The nature of peptide interactions with acid end-group PLGAs and facile aqueous-based microencapsulation of therapeutic peptides

Abstract

An important poorly understood phenomenon in controlled-release depots involves the strong interaction between common cationic peptides and low Mw free acid end-group poly(lactic-co-glycolic acids) (PLGAs) used to achieve continuous peptide release kinetics. The kinetics of peptide sorption to PLGA was examined by incubating peptide solutions of 0.2-4mM octreotide or leuprolide acetate salts in a 0.1M HEPES buffer, pH7.4, with polymer particles or films at 4-37°C for 24h. The extent of absorption/loading of peptides in PLGA particles/films was assayed by two-phase extraction and amino acid analysis. Confocal Raman microspectroscopy, stimulated Raman scattering (SRS) and laser scanning confocal imaging, and microtome sectioning techniques were used to examine peptide penetration into the polymer phase. The release of sorbed peptide from leuprolide-PLGA particles was evaluated both in vitro (PBST+0.02% sodium azide, 37°C) and in vivo (male Sprague-Dawley rats). We found that when the PLGA-COOH chains are sufficiently mobilized, therapeutic peptides not only bind at the surface, a common belief to date, but also can be internalized and distributed throughout the polymer phase at physiological temperature forming a salt with low-molecular weight PLGA-COOH. Importantly, absorption of leuprolide into low MW PLGA-COOH particles yielded ~17 wt.% leuprolide loading in the polymer (i.e., ~70% of PLGA-COOH acids occupied), and the absorbed peptide was released from the polymer for >2 weeks in a controlled fashion in vitro and as indicated by sustained testosterone suppression in male Sprague-Dawley rats. This new approach, which bypasses the traditional encapsulation method and associated production cost, opens up the potential for facile production of low-cost controlled-release injectable depots for leuprolide and related peptides.

Keywords: Controlled release; Kinetics; Microencapsulation; PLGA; Peptide; Sorption.

Fig. 1

Characterization of peptide sorption to PLGA-COOH 50:50 at 37 °C. A) Effect of peptide type [leuprolide (○) vs. octreotide (●)] on sorption kinetics. B) 24-h sorption isotherm of leuprolide (○) and octreotide (●); C_f = final peptide concentration. C) Effect of ionic strength at pH 7 (0.1 M phosphate buffer (236 mM, ●), 0.1 M HEPES buffer (49 mM, ▲), 10 mM phosphate buffer (23 mM, ○) and 10 mM HEPES buffer (4 mM, Δ)) on the kinetics of octreotide sorption from 0.42 mM initial peptide concentration. D) Effect of pH (0.1 M HEPES buffer, pH 7.4 (▲), 0.1 M MES buffer, pH 5.5 (■), and 0.05 M DEPP buffer, pH 4.0 (●) on 24-h octreotide sorption isotherms. Sorption studies of A and B were conducted in 0.1 M HEPES buffer, pH 7. Initial peptide concentration was 0.4 mM in A and C and in the range of 0.2-4.0 mM in B and D. Symbols represent mean of 3 determinations. Dashed lines represent fits to a biexponential (A and C), and Langmuir models (B and D).

Fig. 2

Effect of polymer M_w [A: Boehringer-Ingelheim Resomer® RG 502H (●), RG 503H (▲), and RG 504H (□)], incubation temperature (B: 37 (Δ), 25 (▲), and 4 (■) °C) and polymer mass/film thickness (C: octreotide sorption at 22 (●), 30 (▲) and 37 (Δ) °C, and leuprolide sorption at 37 °C (■) after 24 h) on peptide sorption to PLGA, and medium on desorption of peptide from PLGA particles or films (D). Sorption of peptides to PLGA was performed at 37 °C in 0.1 M HEPES buffer (pH 7.4) with an initial peptide concentration of 0.4 (A and B) or 1 (C and D) mM. RG 502H polymer was used in sorption and desorption studies unless otherwise specified. Dashed lines represent fits to a biexponential model. Desorption of octreotide from PLGA 50:50 particles (E-M) or films (N) was performed in 5 wt% SDS in water (E); 50 vol% methanol in water (F); 1 mg/mL PEI in 0.1 M acetate buffer, pH 4.0 (G); 0.1 M HEPES, pH 7.4 (H); 0.1% TFA in 0.1 M HEPES, pH 7.0 (I); 0.1 M DEPP, pH 4.0 (J); 0.1 M HEPES, pH 7.4 (K: particles and N: films); 2 M CaCl₂ in HEPES (L); 0.1 M HEPES, pH 7.4 (M). Desorption studies of E to L, and N were performed at 37 °C, except N, which was at 4 °C. PLGA RG502H was used in all cases, except H, which used RG503H. Desorption of octreotide in case of SDS (E) was assessed by recovering sorbed octreotide via two-phase extraction. Symbols represent mean of 3 determinations.

Fig. 3

Direct visualization of peptide (leuprolide) penetration in PLGA film by SRS imaging. A) Raman spectra from pure PLGA film (red) and leuprolide (green). The peaks at 1764 cm⁻¹ and 1545 cm⁻¹ were identified for selective SRS imaging of PLGA and leuprolide, respectively. B) SRS image of PLGA distribution (red) in the film at 1764 cm⁻¹. C) SRS image of leuprolide distribution (green) at the same location and depth in the film at 1545 cm⁻¹. D) Overlay image of B) and C). E) Average intensity of PLGA and leuprolide in the area indicated by the white box in D as a function of depth along the film. Data was normalized against maximum intensity values. Note that the bell-shaped intensity curves are present for both PLGA and leuprolide, and are an artifact of the SRS imaging method.

Fig. 4

Long-term in vitro leuprolide release behavior (A) and in vivo testosterone suppression ability (B) of leuprolide-absorbed PLGA-COOH formulation. Studies were conducted in PBST + 0.02% sodium azide at 37 °C (A) or male Sprague-Dawley rats (B). Animals were injected subcutaneously with soluble leuprolide at day 0 (●), leuprolide-PLGA particles with various dosing intervals (2 week: days 0, 14, 28, and 42 (▲), 3 week: days 21 and 42 (◇), and 4 week: days 0 and 28 (▼)), and blank PLGA particles (○) along with commercial 1-month Lupron Depot® at days 0 and 28 (□). Leuprolide dose was 100 μg/kg/day. Solid and dashed line respectively represents lower testosterone detection limit (0.1 ng/mL) and castration level (0.5 ng/mL). Symbols represent mean ± SEM [n = 3 (A) or 6 (B)] with error bars not visible when smaller than symbols.