Chondroitin Sulphate-Chitosan Based Nanogels Loaded with Naringenin-β-Cyclodextrin Complex as Potential Tool for the Treatment of Diabetic Retinopathy: A Formulation Study

Abstract

Purpose: The main purpose of the study was the formulation development of nanogels (NHs) composed of chondroitin sulfate (CS) and low molecular weight chitosan (lCH), loaded with a naringenin-β-cyclodextrin complex (NAR/β-CD), as a potential treatment for early-stage diabetic retinopathy.

Methods: Different formulations of NHs were prepared by varying polymer concentration, lCH ratio, and pH and, then, characterized for particle size, zeta potential, particle concentration (particles/mL) and morphology. Cytotoxicity and internalization were assessed in vitro using Human Umbilical Vein Endothelial Cells (HUVEC). The NAR/β-CD complex was prepared and evaluated for morphology, complexation efficiency, and solubility. Finally, the most promising NH prototype was loaded with NAR/β-CD (NH@NAR/β-CD) and further characterized for encapsulation efficiency, loading capacity, opacity and cytotoxicity on HUVEC; in vitro release test and DPPH assay were performed to investigate NH capability to sustain NAR release and NH@NAR/β-CD antioxidant properties, respectively.

Results: NH properties were influenced by polymer concentration, lCH ratio, and pH. N3 (0.5 mg/mL; lCH=1.5:1; pH = 5) and N9 (0.5 mg/mL; lCH=1:1; pH = 5) showed optimal characteristics, including small size (<350 nm) and positive zeta potential, facilitating cellular uptake. The NAR/β-CD complex showed 71% complexation efficiency and enhanced NAR solubility. Since characterized by superior properties and better in vitro biocompatibility, N3 was loaded with NAR/β-CD. N3@NAR/β-CD capability to sustain in vitro NAR release, radical scavenging activity and in vitro biocompatibility were finally demonstrated.

Conclusion: The physico-chemical properties of N3@NAR/β-CD were responsible for their cell uptake, suggesting their potential to target retinal endothelial cells. The high NAR/β-CD complexation efficiency and the sustained NAR release over 72 hours could guarantee the maintenance of an effective drug concentration at the damage site while reducing the injection number. Further studies about the safety and the effectiveness of the intravitreal injection of NHs@NAR/β-CD will be performed on a diabetic animal model.

Keywords: antioxidant properties; cellular uptake; inclusion complex; intravitreal administration; polyelectrolyte complexation.

Graphical abstract

Figure 1

Schematic representation of NAR/β-CD complex preparation: (1) addition of NAR solution to β-CD solution; (2) magnetic stirring of the mixture at 60°C; (3) solvent evaporation through vacuum rotary evaporator; (4) rinsing of the product obtained with MilliQ water and subsequent filtration to remove the uncomplexed NAR (5); (6) freeze-drying of the obtained NAR/β-CD complex.

Figure 2

Schematic representation of NH@NAR/β-CD preparation method.

Figure 3

Hydrodynamic diameter and zeta potential values of lCH/CS nanogels (mean value ± s.d.; n=3). ANOVA one-way; MRT (P value ≤ 0.05): (A) Hydrodynamic diameter: a vs b,c; b vs c; Zeta potential: a vs c; b vs c. (B) Hydrodynamic diameter: a vs a”; a’ vs a”; b vs b’, b”; b’ vs b”; c vs c”; c’ vs c”; Zeta potential: a vs a’, a”. (C) Hydrodynamic diameter: a vs a’, a”; a’ vs a”; b vs b’, b”; b’ vs b”; c vs c’, c”; c’ vs c”; Zeta potential: a vs a’, a”; a’ vs a”; b vs b’, b”; b’ vs b”; c vs c’, c”; c’ vs c”.

Figure 4

Hydrodynamic diameter (A) PDI (B) and zeta potential (C) values at t0 and t7 days of N3 and N9 prototypes (mean value ± s.d.; n=3). ANOVA one-way; T test (p <0.05).

Figure 5

Average size distribution of N3 (dil 1:10,000) and N9 (dil 1:1,000) prototypes.

Figure 6

SEM images (A) at two different magnifications (10.0 kX and 20.0 kX) of freeze-dried prototypes N3 and N9; TEM micrographs (B) at two different magnifications (50 kX and 100 kX) of evaporated prototypes N3 and N9 diluted 1:100 and stained with phosphotungstic acid for 1 minute.

Figure 7

Mitochondrial activity % values calculated for N3 (A) and N9 (B) prototypes after dilution 1:10, 1:25, 1:50, 1:100 v/v in CM. CM was used as reference (mean values ± s.d.; n = 6). ANOVA one-way; Dunnett’s test (p <0.05).

Figure 8

HUVEC cellular uptake studies by means of confocal microscopy on N3 diluted 1:25 v/v (B) as compared to the untreated control (A). Green: FITC-dex (λ_ex= 490 nm) loaded into N3; blue: Hoechst dye (λ_ex= 361 nm/ λ_em= 497 nm) specific for nuclei staining; red: TRITC (λ_ex= 514 nm and λ_em= 580 nm) for actin fibers staining.

Figure 9

SEM images at magnification of 2500 X of NAR/β-CDlyo (A) and NAR@β-CD (B), NAR (C), β-CD (D).

Figure 10

Absorption spectra in the range of 220–400 nm of NAR/β-CDlyo, mixNAR_β-CD, NAR, and β-CD.

Figure 11

Diffraction patterns of NAR/β-CD and references NAR, β-CD, mixNAR_β-CD.

Figure 12

NAR release profiles from N3@NAR/β-CD (mean values ± s.d.; n = 3; CV <12%).

Figure 13

RSA% of N3@NAR/β-CD, N3, NAR/β-CD and β-CD. CTR- and NAR are considered as negative and positive controls respectively (mean values ± s.d.; n = 3). ANOVA one-way; Dunnett’s test (p <0.05).

Figure 14

Mitochondrial activity % values calculated for N3, NAR/β-CD and N3@NAR/β-CD diluted 1:10, 1:25, 1:50, 1:100 v/v in CM. CM was used as reference (mean values ± s.d.; n = 6). ANOVA one-way; Dunnett’s test (p <0.05).