Psoriasis is a chronic immune-mediated disease that affects 2% of the population, comprising different clinical manifestations, which are mainly characterized by skin disorders such as erythematous papules or scaly teardrop-like lesions associated with the characteristic pain, stinging, and even bleeding of an inflammatory process. The most affected areas are usually the elbows, knees, scalp, and lower back [1
The immune system plays a crucial role in the pathogenesis of the disease, and particularly, the deregulation of the crosstalk between the innate and adaptive immune system within the interleukin-23 (IL23)/T helper cell 17 (Th17) axis [1
]. Thus, the therapeutic treatments focus on targeting the Th17 response, either as local or systemic non-specific immunosuppressors (conventional therapies) such as dermocorticoids and methotrexate. The side effects produced by these treatments have boosted the use of antibody-based systemic treatments that recognize, bind, and block the activity of those cytokines involved in the Th17 response (biological therapies), such as IL17A (Ixekizumab, Taltz™; Secukinumab, Cosentyx™), IL23 (Guselkumab, Tremfya™), IL23 and IL12 (Ustekinumab, Stelara™), and TNF-α (Adalimumab, Humira™; Etanercept, Enbrel™; Infliximab, Remicade™) [1
]. However, despite the promising results offered by this type of treatment, they also have limitations such as their high cost, some adverse effects, a loss of efficacy due to the development of anti-drug antibodies, and the inability to avoid occasional symptomatic outbreaks or to act on some poorly accessible locations [1
]. Therefore, there is still a need to use therapeutic agents to alleviate the discomfort derived from the symptoms caused by the psoriatic lesions [1
The main palliative agents for the dermal symptomatology of the disease are keratolytics, to reduce the generation of scales, and analgesics, to relieve the painful and itching sensation. Among the keratolytics, salicylic acid stands out, which acts by lowering the pH of the stratum corneum and thus preventing the adhesion of keratinocytes [11
]. In turn, it also improves the absorption of other topical treatments by reducing the stiffness of the stratum, and also reduces the pruritus [12
In terms of analgesic therapy, one of the most common targets in topical applications is the transient receptor potential cation channel 1 (TRPV1), commonly called the capsaicin receptor. This receptor is mainly found in the nociceptive neurons of the peripheral nervous system, although they have also been described in other non-neuronal tissues, including skin cells. TRPV1 is a non-selective tetrameric cation channel with high permeability to calcium (Ca2+
) that has been involved in the transmission and modulation of pain when triggered by different physical and chemical stimuli such as temperatures above 43 °C, acidic pH, capsaicin, and some endogenous mediators of inflammation [13
]. Capsaicin, an alkaloid present in several species in the genus Capsicum
, and some of its derivatives act as analgesic agents by producing TRPV1 desensitization [13
]. Additionally, this family of compounds hold other biological properties such as cardiovascular stimulator, antioxidant, and anticancer, as well as anti-inflammatory, by for instance suppressing the inducement of TNF-α, which is also a psoriasis mediator [17
]. Methyl salicylate, which is derived from salicylic acid, is another analgesic agent. This compound belongs to the family of non-steroidal anti-inflammatory drugs (NSAIDs), and has also been reported to activate TRPV1 [21
]. In addition, it is easily absorbed through the skin, and is already used for the treatment of inflammation and pain [22
However, for the maximization of the expected benefits of their combined delivery, it would be necessary to have a suitable system for their topical application, and desirably, an encapsulation procedure that allows combining all three compounds together without losing their activity, in addition to being simple and scalable to industrial production. In this sense, recent advances in nanotechnology applied to the field of medicine offer an alternative that meets all these requirements, i.e., polymer nanofibers produced by the electrospinning technique [23
]. In addition, the correct choice of the integrating materials would allow the development of nanostructures with appropriate release properties according the particular dosage needs of their load, which in the specific case of capsaicin should be sustained in order to keep TRPV1 desensitized for a prolonged period.
In this work, based on the progress made in our previous study [26
], the production process of electrospun nanofibers loaded with all three compounds described above is optimized using poly(methyl vinyl ether-alt
-maleic ethyl monoester) (PMVEMA-ES) as the polymeric material. Such a biodegradable, biocompatible, bioadhesive, and low-toxic polymer is already commercialized, and used in other biomedical applications, and has already shown its suitability for the creation of these nanostructured systems with the appropriate release characteristics [26
]. Additionally, this polymer not only allows the possibility of being used as the main building polymer for the creation of nanofibers, but also allows combining with other materials, such as fluorene-based copolymers [31
], in order to combine functional properties within the same nanostructure [26
]. The morphological characterization of these nanofibers, together with their encapsulation capacity, stability over time, and activity of the encapsulated compounds are also analyzed in this study.
2. Materials and Methods
2.1. Polymers, Solvents, Drugs, and Common Materials
The copolymer of poly(methyl vinyl ether-alt-maleic acid) monoethyl ester (PMVEMA-ES) (Mw: ~130 kg/mol) was supplied in a 50% w/w solution in ethanol (Sigma-Aldrich, Saint Louis, MO, USA).
The solvents used were dichloromethane, acetone, ethanol (Merck KGaA, Darmstadt, Germany), methanol (VWR International, Radnor, PA, USA), sulfuric acid, and anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich).
The reagents used were salicylic acid, capsaicin, ruthenium red (RR) (Sigma-Aldrich), magnesium sulfate anhydrous (Honeywell Fluka, Morris Plains, NJ, USA), sodium hydroxide (Panreac AppliChem—ITW Reagents, Cinisello Balsamo, Milan, Italy), and ferric chloride (Sigma-Aldrich). Although it is considered in the text as a unique therapeutic agent, according to the supplier’s analysis certificate, the purity of the commercial capsaicin used in this work was 61.1%, and it contained 31.2% of dihydrocapsaicin, which is a derivative with very similar chemical and biological properties to capsaicin.
Methyl salicylate was synthesized from the esterification of salicylic acid in a solution of methanol and sulfuric acid by following the method proposed by Carrillo-Arcos et al.
]. Briefly, the 2-hydroxybenzoic acid (28.96 mmol) was dissolved in 60 mL of methanol, and then sulfuric acid (98%, 2 mL) was added dropwise to the solution, and the mixture was stirred at reflux for 18 h. Next, the solvent of the reaction was removed by vacuum rotary-evaporation, and the crude product was neutralized to pH 5–6 with a solution of NaOH (1 M, 50 mL), which was extracted three times with dichloromethane (3 × 50 mL). The combined organic phases were dried (magnesium sulfate) and concentrated (rotavap). The crude oil was distilled in a Kugelrohr B-585 glass furnace (Büchi, Flawil, Switzerland) to finally obtain a colorless oil (yield of 75%).
The preparation of nanofibers was performed by means of the methodology described before by Mira et al.
]. After optimization, an electrospinnable solution containing 25% w
of PMVE/MA-ES in ethanol was selected. As for the active agents, a combination of 1.5% salicylic acid, 1% capsaicin (2:1 together with dihydrocapsaicin), and 1% methyl salicylate, all w
with respect to the polymer weight, was used.
The electrospinning process was performed on a device that included a two-mL Discardit II syringe (Becton Dickinson, Franklin Lakes, NJ, USA) through which the polymer solution was introduced, and from which it was pumped through a blunt-end stainless steel hypodermic needle 316 of 20 Gauge (101.6 mm in length, external diameter 0.902 mm, and internal diameter 0.584 mm) (Sigma-Aldrich) at a sustained flow controlled by a KDS 100 infusion pump (KD Scientific, Holliston, MA, USA). The needle and the aluminum foil collector, faced vertically, are connected to a Series FC high voltage source (Glassman High Voltage Inc., Whitehouse Station, NJ, USA), which provides the voltage responsible for the generation of the jet to be deposited on the collector located at a settled distance. Any material that is intended to be covered with a mat of electrospun nanofibers, as for example in this work, the adhesive dressings, would be placed on the aluminum collector. After the optimization of the operational parameters (see corresponding section for further details), values were selected for the elaboration of the batches of nanofibers for the following experiments.
For the observation in optical microscopy, the nanofibers were electropun on a microscope slide (Deltalab, Barcelona, Spain) arranged on top of the aluminum collector. The inverted fluorescence optical microscope that was used was a Microsystems DMI3000B (Leica, Bensheim, Germany) equipped with a Leica EL6000 compact light source and a Leica DFC 3000G digital camera. The images were taken with a 63× objective in phase contrast, and the image processing was done manually using the program Leica Application Suite AF 6000 Module Systems. By this methodology, an initial screening, and then a more exhaustive screening, were carried out.
After the optimization of the electrospinning parameters, selected nanofiber samples were also analyzed by scanning electron microscopy (SEM), without metal coating, in a JSM-6360 LV device (Jeol, Tokyo, Japan). In both cases, from the images obtained for each sample, a total of 100 measurements of nanofiber diameters were taken and analyzed by using the Image J software (National Institutes of Health, NIH, Bethesda, MD, USA).
2.4. Gas Chromatography and Mass Spectrometry (GC-MS)
The amount of the therapeutic agents contained in the nanofibers, which are necessary for analyzing the encapsulation efficiency and the stability of the encapsulation over time, was determined by means of a gas chromatograph coupled to a mass spectrometer, in particular, a GCMS-QP2010 SE equipment with quadrupole detector supplemented with a thermal TD-20 adsorption attachment and an AOC-20i/s automatic sample injector (Shimadzu, Kyoto, Japan). The employed GC column was an Agilent J&W capillary HP-5MS UI (5% diphenyl–95% dimethylpolysiloxane, 30 m × 0.25 mm id, film thickness 0.25 μm) (Agilent Technologies Inc., Santa Clara, CA, USA).
The procedure of this analytical methodology is based on a previous study [36
]. Briefly, the temperatures of the injector and detector were 250 °C and 210 °C, respectively. Helium was used as a carrier gas at a flow rate of 1.5 mL/min. The chosen program was 40 °C for two minutes; then, the temperature was raised at a speed of 10 °C/min to 240 °C and, thereafter, at a rate of 5 °C/min up to 270 °C, which was finally maintained for five minutes.
In each experiment, a calibration curve was created for each of the analyzed compounds whose concentration ranges were between 0.018–1.420 mg/mL for capsaicin, 0.009–0.720 mg/mL for dihydrocapsaicin, 0.04–3.22 mg/mL for methyl salicylate, and 0.161–3.22 mg/mL for salicylic acid. All of the values that were provided for each curve corresponded to the area under the curve that was obtained for each concentration of the corresponding compound. In this way, the curves were preferably adjusted to a quadratic equation correlating area with concentration R2
coefficients greater than 0.99 in all of the cases (see Figure S1 in supplementary information
The analyzed samples corresponded to two independent productions of nanofibers loaded with the three therapeutic agents as described above. Each sample of nanofibers was analyzed when just prepared and at 5 and 15 days post-production. These samples were stored in a drawer, uncovered, protected from light, and at room temperature. Immediately prior to their analysis, the samples were dissolved in dichloromethane, filtered with 0.2-μm nylon membranes (Millipore, Bedford, OH, USA) and injected at a concentration of 10 mg/mL w/v. As controls, nanofibers without encapsulated compounds and fresh polymer solutions (made just prior to the electrospinning process) were used.
2.5. Cell Culture
For in vitro cell assays, human embryonic kidney cells stably expressing TRPV1 (HEK293-VR1) were used. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) (Sigma-Aldrich) and 50 μg/mL gentamicin (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in 25-cm2 flasks at 37 °C in a humidified atmosphere with 5% CO2, and for their harvest they were detached with 0.25% trypsin-EDTA solution. For the assays, cells were seeded in opaque 96-well plates with transparent bottoms (Corning Incorporated, Corning, NY, USA) at a cell density of 40,000 cells three days before treatment.
2.6. TRPV1 Channel Activity Assays
Since the TRPV1 channel allows the non-selective passage of cations such as Ca2+
when activated, a Ca2+
fluorescence probe has been used to indirectly quantify such activation, which might be induced by the experimental treatments. Thus, Ca2+
fluorography assays were performed by using the probe Fluo-4 NW (Molecular Probes-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2.5 mM of probenecid, which improves the permeation of the probe in the cells and inhibits losses of fluorescence by avoiding its exit to the cellular exterior, as was recently reported by Serafini et al.
Briefly, in cell plates prepared as described in the previous section, the culture medium was removed, and 100 μL/well of probe solution was added and incubated for 30 min at 37 °C followed by 30 min at 30 °C, always in the dark. Then, the fluorescence measurements were taken (excitation at 485 nm and emission at 535 nm) with a POLARstar Omega plate reader (BMG LABTECH GmbH, Offenburg, Germany) for 20 cycles (each one corresponding to a period of 156 s) and at a constant internal temperature of 30 °C. After the third cycle, one μL/well of each formulation that had been previously dissolved in DMSO at the corresponding concentration was added manually in order to reach the expected treatment doses in the well. In this sense, nanofibers were dissolved at 50 mg/mL in order to treat cells with 54 μM, 33 μM, and 10 μM of salicylic acid, methyl salicylate, and capsaicin, respectively. A solvent control (DMSO), capsaicin as agonist compound at 10 μM [38
], and RR as a non-competitive inhibitor [39
] at 10 μM were also included. Then, the measurements continued until cycle 20. After cycle 10, one μL of 10 μM capsaicin in DMSO was added to some wells that had been previously treated with RR in order to check the stability and selectivity of the system. Calibration curves for capsaicin and methyl salicylate were also included (range 0.1–30 μM and 15–120 μM, respectively). Each sample was analyzed in triplicate in each assay.
2.7. Preliminary In Vivo Test of Topical Application
An initial in vivo study was carried out with three healthy human volunteers to evaluate, when administered topically, the stability time of the experimental nanofibers, as well as to determine the functionality of the encapsulated TRPV1 agonists by analyzing the responsiveness to them. Each volunteer was given an adhesive dressing with electrospun nanofibers on each arm on the skin surface of the deltoid. This test was blind, since for each volunteer, one of the dressings contained empty nanofibers, and the other contained drug-loaded ones, but they were assigned randomly to each of their arms. Each dressing contained approximately 25 mg of nanofibers distributed over a surface area of 20 cm2. The duration of the treatment was eight hours. The surface of the dressing was kept protected from light throughout the full period. Every two hours, photographs were taken of each dressing to determine the state of the nanofibers and treated skin. At the end of the trial, each volunteer was asked which dressing they thought contained the TRPV1 agonists. This study was approved by the Project Evaluation Board of Miguel Hernández University (approval no. 2018.12.05.FPRL).
2.8. Data Analysis and Graphics
Data were analyzed by either GraphPad Prism v6 and Microsoft Excel software. Both of them were used for creating the graphs. Statistical analysis was performed with GraphPad Prism v6 specifically. Applied statistical methods are stated together with the analyzed data in the Results section.