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Combination of hydrotropic nicotinamide with nanoparticles for enhancing tacrolimus percutaneous delivery

View Article: PubMed Central - PubMed

ABSTRACT

Tacrolimus (FK506), an effective immunosuppressant for treating inflammatory skin diseases, hardly penetrates into and through the skin owing to its high hydrophobicity and molecular weight. The aim of this study was to develop a hybrid system based on nicotinamide (NIC) and nanoparticles (NPs) encapsulating FK506, such as FK506–NPs–NIC, for facilitating percutaneous delivery, which exploited virtues of both NIC and NPs to obtain the synergetic effect. Solubility and percutaneous permeation studies were carried out. The results showed that NIC could increase the solubility and permeability of FK506 and that 20% (w/v) NIC presented higher FK506 permeability and was thus chosen as the hydrotropic solution to solubilize FK506 and prepare FK506–NPs–NIC. Hyaluronic acid (HA) was chemically conjugated with cholesterol (Chol) to obtain amphiphilic conjugate of HA–Chol, which self-assembled NPs in 20% NIC solution containing FK506. The particle size, zeta potential, and morphology of NPs were characterized. The encapsulation efficiency and in vitro percutaneous permeation of NPs were evaluated in the presence and absence of NIC. The results demonstrated that hydrotropic solubilizing FK506 was readily encapsulated into NPs with a higher encapsulation efficiency of 79.2%±4.2%, and the combination of NPs with NIC exhibited a significantly synergistic effect on FK506 deposition within the skin (2.39±0.53 μg/cm2) and penetration through the skin (13.38±2.26 μg/cm2). The effect of the combination of NPs with NIC on drug permeation was further visualized by confocal laser scanning microscope through in vivo permeation studies, and the results confirmed that NPs–NIC synergistically enhanced the permeation of the drug into the skin. The cellular uptake performed in HaCaT cells presented a promoting effect of NPs on cellular uptake. These overall results demonstrated that HA–Chol–NPs–NIC can synergistically improve the percutaneous delivery of FK506, and it is a novel potential strategy based on a nano-sized carrier for FK506 to treat skin diseases.

No MeSH data available.


Confocal laser scanning microscopy of cell uptake of HaCaT cell after 4 hours of administration of (A) DMEM; (B) C6 aqueous suspension; (C) C6–NIC complex suspension; (D) C6–HA–Chol–NP suspension; and (E) C6–HA–Chol–NPs–NIC suspension.Notes: The concentration of C6 in each formulation was 10 μg/mL. Cells were stained for nuclei (in blue) and C6 (in green). Scale bar: 20 μm. Magnification ×400.Abbreviations: C6, coumarin 6; C6–HA–Chol–NP, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticle; C6–HA–Chol–NPs–NIC, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticles containing nicotinamide; C6–NIC complex, coumarin 6–nicotinamide complex; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; h, hours; HaCaT, human keratinocytes.
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f7-ijn-11-4037: Confocal laser scanning microscopy of cell uptake of HaCaT cell after 4 hours of administration of (A) DMEM; (B) C6 aqueous suspension; (C) C6–NIC complex suspension; (D) C6–HA–Chol–NP suspension; and (E) C6–HA–Chol–NPs–NIC suspension.Notes: The concentration of C6 in each formulation was 10 μg/mL. Cells were stained for nuclei (in blue) and C6 (in green). Scale bar: 20 μm. Magnification ×400.Abbreviations: C6, coumarin 6; C6–HA–Chol–NP, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticle; C6–HA–Chol–NPs–NIC, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticles containing nicotinamide; C6–NIC complex, coumarin 6–nicotinamide complex; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; h, hours; HaCaT, human keratinocytes.

Mentions: SC was the main barrier of skin. For the purpose of delivering therapeutic agents to target site of skin by NPs, one of the possibilities was that NPs, as the carrier, could be actively internalized by keratinocytes or other cells of the epidermis.51 Immortalized HaCaT, similar to normal keratinocytes, were chosen to evaluate the cellular uptake of HA–Chol–NPs. Cell uptake studies were carried out using C6 as fluorescence probe, which has been widely used in cell uptake experiments for observation by CLSM.52–54 The uptake of C6-loaded HA–Chol–NPs–NIC, C6-loaded HA–Chol–NPs, C6 in 20% (w/v) NIC solution, and C6 aqueous suspension containing an equal amount of C6 (10 μg/mL) by HaCaT cell was evaluated. As shown in Figure 7A, the cell showed no background fluorescence interference except the nucleus labeled by DAPI in the DMEM group. After 4 hours of incubation, the C6 suspension group presented negligible fluorescence, indicating very little C6 uptake by HaCaT cells (Figure 7B). NIC solution could slightly enhance the cell uptake of C6 compared with that of C6 suspension (Figure 7C). However, when C6 was loaded in HA–Chol–NPs, its uptake by HaCaT cells was significantly enhanced (Figure 7D). It has been reported that CD44 expressed in HaCaT cells55 and HA–Chol–NPs could specifically bind to CD44 receptor expressing on HaCaT cells and resulted in uptake by HaCaT cells through CD44 receptor-mediated endocytosis, which would facilitate the uptake of loaded drugs by cells. When C6 was loaded in HA–Chol–NPs with NIC, the cell uptake presented similar green fluorescence to that of HA–Chol–NPs without NIC (Figure 7E), which indicated that cell uptake was mainly ascribed to HA–Chol–NPs and NIC would not hinder the uptake.


Combination of hydrotropic nicotinamide with nanoparticles for enhancing tacrolimus percutaneous delivery
Confocal laser scanning microscopy of cell uptake of HaCaT cell after 4 hours of administration of (A) DMEM; (B) C6 aqueous suspension; (C) C6–NIC complex suspension; (D) C6–HA–Chol–NP suspension; and (E) C6–HA–Chol–NPs–NIC suspension.Notes: The concentration of C6 in each formulation was 10 μg/mL. Cells were stained for nuclei (in blue) and C6 (in green). Scale bar: 20 μm. Magnification ×400.Abbreviations: C6, coumarin 6; C6–HA–Chol–NP, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticle; C6–HA–Chol–NPs–NIC, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticles containing nicotinamide; C6–NIC complex, coumarin 6–nicotinamide complex; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; h, hours; HaCaT, human keratinocytes.
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Related In: Results  -  Collection

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f7-ijn-11-4037: Confocal laser scanning microscopy of cell uptake of HaCaT cell after 4 hours of administration of (A) DMEM; (B) C6 aqueous suspension; (C) C6–NIC complex suspension; (D) C6–HA–Chol–NP suspension; and (E) C6–HA–Chol–NPs–NIC suspension.Notes: The concentration of C6 in each formulation was 10 μg/mL. Cells were stained for nuclei (in blue) and C6 (in green). Scale bar: 20 μm. Magnification ×400.Abbreviations: C6, coumarin 6; C6–HA–Chol–NP, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticle; C6–HA–Chol–NPs–NIC, coumarin 6-loaded hyaluronic acid–cholesterol nanoparticles containing nicotinamide; C6–NIC complex, coumarin 6–nicotinamide complex; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s Modified Eagle’s Medium; h, hours; HaCaT, human keratinocytes.
Mentions: SC was the main barrier of skin. For the purpose of delivering therapeutic agents to target site of skin by NPs, one of the possibilities was that NPs, as the carrier, could be actively internalized by keratinocytes or other cells of the epidermis.51 Immortalized HaCaT, similar to normal keratinocytes, were chosen to evaluate the cellular uptake of HA–Chol–NPs. Cell uptake studies were carried out using C6 as fluorescence probe, which has been widely used in cell uptake experiments for observation by CLSM.52–54 The uptake of C6-loaded HA–Chol–NPs–NIC, C6-loaded HA–Chol–NPs, C6 in 20% (w/v) NIC solution, and C6 aqueous suspension containing an equal amount of C6 (10 μg/mL) by HaCaT cell was evaluated. As shown in Figure 7A, the cell showed no background fluorescence interference except the nucleus labeled by DAPI in the DMEM group. After 4 hours of incubation, the C6 suspension group presented negligible fluorescence, indicating very little C6 uptake by HaCaT cells (Figure 7B). NIC solution could slightly enhance the cell uptake of C6 compared with that of C6 suspension (Figure 7C). However, when C6 was loaded in HA–Chol–NPs, its uptake by HaCaT cells was significantly enhanced (Figure 7D). It has been reported that CD44 expressed in HaCaT cells55 and HA–Chol–NPs could specifically bind to CD44 receptor expressing on HaCaT cells and resulted in uptake by HaCaT cells through CD44 receptor-mediated endocytosis, which would facilitate the uptake of loaded drugs by cells. When C6 was loaded in HA–Chol–NPs with NIC, the cell uptake presented similar green fluorescence to that of HA–Chol–NPs without NIC (Figure 7E), which indicated that cell uptake was mainly ascribed to HA–Chol–NPs and NIC would not hinder the uptake.

View Article: PubMed Central - PubMed

ABSTRACT

Tacrolimus (FK506), an effective immunosuppressant for treating inflammatory skin diseases, hardly penetrates into and through the skin owing to its high hydrophobicity and molecular weight. The aim of this study was to develop a hybrid system based on nicotinamide (NIC) and nanoparticles (NPs) encapsulating FK506, such as FK506–NPs–NIC, for facilitating percutaneous delivery, which exploited virtues of both NIC and NPs to obtain the synergetic effect. Solubility and percutaneous permeation studies were carried out. The results showed that NIC could increase the solubility and permeability of FK506 and that 20% (w/v) NIC presented higher FK506 permeability and was thus chosen as the hydrotropic solution to solubilize FK506 and prepare FK506–NPs–NIC. Hyaluronic acid (HA) was chemically conjugated with cholesterol (Chol) to obtain amphiphilic conjugate of HA–Chol, which self-assembled NPs in 20% NIC solution containing FK506. The particle size, zeta potential, and morphology of NPs were characterized. The encapsulation efficiency and in vitro percutaneous permeation of NPs were evaluated in the presence and absence of NIC. The results demonstrated that hydrotropic solubilizing FK506 was readily encapsulated into NPs with a higher encapsulation efficiency of 79.2%±4.2%, and the combination of NPs with NIC exhibited a significantly synergistic effect on FK506 deposition within the skin (2.39±0.53 μg/cm2) and penetration through the skin (13.38±2.26 μg/cm2). The effect of the combination of NPs with NIC on drug permeation was further visualized by confocal laser scanning microscope through in vivo permeation studies, and the results confirmed that NPs–NIC synergistically enhanced the permeation of the drug into the skin. The cellular uptake performed in HaCaT cells presented a promoting effect of NPs on cellular uptake. These overall results demonstrated that HA–Chol–NPs–NIC can synergistically improve the percutaneous delivery of FK506, and it is a novel potential strategy based on a nano-sized carrier for FK506 to treat skin diseases.

No MeSH data available.