Development of a gel-in-oil emulsion as a transdermal drug delivery system for successful delivery of growth factors.

Growth factors (GFs) are indispensable in regenerative medicine because of their high effectiveness. However, as GFs degenerate easily, the development of a suitable carrier with improved stability for GFs is necessary. In this study, we developed a gel-in-oil (G/O) emulsion technology for the transdermal delivery of growth factors. Nanogel particles prepared with heparin-immobilized gelatin that can bind growth factors were dispersed in isopropyl myristate. The particle size of the G/O emulsion could be controlled by changing the surfactant concentration, volume ratio of the water phase to the oil phase, and gelatin concentration. In vitro skin penetration studies showed better penetration through the stratum corneum of fluorescent proteins containing G/O emulsions than of the aqueous solution of GF. Similarly, an in vivo study showed an angiogenesis-inducing effect after transdermal application of GF-immobilized G/O emulsion. Angiogenesis in mice was confirmed owing to both an increased blood vessel network and higher hemoglobin content in the blood. Therefore, the G/O emulsion could be a promising carrier for GFs with better stability and can effectively deliver GFs at the target site.

Preparation and Characterization of Microemulsions Based on Antarctic Krill Oil.

Antarctic krill oil is high in nutritional value and has biological functions like anti-inflammation and hypolipidemic effects. But it has and unpleasant smell, and unsaturated fatty acids are prone to oxidative deterioration. Its high viscosity and low solubility in water make it difficult for processing. Microemulsion can be a new promising route for development of krill oil product. We determined a formula of krill oil-in-water microemulsion with krill oil: isopropyl myristate = 1:3 as oil phase, Tween 80:Span 80 = 8:2 as surfactant, ethanol as co-surfactant and the mass ratio of surfactant to co-surfactant of 3:1. After screening the formula, we researched several characteristics of the prepared oil-in-water microemulsion, including electrical conductivity, microstructure by transmission electron microscope and cryogenic transmission electron microscope, droplet size analysis, rheological properties, thermal behavior by differential scanning calorimeter and stability against pH, salinity, and storage time.

Role of sodium l-cysteine alginate conjugate and isopropyl myristate to enhance the permeation enhancing activity of BCS class III drug from TDDS; optimization by central composite design and in vivo pharmacokinetics study.

OBJECTIVE: The objective of the present research was to study the effect and optimization of sodium alginate l-cysteine conjugate and permeation enhancer on permeation of high soluble low permeable ropinirole hydrochloride from the transdermal formulation. METHODS: Sodium alginate l-cysteine conjugate was prepared and characterized and the same was added into a transdermal formulation along with IPM as a permeation enhancer. Twelve primary formulations were prepared by solvent casting method and evaluated. The results were fed into Design Expert® Software to obtain optimized formulation. The optimized formulation was evaluated for physicochemical, ex vivo permeation, stability, skin irritation, and pharmacokinetic studies. RESULTS: The results of the characterization of prepared sodium alginate l-cysteine conjugate confirmed the thiolation process. Stability studies suggested that the drug was compatible with all the excipients. SEM images of the transdermal patch revealed that the amorphous drug was uniformly distributed. From the design space, the optimized formulation from the polymer's ratio (SA: SACC; 4:6) and IPM 9.5%w/w of polymers weight showed target steady state flux 9.004 µg/cm2/h with maximum drug permeation. The increased target flux and maximum drug permeation from an optimized patch suggested that there was an effect of SACC on ropinirole hydrochloride permeation in the presence of IPM as a permeation enhancer. Pharmacokinetic studies in rabbits showed that the optimized patch improved bioavailability as compared to marketed oral tablets. CONCLUSIONS: The study was concluded that there was a positive effect of sodium alginate l-cysteine conjugate and IPM on ropinirole hydrochloride permeation from the transdermal formulation.

Transdermal transport of collagen and hyaluronic acid using water in oil microemulsion.

Collagen and hyaluronic acid (HA) are biopolymers that affect the appearance and condition of the skin. Delivery of these compounds into the skin is highly challenging since have a number of disadvantageous properties, such as high molecular weight and hydrophilicity. Here, we evaluated the transdermal penetration of low and high molecular weight collagen and HA from microemulsions. A number of microemulsion formulations, differing in the content of polymers and surfactants (i.e. penetration promoters), were used for the permeation study. In addition, a correlation was made between the composition of these microemulsions and the polymers transport efficiency. The results indicate that, microemulsions enable transdermal permeation of collagen and HA. The concentration of polymers and the solubilization capacity of microemulsions had the greatest influence on the permeation. Surprisingly, the molecular weight of polymers and the content of other components affected the size of microemulsion particles, and thus these parameters had an indirect influence on the permeation process. This study demonstrated therefore the potential therapeutic use of microemulsion with collagen and HA in improving and regenerating the barrier of aged or diseased skin.

Formulation and Structural Study of a Biocompatible Water-in-Oil Microemulsion as an Appropriate Enzyme Carrier: The Model Case of Horseradish Peroxidase.

A novel biocompatible water-in-oil microemulsion was developed using nonionic surfactants and was investigated as a potential enzyme delivery system for pharmaceutical applications. The system was composed of isopropyl myristate/polysorbate 80 (Tween 80)/distilled monoglycerides/water/propylene glycol (PG), had a low total surfactant concentration (8.3% w/w), and was able to incorporate approximately 3% w/w aqueous phase containing horseradish peroxidase (HRP). Structural and activity aspects of the system were studied using a variety of techniques such as dynamic light scattering (DLS), electron paramagnetic resonance (EPR), and dynamic interfacial tension. The apparent hydrodynamic diameter of the empty droplets was calculated at about 37 nm. Different enzyme concentrations, ranging from 0.01 to 1.39 µM, were used for both DLS and EPR studies to effectively determine the localization of the macromolecule in the microemulsion. According to the results, for high enzyme concentrations, a participation of HRP in the surfactant monolayer of the microemulsion is evident. The number of reverse micelles in the microemulsion was defined by a theoretical model and was used to clarify how the enzyme concentration affects the number of empty and loaded reverse micelles. To assure that the system allows the enzyme to retain its catalytic activity, an oxidative reaction catalyzed by HRP was successfully carried out with the use of the model substrate 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]. The influence of several parameters such as temperature, pH, and PG concentration was examined to optimize the reaction conditions, and a kinetic study was conducted revealing an ordered-Bi-Bi mechanism. Values of all kinetic parameters were determined. The release of the encapsulated enzyme was studied using an adequate receiver phase, revealing the effectiveness of the proposed microemulsion not only as a microreactor but also as a carrier for therapeutic biomolecules.

A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant.

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier.

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug in aqueous solution. To overcome this, a nanostructured lipid carrier (NLC) consisting of glyceryl palmitostearate and isopropyl myristate was developed to encapsulate Dac. NLCs with controlled size were achieved using high shear dispersion (HSD) following solidification of oil-in-water emulsion. The synthesis parameters, including surfactant concentration, the speed and time of HSD were optimized to achieve the smallest NLC with size, polydispersion index and zeta potential of 155 ± 10 nm, 0.2 ± 0.01, and -43.4 ± 2 mV, respectively. The optimal parameters were also employed for Dac-loaded NLC preparation. The resultant NLC loaded with Dac possessed size, polydispersion index and zeta potential of 190 ± 10 nm, 0.2 ± 0.01, and -43.5 ± 1.2 mV, respectively. The drug encapsulation efficiency and drug loading reached 98% and 14%, respectively. This is the first report on encapsulation of Dac using NLC, implying that NLC could be a new potential candidate as drug carrier to improve the therapeutic profile of Dac.

Protective effects of ψ taraxasterol 3-O-myristate and arnidiol 3-O-myristate isolated from Calendula officinalis on epithelial intestinal barrier.

The triterpene esters á´ª taraxasterol-3-O-myristate (1) and arnidiol-3-O-myristate (2) were tested for their ability to protect epithelial intestinal barrier in an in vitro model. Their effects on ROS production and on trans-epithelial resistance were investigated on CaCo-2 cell monolayers both in basal and stress-induced conditions. Both compounds were able to modulate the stress damage induced by H2O2 and INFγ+TNFα, showing a potential use as model compounds for the study of new therapeutic agents for intestinal inflammations.

Tris(2-aminoethyl)amine-based α-branched fatty acid amides - Synthesis of lipids and comparative study of transfection efficiency of their lipid formulations.

The synthesis of a new class of cationic lipids, tris(2-aminoethyl)amine-based α-branched fatty acid amides, is described resulting in a series of lipids with specific variations in the lipophilic as well as the hydrophilic part of the lipids. In-vitro structure/transfection relationships were established by application of complexes of these lipids with plasmid DNA (pDNA) to different cell lines. The α-branched fatty acid amide bearing two tetradecyl chains and two lysine molecules (T14diLys) in mixture with the co-lipid 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphoethanolamine (DOPE) (1/2, n/n) exhibits effective pDNA transfer in three different cell lines, namely Hep-G2, A549, and COS-7. The presence of 10% serum during lipoplex incubation of the cells did not affect the transfection efficiency. Based on that, detailed investigations of the complexation of pDNA with the lipid formulation T14diLys/DOPE 1/2 (n/n) were carried out with respect to particle size and charge using dynamic light scattering (DLS), ζ-potential measurements, and transmission electron microscopy (TEM). Additionally, the lipoplex uptake was investigated by confocal laser scanning microscopy (CLSM). Overall, lipoplexes prepared from T14diLys/DOPE 1/2 (n/n) offer large potential as lipid-based polynucleotide carriers and further justify advanced examinations.

Development and in-vivo evaluation of ondansetron gels for transdermal delivery.

Nausea and vomiting are some of the major side effects caused by certain drug therapies, e.g. chemotherapy, radiotherapy and general anesthesia. Because of the nature of the symptoms, oral delivery is inappropriate, while intravenous administration may be unpractical. The aim of the present study was to develop a transdermal gel (2% Klucel®) for ondansetron, a first line 5-HT3-receptor-antagonist antiemetic. The effects of the penetration enhancer camphor and isopropyl-myristate (IPM) were first investigated in-vitro using modified Franz diffusion-cells and then tested in-vivo in a rabbit model by measuring skin and plasma concentrations. Since a disadvantage of transdermal delivery is a prolonged lag-time, the effect of skin treatment with a micro-needle roller was tested. The in-vitro permeation studies through excised porcine ear skin showed that the presence of 2.5% camphor or IPM increased steady state flux by 1.2- and 2.5-fold, respectively, compared to the control gel. Ondansetron was not detectable in either skin or plasma following in-vivo application of the base-gel, whereas the camphor gel and IPM gel delivered 20 and 81 µg/cm(2) of ondansetron, respectively. Microporation led to an increase in plasma Cmax and AUC by 10.47 ± 1.68-fold and 9.31 ± 4.91-fold, respectively, for the camphor gel, and by 2.31 ± 0.53-fold and 1.59 ± 0.38-fold, respectively for the IPM gel. In conclusion, the 2.5% IPM gel demonstrated optimal in-vivo transdermal flux. Skin pretreatment with a micro-needle roller slightly improved the delivery of the IPM gel, whereas dramatically increased the transdermal delivery of the camphor gel.

Transdermal absorption of natural progesterone from alcoholic gel formulations with hydrophilic surfactant.

OBJECTIVES: The aim of this study was to evaluate the in vitro skin permeation and in vivo transdermal absorption of natural progesterone (Prog) from alcoholic gel-based transdermal formulations containing Prog dissolved stably at a concentration of 3%. METHODS: 3% Prog dissolved gel formulations were prepared containing with water, ethanol, 1,3-butylene glycol, carboxyvinylpolymer, diisopropanolamine, polyoxyethylene (2) oleylether and benzyl alcohol. The gel formulations added different hydrophilic surfactants and isopropyl myristate or propylene glycol dicaprylate (PGDC) as oily solvents were applied in vitro permeation study through excised rat skin on unocclusive condition. The gel formulations added polyoxyethylene (20) oleylether (Oleth-20) as hydrophilic surfactant and PGDC were applied in vivo single- and repeated-dose transdermal absorption study of rat on unocclusive condition. RESULTS: The results of evaluation of the gel formulations by an in vitro skin permeation study revealed a high flux of Prog from the formulation containing Oleth-20 and Oleth-20 with PGDC. The results of single and repeated in vivo transdermal absorption studies confirmed that good plasma levels of Prog were achieved and maintained by Oleth-20 and PGDC containing gel formulation. CONCLUSIONS: The Oleth-20 and PGDC containing ethanolic gel formulation seemed to have the ability to maintain a high activity of Prog and high diffusivity or solubility of Prog in the epidermis on the practical formulation application.

Nitroxide-loaded hexosomes provide MRI contrast in vivo.

The purpose of this work was to synthesize and screen, for their effectiveness to act as T1-enhancing magnetic resonance imaging (MRI) contrast agents, a small library of nitroxide lipids incorporated into cubic-phase lipid nanoparticles (cubosomes). The most effective nitroxide lipid was then formulated into lower-toxicity lipid nanoparticles (hexosomes), and effective MR contrast was observed in the aorta and spleen of live rats in vivo. This new class of lower-toxicity lipid nanoparticles allowed for higher relaxivities on the order of those of clinically used gadolinium complexes. The new hexosome formulation presented herein was significantly lower in toxicity and higher in relaxivity than cubosome formulations previously reported by us.

Formation, thermodynamic properties, microstructures and antimicrobial activity of mixed cationic/non-ionic surfactant microemulsions with isopropyl myristate as oil.

HYPOTHESIS: Modification of the interface by blending of surfactants produces considerable changes in the elastic rigidity of the interface, which in turn affects the physicochemical properties of w/o microemulsions. Hence, it could be possible to tune the thermodynamic properties, microstructures and antimicrobial activity of microemulsions by using ionic/non-ionic mixed surfactants and polar lipophilic oil, which are widely used in biologically relevant systems. EXPERIMENTS: The present report was aimed at precise characterization of mixed cetyltrimethylammonium bromide and polyoxyethylene (23) lauryl ether microemulsions stabilized in 1-pentanol (Pn) and isopropyl myristate at different physicochemical conditions by employing phase studies, the dilution method, conductivity, DLS, FTIR (with HOD probing) and (1)H NMR measurements. Further, microbiological activities at different compositions were examined against two bacterial strains Bacillus subtilis and Escherichia coli at 303 K. FINDINGS: The formation of mixed surfactant microemulsions was found to be spontaneous at all compositions, whereas it was endothermic at equimolar composition. FTIR and (1)H NMR measurements showed the existence of bulk-like, bound and trapped water molecules in confined environments. Interestingly, composition dependence of both highest and lowest inhibitory effects was observed against the bacterial strains, whereas similar features in spontaneity of microemulsion formation were also evidenced. These results suggested a close relationship between thermodynamic stability and antimicrobial activities. Such studies on polar lipophilic oil derived mixed surfactant microemulsions have not been reported earlier.

Efficient delivery and distribution in skin of chlorogenic acid and resveratrol induced by microemulsion using sucrose laurate.

To achieve efficient skin delivery of polyphenols, we prepared a novel oil-in-water (o/w)-type microemulsion (MESL) using sucrose laurate as a surfactant and ethanol, isopropyl myristate and water as other components. We examined its usefulness by in vitro studies on skin delivery of chlorogenic acid and resveratrol as hydrophilic and hydrophobic polyphenols using Yucatan micropig skin, and also examined the difference in the distribution of these polyphenols in skin. MESL significantly improved skin incorporation of these polyphenols at all time points examined (6, 20, 40 h) in the epidermis and at 20 and 40 h in the dermis, compared with the microemulsion using Tween 80 as a surfactant component (MEK), although the solubilization capacity of MESL was lower than that of MEK. Using MESL, the incorporation amount in the dermis of each polyphenol increased with time, while the amount in the epidermis was almost constant during the time examined. Incorporation efficiencies into skin of chlorogenic acid and resveratrol induced by MESL at 40 h after application were about 6-fold and 19-fold higher in the epidermis and 3.5-fold and 15-fold higher in the dermis, respectively, than those by MEK. The increase was more prominent for resveratrol. Hydrophilic chlorogenic acid was distributed slightly more in the epidermis, while hydrophobic and smaller-molecular-weight resveratrol was mainly distributed in the dermis. These findings suggest that MESL could be a promising vehicle for the efficient skin delivery of chlorogenic acid and resveratrol, especially for resveratrol to the dermis.

Development of novel ionic liquid-based microemulsion formulation for dermal delivery of 5-Fluorouracil.

The present study was aimed at synthesizing an imidazole-based ionic liquid 1-butyl-3-methylimidazolium bromide (BMIMBr) and subsequent development of a novel ionic liquid-in-oil (IL/o) microemulsion (ME) system for dermal delivery of a poorly permeating drug 5-fluorouracil (5-FU). A significant enhancement in the solubility of 5-FU was observed in BMIMBr. IL/o MEs of 5-FU were prepared using isopropyl myristate, Tween 80/Span 20, and BMIMBr. Results of ex vivo skin permeation studies through mice skin indicated that the selected IL/o ME exhibited 4-fold enhancement in percent drug permeation as compared to aqueous solution, 2.3-fold as compared to hydrophilic ointment, and 1.6-fold greater permeation than water in oil (w/o) ME. The results of in vivo studies against dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mice skin carcinogenesis demonstrated that the IL/o ME could effectively treat skin cancer in 4 weeks. In addition, the side effects such as erythema and irritation associated with the conventional formulations were not observed. Histopathological studies showed that the use of IL/o ME caused no anatomic and pathological changes in the skin structure of mice. These studies suggest that the use of IL-based ME system can efficiently enhance the solubility and permeability of 5-FU and hence its therapeutic efficacy.

A step toward the development of high-temperature stable ionic liquid-in-oil microemulsions containing double-chain anionic surface active ionic liquid.

Owing to their fascinating properties and wide range of potential applications, interest in nonaqueous microemulsions has escalated in the past decade. In the recent past, nonaqueous microemulsions containing ionic liquids (ILs) have been utilized in performing chemical reactions, preparation of nanomaterials, synthesis of nanostructured polymers, and drug delivery systems. The most promising fact about IL-in-oil microemulsions is their high thermal stability compared to that of aqueous microemulsions. Recently, surfactant-like properties of surface active ionic liquids (SAILs) have been used for preparation of microemulsions with high-temperature stability and temperature insensitivity. However, previously described methods present a limited possibility of developing IL-in-oil microemulsions with a wide range of thermal stability. With our previous work, we introduced a novel method of creating a huge number of IL-in-oil microemulsions (Rao, V. G.; Ghosh, S.; Ghatak, C.; Mandal, S.; Brahmachari, U.; Sarkar, N. J. Phys. Chem. B2012, 116, 2850-2855), composed of a SAIL as a surfactant, room-temperature ionic liquids as a polar phase, and benzene as a nonpolar phase. The use of benzene as a nonpolar solvent limits the application of the microemulsions to temperatures below 353 K. To overcome this limitation, we have synthesized N,N-dimethylethanolammonium 1,4-bis(2-ethylhexyl) sulfosuccinate (DAAOT), which was used as a surfactant. DAAOT in combination with isopropyl myristate (IPM, as an oil phase) and ILs (as a polar phase) produces a huge number of high-temperature stable IL-in-oil microemulsions. By far, this is the first report of a huge number of high-temperature stable IL-in-oil microemulsions. In particular, we demonstrate the wide range of thermal stability of [C6mim][TF2N]/DAAOT/IPM microemulsions by performing a phase behavior study, dynamic light scattering measurements, and (1)H NMR measurements and by using coumarin-480 (C-480) as a fluorescent probe molecule.

A novel ionic liquid-in-oil microemulsion composed of biologically acceptable components: an excitation wavelength dependent fluorescence resonance energy transfer study.

In this work we have reported the formulation of a novel ionic liquid-in-oil (IL/O) microemulsion where the polar core of the ionic liquid, 1-ethyl-3-methylimidazolium n-butylsulfate ([C2mim][C4SO4]), is stabilized by a mixture of two nontoxic nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80) and sorbitan laurate (Span-20), in a biological oil phase of isopropyl myristate (IPM). The formation of the microemulsion droplets has been confirmed from the dynamic light scattering (DLS) and phase behavior study. To assess the dynamic heterogeneity of this tween-based IL/O microemulsion, we have performed an excitation wavelength dependent fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G). The multiple donor-acceptor (D-A) distances, ∼15, 30, and 45 Å, obtained from the rise times of the acceptor emission in the presence of a donor can be rationalized from the varying distribution of the donor, C480, in the different regions of the microemulsion system. With increasing the excitation wavelength from 375 to 408 nm, the contribution of the rise component of ∼240 ps which results the D-A distance of ∼30 Å increases significantly due to the enhanced contribution of the C480 probe molecules closer to the acceptor in the ionic liquid pool of the microemulsion.

In vitro percutaneous absorption enhancement of granisetron by chemical penetration enhancers.

UNLABELLED: Granisetron (GRN), a potent antiemetic agent, is frequently used to prevent nausea and vomiting induced by cancer cytotoxic chemotherapy and radiation therapy. OBJECTIVE: As part of our efforts to further modify the physicochemical properties of this market drug, with the ultimate goal to formulate a better dosage form for GRN, this work was carried out to improve its permeability in vitro. METHODS: The permeation behavior of GRN in isopropyl myristate (IPM) was investigated across excised rabbit abdominal skin and the enhancing activities of three novel O-acylmenthol derivatives synthesized in our laboratory as well as five well-known chemical enhancers were evaluated. RESULTS: It was found that the steady-state flux of granisetron free base (GRN-B) was about 26-fold higher than that of granisetron hydrochloride (GRN-H). The novel enhancer, 2-isopropyl-5-methylcyclohexyl heptanoate (M-HEP), was observed to provide the most significant enhancement for the absorption of GRN-B. When incorporated in the donor solution with the optimal enhancer M-HEP, the steady-state flux of GRN-B increased from (196.44 ± 12.03) µg·cm⁻²·h⁻¹ to (1044.95 ± 71.99) µg·cm⁻²·h⁻¹ (P < 0.01). CONCLUSION: These findings indicated that the application of chemical enhancers was an effective approach to increase the percutaneous absorption of GRN in vitro.

Anti-tumor effects of cationic hybrid liposomes against colon carcinoma along with apoptosis in vitro.

New-type three-component cationic hybrid liposomes (HLs) composed of dimyristoylphosphatidylcholine (DMPC), polyoxyethylene(21)dodecyl ether (C(12)(EO)(21)) and O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine chloride (2C(14)ECl) were produced. Cationic HLs were smaller and more stable than pure DMPC liposomes. It is noteworthy that cationic HLs could remarkably inhibit the growth of human colon cancer (HCT116) cells along with apoptosis in vitro for the first time in this study.

Transdermal delivery of the anti-rheumatic agent methotrexate using a solid-in-oil nanocarrier.

Transdermal delivery of methotrexate (MTX) was investigated by using the solid-in-oil (S/O) technique. Because MTX was coated with nonionic surfactant molecules, the resulting complex was easy to dissolve in various organic solvents and provided a transparent solution in isopropyl myristate (IPM). The stability of MTX-surfactant complexes are enhanced by the addition of a basic amino acid such as L-Arginine (L-Arg) or L-Lysine (L-Lys). The average size of the dispersed complex of MTX and amino acid was reduced to below 100 nm and gave a uniform distribution. A transdermal delivery experiment was conducted using the S/O nanocarrier, and the permeation behavior of MTX through Yucatan micropig (YMP) skin was evaluated with a Franz diffusion cell. The permeation efficiency for the S/O nanocarrier (not urea addition) was two- to threefold increased compared to that of the control aqueous solution because the oil-based nanocarrier is effective for penetrating the stratum corneum. Furthermore, addition of urea has dramatically improved the release property of MTX from the S/O nanocarrier, and the S/O nanocarrier containing urea showed an optimal permeation efficiency of approximately 8.8-fold increased compared to that of the control aqueous solution after 24h (p<0.01).