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

Optical Power of the Isolated Human Crystalline Lens

PURPOSE. To characterize the age dependence of isolated human crystalline lens power and quantify the contributions of the lens surfaces and refractive index gradient. METHODS. Experiments were performed on 100 eyes of 73 donors (average 2.8 Ϯ 1.6 days postmortem) with an age range of 6 to 94 years. Lens power was measured with a modified commercial lensmeter or with an optical system based on the Scheiner principle. The radius of curvature and asphericity of the isolated lens surfaces were measured by shadow photography. For each lens, the contributions of the surfaces and the refractive index gradient to the measured lens power were calculated by using optical ray-tracing software. The age dependency of these refractive powers was assessed. RESULTS. The total refractive power and surface refractive power both showed a biphasic age dependency. The total power decreased at a rate of Ϫ0.41 D/y between ages 6 and 58.1, and increased at a rate of 0.33D/y between ages 58.1 and 82. The surface contribution decreased at a rate of Ϫ0.13 D/y between ages 6 and 55.2 and increased at a rate of 0.04 D/y between ages 55.2 and 94. The relative contribution of the surfaces increased by 0.17% per year. The equivalent refractive index also showed a biphasic age dependency with a decrease at a rate of Ϫ3.9 ϫ 10 Ϫ4 per year from ages 6 to 60.4 followed by a plateau. CONCLUSIONS. The lens power decreases with age, due mainly to a decrease in the contribution of the gradient. The use of a constant equivalent refractive index value to calculate lens power with the lens maker formula will underestimate the power of young lenses and overestimate the power of older lenses. (Invest Ophthalmol Vis Sci. 2008;49:2541-2548) DOI: 10.1167/iovs.07-1385 T he optical power of the crystalline lens is determined by the surface curvatures, the refractive index differences at the aqueous lens and lens vitreous interfaces, and the refractive index gradient distribution within the lens. 1 Studying the optical properties of the lens (i.e., optical power, refractive index distribution, and the surface refractive contributions) in vivo is difficult because of the position of the lens behind the cornea and pupil, as well as the distortions of the posterior lens surface caused by the lens refractive index gradient. Two approaches have been used to measure the lens power in vivo. In the first approach the curvatures of the lens surface and lens thickness are measured by phakometry and ultrasonic or optical biometry. The lens power is then calculated assuming an equivalent uniform refractive index (typically, ϳ1.42). 2,3 In the second approach, the lens power is calculated from measurements of axial eye length, anterior chamber depth, corneal power, and refractive state of the eye. These parameters are input into an eye model to calculate the power required for the lens to produce an optical system that matches the measurements. 3-6 Both techniques derive the lens power from measurements of other ocular parameters. Even though recent studies have cross-validated in vivo lens biometry techniques 9 -15 A comparison of in vivo -21 The isolated lens power has been shown to decrease with age

Atomic Force Microscopy Measurement of the Elastic Properties of the Lens

The goal of this project was to develop techniques and instrumentation to measure the elastic properties of the lens and lens capsule in situ and their changes with age using Atomic Force Microscopy (AFM). The studies include the construction, characterization, and calibration of laboratory-based Atomic Force Microscope (AFM) to measure mechanical properties of ophthalmic tissues. Atomic Force Microscopy is a nanoscale imaging technique that has been applied to mechanical property measurement through nanoindentation. Young\u27s modulus of elasticity is determined by monitoring the cantilever deflections when it contacts the sample. The studies also include the development of tissue preparation techniques to enable measurement of the lens elasticity using AFM. This study found that lens capsule elasticity decreases with age, outer lens cortex elasticity remains constant with age, and the inner lens cortex is stiffer than the outer lens cortex. The effect of the changing biometry and mechanical properties with age was investigated by developing a mathematical model of accommodation. These changes will be the limiting factor to accommodative amplitude. Changes in lens capsule mechanical properties will affect the maximal accommodative amplitude in older eyes

Ex vivo accommodative responses in primates natural and polymer refilled lenses

Purpose: To assess the accommodation amplitude (AA) pre–post lens capsule refilling using viscous fluids and 3 prototype crosslinked siloxane polymers. Methods: The change in dioptric power of postmortem lenses mounted in a lens stretching device (EVAS) was measured pre/post phaco–ersatz technique. Tissue specimens consisting of the lens, zonules, ciliary body and segmented sclera were subject to 2mm of radial stretching. The dioptric power of the lens was measured in the stretched and unstretched states using a Scheiner–based optical system. After measuring AA in the natural lens, lens extraction was performed through a continuous circular minicapsulorrhexis (0.8 to 1.3mm) by manual lens aspiration combined with phacoemulsification techniques. A miniature valve (MCV) was placed in the capsulorhexis to isolate the capsular bag which was refilled with biocompatible polymers and cured using a visible light source. Experiments were conducted on 11 rhesus, 9 cynomolgus and 2 baboon eyes (age range: 1 to 15 years; postmortem time 1 to 24hrs). 9 eyes were refilled with a 10kcS viscosity trimethyl–terminated polydimethylsiloxane (PDMS), 7 eyes with prototype Polymer #1, 2 eyes with Polymer #2 and 2 eyes with Polymer #3. Results: For a 2mm radial stretch, (AA) was 14.2±3D in natural lenses (n=22); 9.2±3.5D after refilling with PDMS (n=15); 6.9 ±3.2D after refilling with Polymer #1 and 5 ±3.6D post cure (n=7), 13.1± 0.6D after refilling with Polymer #2 and 8.9±2.6D post cure (n=2), 13.4±2D after refilling with Polymer #3 and 8.1±0.07 D post cure (n=2). Varying the viscosity from 5 to 100kcS had no statistical effect on the change in power during stretching. Overfilling the capsular bag was found to decrease the change in power by as much as 9D. Conclusions: Lens refilling using the Phaco–Ersatz procedure and a prototype polymer restored most of the initial accommodation in post–mortem primate lenses mounted in a lens stretcher

Noncontact optical measurement of lens capsule thickness ex vivo

Purpose: To design a non-contact optical system to measure lens capsule thickness in cadaver eyes. Methods: The optical system uses a 670nm laser beam delivered to a single-mode fiber coupler. The output of the fiber coupler is focused onto the tissue using an aspheric lens (NA=0.68) mounted on a motorized translation stage. Light reflected from the sample is collected by the fiber coupler and sent to a silicon photodiode connected to a power meter. Peaks in the power signal are detected when the focal point of the aspheric lens coincides with the capsule boundaries. The capsule thickness is proportional to the distance between successive peaks. Anterior and posterior lens capsule thickness measurements were performed on 13 human, 10 monkey, and 34 New Zealand white rabbit lenses. The cadaver eyes were prepared for optical measurements by bonding a PMMA ring on the sclera. The posterior pole was sectioned, excess vitreous was removed, and the eye was placed on a Teflon slide. The cornea and iris were then sectioned. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Results: Central anterior lens capsule thickness was 9.4±2.9 m (human), 11.2±6.6 m (monkey), and 10.3±3.6 m (rabbit) optically and 14.9±1.6 m (human), 17.7±4.9 m (monkey), and 12.6±2.3 m (rabbit) histologically. The values for the central posterior capsule were 9.4±2.9 m (human), 6.6±2.5 m (monkey), and 7.9±2.3 m (rabbit) optically and 4.6±1.4 m (human), 4.5±1.2 m (monkey), and 5.7±1.7 m (rabbit) histologically. Conclusions: This study demonstrates that a non-contact optical system can successfully measure lens capsule thickness in cadaver eyes

Non-contact optical measurement of lens capsule thickness during simulated accommodation

Purpose: To non-invasively measure the thickness of the anterior and posterior lens capsule, and to determine if it significantly changes during accommodation. Methods: Anterior and posterior capsule thickness was measured on post-mortem lenses using a non-contact optical system using a focus-detection technique. The optical system uses a 670nm laser beam delivered to a single-mode fiber coupler. The output of the fiber coupler is focused on the tissue surface using an aspheric lens (NA=0.68) mounted on a translation stage with a motorized actuator. Light reflected from the sample surface is collected by the fiber coupler and sent to a photoreceiver connected to a computer-controlled data acquisition system. Optical intensity peaks are detected when the aspheric lens is focused on the capsule boundaries. The capsule thickness is equal to the distance traveled between two peaks multiplied by the capsule refractive index. Anterior and posterior lens capsule thickness measurements were performed on 18 cynomolgus (age average: 6±1 years, range: 4-7 years) eyes, 1 rhesus (age: 2 years) eye, and 12 human (age average: 65±16, range: 47-92) eyes during simulated accommodation. The mounted sample was placed under the focusing objective of the optical system so that the light was incident on the center pole. Measurements were taken of the anterior lens capsule in the unstretched and the stretched 5mm states. The lens was flipped, and the same procedure was performed for the posterior lens capsule. Results: The precision of the optical system was determined to be ±0.5um. The resolution is 4um and the sensitivity is 52dB. The human anterior lens capsule thickness was 6.0±1.2um unstretched and 4.9±0.9um stretched (p=0.008). The human posterior lens capsule was 5.7±1.2um unstretched and 5.7±1.4um stretched (p=0.974). The monkey anterior lens capsule thickness was 5.9±1.9um unstretched and 4.8±1.0um stretched (p=0.002). The monkey posterior lens capsule was 5.9±2.0um unstretched and 5.1±1.3um stretched (p=0.128). Conclusions: The results indicate that the primate anterior lens capsule thickness changes during accommodation

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS)

Biometry of primate lenses during immersion in preservation media

The purpose of this study was to assess the condition of human lenses (obtained from an eye bank) and of fresh monkey lenses, and to determine the effects of maintaining these lenses in various liquid preservation media. Freshly excised human and monkey lenses were maintained for 5 h in one of four solutions (Balanced Saline Solution [BSS], Ringer's Solution, Dulbecco's Modified Eagle Medium with Ham's F-12 [DMEM/F-12/F-12], and Tissue Culture Medium 199 [TC-199]) using a custom-designed, temperature-regulated testing cell. A modified optical comparator and digital camera were used to photograph magnified lens profiles and measure lens diameter and thickness. Lens volume was then calculated assuming rotational symmetry about the optical axis. Seven of the 33 human lenses exhibited extensive swelling and separation of the capsule from the lens cell mass prior to the incubation. During incubation, for 12/22 of the remaining human and 27/27 of the monkey lenses, thickness increased by 1.0-1.8%, diameter decreased by 0.7-1.6% and the volume was essentially unchanged. Substantial swelling and capsular separation were observed in 10 of the 22 human lenses, 7/10 for those maintained in salt solutions, and 3/12 for those in tissue culture media. Lens volumes increased by an average of 6.8%, due to an 8.7% increase in the thickness, while the diameter decreased by 0.9%. These changes appeared to be independent of postmortem time and donor age. Culture media are more effective than simple salt solutions in maintaining lens physical integrity during short-term incubations. Substantial uptake of water, accompanied by separation of the capsule from the lens cell mass, occurs at various stages during storage and experimental manipulations in >60% of human lenses obtained from the eye bank. Data obtained with such lenses will not be representative of the true ex vivo state. It is recommended that lenses be assessed to determine if swelling has taken place before acceptance of data

Crystalline lens MTF measurement during simulated accommodation

Purpose: To design and test an optical system to measure the optical quality of post mortem lenses during simulated accommodation. Methods: An optical bench top system was designed to measure the point spread function and calculate the modulation transfer function (MTF) of monkey and human ex-vivo crystalline lenses. The system consists of a super luminescent diode emitting at 850nm, collimated into a 3mm beam which is focused by the ex-vivo lens under test. The intensity distribution at the focus (point spread function) is re-imaged and magnified onto a beam profiler CCD camera. The optical quality in terms of spatial frequency response (modulation transfer function) is calculated by Fourier transform of the point spread function. The system was used on ex-vivo lenses with attached zonules, ciliary body and sclera. The sclera was glued to 8 separate PMMA segments and stretched radial by 5mm on an accommodation simulating lens stretching device. The point spread function was measured for each lens in the relaxed and stretched state for 5 human (ages 38-86 years) and 5 cynomolgus monkey (ages 53 - 67 months) fresh post mortem crystalline lenses. Results: Stretching induced measurable changes in the MTF. The cutoff frequency increased from 54.4±13.6 lp/mm unstretched to 59.5±21.4 lp/mm stretched in the post-presbyopic human and from 51.9±24.7 lp/mm unstretched to 57.7±18.5 lp/mm stretched cynomolgus monkey lenses. Conclusion: The results demonstrate the feasibility of measuring the optical quality of ex-vivo human and cynomolgus monkey lenses during simulated accommodation. Additional experiments are underway to quantify changes in optical quality induced by stretching