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Original Article

Influence of nanoparticle size on the skin penetration, skin retention and anti-inflammatory activity of non-steroidal anti-inflammatory drugs

Yokota, Junkoa,b,*; Kyotani, Shojiroa

Author Information
Journal of the Chinese Medical Association: June 2018 - Volume 81 - Issue 6 - p 511-519
doi: 10.1016/j.jcma.2018.01.008

    Abstract

    1. Introduction

    Nanotechnology has been described as a field aimed at creating materials with new functions and beneficial characteristics through the control of material structure/sequence at the nanometer level.1,2 Following advances in nanotechnology, it is now becoming possible to create drug delivery systems (DDSs) using nanoparticles or microparticles as drug carriers. Percutaneous drug delivery is often used as a route of administration for low-molecular-weight drugs. For example, a percutaneous route has been adopted for the administration of non-steroidal anti-inflammatory drugs (NSAIDs) to avoid the limitations of oral administration, such as gastrointestinal symptoms and renal impairment.3 The skin is the largest organ in the human body. If certain chemicals are applied to the skin, the active ingredient can penetrate through the skin barrier and become distributed throughout the whole body via the intradermal capillaries. However, the skin also possesses a barrier mechanism to prevent the invasion of various materials into the body.4,5 For this reason, the penetration of materials across the skin is generally quite low, compared with penetration across other tissues. The skin is composed of the epidermis (including the horny layer), the dermis, and subcutaneous tissue. Accessory structures in the skin, such as sweat glands, are also present, and span the epidermis and the dermis. Of these skin components, the horny layer has the highest barrier function. Therefore, the effective passage of drugs through the horny layer is an issue of particular focus in percutaneous drug delivery.6–10

    NSAIDs are widely used in the clinic because of their excellent anti-inflammatory and analgesic action.11 However, oral-dose NSAIDs are associated with adverse systemic reactions, such as gastrointestinal symptoms and renal impairment. For this reason, percutaneously absorbable forms of NSAIDs are often used when dealing with local inflammatory diseases, making locally and percutaneously absorbable drug formulations important research targets. To improve skin penetration, various penetration-promoting agents12,13 and particle sizes14 have been studied, and different formulations such as liposomes,15 gels,16–19 ointments and creams20,21 have been investigated and utilized. The application of nanoparticles to the skin reportedly elevates drug penetration across the skin barrier. Nanoparticles are therefore expected to provide a means of improving drug penetration through the skin, and local DDSs using nanoparticles have the potential for extensive clinical use. However, differences in particle size can affect nanoparticle skin uptake, retention time during circulation, and the potential for adherence and degradation. Keeping these points in mind, we explored the potential of nanoparticles and microparticles as percutaneous drug carriers by preparing fine particles containing NSAIDs using a planetary centrifugal mixer and evaluating their skin penetration and safety.22 In addition, the anti-inflammatory activity of the NSAID particles was evaluated in animal models of acute and chronic inflammation.

    2. Methods

    2.1. Materials and reagents

    Indomethacin (IMC), ketoprofen (KET), piroxicam (PXC) and diclofenac sodium were purchased from Nacalai Tesque, Inc (Kyoto, Japan). Other reagents used were commercial grade.

    2.2. Preparation of fine NSAIDs particles

    NSAIDs were incorporated into fine particles by processing the drug, zirconia beads (2.5 g), and an aqueous polymer (hydroxypropyl cellulose SSL [HPC]) solution in a planetary centrifugal mixer (NP-100; THINKY Corporation; fitted with a −20 °C freezer) at a rotation rate of 1700 rpm, a mixing time of 10 min, and a medium (zirconia ball) quantity (2.5 g). The particle size of the drug in the suspension was measured using a laser diffraction particle size distribution meter. The active ingredients (ai) used were IMC (IMCai), KET (KETai), and PXC (PXCai), and the fine-particle formulations of these drugs are expressed as IMCnano, KETnano, and PXCnano, respectively.

    2.3. Skin penetration test of fine-particle NSAIDs and measurement of their residual levels in the skin

    2.3.1. Preparation of test drug ointments

    NSAIDai and NSAIDnano were individually suspended in anhydrous ethanol and combined with a hydrophilic ointment. The drug concentration was 1 w/w%.

    2.3.2. In vitro skin penetration of NSAID ointment

    2.3.2.1. Skin penetration test23.

    The excised dorsal skin of hairless mice (Laboskin®, Hos: HR-1 male, 7 weeks; Hoshino Laboratory Animals, Inc., Saitama, Japan) was used. Franz-type diffusion cells (effective diffusional area: 1.77 cm2, receptor volume: 12 mL) were also used. Each test drug (0.2 g) was added to the epidermal side of the Laboskin®. The receptor phase was agitated, and the temperature was maintained at 37 °C. Following the addition of the test drugs, the receptor solution (0.5 mL) was collected over time. The concentration in each sample was measured using HPLC. The skin permeation parameters were calculated using the method reported by Iwaki et al.24

    2.3.2.2. Measurement of residual drug concentration in skin.

    Skin mounted on a Franz-type diffusion cell (1 cm2) was harvested over time in a manner similar to that described in section 2.3.2.1. The harvested skin was cut into small pieces and combined with methanol for homogenization, followed by centrifugation for 30 min (15,000 × g, 4 °C). The supernatant was used as the test sample. The drug concentration in the test sample was measured using HPLC.

    2.3.3. Test drug quantification method

    The HPLC system used was an LC-10 System, Shimadzu Corporation (Kyoto, Japan). The concentration of NSAIDs was quantified in accordance with the Japanese Pharmacopoeia 16th edition.

    2.4. Anti-inflammatory activity

    2.4.1. Experimental animals

    Male Wistar rats (5 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The rats were housed at 23 ± 2 °C and 50 ± 3% humidity. The animals were divided into an untreated group, a drug-free vehicle treatment group, and a treatment group for each test drug (NSAIDai group and NSAIDnano group). The experiment was performed with the approval of the Animal Experiment Review Board of Kochi University and in accordance with the Guidelines for Animal Experiments of Kochi Medical School.

    2.4.2. Test drugs

    Using a method similar to that described in section 2.3.1, NSAID ointments were prepared at concentrations of 0.5, 1, and 3% and a dose-response test was then conducted using these ointments. To compare efficacies, ointments with the same concentrations as those of commercially available products (1 w/w% IMC, 3 w/w% KET, and 0.5 w/w% PXC) were used.

    2.4.3. Carrageenan-induced footpad edema model25–27

    The left hindlimb pad volume was measured using a foot volume measuring device. A 0.5% carrageenan saline solution (0.1 mL) was injected subcutaneously into the left hindlimb pad, immediately followed by the application of each test drug ointment on gauze (2.0 cm × 2.0 cm, 0.5 g) to the footpad and fixation with surgical tape. After addition of the test drugs, foot volume was measured over time and the percent increase in the footpad volume relative to the pre-injection volume, as well as the percentage of edema (edema coverage) and the AUC, were calculated. In addition, the effective dose 50 ([ED50], %) was calculated from the edema suppression rate (%) at 3 h after carrageenan treatment, and the edema suppression rate at each concentration.

    2.4.4. Adjuvant-induced arthritis model26,27

    The right hindlimb volume was measured using the method described in section 2.4.3. Heat-treated nonviable Mycobacterium butyricum suspended in liquid paraffin (6 mg/mL; volume, 0.1 mL) was then injected intradermally into the hindlimb base. Fourteen days after adjuvant inoculation, the right hindlimb volume was measured; animals that were diagnosed as having definite arthritis (foot swelling rate of 80% or more) were selected and divided into groups so that the extent of right hindlimb swelling would be similar in each group. Beginning at 14 d post adjuvant inoculation, gauze bearing the test drug ointment (2.0 cm × 2.0 cm, 0.5 g) was applied once daily to the skin over the right hindlimb joint for 7 consecutive days. The right hindlimb volume was measured at 1, 3, 5 and 7 d after the start of drug treatment. The magnitude of change in the right hindlimb volume was determined as the difference relative to the volume before adjuvant inoculation. In addition, the healing rate at 7 d after the start of drug treatment was calculated. The ED50 (%) at each concentration was calculated from the healing rate (%) at each concentration level at 7 d after the start of the test drug treatment. Healing rate (%) = (V14-Vt7)/(V14-V0) × 100.

    2.5. Statistical analysis

    The obtained values are shown as mean ± standard error. For all other measurements, a one-way ANOVA was used for dispersion analysis. Differences in the efficacy of each particle size preparation between two groups were tested using a t-test. Comparisons of each test drug treatment group with the untreated group or the drug-free vehicle treatment group were performed using Dunnet's multiple comparison.

    3. Results

    3.1. Formation of fine particles of NSAIDs

    Fine particle formulations of IMC, KET and PXC were successfully created. However, diclofenac sodium could not be made into fine particles because it was soluble in the suspending agent. The particle sizes of the fine-particle NSAIDs are shown in Table 1, and the distribution of the particle sizes is shown in Fig. 1. The particle sizes of the active ingredient NSAIDs (IMCai, KETai, and PXCai) were in the micrometer range. Table 1 shows the stability data after crushing using the mixer. Particle size measured 14 d post crushing, was unchanged from that recorded immediately after crushing.

    T1-4
    Table 1:
    Changes in NSAID particle size in formulations of particle dispersions containing NSAIDs, 14 d after treatment.
    F1-4
    Fig. 1.:
    Particle size distribution and frequency in NSAID particles. a)Particle size distribution and frequency for Indomethacin particles, b) Ketprofen particles and c) Piroxicam particles. … is NSAIDai, — is NSAIDnano.

    3.2. Skin penetration test

    The influence of particle size on the cumulative skin penetration of each NSAID is shown in Fig. 2. All of the NSAIDs showed a linear increase in the concentration of the penetrating drug. Compared with the active ingredient formulations, the nanoparticle preparations promoted penetration. A cumulative increase in the quantity of penetrated drug, compared with that of the respective active ingredients, was observed at 4, 2, and 4 h after the application of the IMC, KET and PXC nanoparticles, respectively. Skin penetration data are shown in Table 2. IMCnano showed significantly higher penetration rate (Jc), drug penetration coefficient (Kp), and AUC (0 → 24) than IMCai. The penetration lag time (Tlag) was significantly shorter for IMCnano. Similar results were obtained for the KET and PXC preparations.

    F2-4
    Fig. 2.:
    The influence of particle size on the cumulative skin penetration of each NSAID. Results are expressed as mean ± SE. Differences in the efficacy of each particle size preparation between the two groups were tested using an unpaired Student's t-test. *p<0.05, **p<0.01 compared with the corresponding NSAIDai group.
    T2-4
    Table 2:
    Pharmacokinetic parameters for the skin penetration of NSAIDs.

    3.3. Residual drug concentration in the skin

    Fig. 3 shows the influence of particle size on the concentration of each NSAID remaining in the skin. The concentration reached a steady state at 4–6 h after the application of each active ingredient or nanoparticle formulation. The quantity remaining in the skin was larger for the nanoparticle formulation of each NSAID compared with the active ingredient forms. The quantity remaining in the skin began to show a significant increase at 4 h (IMC), 2 h (KET), and 4 h (PXC) after the application of the nanoparticles.

    F3-4
    Fig. 3.:
    The influence of particle size on the concentration of each NSAID remaining in the skin. Results are expressed as mean ± SE. Differences in the efficacy of each particle size preparation between the two groups were tested using an unpaired Student's t-test. *p<0.05, **p<0.01 compared with the corresponding NSAIDai group.

    3.4. Carrageenan-induced footpad edema model

    Edema peaked at 3 h after carrageenan treatment, with an edema coverage of 66.3 ± 2.2%, evaluated by analysis of edema coverage calculated from the foot volume (Fig. 4). In each NSAID group, edema coverage began to decrease significantly at 1 h post treatment with the active ingredient or nanoparticle formulation, compared with the untreated group and the drug-free vehicle treatment group; this efficacy persisted until 6 h after treatment. Comparing the NSAIDai formulations with the NSAIDnano analogues, showed that edema coverage began to decrease significantly 2 h after treatment with IMCnano or PXCnano, demonstrating the edema-suppression effects of these preparations. Following KET treatment, edema coverage began to show a significant decrease at 1 h post treatment using the nanoparticle, compared with treatment using the active ingredient form, thereby demonstrating the edema-suppression effects of KETnano. In addition, AUC analysis demonstrated that the AUC was significantly lower following treatment using the nanoparticle form of each NSAID compared with treatment using the bulk analog (Table 3).

    F4-4
    Fig. 4.:
    Time course of edema after topically applied NSAIDs in carrageenan-induced footpad inflammation in rats. Results are expressed as mean ± SE of the data from six animals.*p < 0.05, **p < 0.01 compared with the corresponding control group (analysis of variance followed by Dunnett's test). #p < 0.01 compared with the corresponding NSAIDai group (analysis of variance followed by unpaired Student's t-test). ♦ Control, ⋄ Ointment base, ○ IMCai, • IMCnano, Δ KETai, Δ KETnano, □ PXCai, ▪ PXCnano.
    T3-4
    Table 3:
    Effect of NSAIDs on carrageenan-induced footpad inflammation edema in rats.

    3.5. Adjuvant-induced arthritis model

    When each test drug was administered for 7 d after the induction of arthritis, the IMC groups exhibited a significant suppression of foot swelling on the first day of treatment, compared with the untreated group and the drug-free vehicle treatment group. On the fifth and seventh day of treatment, IMCnano showed a significant suppression of foot swelling, compared with IMCai (Fig. 5a). The KET groups showed a significant suppression of foot swelling on the third day of treatment, compared with the untreated and drug-free vehicle treatment groups. On the seventh day of treatment, KETnano showed a significant suppression of foot swelling, compared with KETai (Fig. 5b). In the PXC evaluation, PXCnano showed a significant suppression of foot swelling on the first day of treatment, while PXCai showed a significant suppression of foot swelling on the fifth day of treatment; when each of these groups was compared with the untreated and drug-free vehicle treatment groups. Compared with PXCai, PXCnano showed significant suppression of foot swelling from the third day following the start of treatment (Fig. 5c). The healing rates for 7 d following the start of the test drug treatment are shown in Table 4. The rates were 40.3, 43.0 and 35.8% for IMCai, KETai and PXCai, respectively, while they were 53.0, 57.4 and 66.8% for IMCnano, KETnano and PXCnano, respectively. A comparison of the three nanoparticle preparations revealed a tendency for PXCnano to be more effective than the other nanoparticle preparations.

    F5-4
    Fig. 5.:
    Time course of edema after topically applied NSAIDs in adjuvant-induced arthritis in rats. Results are expressed as mean ± SE of the data from six animals. *p<0.05, **p<0.01 compared with the corresponding control group (analysis of variance followed by Dunnett's test). #p < 0.05, ##p < 0.01 compared with the corresponding NSAIDai group (analysis of variance followed by unpaired Student's t-test). ♦ Control, ⋄ Ointment base, ○ IMCai, • IMCnano, Δ KETai, Δ KETnano, □ PXCai, ▪ PXCnano.
    T4-4
    Table 4:
    Therapeutic effect of NSAIDs on adjuvant-induced arthritis in rats 7 d after treatment.

    4. Discussion

    The present study was designed to investigate the potential of ointments containing NSAID nanoparticles in terms of skin penetration, safety and anti-inflammatory activity. Nanoparticles are colloidal dispersion systems. Because they are thermodynamically unstable, it is difficult to maintain a dispersion of nanoparticles in water for a long period, and aggregation and sedimentation are likely to occur.28,29 The nanoformulations prepared were free of sedimentation at 14 d post preparation, and the particle size remained unchanged. The ointments containing NSAIDs were prepared to give resulting NSAID concentrations equivalent to those of commercially available NSAID products in Japan. In the in vitro skin penetration test, the cumulative penetration increased linearly for each NSAID ointment during the 24 h after application. The skin surface can be damaged by the application of several drugs, and drug penetration can increase as a result.30 For each of the NSAID ointments prepared in this study, a primary skin test was conducted in rabbits (data not shown). Following the application of the active ingredient drug or nanoparticle, both untreated intact skin and damaged skin, showed no stimulus-induced reaction throughout the observation period. The present study revealed that NSAIDnano formulations enabled greater penetration of drug into the skin compared with NSAIDai. These results indicate differences in the skin penetration profiles of NSAIDai and NSAIDnano and suggest a higher local activity of NSAIDnano, compared with NSAIDai, when applied to the skin. The horny layer, the outermost layer of the skin, is the most prominent skin barrier, and the intercellular spaces in this layer are considered to have dimensions in the 50–100 nm range. Because the NSAIDnano formulations produced in this study had a particle size of 70–80 nm, it was assumed that percutaneous penetration of the nanoparticle formulations would be enhanced. To achieve systemic delivery of active ingredients applied to the skin, these ingredients must penetrate the epidermis and enter blood vessels located in tissues below the dermis. In this study, because concentrations of residual drugs in the skin were higher for the nanoparticle formulations, an increase in their percutaneous permeability can reasonably be assumed. In the acute inflammation model, the edema coverage after treatment with each NSAIDnano formulation was significantly lower compared with those after treatment with each NSAIDai preparation. Carrageenan-induced footpad inflammation caused by the release of serotonin and histamine, arises during the first phase, while kinins and PGs are involved in the second phase.31–33 NSAIDs suppress edema during both the first and second phases. The chronic model is also known to involve two inflammatory processes. Primary inflammation begins on the day following the injection of the adjuvant into the hindlimb. Secondary inflammation begins at 7 d after the adjuvant injection.34,35 The pathogenic processes and the histopathological features of the resulting lesions, are similar to those observed in human rheumatoid arthritis.36,37 Both NSAIDai and NSAIDnano suppressed foot swelling in a time-dependent manner. The healing rates at 7 d post treatment were significantly higher following treatment with each NSAIDnano preparation compared with the NSAIDai preparations. The concentrations of NSAIDnano required to suppress edema were much lower than those of the NSAIDai, indicating the potency of the NSAIDnano formulations. A comparison among the nanoparticle preparations revealed a tendency for PXCnano to be more effective than the other nanoparticle preparations. Regarding the effects of NSAIDs on acute inflammation; after the onset of carrageenan-induced inflammation, cyclooxygenase levels reportedly reach a plateau 3–5 h after administration of NSAIDs, therefore the mode of action of the test drugs appears to be inhibition of expressed cyclooxygenase. In contrast, because the immune system is involved in the pathogenic mechanisms underlying chronic inflammation, it was assumed that the test drugs exerted their effects through not only inhibition of cyclooxygenase, but also inhibition of leukocyte migration and plasma membrane stabilization. Thus, NSAIDs appear to be effective for not only acute, but also chronic inflammation because they have various modes of action. NSAIDnano preparations are expected to serve as percutaneously absorbable preparations with excellent efficacy for a variety of diseases and conditions. Considering that the skin penetration potential, the residual drug levels in the skin, and the efficacy at the inflamed site, were higher for NSAIDnano than for NSAIDai, the NSAIDnano formulations could potentially serve as the basis for new pharmaceutical products. Furthermore, the results from the current study suggest that NSAIDnano preparations can exert anti-inflammatory effects at lower drug concentrations than those currently used, and may enable longer-lasting activity. These features could help to reduce healthcare expenditure.

    However, products containing NSAIDnano might undergo changes in terms of pharmacokinetics and safety, and they would likely be required to undergo evaluations such as those currently required for new drugs before clinical use. The exact skin penetration mechanism of these formulations must be clarified, and further research into the development of appropriate ad ditives and formulations is needed. The NSAIDs used in this study have been investigated in conjunction with nanoparticles in other studies,14,17,38–40 however the method of nanoparticle synthesis used here has the following advantages over those described in previous reports: 1) No organic solvents were used in nanoparticle synthesis, and no stabilizer was added. 2) No specialized equipment was used, with the exception of a rotating and revolving crusher, which is easy to obtain. 3) The ointment formulation was prepared by dissolving the nanoparticles in a small amount of ethanol, followed by mixing with an ointment base, and contains few substances such as gels, polymers, and surfactants that are irritating to the skin. The preparation cost of the nanoparticles is therefore expected to be low, and basic safety considerations have been made.

    These nanoparticle NSAIDs could provide an effective and valid means of treatment, while avoiding undesirable adverse reactions. The utilization of nanoparticles may lead to the development of new pharmaceutical products for percutaneous use.

    Acknowledgments

    The authors would like to thank Department of Pharmacy, Kochi Medical School Hospital for technical assistance with the experiments. We thank Sarah Dodds, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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    Keywords:

    Drug delivery; Nanoparticles; Nanotechnology; Non-steroidal anti-inflammatory drugs (NSAIDs); Percutaneous

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