Loading of Coal Tar in Polymeric Nanoparticles as a Potential Therapeutic Modality for Psoriasis.

Coal tar (CT) is a commonly used therapeutic agent in psoriasis treatment. CT formulations currently in clinical use have limitations such as toxicity and skin staining properties, leading to patient nonadherence. The purpose of this study was to develop a nanoparticle (NP) formulation for CT based on biocompatible poly(lactide-co-glycolide) (PLGA). CT was entrapped in PLGA NPs by nanoprecipitation, and the resulting NPs were characterized using dynamic light scattering and high-performance liquid chromatography (HPLC) to determine the particle size and CT loading efficiency, respectively. In vitro biocompatibility of the NPs was examined in human dermal fibroblasts. Permeation, washability, and staining experiments were carried out using skin-mimetic Strat-M membranes in Franz diffusion cells. The optimal CT-loaded PLGA NPs achieved 92% loading efficiency and were 133 nm in size with a polydispersity index (PDI) of 0.10 and a zeta potential of -40 mV, promoting colloidal stability during storage. CT NPs significantly reduced the cytotoxicity of crude CT in human dermal fibroblasts, maintaining more than 75% cell viability at the highest concentration tested, whereas an equivalent concentration of CT was associated with 28% viability. Permeation studies showed that only a negligible amount of CT NPs could cross the Strat-M membrane after 24 h, with 97% of the applied dose found accumulated within the membrane. The superiority of CT NPs was further demonstrated by the notably diminished staining ability and enhanced washability compared to those of crude CT. Our findings present a promising CT nanoformulation that can overcome its limitations in the treatment of psoriasis and other skin disorders.

Introduction

Psoriasis vulgaris is a chronic inflammatory condition that involves the development of raised plaques that continually shed scales due to excessive proliferation of skin epithelial cells.[1,2] The disorder is characterized by hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia, and penetration of T-lymphocytes, neutrophils, and leukocytes.[3,4] Plaque psoriasis is the most widely recognized type of psoriasis vulgaris and traditionally manifests as discrete, erythematous plaques with an overlying silvery scale on extensor surfaces. Psoriasis can also include the scalp and nail or progress to erythroderma. Less-common clinical presentations include guttate, reverse, or pustular psoriasis.[5]

Psoriasis is a multifactorial relapsing and remitting inflammatory skin disease that affects approximately 2–4% of the population in western countries.[6,7] Many treatments help relieve psoriasis symptoms and are prescribed depending on the type and severity of the illness. The most popular treatments include steroids, moisturizers for dry skin, vitamin D, retinoids, and psoralen-UV-A (PUVA), which slow the growth of skin cells.[8] Methotrexate can work as an immunosuppressant, but it has serious side effects, so it is only used for severe cases.[9] Biologics such as adalimumab work by blocking the body’s immune system.[10] Phosphodiesterase 4 (PDE4) inhibitors such as apremilast which help in psoriatic arthritis can also be used[11] and so can be crude coal tar (CT) which is available as lotion, shampoo, bath solution, cream, and foam.[12]

CT is a byproduct of the process of making coke from coal.[13] Coal is heated in the absence of air at very high temperatures producing an amorphous coke mass, while the gases condense into a liquid, forming crude CT.[14,15] CT has close to 10,000 ingredients, of which only about 400 have been identified and were found to consist mainly of carbon, water, and polycyclic aromatic hydrocarbons (PAHs).[16] CT is used to treat psoriasis as it is recognized to exert antipruritic, keratolytic, anti-inflammatory, and antimitotic actions.[17] It can help in restoring the skin’s appearance by slowing down the rapid growth of skin cells. It can also help reduce psoriasis symptoms such as inflammation, itching, and scaling. Tar products vary in terms of CT concentration, where the higher the concentration, the more potent the product.[18] However, a high concentration of CT can cause irritation, redness, and skin dryness. CT stains the skin, clothing, bedsheets, and light-colored hair. CT can also cause photosensitivity. On top of that, PAHs in CT are known to be carcinogenic and cytotoxic.[16,19,20] For these reasons, CT is used less often in psoriasis treatment.[21]

Over the past decades, there has been a clear and growing interest in the application of nanotechnology in the biomedical and pharmaceutical fields. Many types of nanoparticles (NPs) have been reported such as those based on metals, lipids, polymers, and carbon dots, with proven benefits in the treatment and diagnosis of various diseases, for example, cancer and inflammation.[22−28,55] Poly(lactide-co-glycolide) (PLGA) is a copolymer commonly utilized for NP synthesis because of its biocompatibility and biodegradability. PLGA NPs can entrap and deliver a broad spectrum of drugs. PLGA is approved by the US Food and Drug Administration (FDA) for use in drug delivery systems due to its sustained release properties, low toxicity, and biocompatibility with tissues and cells.[29−32]

Given the known limitations of using CT for psoriasis treatment, it is highly desirable to develop a new formula for CT that can maintain its effectiveness while reducing its side effects. The main objective of this study was to create a polymeric NP formulation for CT based on PLGA. Upon optimizing the formulation parameters, a series of experiments were conducted to demonstrate the superiority of the NP formula compared to crude CT in terms of biocompatibility, skin accumulation, washability, and staining properties.

Results and Discussion

Preparation of CT-Loaded PLGA NPs

CT was loaded in PLGA NPs via nanoprecipitation (Figure ). This technique is usually used to load hydrophobic compounds in polymeric NPs. As the organic phase containing the polymer and the drug is gradually added to the aqueous phase, the hydrophobic drug becomes entangled within the polymer matrix as it precipitates out of solution forming the NPs.[33] Various formulations were attempted where the amount of PLGA was fixed at 50 mg and the amount of CT was varied between 5, 10, or 20 mg, resulting in F1, F2, and F3, respectively. F4 was the blank formulation consisting of only PLGA. The composition of the different formulations prepared in this study is summarized in Table . Upon NP formation, un-entrapped CT is expected to precipitate due to its extreme hydrophobicity. As illustrated in Figure S1 of the Supporting Information, formulations F1 and F2 produced milky colloidal dispersions with no visible precipitates, indicating that most of the loaded CT was successfully entrapped within the NPs. On the other hand, F3 formed yellow-colored aggregates attributed to un-entrapped CT (Figure S1), most likely due to the high amount of CT in the formulation. Thus, F3 was excluded from further investigation. Table summarizes the characteristics of the remaining formulations, namely the particle size, polydispersity, CT loading, and loading efficiency. CT loading efficiency was set as the criteria for choosing the optimal NP formulation for subsequent experiments.

Figure 1

Overview of CT NP preparation by nanoprecipitation.

Table 1

Composition of the NP Formulations Prepared in This Study

	organic phase
formulation	CT (mg)	PLGA (mg)
F1	5	50
F2	10	50
F3	20	50
F4		50

Table 2

Characteristics of CT NPs (Mean ± SD; n = 3)

formulation	particle size (nm)	PDI	zeta potential (mV)	CT loading (% w/w)	loading efficiency (%)
F1	133.1 ± 3.8	0.10 ± 0.01	–39.6 ± 4.2	9.2 ± 0.1	91.8 ± 1.4
F2	148.6 ± 6.0	0.13 ± 0.01	–37.5 ± 0.7	13.4 ± 1.1	67.0 ± 5.3
F3
F4 (empty)	121.0 ± 4.3	0.06 ± 0.02	–36.2 ± 1.9
F1 (1 month)	139.0 ± 3.0	0.14 ± 0.02	–38.0 ± 1.7	8.9 ± 0.2	89.3 ± 2.1
F2 (1 month)	142.3 ± 4.5	0.16 ± 0.03	–38.7 ± 0.6	12.9 ± 0.8	64.5 ± 4.1

As shown in Table , except for F3 which failed to produce NPs, CT could be successfully entrapped within PLGA NPs, most likely through hydrophobic interactions since CT is primarily composed of PAHs. All NPs had particle sizes of <150 nm. Interestingly, there was a gradual increase in particle size going from empty PLGA NPs (121 nm) to F1 (133 nm; Figure ) and F2 (149 nm) as the CT loading increased from 0 to 5 to 10 mg per batch. This is consistent with previous reports where the NP size increased upon drug loading.[34] The NPs also exhibited a narrow size distribution as indicated by the small PDI values ranging from 0.06 to 0.13. In general, PDI values between 0.0 and 0.1 reflect narrowly monodisperse samples, whereas values between 0.1 and 0.4 indicate moderately polydisperse samples.[35]

Figure 2

Representative particle size distribution of CT NPs (F1).

The surface charge of colloidal particles can impact their therapeutic efficacy by affecting their targeting ability and colloidal stability.[36] PLGA NPs typically exhibit a negative surface charge due to the ionization of carboxylic end groups of the polymer chains exposed on the surface.[37] As shown in Table , the zeta potential of F1, F2, and F4 ranged between −36 and −40 mV, which promotes particle stability because the repulsive forces prevent aggregation with aging.[38] This was strongly supported by the excellent colloidal stability exhibited by F1 and F2 after storage for 1 month at 4 °C, where no agglomeration or precipitation was observed. DLS measurements also showed insignificant changes in NP characteristics (particle size, PDI, and zeta potential) upon storage (Table ). As for CT loading, increasing the amount of CT in the formula from 5 to 10 mg led to a decrease in loading efficiency from 92 to 67%, indicating the limited capacity for PLGA to accommodate CT and that F1 composed of 5 mg of CT and 50 mg of PLGA was the optimal formulation.

High-Performance Liquid Chromatography Method Development and Validation

A high-performance liquid chromatography (HPLC) method was developed and validated to quantify the amount of CT loaded in the NPs and the amounts released during in vitro permeation studies. The UV spectrum of CT dissolved in acetonitrile (ACN) was scanned to determine λmax, which was found to be 270 nm. Upon HPLC method development, CT could be detected at 270 nm using ACN as a mobile phase delivered isocratically at 0.5 mL/min for a total run time of 12 min. The CT peak appeared at 6.44 min (Supporting Information, Figure S2). Method validation examined the linearity, accuracy and precision, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, stability, and robustness. The coefficient of determination (R2) values for six CT calibration curves were in the range of 0.9974–0.9993, indicating that the method used was linear (Figure S3 and Table S1). The intraday accuracy ranged from 85.64 to 99.90%, and the intraday precision ranged from 0.47 to 2.49% (Table S2). The interday accuracy ranged from 88.96 to 103.50%, and the interday precision ranged from 2.72 to 4.73% (Table S3). The LOD and LOQ for CT were found to be 0.4 and 1.2 μg/mL, respectively (Table S4), which were much lower than the lowest concentrations used throughout this study.

As for method selectivity, the maximum concentration of each of the additives used during NP preparation was analyzed and tested for any interference with CT analysis. The chromatograms (Figure S4–S8) showed that there were no interfering peaks from these materials at the retention time of CT. Accordingly, the method was determined to be selective for the analyte. Likewise, results from benchtop stability of CT samples indicated that the chosen samples at the tested concentrations were stable for 48 h at room temperature (RT; Table S5). In terms of method robustness (Table S6), the capacity factor (K′) values were above 1 under the nominal condition and after slight variations were made, indicating good retention of the analyte. The number of theoretical plates (N) was more than 2000 for the nominal conditions and throughout all the variations, reflecting the excellent performance of the column. Asymmetry (As) values were found to be less than 1.2 under the nominal conditions and after variations were made. Overall, the results showed that the method was rigid and robust to small variations.

Biocompatibility of CT NPs

In this study, cell viability assays were carried out on human dermal fibroblasts as a representative normal cell line derived from skin cells. The purpose of the experiment was to evaluate the biocompatibility and potential toxicity of CT NPs compared to those of crude CT. For the experiments, cells were treated with increasing concentrations of CT NPs, crude CT, and empty PLGA NPs for 72 h. Note that the concentrations used for crude CT correspond to the concentrations of CT present in CT NPs. As shown in Figure , cells treated with low concentrations of CT NPs did not display any significant cytotoxicity, as the percentage of cell viability was similar to that of empty PLGA NPs at all the concentrations tested. Conversely, crude CT induced a significant reduction in cell viability, which confirms the cytotoxic effects of CT that are mainly attributed to the PAHs.[19,20,39] Notably, treatment with CT NPs at the maximum concentration (1000 μg/mL) maintained a cell viability greater than 75%, while cells treated with an equivalent concentration of CT exhibited only 28% viability. These results confirm that entrapping CT in a protective matrix of PLGA NPs can significantly enhance CT’s biocompatibility and reduce its cytotoxic effects.

Figure 3

Cell viability of human dermal fibroblasts treated with CT NPs (50–1000 μg/mL), equivalent concentrations of CT, and empty PLGA NPs for 72 h. Results represent means ± SD of four replicates per treatment. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test.

In Vitro Permeation Studies

Permeation studies were conducted in Franz diffusion cells (FDCs) to evaluate the release and potential transdermal permeability of CT NPs. FDCs are the most widely used experimental apparatuses to assess the skin permeation and release profile of topical and transdermal formulations.[40] Permeation studies were carried out using a cellulose membrane as a model membrane and the skin-mimetic Strat-M membrane to simulate the passage of CT NPs through human skin. Strat-M is a synthetic membrane made of repetitious polyester sulfone layers that exhibit a morphology comparable to that of human skin. The Strat-M membrane layers have diverse pores with diffusional resistance imparted by the presence of artificial lipids.[41] Recent studies have shown that Strat-M can properly mimic the skin barrier properties, so it is widely used as a transdermal diffusion model in pharmaceutical development.[42]

Figure shows the permeation profile of CT NPs through the cellulose membrane and the Strat-M membrane, expressed as % permeation, that is, the % cumulative amount of CT found in the receiver compartment relative to the initial amount added to the donor compartment, as a function of time. Although minute amounts of CT NPs permeated through the cellulose membrane within the first 3 h, % permeation increased to 37.4 and 63.7% after 8 and 24 h, respectively. On the other hand, permeation of CT NPs through the Strat-M membrane, which is more physiologically relevant than the cellulose membrane, was significantly lower. In fact, no CT was detected in the receiver compartment until 24 h, where only 2.9% permeation was achieved, indicating limited transdermal diffusion of CT NPs and that 97.1% of CT NPs accumulated inside the membrane (Table ). In addition, after 24 h of incubation, the amount of CT NPs in the donor chamber was completely absorbed by the Strat-M membrane as evidenced by the absence of the NP dispersion in the donor chamber.

Figure 4

Comparative permeation profiles of CT NPs through cellulose and Strat-M membranes upon incubation in FDCs up to 24 h (n = 3).

Table 3

Degree of CT Accumulation and Permeation through the Cellulose and Strat-M Membranes after 24 h of Incubation in FDCs (n = 3)

<!—Col Count:3-->membrane	% accumulation	% permeation
cellulose	36.5 ± 0.8	63.5 ± 0.8
Strat-M	97.1 ± 0.1	2.9 ± 0.1

Another indicator of CT permeation was the characteristic odor of CT. In the case of samples incubated with the cellulose membrane, the smoky odor of CT was evident in the withdrawn samples starting from the first hour of sampling. Meanwhile, the smoky odor was only evident after 24 h in the samples incubated with the Strat-M membrane. The particle size of CT NPs (133 nm) likely contributed to their Strat-M membrane accumulation and limited permeation to the receiver solution. This feature renders CT NPs promising therapeutic agents in treating psoriatic plaques, as it is important for the NPs to accumulate within the skin layers rather than permeate transdermally, to maximize CT localization within the skin while limiting systemic exposure to CT.

Washability Study

The skin and cloth staining characteristics of CT can negatively impact patient acceptability, compliance, and satisfaction with CT as a treatment choice. Skin staining was listed as a significant unwanted effect by patients. CT is also associated with irreversible staining of clothes, even if it is only used once, which can lead to termination of therapy.[43] One of the objectives of this study was to develop a CT formulation that can diminish or limit the ability of CT to cause skin discoloration and staining of clothes during therapy. For this reason, Strat-M membranes were incubated with either crude CT or CT NPs in the same setup used for transdermal permeation studies for up to 24 h at 37 °C. After 24 h, visual inspection of the membranes revealed that those incubated with crude CT were markedly stained (Figure ). By contrast, membranes incubated with CT NPs showed very limited staining. Following repetitive washing with water and detergent, crude CT left behind dark-brown stains over the applied areas, whereas CT NPs left no residue (Figure ). These observations strongly indicate that the CT NP formulation exhibits a nonsticky character, which is advantageous in terms of better spreadability and ease of application than the conventional CT preparation. Trapping CT within the polymer matrix prevents it from directly interacting with the patient’s clothes or skin and causing discoloration. At the same time, the NP formulation will control the release of CT inside the skin layers, which can significantly enhance its pharmacodynamic action, further demonstrating the advantages of NP-based systems in reducing drug side effects and enabling a more effective interaction with the affected target site.[44]

Figure 5

Representative images of Strat-M membranes incubated with CT NPs and crude CT for 24 h at 37 °C before and after washing with water and detergent.

Conclusions

CT is an extremely hydrophobic material with significant staining properties to the skin and clothes. It is also known to be cytotoxic with a tendency to produce cancer, particularly when the skin on which CT is applied is exposed to the sun. These reasons have limited its clinical utilization as a drug of choice in the treatment of psoriasis and for other skin disorders. In this study, CT was successfully loaded for the first time in biodegradable and biocompatible polymeric NPs. A rapid and robust HPLC method for CT analysis was developed, validated, and successfully used. CT-loaded PLGA NPs were 133 nm in size and highly monodisperse, achieving more than 90% loading efficiency. The NPs also exhibited a negative surface charge that contributed to their colloidal stability during storage. CT NPs profoundly reduced CT’s cytotoxicity toward human dermal fibroblasts even at high concentrations, demonstrating their excellent biocompatibility. Permeation studies revealed that CT NPs achieved extensive accumulation in the skin-mimetic Strat-M membrane and limited transdermal permeation. This attribute is highly desired in the treatment of localized skin disorders such as psoriasis, where the drug is needed to accumulate in the formed scaly tissues to maximize its effect. The washability and nonstaining properties of CT NPs further support the superiority of this novel nanoscale formulation versus conventional CT as a therapeutic modality for psoriasis. Since the NPs are in the form of an aqueous colloidal dispersion, they may be incorporated in a semisolid vehicle such as a cream or gel for convenient administration and to enhance their skin residence time.

Experimental Section

Materials

CT (batch no. 19023-08, CAS 8036-83) was purchased from Al-Asmah Drug Store (Amman, Jordan). PLGA (MW 10 kDa, 50:50 lactide/glycolide), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and penicillin–streptomycin were supplied by Sigma-Aldrich (St. Louis, MO, USA). Acetone and Tween 20 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Acetonitrile (ACN) (HPLC gradient grade) and isopropanol were supplied by BDH Chemicals (Poole, UK). High-glucose Dulbecco’s modified Eagle medium (DMEM) with ultra-glutamine was supplied from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was obtained from Gibco (Waltham, MA, USA). Amphotericin B was purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). Dimethyl sulfoxide (DMSO) was supplied by Fisher Chemical (Waltham, MA, USA). Hydrochloric acid (HCl) was obtained from Thermo Fisher Scientific (Loughborough, UK). Phosphate-buffered saline (PBS) was purchased from Gibco (Invitrogen, Cergy-Pontoise, France). l-glutamine was obtained from Merck (Darmstadt, Germany). Sodium chloride (NaCl) was supplied by MS Pharma (Sahab, Jordan). Trypsin was obtained from Biochrom AG (Berlin, Germany). Water used in all experiments was distilled. All chemicals were used as supplied without further modification.

Preparation of CT-Loaded NPs

CT-loaded PLGA NPs were prepared using the nanoprecipitation technique as previously described with some modifications.[45−49] Briefly, PLGA and CT at different PLGA: CT ratios (Table ) were weighed and dissolved in 5 mL of acetone. The organic phase was injected using a syringe pump (Kent Scientific Genie Plus, USA) operating at a flow rate of 0.7 mL per minute into 10 mL of distilled water under gentle stirring at RT. The formed NPs were stirred overnight to ensure the total removal of acetone. The concentration of the NP suspension was determined by freeze-drying 1 mL of the suspended NPs and weighing the resulting mass.[49] The NP formulations were kept at 4 °C until further characterization. No surfactants were added at any step of NP preparation. Empty NPs were prepared using the same procedure without adding CT to the organic phase.

Characterization of CT-Loaded NPs

The particle size, polydispersity, and zeta potential of the NPs were measured by dynamic light scattering (DLS) using a Malvern instrument (Zetasizer Nano, UK). All measurements were done in triplicate. Results were reported from at least three NP batches and were expressed as mean ± standard deviation (SD).

HPLC Analysis

HPLC was employed to quantify the amount of CT loaded in the NPs and the amount released during in vitro permeation studies. ACN was selected as the mobile phase because it is one of the few organic solvents that can dissolve both PLGA and CT. First, the UV spectrum of CT dissolved in ACN was scanned using a UV/Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to determine the wavelength of maximum absorption (λmax) for CT. HPLC analysis was performed using a Finnigan Surveyor LC Pump Plus system (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a photodiode array UV detector. The mobile phase consisted of HPLC-grade ACN and was subjected to filtration using a 0.45 μm membrane filter followed by degassing. Elution was performed at 0.5 mL/min and 25 °C on a Hypersil phenyl-BDS HPLC column (250 × 4.6 mm, 5 μm; Thermo Fisher Scientific, Loughborough, UK), and the detection wavelength was set to 270 nm. The elution time was set to 12 min. Data analysis was performed using Chromrequest System Version 8.1. CT calibration curves were constructed by running serial dilutions of CT in the mobile phase. Linearity for CT was tested over the concentration range of 10–1250 μg/mL, and 10 μg/mL was set as the lower limit of the calibration curve. Blank (ACN), PLGA, PLGA NP, PLGA-CT physical mixture, and CT NP samples were prepared to confirm the absence of interferences and the method selectivity. The analytical method was validated according to the FDA’s “Guidance for Industry, Bioanalytical Method Validation”[50] and International Conference on Harmonization (ICH) guidelines[51] using quality control samples with known concentrations. Full details of the method validation are provided in the Supporting Information.

Determination of the CT Loading and Loading Efficiency

The amount of CT loaded in the NPs was determined by a validated HPLC method as described above. To calculate the amount of CT loaded in the NPs, a known weight of freeze-dried CT NPs was dissolved in 1 mL of ACN. After injecting the dissolved NPs in the HPLC column, CT was detected at 270 nm and the peak area was compared against the calibration curve. Results were reported from at least three NP batches. The CT loading and loading efficiency were calculated according to eqs and 2, respectively

Biocompatibility of CT-Loaded NPs

Cell Culture Conditions

Biocompatibility of CT NPs was evaluated in human dermal fibroblasts as a model normal cell line. Cells were kindly provided by the Biotechnology Laboratory at the University of Petra and sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown and cultured in DMEM supplemented with 10% FBS and 1% l-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2.5 μg/mL amphotericin B at 37 °C in a humidified atmosphere containing 5% CO2.

Cell Viability Assays

Cells were trypsinized at 75–85% confluence, centrifuged, resuspended in fresh media, and counted. Cell suspensions were then seeded into 96-well plates at a density of 2–3 × 104 cells per well and incubated for 24 h. Then, cells were incubated with freshly prepared aqueous dispersions of CT NPs (equivalent to 50–1000 μg/mL NPs) and crude CT (at a concentration equivalent to that found in CT NPs). Crude CT was first prepared as a stock solution in DMSO and then diluted appropriately in complete cell culture medium. Cells were also treated with empty PLGA NPs at concentrations equal to those of CT NPs. Cells incubated with media only served as the negative control. The microplates were incubated for 72 h at 37 °C and 5% CO2. After 72 h, the media were removed and replaced with fresh media containing 0.5 mg/mL MTT cell proliferation reagent, and the cells were incubated for another 4 h. After that, the media were withdrawn and replaced with isopropanol to solubilize formazan crystals. The microplates were gently shaken for 2 min before reading the absorbance at 560 nm using a microplate reader (GLOMAX Multidetection System, Promega, Madison, WI, USA). The results were reported as % cell viability, which was calculated according to eq

Transdermal permeation of CT NPs was evaluated in vertical Franz diffusion cells (FDC; SES GmbH, Bechenheim, Germany). The receiver chambers were each filled with 11 mL of PBS (pH 7.4), and the FDCs were equilibrated for 24 h at 37 °C. The air bubbles in the receiver compartments were removed by manual tipping of the cells. In vitro permeation was first performed using regenerated cellulose membranes (molecular weight cutoff of 12–14 kDa, Bel-Art Products, Pequannock, NJ, USA) to ensure the method’s validity. The experiment was then repeated using Strat-M membranes (25 mm, Merck Millipore, Billerica, MA, USA), which are an acceptable in vitro model to simulate and predict diffusion in human skin.[41,52] The same conditions were applied in terms of temperature, media, and mixing speed for both membranes. The membranes were sandwiched between the donor and receiver compartments, and CT NPs (0.5 mL) were placed in the donor chambers in triplicate and wrapped with parafilm. Samples were periodically withdrawn from the receiver solutions up to 24 h and replaced immediately with an identical volume of fresh medium. The extent of CT permeation was analyzed by HPLC. Results were expressed as % cumulative permeation versus time. The percentage of CT NPs accumulated in the membranes after 24 h (% accumulation) was calculated according to eq (53)where A0 is the initial amount of CT NPs and A is the cumulative permeated amount in the receiver solutions after 24 h.

Washability and Staining Property Evaluation

Strat-M membranes were employed to test the staining and washability of CT NPs and crude CT following a previously published procedure with some modifications.[54] Membranes (n = 3 per group) were incubated with CT NPs (0.5 mL of freshly prepared NPs) and crude CT (an amount equivalent to CT NPs) in the same FDC setup described above for 24 h at 37 °C. After 24 h, the membranes were photographed, washed with running water for 1 min at RT, and then soaked in a detergent solution for 2 min, followed by washing using running water again for 1 min. The membranes were finally dried and photographed.

Statistical Analysis

All numerical results were presented as mean ± SD from at least three independent experiments. Statistical analysis was performed in GraphPad Prism 7 using one- or two-way analysis of variance (ANOVA) followed by Tukey or Sidak’s post-hoc tests, respectively, where p < 0.05 was considered statistically significant.