Zengping Liu

Intraocular pharmacokinetics of ranibizumab following a single intravitreal injection in humans

PURPOSE To investigate intraocular concentrations and pharmacokinetics of ranibizumab after a single intravitreal injection in humans. DESIGN Prospective, noncomparative, interventional case series. METHODS We included 18 nonvitrectomized eyes of 18 patients (age range, 61-85 years) that were diagnosed with both clinically significant cataract and macular edema secondary to either exudative age-related macular degeneration, diabetic maculopathy, or retinal vein occlusion. Each eye received a single intravitreal injection of 0.5 mg ranibizumab. An aqueous humor sample was obtained during cataract surgery between 1 and 37 days after injection. Concentrations of unbound ranibizumab in these samples were quantified by enzyme-linked immunosorbent assay. RESULTS Ranibizumab concentration in aqueous humor peaked the first day after injection (range, 36.9-66.1 μg/mL) and subsequently declined in a mono-exponential fashion. Nonlinear regression analysis determined an initial peak concentration (c(max)) of 56.1 μg/mL and an elimination half-life (t(1/2)) of 7.19 days with a coefficient of determination (R(2)) of 0.90. Correction of ranibizumab concentrations for ocular volume as calculated from axial length measurements did not alter regression analysis results significantly (t(1/2), 7.15 days; R(2), 0.89). CONCLUSIONS In human nonvitrectomized eyes, the aqueous half-life of 0.5 mg intravitreally injected ranibizumab is 7.19 days, slightly shorter than the half-life of 9.82 days previously determined for bevacizumab by comparable methods.

Human RPE stem cells grown into polarized RPE monolayers on a polyester matrix are maintained after grafting into rabbit subretinal space.

Summary Transplantation of the retinal pigment epithelium (RPE) is being developed as a cell-replacement therapy for age-related macular degeneration. Human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC)-derived RPE are currently translating toward clinic. We introduce the adult human RPE stem cell (hRPESC) as an alternative RPE source. Polarized monolayers of adult hRPESC-derived RPE grown on polyester (PET) membranes had near-native characteristics. Trephined pieces of RPE monolayers on PET were transplanted subretinally in the rabbit, a large-eyed animal model. After 4 days, retinal edema was observed above the implant, detected by spectral domain optical coherence tomography (SD-OCT) and fundoscopy. At 1 week, retinal atrophy overlying the fetal or adult transplant was observed, remaining stable thereafter. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. Taken together, the xeno-RPE survived with retained characteristics in the subretinal space. These experiments support that adult hRPESC-derived RPE are a potential source for transplantation therapies.

Enhancement of retinal pigment epithelial culture characteristics and subretinal space tolerance of scaffolds with 200 nm fiber topography

Tissue engineered retinal pigment epithelial (RPE) transplantation is a promising cell-based therapy for age-related macular degeneration. The aim of this work is to develop a supportive scaffold with a favorable topography to aid functional RPE monolayer maintenance while being tolerated underneath the retina. To this end, films and electrospun substrates with fiber diameters ranging from 200 to 1000 nm were made of polyethylene terephthalate or poly(L-lactide-co-ε-caprolactone), and then tested using human fetal RPE cells in vitro and transplanted subretinally in rabbits. The results indicated that RPE on both 200 nm fiber variants showed the highest cell densities, adherent monolayers achieved deeper pigmentation, and more uniform hexagonal tight junctions. Facile subretinal implantation of flat 200 nm fiber membranes was achieved by electrospinning them onto a porous rigid-elastic carrier. Spectral-domain optical coherence tomography showed a reattached, slightly thinned retina overlying the implants over 2 weeks observation. Histology demonstrated native RPE variably migrated onto the nanofibers, and a reactive gliosis with some photoreceptor degeneration. In conclusion, scaffolds with 200 nm fiber topography enhanced RPE culture, showed subretinal biocompatibility, and should thus be considered for future cell-based therapies in blinding retinal diseases.

Release And Velocity Of Micronized Dexamethasone Implants With An Intravitreal Drug Delivery System: Kinematic Analysis With a High-speed Camera

Purpose: Ozurdex, a novel dexamethasone (DEX) implant, is released by a drug delivery system into the vitreous cavity. We analyzed the mechanical release aperture of the novel applicator, obtained real-time recordings using a high-speed camera system and performed kinematic analysis of the DEX application. Design: Experimental study. Methods: The application of intravitreal DEX implants (6 mm length, 0.46 mm diameter; 700 &mgr;g DEX mass, 0.0012 g total implant mass) was recorded by a high-speed camera (500 frames per second) in water (Group A: n = 7) or vitreous (Group B: n = 7) filled tanks. Kinematic analysis calculated the initial muzzle velocity as well as the impact on the retinal surface at approximately 15 mm of the injected drug delivery system implant in both groups. A series of drug delivery system implant positions was obtained and graphically plotted over time. Results: High-speed real-time recordings revealed that the entire movement of the DEX implant lasted between 28 milliseconds and 55 milliseconds in Group A and 1 millisecond and 7 milliseconds in Group B. The implants moved with a mean muzzle velocity of 820 ± 350 mm/s (±SD, range, 326–1,349 mm/s) in Group A and 817 ± 307 mm/s (±SD, range, 373–1,185 mm/s) in Group B. In both groups, the implant gradually decelerated because of drag force. With greater distances, the velocity of the DEX implant decreased exponentially to a complete stop at 13.9 mm to 24.7 mm in Group A and at 6.4 mm to 8.0 mm in Group B. Five DEX implants in Group A reached a total distance of more than 15 mm, and their calculated mean velocity at a retinal impact of 15 mm was 408 ± 145 mm/s (±SD, range, 322–667 mm/s), and the consecutive normalized energy was 0.55 ± 0.44 J/m2 (±SD). In Group B, none of the DEX implants reached a total distance of 6 mm or more. An accidental application at an angle of 30 grade and consecutively reduced distance of approximately 6 mm may result in a mean velocity of 844 and mean normalized energy of 0.15 J/m2 (SD ± 0.47) in a water-filled eye. Conclusion: The muzzle velocity of DEX implants is approximately 0.8 m/s and decreases exponentially over distance. The drag over time in vitreous is faster than in water. The calculated retinal impact energy does not reach reported damage levels for direct foreign bodies or other projectiles.

A Nanofibrillar Surface Promotes Superior Growth Characteristics in Cultured Human Retinal Pigment Epithelium

Background: To evaluate the influence of surface topography on the proliferation of the retinal pigment epithelium (RPE) by comparing nanofibrillar and smooth substrates. Methods: Electrospun polyamide nanofibers (EPN) are an engineered surface mimicking native basement membranes. Commonly used plastic (polystyrene, PS) and glass substrates have a smooth topography. All were analyzed by scanning electron microscopy. RPE cultures were established from fetal and adult donors. Growth curves were established on the above substrates. Cell cycle and growth fractions were analyzed with 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI). Results: At a magnification of ×5,000, EPN showed randomly overlapping fibers and pores. The surface of glass was slightly studded yet regular, in contrast to ideally smooth PS. Polygonal cells grew on nanofibers in a colony-like distribution, while randomly spread spindle-shaped cell morphologies were seen on smooth surfaces. This was observed at all donor ages. Initial proliferation rates were higher on EPN, and similar final cell densities were reached in all age groups, compared to an age-related decline on PS. EdU/DAPI revealed faster cell cycles on EPN. Growth fractions were higher and maintained longer on EPN. Observed substrate differences in growth behavior were statistically significant. Conclusion: Surface topography appears to induce distinct RPE proliferation characteristics.

Routes for Drug Delivery to the Eye and Retina: Intravitreal Injections

The advantage of intravitreal injections is an immediate and increased therapeutic effect in the intended retinal tissue. The accuracy, precision and reproducibility of the delivered volume depend on the size of the syringe and the physicians manual experience. The eyelids and eyelashes are usually disinfected using a povidone-iodine solution (10%); a sterile speculum is placed and drops of povidone-iodine (5%) are applied. The use of adequate anesthetic topical lidocaine 2% is required. The injection site should be located 3.5-4 mm posterior to the limbus. The angle of the incision through the sclera may be directed in an oblique fashion of 30°. The diameter of the needle should be smaller than 25 G, and the injected volume should be limited to 0.15 ml without a routine paracentesis. The incidence of lens injury is 0.006% (2/32,318) and 0.013% (5/35,942) for rhegmatogenous retinal detachments. The rate of suspected endophthalmitis is 0.018% after bevacizumab and 0.027% after ranibizumab injections. Sterile inflammations have been observed after Avastin injections. The concentrations of vascular endothelial growth factor inhibitors decline in a monoexponential fashion. The half-life of unbound bevacizumab is 9.82 days and that of ranibizumab 7.19 days.

Geometry, penetration force, and cutting profile of different 23-gauge trocars systems for pars plana vitrectomy.

Background: To investigate the geometry, penetration force, and cutting profile of 23-gauge trocar systems for pars plana vitrectomy based on their grinding methods in a standardized laboratory setting. Methods: In this experimental study, Eleven different commercially available 23-gauge sclerotomy trocar systems were divided into 4 groups according to their needle grinding and deburring: “back” bevel, “spear” bevel, “lancet” bevel, and “spatula” bevel. The normative geometrical data of the trocar systems were systematically analyzed according to nomenclature ISO 7864 and ISO 9626. Force to penetrate a 0.4-mm thick polyurethane foil was measured by a Penetrometer, when the trocar needle was piercing, cutting, and sliding through the foil at different defined loading phases and plotted as a load–displacement diagram. Magnified images of the consecutive cut were taken under a microscope after the entire penetration through the foil. Three physicians used all trocar systems in a masked fashion on human sclera to evaluate the manual penetration force in 30° and 90°. Results: The mean outer diameter of the trocar systems was 0.630 ± 0.009 mm, and the mean outer diameter of the trocars was 0.750 ± 0.013 mm. The mean point length was 3.11 ± 0.49 mm, and the mean length of the bevel was 1.46 ± 0.23 mm. The primary bevel angle was 10.75 ± 0.41°, and the secondary bevel angle was 65.9 ± 42.56°. The piercing forces of the back bevel and spear-pointed trocars/needles were at the same level (0.087 ± 0.028 N). The lancet-pointed needle had remarkable low piercing and cutting forces with 0.41 N (range, 0.35–0.47 N). The spatula bevel tip showed the highest penetration piercing force with 1.6 N (range, 1.59–1.73 N). The back bevel systems induced frequently triangular-shaped incisions, with two nearly rectangular cuts of short length. The spear bevels produced a regular characteristic linear cut. Especially, the lancet blade created straight cut with a linear wound apposition. Spatula trocar systems caused usually an arched accurate incision. The manual force to penetrate the human sclera in an angled and rectangular angle appeared in the surgeons hand lower with a back bevel, lancet, or spear tip, whereas higher with spatula bevel systems. Conclusion: Lancet and back bevel systems show less penetration force of inner needles than the spatula systems. The results of the penetration force experiments correlated well with the manual force on sclera.

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision. Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology. The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Indocyanine green concentrations used in chromovitrectomy cause a reversible functional alteration in the outer blood–retinal barrier

To assess tight junction integrity in cultured human foetal retinal pigment epithelium (RPE) after exposure to clinically relevant indocyanine green (ICG) concentrations.

Effect of Novel Vital Dyes on Outer Blood-Retina Barrier Function in Cultured Human Retinal Pigment Epithelium

Purpose: To assess tight junction (TJ) integrity in cultured human fetal retinal pigment epithelium (HFRPE) after exposure to clinically relevant novel vital dyes. Methods: HFRPE floater cells were harvested from RPE primary cultures of 4 donor eyes and seeded on polyester Transwell® for 4-6 weeks. The apical compartments of well-differentiated cultures were exposed to 0.005 mg/ml Coomassie violet R200 (CVR), methyl 2B (M2B) or Orange II. Periods of 30-300 s were chosen to mimic surgical exposure times, while 3 h was used for toxicity assays, with subsequent washout. Cell-cell junctions were studied by immunofluorescence (zonula occludens-1, ZO-1). Transepithelial electrical resistance (TER) was measured regarding blood-retina barrier (BRB) function. Results: At 4-6 weeks after confluence, HFRPE had grown into pigmented hexagonal monolayers with stable TER values (451-1,520 Ω·cm2). After 300-second dye treatments, a continuous ZO-1 signal was detected in all vital dye-treated groups 1.5 h after exposure, whereas trypsin controls showed patchy loss of the TJ stain. TER of CVR-, M2B- and Orange-II-treated groups had dropped 1.5 h after exposure to 148 ± 58.4, 162 ± 23.7 and 164 ± 18.5 Ω·cm2, respectively, compared to 73 ± 44.9 Ω·cm2 in positive controls. After 3 h of exposure to 0.005 mg/ml vital dyes in thick drops, TER maintained similar levels to those prior to exposure (90.8 ± 4.7% of the original values, 93.8 ± 6.5 and 91.9 ± 3.6%, respectively), together with no difference from the vehicle controls (94.8 ± 6.6%). TER values recovered in all groups to prior levels within 3 days. Conclusion: Novel vital dyes (CVR, M2B and Orange II) caused no outer BRB function alteration.