to a subject, said method comprising administering or implanting a hydrogel of any one of claims 11-14 into said subject, wherein said hydrogel comprises and / or encapsulates said therapeutic agent or cell, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

26. A method of detecting nitric oxide in a subject, said method comprising administering or implanting a hydrogel of any one of claims 11-14 into said subject and detecting fluorescence from the hydrogel, wherein said hydrogel comprises and / or encapsulates a carbon nanotube, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

27. The method of claim 26, wherein said carbon nanotube comprises a single-walled carbon nanotube (SWNT) or a DNA-wrapped SWNT.

28. A method for producing a cell spheroid, said method comprising culturing cells on a hydrogel of any one of claims 11-14, thereby producing said cell spheroid, optionally wherein the hydrogel further comprises an ionic salt and / or conductive filler.

29. The method of claim 28, further comprising isolating said cell spheroid.

30. The method of claim 28, wherein said hydrogel is a tissue culture well insert.

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