In Vitro Hemoglobin Binding and Molecular Docking of Synthesized Chitosan-Based Drug-Carrying Nanocomposite for Ciprofloxacin-HCl Drug Delivery System

Abstract

Understanding the intermolecular interactions between antibiotic drugs and hemoglobin is crucial in biological systems. The current study aimed to investigate the preparation of chitosan/polysorbate-80/tripolyphosphate (CS-PS/TPP) nanocomposite as a potential drug carrier for Ciprofloxacin-HCl drug (CFX), intended for controlled release formulation and further used to interact with bovine hemoglobin. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis-differential thermal analysis (TGA-DTA), scanning electron microscopy (SEM), dynamic light scattering (DLS), and X-ray diffraction analyses were used to characterize the CS-PS/TPP nanocomposite and its CFX-loaded nanocomposite. The second series of biophysical properties were performed on the Ciprofloxacin-loaded CS-PS/TPP (NCFX) for interaction with bovine hemoglobin (BHb). The interactions of (CFX and NCFX) with redox protein hemoglobin were investigated for the first time through a series of in vitro experimental techniques to provide comprehensive knowledge of the drug-protein binding interactions. Additionally, the effect of inclusion of PS-80 on the CFX-BHb interaction was also studied at different concentrations using fluorescence spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, and circular dichroism (CD) under physiological conditions. The binding process of CFX and NCFX was spontaneous, and the fluorescence of BHb was quenched due to the static mechanism formation of the (CFX/BHb) and (NCFX/BHb) complexes. Thermodynamic parameters ΔG, ΔH, and ΔS at various temperatures indicate that the hydrogen bonding and van der Waals forces play a major role in the CFX-BHb association.

Figure 1

(a) Scheme representing the encapsulation routes of CFX in the presence and absence of PS-80. (b) Molecular structure of a ciprofloxacin–HCl.

Figure 2

(a) FT-IR spectra of CFX, CFX@CS/PS-TPP, and CS/PS-TPP; (b) XRD patterns of CFX and CS/PS-TPP before and after loading of CFX

Figure 3

(a) TGA-DTA studies of CS/PS-TPP. (b) TGA-DTA analysis of CFX@CS/PS-TPP

Figure 4

SEM images of CS/PS-TPP before (a) and after (b) loading with CFX.

Figure 5

(DLS) Particle size distribution curves of CS/PS-TPP before (a) and after (b) loading with ciprofloxacin.

Figure 6

Release profiles of CFX from CS/PS-TPP nanocomposite at various pH values (a). Calibration curve of CFX from CS/PS-TPP nanocomposite at 298 K (b).

Figure 7

Emission spectra of BHb C_(BHb) = 10 μM in the presence of various concentrations of (a) CFX (0.5–5) × 10^–5 M and (b) NCFX (0.5–5) 10^–5 M; effect the inclusion of (c) 1 μM PS-80 and (d) 2 μM PS-80 on the interaction of BHb with various concentrations of CFX C_(BHb) = 10 μM; C_CFX; (0.5–4) μM.

Figure 8

(a) Stern–Volmer plots describing BHb quenching of CFX, C_BHb = 10 μM; CFX (0.5–5) μM at 298 °K, pH 7.4, λ_ex = 280 nm. (b) Plots of log(F₀ – F)/F versus log[Q].

Figure 9

Synchronous fluorescence spectra of (a) BHb-CFX at Δλ = 15 nm, (b) BHb-CFX at Δλ = 60 nm, (c) BHb-NCFX at Δλ = 15 nm, (d) BHb-NCFX at Δλ = 60 nm, (e) BHb-PS-CFX at Δλ = 15 nm, and (f) BHb-PS-CFX at Δλ = 60 nm.

Figure 10

(a, c) Modified Stern–Volmer plots with log(F₀ – F)/F v/s log[Q], hence C_BHb = 10 μM; C_CFX and C_NCFX (0.5–5) μM at various temperatures, pH = 7.4. (b, d) Stern–Volmer plots for describing BHb quenching of CFX and NCFX, hence C_BHb = 10 μM, C_CFX and C_NFCX (0.5–5) μM at various temperatures (pH = 7.4).

Figure 11

(a, b) Overlapping of the absorbance spectra of CFX and NCFX with the emission spectrum of HBb (λ_ex = 280 nm). (c, d) UV–vis spectra of BHb in the presence of (a) CFX; C_(BHb) = 10 μM, C_(CFX) = (0.5–5) μM (b) NCFX- BHb C_(BHb) = 10 μM and C_(CFX) = (0.5–5) μM.

Figure 12

CD spectra of (A) HBA (10 μM) in pH 7.40 phosphate buffer at different concentrations of (a) CFX, (b) NCFX, and (c) CFX/PS at 298 K.

Figure 13

Three-dimensional fluorescence diagrams of (a) BHb only, (b) CFX-BHb system, (c) CFX/PS-BHb system, and (d) NCFX-BHb system.

Figure 14

(A–D) Lowest binding energy docked conformation for CFX on BHb (A); amino acid residues surrounding CFX (B) binding pocket for CFX-BHb system; (C) 2D binding for CFX-BHb system; and (D) 3D binding sites for CFX on BHb.