Nanoscaled buffering zone of charged (PLGA)n-b-bPEI micelles in acidic microclimate for potential protein delivery application

Abstract

Poly(lactide-co-glycolide) (PLGA) has most often been employed for the controlled release of protein formulations because of its safety profile with non-toxic degradation products. Nevertheless, such formulations have been plagued by a local acidic microenvironment and protein-polymer interactions, which result in chemical and physical denaturation of loaded proteins and often unfavorable release profiles. This study investigated the pH change of inner PLGA microsphere (MS) using charged (PLGA)(n)-b-branched polyethyleneimine (bPEI) micelles. The designed micelles can be transformed into either micelle or reverse micelle (RM) depending on the solvent and RM can form microspheres. In addition, (PLGA)(n)-b-bPEI can be modified into (PLGA)(n)-b-(carboxylated bPEI) via carboxylation of the primary amines. Cationic micelle (CM) or anionic micelle (AM) was complexed with counter-charged proteins leading to nanosized particles (approximately 100nm). In the micelle/protein complexes, the micelles mostly maintained their proton buffering capacity, and consequently, prevented or delayed the typical decrease in pH caused by degradation of PLGA in aqueous solution. Reconstitutable micelle/protein complexes allowed for increased and fine-tuned protein loading (~20wt.% when using CM1 (CM prepared from PLGA(36kDa)-b-bPEI(25kDa))/insulin complexes) in PLGA MS. In CM2 (CM prepared from (PLGA(36kDa))(2)-b-bPEI(25kDa))/insulin (4 of weight ratio (WR) of micelle to protein; WR4)-loaded PLGA MS, CM2 strongly prevented the micellar nanoenvironmental pH (pH 6.6 within 5days and then approximately pH 8.5) to be acidified in PLGA MS for 9weeks, unlike CM2-free PLGA MS. In conclusion, our findings propose that the proton buffering capacity and protein loading in PLGA MS can be tuned by controlling the complexation ratios of micelles and proteins, polymeric architectures of (PLGA)(n)-b-bPEI copolymers and WR of micelle/protein complexes and PLGA (or RM).

Fig. 1

(a) Synthetic scheme of (PLGA)_n-b-bPEI block copolymers and their carboxylated copolymers and (b) schematic representation of the structures of the block copolymers, micelles, micelle/protein complexes, and micelle/protein-loaded PLGA microspheres.

Fig. 2

¹H-NMR spectra and micellar structures of (PLGA)_n-b-bPEI_25kDa block copolymers in D₂O and CD₂Cl₂.

Fig. 3

Size and surface charge of charged micelles derived from ± (PLGA)_n-b-bPEI_25kDa block copolymers.

Fig. 4

(a) CM- and WR-dependent hydrodynamic (Z_avg) particle sizes and surface charges of the CM/insulin complexes (n=3, Mean±SD) and (b) number-based particle sizes (n=3, Mean±SD), (c) surface charges of fresh and reconstituted CM2/insulin complexes (n=3, Mean±SD), and (d) WR-dependent estimated configuration of CM/insulin complexes.

Fig. 5

Hydrodynamic particle sizes and surface charges of the CM2/IgG complexes and the AM2/Lysozyme complexes. (n=3, Mean±SD)

Fig. 6

Acid titration curves of micelles (CM or AM) and their protein complexes (CM/insulin or AM/lysozyme). Protein concentration was 1 mg/mL. (n=3, Mean±SD)

Fig. 7

Solution pH-dependent fluorophore intensity ratios of fluorophore solutions (FITC and RITC) and fluorophore-labeled CM2

Fig. 8

Time-dependent fluorophore intensity ratios, solution pH and estimated micellar pH of fluorophore-labeled CM2 incubated in DPBS (initial pH 7.4) at 37°C. (n=3, Mean±SD)

Fig. 9

Time-dependent fluorophore intensity ratios, solution pH and estimated nanoenvironmental pH of CM2/insulin (WR 4)-loaded PLGA_36kDa MS incubated in DPBS (initial pH 7.4) at 37°C. (n=3, Mean±SD)