Transdermal Cubic Phases of Metformin Hydrochloride: In Silico and

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Transdermal Cubic Phases of Metformin Hydrochloride: In Silico and In Vitro Studies of Delivery Mechanisms Xiang Yu, Yiguang Jin, Lina Du, Mengchi Sun, Jian Wang, Qiu Li, Xiangyu Zhang, Zisen Gao, and Pingtian Ding Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00209 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Molecular Pharmaceutics

Transdermal Cubic Phases of Metformin Hydrochloride: In Silico and In Vitro Studies of Delivery Mechanisms

Xiang Yu†,‡, Yiguang Jin*,†,‡, Lina Du‡，Mengchi Sun†, Jian Wang†, Qiu Li§, Xiangyu Zhang†, Zisen Gao†, and Pingtian Ding †

School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

Shenyang 110016, China, e-mail address: [email protected] ‡

Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27

Taiping Road, Beijing 100850, China §

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese

Medical Sciences, University of Macau, Avenida Padre Tomas Pereira, Taipa, Macao SAR, China

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Transdermal delivery is one of important controlled drug release strategies for drug development. Cubic phases are the assemblies of amphiphilic molecules in water with the hydrophilic-hydrophobic interpenetrating network for transdermal delivery of both hydrophilic and hydrophobic drugs. However, many details about the transdermal delivery of drugs from cubic phases remain unclear. Here, metformin hydrochloride (Met) cubic phases were prepared with glyceryl monooleate (GMO), ethanol and water. The cubic structure was identified with the polarizing light microscopy and small-angle X-ray scattering method. Dissipative particle dynamics (DPD) was used for building the microstructures of the cubic phases to explore the mechanism of drug release that mainly depended on drug diffusion from the water channels of cubic phases in accordance with the Higuchi equation of in vitro release experiments. The coarse-grained model and molecular docking method showed that GMO could enhance drug permeation through the skin by disturbing the interaction between Met and the skin proteins, and increasing the fluidity of skin lipids, which was confirmed with the FT-IR spectroscopy, Langmuir monolayer, and immunohistochemistry. Furthermore, in vitro permeation experiments showed the high Met transdermal improvement of cubic phases. Cubic phases are an ideal transdermal delivery system of Met. In silico methods are very useful for analyzing the molecular mechanisms of transdermal formulations. Keywords: computational simulation, cubic phase, dissipative particle dynamics, metformin hydrochloride, molecular interaction, transdermal delivery 2


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1. INTRODUCTION Metformin hydrochloride (Met) is a commonly applied anti-diabetic drug, which is reported to have multiple pharmacological functions such as anti-skin cancer and anti-oxidation.1-2 In spite of the potent and superior therapeutic effect, Met is only available in the form of tablets nowadays. However, oral administration of Met is associated with gastrointestinal adverse effects such as indigestion and abdominal pain, which limits its clinical application. Transdermal drug delivery systems (TDDS) are a promising alternative to avoid the above limitations associated with oral administration. It exhibits many advantages such as bypassing first-pass liver metabolism, increasing bioavailability, controlling release of drugs and good patient compliance.3 Stratum corneum (SC) is the major barrier to hinder drug permeating into the skin.4 A variety of techniques are tried to enhance the transdermal efficiencies of drugs, such as permeation enhancers,5 nanoemulsions,6 β-cyclodextrin inclusions,7 and nanoparticles.8 However, the transdermal delivery mechanisms of these methods are complex processes and more comprehensive description is needed, which further takes a lot of ineffective and unproductive experimental studies and highly affects the development of TDDS. Cubic phases have been applied as a transdermal drug delivery system for decades.9-11 Cubic phases are the highly thermodynamically stable complex spatial structures characterized by bicontinuous interpenetrating networks that consist of water and amphiphiles.10, 11 The unique advantage of cubic phases is to load a variety of polar or nonpolar regimens, such as small molecular drugs, proteins and vaccines. 3



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In the previous work, cubic phases were often selected as transdermal carriers to enhance the percutaneous absorption of drugs for treatment of topical inflammation, pigmentation, skin cancer, and so on.12-15 Celecoxib-loaded cubic phases showed higher anti-inflammatory activity compared to a marketed product, ketoprofen gels. Our previous work demonstrated that a cubic phase of mitoxantrone was satisfied for topical therapy of melanoma. Moreover, cubic phases were potential carriers to deliver 5-aminolevulinic acid for topical application in photodynamic therapy. Paeonol-loaded cubic phases showed significant therapeutic effect on topical pigmentation. Furthermore, the evidence of cubic phases as good transdermal carriers was shown by the two-photon microscopic observation of drugs distribution in the skin, and micro-fissures were the major permeation route.

16

However, the whole

release and permeation of drugs from transdermal cubic phases keeps unclear. Transdermal delivery of drugs from cubic phases mainly involves two procedures of drug release and skin permeation. Drug release from cubic phases is reported to mainly accord with the Higuchi equation.17 Namely, the release is mainly based on diffusive control. The enhanced permeation mechanism of cubic phases is currently regarded as the insertion of amphiphiles into the extracellular lipid matrix of the skin to lead to disorder of the highly compacted structure.18 Unfortunately, these theories just result from one release experiment or one transdermal permeation experiment, which are not confirmed by other researchers. More importantly, the molecular mechanism remains unclear for transdermal delivery of drugs from cubic phases because of limited experimental techniques and the complicacy of transdermal 4


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procedure. Therefore, novel techniques and methods are urgently needed to uncover the molecular mechanisms, favoring the understanding of this dosage form and the development of hydrophilic drug-loaded cubic phases. Reliable theoretical approximations and computational techniques are widely used in pharmaceutical research with the development of fast computing technologies.19, 20 They can support a bridge between molecules and microstructures of drug delivery systems.21, 22 Here, Met-loaded cubic phases were prepared with glyceryl monooleate (GMO), ethanol and water. Polarizing light microscopy (PLM) and small-angle X-ray scattering (SAXS) were performed to validate the structures of the cubic phases. The simulations of totally atomic molecular dynamics (MD) and dissipative particle dynamics (DPD) were firstly introduced to build the microstructures of Met-loaded cubic phases for exploring the drug release mechanism. Release mechanism was further validated based on in vitro release profiles. The effect of cubic phases on Met skin permeation was studied by in vitro permeation experiments. In addition, DPD simulation and molecular docking were applied to explore the enhanced permeation mechanisms of cubic phases for Met at the molecular or mesoscopic level, which was also confirmed by the FTIR spectroscopy, Langmuir monolayers, and immunohistochemistry. To our knowledge, this is the first study to explore the transdermal mechanism of cubic phases based on the molecular level.

2. MATERIALS AND METHODS 5



2.1. Materials. Metformin hydrochloride (Met) was purchased from Jiang Su Heng Rui Medicine Co., Ltd. (Lianyungang, China). Glycerol monooleate was supplied by Dansico Co., Ltd. (Boston, USA). The anti-keratin K5 antibody was bought from Bioss Biotechnology Co., Ltd. (Bejing, China). Ceramide NS was purchased from Sheng li De Biotechnology Co., Ltd. (Nanjing, China). All other materials were of pharmaceutical or analytical grade.

2.2. Animals. Male Balb/c nude mice (7−9 weeks old) were supplied by the Sipeifu Co., Ltd. (Bejing, China). All the procedures were performed in line with the NIH Guidelines for the Care and use of Laboratory Animals.

2.3. Preparation of Met-Loaded Cubic Phases. A Met aqueous solution was prepared by dissolving 0.5 g of Met in 3 g of water at 55 °C. The Met solution was dropped into a 6.4 g of GMO solution in 0.6 g of ethanol at 55 °C under homogeneously vortexing. The obtained cubic phases were sealed in a tube and ready for structural identification.

2.4. Characterization of Cubic Phase Structures. The structure of the sample was identified with the polarized light microscopy (PLM, DMLP, Leica, Germany) and small-angle X-ray scattering (SAXS, SAXSee, Anton Paar, Austria) method referred to other reports.23 Scattering intensities were plotted as a function of the reciprocal spacing. The plot of reciprocal spacing for the observed reflections was obtained 6


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against Miller indexes, and the lattice constants of the sample was calculated from the linear slope.

2.5. Observation of Cubic Phases in Silico. Here, MD and DPD simulations were successively performed to build the microstructures of the cubic phases at 328 K. 2.5.1. Molecular Dynamics Simulation. One GMO molecule was divided into Fragments A and B that represented the hydrophilic glycerol and carboxyl group and the hydrophobic long carbon chain, respectively (Figure 1). MD simulations are usually applied to calculate solubility parameters (δ) and Flory-Huggins parameters (χ) of pure and binary components.24, 25 In this study, all the systems were constructed with the Amorphous Cell under the COMPASS force field in Materials Studio 7.0 (Accelrys Co., US). It should be noted metformin was protonated at secondary amine. To electrically neutralize the charges on metformin, chloride ions were included in the system of metformin. The initial configurations of systems were subjected to 10000 steps of energy minimization with an energy convergence threshold of 1 × 10−4 kcal·mol−1 and a force convergence of 0.005 kcal·mol−1·Å−1. And then, thermal annealing was performed from 1000 to 300 K. Five hundred ps MD equilibrium simulations of one pure component were conducted under the isothermal-isobaric (NPT) condition, whose trajectory files were generated every 5 ps, and the final 10 configurations were used to calculate δ and cohesive energy densities via the Forcite module. Similarly, binary components were subjected to the above MD equilibrium simulations under the isothermal-isochoric (NVT) condition and their trajectory files 7



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and cohesive energy densities were also generated as above. The temperature was maintained at 328 K (55 °C) in the whole MD simulation process.

Figure 1. Chemical structure of GMO divided into Fragments A and B.

The Flory-Huggins parameter χ ij between the components i and j in nonpolar systems can be calculated with equation (1) if no special interactions such as hydrogen bonding occurs.24

χij =

(δi − δ j ) 2Vref

(1)

RT

where δi and δj are solubility parameters of i and j, respectively, equal to the square root of cohesive energy densities; Vref is reference volume; R is gas constant; and T is temperature. The Flory-Huggins parameter χij of polar components or the components with special interactions can be approximately estimated from equations (2) and (3). ∆ E mix = Φ i (

χij =

E coh E E ) i + Φ j ( coh ) j − ( coh ) mix V V V

∆EmixVref

(2)

(3)

RT

where ∆Emix is the energy of mixing i and j; Φi and Φj are the volume fraction of i and j; (

E coh E E ) i , ( coh ) j and ( coh ) mix are the cohesive energy densities of i, j and their V V V 8


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mixture, respectively.

2.5.2. Dissipative Particle Dynamics Simulation. In this study, DPD simulations were used to build the microstructures of Met/GMO/Ethanol/Water systems for investigating the mechanism of drug release. The coarse-grained bead volume is set in the beginning of DPD simulation.25, 26 Here, one water molecule was considered as one DPD bead. Three DPD beads and five DPD beads represented one ethanol molecule and one Met molecule, respectively. A GMO molecule consisted of total 21 DPD beads with 5 for Fragment A and 16 for Fragment B. Interaction parameters of different components aij can be estimated with the following equation (4).

aij = 16.5 + 1.45 χ ij (ρ = 5，T = 328 K)

(4)

where aij is a repulsive parameter between i and j beads. In the context, equation (4) bridges the gap between atomistic MD and DPD simulations. For each system, the simulation box size was 20×20×20 rc with a periodic boundary condition. The total beads were 27302 and the spring constant C was chosen as 4.0. To obtain the steady results, 50000 DPD steps had been adopted with a time step of 1.0 ps.

2.6. In Vitro Drug Release Experiments. Vertical Franz diffusion cells were used with the effective diffusion area of 0.785 cm2 for the drug release study of cubic phases. The receptor compartment filled with normal saline of 10 mL was kept in a 32 °C water bath, which was satisfied for the release sink condition of the cubic 9



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phases containing 47.6 mg of Met because the the solubility of Met in saline was 178 mg/mL measured by us. The saline was magnetically stirred at 300 rpm. The single dialysis membrane (cut MW, 3500, American Carbon Chemical Co.) was assembled between the donor cell and receptor cell. 1 g of the Met-loaded cubic phase was put into the donor cell, which directly contacted the single dialysis membrane. At the predetermined time points (0.5, 1, 2, 3, 4 and 5 h), 1 mL of normal saline (0.9% NaCl solutions) was withdrawn from the receptor cell and the equal volume of fresh saline was supplemented to achieve the sink condition. The withdrawn samples were measured with the HPLC method as the reference.27 The cumulative release amount of Met (Qr) was calculated and plotted versus time. The release of Met solutions was also conducted as control. The mechanism of Met release from the cubic phases was analyzed by fitting Qr to the following Higuchi equation (5). 1

Qr = kt 2

(5)

where t is the release time and k is the slope. 2.7. Permeation Experiments. The dorsal skins of Balb/c nude mice were obtained after sacrifice. Here, we used the skin to replace a single dialysis membrane in the Franz diffusion cell with the effective diffusion area of 0.785 cm2. The skin was assembled between the donor cell and receptor cell with the stratum corneum (SC) side facing toward the donor cell, and two cells were carefully clamped. A Met solution or a Met-loaded cubic phase was put into the donor cell, which directly contacted the surface of SC. The administered dose was 1.0 g, containing 47.6 mg of Met and 57 mg of ethanol. The other experimental procedure was the same as the 10


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above release test. The cumulative Met permeation amount (Qn, µg/cm2) was calculated according to the equation (6). n −1

Qn =

[C n ⋅ V + V0 ∑ C i ] i =1

A

(6)

where V (ml) is total volume of saline in the receptor cell, Cn (µg/ml) is the concentration of Sample n, Ci (µg/ml) is the concentration of Sample i, V0 (ml) is the volume of withdrawn saline and A (cm2) is the effective diffusion area. The Qn was plotted as a function of time. The slop and intercept of the linear portion of the profile were presented as (Jss, µg·cm-2·h-1) and lag time (Tlag, h), respectively. The activity of cubic phases was expressed as enhancement ratio (ER) that was the ratio of Q5h in the cubic phase group to that in the solution group. After the permeation experiments were finished, the skins were thoroughly washed with saline. The clean skin was immersed in 100 mL of normal saline and sonicated for 1 h. The solution was filtered through 0.22 µm filters and Met in the filtrate was measured with the HPLC method. The skin cumulative retention amount (Qskin) of Met was obtained.

2.8. Transepidermal Water Loss Experiments. An open-chamber Tewameter (MPA4, Courage & Khazaha Co., Germany) was applied to determine the transepidermal water loss (TEWL) value. The mice were anesthetized with isoflurane. The probe of Tewameter was tightly pressed on the dorsal skin for 30 s until to a stable value. The initial TEWL values of the mice were measured as TEWL0. The 11



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dorsal skins of the mice were administered with 1.0 g of saline, azone, Met solutions or cubic phases for 1 h, respectively. These regimens were completely absorbed by the skins within 1 h. Then, after 0-200 min, the TEWL values were measured as TEWLt. Each experiment was repeated three times. △TEWL was calculated as equation (7).28

△TEWL = TEWLt–TEWL0

(7)

2.9. Bilayer Model. The lipids in the stratum corneum (SC) are mainly composed of ceramides, free fatty acids and cholesterol, whereof ceramides are considered as the essential component for maintaining the skin barrier function.29 GMO is reported to be the key for enhancing the permeation of cubic phases.30 Here, a common type of ceramides, ceramide NS was selected for building a coarse-grained bilayer model to investigate the effect of GMO on the skin lipids. Three molecules were involved in the simulation system, including ceramide NS, GMO and water, whose force field parameters were based on the Martini force field according to the literature.31 The molecules were represented by grouping four heavy atoms into a particle (Figure 2), which was reported to reduce the number of degrees of freedom.32 Their chemical structures and coarse-grained (CG) mappings were shown in Figure 2. The parameter files of the CG models of ceramide NS and water were available in the Martini website (Figure 2B and 2C).33 The hydrophilic group of GMO was represented by a polar particle (P4), the ester group was represented by a nonpolar particle (Na), and the hydrocarbon chain was represented by one particle with low apolarity (C1), two particles with low apolarity (C2) and one particle with high apolarity (C4). 12


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The initial coordinates of ceramide NS bilayer membrane composed of ceramide NS (325 lipid molecules) was built based on the Martini force field using Material Studio 7.0. The appropriate amount of water molecules (4207 H2O molecules) was filled into the box. This system was energy-minimized using the method of steepest to remove the bad contacts between molecules. After setting the temperature of the simulation systems at 305 K by Nose-Hoover, the MD simulations were performed for 500 ns NVT ensemble. The simulations were done on GMO (55 molecules) in the presence of appropriate number of water molecules (4203 H2O) and the equilibrated ceramide NS bilayer as the above procedure. Finally, all trajectories were analyzed to characterize the dynamic properties of the lipid membrane.

Figure 2. Chemical structures, CG mapping and interaction parameters for GMO (A), ceramide NS (B) and water (C).

2.10. FT-IR Spectroscopy. The skin samples were treated with normal saline, Met solutions and Met-loaded cubic phases in the transdermal permeation study. Some of 13



them were washed with normal saline and directly placed into an FT-IR instrument equipped with an ATR tool and a MCT detector (Bruker, Ettlingrn, Germany). The effects of the regimens on the surfaces of the skins were investigated. The spectra were obtained in the range of 4000-1000 cm-1. The spectra data were analyzed using the Peakfit 4.12 software.

2.11. Langmuir Monolayers. Ceramide NS and GMO were separately dissolved in chloroform to 1 mg/mL. They were mixed with various molar ratios to prepare the mixture solutions. Surface pressure-molecular area (π-A) isotherms of all the solutions were measured using a Minitrough film balance (KSV, Finland) equipped with dual barriers and a Pt Wilhelmy plate-sensing device. The Teflon trough has a width of 75 mm and an area of 24300 mm2. The subphase was purified water (pH 6.0). The experiments were performed at 25 °C. The above solutions were separately deposited onto the water subphase with a microsyringe, and 15 min were allowed for solvent evaporation. Monolayers were then compressed until the monolayers collapsed. The compression rate was 10 mm/min. Subsequently, all isotherms used for miscibility calculations were acquired.

2.12. Molecular Docking. We selected the initial file for the 2B regions of keratin K5 referred to the RCBS Protein Data Bank (PDB, 3TNU).34 The structural files of other molecules were built after structural minimization and dynamic optimization with the Sybyl 6.9.1 software package (Tripos Associates, St. Louis, US).35 Docking 14


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calculations were performed with the Auto Dock program 4.2.36 The keratin models were refined after removal of hydrogen atoms. Polar hydrogen atoms were then added followed by the assignment of Kollman charges, fragmental volumes and atomic solvation parameters to keratin. As for the ligands, GMO and Met were refined by removing and subsequently adding hydrogen atoms similarly to that for keratin, respectively. Then, Gasteiger partial charges were assigned to the ligands and nonpolar hydrogen atoms were merged. All torsions were allowed to rotate during docking. The LGA was used to find the appropriate binding positions, orientations, and conformations of the ligands. Default parameters were used, except for the number of generations that was set at 100. The best docking type of the receptor– ligand complex was chosen based on the binding energy scores. The corresponding heat of formation was calculated. Additionally, GMO and Met were selected as receptor and ligand, respectively. Docking calculations were performed again. All the procedures were carried out as mentioned above.

2.13. Immunohistochemistry. The above Met solution- or cubic phase-treated skin samples were immunohistochemically processed as follows. The samples were frozen and cut into sections that were masked with the phosphate buffered solutions (PBS) containing 10% sodium nitroprusside, 10% ferricyanatum kalium and 10% sodium hydroxide for 1 h at 37 °C. The keratin K5 antibodies were diluted with PBS, added onto the section slides and preserved overnight at 4 °C. The slides were washed with PBS three times, incubated with the fluorescence-labeled secondary antibodies for 1 h 15



at room temperature, and finally washed three times before microscopic investigation.

2.14. Statistical Analysis. The correlation analysis was performed using the Pearson Correlation with SPSS 10.0 and p < 0.05 denoted significant variance. All data were expressed as mean ± SD. The data were subjected to analysis of variance through one way ANOVA test using the SPSS 10.0 software with the significant level of p < 0.05.

3. RESULTS 3.1. Characteristics of Cubic Phases. The sample was a homogeneous transparent gel that was stiff and could not flow. The image of the sample just displayed dark background without birefringent phenomenon under PLM (Figure 3A). The values of the scattering vector q corresponding to the scattering peaks appeared in the ratio of

√6: √3

which seemed to be consistent with Pn3m space group of cubic symmetry

(Figure 3B). The lattice parameter of the sample was calculated from the linear slope (Figure 3C), and the value was 134 Å. These results demonstrated the sample was cubic phase with the internal structure of Pn3m space group.

16


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Figure 3. Characterization of liquid crystalline systems by experimental measurements. PLM images of cubic phases (A) at room temperature (400×). SAXS diffraction patterns obtained from cubic phases (B). Plots of the reciprocal d-spacings (q) as a function of the Miller indices, (h2+k2+l2)1/2, from the observed reflections in the SAXS diffraction patterns for cubic phases (C).

3.2. Flory-Huggins Parameters. MD simulations are usually applied to estimate Flory-Huggins parameters. Firstly, the solubility parameters (δ) of the pure 17



components in liquid crystalline systems at 328 K were obtained (Table 1). δH2O and δEthanol were 43.5 and 24.6, respectively. |δi−δj| < 2 (J/cm3)0.5 indicated that two

components i and j were miscible.24 However, |δH2O−δEthanol| was equal to 24.59 (J/cm3)0.5 so that ethanol seemed to be insoluble in H2O; but this result was not actual. Hence, the equation (1) did not involve the hydrogen bonding interaction between ethanol and water. Equation (1) was only applied to calculate the Flory-Huggins parameters between the components without hydrogen bonding such as water and Fragment B. Equations (2) and (3) were applied for the components with hydrogen bonding such as water and Fragment A.

18


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Table 1. Molecular parameters in MD simulations at 328 K Components

Number of

Density

δ

Molecules

(g/cm3)

(J/cm3)0.5

H2 O

700

0.97

43.5

Ethanol

200

0.78

24.6

Met

80

1.28

42.9

Fragment A1

80

1.28

28.7

80

0.79

16.2

H2O/Met

664/12

1.00

0.19

16.8

H2O/Fragment A

664/72

1.08

–1.1

15.0

H2O/Fragment B2

664/72

0.86

5.1

23.9

H2O/Ethanol

664/51

0.93

–0.07

16.4

Met/Fragment A

48/288

1.28

–1.22

14.7

Met/Fragment B

48/288

0.820

4.87

23.5

Met/Ethanol

48/68

1.22

–0.97

15.1

Fragment A/Fragment

72/72

0.969

1.07

18.0

Fragment A/Ethanol

72/51

1.12

0.34

17.0

Fragment B/Ethanol

72/85

0.786

0.48

17.2

Fragment B1

χ

a

B

1

The meanings of Fragments A and B of GMO are referred to Figure 1.

2

The Flory-Huggins parameter of H2O/Fragment B is present as χB/H2O. Other χ are

present as the similar mode. Absolutes of the Flory-Huggins parameters (χ) less than 0.5 mean the binary components miscible, and the minus means the attraction of binary components. Table 1 showed all the Flory-Huggins parameters of components in cubic phases. The estimated χH2O/Ethanol was –0.07, suggesting H2O/Ethanol was miscible as 19



experimentally observed. The χH2O/A was predicted to be –1.1, suggesting Fragment A was insoluble in H2O but there was a certain amount of attraction force between them. The estimated χH2O/B equal to 5.1 indicated that Fragment B was hydrophobic, which made up the lipid lyophobic region of the liquid crystalline systems below. Fragment A/Ethanol was miscible as illustrated by χA/Ethanol = 0.34. The estimated χB/Ethanol was 0.48, suggesting that Fragment B/Ethanol was weakly miscible. Met in the system could be immiscible with other components except water according to the Flory-Huggins parameters, indicating that Met mainly distributed in the water channels of the cubic phases.

3.3. DPD Modeling of Cubic Phases. The “dot-and-line” model showed the system exhibited complex spatial organization with the bicontinuous interpenetrating networks of GMO and water (Figure 4A), which was the typical characteristic of cubic phases. The hydrophilic groups of GMO were inward while the hydrophobic group were outward. The isodensity surfaces reflected the internal structure and drug distribution of the cubic phases. The inner structure of cubic phases contained two congruent networks of water channels where Met mainly distributed (Figure 4B). Moreover, the water channels were independent and some of them opened to the outside (Figure 4B). These findings indicated that Met could diffuse from the water channels to the outside.

20


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Figure 4. DPD simulation characteristics of cubic phases. (A) The “dot-and-line” model (Fragment A, Fragment B, Met, ethanol and water particle are presented in black, red, pink, blue and light blue respectively). (B) The isodensity surfaces of cubic phases composed of Met and water.

3.4. In Vitro Release Profile of Met from Cubic Phases. The cubic phases could provide a sustained release system for Met (Figure 5A). A high cumulative release amount of Met from the cubic phases, 3880±229 µg/cm2, was achieved after 5 h. In addition, the release amount of solutions was extremely higher than that of cubic phases (Figure S1). Therefore, drug release from cubic phases played a decisive role while the barrier effect of the dialysis bag was neglected. The linear relationship was found between the amount of Met per unit area released and the square root of time (Figure 5B, r = 0.9995 > 0.99), suggesting that the release kinetics was consistent with the Higuchi equation. Therefore, the release of Met was based on diffusive control.

21



Figure 5. Release and permeation experiments of Met from the formulations. In vitro drug release profiles of Met from the cubic phases (A). The released amount of Met from the cubic phases per area as a function of the square root of time (B). In vitro skin permeation profiles of Met from the cubic phases and the solutions (C). Skin cumulative retention amount of Met from the formulations (D).

3.5. Improved Met in Vitro Permeation through the Skin by Cubic Phases. Figure 5C showed that the permeation profiles of the two formulations were increasing along time. However, the permeation amount of Met from the cubic phases was higher than that from the solutions. As listed in Table 2, Significant difference appeared between 22


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the cumulative permeation amount of Met from the cubic phases (Q5h = 1890 ± 182 µg/cm2) and that from the solutions (Q5h = 1279 ± 126 µg/cm2，p < 0.05). Compared with the solutions (Jss = 247.5 ± 24.7 µg·cm-2·h-1), the steady-state flux of Met from the cubic phases (Jss =367.7 ± 29.6

µg·cm-2·h-1) was obviously higher (p < 0.05).

Two conclusions can be deduced from the above results: (a) Met has good ability to penetrate the skin though of hydrophilicity; (b) the cubic phase improves Met permeation through the skin. Additionally, the cumulative drug retention amounts of two groups were compared (Figure 5D). The cumulative Met retention amount of the solutions (Qskin = 8847 ± 1442 µg/g) was significantly higher than that of the cubic phases (Qskin = 4808 ± 899 µg/g, p < 0.05) (Table 2).

Table 2. The permeation parameters and cumulative retention amounts of Met from various formulations Samples

Q5h

Jss

(µg/cm2)

(µg/cm2/h)

Cubic phase

1890±182*

367.7±29.6*

1.48

0.04

4808±899*

Solution

1279±126

247.5±24.7

1

0.11

8847±1442

ER

Tlag

Qskin

(h)

(µg/g)

Each value was presented as mean ± SD (n = 3). * p < 0.05.

3.6. Reverse Action of Cubic Phases on the Barrier Function of SC. TEWL was measured to investigate the effect of cubic phases on the barrier function of SC. The

△TEWL value of the AZ group reached 75.37±7.83 (g·m-2·h-1) at 20 min, and then 23



maintained a plateau to the end of the experiment (Figure 6). Both the saline and the solutions showed the similar trends in the state of SC according to the TEWL results. The △TEWL value of the cubic phase group initially increased, and then decreased after 60 min, and finally reached the control level. These findings suggest that cubic phases affect the barrier function of SC, but this effect is reversible.

Figure 6. The profiles of △TEWL of the skins treated with various regimens.

3.7. Effect of Cubic Phases on the Fluidity of Intercellular Lipids in SC. 3.7.1 Modeling of Skin Lipid Molecules. To explore the interaction between the GMO

and the intercellular lipids of SC, DPD simulation was introduced to investigate the dynamic properties of the lipid membrane.37 The snapshots of the bilayer packing with GMO showed that the tail-end hydrophobic part of GMO molecules located in the hydrophobic lipid tails in the membrane (Figure 7A). In addition, mean square displacement (MSD) of ceramides was calculated to evaluate the molecular lateral 24


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movement.38, 39 The diffusion coefficient of the lipid was obtained from the slope of the curves (Figure 7B), meaning the membrane’s fluidity. The average diffusion coefficient of lipids mixed with GMO (3.38×10-11 m2/s) was significantly higher than that of pure lipids (1.527×10-11 m2/s). These results demonstrated that GMO could increase the fluidity of lipid membranes.

Figure 7. Snapshots of the simulated systems containing GMO (A), and mean square displacement of lipids in different systems (B).

3.7.2 Molecular Organization of Skin Lipids. The FT-IR spectroscopy was applied to

investigate the effect of cubic phases on the molecular organization of the lipid alkyl chains according to the reference. In this work, compared with the saline, there were blue shifts in the peaks of the CH2 asymmetric vibration (vas CH2) and symmetric 25



Page 26 of 57

stretching vibration (vs CH2) in the cubic phases while both νs CH2 and vas CH2 in the solutions slightly changed (Figure 8A, Table 3，p > 0.05). The results indicated that GMO could insert into the hydrophobic chains of the SC lipids to increase the fluidity of lipids in the SC.

Table 3. Peaks in the FT-IR spectra of skin samples after 5 h post-treatment Samples

vas CH2

vs CH2

Amide I

Normal saline

2924.45±0.27

2853.18±0.10

1630.91±0.07

1549.06±0.40

Cubic phases

2925.48±0.56*

2853.90±0.20*

1630.88±0.14

1550.32±1.32

Solutions

2924.61±0.19

2853.25±0.19

1633.28±1.01*

1545.13±1.84*

Each value was presented as mean ± SD (n = 3).

* p < 0.05.

26


Amide II

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Figure 8. FTIR spectra of vas CH2 and vs CH2 regions after treatment with normal saline, the cubic phase, the solution (A). The π-A isotherms (B), the compression modulus (Cs-1) (C), and the excess Gibbs energy (△Gex) (D) of the Langmuir monolayers of ceramide, GMO, and their mixtures with the GMO molar percentages of 20%, 40%, 60%, 80%, respectively. 3.7.3. Isotherms, Compressibility and Miscibility of Ceramide and GMO Monolayers.

The Langmuir monolayers of ceramide and GMO showed the similar isotherm at the air/water interface (Figure 8B). However, the ceramide monolayer had the larger extrapolated molecular area (0.741 nm2) than the GMO monolayer (0.512 nm2), 27



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indicating a larger polar head of ceramide than GMO, which was in agreement to the molecular structures of them (Figure 2). The high collapse pressures of about 45 mN/m of ceramide and GMO monolayers indicated they formed condensed monolayers at the air/water interface. The monolayer compression modulus (Cs−1) was a measure of the film stiffness/elasticity and calculated from π−A isotherms with the equation (8).

C

−1 s

= − A( dπ / dA)

(8)

where A was the mean molecular area (MMA) and π was the corresponding surface pressure under constant temperature. A low compression modulus indicates a monolayer with low interfacial stiffness or high elasticity. After the surface pressure was over 10 mN/m, ceramide monolayers displayed higher Cs−1 than GMO monolayers (Figure 8C). Moreover, the mixture monolayers of GMO and ceramide also had low Cs−1, similar to that of GMO. Therefore, GMO enhanced the elasticity or fluidity of the monolayers of ceramide/GMO mixtures. Excess Gibbs energy (△Gex) can well exhibit the miscibility of amphiphilic mixtures. It can be calculated from the equation (9). π

∆Gex = N ∫ [ A12 − ( X1 A1 + X 2 A2 ]dπ 0

(9)

where N was Avogadro’s constant. △Gex represented the energy contribution from mixing of components in the monolayer. A1 and A2 represented the MMAs for the pure monolayers of component 1 (ceramide) and component 2 (GMO), respectively, under the same surface pressures, while X1 and X2 were the mole fractions of each component in the mixture. Mixtures with more negative ∆Gex were 28


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more thermodynamically stable. All the ∆Gex of the ceramide/GMO mixture monolayers was negative and the mixture of 40% (mol/mol) GMO showed the lowest ∆Gex (Figure 8D). Therefore, the mixture of ceramide/GMO is thermodynamically stable, and GMO can be miscible with ceramide.

3.8. Effect of Cubic Phases on the Interaction between Met and Keratin in SC 3.8.1 Molecular Modeling of Keratin. Both glutamine of keratin and hydroxyl group of GMO could form the hydrogen bonds with the amino group of Met (Figure 9B and 9C). The binding energies between GMO and Met, keratin and Met were -2.4 kcal/mol and -3.3 kcal/mol, respectively. Additionally, there was a weak hydrogen bond (-2.1 kcal/mol) between keratin and carbonyl group of GMO molecule (Figure 9A). These results indicated that there might be a competition between GMO and keratin when they formed hydrogen bonds with Met (Figure 9D).

29



Figure 9. Molecular docking of GMO and skin proteins (A), Met and skin proteins (B), GMO and Met (C). Schematic illustration of mechanic exploration (D).

3.8.2 Spectral Features of Met/Keratin in SC. The amide I (~1630 cm-1) and amide II (~1550 cm-1) stretching vibrations are commonly used as the parameters for evaluating the interaction between chemicals and SC skin proteins.25 Compared with saline, the Met solutions led to a significant change at the peak of amide I and amide 30


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II while the cubic phases took little change (Figure 10A，Table 3). The change of amide I peak and amide II peak showed the change of skin protein second structures.40 Therefore, the solution formulation of Met had great influences on the skin proteins but the cubic phase formulation of Met did not. 3.8.3 Distribution of Met in the Skin. Met can react with the mixed solution of sodium nitroprusside and ferricyanatum kalium under alkaline condition to produce pink precipitation of iron hydroxide. A large amount of pink precipitates appeared around the keratin K5 of the solutions (Figure 10B), but a little of pink precipitates emerged homogenously in the skin tissue of the cubic phases (Figure 10C). These results indicated that Met in the solution mainly located around the keratin while Met in the cubic phase homogenously distributed in the skin tissue.

Figure 10. FTIR spectra of amide I and amide II regions after treatment with saline, the cubic phases, the solutions (A). The immunohistochemical images of the solutions 31



group (B) and the cubic phases group (C) after 5 h post-treatment (B, C, 400×).

4. DISCUSSION Cubic phases are always focused on and very promising transdermal liquid crystalline systems. The drugs loaded in cubic phases have not only high stability due to chemical and physical protection but also enhanced permeation across the skin.12 Met is an important anti-diabetes hydrophilic drug, and oral administration is the major method. Here, we prepared Met-loaded transdermal cubic phases to explore the facilitated release and enhance percutaneous absorption of Met using the computer and experimental methods. The Flory–Huggins parameter is an extremely important index in computational simulations, indicating the miscibility of binary mixtures. MD simulation is a key method to obtain the Flory–Huggins parameter that is used for the mesoscale simulations of the DPD technique. Therefore, the Flory–Huggins parameter is the firstly calculated value for the next computer simulation, also a bridge between MD simulation and DPD simulation. The solubility parameter method (shown by equation (1)) is commonly used for calculation of the Flory–Huggins parameter.41 This method is simple, although it is just suitable for non-polar components or the components without some specific interactions such as hydrogen bonding and ionic binding. Furthermore, the method based on the energies of mixing (shown by equations (2) and (3)) can be suitable for

32


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various components but tedious. Here, two methods were together used to estimate the Flory–Huggins parameters (χ), depending on the properties of components. The miscibility of binary components in cubic phases can be approximately predicted with MD simulation. Here, Met in the cubic phase was proved to be immiscible with other components except for water according to the values of χ. Therefore, Met mainly distributed in the water channels of cubic phases (Figure 4B). Because some water channels in cubic phases were open to the outside according to the DPD simulation and the reference,42 Met could diffuse through the water channels to reach the skin surface. Met release from the cubic phase was a controlling profile in accordance with the Higuchi’s equation, so that the release of Met mainly depended on drug diffusion through the water channels. Because the release amount of Met from cubic phases was 3880 ± 229 µg/cm2, much higher than the penetration amount of Met, the permeation process was the major limited step in the whole transdermal delivery. Furthermore, the in vitro permeation experiments demonstrated the advantage of cubic phases on enhanced permeation of Met compared with the solution formulation. The cubic phases had significantly higher (ER = 1.48) and faster (Tlag = 0.04 h) enhancement of Met permeation than the solutions (Table 2). The SC is the main barrier of transdermal delivery. It is a thin (15-20 µm) heterogeneous layer and composed of terminally differentiated and keratinized epidermal cells separated by an intercellular lipid-protein matrix.43,

44

This

arrangement of the corneocytes within the lipid-protein matrix can be regarded as a 33



brick wall with the corneocytes being the bricks and the lipid-protein matrix the mortar.45 Drugs are generally in the form of reversible/irreversible binding to the SC keratin, affecting their partition and diffusivity.46 Here, the cubic phases led to significantly lower cumulative Met retention amount in the skin than the solutions (Figure 5D). According to the computer and experimental result, Met band to the skin proteins but GMO interfered with the binding between Met and skin proteins, leading to Met diffusion into the deep skin layers. The effect of transdermal formulations on the skin structure and components is an important factor for development of them. The lipids and keratin of SC can modulate the skin permeability of water and small molecules.47 The TEWL technique was used to evaluate the effect of cubic phases on the skin barrier function. Although a significant increase of ∆TEWL occurred before 60 min post-treatment of the cubic phases, a normal level was achieved at the late stage (Figure 6). Therefore, the cubic phases are a safe transdermal formulation due to the reversible effect on the skin. The transdermal drug delivery process is complicated and influenced by many factors, such as the condition of skins, the properties of drugs, and the diffusion and partition of drugs in the skin.48 The SC is the main permeation barrier. There are different routes by which a molecule can penetrate the SC, including the intercellular, transcellular and appendageal (sweat glands or hair follicles).48 The appendageal route is not a typical permeation pathway because sweat glands and hair follicles occupy only 0.1% of the total surface area of human skin.48 Here, the skin of nude mice was selected as the model, which had few hair follicles and sweat glands compared to 34


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other kinds of skins. Hence, the transcellular pathway and intercellular lipid pathway were considered as the main permeation pathways through the skin. GMO is the major component of the Met-loaded cubic phase. It has been proved that GMO can enhance the transdermal permeation of drugs based on the ability of GMO to remove skin ceramides and increase the lipid fluidity in the SC.18 However, this work proposes a new mechanism involving the effect of GMO on the intercellular pathway and transcellular pathway at the molecular level. We demonstrated the alterations of the SC intercellular lipid organization and keratin of corneocytes with a series of computer simulations and experiments including MD simulations, FT-IR spectroscopy, Langmuir monolayer, molecular docking and immunohistochemistry. The interaction between GMO molecules and the components of the skin directly determines the percutaneous absorption of drugs. The tail-end hydrophobic part of GMO molecules located in the hydrophobic lipid tails in the membrane composed of ceramide after the dynamic simulation for 50 ns, so that the hydrophobic part of GMO could theoretically insert into the lipid layers of SC. Both the fluid and solid lipid phases in the SC simultaneously existed, where the solid lipids contribute to the barrier function and the fluid lipids constitute a major transport route. The permeability of water and other hydrophilic molecules are much higher through the fluid lipid region than the solid lipid region.46 GMO increased the mobility of the SC lipids according to the computer simulations, leading to the enhanced skin permeation of the hydrophilic Met. The FTIR technique is generally used to evaluate the changes of lateral 35



organization of the SC lipids by the topical application of products and provide information about the molecular basis of the interaction.46 Compared to the saline group, the blue shift of νs CH2 and νas CH2 of skin lipids occurred after treatment of the cubic phase while no significant changes appeared in the solution group. Therefore, GMO could interact with the lipid alkyl chains to increase the fluidity of skin lipids, which was in agreement with the results of the above simulations. The miscibility and interaction of GMO and SC lipids can be explored by comparing a series of π−A isotherms of ceramide and GMO monolayers. Firstly, the addition of GMO to SC lipid (i.e., ceramide) monolayers decreased the monolayer compression modulus, indicating the significant fluidizing effect of GMO on SC lipid monolayers. Furthermore, ∆Gex was negative for all the mixtures of GMO/ceramide, suggesting that GMO could well insert into ceramide molecules to form stable monolayer. Molecular docking is a good method to evaluate molecular interaction.39 The interaction of GMO/keratin, Met/keratin and Met/GMO was investigated, respectively. Both the glutamine of keratin and hydroxyl group of GMO could form the hydrogen bonds with the amino group of Met. However, there was only a weak hydrogen bond between keratin and carbonyl group of GMO molecule. GMO had effect on the hydrogen bonding of Met/keratin to improve the percutaneous penetration of Met (Figure 9D). In fact, the drug permeation is limited due to the interactions between drugs and the different skin layers.48 These interactions may be reversible or irreversible. A drug reservoir may form in the skin to slow down drug diffusion. In 36


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this study, GMO could pull drugs to go into the deep skin due to the disturbance of GMO on the interaction of Met/keratin. The FT-IR spectra can provide the information about the SC skin proteins with the bands of amide I (~1650 cm-1) and amide II (~1550 cm-1).28 Met interacted with the amide I and amide II of skin proteins, which further indicated that the solutions led to the higher cumulative Met retention in the skin than the cubic phases. Here, the cubic phases did not affect the IR peaks of amide I and amide II groups; whereas, the solutions had significant effects on the peaks of amide I and amide II (Table 3). The disturbance of GMO on the interaction of Met/keratin was also proved by the immunohistochemical experiments. The solutions made Met mainly distributing around the skin proteins due to the interaction of Met/keratin. In contrast, the cubic phases improved Met penetration into the deep skin layers due to the disturbance of GMO. These results were in accordance with molecular docking. Therefore, the binding of Met/skin proteins was one important factor to affect drug distribution and diffusion in the skin and GMO’s disturbance improved the transdermal permeation and absorption of Met. Liquid crystalline systems mainly include cubic phases, lamellar phases and hexagonal phases, where GMO is usually the major component.49 Both cubic phases and hexagonal phases have subcategories of gels and liquid crystalline particles.50, 51 Liquid crystalline systems are made up of the water channels and lipid channels where some water channels are open to the outside. According to the result of our study, if one hydrophilic drug is immiscible with GMO, its release could mainly 37



depend on drug diffusion from the water channels of liquid crystalline systems. Moreover, GMO plays a key role in the whole percutaneous absorption of hydrophilic drugs. Therefore, GMO-contained liquid crystalline systems are promising transdermal carriers for hydrophilic drugs.

5. CONCLUSIONS The delivery mechanisms of drug formulations are the key to research and development of drug delivery systems. Cubic phases are one of the important transdermal delivery systems though the delivery mechanisms remain unclear. Drug transdermal permeation in cubic phases consists of two processes: drug release from cubic phases and its percutaneous absorption. Firstly, Met diffuses from the water channels of cubic phases to the surface of SC. Next, GMO enhances Met permeation through skin by disturbing the interaction between Met and skin proteins, and increasing the fluidity of skin lipids. The above conclusions are useful for extending our understanding of the mechanisms of transdermal delivery of drugs from cubic phases. More importantly, cubic phases are an ideal transdermal delivery system of Met. In addition, GMO may have the same action in other formulations than cubic phases. Our findings can serve as a theoretical and experimental reference for transdermal delivery of hydrophilic drugs.

AUTHOR INFORMATION Corresponding Author: Yiguang Jin (ORCID: 0000-0002-3528-1397) 38


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E-mail: [email protected] Notes The authors declare no competing financial interest.

REFERENCES (1) Janjetovic, K.; Harhaji-Trajkovic, L.; Misirkic-Marjanovic, M.; Vucicevic, L.; Stevanovic, D.; Zogovic, N.; Sumarac-Dumanovic, M.; Micic, D.; Trajkovic, V., In vitro and in vivo anti-melanoma action of metformin. Eur. J. Pharmcol. 2011, 668, 373-382. (2) Ashutosh, M.; Rajesh, P.; Mukesh, G., Formulation, development and evaluation of patient friendly dosage forms of metformin. Part-i: Orally disintegrating tablets. Asian J. Pharm. Sci. 2014, 3, 25-28. (3) Prausnitz, M. R.; Langer, R., Transdermal drug delivery. Nat. Biotechnol. 2008, 26, 1261-1268. (4) Cao, Y.; Kakar, P.; Hossen, N.; Wu, M. X.; Chen, X., Sustained epidermal powder drug delivery via skin microchannels. J. Control. Rel. 2017, 249, 94-102. (5) Chang, M.; Li, X.; Sun, Y.; Cheng, F.; Wang, Q.; Xie, X.; Zhao, W.; Tian, X., Effect of cationic cyclopeptides on transdermal and transmembrane delivery of insulin. Mol. Pharmaceut. 2013, 10, 951-957. (6) Rai, V. K.; Mishra, N.; Yadav, K. S., Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications. J. Control. Rel. 2017, 270, 203-225. 39



(7) Anirudhan, T. S.; Nair, S. S.; Sasidhara, A. V., Methacrylate stitched β-cyclodextrin embedded with nanogold/nanotitania-a skin adhesive device for enhanced transdermal drug delivery. ACS Appl. Mater. Interfaces 2017, 9, 44377-44391. (8) Marimuthu, M.; Bennet, D.; Kim, S., Self-assembled nanoparticles of PLGA-conjugated glucosamine as a sustained transdermal drug delivery vehicle. Polym. J. 2013, 45, 202-209. (9) Zhai, J.; Tran, N.; Sarkar, S.; Fong, C.; Mulet, X.; Drummond, C. J., Self-assembled lyotropic liquid crystalline phase behaviour of monoolein-capric acid-phospholipid nanoparticulate systems. Langmuir 2017, 33, 2571–2580. (10) Chong, J. Y.; Mulet, X.; Keddie, D. J.; Waddington, L.; Mudie, S. T.; Boyd, B. J.; Drummond, C. J., Novel steric stabilizers for lyotropic liquid crystalline nanoparticles: Pegylated-phytanyl copolymers. Langmuir 2015, 31, 2615-2619. (11) Yariv, D.; Efrat, R.; Libster, D.; Aserin, A.; Garti, N., In vitro permeation of diclofenac salts from lyotropic liquid crystalline systems. Colloids Surf. B Biointerfaces 2010, 78, 185-192. (12) Dante, M. D. C. L.; Borgheti-Cardoso, L. N.; de Abreu Fantini, M. C.; Praça, F. S. G.; Medina, W. S. G.; Pierre, M. B. R.; Lara, M. G, Liquid crystalline systems based on glyceryl monooleate and penetration enhancers for skin delivery of celecoxib: characterization, in vitro drug release, and in vivo studies. Journal of pharmaceutical sciences. J. Pharm. Sci. 2017, 30, 1-9. (13) Yu, X.; Du, L.; Zhu, L.; Liu, X.; Zhang, B.; Fu, G.; Jin, Y., Melanoma therapy 40


Page 40 of 57

Page 41 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with transdermal mitoxantrone cubic phases. Drug Del. 2016, 23, 1565-1570. (14) Turchiello, R. F.; Vena, F. C. B.; Maillard, P. H.; Souza, C. S.; Bentley, M. V. B. L.; Tedesco, A. C., Cubic phase gel as a drug delivery system for topical application of 5-ALA, its ester derivatives and m-THPC in photodynamic therapy (PDT). J. Photochem. Photobiol. B: Biology, 2003, 70, 1-6. (15) Li, J. C.; Zhu, N.; Zhu, J. X.; Zhang, W. J.; Zhang, H. M.; Wang, Q. Q.; Hao, J. F., Self-assembled cubic liquid crystalline nanoparticles for transdermal delivery of Paeonol. Med. Sci. Monit, 2015, 21, 3298-3310. (16) Bender, J.; Simonsson, C.; Smedh, M., Engström, S.; Ericson, M. B, Lipid cubic phases in topical drug delivery: visualization of skin distribution using two-photon microscopy. J. Control. Rel., 2008, 129, 163-169. (17) Peng, X.; Wen, X.; Pan, X.; Wang, R.; Chen, B.; Wu, C., Design and in vitro evaluation of capsaicin transdermal controlled release cubic phase gels. AAPS. PharmSciTech. 2010, 11, 1405-1410. (18) Simonetti, L. D. D.; Gelfuso, G. M.; Barbosa, J. C. R.; Lopez, R. F. V., Assessment of the percutaneous penetration of cisplatin: The effect of monoolein and the drug skin penetration pathway. Eur. J. Pharm. Biopharm. 2009, 73, 90-94. (19) Comer, J.; Chen, R.; Poblete, H.; Vergara-Jaque, A.; Riviere, J. E., Predicting adsorption affinities of small molecules on carbon nanotubes using molecular dynamics simulation. ACS Nano 2015, 9, 11761–11774. (20) Kulkarni, A.; Pandey, P.; Rao, P.; Mahmoud, A.; Goldman, A.; Sabbisetti, V.; Parcha, S.; Natarajan, S. K.; Chandrasekar, V.; Dinulescu, D.; Roy, S.; Sengupta, S., 41



Algorithm for designing nanoscale supramolecular therapeutics with increased anticancer efficacy. ACS Nano 2016, 10, 8154-8168. (21) Xu, Z.; Yang, X.; Yang, Z., A molecular simulation probing of structure and interaction for supramolecular sodium dodecyl sulfate/single-wall carbon nanotube assemblies. Nano Lett. 2010, 10, 985-991. (22) Angioletti-Uberti, S., Theory, simulations and the design of functionalized nanoparticles for biomedical applications: A soft matter perspective. npj Comput. Mat.

2017, 3, 48. (23) Estracanholli, É. A.; Praça, F. S. G.; Cintra, A. B.; Pierre, M. B. R.; Lara, M. G., Liquid crystalline systems for transdermal delivery of celecoxib: In vitro drug release and skin permeation studies. AAPS. PharmSciTech. 2014, 15, 1468-1475. (24) Luo, Z.; Jiang, J., pH-sensitive drug loading/releasing in amphiphilic copolymer PAE–PEG: Integrating molecular dynamics and dissipative particle dynamics simulations. J. Control. Rel. 2012, 162, 185-193. (25) Zhao, Y.; You, L.; Lu, Z.; Sun, C., Dissipative particle dynamics study on the multicompartment micelles self-assembled from the mixture of diblock copolymer poly (ethyl ethylene)-block-poly(ethylene oxide) and homopolymer poly(propylene oxide) in aqueous solution. Polymer 2009, 50, 5333-5340. (26) Li, Q.; Yao, W.; Yu, X.; Zhang, B.; Dong, J.; Jin, Y., Drug-loaded pH-responsive polymeric micelles: Simulations and experiments of micelle formation, drug loading and drug release. Colloids Surf. B Biointerfaces 2017, 158, 709-716. (27) Charles, B. G.; Jacobsen, N. W.; Ravenscroft, P. J., Rapid liquid-chromatographic 42


Page 42 of 57

Page 43 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


determination of metformin in plasma and urine. Clin. Chem. 1981, 27, 434-436. (28) Liu, X.; Quan, P.; Li, S.; Liu, C.; Zhao, Y.; Zhao, Y.; Fang, L., Time dependence of the enhancement effect of chemical enhancers: Molecular mechanisms of enhancing kinetics. J. Control. Rel. 2017, 248, 33-44. (29) Chen, Y.; Wang, J.; Cun, D.; Wang, M.; Jiang, J.; Xi, H.; Cui, H.; Xu, Y.; Cheng, M.; Fang, L., Effect of unsaturated menthol analogues on the in vitro penetration of 5

一 fluorouracil through rat skin. Int. J. Pharm. 2013, 443, 120-127. (30) Lim, D. G.; Jeong, W.-W.; Kim, N. A.; Lim, J. Y.; Lee, S.-H.; Shim, W. S.; Kang, N.-G.; Jeong, S. H., Effect of the glyceryl monooleate-based lyotropic phases on skin permeation using in vitro diffusion and skin imaging. Asian J. Pharm. Sci. 2014, 9, 324-329. (31) Chang, C.-P., Effect of simulation temperature on phospholipid bilayer-vesicle transition studied by coarse-grained molecular dynamics simulations. Soft Matter

2013, 9, 7294–7301 (32) Baoukin, S.; Monticelli, L.; Risselada, H. J.; Marrink, S. J.; Tieleman, D. P., The molecular mechanism of lipid monolayer collapse. Proc. Nat. Acad. Sci. USA 2008, 105, 10803-10808. (33) Http://md.Chem.Rug.nl/cgmartini/index.php/paramet-rzining-new-molecule. (34) Kumar, S.; Zakrewsky, M.; Chen, M.; Menegatti, S.; Muraski, J. A.; Mitragotri, S., Peptides as skin penetration enhancers: Mechanisms of action. J. Control. Rel.

2015, 199, 168-178. (35) Sun, M.; Wu, C.; Fu, Q.; Di, D.; Kuang, X.; Wang, C.; He, Z.; Wang, J.; Sun, J., 43



Solvent-shift strategy to identify suitable polymers to inhibit humidity-induced solid-state crystallization of lacidipine amorphous solid dispersions. Int. J. Pharm.

2016, 503, 238-246. (36) Elokely, K. M.; Doerksen, R. J., Docking challenge: Protein sampling and molecular docking performance. J. Chem. Inf. Modol. 2013, 19, A-L. (37) Kang, M.; Loverde, S. M., Molecular simulation of the concentration-dependent interaction of hydrophobic drugs with model cellular membranes. J. Phy. Chem. B

2014, 118, 11965-11972. (38) Anderson, R.; Schweitzer, B.; Wu, T.; Carreon, M. A.; Gómez-Gualdrón, D. A., Molecular simulation insights on xe/kr separation in a set of nanoporous crystalline membranes. ACS Appl. Mater. Interfaces 2018, 10, 582-592. (39) Wan, G.; Dai, X.; Yin, Q.; Shi, X.; Qiao, Y., Interaction of menthol with mixed-lipid bilayer of stratum corneum: A coarse-grained simulation study. J. Mol. Graph Model. 2015, 60, 98-107. (40) Byler, D. M.; Susi, H., Examination of the secondary structure of proteins by deconvolved ftir spectra. Biopolymers 1986, 25, 469-487. (41) Jin, Y.; Yao, W.; Wu, L.; Du, L., Langmuir monolayers of n-acyl derivatives of adefovir phosphonate at the air/water interface and molecular self-assembly in water. Colloids Surf. A Physico-Chem. Asp. 2014, 462, 231-236. (42) Pan, X.; Han, K.; Peng, X.; Yang, Z.; Qin, L.; Zhu, C.; Huang, X.; Shi, X.; Dian, L.; Lu, M.; Wu, C., Nanostructed cubosomes as advanced drug delivery system. Cur. Pharma. Design 2013, 19, 6290-6297. 44


Page 44 of 57

Page 45 of 57 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(43) Horita, D.; Hatta, I.; Yoshimoto, M.; Kitao, Y.; Todo, H.; Sugibayashi, K., Molecular mechanisms of action of different concentrations of ethanol in water on ordered structures of intercellular lipids and soft keratin in the stratum corneum. Biochim. Biophys. 2015, 1848, 1196-1202. (44) Nakazawa, H.; Ohta, N.; Hatta, I., A possible regulation mechanism of water content in human stratum corneum via intercellular lipid matrix. Chem. Phys. Lipids

2012, 165, 238-243. (45) Ramms, L.; Fabris, G.; Windoffer, R.; Schwarz, N.; Springer, R.; Zhou, C.; Lazar, J.; Stiefel, S.; Hersch, N.; Schnakenberg, U.; Magin, T. M.; Leube, R. E.; Merkel, R.; Hoffmann, B., Keratins as the main component for the mechanical integrity of keratinocytes. Proc. Nat. Acad. Sci. USA 2013, 110, 18513–18518. (46) Boncheva, M.; Damien, F.; Normand, V., Molecular organization of the lipid matrix in intact stratum corneum using ATR-FTIR spectroscopy. Biochim. Biophys. Acta. 2008, 1778, 1344-1355. (47) Liu, X.; Liu, M.; Liu, C.; Quan, P.; Zhao, Y.; Fanga, L., An insight into the molecular mechanism of the temporary enhancement effect of isopulegol decanoate on the skin. Int. J. Pharm. 2017, 529, 161-167. (48) Lane, M. E., Skin penetration enhancers. Int. J. Pharm. 2013, 447, 12-21. (49) Nazaruk, E.; Szlęzak, M.; Górecka, E.; Bilewicz, R.; Osornio, Y. M.; Uebelhart, P.; Landau, E. M., Design and assembly of pH-sensitive lipidic cubic phase matrices for drug release. Langmuir 2014, 30, 1383-1390. (50) Feast, G. C.; Lepitre, T.; Tran, N.; Conn, C. E.; Hutt, O. E.; Mulet, X.; Savage, G. 45



P, Inverse hexagonal and cubic micellar lyotropic liquid crystalline phase behaviour of novel double chain sugar-based amphiphiles. Colloids Surf. B Biointerfaces 2017, 151, 709-716. (51) Pirolt, F.; Glatter, O.; Trimmel, G, Reverse-hexosome dispersions in alkane–the challenge of inverting structures. Langmuir 2018, 14.

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For Table of Contents Use Only Cubic Phases for Transdermal Delivery of Metformin Hydrochloride: In Silico and In Vitro Studies of Transdermal Delivery Mechanisms Xiang Yu, Yiguang Jin*, Lina Du, Mengchi Sun, Jian Wang, Qiu Li, Xiangyu Zhang, Zisen Gao, and Pingtian Ding

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204x64mm (117 x 117 DPI)


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139x146mm (149 x 149 DPI)


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Transdermal Cubic Phases of Metformin Hydrochloride: In Silico and

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