Synthesis and Characterization of Nanoparticle-Based Dexamethasone-Polypeptide Conjugates as Potential Intravitreal Delivery Systems

Abstract

The use of dexamethasone for eye disease treatment is limited by its low solubility, bioavailability, and rapid elimination when applied topically. The covalent conjugation of dexamethasone with polymeric carriers is a promising strategy to overcome existing drawbacks. In this work, amphiphilic polypeptides capable of self-assembly into nanoparticles were proposed as potential delivery systems for intravitreal delivery. The nanoparticles were prepared and characterized using poly(L-glutamic acid-co-D-phenylalanine) and poly(L-lysine-co-D/L-phenylalanine) as well as poly(L-lysine-co-D/L-phenylalanine) covered with heparin. The critical association concentration for the polypeptides obtained was in the 4.2-9.4 μg/mL range. The hydrodynamic size of the formed nanoparticles was between 90 and 210 nm, and they had an index of polydispersity between 0.08 and 0.27 and an absolute zeta-potential value between 20 and 45 mV. The ability of nanoparticles to migrate in the vitreous humor was examined using intact porcine vitreous. Conjugation of DEX with polypeptides was performed by additional succinylation of DEX and activation of carboxyl groups introduced to react with primary amines in polypeptides. The structures of all intermediate and final compounds were verified by ¹H NMR spectroscopy. The amount of conjugated DEX can be varied from 6 to 220 µg/mg of polymer. The hydrodynamic diameter of the nanoparticle-based conjugates was increased to 200-370 nm, depending on the polymer sample and drug loading. The release of DEX from the conjugates due to hydrolysis of the ester bond between DEX and the succinyl moiety was studied both in a buffer medium and a vitreous/buffer mixture (50/50, v/v). As expected, the release in the vitreous medium was faster. However, the release rate could be controlled in the range of 96-192 h by varying the polymer composition. In addition, several mathematical models were used to assess the release profiles and figure out how DEX is released.

Keywords: amphiphilic polypeptides; dexamethasone; drug delivery systems; intravitreal delivery; polymer-drug conjugates; self-assembled nanoparticles.

Figure 1

Stability of nanoparticles under simulated physiological conditions (PBS, pH 7.4, containing papain, 37 °C): P[KF]_L nanoparticles with initial D_H of 200 ± 20 nm; P[KF]_D,L nanoparticles with initial D_H of 110 ± 10 nm.

Figure 2

Trajectories of different nanoparticles based in the intact porcine vitreous: anionic P[EF]-2 nanoparticles (a), anionic P[KF]_D,L-2/HEP nanoparticles (b), and cationic P[KF]_D,L-2 nanoparticles (c). Nanoparticles based on P[EF]-2 were labeled with Cy5; P[KF]_D,L-2-based samples were labeled with Cy3 dye. Scale bar corresponds to 2 µm.

Figure 3

Effect of P[EF]_D-2 and P[KF]_D,L-1 nanoparticles on ARPE-19 cell proliferation for 24 h (a) and 72 h (b).

Figure 4

Scheme for the synthesis of DEX-polypeptide conjugates: DEX succinylation (a), P[EF] modification with ethylenediamine (b), and conjugation of polypeptides with succinylated DEX (c).

Figure 5

¹H NMR spectra of P[EF]-EDA(BOC) (a) and P[EF]-EDA (b) (DMSO-d₆).

Figure 6

¹H NMR spectrum of P[EF]-DEX (a) and P[KF]-DEX (b) conjugates (DMSO-d₆).

Figure 7

Schematic representation of the different types of self-assembled nanoparticle-based DEX-conjugates: P[EF]-EX (a), P[KF]-DEX (b) and P[KF]-DEX/HEP (c).

Figure 8

Dependence of DEX-content conjugated to the polypeptide (P[KF]_D,L-1) on the amount of DEX-S taken for conjugation.

Figure 9

TEM images of nanoparticles and nanoparticle-based polypeptide conjugates with DEX (scale bar 0.5 μm): P[EF]-2 (a), P[EF]-2-DEX (b), P[KF]_DL-2 (c), P[KF]_DL-2-DEX (d), P[KF]_DL-2-DEX/HEP (e).

Figure 10

DLS monitoring of hydrodynamic diameters over time for neat nanoparticles and their conjugates with DEX stored at 0.01 M phosphate buffer solution and room temperature (20 °C).

Figure 11

DEX release profiles from the various delivery systems: lysine- and glutamic acid-based conjugates in PBS (a), heparin-covered and non-covered lysine-based conjugates (P[KF]_D,L-2/DEX/HEP and P[KF]_D,L-2/DEX) and encapsulated nanoparticles (P[KF]_D,L-2(DEX)/HEP and P[KF]_D,L-2(DEX)) in 0.01 M PBS (pH 7.4) (b), and lysine- and glutamic acid-based conjugates in the vitreous/PBS mixture (50/50, v/v) (c) (incubation at 37 °C).