Formulation and optimization of transfersomes containing minoxidil and caffeine
Alopecia is one of the common causes of hair loss in the world. Aimed to improve drug delivery to hair follicles, a box benken design was applied to formulate minoxidil and caffeine in liposomes with flexible membrane (transfersome). In particular, the ratio of polysorbate 20 and polysorbate 80, as the edge activators, and the hydration volume were studied as independent variables. Furthermore, entrapment efficiency, release rate, and stability of transfersomes were evaluated as dependent variables. The results showed that using of 22.2% of polysorbate 80 and 9.3 % polysorbate 20 in the formulation enhanced the drug delivery to skin. Also, increasing the ratio of polysorbate enhanced entrapment efficiency of minoxidil and caffeine because of an increase in liposome formation. Finally, Co- delivery of minoxidil and caffeine in transfersomes enhanced the hair length and weight in vivo.
Key word: Transfersome, Caffeine, Minoxidil, Polysorbate 80, Polysorbate 20
Androgenic alopecia is a progressive, age-dependent hair loss that involves both men and women. Systemic and/or topical administration of a variety of agents such as 5-alpha reductase inhibitors, minoxidil and caffeine are indicated for the treatment of alopecia. While systemic administration is limited by adverse drug effects, effective drug delivery is challenging in topical formulations (1).
Dermal and transdermal systems are used for local and systemic drug delivery (2, 3). In these systems, stratum corneum, the outer layer of skin, is the major barrier against drug absorption and production of therapeutic concentration (4). A large body of experiments are conducted to overcome the low permeability of this layer in order to increase drug concentration in hair follicles (5).
Minoxidil, initially developed as an antihypertensive agent, is also approved by FDA for the improvement of hair growth. In fact, when minoxidil was indicated for the treatment of hypertension, hirsutism was reported as a side effect (6). The mechanism of minoxidil-induced hair growth is not clearly understood, but it enhances blood supply to the hair follicles and, thereby, increases oxygen and nutrient delivery to the cells. It, also, shortens the telogen phase and elongates anagen phase (7). Effective therapeutic concentration for the treatment of androgenic alopecia is achieved by formulations containing 2% and 5% of minoxidil in women and men, respectively. For optimal drug delivery, it should be applied regularly on the scalp at least 2 times a day. Hair growth is observable as far as the patient applies the drug (6).
According to the fact that minoxidil is insoluble in water, hydroalcoholic formulations are designed to ensure achievement of effective therapeutic concentrations. These formulations, however, cause irritation and contact dermatitis. In particular, propylene glycol, a co-solvent and absorption enhancer in topical minoxidil formulations, is more frequently reported to cause hypersensitivity and allergic reactions. Minoxidil, also, causes pruritus, dandruff, and local intolerance. In addition, such topical formulations should be applied at least two times a day to produces a notable effect, which may decrease patient’s compliance (6, 8).
Caffeine, available in topical formulations, is also indicated for the treatment of hair loss and stimulation of hair growth (9). In most cases of androgenic alopecia, dihydrotestosterone (DHT) is the main culprit for hair loss. It influences hair follicle and shortens telogen and anagen phases of the hair growth cycle. Therefore, the follicles stop growing after several cycles. Caffeine is shown to inhibit 5-α-reductase, an enzyme responsible for testosterone conversion to DHT. Caffeine, also, inhibits phosphodiesterase (PDE), an enzyme responsible for degradation of cAMP. This increases intracellular levels of cAMP and stimulates cellular metabolism. Furthermore, caffeine causes vasodilation and, thereby, increases blood supply to the follicles. The therapeutic effect of caffeine is observed in formulations containing 0.001% -0.005% of the drug (10).
Write a paragraph about the synergism of minoxidil and caffeine!!!
Liposomal formulations are considered as phenomenal structures due to their ability to entrap both hydrophilic and lipophilic compounds (11). Their structures contain one or more phospholipid bilayer(s), consisted of soy oil, egg yolk or synthetic phospholipids, surrounding an aqueous core (4). These structures are vastly used for systemic or topical drug delivery. New generation of liposomes, transfersomes, contain other surfactants in addition to phospholipids. These include sodium cholate, deoxycholate, Span 80, Polysorbate 80 and dipotassium glycyrrhizinate (12). Surfactants, also known as edge activator, produce more flexible liposomes, and enhances drug permeation through the skin. This increases drug concentrations in deeper layers of the skin even in blood circulation (13). Therefore, liposomal formulations, especially transfersomes, are suitable delivery systems for compounds with low water solubility or skin permeation.
In this investigation, aimed to increase the efficacy of topical minoxidil and caffeine, we designed a transfersome for co-delivery of minoxidil and caffeine.
Materials and methods
Soybean phosphatidylcholine was obtained from lipoid (Canada). Minoxidil and caffeine were purchased from Fagron Ltd (UK) and Merck (Germany), respectively. Lactic acid, polysorbate 80 and polysorbate 20) and dialysis tube with cut off 12000 D were from Sigma-Aldrich (USA). All reagents were at analytical grade.
Transfersomes were prepared by thin film hydration method with some modifications (Bangham et al.,1965)(14). In particular, lecithin was mixed with various amounts of edge activators (table 1), and dissolved in chloroform. Aqueous solutions of minoxidil (2%) and caffeine (0.01 %) was prepared in acidic medium (pH=??). In fact, lactic acid (10% v/v) was used to increase the water solubility of minoxidil. The mixture was then added to round-bottomed flasks containing lecithin and other surfactants, and incubated with glass beads to hydrate the lipid thin layer for 30 min. Organic solvent was eliminated using a rotary evaporator (IKA, RV10).
In vitro drug release
The in vitro drug release studies were performed using diffusion Franz cell. Artificial membrane was mounted between the donor and receptor compartment. Consequently, the donor compartment was filled with 1 ml of each formulation, and the receptor compartment was filled with 33 ml of distilled water. The temperature in Franz cell was maintained at 37 ºC, and the receptor compartment was stirred continuously at 50 rpm using a magnetic stirrer. Samples were taken at regular intervals over 24 h. Finally, the concentration of minoxidil and caffeine in the samples were analyzed with UV spectrophotometry at 230nm and 205 nm, respectively, and quantified using a standard curve with LOD and LOQ of ???.
Stability test was performed to analyze the number of remaining vesicles over time. All formulations were stored at 25ºC for 28 days in glass vials, and samples were taken every week. The number of vesicles were counted using a neubauer chamber and optical microscopy. Subsequently, the number of the vesicles in 1 ml of each formulation was calculated with the formula 1 (15).
concentration= Number of vesicle ×10000Number of square × dilution factor formula 1
The encapsulation efficiency (EE%) of transfersomes were determined using spectrophotometry. In particular, a dialysis bag was filled with 1 ml of each formulation, and submerged in 2 L of distilled water for 4 h at 4 °C. Then, the transfersomes were lysed with ethanol, and their minoxidil content was determined using a spectrophotometer at 230 nm. Finally, encapsulation efficiency (EE%) was calculated according to the formula 2.
EE%=drug contenttotal drug added*100% Formula 2
Male and female Wistar rats, weighting 300-350 gr, were used for the evaluation of hair growth in vivo. Rats were divided into four groups (n=8; 4 male and 4 female), and an area of 4 cm2 from dorsal side was shaved. Subsequently, 1 ml of the each sample was applied on the shaved area once a day for 30 days. Group 1 was treated with optimized transfersomes containing minoxidil and caffeine; group 2 received transfersome placebo; group 3 was treated with aqueous solution of minoxidil and caffeine, and the last group was treated with commercial minoxidil hydroalcoholic solution. The rats were kept at 12/12 h light/dark cycles and had free access to food and water, and animal procedures were approved by the Ethical Committee of Shahid Sadoughi University of Medical Sciences.
The effects of formulations on hair growth was evaluated on days 10, 20, and 30 after topical administration of the drugs. In fact, ten samples of hair were plucked randomly from the shaved area using forceps, and their length and weight were determined using appropriate instruments.
A box behnken method was applied for design, evaluation and optimization of transfersome. In particular, phosphatidylcholine was fixed at 50 mg; the lower (-1) and higher (+1) levels of other ingredients were defined as independent variables. In fact, polysorbate 80 from 0 to 50% of phosphatidylcholine and polysorbate 20 from 0 to 30% of phosphatidylcholine was used in different formulations. In addition, the total aqueous volume of hydration was adjusted from 2.5 to 7.5 ml . Ultimately, 14 runs with 2 center points were designed according to table 1. Entrapment efficiency (EE%), drug release rate from vesicles, and the stability of vesicles were analyzed as dependent variables. Finally, linear regression with ANOVA was used to choose the best fitted model, and p values less than 0.05 were considered as statistically significant. In addition, the data of in vivo study were analyzed using two-way ANOVA followed by Bonferoni post-test.
Results and discussion
Minoxidil base is insoluble in water but soluble in alcohol (Fig. 1). Aqueous solubility of the drug was enhanced with ionization of the molecule at acidic medium (pH: 3.8 – 4) with lactic acid. Ionization of minoxidil enhances water solubility. However, it decreases drug absorption, because lipid membranes such as skin allow only the uncharged molecules to cross the barrier. Therefore, appropriate vehicles should be used to enhance skin penetration of charged molecules (16).
To increase the skin penetration of charged minoxidil, highly flexible liposome for co-delivery of minoxidil and caffeine was formulated using a box behnken design. A total of 14 runs were experimented to evaluate several independent and dependent variables. However, the concentrations of caffeine and minoxidil were fixed at 0.01% and 2%, respectively. Aimed to increase the efficacy and flexibility of lecithin based liposomes, polysorbate 80 and polysorbate 20 were used as edge activator.
Drug entrapment efficiency
The overall entrapment efficiency of transfersomes varied from 13.62 to 48.82% in different formulations (Table 2). In fact, the data of entrapment efficieny was best fitted to a modified quadratic model; EE% = +37.03 +5.01*A +0.45*B +4.29*C -4.53*A*C -3.87*B*C +2.09*A2 -9.67*C2; where A, B, and C are polysorbate 80, polysorbate 20, and hydration volume, respectively. Indeed, the amount of entrapped drug in vesicles was dependent on the ratio of polysorbate 80 (β: 5.01, p= 0.012) as well as the hydration volume (β: 4.29 , p= 0.023). In contrast, polysorbate 20 (β: 0.45, p >0.05) didn’t produce any significant effect on drug entrapment in any concentration. Furthermore, there was a significant interaction between polysorbate 80 and hydration volume (β: -4.53, p<0.05).
There was a positive correlation between hydrophilic-lipophilic balance (HLB) and entrapment efficiency (Fig. 2). In fact, higher entrapment efficiency was obtained in formulations with higher HLB. This is due to facilitation of vesicle formation because of an increase in total surfactant content. Accordingly, Van den Bergh et al., (2001) showed that increasing the content of edge activator in transfersomes enhanced entrapment efficiency due to formation of larger vesicles (17).
Hydration volume produced a biphasic effect on EE. In fact, EE increased to 36%, when hydration volume raised from 2.5 to 6.25 ml (Fig. 3). However, further increase of hydration volume caused a perturbation of EE. This is explained by the fact that higher lipid content of the formulation restricts the membrane hydration and, therefore, decreases entrapment of water soluble drugs (Ref.??). Furthermore, higher concentration of total lipid in aqueous phase hampers surfactant-water interaction leading to formation of surfactant micelles or other mixed micelle systems (Ref??). In addition, too much edge activators in liposomes may cause the formation of pores in lipid bilayer (20), hence, decrease the entrapment efficacy. In this regard, encapsulation of ﬂurbiprofen into proniosomes (18) and diclofenac into transfersomes (19) required optimization of total lipid content and hydration of the formulation. taken together, for maximal EE, the concentration of edge activators and hydration volume should be optimized based on the physicochemical properties of the drug.
Stability of transfersomes
Stability of transfersomes as an essential factor of formulation was evaluated by vesicle counting at various time intervals. At time 0, the data was best fitted to a 2FI model; Vesicle count = +2555.00 +272.87*A -1021.25*B -216.63*C -53.50*AB –109.75*A*C +221.00*B*C -1109.37*A2; where A, B, and C are polysorbate 80, polysorbate 20, and hydration volume, respectively. This indicates that only the concentration of polysorbate 20 negatively influenced the vesicle count (β: -1021.25, p<0.01, table 3). Similarly, vesicle counting 7 days (β: -599.75) and 28 days (β: -324.88) after formulation revealed polysorbate 20 as the main influencing factor. This indicates that polysorbate 20 not only decreased vesicle formation at the time of formulation, but also caused perturbation of the vesicle stability. Interestingly, formulations without polysorbates, run13, showed good stability over the time of study compared with those containing different amounts of polysorbates (Fig. 4).
Proper formation of liposomes depends on efficient hydration of lipid film. Therefore, surface active agents with high HLB value ought to enhance vesicle count. In contrast, we observed no benefical effect of polysorbate 80 and even a negative effect of polysorbate 20 on lipid bilayer formation and stability. This can be explained by the fact that phosphatidylcholine becomes ionized in acidic medium which was used to increase water solubility of minoxidil. Therefore, the similarity of charges between the polysorbates, especialy with polysorbate 20, and other membrane ingredients such as phosphatidylcholine in acidic medium hampers liposome formation and stability. It seems that surfactants interact with membrane according to Lichtenbergs three-step model (21). Indeed, that increase of the surfactant concentration gradually dissolves the liposome into mixed micelles. Further increase in surfactant concentration may completely dissolve liposomal bilayer structure and cause instability. Therefore, proper selection of the surfactant as well as its concentration and composition of the lipid bilayer ingredients are critical for optimal vesicle count (18, 22).
The rate and pattern of drug release from various formulations are summerized in table 4. Overall, the first order kinetics was the best fitted model for the release pattern of both minoxidil and caffein in all formulations. However, the rate of release was different in formulations containing different amounts of excipients.
Both caffeine and minoxidil were dissolved in water. Therefore, they were incorporated in the internal aqueous compartment, and exhibited similar patterns of release. In particular, A modified quadratic model was fitted to minoxidil release; minoxidil release = +341.30 -22.00*A -7.13*B 1.88* C -23.00*A*B -73.00*A*C -75.45*A2 -17.70*B2; where A, B, and C are polysorbate 80, polysorbate 20, and hydration volume, respectively. This indicates polysorbate 80 (β: -22, p=0.02) as the main factor influencing the release rate. Moreover, a synergist effect was seen between polysorbate 80 and polysorbate 20 (Fig. 6) as well as between polysorbate 80 and hydration volume (Fig. ??). Taken together, formulations containing 22.2% of polysorbate 80 and 9.03 of polysorbate 20 exhibitted the lowest (highest ??) release rate.
The general first-order kinetics for drug release from liposomes indicates concentration-dependent diffusion across the membrane. On the other hand, zero-order kinetics reveals concentration-independent release and possible disintegration of liposomal membrane resulting in sudden drug release. In this regard, surfactants enhance the membrane fluidity and deformity of liposome (Ref??). Moreover, phosphatidylcholine produces more hydrogen bonds with aqueous solutions in acidic medium, hence, increases the stability of membrane. Therefore, application of acidic medium not only enhances minoxidil solubility in water, but also increases the membrane integrity, resulting in simple diffusion of the drug from membrane in a first-order kinetics. However, as discussed in previous sections, such a phenomenon requires proper selection of the type and amount of the surfactant.
Surfactants produce different behaviors in liposome membrane. In particular, surfactants at high concentrations may cause pore formation resulting in burst release of encapsulated drug from the liposome (23, 24) (4). In contrast, they may improve membrane integrity, thus, cause a uniform slow rate of release (ref???). We found an inverse correlation between the concentration of polysorbate 80 (β: -22, p=0.02) and drug release rat as well as an insignificant effect for polysorbate 20. Therefore, the uniform release of caffeine and minoxidil from the liposomes without a burst release shows no solubilizing effect or pore formation for the surfactants at concentrations applied. In general, the release of drugs from liposomes can be increased by the existence of bilayer softening materials (25, 26). However, the precise selection of the type and concentration of surfactants and presence of other excipients which can influence membrane integrity are also critical for drug release from the liposomes (Ref??).
The optimized formulation was designed based on the effect of independent variables on each dependent variable. In particular, independent factors at pervious ranges were chosen to result in maximum EE and stability after 28 days and minimum release rate. The final optimized formulation contained minoxidil 2%, caffeine 0.01%, 50 mg phosphatidylcholine, 22.2% polysorbate 80, 9.3% polysorbate 20 and the hydration volume of 6.33 ml.
In vivo study
Hair length and weight was studies over a period of 30 days to evaluate the therapeutic efficacy of minoxidil and caffeine in optimized transfersomes. As shown in figure 7 and 8, all groups even those receiving placebo showed an increase in hair growth over the period of study. However, the growth rate was significantly different among the groups. In particular, rats treated with optimized formulation showed the fastest hair growth in terms of length (mean difference with placebo: ??, p<??) and weight (mean difference with placebo: ??, p<??) 10 days after topical administration of the drug. Accordingly, aqueous solution as well as hydroalcoholic solutions also increased hair length and weight, though with less magnitude of effect compared to the minoxidil-caffeine transfersomes. This pattern, also, repeated in other time intervals of study (20 and 30 days after topical administration).
Interestingly, the effect of transfersome on hair length was more evident in the first 10 days (mean difference with placebo: ??, p<??) compared to days 20 (mean difference with placebo: ??, p<??) or 30 (mean difference with placebo: ??, p<??) after drug administration. The similar pattern was also observed in terms of hair weight. This indicates different growth rate in measured time intervals. This may be explained by the fact that hair growth reached a maximum after 30 days. In fact, hair development in rats follows 3 cycles anagen, catagen and telogen every 35 days. Generally, hair growth in length and weight happens in anagen, whereas pigmentation and deposition of germ and club occurs in catagen; finally, in telogen hair follicles will undergo a rest period (27).
The better efficacy of transfersomes over aqueous solution revealed higher efficacy of the former for drug delivery to follicles. This is achieved by means of two distinct mechanisms. Firstly, surfactants in lipid bilayer increase penetration through stratum corneum. Furthermore, the transfersome vesicles disturb the intercellular lipid layers, and improve penetration into the deeper layers of the skin. Therefore, transfersomes enhance minoxidil and caffeine bioavailability in cutaneous targeting (24). Consequently, minoxidil and caffeine promote hair growth through better nourishment of hair follicles and entry of more cells to anagen phase (5)(9).
Transfersomes are suitable systems for co-delivery of minoxidil and caffeine to the deeper layers of the skin. However, the type and amount of excipients are critical for optimal drug deposition and release. In this study, a transfersome system was formulated for the delivery of minoxidil and caffeine to hair follicles. The optimal formulation contained minoxidil 2%, caffeine 0.01%, 50 mg phosphatidylcholine, 22.2% polysorbate 80, 9.3% polysorbate 20, with the hydration volume of 6.33 ml. Topical administration of this formulation more effectively promoted hair growth in rats compared to commercially available hydroalcoholic and aqueous solutions.
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