Uday B. Kompella

Nanomedicines for back of the eye drug delivery, gene delivery, and imaging

Treatment and management of diseases of the posterior segment of the eye such as diabetic retinopathy, retinoblastoma, retinitis pigmentosa, and choroidal neovascularization is a challenging task due to the anatomy and physiology of ocular barriers. For instance, traditional routes of drug delivery for therapeutic treatment are hindered by poor intraocular penetration and/or rapid ocular elimination. One possible approach to improve ocular therapy is to employ nanotechnology. Nanomedicines, products of nanotechnology, having at least one dimension in the nanoscale include nanoparticles, micelles, nanotubes, and dendrimers, with and without targeting ligands. Nanomedicines are making a significant impact in the fields of ocular drug delivery, gene delivery, and imaging, the focus of this review. Key applications of nanotechnology discussed in this review include a) bioadhesive nanomedicines; b) functionalized nanomedicines that enhance target recognition and/or cell entry; c) nanomedicines capable of controlled release of the payload; d) nanomedicines capable of enhancing gene transfection and duration of transfection; f) nanomedicines responsive to stimuli including light, heat, ultrasound, electrical signals, pH, and oxidative stress; g) diversely sized and colored nanoparticles for imaging, and h) nanowires for retinal prostheses. Additionally, nanofabricated delivery systems including implants, films, microparticles, and nanoparticles are described. Although the above nanomedicines may be administered by various routes including topical, intravitreal, intravenous, transscleral, suprachoroidal, and subretinal routes, each nanomedicine should be tailored for the disease, drug, and site of administration. In addition to the nature of materials used in nanomedicine design, depending on the site of nanomedicine administration, clearance and toxicity are expected to differ.

Drug and gene delivery to the back of the eye: from bench to bedside.

The ARVO 2012 Summer Eye Research Conference (SERC 2012) on “Drug and Gene Delivery to the Back of the Eye: From Bench to Bedside” was held June 15 and 16, 2012, at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The SERC provided a diverse group of approximately 150 scientists and physicians representing industry and academia from 14 countries with a unique opportunity to explore the latest approaches to drug and gene delivery to the posterior segment of the eye. Unlike the 2009 SERC meeting, which focused on novel drug delivery platforms while elucidating the anatomic barriers to reach the posterior segment,1 the most recent meeting explored strategies for bypassing ocular barriers using novel materials, nanoparticulate delivery systems, and gene therapy. It brought together experts in both ophthalmology and tangentially related areas to discuss the application and inherent technical challenges for translating experimental results from the laboratory bench to dependable medical therapies at the bedside and, where possible, it exemplified findings in ocular models with methods and results gleaned from disciplines outside of ophthalmology. The present review of the SERC provides investigators with tools to navigate these nascent approaches by exploring strategies from key laboratory investigations, drug development specialists, and clinical trials. The 2-day conference comprised the following six sessions: (1) barriers to drug delivery and transporter-guided drug design; (2) drug/gene delivery systems and cell therapies for the eye; (3) pharmacokinetics (PK), pharmacodynamics, and alternative routes of drug delivery; (4) nanotechnology for diagnosis and treatment of posterior eye disease; (5) translation of gene delivery for posterior eye disease; and (6) clinical trials. Rather than being a deliberate summary of each presentation, this review describes the common themes expressed during the six sessions.

Functionalized Nanosystems for Targeted Mitochondrial Delivery

Mitochondrial dysfunction including oxidative stress and DNA mutations underlies the pathology of various diseases including Alzheimers disease and diabetes, necessitating the development of mitochondria targeted therapeutic agents. Nanotechnology offers unique tools and materials to target therapeutic agents to mitochondria. As discussed in this paper, a variety of functionalized nanosystems including polymeric and metallic nanoparticles as well as liposomes are more effective than plain drug and non-functionalized nanosystems in delivering therapeutic agents to mitochondria. Although the field is in its infancy, studies to date suggest the superior therapeutic activity of functionalized nanosystems for treating mitochondrial defects.

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

PURPOSE The chaperone proteins, α-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer α-crystallin-derived mini-chaperone peptides (α-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer αA- or αB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, α-crystallin-derived mini-peptides and recombinant full-length αB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for α-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated αB-crystallin mini-chaperone peptides from H2O2-induced cell death was studied. RESULTS Primary hfRPE cells exposed to oxidative stress and either αA- or αB-crystallin mini-chaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length αB-crystallin was minimal while a time-dependent uptake of αB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing αB-crystallin mini-chaperone were also taken up and protected hfRPE from H2O2-induced cell death at significantly lower concentrations than free αB-crystallin mini-chaperone peptide. CONCLUSIONS αA- and αB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of αB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.

Influence of choroidal neovascularization and biodegradable polymeric particle size on transscleral sustained delivery of triamcinolone acetonide.

PURPOSE One objective of this study was to determine whether polymeric nanoparticles and/or microparticles sustain transscleral choroidal and retinal delivery of triamcinolone acetonide (TA) for two months in therapeutically effective concentrations after single periocular administration. Another objective of this study was to assess the influence of choroidal neovascularization on transscleral delivery of TA. METHODS Polymeric nano- and micro-particles of TA were prepared by o/w emulsion-solvent evaporation method using poly-l-lactide (PLA). Particles were characterized for drug loading, size, surface morphology, and the in vitro drug release profile. Choroidal neovascularization (CNV) was induced in brown Norway (BN) rats using a 532 nm diode argon laser and the CNV induction was assessed using fluorescein angiography. In vivo delivery was assessed in control and CNV induced rats at 2 months after periocular injection of TA loaded nano- or micro-particle suspension, or plain TA suspension in PBS (pH 7.4). Ocular tissue levels of TA were estimated using LC-MS/MS following liquid-liquid extraction of drug from tissue samples. Nile red loaded microparticles entrapped in periocular tissue at the end of the study was visualized using scanning electron microscopy and confocal microscopy. Inhibitory effect of TA on VEGF secretion was evaluated in ARPE-19 cells. RESULTS Triamcinolone acetonide-PLA nano- (551 nm) and micro-particles (2090 nm), with 14.7 and 29.5% drug loading, respectively, sustained in vitro TA release for about 45 and 120 days. After subconjunctival injection, microparticles were able to sustain the delivery in all intraocular tissues for 2 months; whereas no drug levels were detected for TA loaded nanoparticles and plain suspension of TA. Intraocular delivery of TA from microparticles was higher in CNV induced rats when compared to control rats. Significant amount of microparticles remained in periocular tissue at 2 months after injection, and maintained spherical shape. TA decreased VEGF secretion by 50% at 0.07 μM. At the end of the in vivo study, choroid-RPE and retina TA levels in CNV induced rats were 16- and 5-fold higher than the IC(50) for VEGF secretion. CONCLUSIONS Single periocular injection of polymeric microparticles but not nanoparticles sustained effective levels of TA in choroid-RPE and retina for 2 months, with the TA delivery being greater in CNV induced rats than the control rats.

Anti-inflammatory and antiangiogenic effects of nanoparticle-mediated delivery of a natural angiogenic inhibitor.

PURPOSE The purpose of this study was to evaluate the inhibitory effects of the nanoparticle-mediated delivery of plasminogen kringle 5 (K5) on choroidal neovascularization (CNV) and retinal inflammation. METHODS CNV was induced by laser in adult rats. Nanoparticles with an expression plasmid of K5 (K5-NP) were injected into the vitreous. K5 expression was detected by immunohistochemistry. The CNV area was measured after fluorescein angiography. Retinal vascular permeability was quantified with Evans blue as a tracer. Expression of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 was measured by Western blot analysis or ELISA and real-time RT-PCR. RESULTS Intense K5 expression was detected in the retina 2 weeks after the injection of K5-NP. Areas of CNV were significantly decreased in the K5-NP treatment group compared with that in the control-NP group. The K5-NP injection also significantly reduced vascular permeability. The expression of VEGF was downregulated by K5-NP at both the protein and mRNA levels. Moreover, K5-NP also inhibited expression of TNF-α and ICAM-1. Similarly, K5-NP decreased retinal levels of total β-catenin. In cultured cells, K5-NP suppressed hypoxia-induced secretion of MCP-1 and TNF-α. CONCLUSIONS K5 has a novel anti-inflammatory activity. K5-NP mediates a sustained inhibitory effect on CNV and thus has therapeutic potential for age-related macular degeneration.

Pirfenidone nanoparticles improve corneal wound healing and prevent scarring following alkali burn.

Purpose To evaluate the effects of pirfenidone nanoparticles on corneal re-epithelialization and scarring, major clinical challenges after alkali burn. Methods Effect of pirfenidone on collagen I and α-smooth muscle actin (α-SMA) synthesis by TGFβ induced primary corneal fibroblast cells was evaluated by immunoblotting and immunocytochemistry. Pirfenidone loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were prepared, characterized and their cellular entry was examined in primary corneal fibroblast cells by fluorescence microscopy. Alkali burn was induced in one eye of Sprague Dawley rats followed by daily topical treatment with free pirfenidone, pirfenidone nanoparticles or vehicle. Corneal re-epithelialization was assessed daily by flourescein dye test; absence of stained area indicated complete re-epithelialization and the time for complete re-epithelialization was determined. Corneal haze was assessed daily for 7 days under slit lamp microscope and graded using a standard method. After 7 days, collagen I deposition in the superficial layer of cornea was examined by immunohistochemistry. Results Pirfenidone prevented (P<0.05) increase in TGF β induced collagen I and α-SMA synthesis by corneal fibroblasts in a dose dependent manner. Pirfenidone could be loaded successfully within PLGA nanoparticles, which entered the corneal fibroblasts within 5 minutes. Pirfenidone nanoparticles but not free pirfenidone significantly (P<0.05) reduced collagen I level, corneal haze and the time for corneal re-epithelialization following alkali burn. Conclusion Pirfenidone decreases collagen synthesis and prevents myofibroblast formation. Pirfenidone nanoparticles improve corneal wound healing and prevent fibrosis. Pirfenidone nanoparticles are of potential value in treating corneal chemical burns and other corneal fibrotic diseases.

Supercritical fluid technology based large porous celecoxib–PLGA microparticles do not induce pulmonary fibrosis and sustain drug delivery and efficacy for several weeks following a single dose

Although pulmonary dosing of large porous particles has been shown to sustain drug delivery for a few days, there are no reports on safety or long term delivery. In this study we prepared large porous poly(lactide-co-glycolide) (PLGA) microparticles of celecoxib using supercritical fluid pressure-quench technology and demonstrated 4.8-, 15.7-, and 2.1-fold greater drug levels in lung, bronchoalveolar lavage fluid (BAL), and plasma compared to conventional microparticles on day 21 after a single intratracheal dosing of dry powders in A/J mice. Porous particle based delivery was 50.2-, 95.5-, and 7.7-fold higher compared to plain drug in the lung, BAL, and plasma, respectively. Toxicity of the formulations was assessed on day 21 following a fibrosis assessment protocol in A/J mice. There was no significant change in lactate dehydrogenase (LDH), total protein, and total cell counts in the BAL, and soluble collagen levels in the lung tissue following particle or drug treatments. Lung histology indicated no significant hyperplasia, granuloma, or collagen deposition in the treated groups. Chemopreventive potential of celecoxib porous particles was assessed in a benzo[a]pyrene (B[a]P) induced lung cancer model in A/J mice, on day 60 following a single intratracheal dose with or without single intravenous paclitaxel/carboplatin treatment. The combination group was more effective than individual groups, with the inhibition of tumor multiplicity and reduction of vascular endothelial growth factor in the BAL being 70 and 58%, respectively. Thus, large porous celecoxib-PLGA microparticles prepared using supercritical fluid technology exhibited sustained drug delivery and anti-tumor efficacy, without causing any significant toxicity.

Comparison of suprachoroidal drug delivery with subconjunctival and intravitreal routes using noninvasive fluorophotometry.

Purpose To determine whether exposure of sodium fluorescein (NaF) to the choroid-retina region in the posterior segment of the eye is greater with suprachoroidal injection when compared to intravitreal and transscleral routes. Methods Suprachoroidal injection, a new approach for drug delivery to the posterior segment of the eye was validated using a 34 G needle and Indian ink injections in Sprague Dawley rats, followed by histology. Delivery of NaF was compared in Sprague Dawley rats after suprachoroidal, posterior subconjunctival, or intravitreal injections. NaF levels were monitored noninvasively up to 6 hours using Fluorotron Master™, an ocular fluorophotometer Pharmacokinetic parameters were estimated using WinNonlin. Results Histological analysis indicated localization of India ink to the suprachoroidal space below sclera, following injection. NaF delivery to choroid-retina was in the order: suprachoroidal > intravitreal >posterior subconjunctival injection. Peak NaF concentration (Cmax) in choroid-retina was 36-fold (p = 0.001) and 25-fold (p = 0.001) higher after suprachoroidal (2744±1111 ng/ml) injection when compared to posterior subconjunctival (76±6 ng/ml) and intravitreal (108±39 ng/ml) injections, respectively. NaF exposure (AUC0–360min) to choroid-retina after suprachoroidal injection was 6-fold (p = 0.001) and 2-fold (p = 0.03) higher than posterior subconjunctival and intravitreal injections, respectively. Choroid-retina Tmax was observed immediately after dosing with suprachoroidal injections and at 10 and 27.5 minutes, respectively, with subconjunctival and intravitreal injections. Conclusions Suprachoroidal injections are feasible in a rat model. Suprachoroidal injections resulted in the highest bioavailability, that is, the extent and rate of delivery of NaF to choroid-retina, when compared to intravitreal and posterior subconjunctival injections. Ocular fluorophotometry is useful for noninvasive monitoring of NaF in rats following administration by various routes including suprachoroidal route.

Intraocular distribution of melanin in human, monkey, rabbit, minipig and dog eyes

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity.