increased, the size of the micelles increased. All micelles fabricated with a molar ratio of e-CL to MePEG of 50, 75 and 100 exhibited ~30% cumulative release at day 14, micelles with a ratio of 25 exhibited 15% release at day 6 and plain indomethacin had nearly 100% cumulative release at day 2. Interestingly, micelles were capable of prolonging the release of their contents by a significant factor.

Very few researchers have tested the ability of micelles to improve drug pharmacokinetics in ocular tissues. Gupta et al. in 2000 are among the first to investigate the permeability of ketorolac-loaded micelles made with N-isopropylacrylamide (NIPAAM) copolymer, vinyl pyrrolidone, and acrylic acid (AA) crosslinked with N¢,N¢-methylene bis-acrylamide across the cornea of excised rabbit cornea. The micelles were of < 50 nm in size and the release of drug from the micelles was pHdependent. Slowest release (approximately 40% in 8 h) was seen for acidic pH (pH 5) while pH 7.2 and 10 exhibited faster release. At pH 7.2, about 50% drug was released at 6 h and at pH 10, about 75% drug was released in 6 h (Gupta et al. 2000). The micellar formulation of ketorolac and the aqueous suspension of ketorolac had ~ 7% and 4% cumulative amount of keterolac that permeated the cornea, respectively, after 60 min. The study also found that the micellar formulations were able to prevent ocular inflammation more quickly than plain ketorolac as denoted by lid closure induced by prostaglandin E2 in a rabbit eye. Lid closure was rated as: 0 – fully open, 2 2/3 – open, and 3 – fully closed. At 30 min, the lid closure rating for the micellar formulation and plain ketorolac was ~0.5 and 1.7, respectively. The lid closure rating for the micelle-treated rabbits was consistent up to 3 h after which the lid closure rating was 0 for 4 h and 5 h time points. The lid closure rating for the plain ketorolac did not drop to ~0.5 until 3 h and decreased to 0 at 5 h. The micellar formulation of ketorolac showed increased residence time of the drug in ocular tissues as well as sustained release of the drug from the formulation.

11.2.4  Protein Nanoparticles

Nanoparticles or nanosystems can be prepared using a variety of naturally occurring or synthetic proteins. While naturally occurring proteins are likely safer, all proteins suffer from the potential for immunogenicity, especially when administered in forms that are altered when compared to their endogenous forms in the human body. While immunogenicity remains a challenge, protein-based delivery systems are still viable, given the success of AbraxaneTM, an albumin-based, intravenously administered paclitaxel nanoparticle for cancer therapy. Eye, being relatively immuno-privileged, might tolerate protein-based nanosystems better than some other parts of the body.

Albumin is a commonly assessed protein for drug delivery due to ease of synthesis (Zimmer et al. 1994) and knowledge regarding its biocompatibility, ability to bind various drug molecules, and its nontoxic nature. In fact, one study has evaluated the ocular disposition and tolerance of ganciclovir-loaded albumin nanoparticles after intravitreal injections in rats (Merodio et al. 2002). Albumin nanoparticles were detected in the vitreous up to 2 weeks after injection, and the histopathology of the

retina, ciliary muscle, neuronal interplay area, outer and inner nuclear layers, and the vitreous cavity showed no signs of inflammation after 2 weeks. Further, the cytoarchitecture of the retina showed no signs of alteration in photoreceptors or neuronal layers. In addition, the mechanism of degradation of albumin nanoparticles is known to involve phagocytosis by the RES (Schafer et al. 1994). Not only are protein nanoparticles safe and biocompatible, they have a unique inherent ability to bind drugs with various physiochemical properties due to the wide-range of charged, lipophilic, and hydrophilic amino acids.

Pioneer research completed by Merodio and colleagues has focused on the ocular use of albumin nanoparticles in sustaining the release of ganciclovir in the treatment of cytomegalovirus retinitis (Merodio et al. 2000). Drug release from albumin nanoparticles followed a biphasic model whereby an initial rapid release of drug was followed by a period of slow release. The nature of the concentration vs. time curve for ganciclovir directly depended on the method of synthesis and the addition of excipients. Three different methods of synthesis were used: Model A, B and C. For Model A nanoparticles, ethanol was added dropwise to a 2% (w/v) albumin solution while continuously stirring. Glutaraldehyde was then added to harden the coacervates. The nanoparticles were then purified by centrifugation to remove unreacted gluteraldehyde and albumin. The pelleted albumin nanoparticles were suspended with a ganciclovir solution and allowed to incubate up to 4 h. Unencapsulated drug was removed by centrifugation. Model B nanoparticles were made by adding ganciclovir directly to a 2% (w/v) albumin solution up to 4 h and afterwards the pH was adjusted to the isoelectric point of albumin (pI 5.5). The coacervates were dissolved with ethanol and then hardened with glutaraldehyde for 2 h. Finally, centrifugation was completed to remove unreacted glutaraldehyde, albumin, and ganciclovir. Model C nanoparticles were made by adding ganciclovir to a 2% (w/v) albumin solution containing a crosslinking agent and incubated up to 4 h. The pH of the solution was then adjusted to 5.5 (the pI of albumin) and afterwards ethanol was added. Again, centrifugation was used lastly as a purification step to remove unreacted compounds.

Addition of ganciclovir to albumin nanoparticles formed 4 h prior to the addition of ganciclovir (Model A nanoparticles) resulted in release of 60% of encapsulated drug within 1 h; however, addition of ganciclovir directly to the albumin solution in the initial step (Model B nanoparticles) decreased the amount of drug released to 40% and only 20% of drug was released from Model C nanoparticles. However, for all formulations of ganciclovir-loaded albumin nanoparticles, percent cumulative release of drug after 1 h remained constant over 5 days. The mechanism of drug release was found to be directly dependent on pH whereby increased ganciclovir release was observed under extremely basic and acidic conditions, but minimal release was observed near pH 7. Thus, sustained release properties in the order of a few days can easily be obtained using albumin nanoparticles by optimizing formulation pH and excipients.

Albumin nanoparticles also demonstrated superiority over lipofectamine in gene therapy. Human serum albumin nanoparticles loaded with the Cu, Zn superoxide dismutase (SOD1) gene were prepared (Fig. 11.4) and tested for their safety,

Fig. 11.4  Scheme depicting the methods for preparing human serum albumin (HSA) nanoparticles loaded with the plasmid capable of expressing superoxide dismutase 1 (pSOD1). Degree of crosslinking controls particle size (Mo et al. 2007)

release profiles, and efficacy (gene expression) by Mo et al. (2007). The albumin nanoparticles loaded with the SOD1 gene were synthesized using a modified ­desolvation-crosslinking method: a 2% (w/v) albumin solution was mixed with pSOD1 (plasmid encoding the SOD1 gene) in a Tris-EDTA solution at pH 8.0 for 5 min at room temperature. The solution was then added dropwise to an ethanol solution while stirring and the nanoparticles were crosslinked by adding 1% glutaraldehyde and stirring for 12 h. Excess glutaraldehyde was removed by addition of ethanol and centrifuging. The SOD1-loaded albumin nanoparticles were ~120 nm with 20 mL glutaraldehyde and ~160 nm if only 1 mL of glutaraldehyde was added as a crosslinker agent. The larger nanoparticles (160 nm) exhibited a biphasic release profile with release of 65% of the DNA in the first 6 h followed by sustained release for the next 44 h. The smaller nanoparticles (120 nm) had a slightly less drastic burst effect by which only 23% of the DNA was released in 6 h, followed by sustained release for 6 days. The nanoparticles were shown to be protective against DNAse I-induced degradation of the plasmid and were noncytotoxic to retinal pigment epithelial (ARPE-19) cells over 96 h at nanoparticle concentrations up to 5 mg/mL. The in vitro data clearly demonstrates that albumin pSOD1-loaded nanoparticles have higher SOD1 activity due to gene expression than pSOD1 + lipofectamine. Intravitreal injection of albumin pSOD1-loaded nanoparticles into mice had high protein levels of SOD1 compared to intravitreal injection of pSOD1 only.

Most recently, the effects of surface charge on albumin nanoparticle disposition within the vitreous and retina of rat eyes were determined (Kim et al. 2009b). Anionic nanoparticles were found to penetrate the retina after intravitreal injection; however, these nanoparticles could not penetrate the blood–retinal barrier. Cationic albumin nanoparticles were not able to efficiently penetrate the retina as only few

particles made it to the retina after 5 h due to the aggregates formed in the vitreous. Neither albumin nanoparticle formulations were found in the choroid or Bruch’s membrane. Depending on the target for the disease to be treated, albumin nanoparticles may not be an appropriate delivery device. For example, for the treatment of wet age-related macular degeneration (AMD), the drug must be able to reach the choroid and therefore, albumin nanoparticles may not be desirable if they are unable to reach the choroid. Further studies are needed to investigate the ability for albumin nanoparticles to deliver drug over extended periods of time and to compare albumin nanoparticles to current treatment regimes. This field of ocular drug delivery is relatively new and will likely expand within the next decade as the necessity for ocular drug delivery vehicles increases.

11.2.5  Carbohydrate Nanoparticles

Chitosan, a polysaccharide, in particular has been investigated extensively over the past three decades as a material for making drug delivery devices (Paolicelli et al. 2009). Chitosan is an acetylated form of chitin, which is found in lobster, crab and shrimp shells as well as in other insects and fungi. In addition, degraded forms of chitin are also found in plant soil to help plants defend against bacterium and other pests including the pine beetle. The chemical structure of chitosan consists of randomly oriented units of b-(1→4)-D-glucosamine and N-acetyl-D-glucosamine.

The method of biodegradation of chitin within the body is relatively well understood and involves both deacetylation and lysosomal degradation (Pangburn et al. 1982). The rate of degradation and cellular toxicity is highly dependent on the percentage of N-acetylation of chitosan (Freier et al. 2005). With 30–70% acetylation, 50% of the chitosan mass was lost by lysosomal degradation over 4 weeks. Samples with extremely high or low percentages of acetylation showed minimal weight loss over 4 weeks. Chitosan with an extremely low percentage of acetylation (0.5%) had the highest cell viability compared to chitosan with more acetylation. Both toxicity and rate of degradation can be controlled by synthesizing chitosan with specific amounts of N-acetylation.

In 2001, it was proposed by De Campos that chitosan nanoparticles may be effective ocular drug delivery vehicles (De Campos et al. 2001). Cyclosporin A (CyA)-loaded chitosan nanoparticles were shown to have at least twofold higher corneal concentrations than CyA in solution at all time points assessed up to 48 h (De Campos et al. 2001). In the conjunctiva, chitosan CyA nanoparticles had ~4,000 ng/g at 2 h compared with only ~900 ng/g at 2 h for the aqueous solution of CyA. At 6 and 24 h, chitosan CyA nanoparticles had twofold higher concentrations than the aqueous solution. The amount of drug in blood, iris/ciliary body, and aqueous humor were nearly indifferent. The same research group reported in 2006 that the cell viability in the presence and absence of chitosan nanoparticles was the same, with no signs of inflammation after cell uptake of nanoparticles (Salamanca et al. 2006). Chitosan nanoparticles are emerging as a new class of drug delivery