Porous Silicon Nanoparticles Embedded in Poly(lactic‐ co ‐glycolic acid) Nanofiber Scaffolds Deliver Neurotrophic Payloads to Enhance Neuronal Growth

Scaffolds made from biocompatible polymers provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. The inclusion of neurotrophic payloads in these scaffolds can substantially enhance regrowth and repair processes. However, many promising neurotrophic candidates are excluded from this approach due to incompatibilities with the polymer or with the polymer processing conditions. This work provides one solution to this problem by incorporating porous silicon nanoparticles (pSiNPs) that are pre-loaded with the therapeutic into a polymer scaffold during fabrication. The nanoparticle-drug-polymer hybrids are prepared in the form of oriented poly(lactic-co-glycolic acid) nanofiber scaffolds. We test three different therapeutic payloads: bpV(HOpic), a small molecule inhibitor of phosphatase and tensin homolog (PTEN); an RNA aptamer specific to tropomyosin-related kinase receptor type B (TrkB); and the protein nerve growth factor (NGF). Each therapeutic is loaded using a loading chemistry that is optimized to slow the rate of release of these water-soluble payloads. The drug-loaded pSiNP-nanofiber hybrids release approximately half of their TrkB aptamer, bpV(HOpic), or NGF payload in 2, 10, and >40 days, respectively. The nanofiber hybrids increase neurite extension relative to drug-free control nanofibers in a dorsal root ganglion explant assay

Development of Atomic Force Microscopy Lithography Techniques for Retinal Ganglion Cell Axon Guidance in an In Vitro Model of the Optic Nerve

For decades, the murine model has been the &ldquo;gold standard&rdquo; in retinal research; however, recent advances in retina-on-a-chip and retinal organoid technologies that lack many of the drawbacks of using mice are poised to supplement or even supplant the mouse in modeling human disease or evaluating prospective therapies. However, much work remains to be done in further developing these tools. To create better in vitro models, an atomic force microscope-based lithography device optimized for retinal tissue engineering was designed and constructed. High resolution computer controlled piezoelectric actuators were installed and programmed with control software written in IGOR Pro. The device was calibrated for resolution and functionality by patterning gold coated glass petri dishes and imaging them with scanning electron microscopy. Feature patterning modalities for guiding retinal ganglion cell axons were then developed, including patterning on soft gels. Finally, primary mouse retinal ganglion cells were seeded onto a patterned petri dish. It was shown that features patterned using this custom atomic force microscope-based device could be successfully created, and were compatible with neurons. These methods could potentially be very useful in the emerging field of retina-on-a-chip science.</p

Characterization of Trabecular Meshwork Mechanical Property Modulation After Application of Lipids

Treatment of lipids endogenous to the aqueous humor of the eye could serve as a potential therapy to slow the progression of glaucoma. Herein, we describe the method to treat trabecular meshwork samples in vitro with lipids and characterize changes in the samples' stiffness

Biomechanical properties of porcine meniscus as determined via AFM: Effect of region, compartment and anisotropy

The meniscus is a fibrocartilaginous tissue that plays an essential role in load transmission, lubrication, and stabilization of the knee. Loss of meniscus function, through degeneration or trauma, can lead to osteoarthritis in the underlying articular cartilage. To perform its crucial function, the meniscus extracellular matrix has a particular organization, including collagen fiber bundles running circumferentially, allowing the tissue to withstand tensile hoop stresses developed during axial loading. Given its critical role in preserving the health of the knee, better understanding structure-function relations of the biomechanical properties of the meniscus is critical. The main objective of this study was to measure the compressive modulus of porcine meniscus using Atomic Force Microscopy (AFM); the effects of three key factors were investigated: direction (axial, circumferential), compartment (medial, lateral) and region (inner, outer). Porcine menisci were prepared in 8 groups (= 2 directions x 2 compartments x 2 regions) with n = 9 per group. A custom AFM was used to obtain force-indentation curves, which were then curve-fit with the Hertz model to determine the tissue's compressive modulus. The compressive modulus ranged from 0.75 to 4.00 MPa across the 8 groups, with an averaged value of 2.04±0.86MPa. Only direction had a significant effect on meniscus compressive modulus (circumferential > axial, p = 0.024), in agreement with earlier studies demonstrating that mechanical properties in the tissue are anisotropic. This behavior is likely the result of the particular collagen fiber arrangement in the tissue and plays a key role in load transmission capability. This study provides important information on the micromechanical properties of the meniscus, which is crucial for understanding tissue pathophysiology, as well as for developing novel treatments for tissue repair

Age-dependency of molecular diffusion in the human anterior lens capsule assessed using fluorescence recovery after photobleaching

To quantify the partition coefficient and the diffusion coefficient of metal-carrier proteins in the human lens capsule as a function of age. Whole lenses from human donors were incubated overnight in a solution of fluorescently labeled transferrin, albumin, or ceruloplasmin. In the central plane of the capsule thickness, fluorescence recovery after photobleaching (FRAP) experiments were conducted to measure the diffusion of the protein within the lens capsule. The anterior portion of the lens was recorded before the FRAP experiments to locate the boundaries of the anterior lens capsule and to measure the partition coefficient of the labeled proteins. The partition coefficient (P), the time to half maximum recovery of the fluorescent intensity (τ ), and the diffusion coefficient (D) for each protein were analyzed as a function of donor age. There was no statistically significant relationship between the half maximum recovery time or the diffusion coefficient and age for transferrin (molecular weight [MW]=79.5 kDa, τ =17.26±4.840 s, D=0.17±0.05 μm /s), serum albumin (MW=66.5 kDa, τ =18.45±6.110 s, D=0.17±0.06 μm /s), or ceruloplasmin (MW=120 kDa, τ =36.57±5.660 s, D=0.08±0.01 μm /s). As expected, the larger protein (ceruloplasmin) took longer to recover fluorescent intensity due to its slower movement within the lens capsule. The partition coefficient statistically significantly increased with age for each protein (P : 0.09-0.71, P : 0.42-0.95, P : 0.19-1.17). The diffusion of heavy-metal protein carriers within the anterior lens capsule is not dependent on age, but it is dependent on the size of the protein. The permeability of the lens capsule to these heavy-metal protein carriers increases with age, suggesting that there will be a higher concentration of heavy metals in the older lens. This behavior may favor the formation of cataract, because heavy metals enhance protein oxidation through the Fenton reaction

Measuring the effects of postmortem time and age on mouse lens elasticity using atomic force microscopy

The mouse lens is frequently used both in vivo and ex vivo in ophthalmic research to model conditions affecting the human lens, such as presbyopia. The mouse lens has a delicate structure which is prone to damage and biomechanical changes both before and after extraction from the whole globe. When not properly controlled for, these changes can confound the biomechanical analysis of mouse lenses. In this study, atomic force microscopy microindentation was used to assess changes in the Young's Modulus of Elasticity of the mouse lens as a function of mouse age and postmortem time. Old mouse lenses measured immediately postmortem were significantly stiffer than young mouse lenses (p = 0.028). However, after 18 h of incubation, there was no measurable difference in lens stiffness between old and young mouse lenses (p = 0.997). This demonstrates the need for careful experimental control in experiments using the mouse lens, especially regarding postmortem time. •Long postmortem times can obscure changes in the stiffness of the mouse lens ex vivo.•Young mice lenses are less stiff than old lenses when measured immediately.•Young mice lenses and old mice lenses have comparable stiffness after incubation for 18 hours

Porous Silicon Nanoparticles Embedded in Poly(lactic‐ co

Scaffolds made from biocompatible polymers provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. The inclusion of neurotrophic payloads in these scaffolds can substantially enhance regrowth and repair processes. However, many promising neurotrophic candidates are excluded from this approach due to incompatibilities with the polymer or with the polymer processing conditions. This work provides one solution to this problem by incorporating porous silicon nanoparticles (pSiNPs) that are preloaded with the therapeutic into a polymer scaffold during fabrication. The nanoparticle- drug- polymer hybrids are prepared in the form of oriented poly(lactic- co- glycolic acid) nanofiber scaffolds. Three different therapeutic payloads are tested: bpV(HOpic), a small molecule inhibitor of phosphatase and tensin homolog (PTEN); an RNA aptamer specific to tropomyosin- related kinase receptor type B (TrkB); and the protein nerve growth factor (NGF). Each therapeutic is loaded using a loading chemistry that is optimized to slow the rate of release of these water- soluble payloads. The drug- loaded pSiNP- nanofiber hybrids release approximately half of their TrkB aptamer, bpV(HOpic), or NGF payload in 2, 10, and >40 days, respectively. The nanofiber hybrids increase neurite extension relative to drug- free control nanofibers in a dorsal root ganglion explant assay.Porous silicon nanoparticles are loaded with bpV(HOpic), a tropomyosin- related kinase receptor type B RNA aptamer, or nerve growth factor using three distinct loading chemistries. They are incorporated into aligned poly(lactic- co- glycolic acid) nanofibers using an airbrush, and the nanofiber hybrids release their payloads over varying timescales. The three released payloads maintain their bioactivity as shown by enhanced neurite extension of dorsal root ganglion explants cultured on the hybrid nanofiber scaffolds.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155880/1/adfm202002560.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155880/2/adfm202002560_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155880/3/adfm202002560-sup-0001-SuppMat.pd

Effect of compound treatments on mouse lens viscoelasticity

Previous studies have shown that pharmaceutical agents such as lipoic acid have the ability to soften the lens, presenting a promising avenue for treating presbyopia. One obstacle encountered in the preclinical stage of such agents is the need for precise measurements of lens elasticity in experimental models. This study aimed to evaluate the effects of 25-hydroxycholesterol, lipoic acid, and obeticholic acid on the viscoelastic properties of mouse lenses using a custom-built elastometer system. Data were acquired on lenses from C57BL/6J female mice from two age groups: young (age: 8-10 weeks) and old (age: 32-43 weeks). OD lenses were used as the control and OS lenses were treated. Control lenses were immersed in Dulbecco's Modified Eagle Medium (DMEM) and treatment lenses were immersed in a compound solution containing 25-hydroxycholesterol (5 young and 5 old), lipoic acid at 2.35 mM (5 young and 5 old), lipoic acid at 0.66 mM (5 old), or obeticholic acid (5 old) at 37ºC for 18 hours. After treatment, the mouse lenses were placed in a DMEM-filled chamber within a custom-built elastometer system that recorded the load and lens shape as the lens was compressed by 600 μm at a speed of 50 μm/s. The load was continuously recorded during compression and during stress-relaxation. The compression phase was fit with a linear function to quantify lens stiffness. The stress-relaxation phase was fit with a 3-term exponential relaxation model providing relaxation time constants (t1, t2, t3), and equilibrium load. The lens stiffness, time constants and equilibrium load were compared for the control and treated groups. Results revealed an increase in stiffness with age for the control group (young: 1.16 ± 0.11 g/mm, old: 1.29 ± 0.14 g/mm) and relaxation time constants decreased with age (young: t1 = 221.9 ± 29.0 s, t2 = 24.7 ± 3.8 s, t3 = 3.12 ± 0.87 s, old: t1 = 183.0 ± 22.0 s, t2 = 20.6 ± 2.6 s and t3 = 2.24 ± 0.43 s). Among the compounds tested, only 25-hydroxycholesterol produced statistically significant changes in the lens stiffness, relaxation time constants, and equilibrium load. In conclusion, older mouse lenses are stiffer and less viscous than young mouse lenses. Notably, no significant change in lens stiffness was observed following treatment with lipoic acid, contrary to previous findings.Previous studies have shown that pharmaceutical agents such as lipoic acid have the ability to soften the lens, presenting a promising avenue for treating presbyopia. One obstacle encountered in the preclinical stage of such agents is the need for precise measurements of lens elasticity in experimental models. This study aimed to evaluate the effects of 25-hydroxycholesterol, lipoic acid, and obeticholic acid on the viscoelastic properties of mouse lenses using a custom-built elastometer system. Data were acquired on lenses from C57BL/6J female mice from two age groups: young (age: 8-10 weeks) and old (age: 32-43 weeks). OD lenses were used as the control and OS lenses were treated. Control lenses were immersed in Dulbecco's Modified Eagle Medium (DMEM) and treatment lenses were immersed in a compound solution containing 25-hydroxycholesterol (5 young and 5 old), lipoic acid at 2.35 mM (5 young and 5 old), lipoic acid at 0.66 mM (5 old), or obeticholic acid (5 old) at 37ºC for 18 hours. After treatment, the mouse lenses were placed in a DMEM-filled chamber within a custom-built elastometer system that recorded the load and lens shape as the lens was compressed by 600 μm at a speed of 50 μm/s. The load was continuously recorded during compression and during stress-relaxation. The compression phase was fit with a linear function to quantify lens stiffness. The stress-relaxation phase was fit with a 3-term exponential relaxation model providing relaxation time constants (t1, t2, t3), and equilibrium load. The lens stiffness, time constants and equilibrium load were compared for the control and treated groups. Results revealed an increase in stiffness with age for the control group (young: 1.16 ± 0.11 g/mm, old: 1.29 ± 0.14 g/mm) and relaxation time constants decreased with age (young: t1 = 221.9 ± 29.0 s, t2 = 24.7 ± 3.8 s, t3 = 3.12 ± 0.87 s, old: t1 = 183.0 ± 22.0 s, t2 = 20.6 ± 2.6 s and t3 = 2.24 ± 0.43 s). Among the compounds tested, only 25-hydroxycholesterol produced statistically significant changes in the lens stiffness, relaxation time constants, and equilibrium load. In conclusion, older mouse lenses are stiffer and less viscous than young mouse lenses. Notably, no significant change in lens stiffness was observed following treatment with lipoic acid, contrary to previous findings