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Lipid nanoparticles for cyclosporine A administration: development, characterization, and in vitro evaluation of their immunosuppression activity.

Guada M, Sebastián V, Irusta S, Feijoó E, Dios-Viéitez Mdel C, Blanco-Prieto MJ - Int J Nanomedicine (2015)

Bottom Line: Some of these issues are associated with the drug or excipients and others with the dosage forms.CsA was successfully incorporated into LN using the method of hot homogenization followed by ultrasonication.Finally, the new CsA formulations showed in vitro dose-dependent immuno-suppressive effects caused by the inhibition of IL-2 levels secreted from stimulated Jurkat cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona ; Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona.

ABSTRACT
Cyclosporine A (CsA) is an immunosuppressant commonly used in transplantation for prevention of organ rejection as well as in the treatment of several autoimmune disorders. Although commercial formulations are available, they have some stability, bioavailability, and toxicity related problems. Some of these issues are associated with the drug or excipients and others with the dosage forms. With the aim of overcoming these drawbacks, lipid nanoparticles (LN) have been proposed as an alternative, since excipients are biocompatible and also a large amount of surfactants and organic solvents can be avoided. CsA was successfully incorporated into LN using the method of hot homogenization followed by ultrasonication. Three different formulations were optimized for CsA oral administration, using different surfactants: Tween(®) 80, phosphatidylcholine, taurocholate and Pluronic(®) F127 (either alone or mixtures). Freshly prepared Precirol nanoparticles showed mean sizes with a narrow size distribution ranging from 121 to 202 nm, and after freeze-drying were between 163 and 270 nm, depending on the stabilizer used. Surface charge was negative in all LN developed. High CsA entrapment efficiency of approximately 100% was achieved. Transmission electron microscopy was used to study the morphology of the optimized LN. Also, the crystallinity of the nanoparticles was studied by X-ray powder diffraction and differential scanning calorimetry. The presence of the drug in LN surfaces was confirmed by X-ray photoelectron spectroscopy. The CsA LN developed preserved their physicochemical properties for 3 months when stored at 4°C. Moreover, when the stabilizer system was composed of two surfactants, the LN formulations were also stable at room temperature. Finally, the new CsA formulations showed in vitro dose-dependent immuno-suppressive effects caused by the inhibition of IL-2 levels secreted from stimulated Jurkat cells. The findings obtained in this paper suggest that new lipid nanosystems are a good alternative to produce physicochemically stable CsA formulations for oral administration.

No MeSH data available.


Related in: MedlinePlus

Crystallinity and thermal characterization of the lipid nanoparticles.Notes: (A) X-ray diffractograms of: (a) cyclosporine A (CsA), (b) Precirol, (c) LN Lec:TC-Blank, (d) LN Lec:TC-CsA. Gray shadow corresponds to Bragg-spacing. (B) Differential scanning calorimetry thermograms of: (a) Precirol, (b) cyclosporine A, (c) L-α-phosphatidylcholine, (d) taurocholic acid sodium salt hydrate, (e) LN Lec:TC-Blank, (f) LN Lec:TC-CsA.Abbreviations: LN, lipid nanoparticles; Lec, L-α-phosphatidylcholine; TC, taurocholic acid sodium salt hydrate.
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f2-ijn-10-6541: Crystallinity and thermal characterization of the lipid nanoparticles.Notes: (A) X-ray diffractograms of: (a) cyclosporine A (CsA), (b) Precirol, (c) LN Lec:TC-Blank, (d) LN Lec:TC-CsA. Gray shadow corresponds to Bragg-spacing. (B) Differential scanning calorimetry thermograms of: (a) Precirol, (b) cyclosporine A, (c) L-α-phosphatidylcholine, (d) taurocholic acid sodium salt hydrate, (e) LN Lec:TC-Blank, (f) LN Lec:TC-CsA.Abbreviations: LN, lipid nanoparticles; Lec, L-α-phosphatidylcholine; TC, taurocholic acid sodium salt hydrate.

Mentions: XRD analysis enables us to identify the crystalline or amorphous state of LN, as well as revealing the spacings in the solid lipid lattice.18 This is particularly important since lipids are very often polymorphic substances. XRD patterns of pure lipid and CsA were used as references for the evaluation of LN spectra (Figure 2A). Reference spectra indicated that Precirol and CsA existed in a crystalline state before being processed to give rise to the LN Lec-TC formulation. LN prepared with and without CsA and further lyophilized exhibited the same peaks with the starting lipid material, despite the hot homogenization conditions. On the other hand, the intensity peaks that belong to CsA could not be identified in the diffractograms of CsA loaded LN, suggesting the presence of an amorphous CsA payload. The Bragg-spacing values calculated for the reflections show the presence of two types of spacings: long spacings (depending on the fatty acid chain length and the angle tilt) and short spacings (non-dependent on fatty acid chain length). Short spacings correspond to reflections at high angles, originating from the packing of lipids, and are related with the presence of polymorphs. The most stable form, the β polymorph, has a triclinic subcell with a characteristic spacing at 4.6 Å. The β′ form has an orthorhombic subcell structure with characteristic spacings at 3.8 Å and 4.2 Å. Finally, the α polymorph has a hexagonal subcell with a characteristic spacing at 4.15 Å.25Figure 2A shows the existence of 4.61, 4.22, and 3.86 Bragg-spacings, which implies that both the bulk lipid and the nanoscale counterpart are a mixture of β and β′ polymorphs. The polymorphs differ in stability, melting point, density, and melting enthalpy. The β polymorph is the most stable and has the highest melting point and melting enthalpy. XRD results can be corroborated by DSC analysis. The DSC thermogram of pure Precirol exhibited a melting endothermic peak at 58°C, while pure CsA, Lec, and TC peaked at 140°C, 225°C, and 140°C, respectively (Figure 2B). The observed melting peak of LN, with and without CsA payload, was found to be 53°C. This depression cannot be attributed to any polymorph transition, since XRD did not reveal any Bragg-spacing modification. On the other hand, it is stated that the presence of surfactants in the melted lipid phase during the production process could distort crystals resulting in a lower melting energy.26 This fact can also be explained by the Kelvin effect, where a reduced particle size and increased surface area led to a decrease in the melting enthalpy compared to the bulk lipid.26 The CsA melting endothermic peak of loaded LN disappeared, indicating the existence of amorphous CsA or that it has been molecularly dispersed within the Precirol matrix, confirming the results obtained by XRD.


Lipid nanoparticles for cyclosporine A administration: development, characterization, and in vitro evaluation of their immunosuppression activity.

Guada M, Sebastián V, Irusta S, Feijoó E, Dios-Viéitez Mdel C, Blanco-Prieto MJ - Int J Nanomedicine (2015)

Crystallinity and thermal characterization of the lipid nanoparticles.Notes: (A) X-ray diffractograms of: (a) cyclosporine A (CsA), (b) Precirol, (c) LN Lec:TC-Blank, (d) LN Lec:TC-CsA. Gray shadow corresponds to Bragg-spacing. (B) Differential scanning calorimetry thermograms of: (a) Precirol, (b) cyclosporine A, (c) L-α-phosphatidylcholine, (d) taurocholic acid sodium salt hydrate, (e) LN Lec:TC-Blank, (f) LN Lec:TC-CsA.Abbreviations: LN, lipid nanoparticles; Lec, L-α-phosphatidylcholine; TC, taurocholic acid sodium salt hydrate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4621240&req=5

f2-ijn-10-6541: Crystallinity and thermal characterization of the lipid nanoparticles.Notes: (A) X-ray diffractograms of: (a) cyclosporine A (CsA), (b) Precirol, (c) LN Lec:TC-Blank, (d) LN Lec:TC-CsA. Gray shadow corresponds to Bragg-spacing. (B) Differential scanning calorimetry thermograms of: (a) Precirol, (b) cyclosporine A, (c) L-α-phosphatidylcholine, (d) taurocholic acid sodium salt hydrate, (e) LN Lec:TC-Blank, (f) LN Lec:TC-CsA.Abbreviations: LN, lipid nanoparticles; Lec, L-α-phosphatidylcholine; TC, taurocholic acid sodium salt hydrate.
Mentions: XRD analysis enables us to identify the crystalline or amorphous state of LN, as well as revealing the spacings in the solid lipid lattice.18 This is particularly important since lipids are very often polymorphic substances. XRD patterns of pure lipid and CsA were used as references for the evaluation of LN spectra (Figure 2A). Reference spectra indicated that Precirol and CsA existed in a crystalline state before being processed to give rise to the LN Lec-TC formulation. LN prepared with and without CsA and further lyophilized exhibited the same peaks with the starting lipid material, despite the hot homogenization conditions. On the other hand, the intensity peaks that belong to CsA could not be identified in the diffractograms of CsA loaded LN, suggesting the presence of an amorphous CsA payload. The Bragg-spacing values calculated for the reflections show the presence of two types of spacings: long spacings (depending on the fatty acid chain length and the angle tilt) and short spacings (non-dependent on fatty acid chain length). Short spacings correspond to reflections at high angles, originating from the packing of lipids, and are related with the presence of polymorphs. The most stable form, the β polymorph, has a triclinic subcell with a characteristic spacing at 4.6 Å. The β′ form has an orthorhombic subcell structure with characteristic spacings at 3.8 Å and 4.2 Å. Finally, the α polymorph has a hexagonal subcell with a characteristic spacing at 4.15 Å.25Figure 2A shows the existence of 4.61, 4.22, and 3.86 Bragg-spacings, which implies that both the bulk lipid and the nanoscale counterpart are a mixture of β and β′ polymorphs. The polymorphs differ in stability, melting point, density, and melting enthalpy. The β polymorph is the most stable and has the highest melting point and melting enthalpy. XRD results can be corroborated by DSC analysis. The DSC thermogram of pure Precirol exhibited a melting endothermic peak at 58°C, while pure CsA, Lec, and TC peaked at 140°C, 225°C, and 140°C, respectively (Figure 2B). The observed melting peak of LN, with and without CsA payload, was found to be 53°C. This depression cannot be attributed to any polymorph transition, since XRD did not reveal any Bragg-spacing modification. On the other hand, it is stated that the presence of surfactants in the melted lipid phase during the production process could distort crystals resulting in a lower melting energy.26 This fact can also be explained by the Kelvin effect, where a reduced particle size and increased surface area led to a decrease in the melting enthalpy compared to the bulk lipid.26 The CsA melting endothermic peak of loaded LN disappeared, indicating the existence of amorphous CsA or that it has been molecularly dispersed within the Precirol matrix, confirming the results obtained by XRD.

Bottom Line: Some of these issues are associated with the drug or excipients and others with the dosage forms.CsA was successfully incorporated into LN using the method of hot homogenization followed by ultrasonication.Finally, the new CsA formulations showed in vitro dose-dependent immuno-suppressive effects caused by the inhibition of IL-2 levels secreted from stimulated Jurkat cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona ; Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona.

ABSTRACT
Cyclosporine A (CsA) is an immunosuppressant commonly used in transplantation for prevention of organ rejection as well as in the treatment of several autoimmune disorders. Although commercial formulations are available, they have some stability, bioavailability, and toxicity related problems. Some of these issues are associated with the drug or excipients and others with the dosage forms. With the aim of overcoming these drawbacks, lipid nanoparticles (LN) have been proposed as an alternative, since excipients are biocompatible and also a large amount of surfactants and organic solvents can be avoided. CsA was successfully incorporated into LN using the method of hot homogenization followed by ultrasonication. Three different formulations were optimized for CsA oral administration, using different surfactants: Tween(®) 80, phosphatidylcholine, taurocholate and Pluronic(®) F127 (either alone or mixtures). Freshly prepared Precirol nanoparticles showed mean sizes with a narrow size distribution ranging from 121 to 202 nm, and after freeze-drying were between 163 and 270 nm, depending on the stabilizer used. Surface charge was negative in all LN developed. High CsA entrapment efficiency of approximately 100% was achieved. Transmission electron microscopy was used to study the morphology of the optimized LN. Also, the crystallinity of the nanoparticles was studied by X-ray powder diffraction and differential scanning calorimetry. The presence of the drug in LN surfaces was confirmed by X-ray photoelectron spectroscopy. The CsA LN developed preserved their physicochemical properties for 3 months when stored at 4°C. Moreover, when the stabilizer system was composed of two surfactants, the LN formulations were also stable at room temperature. Finally, the new CsA formulations showed in vitro dose-dependent immuno-suppressive effects caused by the inhibition of IL-2 levels secreted from stimulated Jurkat cells. The findings obtained in this paper suggest that new lipid nanosystems are a good alternative to produce physicochemically stable CsA formulations for oral administration.

No MeSH data available.


Related in: MedlinePlus