Improved in vitro and in vivo collagen biosynthesis by asiaticoside-loaded ultradeformable vesicles

https://doi.org/10.1016/j.jconrel.2012.05.050Get rights and content

Abstract

The potentiality of ultradeformable vesicles as a possible topical delivery system for asiaticoside, a natural compound obtained from Centella asiatica was evaluated, because this compound exhibits collagen biosynthesis promoting activity. Ultradeformable vesicles were prepared by the extrusion technique; these vesicles were composed of Phospholipon 100® and different molar fractions of sodium cholate as the edge activator. The physicochemical properties of the ultradeformable vesicles were investigated through differential scanning calorimetry and light scattering techniques. The potential cyctotoxicity and biological activity of asiaticoside-loaded ultradeformable vesicles were evaluated on primary human dermal fibroblast cells by determining the extracellular lactic dehydrogenase activity, the cellular viability and the biosynthetic production of collagen. In vitro permeation experiments through human stratum corneum and epidermis membranes were also carried out. Ultradeformable vesicles having sodium cholate molar fraction of 0.2 proved to be the most suitable topical carriers for asiaticoside. A sodium cholate content of > 0.2 was observed to be cytotoxic probably due to its co-existence with other lipid aggregates, an example being mixed micelles. Asiaticoside-loaded ultradeformable vesicles with a sodium cholate molar fraction of 0.2 elicited the greatest degree of collagen biosynthesis in human fibroblasts. Ultradeformable vesicles provided the greatest in vitro skin permeation of asiaticoside showing a 10-fold increase with respect to the free drug solution and favoured an increase in in vivo collagen biosynthesis. Ultradeformable vesicles are therefore suitable carriers for the pharmaceutical and cosmetic application of the natural agent asiaticoside.

Introduction

Collagen proteins are one of the major constituents of extracellular matrices. These large heterogeneous classes of molecules provide several patterns with respect to structural and bio-physical properties. Particularly, collagen is involved, either directly or indirectly, in cell attachment and differentiation, activation of chemotactic agents, immunoresponsive processes and certain pathological conditions. The impairment of collagen biosynthesis is characteristic both for serious diseases requiring a specific treatment (such as squamous cell carcinoma, actinic keratosis and melanoma) and for wrinkle development, photo-damage, skin aging, wound healing, and scar formation in post-surgery events.

It is well known that Centella asiatica or its active component, asiaticoside, promotes fibroblast proliferation and collagen biosynthesis [1]. In vitro and in vivo experiments showed that the expression of genes coding for type I to type III collagens gradually changed following the administration of asiaticoside and the up-regulation of encoding enzymes, directly involved in collagen biosynthesis, occurred in the process of asiaticoside stimulation [2]. However, the effect of asiaticoside on the skin tissue depended both on the administered dose and the incubation time, thus showing different activity of this compound on collagen biosynthesis. Particularly, in the keloid pathogenesis, a fibrotic disorder of fibroblasts mediated by TGF-β phosphorylated receptors, an alteration of the Smad cascade (related to the collagen biosynthesis) occurs. Asiaticoside can negatively regulate the expression of TGF-βRI and TGF-βRII and increase the expression of Smad7, thereby altering fibroblast proliferation and collagen production. In these conditions, asiaticoside prevented keloid formation and excessive scaring [3]. Therefore, asiaticoside has a potential role in the treatment of diseases/traumas associated with a loss or an impairment of collagen production. Efforts are recently being made to prepare and develop pharmaceutical formulations able to promote healing, particularly those containing natural agents [4].

Suitable percutaneous penetration is an important requisite for the successful topical application of asiaticoside, especially in the treatment of skin conditions that are not characterized by a lack of cutaneous integrity. Thus, an effective topical drug delivery device is desirable. In particular, vesicular drug delivery systems seem to be the most promising topical carriers [5]. The most representative vesicular system is the liposomal carrier, the composition of which seems to determine the type of interaction and the passage through the outer skin layers [6], [7], [8]. Rigid liposomes were shown to be unsuitable vesicular carriers in the penetration of the skin barrier [9], [10]; conversely, highly elastic and deformable vesicles demonstrated the ability to increase the percutaneous permeation of loaded drugs, and penetrate the skin while remaining unfragmented, although this result is controversial [11].

The in vivo non-occlusive application of ultradeformable vesicles allows an effective percutaneous delivery of encapsulated drugs, even in the case of macromolecular vesicles [12]. For the preparation of ultradeformable vesicles, the so-called edge activators were incorporated into the phospholipid bilayers in suitable amounts, with sodium cholate often being used for this purpose. In fact, it has been suggested that the transdermal transport of these devices is driven by the osmotic gradient and occlusion would eliminate this action, thus compromising the activity of the deformable vesicles [13].

The aim of this work was the preparation and characterization of ultradeformable vesicular carriers containing asiaticoside. The asiaticoside-loaded ultradeformable vesicles were tested in vitro by evaluating the amount of asiaticoside permeated through excised human skin. Some in vitro experiments were carried out on primary human dermal fibroblast cells in order to evaluate both the possible cytotoxicity of ultradeformable vesicles as a function of their composition and their efficacy in the stimulation of collagen biosynthesis. An in vivo animal model was also used to explore the real therapeutic effectiveness and clinical use potentialities of the systems.

Section snippets

Chemicals

Phosphatidylcholine (Phospholipon® 100G) from soybean (purity > 98%) was a kind gift of Nattermann Phospholipid (Koln, Germany). 1,2-dipalmitoyl-sn-glycero-phosphocholine monohydrate (DPPC) was a Genzyme product (Suffolk, England). Sodium cholate was purchased from Merck (Darmstadt, Germany). Asiaticoside, Ehrlich reagent and chloramine-T were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lissamine rhodamine B 1,2 dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt

Ultradeformable vesicular carrier characterization

The mean size and size distribution of the ultradeformable vesicles were evaluated as a function of the molar fraction of the sodium cholate used in their preparation. They showed a mean size of ~ 100 nm with a narrow size distribution (polydispersity index values lower than 0.2) up to the sodium cholate molar fraction of 0.2 (Table 1). These findings are suitably correlated to those expected from the preparation method applied in order to obtain small unilamellar vesicles, in which the vesicles

Conclusions

The capacity of ultradeformable vesicles to act as a topical carrier for natural agents such as asiaticoside was demonstrated. A fundamental formulation parameter for ultradeformable vesicles both in terms of drug delivery and carrier toxicity/tolerability is the content of the edge activator; that is, a high amount can determine a reduction of encapsulation efficiency and an increase in cellular cytotoxicity. The results herein reported show that ultradeformable vesicles were not only able to

Acknowledgements

The authors are very grateful to Dr. Lynn Whitted for her revision of the language of this paper, and to Dr. Nicola Costa for his excellent and valuable support in the animal department.

References (34)

Cited by (0)

View full text