Curcumin Transferosome-Loaded Thermosensitive Intranasal in situ Gel as Prospective Antiviral Therapy for SARS-Cov-2

Abstract

Purpose: Immunomodulatory and broad-spectrum antiviral activities have motivated the evaluation of curcumin for Coronavirus infection 2019 (COVID-19) management. Inadequate bioavailability is the main impediment to the therapeutic effects of oral Cur. This study aimed to develop an optimal curcumin transferosome-loaded thermosensitive in situ gel to improve its delivery to the lungs.

Methods: Transferosomes were developed by using 3³ screening layouts. The phospholipid concentration as well as the concentration and type of surfactant were considered independent variables. The entrapment efficiency (EE%), size, surface charge, and polydispersity index (PDI) were regarded as dependent factors. A cold technique was employed to develop thermosensitive in-situ gels. Optimized transferosomes were loaded onto the selected gels. The produced gel was assessed based on shape attributes, ex vivo permeability enhancement, and the safety of the nasal mucosa. The in vitro cytotoxicity, antiviral cytopathic effect, and plaque assay (CV/CPE/Plaque activity), and in vivo performance were evaluated after intranasal administration in experimental rabbits.

Results: The optimized preparation displayed a particle size of 664.3 ± 69.3 nm, EE% of 82.8 ± 0.02%, ZP of -11.23 ± 2.5 mV, and PDI of 0.6 ± 0.03. The in vitro curcumin release from the optimized transferosomal gel was markedly improved compared with that of the free drug-loaded gel. An ex vivo permeation study revealed a significant improvement (2.58-fold) in drug permeability across nasal tissues of sheep. Histopathological screening confirmed the safety of these preparations. This formulation showed high antiviral activity against SARS-CoV-2 at reduced concentrations. High relative bioavailability (226.45%) was attained after the formula intranasally administered to rabbits compared to the free drug in-situ gel. The curcumin transferosome gel displayed a relatively high lung accumulation after intranasal administration.

Conclusion: This study provides a promising formulation for the antiviral treatment of COVID-19 patients, which can be evaluated further in preclinical and clinical studies.

Keywords: SARS-CoV-2; coronavirus 2; curcumin; in situ gels; intranasal delivery; transferosomes.

Graphical abstract

Figure 1

(A) Curcumin (in bright Orange sticks) docked on main protease enzyme (cyan cartoon) and overlapped with GC-376 (in mauve sticks). H-bond interactions formed with protease amino acids are shown in blue dots. (B) interactions of curcumin with SARS-CoV-2 main protease.

Figure 2

(A) Overlay of curcumin (lime green sticks) and remdesivir (blue sticks) in the RNA dependent RNA polymerase PDB code 7bv2. (B) docking of curcumin in (RdRp) PDB code 7bv2. (C) 2D illustration of the interaction mode of remdesivir on RNA dependent RNA polymerase PDB code 7bv2. (D) 2D illustration of the mode of binding of curcumin on RNA dependent RNA polymerase PDB code 7bv2.

Figure 3

(A) binding interactions between RBD of SARS-CoV-2 (shown in salmon) and ACE2 (shown in green). (B) curcumin (in Orange sticks) is docked on RBD of SARS-CoV-2 (mauve cartoon) complexed with human ACE2 (cyan cartoon), H-bonds are shown in blue dots. (C) 2D representation of the interaction between docked curcumin and SARS-CoV-2 RBD complexed with human ACE2 (PDB code 6vw1).

Figure 4

Three-dimensional response surface plots, representing the effect of lipid amount (A) and surfactant amount (B) on (A) entrapment efficiency (EE%), (B) vesicle size, (C) zeta potential, (D), polydispersity index (PDI) and (E) desirability of the prepared transferosomes formulations.

Figure 5

Rheological profiles of in-situ gel formulations. (A) at 4 ^◦C, (B) at 25 ^◦C, and (C) at gelling temperature.

Figure 6

FTIR spectra of (A); Curcumin powder, (B) Physical mixture (Phospholipon^® 90G + Polyoxyl 40 Hydrogenated Castor Oil + curcumin), (C) Plain freeze-dried transferosomes, (D) Curcumin transferosomes freeze-dried powder, (E) Freeze dried free curcumin gel, (F) Freeze dried plain gel, and (G) Freeze dried curcumin transferosomes gel. (E-G) show similar absorption bands because all of them share the same composition of gel (Poloxamer 407 and Poloxamer 188, and CP 934P). The absence of characteristic bands of curcumin in the free drug loaded gel formulation (E) may indicate drug-polymer molecular interactions and solid solution formation of curcumin in the polymeric network of the gel. Besides, the disappearance of all the distinguishing bands of curcumin in the freeze-dried curcumin transferosome-loaded gel formulation (G) may be explained by drug dissolution in lipids and encapsulation within the transferosome-loaded gel.

Figure 7

DSC thermogram of (A); Curcumin powder, (B) Freeze dried free curcumin gel, (C) Freeze dried plain gel, and (D) Freeze dried curcumin transferosomes gel.

Figure 8

(A) Cumulative in vitro release patterns of curcumin from in-situ gel (F7), curcumin suspension, transferosomes dispersion (Polyoxyl 40 Hydrogenated Castor Oil), and transferosomes- in-situ gel (Polyoxyl 40 Hydrogenated Castor Oil) formulations in artificial nasal fluid (SNF, pH 5.5, 5% tween 80) at 37 ^◦C. (B) Cumulative in vitro release profiles of curcumin transferosomes dispersion (Span 60), and curcumin transferosomes- in-situ gel (Span 60) compared with curcumin transferosomes- in-situ gel (Polyoxyl 40 Hydrogenated Castor Oil) formulation in artificial nasal fluid (SNF, pH 5.5, 5% tween 80) at 37 ^◦C. Data are represented as mean ± SD (n= 3).

Figure 9

Representation showing SEM micrographs of curcumin-loaded transferosomes ((A), × 15,000) and curcumin transferosomes loaded in-situ gel ((B), × 15,000).

Figure 10

Profile of ex vivo transport of the optimum curcumin transferosomes-loaded intranasal in-situ gel in comparison to free curcumin in-situ gel in SNF, pH 5.5, 5% tween 80 at 37 °C (findings displayed as average ± SD, n = 3).

Figure 11

% Cell viability and % viral inhibition of the investigated curcumin transferosomes in-situ gel at various concentrations in Vero-E6 cells and represented as % cell viability or % viral inhibition versus log₁₀ concentrations. The test sample expressed an enhanced antiviral efficacy against SARS-CoV-2.

Figure 12

Biodistribution of curcumin transferosomes in-situ gel and free curcumin in-situ gel in rabbits after 3 h of administration (A), and after 12 h of administration (B). *Significantly different from free curcumin in-situ gel control (p < 0.05). ***Extremely significant different (p < 0.001). ****Extremely significant different (p < 0.0001). ^nsNon-significant difference compared to free curcumin in-situ gel (p > 0.05). The statistical significance was computed by Student’s t-test.