Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery

Abstract

In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)-polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen's egg test-chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery.

Keywords: anti-inflammatory; controlled release; flurbiprofen; ocular tolerance; peptide for ocular delivery.

Figure 1

Synthesis of (A) PLGA-NPs-PEG-peptide and (B) PLGA-PEG-peptide polymer followed NPs preparation. Abbreviations: EDC, ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; FB, flurbiprofen; NHS, N-hydroxysuccinimide; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); SH, sulfhydryl group.

Figure 2

The ¹H-NMR spectra of PLGA-PEG-POD and PLGA-PEG-HIV-Tat confirm the peptide coupling to PLGA-PEG-maleimide polymer. Notes: ¹H-NMR spectra in DMSO-d₆ at 25°C of (A) PLGA-PEG-maleimide-POD copolymer and POD peptide. We used the specified amide/amine side chain region for peptide signal integration. (B) For PLGA-PEG-maleimide-HIV-Tat and PLGA-PEG-maleimide, we clearly see a reduction of the maleimide methine protons signal intensity and the appearance of Tyr aromatic protons from HIV-Tat peptide after conjugation. Abbreviations: ¹H-NMR, proton nuclear magnetic resonance; DMF, N,N-Dimethylformamide; DMSO, dimethyl sulfoxide-d₆; HIV-Tat, human immunodeficiency virus transactivator; Mal, maleimide; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; Tyr, tyrosine.

Figure 3

In vitro release profiles of FB from (A) PLGA-NPs-PEG-peptide, (B) PLGA-PEG NPs, and PLGA-PEG-peptide NPs. Abbreviations: FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PEG^®, Resomer^® RGP type d5055; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure 4

CLSM images of a cross-section of (A) corneal epithelium at depth of 10 μm, (B) pretreated with PLGA-PEG-HIV-Tat-Rho NPs, (C) pretreated with PLGA-PEG-POD-Rho NPs, (D) their corresponding phase contrast with nucleus, and (E) cell membranes. Abbreviations: CLSM, confocal laser scanning microscopy; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; Rho, 5(6)-carboxytetramethylrhodamine.

Figure 5

Cytotoxicity of PLGA-PEG-peptide NPs on (A) HepG2 and (B) HeLa cells lines using the MTT assay, and photographs of (C) HepG2 and HeLa cells incubated with NPs to LD₅₀ and LD₁₀₀ concentrations. Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; LD₅₀, median lethal dose; LD₁₀₀, lethal dose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure 6

Comparison of anti-inflammatory efficacy of PLGA-PEG NPs, PLGA-PEG-peptide NPs, and Ocufen^® in the (A) treatment and (B) prevention of ocular inflammation induced by SA in the rabbit eye. Notes: Values are expressed as mean ± SD; *P<0.05, **P<0.01, and ***P<0.001 significantly lower than the inflammatory effect induced by SA; ^$P<0.05, ^$$P<0.01, and ^$$$P<0.001 significantly lower than anti-inflammatory efficacy of Ocufen^®. Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SA, sodium arachidonate; SD, standard deviation.