Mucoadhesive PLGA Nanospheres and Nanocapsules for Lactoferrin Controlled Ocular Delivery

Abstract

Background: the present work describes the preparation, characterization and optimization of eight types of PLGA-based nanosystems (nanospheres and nanocapsules) as innovative mucoadhesive drug delivery systems of lactoferrin, in order to achieve a preclinical consistent base as an alternative pharmacological treatment to different ocular syndromes and diseases.

Methods: All different nanoparticles were prepared via two modified nanoprecipitation techniques, using a three-component mixture of drug/polymer/surfactant (Lf/PLGA/Poloxamer), as a way to overcome the inherent limitations of conventional PLGA NPs. These modified polymeric nanocarriers, intended for topical ophthalmic administration, were subjected to in vitro characterization, surface modification and in vitro and in vivo assessments.

Results: An appropriate size range, uniform size distribution and negative ζ potential values were obtained for all types of formulations. Lactoferrin could be effectively included into all types of nanoparticles with appropriate encapsulation efficiency and loading capacity values. A greater, extended, and controlled delivery of Lf from the polymeric matrix was observed through the in vitro release studies. No instability or cytotoxicity was proved for all the formulations by means of organotypic models. Additionally, mucoadhesive in vitro and in vivo experiments show a significant increase in the residence time of the nanoparticles in the eye surface.

Conclusions: all types of prepared PLGA nanoparticles might be a potential alternative for the topical ophthalmic administration of lactoferrin.

Keywords: PLGA; corneal ecstatic disorder; keratoconus; lactoferrin; nanoparticles; nanoprecipitation; protein nanocarriers; topical ophthalmic administration.

Figure 1

Simplified representation of the PLGA-based nanospheres elaboration process.

Figure 2

Simplified representation of the PLGA-based nanocapsules elaboration process.

Figure 3

Comparison of size and ζ potential differences for blank and lactoferrin-loaded PLGA-based nanoparticles. A one-way ANOVA statistical analysis was applied, and no statistically significant differences were found among the formulations (p > 0.05), regardless of the polymer used.

Figure 4

TEM images of lactoferrin-loaded PLGA NSs and lactoferrin-loaded PLGA NCs.

Figure 5

PY, EE, and LC values for PLGA-nanoparticles. No statistically significant differences were found in terms of PY and EE (p > 0.05). Nevertheless, considerable differences were observed for LC values, where NCs-based formulations permitted the obtention of higher Lf-embedded content into the polymer matrix, compared to the NSs-based formulations (p < 0.0001).

Figure 6

Stability-to-storage study for PLGA-based NSs at three different temperature conditions: (I) 4 ± 2 °C, (II) 25 ± 2 °C and (III) 37 ± 2 °C, respectively.

Figure 7

Stability-to-storage study for PLGA-based NCs at three different temperature conditions: (I) 4 ± 2 °C, (II) 25 ± 2 °C and (III) 37 ± 2 °C, respectively.

Figure 8

Changes in size and ζ potential values of lactoferrin-loaded PLGA NSs over the studied pH interval.

Figure 9

Changes in size and ζ potential values of lactoferrin-loaded PLGA NCs over the studied pH interval.

Figure 10

Changes in size and ζ potential values of lactoferrin-loaded PLGA NSs over the studied ionic strength interval.

Figure 11

Changes in size and ζ potential values of lactoferrin-loaded PLGA NCs over the studied ionic strength interval.

Figure 12

In vitro release study of Lf from PLGA NSs (a) and NCs (b). The first graph embodies the raw amount of Lf corrected by the available surface released from the PLGA NSs over the 24 h period. The second graph represents the percentage of Lf released from the PLGA NSs over the 24 h period. No statistically significant differences were observed among all the PLGA-based formulations (p > 0.05).

Figure 13

Resulting data of the BCOP test for PLGA-based NSs (top) and NCS (bottom). (a) final corneal transparency values measured by UV-Vis spectrophotometry; (b) final corneal transparency values measured by luxmetry, and (c) fluorescein permeability measured by UV-Vis spectrophotometry.

Figure 14

Resulting images of CAM membranes after the PLGA-based nanoparticles administration during the HET-CAM test, compared to the control solutions: (A) 502 PLGA NSs; (B) 502H PLGA NSs; (C) 503 PLGA NSs; (D) 503H PLGA NSs; (E) 502 PLGA NCs; (F) 502H PLGA NCs; (G) 503 PLGA NCs; (H) 503H PLGA NCs; (I) NaCl aqueous solution; and (J) NaOH aqueous solution.

Figure 15

Ex vivo mucoadhesion data for PLGA-based nanoparticles. A one-way ANOVA analysis was applied for the PLGA NSs, showing no differences among the formulations (p > 0.05), except for the 502 PLGA NSs, which showed a higher mucoadhesion than the rest, being statistically significant (p < 0.02). Nevertheless, the same one-way ANOVA analysis was applied for the PLGA NCs, and no significant differences were observed between the prepared formulations, even for the 502 PLGA NCs compared to the others (p > 0.05).

Figure 16

¹⁸F-Choline (top), ¹⁸F-FDG (middle) and ⁶⁸Ga-DOTA (bottom) radiolabeling stability and efficiency for PLGA-based nanoparticles over time.

Figure 17

Ocular biopermanence of a ¹⁸F-Choline radiolabeled PLGA-based nanoparticles and a ¹⁸F-FDG aqueous solution (control), assessed pondering the primary biopermanence data (%) in the ROI. Statistically significant differences (p < 0.05) were observed regarding PLGA-based formulations and 18F-FDG solution.

Figure 18

Fused PET/CT images during the 5 h studied interval. (I) ¹⁸F-FDG buffered aqueous solution, (II) ¹⁸F-Choline radiolabeled PLGA NSs and (III) ¹⁸F-Choline radiolabeled PLGA NCs.