Lithium Acylphosphinate Research Articles

Photo-Cross-Linkable, Injectable, and Highly Adhesive GelMA-Glycol Chitosan Hydrogels for Cartilage Repair.

Hydrogels provide a promising platform for cartilage repair and regeneration. Although hydrogels have shown some efficacy, they still have shortcomings including poor mechanical properties and suboptimal integration with surrounding cartilage. Herein, hydrogels that are injectable, cytocompatible, mechanically robust, and highly adhesive to cartilage are developed. This approach uses GelMA-glycol chitosan (GelMA-GC) that is crosslinkable with visible light and photoinitiators (lithium acylphosphinate and tris (2,2'-bipyridyl) dichlororuthenium (II) hexahydrate ([RuII(bpy)3 ]2+ and sodium persulfate (Ru/SPS)). Ru/SPS-cross-linked hydrogels have higher compressive and tensile modulus, and most prominently higher adhesive strength with cartilage, which also depends on inclusion of GC. Tensile and push-out tests of the Ru/SPS-cross-linked GelMA-GC hydrogels demonstrate adhesive strength of ≈100 and 46kPa, respectively. Hydrogel precursor solutions behave in a Newtonian manner and are injectable. After injection in focal bovine cartilage defects and in situ cross-linking, this hydrogel system remains intact and integrated with cartilage following joint manipulation ex vivo. Cells remain viable (>85%) in the hydrogel system and further show tissue regeneration potential after three weeks ofin vitroculture. These preliminary results provide further motivation for future research on bioadhesive hydrogels for cartilage repair and regeneration.

Engineering Microvascular Networks in LED Light-Cured Cell-Laden Hydrogels.

The success of tissue engineering inevitably depends on the fabrication of tissue constructs that can be vascularized and that anastomose with the host vasculature. In this report, we studied the effects of light-emitting diode (LED) photopolymerized gelatin methacryloyl hydrogels (GelMA), encapsulated with stem cells from the apical papilla (SCAP) and human umbilical vein endothelial cells (HUVECs), in promoting vasculature network formation as a function of hydrogel physical and mechanical properties, as well as total cell density. Lithium acylphosphinate (LAP) was used as the photoinitiator in concentrations of 0.05, 0.075, 0.1% (w/v). GelMA hydrogel precursors of 5% (w/v) were encapsulated with cocultures of SCAPs and HUVECs at different cell densities (1×, 5×, and 10 × 106 cells/mL) and photo-cross-linked for 5 s. Results suggested that the compressive modulus of GelMA hydrogels increased as a function of LAP concentration, and had a maximum stiffness of 3.2 kPa. GelMA hydrogels photopolymerized using 0.05 or 0.075% LAP, which had an average of 1.5 and 1.6 kPa of elastic modulus respectively, had the most efficient vasculature formation after 5 days, and these results were further enhanced when the highest cell density (10 × 106 cells/mL) was used. Immunofluorescence images showed that SCAP cells spread in close contact with endothelial networks and expressed alpha smooth muscle actin (αSMA), which is suggestive of their differentiation into pericyte-like cells. αSMA expression was also apparently higher in hydrogels polymerized with 0.05% LAP and 10 × 106 cells/mLl. In conclusion, photopolymerization of GelMA hydrogels using an LED-light source can be an effective method for potential chair-side/in situ procedures for engineering of vascularized tissue constructs in regenerative medicine.

Solution-Phase Photochemical Nanopatterning Enabled by High-Refractive-Index Beam Pen Arrays.

A high-throughput, solution-based, scanning-probe photochemical nanopatterning approach, which does not require the use of probes with subwavelength apertures, is reported. Specifically, pyramid arrays made from high-refractive-index polymeric materials were constructed and studied as patterning tools in a conventional liquid-phase beam pen lithography experiment. Two versions of the arrays were explored with either metal-coated or metal-free tips. Importantly, light can be channeled through both types of tips and the appropriate solution phase (e.g., H2O or CH3OH) and focused on subwavelength regions of a substrate to effect a photoreaction in solution that results in localized patterning of a self-assembled monolayer (SAM)-coated Au thin film substrate. Arrays with as many as 4500 pyramid-shaped probes were used to simultaneously initiate thousands of localized free-radical photoreactions (decomposition of a lithium acylphosphinate photoinitiator in an aqueous solution) that result in oxidative removal of the SAM. The technique is attractive since it allows one to rapidly generate features less than 200 nm in diameter, and the metal-free tips afford more than 10-fold higher intensity than the tips with nanoapertures over a micrometer propagation length. In principle, this mask-free method can be utilized as a versatile tool for performing a wide variety of photochemistries across multiple scales that may be important in high-throughput combinatorial screening applications related to chemistry, biology, and materials science.

Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells

ABSTRACTPhotopolymerization techniques have been widely used to create hydrogels for biomedical applications. Visible light‐based photopolymerizations are commonly initiated by type II (i.e., noncleavage‐type) photoinitiator in conjunction with a coinitiator. On the other hand, type I photoinitiators (i.e., cleavage type) are rarely compatible with visible light‐based initiation due to their limited molar absorbability in the visible light wavelengths. Here, we report visible light initiated orthogonal photoclick crosslinking to fabricate gelatin‐norbornene and poly(ethylene glycol)‐tetra‐thiol hydrogels using either cleavage‐type (i.e., lithium acylphosphinate, LAP) or noncleavage‐type photoinitiator (i.e., eosin‐Y, EY) without the use of a coinitiator. Regardless of the initiator type, the step‐growth gelatin‐PEG hybrid hydrogels crosslinked and degraded similarly. While both systems exhibited similar cytocompatibility for hepatic progenitor HepaRG cells, gelation initiated by noncleavage‐type initiator EY afforded slightly higher degree of hepatic gene expression. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44585.

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol−ene and thiol−yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol−X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol−ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol−ene and thiol−yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol−X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol−ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

Spatially controlled photo-patterning of multi-material bioactive hydrogels

Event Abstract Back to Event Spatially controlled photo-patterning of multi-material bioactive hydrogels Bagrat Grigoryan1*, Jonathon D. Roybal2, Paul T. Greenfield1, Alexander J. Zaita1, Samantha J. Paulsen1, Jacob L. Albritton1, Anderson Ta1, Don L. Gibbons2, 3 and Jordan S. Miller1 1 Rice University, Bioengineering, United States 2 The University of Texas M.D. Anderson Cancer Center, Thoracic/Head and Neck Medical Oncology, United States 3 The University of Texas, MD Anderson Cancer Center, Molecular and Cellular Oncology, United States Introduction: While fabrication of simple microenvironments to study cell fate and function has been achieved using soft lithography and photolithography techniques, mimicking the complex, heterogeneous patterns of native tissue in vitro remains challenging due to lack of available simple, automated patterning technologies[1]. To this end, we developed a stereolithography (SLA)-based 3D printer for automated fabrication of hydrogels containing microstructures composed of multi-materials for applications towards understanding the lung tumor microenvironment. Materials and Methods: We developed and characterized our custom SLA-based 3D printer to spatially control the patterning of multiple materials within a single layer and across successive layers. We performed photorheological characterization of prepolymer formulations containing poly(ethylene glycol) diacrylate (PEGDA) and lithium acylphosphinate (LAP) photoinitiator to quantitatively understand the gelation kinetics and modulus of a single layer of hydrogel. Patterned entrapped fluorophore molecules facilitated characterization of the fidelity of multi-material patterns. To demonstrate biocompatibility of our technology, we added hMSCs into the prepolymer solution and performed LIVE/DEAD staining of printed cell-laden hydrogels at various time points. We further demonstrate the biocompatibility of our technology by adding a murine metastatic lung adenocarcinoma cell line, 344SQ, into the prepolymer solution to fabricate bioactive PEG-based gels containing cell-adhesive CGRGDS ligand and MMP-sensitive peptides incorporated into the polymer backbone[2]. We investigated cell viability and function by monitoring the innate ability of 344SQ cells to form into aggregates and lumenize over time in the proper microenvironment. Results and Discussion: The photorheological assay provided us with information on the exposure duration needed for a certain layer thickness as a function of PEG molecular weight, PEG concentration, and LAP concentration in the prepolymer formulation for reproducible fabrication of hydrogels (Figure 1A). Fluorescent microscopy images demonstrated fabrication of features as small as 300µm in diameter as well as precise spatial control in photo-patterning of multiple materials within the same plane (Figure 1B). Encapsulation of cells in bioactive PEG-based gels demonstrated cell viability greater than 90% after 2 days of culture (Figure 1C-Top). Encapsulated 344SQ cells retained their biological activity by forming into aggregates and lumenizing throughout the 2-week long culture (Figure 1C-Bottom). Conclusion: We have developed an open-source SLA-based 3D printer that enables us to automatically fabricate hydrogels containing bioactive microstructures with spatial control of multiple materials at a medium-high throughput. We utilized this technology to encapsulate hMSCs and metastatic cells in PEG-based hydrogels with high cell viability and function. We believe the automated nature of our developed system will enable researchers to recapitulate intricate microenvironments for studying complex cellular interactions in vitro without facing difficulties associated with precisely aligning masks with traditional photolithographic techniques. Thanks to the Open Source Hardware Association, RepRap.org, and related projects and companies that support worldwide, open standardization of 3D printing. This work was supported in part by NSF Graduate Research Fellowship (BG).

Synthesis of MMP sensor microgel for spatial and temporal monitoring of MMP activities

Event Abstract Back to Event Synthesis of MMP sensor microgel for spatial and temporal monitoring of MMP activities Della S. Shin1, Emi Y. Tokuda1 and Kristi S. Anseth1, 2, 3 1 University of Colorado, Department of Chemical and Biological Engineering, United States 2 BioFrontiers Institute, United States 3 Howard Hughes Medical Institute, United States Statement of Purpose: Matrix Metalloproteinase (MMPs) are a group of endopeptidases that are known to be involved in various cellular processes related to migration, such as invasion by cancer cells through remodeling of their surrounding matrix [1]. Here, we developed a method to monitor MMP activity in real time at the site of action using a microgel sensor which contains covalently coupled MMP degradable peptides with FRET fluorophore-quencher pairs at each end. With this sensor, it is possible to measure MMP activity in the range of 10 ng/ml to 50 µg/ml in real time over 100 min and analyze spatially detailed MMP activity of melanoma cancer cells. For 4-dimensional monitoring of MMPs inside the artificial extracellular matrix (ECM) (3-D space and time), the size of the microgels was selected to be over 10 µm to efficiently evade uptake by the cells and prevent rapid diffusion into the artificial ECM. This MMP activity monitoring microgel sensor can potentially serve as a platform for not only studying cancer cells, but also for monitoring cell-ECM interaction and migration inside various ECM mimicking scaffolds for tissue regeneration. Methods: A quenched fluorigenic peptide(Dabcyl-GGPQG↓IWGQK-Fluorescein-AhxC) was prepared as reported previously [2]. Briefly, a fluorophore, fluorescein and a quencher, dabcyl were both covalently functionalized at the end of MMP degradable peptide substrate, GPQG↑IWGQ (where ↑ denotes the cleavage site). Microgels were prepared using an 8-arm poly (ethylene glycol) (20 mM PEG) end functionalized with norbornene, crosslinked by dithiothreitol (36 mM DTT) through a photo-initiated (10 mM lithium acylphosphinate (LAP)) polymerization. The sensor peptide was conjugated as a pendant group (4 mM). As a control, fluorescence change was monitored in response to various collagenase solutions with an automated Operetta confocal microscopy assay. Microgel solutions were exposed to each target collagenase concentration, and the fluorescence was taken automatically every 5 minutes. For 3-D monitoring of collagenase activity, microgels were encapsulated in a matrix-mimicking hydrogel. Imaging from the bottom to top of this hydrogel gave spatial information related to the collagenase activity. Results: Microgels were synthesized and their size was characterized based on their fluorescence image. The averaged diameter of the synthesized microgels was 40 ± 5 μm or 28 ± 5 μm each when the starting monomer was 5kDa-4-arm or 10kDa 8-arm PEG-norbornene, respectively. Figure 1. (a) Fluorescence microscope image and schematic illustration of the MMP sensitive microgel before and after addition of collagenase solution which contains MMPs. (b) Fluorescence change (F/F0) after addition of various concentration of collagenase solution. Next, the fluorescence changes in the microgels was monitored during exposure to various concentration of a collagenase solution. Before exposure to the collagenase solution, the fluoresein in the microgel was quenched by dabcyl and showed only low background levels. After cleavage by MMP, the quencher molecules diffused out from the microgel, and the microgel showed higher fluorescence as shown in Figure 1-(a). This fluorescence change was dependent on the concentration of collagenase in the solution and the time of exposure to collagenase solution (Fig 1-(b)). Current research is underway to examine local changes in MMP-activity in the presence of cell-laden hydrogels. This work was supported in part by the the Howard Hughes Medical Institute and grants from the National Institutes of Health (5R21EB018505-02).

Three dimensional live cell lithography

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.

Photodegradable, Photoadaptable Hydrogels via Radical-Mediated Disulfide Fragmentation Reaction

Various techniques have been adopted to impart a biological responsiveness to synthetic hydrogels for the delivery of therapeutic agents as well as the study and manipulation of biological processes and tissue development. Such techniques and materials include polyelectrolyte gels that swell and deswell with changes in pH, thermosensitive gels that contract at physiological temperatures, and peptide cross-linked hydrogels that degrade upon peptidolysis by cell-secreted enzymes. Herein we report a unique approach to photochemically deform and degrade disulfide cross-linked hydrogels, mitigating the challenges of light attenuation and low quantum yield, permitting the degradation of hydrogels up to 2 mm thick within 120 s at low light intensities (10 mW/cm2 at 365 nm). Hydrogels were formed by the oxidation of thiol-functionalized 4-armed poly(ethylene glycol) macromolecules. These disulfide cross-linked hydrogels were then swollen in a lithium acylphosphinate photoinitiator solution. Upon exposure to light, photogenerated radicals initiate multiple fragmentation and disulfide exchange reactions, permitting and promoting photodeformation, photowelding, and photodegradation. This novel, but simple, approach to generate photoadaptable hydrogels portends the study of cellular response to mechanically and topographically dynamic substrates as well as novel encapsulations by the welding of solid substrates. The principles and techniques described herein hold implications for more than hydrogel materials but also for photoadaptable polymers more generally.

Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility

Due to mild reaction conditions and temporal and spatial control over material formation, photopolymerization has become a valuable technique for the encapsulation of living cells in three dimensional, hydrated, biomimetic materials. For such applications, 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (I2959) is the most commonly used photoinitiator (by virtue of its moderate water solubility), yet this initiator has an absorption spectrum that is poorly matched with wavelengths of light generally regarded as benign to living cells, limiting the rate at which it may initiate polymerization in their presence. In contrast, acylphosphine oxide photoinitiators, generally exhibit absorption spectra at wavelengths suitable for cell encapsulation, yet commercially available initiators of this class have low water solubility. Here, a water soluble lithium acylphosphinate salt is evaluated for its ability to polymerize diacrylated poly(ethylene glycol) (PEGDA) monomers rapidly into hydrogels, while maintaining high viability during direct encapsulation of cells. Through rheometric measurements, the time to reach gelation of a PEGDA solution with the phosphinate initiator is one tenth the time for that using I2959 at similar concentrations, when exposed to 365 nm light. Further, polymerization with the phosphinate initiator at 405 nm visible light exposure is achieved with low initiator concentrations and light intensities, precluded in polymerizations initiated with I2959 by its absorbance profile. When examined 24 h after encapsulation, survival rates of human neonatal fibroblasts encapsulated in hydrogels polymerized with the phosphinate initiator exceed 95%, demonstrating the cytocompatibility of this initiating system.