Tacrolimus nanoparticles based on chitosan combined with nicotinamide: enhancing percutaneous delivery and treatment efficacy for atopic dermatitis and reducing dose

Abstract

Background: Topical application of tacrolimus (FK506) was effective in the treatment of atopic dermatitis (AD); however, adverse effects frequently occurred with the increase of FK506 dose during long-term treatment.

Objective: The objective of this project was to develop a hybrid skin targeting system encapsulating FK506 based on nicotinamide (NIC) and chitosan nanoparticles (CS-NPs), ie, FK506-NIC-CS-NPs, which took advantages of both of NIC and CS-NPs to obtain the synergetic effects of percutaneous delivery and treatment efficacy enhancement along with dose reduction.

Methods: The formulation of FK506-NIC-CS-NPs was optimized and characterized. In vitro and in vivo skin permeation studies were performed. AD-like skin lesions were constructed with BALB/c mice by 1-chloro-2, 4-dinitrobenzene (DNCB)-induced, and FK506-NIC-CS-NPs containing different dose of FK506 were topically administered to treat AD-like skin lesions in comparison with Protopic.

Results: NIC was found to significantly increase the FK506 EE to 92.2% by CS-NPs. In comparison with commercial FK506 ointment (Protopic), in vitro and in vivo skin permeation studies demonstrated that NIC-CS-NPs system significantly enhanced FK506 permeation through and into the skin, and deposited more FK506 into the skin. The treatment efficacy on clinical symptoms, histological analysis, and molecular biology of the AD-mice demonstrated that NIC-CS-NPs with ~1/3 dose of FK506 of Protopic was superior to that of Protopic, and NIC-CS-NPs vehicle exhibited the adjuvant therapy and moderate anti-AD effects.

Conclusion: The system of NIC-CS-NPs enhances the permeability of FK506, plays an adjuvant role in anti-AD, reduces the dose of FK506 in treating AD, and is therefore a promising nanoscale system of FK506 for the effective treatment of AD.

Keywords: atopic dermatitis; chitosan; nanoparticles; nicotinamide; percutaneous delivery; reducing dose; tacrolimus.

Figure 1

Stability of FK506–NIC-CS-NPs with 0.1% (w/v) FK506 at different pH values in size (A) and PDI (B). Note: Each symbol and bar represented the mean ± SD of three determinations. Abbreviations: FK506, tacrolimus; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; PDI, polydispersity index; SD, standard deviation.

Figure 2

Permeation profiles of FK506 through rat skin from different formulations in vitro (A) and FK506 skin retention after 24-h permeation of different formulations in vitro (B). Notes: Each symbol and bar represented the mean ± SD of six determinations. Significant differences were calculated using ANOVA test (*P<0.05). The concentration of FK506 in each formulation was 1 mg/mL (g). Abbreviations: FK506, tacrolimus; FK506–NIC, FK506 dissolved in 20% nicotinamide aqueous solution; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; h, hours; SD, standard deviation.

Figure 3

In vivo skin retention of FK506 from different formulations in different skin layers of SC, E+D, and total amount in skin (Total). Notes: Each symbol and bar represented the mean ± SD of six determinations. Significant differences were calculated using ANOVA test (*P<0.05). The concentration of FK506 in each formulation was 1 mg/mL (g). Abbreviations: E+D, epidermis and dermis; FK506, tacrolimus; FK506–NIC, FK506 dissolved in 20% nicotinamide aqueous solution; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; h, hours; SC, stratum corneum; SD, standard deviation.

Figure 4

Schematic diagram of the experimental protocol in a mouse model (A). Dermatitis scores of AD-model mice treated with different formulations (B) and representative clinical features of AD-like skin lesions at the end of the experiment (C). Notes: Each bar represented the mean ± SD of six determinations. Significant differences were calculated using ANOVA test. *P<0.05 in comparison with the DNCB group; #P<0.05 in comparison with the DNCB+Protopic group. Abbreviations: AD, atopic dermatitis; C–0.01, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.01% (w/v) FK506; C–0.03, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.03% (w/v) FK506; C–0.05, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.05% (w/v) FK506; C–0.1, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.1% (w/v) FK506; DNCB, 1-chloro-2,4-dinitrobenzene; FK506, tacrolimus; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; NIC, nicotinamide.

Figure 5

The ear thickness (A) and spleen index (B) on day 29 of mice treated with different formulations. Notes: Each bar represented the mean ± SD of six determinations. Significant differences were calculated using ANOVA test. *P<0.05 in comparison with DNCB group; #P<0.05 in comparison with the DNCB+Protopic group. Abbreviations: C–0.01, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.01% (w/v) FK506; C–0.03, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.03% (w/v) FK506; C–0.05, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.05% (w/v) FK506; C–0.1, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.1% (w/v) FK506; DNCB, 1-chloro-2,4-dinitrobenzene; FK506, tacrolimus; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; NIC, nicotinamide.

Figure 6

Histological features of skin after treatment with different formulations in the AD mouse model. The sliced sections were stained with hematoxylin and eosin (A) and toluidine blue (B) (magnification 100×). Scale bar: 200 μm. Abbreviations: AD, atopic dermatitis; C–0.01, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.01% (w/v) FK506; C–0.03, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.03% (w/v) FK506; C–0.05, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.05% (w/v) FK506; C–0.1, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.1% (w/v) FK506; DNCB, 1-chloro-2,4-dinitrobenzene; FK506, tacrolimus; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; NIC, nicotinamide.

Figure 7

Effects of NPs with low-dose FK506 on serum total IgE levels. Notes: Each bar represented the mean ± SD of six determinations. Significant differences were calculated using ANOVA test. *P<0.05 in comparison with the DNCB group; ^#P<0.05 in comparison with the DNCB+Protopic group. Abbreviations: C–0.01, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.01% (w/v) FK506; C–0.03, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.03% (w/v) FK506; C–0.05, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.05% (w/v) FK506; C–0.1, FK506–NIC-CS-NPs containing 20% (w/v) NIC and 0.1% (w/v) FK506; DNCB, 1-chloro-2,4-dinitrobenzene; FK506, tacrolimus; FK506–NIC-CS-NPs, tacrolimus-loaded chitosan nanoparticles containing nicotinamide; IgE, immunoglobulin E; NIC, nicotinamide.