10.7  In Vivo Studies

10.7.1  In Vivo Injection of Microparticles

Local administration of microand nanoparticles has been carried out by intravitreal and periocular injections (Herrero-Vanrell and Refojo 2001; Kompella et al. 2003; Amrite and Kompella 2005; Ranta and Urtti 2006).

Injection of microparticles suspension has been usually made using conventional needles: 33, 30, 27, 26, 25, 23, 20, and 18-gauge. In some cases, the needle has been connected to a glass micropipette (tip diameter of 40 mm) (Herrero-Vanrell and Refojo 2001). Needles most frequently employed are 25–30 G for sizes 1–106 mm and 18 G for particle sizes up to 500 mm (Veloso et al. 1997; Urata et al. 1999; Lee et al. 2002; He et al. 2006).

The microspheres are suspended in a physiological solution that acts as the vehicle for the injection of the particles into the eye. The most frequent vehicles used to suspend microspheres for intraocular or periocular injections are isotonic phosphate buffer solution (PBS) or balanced salt solution (BSS) (pH 7.4). Because a significant proportion of the dose of microparticles suspended in PBS or BSS tended to adhere and remain in the syringe after its injection, some investigators have employed viscous vehicles to retain the particles in suspension better than the less viscous PBS and BSS (Veloso et al. 1997). Microparticles have been suspended in physiological solutions of hyaluronic acid (HA) or hydroxypropylmethyl cellulose (HPMC). These vehicles are commonly used as surgical aids in ophthalmology (Chan et al. 1984). Furthermore, these polymers form solutions that are transparent and biocompatible, and are rapidly diluted in the intraocular fluids and are eventually eliminated from the eye (Chan et al. 1984; Tolentino et al. 1989).

10.7.2  Ocular Disposition and Cellular Uptake

PLGA microparticles suffer aggregation after their intravitreal injection, diminishing the surface area and prolonging the drug release time. This effect has been already observed in animal models (rabbits) and humans (Giordano et al. 1995; Cardillo et al. 2006).

One concern of intravitreal injection of microspheres is regarding the behavior of the particles in the site of administration and the possibility of causing a visual impairing and/or vitreous haze following a single injection. However, preliminary investigation using triamcinolone PLGA microspheres for the treatment of diabetic macular edema in 25 human eyes has showed the opposite. In fact, in contrast to initial fears, the tendency of the microspheres to aggregate and condensate at the site of the injection and leave a free visual axis was clinically observed in all patients (Cardillo et al. 2006; Herrero-Vanrell and Cardillo 2010). However, when used in the eye, care should be taken to inject the microspheres such that they do not interfere with the visual pathway.

The movement of microspheres (7–10 mm size) was studied in phakic and ­aphakic eyes. The intravitreous injected microparticles were retained in the vitreous cavity in phakic and aphakic eyes of rabbits. On the contrary, some particles moved to the anterior chamber although most of them remained in the vitreous for long periods of time (Algvere and Martini, 1979).

In the case of periocular injection, the size of particles affects their ocular distribution.

Subconjunctival injection of nanoparticles (20 and 200 nm) and microparticles (2 mm) demonstrated that particles higher than 200 nm are retained in the site of injection up to 60 days (Amrite and Kompella 2005). The study was carried out with non biodegradable fluorescent polystyrene particles. The results obtained in this work have suggested the potential use of particulate systems of 200 nm and above for sustained drug delivery to the retina after periocular injection. On the contrary, if delivering the entire particle is desired, the subconjuctival administration is not a good approach.

Moritera et al. (1994) studied the cellular uptake of PLGA microand nanoparticles by RPE. The authors demonstrated that PLA and PLGA microparticles up to 1–2 mm size are susceptible to suffer phagocytosis by RPE cells.

10.7.3  Tolerance of Microparticles

Several reactions have been described after intraocular administration of PLGA microspheres. Khooebi et al. (1991) have pointed out the presence of whitish material in the vitreous cavity during the 10 days after injection of fluorescein PLGA microparticles that disappeared at 20–25 days after injection in rabbits (Veloso et al. 1997). However, histologically retinal and choroidal damage were not reported after 35 days of administration.

Regarding to other ocular reactions to microparticles, Veloso et al. (1997) described a mild localized foreign body reaction surrounding partially degraded ganciclovir loaded microspheres after their injection in rabbits. Histopathologic analysis at 4 and 8 weeks, after injection of microparticles, showed mononuclear cells and multinucleated giant cells with no involvement of the retina or other ocular structures. In the last case, biodegradation was virtually completed by day 63. In general, the foreign body reaction, associated with these polymers in the eye as well as intramuscular, gradually decreased with time. According to some authors, 12 weeks after surgery only degraded pieces of microparticles could be recognized remaining at the implantation site (Gould et al. 1994; Moritera et al. 1992).

Signs of inflammation have been described after intravitreal injection of microparticles in rabbits. These signs were similar to the ones reported for sutures and disappeared 2–4 weeks after administration (Giordano et al. 1995). Moreover, the reaction is similar to that described for microspheres injected intramuscularly in rabbits (Visscher et al. 1985; Park and Park, 1996). On the other hand, Amrite et al. (2006) described no signs of inflammation in the retina at 60 days after administration of the PLGA microspheres loaded with celecoxib. Furthermore, no significant changes were

observed in the thickness of retinal layers between untreated normal rats and normal animals treated with celecoxib microparticles. In this study, the visual inspection of the site of action (periocular tissue) did not reveal the presence of inflammation including redness and edema. The difference in terms of inflammation due to PLGA microparticles can be attributed to the different animals tested. It is well known that rabbits are more susceptible to inflammation than other animal species.

PLGA microspheres loaded with triamcinolone as potential treatment for diabetic macular edema have already been injected intravitreally in humans. Preliminary results have reported good tolerance after microparticle injection (Cardillo et al. 2006).

Periocular injection of poly(d,L lactide-co-glycolide) glucose microspheres nonloaded and loaded with 25% (42.5 mm) or 50% (67.7 mm) PKC412 caused mild conjunctival injection that resulted similar among the three groups. The authors reported discernible signs of inflammation or irritation. Gross pathologic examination of the eyes showed microspheres outside of sclera (Saishin et al. 2003).

Nonloaded PLGA microspheres and dexamethasone microspheres have been administered by periocular injection in rabbits. An amount of 5 mg suspended in BSS was administered by yuxtaescleral injection. IOP remained unchanged before, and 24 h, 1, 2, and 4 weeks after blank MP and Dx-MP administration. No adverse signs were observed after injection of formulations in terms of conjunctival discharge, conjunctival swelling, aqueous flare, light reflex, iris involvement, cornea, surface of cornea cloudiness, pannus, fluorescein stain, lens, vitreous opacity, vascular congestion, vitreal and retinal hemorrhage, and retinal detachment (RD). The only sign observed was a conjunctival congestion at the injection site at 24 h and 2 weeks postinjection for unloaded microspheres and 24 h and 1 week for dexamethasone microspheres. Authors concluded that PLGA microparticles unloaded and loaded with dexamethasone are suitable for juxtascleral injection with no adverse effects (Barbosa et al. 2010).

In terms of intraocular tolerance, the nature of the polymer is critical. Rincon et al. (2005) studied the response to microparticles prepared from an elastin derivative poly (valine-proline-alanine-valine-guanine) (VPAVG). Although no inflammatory response was observed after subcutaneous injection in the hind-paw of the rat and only a few eyes (2/11) of the experimental group presented inflammation signs after intravitreal injection of 2.5 mg of poly (VPAVG) microparticles, 45% (5/11) of the animals showed tractional retinal detachment (TRD). This adverse effect was related to certain fibroblastic activity induced by the polymer.

10.7.4  In Vivo Degradation of PLA and PLGA Microparticles

Experience has demonstrated that the PLA and PLGA polymers are biocompatible. Their biodegradation products, lactic acid and glycolic acid, are also biocompatible and easily eliminated from the body (Colthrust et al. 2000).

As cited previously, the rate of polymer biodegradation mainly depends on the polymer composition (Thomas et al. 1993; Robinson 1993) and its molecular weight (Miller et al. 1977). Others factors such as particle size (total surface area)