Therapeutic Benefits from Nanoparticles: The Potential Significance of Nanoscience in Retinal Degenerative Diseases

Abstract

Several nanotechnology podiums have gained remarkable attention in the area of medical sciences, including diagnostics and treatment. In the past decade, engineered multifunctional nanoparticles have served as drug and gene carriers. The most important aspect of translating nanoparticles from the bench to bedside is safety. These nanoparticles should not elicit any immune response and should not be toxic to humans or the environment. Lipid-based nanoparticles have been shown to be the least toxic for in vivo applications, and significant progress has been made in gene and drug delivery employing lipid-based nanoassemblies. Several excellent reviews and reports discuss the general use and application of lipid-based nanoparticles; our review focuses on the application of lipid-based nanoparticles for the treatment of ocular diseases, and recent advances in and updates on their use.

Introduction

The human body has specialized sensory organs for sight, hearing, smell, touch, and taste. Our eyes are one of the sensory organs. These organs respond to light rays and convert the energy in the light rays to biological nerve impulses that are carried by the fibers of the nerve cells in the eye to the part of the brain specialized for the perception of visual images called the visual cortex. The retina - the thin, fragile layer of tissue lining the back of the eye - aids in vision. The retina is an extension of the brain and, like the brain, is comprised of nerve cells. Unlike most other cells in our body, mature nerve cells do not divide, which means the cells in our retina and brain must last a lifetime! Once lost, they cannot be replaced.

In mammalian retina, photoreceptors include rods, cones, and photosensitive ganglion cells (1). Both rods and cones support vision, whereas photosensitive ganglion cells mediate circadian rhythms and pupillary reflex (1). We use rod photoreceptors in dim light, whereas cones support color vision. In the human retina, approximately 120 million rod cells, 6 million cones, and 2–3 million ganglion cells, of which 1–2% are photosensitive, are present.

The nerve cells in the retina die in retinal diseases such as retinitis pigmentosa (RP) (2–5), age-related macular degeneration (AMD), diabetic retinopathy (DR) (6–8), and glaucoma (9,10). Since they cannot be replaced, their loss leads to reduced vision, and often blindness. Significant progress has been made in the last 20 years on the role of genetics and environmental factors contribute to the retinal degenerative diseases. However, the knowledge regarding how to successfully treat retinal degeneration has been limited. To treat retinal diseases, several approaches have been used, including gene therapy (11–30). antioxidant administration (31–34), cytokine administration (35–39), tissue transplantation (40), optogenetics (41–43), surgical implantation of light-sensitive photocells and prosthesis (44,45), microRNA administration (46), and stem cell therapy (47,48). Gene therapy is a promising treatment option for several diseases, including inherited disorders, blinding eye diseases, some types of cancer, and certain viral infections. Currently, this approach is being tested only for diseases that have no other cures. Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

The retina has a blood-retinal barrier (BRB). Agents (drugs or genes) that are injected systemically must cross the BRB before reaching the eye. By the time the agent reaches the eye, the quantity and efficacy of the agent could be significantly reduced. Fortunately, it is easy to deliver agents without going through systemic circulation. The agents can be easily administered subretinally or intravitreally due to the eye’s self-containment, the relatively low doses of agent required, and the fact that the eye is immune-privileged (49).

. A gene without a promoter that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene, and the promoter located upstream of a gene in the vector drives the gene expression. A gene carrying a functional promoter can be inserted directly into cells through electroporation, and this electrogene therapy has been successful in both eye (50,51) and other tissues (52). DNA degradation is a fundamental problem for any gene therapy, especially after naked DNA injection. Instead, a carrier called a vector is genetically engineered to deliver the gene. There are two types of carriers that are commonly used to introduce genes into tissues. These gene therapy vehicles or transporters are broadly divided into two classes: viral and non-viral gene delivery vehicles (53–55). Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. Researchers have used many viruses, including retrovirus (54), adenovirus (56), herpes simplex virus (HSV) (57), adeno-associated virus (AAV) (58), pox virus (59), lentivirus (30), as highly efficient gene transduction can be achieved with viruses. The viruses are modified so that they cannot cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. The disadvantages of this approach include the associated biosafety issues, toxicity, induction of immune reaction, and potential recombination of virus that is harmful to the recipient and the environment. Another limitation is the size of the insert that can be cloned into the viral vector. Gene(s) with a size of >5 kb are not suitable for viral-mediated gene delivery. Moreover, multiple genes cannot be transduced with one viral vector, and transduction of multiple genes require multiple viral vectors. The advantage of the non-viral system allows us to simultaneously introduce multiple biomolecules with one vector. Often, treatment of retinal degenerative diseases would involve the replacement of more than one gene. For example, more than 140 mutations have been mapped on human rhodopsin gene, and if we correct every mutation, individual viral constructs must be generated. This would be a laborious process.

Often, the gene introduced into cells or tissues by the virus may not express the protein and is due to a tissue tropism. For example, currently, there are 13 serotypes of adeno-associated virus (1–13). This list may grow as new serotypes are identified (60). A serotype is a serologically distinguishable strain of a microorganism. Tissue tropism is the cells and tissue of a host that support the growth of a virus or bacterium. Some bacteria and viruses have a broad tissue tropism and can infect many types of cells and tissues. Other viruses may infect primarily a single tissue. For example, rabies virus affects primarily neuronal tissue.

Therefore, researchers have begun to design non-viral vectors for efficient gene therapy. These vectors must overcome size limitations, achieve safety, and create effective gene therapy. Ideal vectors must accommodate genes with no size restrictions, have high transduction competence, elicit no immune reaction, be affordable, well-tolerated and biodegradable, and have controlled and specific target tissue expression while preserving the well-being of both the patient and the environment. The inherent limitations associated with viral vectors and the disadvantages associated with viral gene transduction have encouraged many researchers to focus on the development of non-viral gene delivery systems. Researchers have designed non-viral vectors (61) for the delivery of genes for diseases, such as cystic fibrosis (62), respiratory diseases (63), cardiovascular diseases (64), and also to carry vaccines (65). Some of these nonviral vectors are currently using in clinical trials.

Metal-based nanoparticles

Aging and oxidative stress play important roles in many diseases, including neurodegenerative diseases (66). Oxidative stress has been shown to be an important player in the pathogenesis of retinal degenerative diseases (67–71). Researchers have taken advantage of nanoparticles that have high redox scavenger activity to neutralize the toxic effects of oxygen free radicals. For ocular applications, cerium oxide (CeO₂) and yttrium oxide (Y₂O₃) metal-based nanoparticles have been tested in several retinal degenerative and light-stress animal models (72). Cerium is a rare earth element of the lanthanide series; cerium oxide is an inorganic and insoluble in water, although it does possess intrinsic antioxidant activity (73,74). It has been further shown that nanoceria mimic the enzymatic activities of superoxide dismutase (SOD) and catalase (75–77). These particles have no adverse effects on retinal structure and function, and their half-lives in the retina are more than one year (75–77).

Light stress is commonly used as a means to examine the neuroprotective potential of the retina (74). Exposure of animals to increasing amounts of light causes the retinas to undergo retinal degeneration (78,79). This light-induced retinal degeneration is exacerbated in the presence of mutations (80,81). This model is well suited to screening drugs to treat retinal degenerative diseases (82,83). The light-stress model also mimics the oxidative stress model, and nanoceria has been shown to prevent light-stress-induced retinal degeneration (74). Nanoceria also attenuated blood vessel leakage into the retina and oxidative stress-induced neovascularization (77,84), and inhibited the growth of retinoblastoma tumors (85,86). Retinoblastoma is a rare form of cancer of the retina and the most common malignant cancer of the eye in children (87).

Polymer-based nanoparticles

Polymer-based nanoparticles have been used as gene delivery vehicles for ocular gene therapy (55). Polyethylene-glycol nanoparticles efficiently transfect both retinal pigment epithelium and photoreceptor cells (12,55). These particles have been used to correct vision deficiencies and prevent retinal degeneration in mouse models of Leber’s congenital amaurosis (LCA), Stargardt disease, and retinitis pigmentosa (19,22,24,88,89). Polymer-based nanoparticles have also been used to target microRNA for the treatment of diabetic retinopathy [95]. No adverse effects or activation of molecules that induce inflammation or immune reaction were reported in any of the above-cited studies with polymer-based nanoparticles (19,22,24,88,89).

Pentablock polymers and nanomicellar formulations

Drug delivery to the eye involves intravitreal injections, which can have several undesirable side effects. To overcome these barriers, researchers have formulated novel nanotechnology-based strategies for safe and long-term drug release. Due to their small particle size and large and modifiable surface, nanoparticles have unique advantages over other drug carriers. Thus, novel pentablock copolymers (polylactide-polycaprolactone-polyethylene glycol-polycaprolactone-polylactide; PLA-PCL-PEG-PCL-PLA), which provide continuous delivery of drugs over a longer duration with minimal burst effect, have been formulated (90). These novel tailor-made pentablock polymers with thermosensitive gel properties have attracted attention for practical biomedical or pharmaceutical applications. Thermosensitive gels begin as aqueous polymer solutions and are transformed into gels by changes in environmental conditions, such as temperature and pH, resulting in in situ hydrogel formation. Utilization of pentablock copolymers for the controlled and non-invasive delivery of the drug is a promising platform for ocular delivery of therapeutic macromolecules and can minimize the side effects associated with frequent intravitreal injections. Nanomicellar formulations are also available; these particles possess the advantages of small size, low toxicity, the ability to increase the solubility of hydrophobic drugs, and the ability to achieve therapeutic concentrations (91). The nanomicellar drug delivery platform has been successfully applied for topical administration of hydrophobic drugs (91).

Lipid-protamine-DNA (LPD) complexes for ocular diseases

The backbone of lipid-based nanoparticles is derived from liposomes. British hematologist Dr. Alec D. Bangham first described liposomes in 1961 (92). Liposomes are circular vesicles with a minimum of one lipid bilayer. These vesicles have long been known to be effective cargo vehicles for transporting biomolecules, nutrients, and therapeutic agents. Disruption of natural membranes by sonication results in the formation of liposomes. Naturally occurring liposomes have bilayers consisting of phospholipids and/or cholesterol, which are non-toxic and inert under physiological conditions.

Researchers have found that liposomes prepared from cationic lipids are best suited to gene delivery since they are easy to prepare, can be produced in large quantities, have minimal-to-no toxicity, are environmentally friendly, are bio-decomposable, and cannot elicit an immune response (93–95). Guo and Huang demonstrated that inclusion of a cationic polymer into cationic lipid-DNA complexes enhances the lipofectin in vitro (93). They found that the enhanced transfection is due to the protection of DNA against nucleases; this also enhanced the nuclear transport of the plasmid DNA (96). Based on the excellent gene transduction efficiency of cationic lipid and cationic polymers, liposome-polycation-DNA complexes (LPD) have been formulated for both in vitro and in vivo gene transduction (93,96). In addition, the DNA condensing agent protamine, an arginine-rich nuclear protein, has been incorporated into liposome to improve gene transduction efficiency (97,98). These earlier studies were performed with animals by examining reporter gene expression (96), but no target gene delivery or gene corrections were conducted.

Serum sensitivity is a potential problem with lipofection, as lipids are sequestered by serum proteins (96). This serum sensitivity can be overcome by increasing the charge ratio of cationic lipid-DNA (99). In general, gene transduction vectors require two important features: 1) the vector must cross the cell membrane, and 2) the vector must be efficiently transported into the nucleus by crossing the nuclear membrane. Protein transduction domains (PTD) have been shown to be efficiently cross the lipid bilayer (100). Recently, it has been shown that one of the PTD domains, TAT peptide, from the HIV-1 virus has been shown to effectively transduce LPD-mediated delivery of transgenes (100).

LPD efficiency is further enhanced by deliberately adding cell-specific targeting signals for improved gene transduction. Cell-targeting and cell-internalization peptides have been extensively used for efficient drug delivery and for image analysis (98,101). A peptide derived from TAT-protein from human immunodeficiency virus (HIV)-1, has been utilized to deliver exogenous proteins that translocate through the cell membranes and accumulate in the nucleus (102). These protein transduction domains have been extensively used for the delivery of biologically active proteins in vivo (103–105), and protein therapy has been used as a treatment for various diseases (106–108). Adding the nuclear localization signal peptide from SV40 large T antigen to LPD increases gene transduction (98). Similarly, the addition of homing peptides increases gene transduction efficiency (98,101). A recent study showed that synthetic steroidal glucocorticoid dexamethasone (DEX) complexed with LPD improved gene expression. DEX has been shown to dilate the nuclear pore up to 60 nm and facilitate the uptake of transfected DNA into the nucleus (109,110).

Fascinating improvements have been made in LPD, such as a recent study reporting a lipid-based nanoparticle-like protocell, or protobiont (110). A protocell is a tool with which to understand the origin of life, as this nanoparticle is a self-organized, globular collection of lipids (111).The protocell is a nano-bioreactor mimicking the cell membrane formed by neutral lipid bilayer (111). Protocells do not elicit an immune response or cytotoxicity, as they are exclusively composed of neutral lipids without cationic charge (110). They can be used widely for gene transduction studies.

The liposomes complexed with PTD, nuclear localization signal peptide, and protamine ranged in size from 80–120 nm (100). LPD nanoparticles (Fig.1) have been successfully used to deliver Rpe65 gene to mice deficient in Rpe65 protein, and corrected the vision in a mouse model of Leber’s congenital amaurosis (LCA) (112). The Rpe65 gene delivery to Rpe65 knockout mice is comparable to AAV (25) and lentiviral (17) gene transfer of Rpe65 to Rpe65 knockout mice. Researchers have observed a long-term expression of transgene by a single injection of LPD complexed with transgene to eye tissues (112). Mechanistic target of rapamycin (mTOR) activation is needed for cone photoreceptor survival (113,114), and constitutively activated form of mTOR delivery to the retina by LPD nanoparticles delayed the death of cone photoreceptors in a mouse model of retinitis pigmentosa (115). It has been shown that activation of Wnt signaling has been implicated in the pathogenies of diabetic retinopathy (46). Targeting a microRNA 184 mimic with LPD inhibited Wnt signaling in an oxygen-induced retinopathy mouse model (46).

Figure 1. Formulation of liposome-protamine-DNA (LPD) complex for in vitro and in vivo application.

Lipid nanoparticles (LPD) can be prepared from liposome (A) combined with protamine, protein transduction domain (PTD) peptide, SV40 nuclear localization (NLS) peptide (B), and plasmid DNA (C). LPD can also deliver miRNA (D) and lipids (E). Panel F represents the expression of enhanced green fluorescent protein (eGFP) in human embryonic kidney cells (HEK-293T) delivered through LPD nanoparticles. Panel G represents the expression of eGFP in mouse eye delivered through LPD nanoparticles. Fluorescent fundoscopy was used to visualize GFP fluorescence.

Peroxisome proliferator-activated receptor alpha (PPARα) is an important receptor that is implicated in diabetic retinopathy and is downregulated in this disease (116).The mechanism of PPARα downregulation in diabetic retinopathy occurs partly through upregulation of miR-21 (116). Intravitreal delivery of LPD-mediated miR-21 inhibitor attenuated PPARα downregulation and suppressed retinal inflammation in db/db mice (116). These studies suggest that LPD could be used to deliver defective genes and microRNA for retinal degenerative diseases. Further, LPD administration, either systemically or intravitreally, has been shown to have no adverse effects on the eye in terms of inflammation (100,112,117).

LPD-mediated delivery of agents to specific retinal cell type

One of the problems associated with the LPD is achieving cell and tissue-specificity. Expression of genes to unwanted tissues would cause off-target effects. Researchers have recently overcome this problem by developing cell-specific promoter vectors that, coupled with LPD, enabled the expression of transgene to the specific cell type (100,118). Researchers have demonstrated the delivery of genes to retinal pigment epithelium, rod photoreceptor cells, cone photoreceptor cells, and retinal ganglion cells (100,118) (Fig.2). These cells are commonly affected in age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and glaucoma. Targeting genes to specific cell type(s) will help to save the dying neurons in retinal degenerative diseases.

Figure 2. Cell-specific promoters enable LPD nanoparticles to deliver genes to specific cells of the retina in vivo.

The retina is composed of various retinal cell types (A). Cell-specific promoters (B) enable LPD nanoparticles to RPE cells with vitelliform macular dystrophy (VMD2) promoter, rod cells with mouse rhodopsin promoter, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen (Thy 1.2) promoter.

Lipid-based nanoparticles for drug delivery to ocular tissues

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been recognized as effective non-viral gene transduction vectors for the treatment of genetic and non-genetic diseases (119). These particles have been used and proposed for the future clinical application to ocular diseases, infectious diseases, lysosomal storage disorders, and cancer (119). For example, fungal keratitis is a corneal infection in which pathogenic fungi infiltrate the ocular surface. Chitosan-modified nanostructured lipid carriers complexed with amphotericin B are a promising targeted therapy for fungal keratitis (120). Nanostructured lipid carriers modified with chitosan oligosaccharide lactate complexed with ofloxacin are effective in the treatment of bacterial keratitis in vivo (121). Solid lipid nanoparticles, nanostructured lipid carriers, and lipid drug conjugates are effective drug and gene delivery systems for retinal diseases (122). Protamine and hyaluronic acid (HA) -containing solid lipid nanoparticles are effective carriers in delivering genes to ARPE-19 and HEK-293 cells (123). The HA in these particles has been shown to modulate the condensation of DNA by protamine (123). In addition, HA-modified core-shell liponanoparticles have been specifically targeted to retinal pigment epithelium; this specificity is achieved through the interaction of HA and the CD44 receptor expressed on the RPE cells (124). Solid lipid nanoparticles containing dextran and protamine improved expression of reporter and target genes into ARPE-19 cells (125).

Corticosteroid is commonly designated for the treatment of macular edema, neovascularization, and other ocular inflammatory disorders (126). Nanostructured lipid carriers (NLC) have been used for sustained release of corticosteroid (triamcinolone acetonide) for ocular diseases of the posterior segment (126). The key limitations for the treatment of eye diseases and disorders are extensive pre-corneal retention time and high diffusion into aqueous humor and intraocular tissues (127). To overcome these limitations, researchers have formulated thiolated nanostructured lipid carriers (Cys-NLC) as potential drug delivery vehicles for ocular tissue (127). These studies show that the Cys-NLC group has a higher retention time in aqueous humor, tears, and eye tissue than does non-thiolated NLC (127).

Another major challenge in ocular drug therapy is improving the penetration of topically applied drugs and increasing drug absorption and bioavailability of drugs (128). The use of submicron-meter-sized colloidal carrier systems has overcome the barriers imposed by the eye on drug absorption (128). Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have been shown to be superior for eye disease treatment compared with nanoemulsions, liposomes, and polymeric nanoparticles (128). SLN and NLC carriers have been used to deliver both non-steroidal and steroidal anti-inflammatory drugs for the treatment of ocular inflammatory diseases (128).

Topical application of drugs to the anterior segment of the eye is widely used. In many instances, this type of application results in less drug availability due to washout by tears. Lipid-based nanocarriers and lipid nanoparticles have the ability of mucoadhesion with increased corneal holding time for a drug that is administered topically (129,130). To improve the pharmacological efficacy of drugs that combat the oxidative stress and inflammation in diseases such as age-related macular degeneration, diabetic retinopathy, and inflammation, cationic lipid nanoparticles (LNs) have been loaded with a polyphenol, epigallocatechin gallate (EGCG) (131). These particles are effective, safe, and do not irritate the eye (131). Drug oxidation and epimerization can also be avoided through encapsulation of EGCG in lipid nanoparticles (132). Solid lipid nanoparticles have been used for the delivery of calendula officinalis extract to improve epithelium repair on the ocular surface (133). Lipid nanovesicles have been successfully used for the delivery of photoreceptor-specific proteins and antibodies against photoreceptor proteins to investigate the distribution of proteins in vivo (134). These studies set the stage for lipid nanoparticle-mediated protein therapy for retinal diseases.

Lipid nanoparticle applications in lipid therapy

Lipid replacement therapy (LPT) is a natural medicine approach to restore cellular processes that were damaged due to damaged lipids in cellular membranes and organelles (135). The process includes oral supplementation with cell membrane phospholipids and anti-oxidants to replace oxidized membrane glycerophospholipids that accumulate during the aging process (135). Once these lipids arrive at the membrane sites, the damaged lipids will be replaced and removed (135). Clinical trials of LPT have yielded promising results in the restoration of mitochondrial function and demonstrated reduced fatigue in aged individuals and patients with clinical manifestations of loss of mitochondrial function, including fatigue (135).

Glycerophospholipids are the most common lipids in the membrane structure of living organisms (136,137), except for phospholipids in which sphingosine substitutes for glycerol (sphingomyelins or ceramide-1-phosphorylcholines) that are present on the exterior side of the cell membranes (136,137). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG) are the major structural eukaryotic glycerophospholipids (136–138). Cholesterol is an abundant membrane constituent and the only sterol found in the membranes (136,137,139,140). These membranes also contain acylglycerol, fatty acids, and minor lipid constituents, the function of which is not known at this time (136,137). Lipid therapies have great potential for the treatment of several diseases, including metabolic disorders, diabetes, inflammation, infectious and autoimmune diseases, cancer, and cardiovascular and neurodegenerative disorders (141–144).

Lipids and lipid-soluble compounds are indispensable elements of the cells and tissues that encompass the eye. Defects in lipid transport, biosynthesis, trafficking, and turnover contribute to devastating eye diseases (145). Lipids and lipid metabolism in the retina are vital to membrane organization and turnover in the rod and cone photoreceptor neurons (145–147). Recent evidence suggests that either overabundance or deficiency of specific lipids causes retinal cell dysfunction and, ultimately, the death of post-mitotic retinal cells (145,148–152). Further, the lipid second messengers in the retina play an important role in recruiting protein complexes at the plasma membrane and regulating the survival signaling pathways in both rod and cone photoreceptor cells (153–155).

Phosphoinositide (PI) lipids are less abundant in the cell membrane (156–159). The parent molecule phosphoinositide or phosphatidylinositol (PI) is composed of a D-myoinositol head group, a glycerol backbone, and two fatty acids at the C1 and C2 acyl positions of glycerol. The PI undergoes phosphorylation on multiple free hydroxyl groups in the inositol head group and generates multiple phosphorylated PIs, commonly called phosphoinositides (153,160,161). Seven distinct phosphoinositides are generated and present in the cell membranes of higher eukaryotes (153,156). These seven PIs are formed from the rapid conversion of one PI to another through the action of specific phosphoinositide kinases and phosphoinositide phosphatases (160,162–164). In addition, these PIs serve as site-specific signals on membranes that recruit and regulate protein complexes at the interface of the cytosol and activate the downstream signaling cascades (153,160,162–164). Studies showed that phosphoinositide 3-kinase class-I (PI3K) phosphorylates PI on position 3 of the inositol ring generates 3’-PIs (160). This enzyme has been shown to be downregulated in retinal tissues from diabetic mice (165–167). Further, deletion of class I-PI3K in cone photoreceptors resulted in age-related cone degeneration (150). Similarly, deletion of class III PI3K resulted in rod photoreceptor degeneration (151), suggesting the importance of these enzymes in photoreceptor survival. These PIs also regulate autophagy; failure to execute autophagy leads to cell death (168–170). Researchers have shown that the introduction of specific PI-lipids to cells activates the defective autophagy process (169). Lipid nanoparticles prepared with specific PIs would be able to rescue the retinal degeneration and activate the signaling pathways. This approach would eliminate the need to introduce multiple proteins of the PI3K pathway, the receptor, regulators, and co-factors to activate the PI3K pathway.

Autosomal dominant stromal dystrophy has been shown to result from mutations in the FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) gene on chromosome 2 (2q35) (171–174). A variety of missense, frameshift, and protein truncation mutations have been observed in the PIKfyve gene (173). PIKfyve generates phosphoinositide-5-P (PI-5-P) and phosphoinositide 3,5-diphosphate (PI-3,5-P2) (169). LPD-mediated delivery of these lipids to patients with corneal dystrophy may have therapeutic benefits.

In the retina, mutations in ELOVL4 (Elongation of Very Long Chain Fatty Acids 4) have been shown to cause Stargardt-like macular dystrophy (STGD3), a juvenile onset of the autosomal dominant form of macular degeneration (175). ELOVL4 is a fatty acid elongase that is involved in the formation of very long chain polyunsaturated fatty acids (VLC-PUFAs) (149). It has been suggested that preventing the formation of VLC-PUFAs causes photoreceptor degeneration. The LPD-mediated delivery of these lipids in animal models and patients suffering from Stargardt-like macular dystrophy may have therapeutic benefits. Lipid nanoparticle therapy is advantageous over gene therapy. In disease conditions, the introduced gene must be functional. If it is an enzyme, the substrate must be available for product formation. Directly introducing the lipid product may activate the pathways without going through transcription and translation, and may properly target the lipids to the specific compartments.

Conclusions

Myles Munroe said that “vision is the Source and hope of life. The greatest gift ever given to mankind is not the gift of sight, but the gift of vision. Sight is a function of the eyes; vision is a function of the heart. ‘Eyes that look are common, but eyes that see are rare.’ Nothing noble or noteworthy on earth was ever done without vision” (http://www.azquotes.com/quote/770789). Mammalian retinal neurons have the remarkable ability to survive in a hostile environment where they are subjected constantly to high levels of oxygen and bombarded by photons, while containing large amounts of highly oxidizable polyunsaturated fatty acids (PUFAs) and occasional lethal mutations most often expressed only in rods and/or cones. Researchers have shown that retinal neurons possess sophisticated endogenous neuroprotective mechanisms (35,176–178), some of which can be upregulated by pre-conditioning these neurons to sub-lethal stress, e.g., light (78) and products of lipid peroxidation (179,180). These pathways, developed over millions of years of evolution, are remarkably successful in protecting these post-mitotic neurons from the daily insults that they must endure.

Age-related macular degeneration (AMD) is a retinal degenerative disease that causes progressive loss of central vision. Retinitis pigmentosa (RP) is a group of genetic eye conditions. In the progression of symptoms in RP, night blindness generally precedes tunnel vision by years or even decades. Stargardt disease is the most common form of inherited juvenile macular degeneration. The progressive vision loss associated with Stargardt disease is caused by the death of photoreceptor cells in the central portion of the retina called the macula. Usher syndrome is an inherited condition characterized by hearing impairment and progressive vision loss. Vision loss also occurs in diabetic retinopathy and glaucoma. In some instances, these neurons survive for decades, but ultimately succumb, leading to loss of vision and, in many instances, blindness.

Much research has been aimed at elucidating these pathways and identifying strategies to either upregulate neuroprotective pathways or downregulate cell death pathways, to develop rational therapeutic interventions for treating retinal degenerations, especially those associated with age, e.g., AMD. These are important goals, since prolonging the life of foveal cones for even one decade would have a beneficial effect on usable vision in an aging population.

Delivering the agents through a suitable system is desirable; however, the knowledge base in this area remains limited, and such interventions have yet to emerge. Gene therapies using viral vectors have been successfully applied to the treatment of eye diseases, but safety is a major concern for both patients and the environment. Many non-viral vectors, including lipid nanoparticles, have been used to treat eye diseases. Combining the power of lipid nanoparticles with cell-specific promoters enables the delivery of LPD to specific cells of the retina. These approaches may inspire investigators in other fields to couple cell-specific promoters with LPD to directly target genes to specific tissue and cell types. The greatest advantage of LPD nanoparticles is their ability to deliver specific lipids and lipid second messengers to activate defective pathways. These unique characteristics make LPD a promising delivery system for ocular tissues.