An Injectable Nano-Enabled Thermogel to Attain Controlled Delivery of p11 Peptide for the Potential Treatment of Ocular Angiogenic Disorders of the Posterior Segment

Abstract

This investigation focused on the design of an injectable nano-enabled thermogel (nano-thermogel) system to attain controlled delivery of p11 anti-angiogenic peptide for proposed effective prevention of neovascularisation and to overcome the drawbacks of the existing treatment approaches for ocular disorders characterised by angiogenesis, which employ multiple intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) antibodies. Synthesis of a polyethylene glycol-polycaprolactone-polyethylene glycol (PEG-PCL-PEG) triblock co-polymer was undertaken, followed by characterisation employing Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and differential scanning calorimetry (DSC) to ascertain the chemical stability and integrity of the co-polymer instituted for nano-thermogel formulation. The p11 anti-angiogenic peptide underwent encapsulation within poly(lactic-co-glycolic acid) (PLGA) nanoparticles via a double emulsion solvent evaporation method and was incorporated into the thermogel following characterisation by scanning electron microscopy (SEM), zeta size and zeta-potential analysis. The tube inversion approach and rheological analysis were employed to ascertain the thermo-sensitive sol-gel conversion of the nano-thermogel system. Chromatographic assessment of the in vitro release of the peptide was performed, with stability confirmation via Tris-Tricine PAGE (Polyacrylamide Gel Electrophoresis). In vitro biocompatibility of the nano-thermogel system was investigated employing a retinal cell line (ARP-19). A nanoparticle size range of 100-200 nm and peptide loading efficiency of 67% was achieved. Sol-gel conversion of the nano-thermogel was observed between 32-45 °C. Release of the peptide in vitro was sustained, with maintenance of stability, for 60 days. Biocompatibility assessment highlighted 97-99% cell viability with non-haemolytic ability, which supports the potential applicability of the nano-thermogel system for extended delivery of peptide for ocular disorder treatment.

Keywords: angiogenesis; nanoparticles; ocular drug delivery; peptide; thermosensitive hydrogel.

Figure 1

Schematic depiction of the componential formulation and proposed delivery of the thermo-nanogel system.

Figure 2

(a) Particle size and zeta potential analysis of the native and FITC-p11 peptide-encapsulated nanoparticles. (b) (i) SEM (scanning electron microscopy) image of nanoparticle. (ii) High magnification SEM image. (iii) Confocal microscopic image of the FITC-P11 peptide-encapsulated nanoparticles.

Figure 3

(a) Fourier-transform infrared spectroscopy of PEG, ε-caprolactone and PEG-PCL-PEG (polyethylene glycol-polycaprolactone-polyethylene glycol). (b) NMR spectroscopy of PEG-PCL-PEG.

Figure 4

(a) Graphical depiction of sol-gel conversion of Pluronic F127 (red) and PEG-PCL-PEG hydrogel (blue). (b) Photograph of tube inversion method for sol-gel conversion of the hydrogels at different concentrations. Rheological analysis of (c) PEG-PCL-PEG hydrogels at different concentrations and (d) comparative analysis of Pluronic, PEG-PCL-PEG and PEG-PCL-PEG/PLU hydrogels at a concentration of 25% w/v.

Figure 5

DSC (differential scanning calorimetry) thermograms of synthesised and lyophilised hydrogels (a) PEG-PCL-PEG, (b) Pluronic and (c) PEG-PCL-PEG/PLU.

Figure 6

Graphical depiction of swelling ratio of the investigated hydrogel systems.

Figure 7

(a) Plot depicting the in vitro p11 peptide release from PLGA nanoparticles, Pluronic hydrogel, PLU/PEG-PCL-PEG hydrogel, PEG-PCL-PEG hydrogel, nanoparticle-loaded PEG-PCL-PEG hydrogel and nanoparticle-loaded PEG-PCL-PEG/PLU hydrogel at 37 °C, in PBS for a period of 60 days. (b) SDS–PAGE results of p11 peptide in vitro release profile (Lane 1, marker; Lane 2, 5days; Lane 3, 10 days; Lane 4, 20 days; Lane 5, 35 days; Lane 6, 45 days; Lane 7, 55 days; Lane 8 control p11 peptide.

Figure 8

(a) Assessment of percentage cell viability after incubation with 30 days and 60 days release medium using MTT assay. (b) Analysis of haemolytic potential of different hydrogel systems and PLGA nanoparticles. Inset: Representative photograph showing no significant haemolysis in the hydrogel systems and PLGA nanoparticles in comparison to positive control (Triton × 100).