Identification of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffolds as potent Lck inhibitors as anti-cancer agents.

Lymphocyte-specific protein tyrosine kinase (Lck) plays vital roles in the T-cell receptor- mediated development, function, and differentiation of T-cells. Given its substantial involvement in T cell signaling, irregularities in the expression and functionality of Lck may lead to various diseases, including cancer. In this study, we found that compound 12a exerted significant inhibitory potency against Lck with an IC50 value of 10.6 nM. In addition, 12a demonstrated high efficacy in various colon cancer cell lines as indicated by GI50 values ranging from 0.24 to 1.26 µM. Notably, 12a inhibited the phosphorylation of Lck in Colo201 cells. Overall, the anti-proliferative effects of 12a on diverse cancer cell lines highlights its potential application for the treatment of various cancer types.

Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration.

Molding processes with molds containing topographical structures have been used for fabrication of hydrogel and cryogel particles. However, they can involve difficulties in separation of fabricated particles with complex shape from the molds or repeated fabrication of the particles although the overall processes do not require much skill and equipment. In this study, molds with etched superhydrophobic patterns have been developed by etching polytetrafluoroethylene (PTFE) blocks in user-defined designs with a femtosecond (FS) laser-based etching system. Lyophilized cryogel particles with various designs and sizes were fabricated by molding precursors with these PTFE molds. Additionally, the clean and easy separation of particles from the molds allowed repeated fabrication of the particles. For an application, relatively 'big' gelatin-norbornene (GelNB) cryogel particles prepared via molding with polydimethylsiloxane (PDMS) molds, swelling in phosphate buffered saline (PBS) and slicing height in half and 'small' GelNB cryogel particles fabricated with the PTFE molds were fabricated. Then, they were used to study scaffold size effect on calvarial bone regeneration. The molds generated with the FS laser-based etching system can be useful for various applications that require the mass production of cryogel particles in various geometries.

Nanomaterials to improve cancer immunotherapy based on ex vivo engineered T cells and NK cells.

Recent clinical successes of chimeric antigen receptor (CAR) T cell therapy have led the booming of developments in cancer immunotherapy utilizing ex vivo engineered immune cells such as T cells and natural killer (NK) cells. However, a number of issues need to be resolved for this novel therapy to become widely applicable to cancer patients as current CAR-T cell therapies are only successful in treating some blood cancers, and economically not feasible for many patients. In this review, we describe various nanomaterial-based approaches developed to overcome current limitations in ex vivo engineered T/NK cells, along with key biological principles underlying each approach. First, nanomaterials developed to improve ex vivo expansion of T/NK cells and the basic principles of T/NK cell activation for designing nanomaterials are summarized. Second, nanomaterial-based gene delivery methods to generate genetically engineered T/NK cells are discussed with an emphasis on challenges in improving transfection efficacy. Third, nanomaterials loaded to T/NK cells to enhance their anti-tumor functions and to overcome tumor microenvironment are described with key biological characteristics of T/NK cells, which are essential for nanomaterial loading and drug release from the nanomaterials. In particular, we comment on similarities and differences of methods developed for T cells and NK cells based on the biological characteristics of each cell type.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFß) signaling. Initiation of the TGFß signaling cascade results from binding of TGFß to the labile type I TGFß receptor (TGFßRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFß signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFß1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFß1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Intrastromal Injection of Hyaluronidase Alters the Structural and Biomechanical Properties of the Corneal Stroma.

Purpose: Glycosaminoglycans (GAGs) are important components of the corneal stroma, and their spatiotemporal arrangement regulates the organization of collagen fibrils and maintains corneal transparency. This study was undertaken to determine the consequences of hyaluronidase (HAse) injected into the corneal stroma on stromal stiffness and ultrastructure. Methods: Equal volumes of HAse or balanced salt solution (vehicle) were injected intrastromally into the corneas of New Zealand white rabbits. Ophthalmic examination and multimodal imaging techniques, including Fourier-domain optical coherence tomography and in vivo confocal microscopy (IVCM), were performed at multiple time points to evaluate the impact of HAse treatment in vivo. Atomic force microscopy and transmission electron microscopy (TEM) were used to measure corneal stiffness and collagen's interfibrillar spacing, respectively. Results: Central corneal thickness progressively decreased after HAse injection, reaching its lowest value at day 7, and then returned to normal by day 42. The HAse did not impact the corneal endothelium but transiently altered keratocyte morphology at days 1 and 7, as measured by IVCM. HAse-injected corneas became stiffer by day 1 postinjection, were stiffest at day 7, and returned to preinjection values by day 90. Changes in stromal stiffness correlated with decreased interfibrillar spacing as measured by TEM. Conclusions: Degradation of GAGs by HAse decreases the corneal thickness and increases stromal stiffness through increased packing of the collagen fibrils in a time-dependent manner. Translational Relevance: Intrastromal HAse injection appears relatively safe in the normal cornea, but its impact on corneal biomechanics and structure under pathologic conditions requires further study.