To investigate the ability of transfersomal gel carrying the antiscarring agent (5-FU) to permeate hypertrophic scars
Hypertrophic scars (HS) are common pathological scars, which result from the excessive healing of wounds and lead to malformed appearance, paresthesia, and even organic dysfunctions. The current nonsurgical treatment for hypertrophic scar focuses on medication, predominantly local injection, and cutaneous application. However, local injections cause obvious pain and require long-term repetitive course, which is associated with a poor compliance. Topical application is easy to operate and has low drug side effects, but the penetration efficacy depends on the permeability of the agent, as well as being limited due to the thickening and dense structure of HS dermis layer and the decreasing of skin’s appendages [
A number of agents have been studied and reported to have the ability to treat hyperplastic scar, for example, corticosteroids, interferon, and 5-fluorouracil (5-FU) [
In our previous studies we investigated the efficacy of transfersome suspensions in HS. We observed a high permeability in hypertrophic scar tissues compared to hydroethanolic solutions. We also reconfirmed what has been reported before: transfersomes are vesicles with an excellent permeability and high retention effect due to their dynamics and high lipid affinity [
This study was performed in accordance with the 1975 Declaration of Helsinki ethical guidelines and approved by Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. Samples were collected from 4 patients who underwent resection and dermoplasty for hypertrophic scald scars (chest and shoulder) in our reconstructive surgery department of Shanghai Ninth People’s Hospital. All patients were female of 22–31 years of age, with a disease course of 6 months−1 year, who never received pharmacologic or radiation HS therapy. Those scars were intact with areas of >5 cm × 5 cm but no ulceration. General anesthesia was used to eliminate the interference of local anesthetics.
A modified preparation protocol by Touitou et al., called improved film dispersion method [
To bind rhodamine to rhodamine 6GO 5-FU suspension PBS pH 7.4 (0.8% (w/v)) was mixed with an equal volume of the carbopol gel under continuing stirring to form rhodamine 5-FU gel (Rho 5-FU CG) (rhodamine 0.03% (w/v)). It has been shown that rhodamine is not changing the affinity or pharmacokinetics of 5-FU [
5-FU TG was diluted 10 times with distilled water. 20
Modified Franz drug percutaneous permeability diffusion tester was applied to perform
In a modified Franz drug percutaneous permeability diffusion tester, 0.5 g 5FU-gel was coated on scars stratum corneum of and tested at 200 r/min,
Rabbit ear hypertrophic scar models were constructed (Morris’s method) [
An ear of each big-ear white rabbit was randomly selected to receive Rho 5-FU TG on the successfully constructed scar wounds. The respective other ear was treated with Rho 5-FU CG and massaged for 30 min. The ear was given, once a day, 0.05 g each time (working concentration of 5-FU 0.2 g/day/scar) [
HE staining was used to observe morphologic changes of 5-FU TG on hypertrophic scars in rabbit ears. 14 days after drug administration, fresh scar tissues in rabbit ears in each group were internally fixed in 4% paraformaldehyde for 48 h. 20~30% sucrose solution was used for gradient dehydration for 24, placed into paraffin blocks, frozen for 30 min, and cut into 5~10
SEI reflects the scars’ hyperplasia. SEI: dermal thickness after wound healing/dermal thickness of adjacent normal tissues. SEI > 1.5 indicates hypertrophic scars.
Fibroblasts derived from scar tissues were enzymatically digested and cultivated in DMEM with 10% fetal bovine serum at 37°C in a humidified incubator with 5% carbon dioxide [
Continuous variables were reported as mean ± SD. Then intercomparison was performed with a one-way ANOVA. SPSS Version 18 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. A
Transmission electron microscope (TEM) images in Figure
5-FU TGs in transmission electron microscopy (TEM): spherical or elliptical vesicles, sizes < 100 nm (
At 6 h of transfersomal gel permeation, retention amount of 5-FU in hypertrophic scar tissues was detected by HPLC. The results were expressed by retention amount per tissue unit. Retention amount in Rho 5-FU TG group was higher (
The retention of 5-FU (6 h administration) in each of the gel groups (
Rho labeled 5-FU TG and CLSM were used to assess scar permeability of two kinds of liposome gels (Figure
CLSM images of cross sections in each gel group containing rhodamine 6GO at 1 h and 6 h.
Fluorescence analysis in human skin and hypertrophic scars (ROI chest and shoulder) in both gel groups containing rhodamine 6GO at 6 h (
On day 14, scar wounds gradually healed with scabs at the surface and no significant eminence. On day 28, wounds showed a complete epithelization. Macroscopic findings included obvious eminence in skin and thick, hard hyperplasia (Figure
Rabbit ear hypertrophic scar model: (a) 2 weeks post-op; (b) 4 weeks post-op.
Depth of Rho permeation in hypertrophic scars of rabbit ears at two time points (1 h and 6 h) was compared. At 1 h after administration, Rho in Rho 5-FU TG group and Rho 5-FU CG group gathered in superficial skin layer, with Rho 5-FU TG group showing the tendency of permeating to dermal layer. At 6 h, Rho 5-FU TG group gel permeated into the superficial layer of dermis and diffused to deep layers.
5-FU CG group gels were retained in superficial skin layers, with no trend of a continuous permeation at 6 h after administration. These results indicate that transfersomal gel percutaneously permeates into dermal layer of body scar tissues (Figure
After HE staining, morphologic changes of rabbit ear hypertrophic scar tissues and their surrounding normal tissues were observed on day 14 after administration; SEI in each group was compared. Numerous fibroblasts were seen in dermal layers in both groups with disordered arrangement and circinate structure, while superficial collagen fibers showed nodular or circinate arrangement. Hyperplasia and several new chondrocytes (reconstruction of cartilage tissues) were found in central scar tissues (Figure
Morphological changes of hypertrophic scars in rabbit ears in both groups on day 14 after administration. (a) and (c) showed the comparison of the hypertrophic scars of before and after administration. Similarly, (b) and (d) showed the changes of 5-FU TG group. Numerous fibroblasts and collagen fibers with disordered arrangement were seen in dermal layer in (a) and (b). Hyperplasia of dermal tissue in 5-FU TG group was alleviated more than in Rho 5-FU CG group.
Comparison of hypertrophic scar index in rabbit ears of each group 14 days after administration,
Compared to all control groups, without 5-FU, each experimental group with 5-FU (10
Over the past few decades, there was a tremendous development of new technologies that enhance the dermal permeation of drugs. The application of transdermal drug delivery is usually limited by the inability to permeate into deep skin and retention in superficial skin, with merely local effects [
Protocols for liposomes with improved components, which greatly improve their ability of transdermal drug delivery, have been reported. The most representative ones are transfersomes, also known as deformed liposome, developed by Cevc and Blume [
Our previous studies have found that permeability of ethosomes with a particle diameter of 65 nm is higher than that of 200 nm [
For percutaneous administration for therapy of skin diseases, the permeation of drugs into skin tissue through intercellular spaces, hair follicles, sweat glands, and other subsidiary organs and retention in local tissues are premises for the drugs’ therapeutic effect. In our
SEI in each group was measured by HE staining and tissue sections. In our
Effects of gels on cell proliferation. Data expressed as the mean ± SD are representative of 4 independent experiments with similar results. Cell survival was assessed at 24 h, 48 h, and 72 h (
The strong advantage in our study is the use of not only
Our results allow the conclusion that 5-FU transfersomal gel is a safe, convenient, and effective drug carrier for transdermal administration.
Transfersomes hold a variety of advantages, such as nontoxicity, good compatibility with organisms, and penetration enhancement. They can wrap hydrosoluble or liposoluble drugs. The gel preparation can increase drugs’ accumulation on the local skin without affecting the structure of transfersomes and transported drugs, enhance the targeting of drugs towards skin, and reduce the stimulation of drugs themselves to skin. Thus, studies of transdermal scar permeation with the transfersomal gels as a drug carrier have important significance.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Z. Zhang and X. Wang contributed equally to this paper.
This research was supported by National Natural Science Foundation of China (NSFC), Grant nos. 81402618, 81272114, and 81301672 (to Z. Zhang, Y. Zhang, and Y. Wo), Ph.D. innovation fund from Shanghai Jiao Tong University School of Medicine, Grant no. BXJ201334 (to Z. Zhang).