Additives such as pluronic F68, polyethylene glycol (PEG) 30%, and migliol have been used to improve the release rate of the drug and diminish the initial burst (He et al. 2006). Addition of an appropriate amount of gelatin in the external phase of the emulsion of PLGA microspheres loaded with acyclovir allowed diminishing the dose of microspheres to be administered by intraocular injection (MartinezSancho et al. 2003b).

A significant challenge in intravitreal drug delivery is the use of additives with therapeutic activity. This is the case of oils such as retinoic acid (RA) (vitamin A) and a-tocopherol (vitamin E) that have antiproliferative and antioxidant properties. Vitamin A was added to the acyclovir PLGA 50:50 microspheres resulting in a more prolonged release of the acyclovir. In this formulation, RA improved the release of acyclovir and potentially prevents the adverse effects of intravitreal injections (Martinez-Sancho et al. 2006).

A novel concept of “combo” microspheres in which more than one active substance is encapsulated has been recently introduced in intraocular drug delivery for the treatment of glaucoma. Checa et al. (2011) have prepared microspheres loaded with a neurotrophic factor GDNF and vitamin E for glaucoma treatment. Under the technological point of view, the addition of the oil produces an increase of GDNF encapsulation efficiency and prolongs its release rate up to 19 weeks. Furthermore, vitamin E is released from the microparticles. Microspheres loaded with GDNF and Vitamin E have been injected in humans (Fig. 10.10).

10.4  Sterilization of Microparticles

Sterility is a critical factor for the intraocular systems. A final sterilization is preferred over aseptic conditions. Nevertheless, PLGA particles are sensitive to most sterilization methods usually employed (heat and ethylene chloride). Gamma irradiation has a high capacity for penetration. The dose required to assure sterilization of a pharmaceutical product is 25 kGy. This procedure has been employed to sterilize microparticles. However, gamma-irradiation of bioresorbable polyesters induces dose-dependent chain scission as well as molecular weight reduction, affecting the properties of the final product (Nijsen et al. 2002). The reduction of the polymer molecular weight accelerates degradation of the polyester. Furthermore, the degradation rate of polymeric biomaterials as PLGA due to gamma-irradiation has been linked to radical formation (Sintzel et al. 1998) and a decrease of Tg values of PLGA favoring subsequent reactions of free radicals due to a higher mobility of the polymer chains (Bittner et al. 1999). This technological problem can be solved by using low temperatures during the exposure time of the microparticles to gamma-radiation. Sterilization by gamma radiation at low temperature has presented optimal results with formulations including ganciclovir, acyclovir, and celocoxib for intravitreal injection ((Herrero-Vanrell et al. 2000; Martinez et al. 2004;

Fig. 10.11  Percentage of ganciclovir released in PBS (1.5 ml) from 10 mg of PLGA microspheres. Size of particles (300–500 mm). Sterilized (filled triangles) nonsterilized (filled cirle). Adapted from Herrero-Vanrell et al. (2000)

Amrite et al. 2006). In the case of ganciclovir, the release rate of the drug from PLGA 50:50 microspheres was not significantly affected by gamma radiation exposure at low temperature. In this work, the release of the drug was compared before and after sterilization by gamma radiation (25 kGy). Release profiles before and after sterilization were compared using the similarity factor (f2). The values of this factor range from 0 to 100 with a higher similarity factor value indicating higher resemblance between two release curves. In the case of the reported work the release rate of the active substance was not significantly affected by the sterilization procedure with a value of f2 higher than 85 (Fig. 10.11).

Change in particle size due to aggregation after gamma irradiation exposure can be avoided with low temperatures. Martinez et al. (2004) reported similar mean diameters of sterilized (45.47 ± 13.36 mm) and nonsterilized (46.38 ± 12.79 mm) microspheres. The authors reported no morphological change in acyclovir microspheres after gamma-irradiation treatment because samples were protected with dry ice during irradiation exposure.

Microparticles loaded with celecoxib (14.93%) were sterilized by gamma radiation at 25 kGy at low temperature. The sterilization process was not significantly affected by the release profile of the active substance from celecoxib PLGA loaded microspheres. The release was slightly lower at a few intermediate time points for the nonsterilized microspheres although the differences were not significant (Amrite et al. 2006).

10.5  Calculation of the Dose of Microparticles for Injection

The amount of microspheres to be injected in the vitreous can be theoretically calculated according to the following mathematical equation:

K0 =Css ´Cl

where Css is the effective drug concentration that has to be maintained in the vitreous and Cl is the drug clearance in the vitreous. These two parameters allow calculating K0 that is the theoretical zero-order drug release rate from the microspheres to achieve therapeutic levels in the vitreous.

An estimation of the drug clearance from the vitreous can be calculated according to the general equation:

Cl =Vd ´ Ke =Vd ´ 0.693 ,

t1/ 2

in which Vd is the volume of the vitreous (i.e., 1.5 ml in rabbits, 4.5 ml in humans, 5 ml in rats, etc.) and Ke is the drug intravitreal elimination rate constant which can be easily derived from the half-life of the drug.

Once calculated, K0 is employed to determine the minimum amount of microspheres necessary to provide effective concentrations in the vitreous and represents the minimum amount of drug per time released from the microspheres to achieve Css . Generally, the release rate is expressed in mg/day.

10.6  Injectability Studies

Injectability of microspheres is an important criterion because it allows testing the minimum needle diameter for a successful intravitreal injection. Application of a maximum ejection force of 12 Newtons over 10 s can be considered as suitable for a properly intraocular injection. Tests are carried out on a suspension of microspheres in an aqueous vehicle employed in the clinical practice to inject the particles (e.g., saline solution, BSS, or phosphate buffer pH 7.4). Then, suspensions of microparticles are injected through different needle diameters. Injectability values lower than 12 s indicate neither partial nor complete blockage of the suspension flow.

Martinez et al. (2004) evaluated the injectability of sterilized acyclovir PLGA 50:50 (15,000 Da) size 20–40 mm. To this, particles were injected through different needle diameters (27G, 25G, and 21G). Data (12.5, 8.4, and 5.5 N, respectively) indicated neither partial nor complete blockage of the suspension flow. Thus, the developed microspheres were considered suitable for intraocular injection through a 27G needle.