Expanding Transdermal Delivery with Lipid Nanoparticles: A New

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Expanding transdermal delivery with lipid nanoparticles: a new drug-in-NLC-in-adhesive design M. Mendes, S. C.C. Nunes, J. J. Sousa, A. A.C. C. Pais, and C. Vitorino Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 9, 2017

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Molecular Pharmaceutics

Expanding transdermal delivery with lipid nanoparticles: a new drug-in-NLC-in-adhesive design M. Mendes †||, S.C.C. Nunes‡, J. J. Sousa†||, A. A. C. C. Pais‡, C. Vitorino†||* †Faculty of Pharmacy, University of Coimbra, Portugal

||Pharmacometrics Group of the Centre for Neurosciences and Cell Biology (CNC), University of

Coimbra, Portugal ‡Coimbra Chemistry Center, Department of Chemistry, University of Coimbra, Portugal

*E-mail: [email protected] KEYWORDS Co-encapsulation, nanostructured lipid carriers, transdermal patches, chemical permeation enhancers, molecular dynamics, Dermaroller®pre-treatment.

ABSTRACT

A monolithic drug-in-NLC-in-adhesive transdermal patch, with a novel design, was developed for co-delivery of olanzapine (OL) and simvastatin (SV). Nanostructured lipid carriers (NLC) and enhancers were used as passive strategies, while the pre-treatment of the skin with Dermaroller® pre-treatment was tested as active approach. The formulation was optimized for composition in a quality by design basis, in terms of enhancer and adhesive, with focus on

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permeation behavior, adhesion properties and cytotoxicity. Propylene glycol promoted the best permeation rate for both drugs, with enhancement ratios of 8.05 and 12.89 for OL and SV, respectively, relative to the corresponding Combo-NLC patch without enhancer. Molecular dynamics results provided a rationale for these observations. The adhesive type displayed an important role in skin permeation, reinforced by the presence of the enhancer. The permeation rates obtained for the optimized transdermal patch largely exceed (by factors of 4.4 and 12.3, for OL and SV, respectively) the target values to ensure therapeutic drug concentrations. Finally, Dermaroller® pre-treatment did not promote a significant improvement in permeation, which highlights the role of the combination of NLC with chemical enhancer in the transdermal patch as the main driving force in the process. It is also observed that NLC are able to reduce cytotoxicity, especially that associated to SV. This work provides a promising in vitro-in silico basis for a future in vivo development.

1. Introduction The transdermal drug delivery is an alternative pathway that offers several advantages over conventional routes of administration. In particular, it avoids the liver first pass and the effect of gastrointestinal tract (GIT) conditions, and provides sustained drug levels over a prolonged period of time, reduced side effects and is a convenient, painless, noninvasive way to selfadminister drugs1. However, it also presents some drawbacks, such as intra- and inter-variability associated to the permeation of skin, the presence of enzymes in the skin that can lead to metabolization of the drug, skin irritation and sensitization. In addition, this route limits the drugs that can be administrated, due to the natural complexity of skin layers. Drugs need specific physicochemical characteristics to have an improved permeation through the skin, including having a small molecular weight (lower than 500 Da), a log P comprised between 1 and 3, being


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unionizable, possessing a reasonable aqueous solubility (> 1 mg/L), a melting point lower than 200 °C and a daily dose < 20 mg2. The main barrier to skin permeation is the stratum corneum (SC), which is a non-viable layer consisting on 10-15 layers of corneocytes, rich in keratin, embedded in a lipid matrix, composed by ceramides, long chain fatty acids, triglycerides and cholesterol3, 4. In order to overcome the complex structure of SC, different strategies, comprising passive and active approaches, may be implemented in transdermal patches to improve the drug permeation through the skin layers. Examples of passive strategies include the use of colloidal systems, which interfere on thermodynamic activity, and chemical penetration enhancers (CPE), which are able to change the compact structure of the SC. In turn, active methods involve the use of external energy to act as a drug driving force and/or to reduce the natural barrier of SC, e.g by creating transient microchannels that allow an increased penetration5. Lipid nanoparticles (LN), especially nanostructured lipid carriers (NLC), consist of a matrix composed of a blend of solid and liquid lipids (oils), stabilized by an aqueous surfactant solution, being considered an appealing approach to promote drug penetration through SC due to their peculiar features6-8. Some of these characteristics are the small particle size, which facilitates the contact of encapsulated drugs with the SC and their particular composition in lipids, which prompts the formation of a film on the skin surface, resulting in an occlusive effect and a local increase in skin hydration9-11. Also, LN are suitable for use on damaged or inflamed skin, because they are composed by biodegradable and biocompatible excipients. The drug distribution through the skin layers depends on the relative solubility of the drug in the components of the delivery system and in the SC. Thus, the natural interface established by the LN influences the depth of penetration of the drug, since this is governed to a large extent by the thermodynamic activity of the drug in the vehicle. To overcome the low partition rate of the drug


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in the SC, it is possible to add CPE to the delivery system, so as to modify the intrinsic diffusional barrier properties of this structure. CPE exert their function by spatial disruption of the ordered arrangement of the intercellular molecules, decrease in the diffusional resistance of the barrier and modification of the intracellular environment of the corneocytes, thus influencing the penetration of certain classes of drugs6,

12, 13

. Some examples are glycols (e.g. propylene

glycol), glycol ethers (e.g. Transcutol®), fatty acids (e.g. oleic acid), pyrrolidones (e.g. N-methyl pirrolidone), surfactants (e.g. Tween® 80) and terpenes (e.g. limonene)14. Another method to improve the transport of drugs through the skin is the use of microneedles as a mechanical technique. It has raised particular interest and has been broadly investigated for several research groups and companies15-17. As a simple mechanical device, it is expected to be easy to use and minimally invasive 18. All of these strategies can be integrated in a patch, as a sole transdermal drug delivery system (TDDS)19-22. A TDDS is a flexible, multi-layered, pharmaceutical single dose preparation of varying size, containing one or more active substances to be applied to the intact skin for systemic absorption. They can be mainly classified into three types, monolithic or multilaminate drug-in-adhesive (DIA), liquid reservoir and polymer matrix22. Combining the previous strategies to maximize the administration of drugs through the skin into a monolithic drug-in-adhesive transdermal patch arises as a challenging approach. DIA systems usually control drug delivery and adhesion to the skin through a single polymer layer, in which the drug is contained. Patches of this type are particularly thin and flexible, characteristics which favor patient compliance. The present work aims at developing a differentiated transdermal formulation based on NLC, for concomitant administration of SV and OL (Fig.1 A and B, respectively). The use of atypical antipsychotics offers many benefits and may reduce some of the factors related to the morbidity


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and mortality in schizophrenia. However, these drugs have been associated with varying degrees of metabolic adverse effects, such as weight gain, impaired glucose metabolism, dyslipidemia and, in some cases, more serious morbidity such as cardiovascular disease. This is the case for OL, a second-generation antipsychotic, which promotes increased levels of triglycerides and low-density lipoprotein cholesterol, LDL-C, while decreasing high-density lipoprotein cholesterol, HDL-C. This leads to an increased cardiovascular risk23. In order to reduce some of these unwanted effects, the use of a combined therapy with statins, such as SV, might be required. SV is a potent inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, involved in the cholesterol synthesis. This inhibition is mainly responsible for reducing LDL-C levels, but SV has also been shown to reduce the levels of triglycerides and increase the levels of HDL-C24. Additionally, it should be noted that for patients with schizophrenia, the risk of relapse (reemergence of previously controlled symptoms) is high, having nonadherence to the antipsychotic regimen as one of the major factors contributing to this risk. It is estimated that among patients with psychiatric disorders, only about one third take medication as prescribed. For the remaining, one third take medication erratically, either with lower dose or missing doses, and the rest do not take their medication25, 26. In this context, a transdermal patch can be considered a solution to improve adherence to medication, and one means of preventing, or at least postponing, relapse. Innovation relies on the design of a drug-in-NLC-in-adhesive patch, for which the impact of the passive and active strategies previously described is assessed. For that, the screening of the best enhancer to combine with the NLC formulation was carried out, supported on permeation studies. Subsequently, the effect of different adhesives in the NLC-DIA system was evaluated based on in vitro and in vivo adhesion studies. A factorial planning was established so as to


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estimate the contribution of each component and the respective interaction on the quality target product. The potential toxic effects of the transdermal patch were investigated through in vitro cytotoxicity studies. Finally, the preliminary use of a microneedle roller device along with the optimal formulation was assessed in terms of permeation enhancement.

Figure 1. Chemical structures of (A) simvastatin and (B) olanzapine.

2. Materials and methods 2.1. Materials Simvastatin was kindly provided by Labesfal (Santiago de Besteiros, Portugal). Olanzapine was purchased from Zhejiang Myjoy (Hangzhou, China). Glyceryl tripalmitate (tripalmitin, T8127, melting point 66ºC), polysorbate 80 (Tween® 80), polyethylene glycol 400 (PEG 400), polyvynilpyrrolidone K30, menthol, dibutyl phthalate, dibutyl sebacate, 9-diethylamino-5benzo[α]phenoxazinone (Nile-Red) and fluorescein isothiocyanate (FITC, 3',6'-dihydroxy-6isothiocyanatospiro[2-benzofuran-3,9'-xanthene]-1-one) were provided by Sigma (Missouri, USA). Eudragit® E100 was kindly donated by Evonik (Essen, Alemanha). Dermacryl® 79 (acrylates/octylacrylamide copolymer) was acquired from National Starch PersonalCare (Sweden). Isopropyl myristate (IPM) was a gift from BASF (Ludwigshafen, Germany).


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Propylene glycol, Transcutol® (diethylene glycol monoethyl ether), squalene, oleic acid, hydroxypropylmethylcellulose (HPMC) and limonene were acquired from Fluka (Missouri, USA). Water was purified (Millipore®) and filtered through a 0.2 µm nylon filter before use. All other reagents and solvents were from analytical or high performance liquid chromatography (HPLC) grade.

2.2. Methods 2.2.1. Preparation of lipid nanoparticles The NLC were prepared by the hot high pressure homogenization (HPH) technique, following an optimized procedure, previously reported27-29. This was carried out at 80 ºC, a temperature above the melting point of the solid lipid. Firstly, the molten lipid phase (7.5 % w/V), containing the oleic acid and tripalmitin (at a ratio of 50:50), was emulsified in an aqueous solution of Tween® 80 (3 % w/V, 150 mL) at the same temperature, using an Ultra-Turrax for 1 min (Ystral GmbH D-7801, Dottingen, Germany). A pre-emulsion was, thus, obtained. Secondly, the preemulsion was processed in a pre-heated high pressure homogenizer (HPH) (Emulsiflex®-C3, Avestin, Inc., Ottawa, Canada), at a pressure of 1000 bar for 12.5 min. In the drug loaded formulation, Combo-NLC, the addition of OL and SV was carried out in the initial lipid molten phase. In what concerns the thermodynamic activity, in the case of SV, the amount clearly exceeds saturation in tripalmitin, while for OL the respective amount is close to saturation in oleic acid. NLC containing 0.2 % (w/w) (in relation to lipid content) of the fluorescent dyes, Nile-Red and FITC, were prepared at the same conditions as the Combo-NLC.


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2.2.2. Characterization of the NLC dispersions 2.2.2.1.Particle size analysis Important parameters including particle size (PS) and polydispersity index (PI) need to be assessed for a proper quality control of lipid nanoparticles. The measurement of these parameters allows predicting the performance in biological environment and stability profile of formulations. Dynamic light scattering (DLS, also known as photon correlation spectroscopy) was used for their determination, using a Zetasizer Nano ZS, Malvern (Malvern, Worcestershire, UK) set at 173⁰ detection angle and at a temperature of 25 ºC. The samples were diluted 100 times in ultrapurified water and analyzed in triplicate. The cumulants method was used to data analysis.

2.2.2.2.Drug loading and entrapment efficiency The drug loading (DL) and entrapment efficiency (EE) were determined indirectly by measuring concentration of free drug in the aqueous phase of the dispersion, after separation through the ultrafiltration-centrifugation method using centrifugal filter units (Amicon® Ultra 4-, Millipore, Germany) with a 100 kDa molecular weight cut-off. Briefly, 500 µL of Combo-NLC were placed in the inner chamber of the centrifuge filter unit and centrifuged at 3000 x g for 45 min at 4 ⁰C. The free drug in aqueous phase collected in the outer chamber of the centrifuge filter unit was suitably diluted in mobile phase, filtered by a 0.22 µm membrane and determined by HPLC. The total amount of drug was estimated using a specific volume of the NLC


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dispersion, after being adequately diluted in mobile phase and heated at 60 ⁰C for 15 min. The dispersion was then centrifuged for 10 min at 11 740 x g in a Minispin® (Eppendorf Ibérica S.L., Madrid, Spain). The supernatant was collected, filtered by a 0.22 µm membrane and determined by HPLC.

2.2.3. Preparation and characterization of the transdermal patches Transdermal patches must be designed to provide a controlled delivery of the active substances through the skin, essentially based on diffusion, which may result in a defined rate and extent of systemic delivery of drug at a therapeutic concentration. This can be achieved taking into account permeation enhancement strategies, involving either the manipulation of the formulation by passive penetration enhancement (e.g. CPE, nanocarriers), the use of physical techniques (e.g. microneedles), or the combination of both strategies in the definition of the quality target product profile.

2.2.3.1. Design of drug-in-NLC-in-adhesive transdermal patches: composition of the formulations and preparation For preparation of the transdermal patches, lipid nanoparticles were dispersed in different adhesives and ethanol as co-solvent. Transdermal patches were manufactured by the solvent evaporation technique and prepared according to the composition presented in Tables 1 and 2. The different enhancers were tested at a concentration of 3.77 % (w/w). The selection of this concentration was based on previous work described elsewhere27. They were dissolved in adhesive and added to NLC dispersions. All compounds were homogenized under magnetic stirring. The formulations were placed in a backing layer (3M Scotchpak™ 9748 Fluoropolymer


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Coated Polyester Film Release, USA), with an area of 25 cm2, followed by solvent evaporation in an oven at 35 ºC for 48 h. The coating weight of the optimal dried patch was 3.84 g, corresponding to 13.6% of the total initial weight. Note that the ethanol content in the patch was lower than 2% after the drying procedure.


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Table 1. Notation for the formulations with different enhancers and respective composition (% w/w). Key: F = formulation; R = reference; A = adhesive; E = enhancer; 0 = absence; 1 – 8 = identification of the enhancer(s)/adhesive tested (see composition); C = Combo-NLC dispersion, as indicated in the text; PVP = polyvinylpirrolidone K30; PEG = polyethylene glycol 400; ET = ethanol; PG = propylene glycol; TCol = Transcutol®; L = limonene; M = menthol; OA = oleic acid; IPM = isopropyl myristate; S = squalene. F#

C

PVP

PEG

ET

PG

TCol

L

M

OA

IPM

S

R

100

-

-

-

-

-

-

-

-

-

-

A1E0

75.35

1.05

0.35

23.25

-

-

-

-

-

-

-

A1E1

75.35

1.05

0.35

19.48

3.77

-

-

-

-

-

-

A1E2

75.35

1.05

0.35

19.48

-

3.77

-

-

-

-

-

A1E3

75.35

1.05

0.35

19.48

-

-

3.77

-

-

-

-

A1E4

75.35

1.05

0.35

19.48

-

-

-

3.77

-

-

-

A1E5

75.35

1.05

0.35

15.71

3.77

-

-

3.77

-

-

-

A1E6

75.35

1.05

0.35

19.48

-

-

-

-

3.77

-

-

A1E7

75.35

1.05

0.35

19.48

-

-

-

-

-

3.77

-

A1E8

75.35

1.05

0.35

19.48

-

-

-

-

-

3.77


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Table 2. Notation for the formulations with different adhesives and respective composition (% w/w). Key: F = formulation; R = reference; A = adhesive; E = enhancer; 0 = absence; 1 – 5 = identification of the adhesive/enhancer tested (see composition); C = Combo-NLC, as indicated in the text; PVP = polyvinylpirrolidone K30; PEG = polyethylene glycol 400; DERM = Dermacryl 79; DBP = dibutyl phthalate; ET = ethanol; PG = propylene glycol.

F#

C

PVP

PEG

DERM

DBP

ET

PG

R

100

-

-

-

-

-

-

A0E0

100

-

-

-

-

-

-

A1E0

75.35

1.05

0.35

-

-

23.25

-

A2E0

75.35

-

-

1

-

23.65

-

A3E0

75.35

-

-

2.5

-

22.15

-

A4E0

75.35

-

-

-

2.5

22.15

-

A0E1

75.35

-

-

20.88

3.77

A1E1

75.35

1.05

0.35

-

-

19.48

3.77

A2E1

75.35

-

-

1

-

19.88

3.77

A4E1

75.35

-

-

-

2.5

18.38

3.77

A5E1

75.35

-

-

1

2.5

17.38

3.77


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2.2.3.2. Assay of OL and SV into transdermal patches For the quantification of OL and SV included into transdermal patches, a pre-defined area of the TDDS (1.44 cm2) was cut and suitably diluted in the mobile phase under sonication until complete redispersion. The samples were filtered (0.45 µm) and drugs quantified by HPLC.

2.2.4. Simulation details 2.2.4.1. System The effect of propylene glycol and Transcutol® (selected on the basis of the respective performance) in terms of membrane perturbation was investigated by molecular dynamics (MD) simulation, using a fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer. DPPC is commonly employed as a model lipid membrane to explore the mechanisms of drug permeation enhancement examining the influence of external agents on the structure and dynamics of the membrane27,

30-32

. All simulations were performed resorting to the Gromacs package, version

4.5.5 employing the GROMOS 53a6 force field33. The topology of each molecule (propylene glycol and Transcutol®) was generated by the ATB server34. A DPPC bilayer, consisting of 128 phospholipid molecules equally distributed by two leaflets and 3655 SPC water molecules, was employed. Six molecules of each enhancer were considered in each system, so as to allow studying the respective interaction with the surrounding lipids, as well as evaluating the preferential positioning and effect upon the bilayer.

2.2.4.2. Parameters and data analysis All MD simulations were performed in the NpT ensemble and considering periodic boundary conditions. A standard time step of 2 fs was set for both the equilibration and production runs. Non-bonded interactions were established on the basis of a neighbor list, updated every 10 steps.


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Long-range electrostatics was set using the particle mesh Ewald (PME) method. For LennardJones energies, a cut-off of 1.4 nm was applied. Temperature and pressure were coupled to the v-rescale and Berendsen external baths kept at 325 K and 1 bar, respectively. To compute a starting configuration, each system was firstly subjected to an energy minimization step. The systems were then left to evolve up to 400 ns, using the LINCS algorithm35. The first 40 ns were considered sufficient to attain equilibrated systems, while the last 360 ns of production runs were further subjected to standard analysis, including the determination of density probability distributions, order parameters, mean square displacements and radial distribution functions (rdf). MD trajectories were inspected and configuration images extracted using the VMD 1.9 software36.

2.2.5. Adhesion properties The adhesive of the TDDS is critical to the safety, efficacy and quality of the product. Different tests can be accomplished to assess the interaction skin/adhesive. These are based on the evaluation of several properties, such as initial and long-term adhesion, lift and residue. 2.2.5.1.Probe tack test The Texture Analyzer TA.XT Plus (Stable Micro Systems Ltd., Surrey, UK) was used to examine adhesive properties (adhesiveness, adhesion energy and separation distance of adhesives) of different transdermal patches. The test "Transdermal Adhesive Tape” was performed, using the analytical probe P/1S to ensure consistent contact with the adhesive attached to a double-sided tape37. The measurements involve both the adhesion and separation mechanisms between the probe and adhesive. The separation distance is related to the energy needed to remove the patch from a defined surface, elucidating about the elongation required to


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promote the detachment. The area under the curve represents the energy of adhesion and the higher the value, the greater the work required to separate the adhesive probe. The adhesiveness is the work required to overcome the forces of attraction between the adhesive surface and the probe. Six replicates were carried out. Data collection and calculation were performed using the Texture Exponent 3.0.5.0 software package of the instrument.

2.2.5.2. In vivo adhesion studies The in vivo evaluation of the bioadhesive properties of the transdermal patches, including peak adhesion force (PAF) and work of adhesion (WA), was performed using a TA.XTPlus Texture analyzer (Stable Micro Systems, Surrey, UK). The test was performed using an analytical probe P10 (10 mm Delrin) to ensure reproducible contact with the adhesive and the skin. The patch was fixed by means of a double sided adhesive tape on the movable carriage of the apparatus. The carriage was moved until contact between the skin of the subject forearm and the movable carriage was established. A preload of 3 N was applied and the contact time between the holder and the skin was 60 s. Afterwards, the movable carriage was moved backward at a constant speed test of 10 mm/s until complete separation of the two surfaces. The curves of displacement (mm) versus adhesive force (mN) were recorded simultaneously. The WA is given by the integral on the range of positive force. The force required to detach the attached film from the human forearm skin was used to represent the magnitude of bioadhesive force of the tested film specimen. Data collection and calculation were performed using the Texture Exponent 3.0.5.0 software package of the instrument.


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2.2.6. Scanning electron microscopy (SEM) The scanning electron microscopy (SEM) analysis was performed in order to investigate the topography of the particles in transdermal patch. It was also used to inspect morphological changes in skin after in vitro permeation studies. Skin samples were placed in a plastic mold and were submersed in Tissue-Tek O.C.T. compound. Transversal sections (100 µm thickness) were obtained using a cryostat (Carl-Zeiss, Mainz, Germany). The samples were thawed, rinsed with deionized water and fixed with 4% formaldehyde during 1.5 h. Subsequently, they were rinsed and placed in deionized water during 2 h at room temperature. Afterwards, samples were dehydrated using different solutions of ethanol in water (30%, 50%, 75% and 95%) for 25 min each. Finally, skin samples were placed in absolute ethanol for 2 h at room temperature. Prior to analysis, the samples were suitably spread on a double-side carbon tape mounted onto an aluminum stud, and coated with gold at a flow rate of 0.6 nm/s for 15 s, in order to make them conducting. SEM images were recorded on a JSM 6010LV/6010LA (Jeol, Tokyo, Japan) scanning electron microscope, at acceleration voltages of 20 kV.

2.2.7. Attenuated total reflectance infrared spectroscopy (ATR-FTIR) In order to understand the interactions between components of transdermal patch, encompassing adhesive, NLC or Combo-NLC and enhancers, ATR infrared spectra of the patches were recorded using a FT-IR/FT-NIR spectrometer (Spectrum 400, Perkin-Elmer, MA, USA) with an ATR accessory fitted with a Zn-Se crystal plate. Pure compounds and transdermal patches were placed in the ATR device and measured using 16 scans for each spectrum, with a resolution of 4 cm-1 and a scan speed of 1 cm/s. The spectra were collected between 4000 and 600 cm-1.


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2.2.8. In vitro release studies In vitro release studies were performed using static Franz diffusion cells (PermeGear, Inc., PA, USA) with a diffusion area of 0.636 cm2 and a receptor compartment of 5 mL. A dialysis cellulose membrane (MWCO~12,000, avg. flat width 33 mm, D9652, Sigma-Aldrich), as artificial membrane, was placed between both compartments and a receptor solution composed of phosphate buffered saline (PBS, pH 7.4) with 30 % (V/V) of ethanol was used to ensure sink conditions38, 39. Note that the percentage of ethanol used is in good agreement with the USP and WHO recommendations for in vitro tests for dermal absorption40, 41. This receptor phase was stirred at 600 rpm and maintained at 37±0.5 °C by a thermostatic water pump, which circulated water through each chamber jacket. By maintaining the receptor solution at 37 °C, a temperature of 32 °C at the surface was assured, thus mimicking human skin conditions. A piece of transdermal patch formulations was applied in the donor compartment. Subsequently, 300 µL of receptor medium were collected at 0, 1, 3, 6, 12, 24, 30, 36 and 48 h, and immediately replaced with the same volume of fresh solution. All drugs were determined using the HPLC method described below.

2.2.9. In vitro permeation studies: skin preparation and integrity tests In vitro permeation studies were performed in static Franz diffusion cells, in the same conditions of the in vitro release studies, but using newborn pig epidermis as skin model, clamped between the donor and receptor compartments, with the SC side facing up. The newborn pig (3 weeks) was provided by a local slaughterhouse. The subcutaneous fat was sectioned off, and the epidermis separated from the underlying dermis using the heat separation technique. The use of heat-separated epidermal membranes is more appropriate for permeants that are poorly water soluble. Briefly, the skin was immersed into hot water at 60 °C, for 1 min


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and, subsequently, the epidermal layer was gently separated from the dermis42. The epidermis sheets were cut into pieces, wrapped with aluminum foil and stored at -20 °C until used. Prior to the experiments, the frozen skin pieces were thawed, and hydrated by placing them in water overnight in a refrigerator (at about 4 °C). The barrier function of the skin was monitored by measuring the transepidermal water loss (TEWL). Only those skin samples that were found below 20 g/m2·h, the usual limit of TEWL values for such measurements, were used for the experiments43. According to Fick’s first law of diffusion, the steady state flux (µg/cm2/h), Jss, can be expressed by Jss=DC0P/h= C0Kp wherein D is the diffusion coefficient of the drug in the SC, C0 represents the drug concentration in the donor compartment, P is the partition coefficient between vehicle and the skin, h is the diffusional path length, and Kp stands for the permeability coefficient. The flux from formulations with enhancers and/or adhesives was measured and compared to the flux from formulations without enhancer/adhesive to obtain the enhancement ratio of the drugs accordingly. The enhancement ratio (ER) for flux was calculated as the ratio between the flux for treated skin with tested component(s) and flux for treated skin without tested component(s).

2.2.10. In vitro full skin permeation studies: drug extraction and quantitation in different skin layers In vitro permeation studies were performed as described in section 2.2.9., but using full newborn pig as skin model, clamped between the donor and receptor compartments, with the SC side facing up. Only the formulation with the optimal therapeutic potential, that is, highest permeation, was assessed. After the experiments, the donor phase was removed and the skin


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washed with purified water, in order to eliminate the donor formulation remaining at the surface44, 45. The skin was carefully dried with a cotton wool and the tape-stripping technique was used to separate the SC from the remaining layers, the epidermis and dermis (Ep+D). The SC and the remaining tissue containing the Ep+D (without SC) were put into individual glass tubes to continue the drug extraction. The tissues were left in contact with methanol, as extraction solvent. Then, the SC and Ep+D fractions were homogenized using an Ultra-Turrax for 3 min (Ystral GmbH D-7801, Dottingen, Germany), sonicated for 20 min, centrifuged for 5 min at 3000 rpm, filtered through a 0.45 µm pore membrane and assayed for drugs content by HPLC.

2.2.11. Experimental Design A two-level full factorial design, 2k, with two variables was used to estimate the influence of the enhancer and the adhesive in transdermal patches over the drug permeation and adhesion properties. This mathematical tool allows obtaining a high amount of information using a reduced number of experiments, when the study of the effect of several factors is deemed necessary. The following mathematical model was applied to describe the main effects and the respective interaction among the variables considered y= β0+ β1 x1+ β2 x2+ β12 x1 x2 where y is the response, β0 corresponds to the arithmetic mean of the response, β1 and β2 are the coefficients of the respective independent variables (main effects) and β12 is the interaction term. For each variable, a high (+1) and a low (-1) level were established (Table 3). The two independent variables were the presence of adhesive and enhancer on the transdermal patches. As responses or dependent variables, the amount of drug permeated after 24 h (Q24) and 48 h (Q48) or adhesion properties were considered. This yielded 4 different experiments. The


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experimental design was solved using the GNU Octave software, including Student’s t-test applied to the fitted models.

Table 3. Experimental design independent variables and respective codification. Components

Independent Variables

Level -1

Level +1

Adhesive

x1

Absence

presence

Enhancer

x2

Absence

presence

2.2.12. Labelled NLC based TDDS: Confocal laser scanning microscopy In order to investigate the distribution of drug/NLC over the skin, patches containing dyes with distinct lipophilicities (Table 4), that is, Nile-red loaded NLC and FITC loaded NLC (0.20 % w/w each), were formulated with ethanol, adhesive and enhancer (NR-NLC and FITCNLC patches) as indicated in section 2.2.1 and were applied for 1 h and 48 h using full newborn pig skin, following the same procedure described in section 2.2.9. For both patches, the effect of Dermaroller® pre-application was also assessed. After the pre-determined time points, the NR-NLC and FITC-NLC transdermal patches were removed and the skin surface was collected. Each piece of skin was embedded in Tissue-Tek O.C.T. compound and frozen at -20 °C. Transversal sections from the dermis to SC with a thickness of 50 µm were obtained using a cryostat (Carl-Zeiss, Mainz, Germany) and mounted in Dako fluorescent medium (Dakocytomation., Carpinteria, CA, U.S.A.) for preservation of the fluorescence signal until confocal laser scanning microscopy (CLSM) imaging analysis.


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Fluorescence images were acquired using a confocal microscope (LSM 510 Meta, Carl Zeiss). Digital images were obtained with an EM charge-coupled device camera (Rolera EMC2;QImaging) by using ZEN software (CarlZeiss). For each condition, a z-stack in three different fields was imaged in each piece of skin.

Table 4. Physicochemical properties of OL, SV, FITC and NR OL46

SV47

FITC48

NR49

312.433 g/mol

418.566 g/mol

389.47 g/mol

318.37 g/mol

Log P/Lipophilicity

2.8

4.7

4.8

3.8

Aqueous solubility

3-5 µg/mL

30 µg/mL

100 µg/mL

-

Tripalmitin

120 µM, respectively (Table 14). These results are in agreement with those previously reported27. OL is not cytotoxic on the range of concentrations tested. For this reason, the following assays were based on the concentration of SV in the systems. Secondly, the cytotoxicity of the optimal patch (A1E1) was evaluated (Fig. 13). The estimated IC50 was 87.27 µM (Table 14). This result suggest that lipid nanoparticles are able to reduce cytotoxicity associated to the drug in solution. The cytotoxicity of the different components of the patch was further assessed at three concentrations: 40 µM, 70 µM and 100 µM (Fig. 15). As expected, at


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40 µM, the concentration below to IC 50, cell viability was higher than 90 % (Fig. 15), irrespective of the formulation. In turn, at 70 µM and 100µM, different cytotoxic effects were observed depending on the components. It can be seen that the isolated effect of adhesive in terms of cytotoxicity was generally higher than the one induced by the enhancer (see B-D, Fig. 15). Moreover, the combination of both components led to a pronounced cytotoxic effect, particularly visible in Fig. 15A, for 100 µM. This probably corresponds to a higher exposition of the cells to the compounds, in the presence of the fluid, than that found under in vivo conditions. Again, cell viability was lower in formulations containing only SV, in comparison to the corresponding ones with OL (Fig. 15 B and C). In Combo-NLC based formulations, cytotoxicity is mainly attributed to SV, and to a lower extent to the individual components, that is, enhancer and adhesive (Fig. 15 D). Despite these aspects, it can be claimed that according to the in vitro cytotoxicity studies, concentration of the formulation should not, optimally, exceed 60 µM.

Figure 13. Cytotoxic effect of drugs in solution, OL and SV, incubated with HaCaT cells for 48 h. Data are expressed as mean ± SD (n = 12).


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Figure 14. Cytotoxic effect of A1E1 incubated with HaCaT cells for 48 h. Data are expressed as mean ± SD (n = 12).

Figure 15. Cytotoxic effect of different components of the optimal formulation (A1E1) incubated with HaCaT cells, for 48 h, at concentrations of 40 µM, 70 µM and 100 µM.


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A) Cytotoxicity of unloaded (blank) NLC patches. B) Cytotoxicity of OL-NLC patches. C) Cytotoxicity of SV-NLC patches. D) Cytotoxicity of Combo-NLC patches. The results were represented as mean ± SD. Key: B = blank NLC; OL = NLC containing only olanzapine; SV = NLC containing only simvastatin; Key: B = unloaded (blank) NLC; A = adhesive; E = enhancer; 0 = absence; 1 = PEG:PVP or PG, if it corresponds to the adhesive or enhancer, respectively. Data are expressed as mean ± SD (n = 18).


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Table 14. The IC50 values of SV and OL in solution and A1E1 transdermal patch after incubation with HaCaT cells, for 48 h. Formulation

IC 50 (µM)

Drug in solution (DMEM in OL 1% DMSO) >120 A1E1 transdermal patch

4.

SV 103.6

87.27

Conclusions In the present work, a new drug-in-NLC-in-adhesive transdermal patch was successfully

developed for the co-delivery of OL and SV, encompassing the assessment of different passive and active strategies to improve drug permeation across the skin. Development was mainly directed by permeation and adhesion studies, as critical quality attributes. From the screening of enhancers, propylene glycol led to the higher permeation rate. Results were supported by molecular dynamics simulations. From adhesive screening, PVPK30:PEG 400 was selected as the best adhesive composition, because it provided the best in vitro-in vivo adhesive properties. The impact of the enhancer and the adhesive on the performance of the NLC based transdermal patch was rationalized using a factorial design. The monitoring of labeled NLC based transdermal patches allowed to study the behaviour and to visualize the distribution of dyes with different lipophilicities throughout the skin, thus widening the application range of the compounds suitable to be incorporated in the system. Finally, the application of Dermaroller® did not result in a significant increase in the dye transport through the skin. This may indicate that the formulation on its own ensures the major driving force for drug permeation. Cytotoxicity studies revealed that lipid nanoparticles were able to reduce cytotoxicity associated to the SV in solution. In conclusion, this work allowed to demonstrate the versatility in the application of


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NLC for transdermal administration, in an innovative transdermal design, which could be a valuable basis for a future in vivo study. Acknowledgements We thank Prof. Luis Arnaut for providing the conditions to perform the cellular studies. We also thank the assistance of Helder Soares. The authors acknowledge the Fundação para a Ciência e a Tecnologia (FCT), Portuguese Agency for Scientific Research, for financial support through the project PEst-UID/NEU/04539/2013, COMPETE (Ref. POCI-01-0145-FEDER-007440) and the Research Project n.º 016648 (Ref. PTDC/CTM-NAN/2658/2014). The Coimbra Chemistry Centre is supported by FCT through the Project Nº 007630 UID/QUI/00313/2013, co-funded by COMPETE2020-UE.

SCCN

also

acknowledge

the

post-doctoral

research

grant

SFRH/BPD/71683/2010, assigned by FCT. Abbreviations A0, absence of adhesive; ATR-FTIR, Attenuated total reflectante infrared spectroscopy; B, unloaded (blank); C, Combo-NLC; CLSM, Confocal laser scanning microscopy; CPE, Chemical Penetration Enhancer; D, Derme; DERM, Dermacryl; DIA, Drug-in-adhesive; DL, Drug loading; DLS, Dynamic light scattering; DMEM, Dulbecco's Modified Eagle Medium; DPPC, Dipalmitoylphosphatidylcholine; DBP = dibutyl phthalate; E, enhancer; E1, presence of enhancer; E0, absence of enhancer; EE, Entrapment efficiency; Ep, Epidermis; ER, Enhancement ratio; ET, Ethanol; FITC, Fluorescein-5-isothiocyanate; Jss, Flux at steady-state; Kp, Permeability coefficient; HPH, High pressure homogenization; IPM, isopropyl myristate; L, Limonene; LN, Lipid nanoparticles; M, Menthol; MD, Molecular Dynamics; NLC, nanostructure lipid carriers; NR, Nile-Red; OL; Olanzapine; OA, Oleic acid; OTC, optimal cutting


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temperature; PAF, peak adhesion force; PEG, Polietilenoglicol 400; PI, Polydispersity índex; PSA, Pressure sensitive adhesive; PVP, Polyvinylpyrrolidone K 30; PG, Propylene Glycol; RDF, Radial distribution function; RSD, Relative standard deviation; S, squalene; SC, Stratum corneum; SEM, Scanning electron microscopy; SV, Simvastatin; SVA, Simvastatin acid; TDDS, Transdermal drug delivery system; TEER, Transepithelial/transendothelial electrical resistance; TEWL, Transepidermal water loss; TCol, Transdutol; TPA, Texture profile analysis; VE, Viable epidermis; WA, work adhesion; ZP, Zeta potential.

5.

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217x229mm (150 x 150 DPI)


Expanding Transdermal Delivery with Lipid Nanoparticles: A New

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