Table 2 presents the findings of caffeine solubility in different types of lipid phases and surfactants. The solubility of caffeine in stearic acid and GMS (solid lipids) was 47.11± 3.048, 32.67± 2.955 mg/g, respectively. Solubility of caffeine in liquid oils showed high solubility in oleic acid 17.043 ± 0.715 mg/g and lowest solubility in garlic oil (0.15 ± 0.022 mg/g). The solubility of caffeine in oils was arranged in descending order as follow; oleic acid > caproyl 90 > O: G (2:1) > Labrafil M1944 > jojoba oil > O: G (1:1) > amla oil > rosemary oil > CCTG > O: G (1:2) > (IPM) > garlic oil. The results also revealed that caproyl 90, O: G (2:1), Labrafil M1944, jojoba oil, and O: G (1:1) had an intermediate effect on the drug solubility that ranged from 6.37 to 4.07 mg/g.
Mixing garlic oil with oleic acid had a marked effect on reducing the drug solubility in the oil mixture. Increasing the ratio of garlic oil in the oil mixture obviously decreased the amount of drug dissolved. The solubility of caffeine in O: G (2:1), O: G (1:1) and O: G (1:2) was 6.37 ± 1.15, 4.07 ± 0.54, and 0.89 ± 0.093mg/g. These results were related to garlic oil nature that contains oil-soluble organosulfur components .
Hence, the extent of drug solubility in lipid excipients greatly affects the drug loading; it was expected to choose oleic acid for NLCs preparation. In this experimental part, the authors not only focused on the drug solubility, but also on how to maximize the therapeutic efficiency of the applied dosage form. This was accomplished through the drug used and the synergistic effect that could be achieved from vehicles having benefits in hair follicle targeting and hair growth-promoting for alopecia treatment . Accordingly, oleic acid: garlic oil (O: G) mixture at ratio 1:1 v/v was picked as the liquid oil phase for NLCs preparation. Oleic acid acts as a skin permeation enhancer through interaction and modification of SC lipids, thus, forms a new type of lipid domain that can decrease the capacity of skin barrier function. Consequently, it will disorganize and increase the lipid packing fluidity, decrease the diffusional resistance, and facilitates permeation . Garlic oil has different health-promoting benefits, especially its effect as antifungal, antimicrobial, and hair growth promoting properties .
The solubility of caffeine in Tween 80 and Cremophor EL was 8.87 ± 0.49 and 8.66 ± 0.756 mg/g, respectively (Table 2). Both surfactants exhibited similar HLB values which may explain the slight difference in their ability to solubilize the drug. Both are nonionic surfactants that are less toxic and used with low concentrations for effective reduction in interfacial tension. The solubility of caffeine in co-surfactant, Transcutol HP, Span 20, and Span 80, was 25.82 ± 0.0832, 14.2 ± 0.448, and 6.25 ± 0.68 mg/g, respectively (Table 2). The results revealed that Transcutol HP showed the highest solubilization capacity for caffeine and that was attributed to the more hydrophilic properties of Transcutol HP relative to Span 20 or Span 80. Transcutol HP was supposed to raise the interfacial fluidity of a surfactant's external surface in the micelles thus enhancing the solubilization and emulsification .
Upon comparing the solubility level of the drug in various estimated phases, co-surfactants, surfactant, solid and liquid lipid, it was found that the highest solubility of the drug was in the solid lipid (about twice or more drug solubility in liquid lipid or surfactant or cosurfactant). This result was the same informed by Amanda, et al.,  who found that solubility of fluconazole (hydrophilic in nature) was the highest solubility in solid lipid reaching 250 mg/g stearic acid and reaches 45mg/g castor oil.
Miscibility of liquid and solid lipid, surfactant and co-surfactant, and drug solubility in mixtures
The NLCs are modulated by the addition of liquid lipids to the solid lipids which make their difference from SLNs. The solid/liquid lipids mixture imparts imperfection or amorphous structures to NLCs matrices. NLCs lipid molecules generate large distances among fatty acid chains, more drug accommodation occurred, and thus increase drug loading during storage . Accordingly, the miscibility test was carried out for determining the ratio of liquid: solid lipid that showed high miscibility and ensures high drug loading capacity. The solid lipid, stearic acid or GMS, and the liquid lipid, O: G at ratio 1:1, were blended at ratios; 90:10, 80:20. 70:30, 60:40 and 50:50. Results revealed that the blends at ratios 90:10, 80:20, and 70:30 showed very good miscibility, whereas, increase the ratio of liquid lipid in the blend as in the ratios 60: 40 and 50:50 showed clear oil droplets on the filter paper that ensure immiscibility of the two phases. These results previously reported that a liquid lipid content of 30% w/w or less was optimal, as higher oil concentrations resulted in the generation of immiscible mixtures and the possibility of nanostructuring of NLC decreases .
The solubility of caffeine in selected lipid blends containing stearic acid/O: G at ratio 1:1 at ratios 90:10, 80: 20, and 70: 30 was 25, 30, and 35 mg/g, respectively, and was 25, 30, and 40 mg/g, respectively, for lipid blends containing GMS as solid lipid. It was clear that drug solubility in the lipid blends increases with increasing liquid lipid ratio. The increase in the volume of liquid lipid decreases the melting point with keeping the solid-state of the solid lipid, decreases the viscosity of the mixture, and increases the dynamic equilibrium for drug molecules that in turn increases drug solubility and minimizes the drug escaping as well as gives better stability to the blend .
Solubility study of the drug in different aqueous media
This experimental part has potential importance for the preparation of water-soluble drugs as solid lipid nanocarriers, which was accepted to increase the percentage of drug-loaded into the NLCs and reduce the amount of drug escape to the aqueous medium. Solubility of caffeine in different buffer systems, pHs, and ionic strengths are presented in table 3. The results showed that caffeine exhibited the lowest solubility in carbonate buffer at higher pH 10.8 and ionic strength 200 mM that reached 8.01%.
Using phosphate buffer systems at pH 7.4 and 7.8 showed that increasing the pH had an obvious effect on decreasing the drug solubility, especially with increasing the ionic strength of the medium (Table 3). At ionic strength 50mM, the drug solubility was nearly the same at both pHs, 4.565 and 4.541 at pH 7.4 and 7.8, respectively. Upon increasing the ionic strength of phosphate buffer (200mM), the drug solubility progressively decreased to reach 2.146 and 1.413 at pH 7.4 and 7.8, respectively. The same result was clearly observed with carbonate buffer at pH 10.8, whereas the drug solubility decreased from 3.320 % at 50mM ioinc strength to 0.801 at 200mM.
The results revealed that increasing the buffer ionic strength statistically decreased the drug solubility, especially if it is accompanied by an increase in the pH of the buffer system. The lower solubility in buffers with high ionic strength was related to increasing the concentrations of dissolved ionic salts that in turn could decrease the solubility of the weak electrolytes. Moreover, these results could be due to weak basic properties of the drug that showed low solubility at high pH and high solubility at lower pH. At acidic pH, the drug has high solubility due to its ionization, whereas, at basic pH (carbonate buffer at pH 10.8) the drug is in the un-ionized form, consequently the solubility decreased leading to precipitation in a crystalline or amorphous form . These findings were similar to Mayer, et al.  who informed that altering the pH of the buffer system was the best choice for encapsulating doxorubicin (water-soluble drug) inside the liposomes. Accordingly, carbonate buffer at pH 10.8 with ionic strength 200 mM was selected as the optimum aqueous medium for encapsulating caffeine into NLCs.
Optimum excipients for caffeine-NLCs preparation
Table 4; illustrates the composition of caffeine-NLCs formulas. The S/CoS mixture at ratio 3:1 was used at a fixed concentration of 3% w/v. The lipid blends were used at a fixed concentration of 2% w/v whereas, the ratio of solid lipid: liquid lipid was 70:30 and drug concentration was 1.5 and 3% w/v. Carbonate buffer pH 10.8 at ionic strength 200 mM was used as an aqueous phase for the emulsification process due to low drug solubility that is expected to increase the %DL and %EE. The formulas were characterized for DSC, FTIR, %Yield, %DL, %EE, particle size, zeta-potential, PDI, pH, electroconductivity, in-vitro release, and ex-vivo permeation.
Characterization of caffeine loaded NLCs
Thermal analysis by DSC was used for detecting drug-excipient incompatibilities, melting point, and recrystallization performance of bulk lipids used in the formulation of caffeine loaded-NLCs . Fig. 1 showed DSC thermograms of the components of caffeine-loaded NLC; (a) caffeine, (b) GMS, (c) stearic acid, (d) physical mix at ratio (1:1), (e) blank NLC, and (f) optimum NLC formula C3. Caffeine showed a sharp endothermic peak with a melting point of 235ºC which was similar to that reported by Carmelo et al., , Fig. 1(a). DSC thermogram of GMS revealed a sharp endothermic peak at 56.59ºC revealing its melting point (Fig.1(b)).
These findings agreed with that of Freitas and Müller,  who studied the DSC of GMS that exhibited an endotherm melting point around 60ºC. DSC profile of stearic acid showed an endothermic peak at 57.93°C, Fig. 1(c) . DSC thermogram of the physical mix, blank NLC, and optimum NLC formula for caffeine, (Fig. 1(d), 1(e), and 1(f) presented a broad endothermic peak at 300ºC, which revealed the absence of caffeine peak. It is known that when the drug endothermic peak does not appear in thermogram of nanopreparations or in the physical mixture, drug converted from crystalline to amorphous phase that was confirmed by its complete solubilization into NLC .
FTIR spectrum of the drug identifies its nature through clearing the functional groups that are related to the drug structure. Fig. 2(a-F) showed the IR spectra of pure caffeine, GMS, stearic acid, physical mix (1:1), blank NLC, and caffeine-loaded NLC of the optimum formula (C3).
FTIR spectra of pure caffeine showed a broad peak at 3408.42 cm−1 due to N–H stretching vibration, aromatic C–H stretch appeared at 3111.18 cm−1and 2953.02 cm−1, the peak at 1661.29 cm−1 is due to −C=N ring stretching, 1725 cm-1 (C=O of C6-ring stretching), 1548.84 cm-1 (C=C stretching), and 1399 cm-1 (C–N stretching), , Fig. (2a).
FTIR spectrum of GMS showed a broad peak at 3315.7 cm−1 due to (O–H), 2916.37 cm−1 and 2848.86 cm−1 for (C–H) stretching, 1734.8 cm−1 (C=O), 1195.87 cm−1 and 1047.35 cm−1 (C–O), Fig. 2(b) . FTIR spectrum of stearic acid showed the peaks at 2848.86 cm−1 and 2916.37 cm−1 which was due to the stretching vibrations of –CH2 groups and the peak at 1734.8 cm−1 related to a carbonyl group of stearic acid , Fig. 2(c).
FTIR spectrum of caffeine/physical mix at ratio (1:1), Fig. 2(d) and the optimum formulas, Fig. 2(f) showed the distinctive peaks of ingredients (GMS and stearic acid) at 2916.37 cm−1 and 2848.86 cm−1 which reveals the structural integrity of the chosen ingredients in the established formulas. IR spectrum of the optimized formulas (C3) showed peaks at 1734.8 cm−1 and no characteristic peaks related to the drug Fig. 2(f). This result confirms satisfactory solubilization and complete entrapment of drug into lipid phase that evidenced by the low intensity, slight shift, broadening, and disappearance of the drug peaks in the formula .
%Yield, %EE, and %DL
The % yield of caffeine-loaded NLCs (C1-C8) was in the range of 90-98%, these high yield values revealed an optimum method for NLCs preparation. The %DL was in the range of 5.99-22.36, and the %EE was in the range of 38.91-72.55 (Table 4). Further investigation for the effects of several factors on the %EE of caffeine-loaded NLCs was carried out. The data was analyzed using 23 full factorial designs at two levels and three variables strategies.
Data analysis according to the factorial design program was presented in Fig. 3 (a, b, and c). It was found that the lipid type, S/CoS type, and drug concentration had a significant effect on the %EE of caffeine (P<0.005).
With respect to lipid type, the results revealed that the NLCs formulas containing stearic acid showed a high entrapment efficiency (Fig. 3a). The entrapment efficiency of NLCs was improved upon increasing the fatty acid carbon chain length which led to higher hydrophobicity and thus increasing drugs residence within its core matrix .
In accordance with the effect of surfactant type (Fig. 3(b)), those formulas that were prepared with cremophore EL showed the highest capability to encapsulate the hydrophilic drugs at low drug concentration (1.5%). The %EE of formula C3 prepared with cremophore EL as a surfactant (72.55%± 0.12) was more than %EE of formula C1 prepared with Tween 80 (60.13%± 1.34). The same results were observed with C5 and C7 (Table 4). Cremophore EL is identified as oxylated triglycerides of ricinoleic acid, this structure decreases the interfacial tension between the oil and aqueous phases during the emulsification, consequently, improving drug entrapment .
Referring to drug concentration (Fig. 3(c)), the results proved that increasing the drug concentration from 1.5 to 3% decreasing %EE, (P<0.005), especially upon using cremophor EL/Transcutol HP as S/CoS type. The formula C3 has 22.36 and 72.55 %DL and %EE, respectively, that was decreased to 7.58 and 48.96, respectively for the formula (C4) when drug concentration increased from 1.5% to 3% w/v. The same results were also observed upon comparing the results of C7 to C8 (Table 4). These results were contributed to the fact that increasing the amount of the drug resulted in an extra porous structure in the nanostructured lipid particles. The drug readily leaked out to the external medium through the cavities and channels filled with the drug. Moreover, upon raising drug concentration in the nanoformulations, the osmotic pressure difference between the inner and outer phases was altered, as a result of some injuries to the formed quasi-emulsion droplets, which in turn caused the drug to leak from the inner phase .
Particle size analysis
Table 4 presented the results of particle size and PDI of caffeine-loaded NLCs formulas (C1-C8). The particle size and PDI ranges were 240-772 nm and 0.541- 0.958, respectively, revealing the existence of the drug in the nanosize range (<1000nm). Figure 4 presents the data analysis according to the factorial design software. It was found that the lipid type, S/CoS type, and drug concentration had a significant effect on particle size (P<0.005).
Concerning the lipid type, NLCs formulas prepared using GMS possessed the smaller particle size and PDI compared to those prepared using stearic acid (Fig. 4 (a) and Table 4). The addition of GMS could decrease the particle size as it has self-emulsifying property due to its structure which has saturated carbon chain length. This structure enables it to capture more drugs and have a uniform particle size in the nanosize range . These findings were agreed with Gardouh, S. G. et al.,  who found that lipid hydrophilicity and self-emulsifying properties affected by the lipid crystal's appearance (and hence the surface area) had an indirect effect on the final size of NLC dispersions.
The results presented in (Fig. 4(b) and Table 4) showed the important effect of surfactants on the NLCs particle size. The formulas, which contain Cremophore EL as surfactant exhibited particle size smaller than those prepared with Tween 80 at the two levels of drug concentrations 1.5% and 3%w/v. Cremophore EL has more emulsification efficacy than Tween 80 that ensures particles uniformity. Comparing C1 to C5, the particle size was 585 and 400um, respectively, C2 and C6 was 772 and 696 nm, respectively), C3 and C7 was 358 and 240nm, respectively, and (C4 and C6 was 624 and 498nm, respectively. This result was attributed to molecular structure differences between both surfactants whereas; Cremophore EL had a slightly lower degree of ethoxylation and unsaturation. At the level of pharmaceutical industries, they have been dedicated much attention for the application of Cremophor EL is more than Tween 80, due to its higher emulsifying capacity and its capability to encapsulate, solubilize, and protect both lipophilic and hydrophilic compounds .
Concerning the drug concentration, the results showed that increasing drug concentration from 1.5% to 3% w/v had an obvious effect on increasing NLCs particle size (Fig. 4(c), regardless of the lipid or S/CoS types (see C1, C3, C5, and C7 in comparison to C2, C4, C6, and C8 in Table 4). These results may be due to the concept of crystallization theory which is influenced by increasing the drug concentration . Higher drug concentration, higher super-saturation rate leading to higher diffusion-controlled growth and faster crystal growth as a result the agglomeration process was higher, resulting in larger particle size. These findings agreed with Dingler, A. et al.,  who reported that the higher NLCs particle size was attributed to higher drug concentration.
From the analysis of the factorial design, it was found that only the lipid type had a significant effect on PDI (P<0.005), Fig. 5 (a). The formulas C1, C2, C3, and C4 prepared with stearic acid exhibited PDI of 0.839, 0.829, 0.912, and 0.958, respectively, which were higher than that exhibited by the formula prepared with GMS C5, C6, C7, and C8; 0.61, 0.541, 0.556, and 0.665, respectively. These results were attributed to the higher particle size of the formulas prepared with stearic acid more than those prepared with GMS as solid lipid. Large particles form clusters or aggregates which in turn lead to higher PDI and broad size distribution . In addition, the efficacy of GMS to act as an emulsifier, provide stability to NLCs dispersions, and ensure nanoparticles homogeneity.
Zeta potential measurements
All NLCs formulas showed higher negative zeta potential (ZP) values > -19 mv (Table 4), which revealed better stability of the colloidal dispersed nanoparticles. This stability was related to electrostatic repulsion that prevents the nanoparticles from aggregation. The higher values of ZP (±30 or more) indicate higher nanoparticle stability . Table 4 and Fig. 6, formula C3 showed a slightly lower ZP of -24.8± 0.70, than formula C4 -29.1± 1.78. This may be attributed to increasing the dug concentration, decreasing the encapsulation efficiency and drug loading and at lower loading, there is a low affinity between the drug and lipid matrix, consequently, more negative charged particles (ionized drug) are released in the external medium and the drug can be retained by the electrostatic interactions .
pH & Electroconductivity
Since NLCs formulas were developed for topical application, the measurement of pH is an essential process to avoid skin damage or severe irritation. The pH of the prepared caffeine NLCs formulas was measured and was found to be in the range of (5-5.97) that mimic the pH of the cuticle as shown in Table 4. Electrical conductivity is utilized for the evaluation of the electro-conductive behavior of the compounds. The conductivity values of the NLCs formulas were high and ranged from 105.36 to 197.33 µS/cm, Table 4. These elevated values are due to high water content which enables ions to move more freely. It was found that conductivity raised with increasing the drug concentration and diminished with increase % encapsulation efficiency. The formula C2 (197.33 ± 9.29 µS/cm) increased by 0.8 fold than the formula C1 (158.13±7.88 µS/cm). These findings were attributed to the dependence of conductivity on the concentration, mobility, and valence states of the ionized species in a solution. Increasing the amount of the drug led to a higher concentration of free and unbounded drug ions in water, which finally resulted in higher conductivity .
In-vitro drug release studies
The release pattern of caffeine-loaded NLCs formulas compared to the market formula was investigated over 2hr and presented in Fig. 7. The results revealed that all tested formulas allowed for high % drug release during the first 0.25min. The market formula released about 47.8%, whereas, NLCs exhibited % drug release in the range of 35.09- 55.88. At that time, the drug release was expressed as rapid release in which the drug was released from the exterior shell of NLCs, then release occurred from the inner core. This fast release could be due to the increase in the chemical potential gradient, which resulted in escaping of the encapsulated active ingredients gradually from NLCs into the external phase. At this stage, the release rate would be much higher as less energy was required to overcome the phase barrier for it to be released to the continuous phase . From the analysis of the factorial design (Fig. 8 a, b, and c) it was found that the lipid type, surfactant type, and drug concentration had a significant effect on the % release (P<0.005). Regarding the lipid type, the formulas C7 and C8 prepared by using GMS exhibited a % release of 97.34±1.24 and 99.65±9.99, respectively after the first hour. This percentage presents 1.3 and 1.1 fold than that released by the formulas C3 and C4 (stearic acid); 72.8±3.18 and 89.24±3.04, respectively (P***<0.001). All the formulas prepared with stearic acid delay the % drug release than those prepared with GMS.
The effect of surfactant type on the % of drug release appeared clearly with the NLCs formulas containing stearic acid. The result clearly revealed that the formulas prepared with Cremophore EL allowed more rapid drug release. Upon comparing the percentage of drug release after 1 hr, the formula (C3) showed % release (72.8±3.18) that presents 1.08 fold of the formula C1 (67.4±2.20). The formula C4 (89.24±3.04) was 1.05 fold of the formula C2 (85.2±1.72), (P***<0.001). These results were related to the higher emulsifying properties of cremophore EL. The penetration activity of Cremophor EL has a potential role in increasing the fluidity of the lipid bilayer, hence increasing the drug release . All the formulas prepared with GMS as solid lipid showed nearly 100% release after 1hr.
Regarding the drug concentration, upon increasing the drug concentration from 1.5 to 3%, the % drug release slightly increased. After 1h, the % of drug release from formula C2 was greater than C1(85.2±1.72>67.4±2.20) and C4 than C3 (89.24±3.04>72.8±3.18), respectively (P***<0.001). The faster release is achieved by higher drug concentration when the adsorption of the drug particles which are weakly bound to the nanocarriers rather than to the drug incorporated in polymer nanoparticles .
Ex- vivo skin permeation studies
Permeation studies were done on the optimized formula, which had the smallest particle size and reasonable % EE. The formulas C3 and C7 showed particle size 358.6 ±1.45, 240.4± 22.87 nm, respectively, and % EE 72.55%±0.12, 67.20%±5.98. The % of drug release for C3 and C7 was 84.28±3.18 and 100.75±0.77 after 2hr, respectively. Consequently, a permeation study was performed on these optimized formulas in comparison to their market formulas. Permeation profiles present the cumulative amounts of caffeine permeated from NLCs formulas and market per unit time for 24hr are presented in Fig. 9, Table 5.
The formula C3 showed the highest amount permeated drug through 24hr. The tested formula could be arranged discerningly as follows: C3 > C7 > market; the cumulative amount permeated (µg/cm2) was as follows: 150.832> 87.2804>55.514, respectively, Table 5. The amount of drug permeated from formulas C3 and C7 present 2.7 and 1.6 fold of market, respectively. These results were due to the mechanism of oleic acid as a chemical permeation enhancer in the NLCs formulas, Its absorption by stratum corneum reduces lipid temperature changes and so, improves the permeation of the drug. Additionally, it decreases the viscosity of lipids of the superficial layer . Cremophor EL and Transcutol HP as S/CoS mixture in NLCs formulation had an efficient effect on drug permeation, which in turn allow better permeation profile . Cremophor EL increases the epithelial permeation through disturbing connections in the epithelial wall, enabling drug transportation via the membrane. So it efficaciously improved topical permeation of caffeine compared to market missing Cremophor EL .
Permeability parameters for caffeine, including; drug flux (Jss), permeation coefficient (Kp), and enhancement ratio (Er) are presented in Table 5. All parameters significantly increased with NLCs formulas in comparison to the market formula (P < 0.001). The values 6.1375 (μg/cm2 /h), 2.713, and 165×10-2 (cm2/h) were recorded for formula C3 as the highest flux (Jss), enhancement ratio (Er), and permeation coefficient (Kp), respectively.
At the end of the permeation experiment, the efficacy of caffeine NLCs formulas to be accumulated in skin layers was estimated. The amount of accumulated caffeine in rat skin after subjecting to NLCs formulas (C3 and C7) and the market formula for 24hr was determined, Table 5. The amount of caffeine accumulated in the skin following topical delivery of NLCs formulas was clearly high compared to the market formula. The amounts of accumulated caffeine from C3 and C7 were; 4830.4716±41.461 and 4398.359±298.8837 µg/cm2 after 24hr, respectively, These amount presents about 3.1 and 2.8 times enhancement over the market formula (1547.670±125.3430 µg/cm2). According to Fei, H. et al. , NLCs enhance the occlusive effect, film formation, and close contact with SC of skin and thus increase the amount of entrapped drug into the skin, improving its penetration.
Transmission electron microscopy studies
Morphology of caffeine NLCs optimized formula (C3) scanned by TEM revealed that it was successfully formulated with a smooth surface, distinct integrity, and spherical shape particles, (Fig. 10). It also showed the sizes ranges (300–500 nm), without obvious aggregation .
Stability studies of the optimized formula (C3) as per ICH guidelines
Table 6, presents the results of stability studies of the NLCs optimized formula (C3). There was no remarkable alteration in color, pH, particle size, PDI, zeta potential, and % EE. Additionally, it kept its homogeneity without the appearance of any phase separation or clog formation, which proved the good physical stability of the NLCs optimized formula.
Confocal Laser Scanning Microscopy (CLSM)
To evaluate the drug distribution performance when loaded on NLCs within the skin layers (epidermis, dermis, and hypodermis), CLSM was employed. The CLSM images of perpendicular slices of rat skin gotten after 48hr application of caffeine RB-NLCs (C3) are shown in Fig. 11(a). The NLC formula (C3) loaded with RB showed higher fluorescence intensity of RB in the hypodermal layer, whereas the drug was distributed through all layers of the skin, indicating high skin targeting efficiency and highly permeation. These results are consistent with previously reported findings of Pople and Singh . Control formula loaded with RB in which drug was applied as a suspension in aqueous media showed drug distribution in the epidermal layer only with low permeation efficiency, Fig. 11(b). Accordingly, this experimental part clearly proved the potential efficacy of NLCs nanoformulation as a skin targeting drug delivery system with a small mean particle size of 419 nm. Conjugation of drug loaded-NLCs with RB has no effect on the particle size of the NLCs.
Morphological examination of rat’s dorsal skin in different groups
This experimental part has crucial importance for evaluating the efficacy of different tested formulas on rat hair growth. The effect of caffeine NLCs optimum formula (C3), blank, and market formulas on the hair growth were compared to control (un-treated rats) Fig. 12 (a-d). The photographs (a-d) showed the thick hair of dorsal rats' skin at zero-day before chemotherapeutic induction in different rat groups treated with; (a) the optimum formula C3, (b) blank formula, (c) market formula, and (d) control. Fig.12(e-h) the rat's bald skin without hairs after chemotherapeutic induction with etoposide (1.5 mg/kg), once daily for three consecutive days. Fig. 12(i-l), presents hair growth for rats after 30 days of treatment. Control group (Fig. 12(i) presents minimum hair growth. The rat group treated with the formula C3 exhibited complete and heavy hair growth (Fig. 12(j)) in comparison to the market formula (Fig. 12(k) and the blank formula (Fig. 12(l) that showed incomplete hair growth. Hair regrowth was noticed during the whole treatment period. The effective NLCs permeation and fast drug release confirm well therapeutic efficiency. The improvement of drug activity of NLCs formula was attributed to the presence of oleic acid, that acts as a permeation enhancer due to its good wetting and moisturizing properties . Oleic acid permits drug permeation through skin layers and in addition, it moistens the dry hair. The lower efficacy of the market formula was related to solvent evaporation that not only decreases drug permeation but also yields dry skin. The blank formula that was presented in aqueous media lacks to permeation enhancer.
Histopathological evaluation of the skin specimens
The effect of topical application of caffeine-loaded NLCs formulas on hair growth was studied through the histopathological examination of the skin layers, Fig. 13 (a-d). Fig. 13 (a, b) showed the histopathological examination of the normal skin specimens with a normal histopathological structure of the epidermal layer, underlying dermis, sebaceous gland, and subcutaneous tissue. Also, it showed multiple mature hair follicles with hair shaft and bulb that are characterized by spindle-like dermal papilla (DP) and Y-shaped melanin granules around the DP without ectopic melanin in the bulb. While Fig. 13 (c, d) showed the histological examination of skin specimens after 3 days of chemotherapeutic induction, a dystrophic change of hair follicles was observed which is characterized by the presence of ectopic melanin granules and swollen DP. These melanin granules are smaller than keratinocyte nuclei and present as clumps in the bulb and hair shaft. The hair follicles showed a catagen-like structure with a ball-like small DP 
Fig. 14 (a,d,g, and i) presents biopsy specimens of rat skin after 10 days of treatment; (a) the optimum formula C3, (d) blank formula, (g) market formula, and (i) control. The skin of rats in the control group showed reduction and atrophy in the hair follicles (decrease in size and number). The animal group treated with optimum NLCs formula (C3) showed multiple hair follicles of the dermis. The group treated with blank formula showed acanthosis in the epidermis with the appearance of mature hair follicles. The animals treated with market caffeine showed atrophy in most hair follicles. Fig. 14 (b,e,h, and k) presents biopsy specimens of rats' skin after 20 days of topical treatment. The control group (Fig. 14 (k) showed few immature hair follicles of the dermis, and the group treated with optimum NLCs formula (C3) showed multiple numbers of mature and immature hair follicles of the dermis (Fig.14 (b). Skin rats of the group treated with blank formula showed few appearances of immature hair follicles and less mature ones (Fig. 14 (e). The rat group treated with market caffeine showed the appearance of multiple mature hair follicles but less than the group treated with the NLCs optimum formula C3 (Fig. 14 (h).
After 30 days of topical treatment, the animals were sacrificed and the histopathological examination of rats' skin specimens was presented in Fig. 14 (c,f,i, and l). The skin of rats in the control group (Fig. 14 (l) showed focal necrosis and ulceration in the epidermis, acanthosis, and invagination of the epidermis and dermis. The subcutaneous tissue showed the excessive formation of tissue granulation with inflammatory cells infiltration. Fig. 14 (c) is for skin rats of the group treated with NLC formula C3. It showed multiple numbers of mature and immature hair follicles of the dermis. Skin rats of the group treated with the blank of formula C3 showed less appearance of mature hair follicles and less immature ones (Fig. 14 (f). Skin rats of the group treated with market caffeine showed the appearance of multiple mature hair follicles but less than that recognized in the group treated with optimum NLCs formula of caffeine (C3), Fig. 14 (i). The appearance of mature and immature hair follicles for skin rats in different groups can be arranged descendingly as follows; group treated with NLCs optimum formula (C3) > group treated with market formula > group treated with blank formula> group treated with control. The appearance of acanthosis, necrosis and granulation tissue formation with inflammatory cells infiltration for skin rats in different groups can be arranged descendingly as follows; group treated with control > treated with blank formula> treated with market formula > treated with optimum formula (C3).