A microparticle/hydrogel combination drug-delivery system for sustained release of retinoids

Abstract

Purpose: To design and develop a drug-delivery system containing a combination of poly(D,L-lactide-co-glycolide) (PLGA) microparticles and alginate hydrogel for sustained release of retinoids to treat retinal blinding diseases that result from an inadequate supply of retinol and generation of 11-cis-retinal.

Methods: To study drug release in vivo, either the drug-loaded microparticle-hydrogel combination was injected subcutaneously or drug-loaded microparticles were injected intravitreally into Lrat(-/-) mice. Orally administered 9-cis-retinoids were used for comparison and drug concentrations in plasma were determined by HPLC. Electroretinography (ERG) and both chemical and histologic analyses were used to evaluate drug effects on visual function and morphology.

Results: Lrat(-/-) mice demonstrated sustained drug release from the microparticle/hydrogel combination that lasted 4 weeks after subcutaneous injection. Drug concentrations in plasma of the control group treated with the same oral dose rose to higher levels for 6-7 hours but then dropped markedly by 24 hours. Significantly increased ERG responses and a markedly improved retinal pigmented epithelium (RPE)-rod outer segment (ROS) interface were observed after subcutaneous injection of the drug-loaded delivery combination. Intravitreal injection of just 2% of the systemic dose of drug-loaded microparticles provided comparable therapeutic efficacy.

Conclusions: Sustained release of therapeutic levels of 9-cis-retinoids was achieved in Lrat(-/-) mice by subcutaneous injection in a microparticle/hydrogel drug-delivery system. Both subcutaneous and intravitreal injections of drug-loaded microparticles into Lrat(-/-) mice improved visual function and retinal structure.

Figure 1.

Schematic description of the microparticle/hydrogel combination delivery system. Scanning electron microscopic image of PLGA microparticles loaded with 9-cis-R-Ac. Schematic illustrating the preparation of a microparticle (brown) loaded with 9-cis-R-Ac (red) and combined with hydrogel. Chemical structure of the alginate monomer that condenses in the presence of Ca²⁺ to form the hydrogel.

Figure 2.

Retinol concentrations in the plasma of 5-week-old Lrat^−/− mice after a single dose of all-trans-R-Ac. Before administration of all-trans-R-Ac, 5-week-old Lrat^−/− mice were maintained for 1 month on a diet lacking vitamin A. For subcutaneous injection, all-trans-R-Ac in the microparticle/hydrogel delivery system was subcutaneously injected into Lrat^−/− mice in a single dose of 6 mg/mouse. For oral administration, all-trans-R-Ac in soybean oil was delivered by gastric gavage to Lrat^−/− mice at the same single dose of 6 mg/mouse. Inset: an expanded initial time course.

Figure 3.

Improved ERG and retinal histology 1 month after 5-week-old Lrat^−/− mice received subcutaneous injection of 9-cis-R-Ac in the microparticle/hydrogel delivery system. For the subcutaneous injection group, 9-cis-R-Ac in the microparticle/hydrogel suspension was subcutaneously injected into 5-week-old Lrat^−/− mice in a single dose of 6 mg/mouse (n = 9). For the oral administration group, 9-cis-R-Ac in soybean oil was delivered by gavage to Lrat^−/− mice in a single dose of 6 mg/mouse (n = 9). No drug was given to Lrat^−/− mice in the control group (n = 5). All mice were housed in a regular 12-hour light (<10 lux)/12-hour dark environment. (A, B) Illustration of a-wave and b-wave amplitudes, respectively. Data are presented as means ± SDs. (C) The rod outer segment (ROS) layer of the subcutaneous injection group was thicker than that of the untreated control group, and more tightly packed than ROS layers in both the orally gavaged and untreated control groups 1 month after subcutaneous injection of 9-cis-R-Ac in microparticle/hydrogel. Panels from left to right present cross-sections of retinas from representative Lrat^−/− mice after 9-cis-R-Ac subcutaneous injection (C), oral administration (D), and no treatment (control) (E), respectively. Labeled layers of the retina are as follows: ONL, outer nuclear layer; IS, inner segment; OS, outer segment; RPE, retinal pigmented epithelium.

Figure 4.

Improved ERG responses in 5-week-old Lrat^−/− mice 10 days after intravitreal injection of 9-cis-R-Ac in PLGA microparticles at 2% of the 6 mg per mouse dose used for subcutaneous injection and oral administration. ERG responses at high illumination intensities improved in the intravitreal group as compared with the control group and were comparable to those of the other 9-cis-R-Ac−treated groups. For the intravitreal injection group, 9-cis-R-Ac in microparticles was injected into mouse eye at a single dose of 0.06 mg/eye (n = 9). For the subcutaneous injection group, 9-cis-R-Ac in microparticle/hydrogel was subcutaneously injected into the Lrat^−/− mice in a single dose of 6 mg/mouse (n = 9). For the oral administration group, 9-cis-R-Ac in soybean oil was gavaged into Lrat^−/− mice in a single dose of 6 mg/mouse (n = 9). No drug was given to the control untreated group (n = 5). Lrat^−/− mice were housed in a darkroom after these treatments. (A, B) Quantification of a-wave and b-wave amplitudes, respectively. Data are presented as means ± SD.

Figure 5.

Retinal morphology 1 month after 5-week-old Lrat^−/− mice received an intravitreal injection of 9-cis-R-Ac in PLGA microparticles. No retinal damage was detected 1 month after intravitreal injection of these microparticles. (A, B) Representative histologic cross-sections of retinas from the intravitreal injected and uninjected control groups, respectively. (C, D) OCT images of representative retinas from the same two groups. Labeled layers of the retina are as follows: ONL, outer nuclear layer; OS, outer segment; RPE, retinal pigmented epithelium.

Figure 6.

Improved ERG responses 1 month after 5-week-old Lrat^−/− mice received an intravitreal injection of 9-cis-R-Ac in PLGA microparticles. For the intravitreal injection group, 9-cis-R-Ac in microparticles was injected into Lrat^−/− mouse eyes as a single dose of 0.06 mg per each eye (n = 9). For the vehicle group, PLGA microparticles lacking retinoids were intravitreally injected into each Lrat^−/− mouse eye instead (n = 5). (A) Representative ERG traces from 9-cis-R-Ac/microparticle−treated, vehicle-treated, and uninjected control animals. Data are presented as means ± SDs. The control group of Lrat^−/− mice received no injections (n = 5). (B, C) a- and b-wave amplitudes, respectively.