Jiao LUO, Cong WANG, Yuping MA, Shengqian TIAN, Jiayi DING, Lili HE

Department of Pharmacy, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives] To investigate the in vitro transdermal effect of curcumin solid dispersion needles prepared with povidone K 30 (PVP K30), polyethylene glycol 6000 (PEG 6000) and poloxamer 188 (block polyether F68, poloxamer 188, F68) as carrier matrix, respectively. [Methods] Vertical Franz diffusion cell and HPLC method were used to detect the cumulative penetration and skin retention of the microneedles within 12 h. [Results] Within 12 h, the cumulative penetration of PVP K30 microneedles was as high as 7.098 μg/cm2, and the skin retention reached 35.28 μg/cm2; PEG 6000 and poloxamer 188 microneedles all showed good transdermal effect; while normal curcumin solid dispersion (control) had no obvious transdermal effect. [Conclusions] The transdermal penetration of curcumin is improved after prepared into solid dispersion microneedles, and among different matrixes, PVP K30 is the best choice.

Key words Curcumin, Solid dispersion, Microneedle, Transdermal penetration

1 Introduction

Curcumin is a yellow polyphenol compound extracted from the rhizomes of Zingiberaceae and Araceae plants. It is a food additive approved by the Food and Drug Administration of the United States (FDA). It has anti-inflammatory, anti-pulmonary fibrosis, anti-cholesterol, anti-oxidation, anti-hypertensive, anti-tumor, and hypolipidemic effects[1-5]. Curcumin and its modifications can also improve Alzheimer’s disease[6]. It is safe and inexpensive, with little toxic and side effects, and has good clinical application prospects. However, its solubility is extremely poor and its bioavailability is low, which limits its clinical application. Solid dispersion technology is a common method to effectively improve the bioavailability of poorly soluble drugs. Microneedling is a new type of transdermal drug delivery. It penetrates the stratum corneum and forms a micron-level channel to deliver the drug to the subcutaneous layer, characterized by no pain, fast drug effect, high safety, and high patient compliance[7-9]. Preparing curcumin into solid dispersion microneedles can solve the barriers of transdermal administration and the problem of poor solubility of curcumin. In previous study, curcumin solid dispersion microneedles (Fig.1) have been prepared successfully. In this study, the transdermal penetration of curcumin microneedles is investigated, in order to solve the clinical limitation of curcumin and provide a certain research basis for the transdermal preparation of poorly water-soluble drugs.

Fig.1 Self-made curcumin solid dispersion microneedles

2 Materials and methods

2.1 AnimalsInstitute of Cancer Research (ICR) mice [specific pathogen free (SPF) grade, 18-22 g, all male, animal pro-duction license SCXK (Sichuan) 2015-030]were provided by the Institute of Laboratory Animals, Sichuan Academy of Medical Sciences.

2.2 Drugs and reagentsCurcumin solid dispersion microneedles (self-made), curcumin (analytically pure, Chengdu Kelong Chemical Co., Ltd.), curcumin (chromatographically pure, 98% purity, Shanghai Yuanye Biological Technology Co., Ltd.), polyethylene glycol 6000 (PEG 6000, analytically pure, Chengdu Kelong Chemical Co., Ltd.), block polyether F68 (Poloxamer 188, Shanghai Yuanye Biological Technology Co., Ltd.), polyvinylpyrrolidone K30 (PVP K30, analytically pure, Chengdu Kelong Chemical Co., Ltd.), absolute ethanol (analytically pure, Chengdu Jinshan Chemical Reagent Co., Ltd.), absolute ether (analytically pure, Chengdu Jinshan Chemical Reagent Co., Ltd.).

2.3 Apparatus and instrumentsRYJ-12B drug transdermal diffusion tester (Shanghai Huanghai Pharmaceutical Co., Ltd.), KQ5200DA digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), JA3003 precise electronic analytical balance (Shanghai Liangping Instrument Co., Ltd.), ultrapure water device (Sichuan Youpu Ultrapure Technology Co., Ltd.), and high performance liquid chromatograph (Agilent, Hangzhou Ruixi Technology Co., Ltd.).

2.4 Preparation of isolated mouse skinAfter 14 d of adaptive feeding, depilatory cream was applied to the skin of each mouse. After about 3 min, the depilatory cream and hair was wiped off. Subsequently, the mice were put back into the cages and raised for another 48 h, and the skin of their abdomens was observed. Then, the mice were anesthetized to death with an overdose of absolute ether, and their abdominal skin was cut off immediately, placed on a clean glass plate, wiped with ethanol-soaked cotton balls to remove the fascia, rinsed with normal saline, placed in normal saline and stored in refrigerator at -20 ℃ for later use.

2.5 Transdermal diffusion testEthanol solution (30%) was prepared as the absorption solution. After ultrasonicated for 30 min to eliminate air bubbles, the ethanol solution was poured into the diffusion cell and filled it up. A magnetic stirrer was placed in the absorption pool and its speed was set as 200 r/min. The mouse skin was covered on the receiving pool with the stratum corneum facing the absorption pool, and the release pool was covered on the mouse skin. Subsequently, the entire device was fixed with clamps in the order of absorption pool, isolated skin, and release pool, and placed in the RYJ-12B drug transdermal diffusion tester (37 ℃, 30 min). After all the bubbles were removed and the temperature of the liquid in the absorption pool was maintained at a constant temperature, microneedles were pierced into the skin with a drug delivery device. At 2, 4, 8 and 12 h, 1.0×10-3L of the liquid was sampled, respectively, and after each sampling, the same amount of 30% ethanol solution was replenished at 37 ℃.

The samples were centrifuged at 8 000 rmp for 30 min, and the supernatant was collected, passed through 0.45 μm microporous membrane, and detected by HPLC. The peak area was recorded. The cumulative penetration of curcumin per unit area (Q, μg/cm2) was calculated. According to the calculation results, theinvitrorelease of curcumin solid dispersion microneedles was determined.

The isolated mouse skin was removed, and the drug administration part (1×1 cm2) was cut off, rinsed repeatedly with water and ethanol to remove the drug on the surface, wiped with filter paper, cut, and placed in centrifuge tube (5×10-3L). The centrifuge tube was added with 1×10-3L methanol (chromatographically pure), sonicated for 60 min, and centrifuged at 8 000 rpm for 30 min, and the supernatant was collected, passed through 0.45 μm microporous membrane, and detected by HPLC. The peak area was recorded. Based on the content of curcumin calculated, the retention amount of curcumin in the isolated skin was calculated.

A solid dispersion that did not contain microneedle but was the same as microneedle in size was placed on the isolated skin. Using the same method, at 2, 4, 8 and 12 h, 1.0×10-3L of liquid was sampled, respectively. After each sampling, the same amount of 30% ethanol solution was replenished immediately at 37 ℃. The samples obtained were centrifuged at 8 000 rmp for 30 min, and the supernatant was collected, passed through 0.45 μm microporous membrane, and detected by HPLC, respectively. The peak area was recorded.

2.6 Establishment of HPLC method for curcumin transdermal penetration amount

2.6.1Chromatographic conditions. Column, Kromasil C18column; system, Agilent 1260; mobile phase, acetonitrile-4% glacial acetic acid (48∶52); detection wavelength, 430 nm; column temperature, 35 ℃; sample size, 10×10-6L.

2.6.2Specificity test. An appropriate amount of curcumin was dissolved in methanol (chromatographically pure), and ultrasonicated for 5 min to make it dissolve completely. Appropriate amount of PVP K30, PEG 6000 and poloxamer 188 was dissolved in methanol (chromatographically pure) and ultrasonicated for 5 min to make it dissolve completely. Under the chromatographic conditions described above, the solutions were detected.

2.6.3Establishment of standard curve. An accurate amount (10.0 mg) of curcumin was dissolved in methanol (chromatographically pure) to prepare into mother liquor of 1 mg/10-3L, and then curcumin standard solutions with concentrations of 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1 μg/10-3L were prepared. The standard solutions were detected under the chromatographic conditions, respectively. With the concentration of curcumin (C, μg/10-3L) as the abscissa and integralarea (IA) as the ordinate, linear regression was performed, and the standard curve of curcumin was obtained.

2.6.4Precision test. Curcumin standard solutions of 0.3, 1.2 and 2.1 μg/10-3L were detected by HPLC five times within a day (interval of 2 h), respectively. The peak areas of curcumin were recorded, and the inner-day relative standard deviation was calculated. Then, the three standard solutions were detected at the same time point of three consecutive days, respectively, The peak areas of curcumin were recorded, and the inter-day relative standard deviation (n=5) was calculated.

2.6.5Stability test. Curcumin standard solutions of 0.3, 1.2 and 2.1 μg/10-3L were detected by HPLC at 0, 2, 4, 6 and 8 h, respectively, and the peak areas of curcumin at each concentration were recorded.

2.6.6Reproducibility test. Curcumin standard solutions of 0.3, 1.2 and 2.1 μg/10-3L were detected by HPLC five times repeatedly, and the peak areas of curcumin at each concentration were recorded.

2.7 Establishment of HPLC method for curcumin skin retention

2.7.1Specificity test. An appropriate amount of curcumin was dissolved in methanol (chromatographically pure), and ultasonicated for 5 min to make it dissolve completely. A piece of mouse skin (1×1 cm2), was cut off, transferred into methanol, and ultrasonicated for 60 min. Under the chromatographic conditions described above, the liquids obtained were detected, respectively.

2.7.2Establishment of standard curve. An accurate amount (10.0 mg) of curcumin was dissolved in methanol (chromatographically pure) completely to prepare into mother liquor with a concentration of 1 mg/10-3L, and then curcumin standard solutions of 6, 12, 18, 24, 30 and 36 μg/10-3L were prepared. Under the chromatographic conditions described above, the standard solutions were detected, respectively, and the peak areas of curcumin were recorded. With the concentration (C, μg/10-3L) as the abscissa and the integralarea (IA) as the ordinate, linear regression was performed, and the standard curve of curcumin was obtained.

2.7.3Precision test. Curcumin standard solutions of 6, 24 and 36 μg/10-3L were detected by HPLC five times a day (interval of 2 h), respectively. Based on the peak areas of curcumin, the inner-day relative standard deviation was calculated. Then, curcumin standard solutions of 6, 24 and 36 μg/10-3L were detected by HPLC at the same time point of three consecutive days, respectively, and the peak areas of curcumin were recorded, and then the inter-day relative standard deviation was calculated.

2.7.4Reproducibility test. Curcumin standard solutions of 6, 24 and 36 μg/10-3L were detected by HPLC five times repeatedly, and the peak areas of curcumin at each concentration were recorded.

2.8 Cumulative penetration of curcumin per unit area

whereQis the cumulative penetration amount;Cnis the drug concentration measured at thenthsampling time point;Ciis the drug concentration at the ith sampling time point;Vis the volume of the absorption liquid;Viis the sampling volume at theithtime point; andAis the transdermal diffusion area (1×1 cm2).

3 Results and analysis

3.1 Methodological verification of HPLC analysis of curcumin transdermal penetration amount

3.1.1Specificity test. As shown in Fig.2, the retention time of curcumin is about 14 min, and PVP K30, PEG 6000 and poloxamer 188 had no interference around the retention time of curcumin.

Fig.2 Specificity test of curcumin

3.1.2Standard curve of curcumin. The regression equation obtained wasy=116.42x-20.317 (R2=0.999 5). The results show that in the concentration range of 0.3-2.1 μg/10-3L, there is a linear relationship (Fig.3), meeting the requirements of methodology.

Fig.3 Standard curve for curcumin in absorption liquid

3.1.3Precision test. The relative standard deviation values of intra-day precision of curcumin standard solutions at high, middle and low concentrations were all less than 15%, and the relative standard deviation values of inter-day precision of curcumin standard solutions at high, middle and low concentrations were all less than 15% (Table 1), indicating good that the precision of the instrument is good.

Table 1 Intra- and inter-day precision of curcumin (n=5)

3.1.4Stability test. Table 2 shows that theRSDvalues were all less than 15%, indicating the curcumin solid dispersion has good stability within 8 h.

Table 2 Stability of curcumin in absorption liquid (n=5)

3.1.5Reproducibility test. The results (Table 3) show that the method has good reproducibility.

Table 3 Reproducibility test of curcumin (n=5)

3.2 Methodological verification of HPLC analysis of curcumin skin retention

3.2.1Specificity test. The results show that the mouse skin did not interfere with the determination result.

3.2.2Standard curve of curcumin. The regression equation obtained wasy=105.3x+11.207 (R2=0.999 1). The results show there is a linear relationship in the concentration range of 6-36 μg/10-3L (Fig.4), meeting the requirements of methodology.

Fig.4 Standard curve for curcumin in skin tissue

3.2.3Precision test. As shown in Table 4, the relative standard deviation values of intra-day precision of curcumin standard solu-tions at high, middle and low concentrations were all less than 15%, and the relative standard deviation values of inter-day precision of curcumin standard solutions at high, middle and low concentrations were all less than 15%, indicating that the precision of the instrument is good.

Table 4 Intra- and inter-day precision of curcumin (n=5)

3.2.4Reproducibility test. Table 5 shows that the method has good reproducibility.

Table 5 Reproducibility test of curcumin (n=5)

3.3 Transdermal test of curcumin solid dispersion microneedlesAs the concentration of the drug increased, the cumulative penetration amount was basically on the rise. The cumulative penetration amount of curcumin PVP K30 microneedles within 12 h reached 7.098 μg/cm2, and the skin retention amount reached 35.28 μg/cm2. For PEG 6000 microneedles, the cumulative pen-etration amount was 8.532 μg/cm2, and the skin retention amount reached 32.8 μg/cm2. For poloxamer 188 microneedles, the penetration amount was as high as 7.795 μg/cm2, and the skin retention amount was 25.5 μg/cm2(Table 6). However, the solid dispersion of curcumin had no obvious transdermal effect, and no curcumin was detected in the absorption liquid. Different matrixes had no obvious effect on the cumulative penetration of the drug. In terms of skin retention, the retention of PVP K30 was the highest, and those of PEG 6000 and poloxamer 188 were relatively low. Fig.5-7 show that the 12-h cumulative penetration did not reach the maximum yet. This might be because the release time was not long enough and the equilibrium had not been reached yet.

Table 6 Results of transdermal experiment of curcumin microneedles

Fig.5 Relationship between cumulative penetration of PVP K30 microneedle and time

Fig.6 Relationship between cumulative penetration of PEG 6000 microneedle and time

Fig.7 Relationship between cumulative penetration of poloxamer 188 microneedle and time

4 Discussion

In theinvitrotransdermal experiment of curcumin microneedles, a vertical Franz diffusion cell was used, and taking the cumulative penetration and retention per unit area of skin as indicators, the transdermal penetration behavior of curcumin microneedlesinvitrowas investigated, and two analysis methods were established for the curcumin content in absorption liquid and skin tissue. The results show that curcumin solid dispersion microneedles significantly improved the transdermal penetration of curcumin solid dispersion.

It can be seen from this experiment that the drug content that could be loaded by the microneedles was extremely low. Although the microneedles could pierce the stratum corneum, the curcumin content in the absorption fluid was not high, and the skin retention was relatively high. This shows that micro-needling can pierce the stratum corneum and then stay in the skin to release drugs. The highest skin retention of PVP K30 may be because PVP K30 has the best hardness and can penetrate into the skin completely and release in the skin. Although the microneedles of PEG 6000 and poloxamer 188 could pierce the stratum corneum, they melted easily when exposed to water, so they mostly stuck to the skin. Over time, the drug could still be released through the skin through the hole formed by the microneedle.

Although microneedles can help the transdermal penetration of drugs, they have the shortcoming of low drug loading. They are more suitable for the preparation of drugs with low dosage and high efficacy. Otherwise, the blood drug concentration required for treatment may not be reached.