A Hyaluronic Acid Functionalized Self-Nano-Emulsifying Drug Delivery System (SNEDDS) for Enhancement in Ciprofloxacin Targeted Delivery against Intracellular Infection

Abstract

Ciprofloxacin (CIP), a potent anti-bacterial agent of the fluroquinolone family, shows poor solubility and permeability, thus leading to the development of intracellular pathogens induced multi-drug resistance and biofilms formation. To synergistically improve the biopharmaceutical parameters of CIP, a hyaluronic acid (FDA approved biocompatible polymer) functionalized self-nano emulsifying drug delivery system (HA-CIP-SNEDDS) was designed in the present study. SNEDDS formulations were tested via solubility, droplet size, zeta potential, a polydispersity index, thermodynamic stability, surface morphology, solid-state characterization, drug loading/release, cellular uptake, and biocompatibility. The final (HA-CIP-SNEDDS) formulation exhibited a mean droplet size of 50 nm with the 0.3 poly dispersity index and negative zeta potential (-11.4 mV). HA-based SNEDDS containing CIP showed an improved ability to permeate goat intestinal mucus. After 4 h, CIP-SNEDDS showed a 2-fold and HA-CIP-SNEDDS showed a 4-fold permeation enhancement as compared to the free CIP. Moreover, 80% drug release of HA-CIP-SNEDDS was demonstrated to be superior and sustained for 72 h in comparison to free CIP. However, anti-biofilm activity of HA-CIP-SNEDDS against Salmonella typhi was higher than CIP-SNEDDS and free CIP. HA-CIP-SNEDDS exhibited increased biocompatibility and improved oral pharmacokinetics as compared to free CIP. Taken together, HA-CIP-SNEDDS formulation seems to be a promising agent against Salmonella typhi with a strong targeting potential.

Keywords: anti-bacterial activity; biofilms formation; drug delivery; pharmacokinetics.

Figure 1

(A) Hyaluronic acid conjugated with ciprofloxacin to form Hyaluronic acid-Ciprofloxacin (HA-CIP) conjugate, (B) Size exclusion chromatography of HA and HA-CIP conjugate. The decrease in the retention time (RT) of the conjugate peak as compared to HA confirmed the synthesis of the conjugate. (C) FTIR spectra of CIP (a), HA (b) and HA-CIP (c).

Figure 2

¹H NMR spectra of HA-CIP conjugate in DMSO.

Figure 3

Ternary phase diagram representing the emulsification region. (A) With Smix ratio = 1:1, (B) Smix ratio = 1:2, & (C) Smix ratio = 2:1. purple colored area indicates nanoemulsion and yellow colored area indicates microemulsion.

Figure 4

XRD spectra of HA (a), CIP (b), and HA-CIP (c), respectively.

Figure 5

Drug entrapment efficiency of HA functionalized CIP SNEDDS. (A) Dissolution studies from CIP and other formulations (B). Permeation of CIP and SNEDDS; (C) Rheological studies of SNEDDS to determine mucoadhesion and (D). Results are listed as duplicates mean ± S.D (p < 0.05), and statistically significant differences were evaluated by one-way ANOVA with a significance level of p < 0.05.

Figure 6

(A) Fluorescence imaging of FITC-tagged Salmonella typhi biofilms showing the Blank (a), CIP (b), CIP-SNEDDS (c), HA (d), HA-SNEDDS (e), and HA-CIP-SNEDDS (f) growth inhibition of biofilms by treating with SNEDDS (* Scale bars: 50 μm). (B) S. typhi biofilm dispersion graph of various formulations, (C) Determination in vitro hemolysis (%).

Figure 7

(A) Sterilizing effect on Salmonella typhi resistant strains via CIP, CIP-SNEDDS, HA-SNEDDS, and HA-CIP SNEDDS (a–d). (B) Zone of inhibition (ZOI) of SNEDDS formulation and CIP.

Figure 8

(A) FITC tagged step wise increase in florescence from Blank-PBS (a), Blank SNEDDS (b), CIP-SNEDDS (c), HA (d), HA-SNEDDS (e), and HA-CIP-SNEDDS (f) * Scale bars: 50 μm, (B) Biocompatibility assay, (C) In-vivo survival assay of SNEDDS, (D) In vivo pharmacokinetics. Results of all experiments are listed as mean ± S.D.