Besifloxacin liposomes with positively charged additives for an improved topical ocular delivery

Abstract

Topical ophthalmic antibiotics show low efficacy due to the well-known physiological defense mechanisms of the eye, which prevents the penetration of exogenous substances. Here, we aimed to incorporate besifloxacin into liposomes containing amines as positively charged additives and to evaluate the influence of this charge on drug delivery in two situations: (i) iontophoretic and (ii) passive treatments. Hypothesis are (i) charge might enhance the electromigration component upon current application improving penetration efficiency for a burst drug delivery, and (ii) positive charge might prolong formulation residence time, hence drug penetration. Liposomes elaborated with phosphatidylcholine (LP PC) or phosphatidylcholine and spermine (LP PC: SPM) were stable under storage at 6 ºC for 30 days, showed mucoadhesive characteristics, and were non-irritant, according to HET-CAM tests. Electron paramagnetic resonance spectroscopy measurements showed that neither the drug nor spermine incorporations produced evident alterations in the fluidity of the liposome's membranes, which retained their structural stability even under iontophoretic conditions. Mean diameter and zeta potential were 177.2 ± 2.7 nm and - 5.7 ± 0.3 mV, respectively, for LP PC; and 175.4 ± 1.9 nm and + 19.5 ± 1.0 mV, respectively, for LP PC:SPM. The minimal inhibitory concentration (MIC) and the minimal bactericide concentration (MBC) of the liposomes for P. aeruginosa showed values lower than the commercial formulation (Besivance). Nevertheless, both formulations presented a similar increase in permeability upon the electric current application. Hence, liposome charge incorporation did not prove to be additionally advantageous for iontophoretic therapy. Passive drug penetration was evaluated through a novel in vitro ocular model that simulates the lacrimal flow and challenges the formulation resistance in the passive delivery situation. As expected, LP PC: SPM showed higher permeation than the control (Besivance). In conclusion, besifloxacin incorporation into positively charged liposomes improved passive topical delivery and can be a good strategy to improve topical ophthalmic treatments.

Figure 1

In vitro ocular model that simulates the lacrimal flow, maintaining a 20 µL/min-flow of PBS through a peristaltic pump.

Figure 2

TEM images of (A) LP PC and (B) LP PC: SPM, indicated by white arrows (magnifications of ×80,000).

Figure 3

Mucoadhesive effect of the formulations LP PC (A) and LP PC: SPM (B) by hydrodynamic diameter variation of mucin 1% incorporation (t = 5 min). The grey line represents the hydrodynamic diameter of the liposome, the black, mucin 1%, and the dashed, the mixture of the liposome and mucin 1%.

Figure 4

Stability of the liposomes’ formulations. (A) hydrodynamic diameter and PdI, (B) encapsulation efficiency (EE), and (C) zeta potential of LP PC and LP PC: SPM stored at 6ºC for 30 days.

Figure 5

Irritability evaluation of LP PC, LP PC: SPM, and commercial formulation (Besivance 0.6%) in egg chorioallantoic membrane (CAM) compared to the positive control (+) using sodium hydroxide solution (NaOH) (1 mol/L) and negative control (−) using PBS.

Figure 6

DSC curves of liposomes components as supplied, and their equimass mixture.

Figure 7

EPR spectra of liposomes labeled with the TEMPO (A) and 5-DSA (B) spin probes. The arrows (in spectra of A) and the dotted lines (in spectra of B) illustrate how the partitioning parameter (f = H/(H + P)) and the maximum hyperfine splitting parameter (2A_||) were obtained from the experimental line. Large values of f indicate high partitioning, i.e., high spin label-membrane interaction and large values of 2A_|| indicate more restricted molecular motion of the spin probe. EPR spectra of the 5-DSA spin label associated with (C) LP PC and (D) LP PC:SPM-besifloxacin liposomes at different temperatures. The values of the maximum hyperfine splitting parameter 2A_|| are indicated for each spectrum (determined as indicated in the last spectrum). The experimental error associated with the 2A_|| values is 0.5 G. The intensities of the experimental spectra (on the y-axis) are normalized, and the total magnetic field range is 100 G. (E) EPR spectra of different PC liposomes labeled with the TEMPO spin probe at 32 °C and exposed to 0, 2, and 4 mA of electric current intensities for 30 min. The values of the partitioning parameter (f = H/(H + P)) are indicated for each spectrum. The intensities of all spectra, represented on the y-axis, are normalized. The total magnetic field range is 50 G. The experimental error associated with the f determination is 0.3. Spectra (a), (e) and (i) refer to pure LP PC liposomes; spectra (b), (f) and (j) refer to LP PC-besifloxacin liposomes; spectra (c), (g) and (k) refer to LP PC:SPM liposomes; whereas spectra (d), (h) and (l) refer to LP PC:SPM-besifloxacin liposomes.

Figure 8

Besifloxacin retained in the cornea from LP PC, LP PC:SPM, and control (Besivance) after passive (30 min) or iontophoretic (10 or 30 min at 2 mA/cm²) permeation experiment trough excised porcine cornea (n = 4). ^*(p ≤ 0.05, ANOVA); ^**(p ≤ 0.01, ANOVA); ^***(p ≤ 0.001, ANOVA); ^****(p ≤ 0.0001, ANOVA).

Figure 9

Scheme of possible dispositions of the besifloxacin in LP PC (A,B) and LP PC: SPM (C,D), according to the characteristics and the method of preparation of each liposome.