Application of Curcumin Microneedles

Zifan ZHAO, Enlong WANG, Lili HE

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

Abstract [Objectives] To prepare curcumin solid dispersion (Cur SD) by the melting method, using povidone (PVP K30), polyethylene glycol 6000 (PEG 6000), and poloxamer 188 (block polyether F-68, poloxamer 188, F68) as the carrier matrix, the body was directly inverted and pressed to make microneedles, and the dissolution was examined. [Methods] The dissolution in 0.2% sodium lauryl sulfate (SDS) solution within 120 min was studied, and the objects were identified by scanning electron microscopy, differential scanning calorimetry and infrared spectroscopy. [Results] The cumulative dissolution rate of 10% solid dispersion microneedles reached more than 60% in 120 min. The results of object identification also showed that curcumin was dispersed in the carrier matrix in an amorphous state. [Conclusions] Microneedles were well formed and curcumin solid dispersion significantly improved the dissolution of curcumin in 120 min.

Key words Curcumin, Solid dispersion, Microneedles, Dissolution

1 Introduction

Curcumin, a natural yellow polyphenolic substance extracted from the rhizomes of Zingiberaceae and Asteraceae plants, has pharmacological action such as antioxidation, anti-tumor, anti-inflammatory and analgesic effects, and improving Alzheimer’s disease, and very small side effects[1-5]. However, curcumin has poor water solubility, slow oral dissolution rate, and rapid metabolism in the body, resulting in low bioavailability, which greatly affects the bioavailability of curcumin and limits its application in medicine. At present, it is mostly used in food colorants. The preparation of curcumin into a solid dispersion can greatly improve the solubility of curcumin, thereby improving its bioavailability.

Microneedle is a new transdermal drug delivery technology. It pierces through the stratum corneum of the skin, but does not touch the dermis, and forms a micron-scale mechanical channel on the skin to promote faster percutaneous absorption of drug; it has the advantages of slight pain, strong practicality for patients, and accurate drug administration[6-7]. Soluble microneedles can be dissolved directly under the skin, and have no problems of traditional metal microneedles that are difficult to handle if they are broken and stay subcutaneously, medical waste and cross infection[8]. However, the problem of low drug loading in microneedles generally needs to be solved.

2 Materials

2.1 InstrumentsThe main instruments included ST-05 microneedle mold (Singapore Micropoint Technologies), 85-2A digital display two-way constant temperature magnetic stirrer (Jiangsu Jintan Jincheng Guosheng Experimental Instrument Factory), vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), KQ5200DA numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), GKC digital display temperature-controlled water bath (Shanghai Boluo Experimental Equipment Co., Ltd.), DF-101S collector-type constant temperature heating magnetic stirrer (Zhengzhou Hengyan Instrument Co., Ltd.), JA3003 precision electronic analytical balance (Shanghai Liangping Instrument Co., Ltd.), ultrapure water device (Sichuan Youpu Ultrapure Technology Co., Ltd.), UV-1901 ultraviolet and visible spectrophotometer (Shimadzu Corporation, Japan), 80-mesh 0.2 mm national standard inspection sieve (Chengdu Qihang Instrument Co., Ltd.), P100N Gilson pipette gun (Gilson, France), S4800 scanning electron microscope (Hitachi, Japan), and Q2000 differential scanner (TA, USA).

2.2 ReagentsThe main reagents included curcumin (analytical grade, Chengdu Kelong Chemical Co., Ltd.), polyethylene glycol 6000 (analytical grade, Chengdu Kelong Chemical Co., Ltd.), block polyether F-68 (poloxamer 188, Shanghai Yuanye Biological Technology Co., Ltd.), polyvinylpyrrolidone K30 (analytical grade, Chengdu Kelong Chemical Co., Ltd.), sodium lauryl sulfate (analytical grade, Tianjin Kemiou Chemical Reagent Co., Ltd.), anhydrous ethanol (analytical grade, Chengdu Jinshan Chemical Reagent Co., Ltd.).

3 Preparation of microneedles

PEG 6000, poloxamer 188, and PVP K30 were selected as the carrier matrixes to prepare solid dispersions with curcumin content of 10%, 20%, and 30%. The selected matrixes have different properties, so curcumin microneedles were prepared by the melting method and solvent method. Since the melting points of PEG 6000 and poloxamer 188 are low, the melting method was used to prepare curcumin microneedles directly. PVP has a higher melting point, so they were prepared by swelling using the solvent method.

3.1 Preparation of blank microneedlesAfter about 0.5 g of PVP K30 powder was dissolve in 2 mL of purified water, the solution was transferred to a microneedle mold with a pipette gun, and was pressurized to make the solution completely enter the groove of the mold. It was dried at room temperature for 24 h. The solution was supplemented three times, and the volume was about 50 μL each time. The completely dried microneedles was gently taken out of the mold[9]and put in a desiccator for later use.

3.2 Preparation of curcumin microneedles by the melting methodPVP K30 was heated to melt in a water bath at 60 ℃, and then the curcumin drug powder was poured into a crucible and thoroughly stirred until it was completely miscible. The miscible material was poured directly into a microneedle mold, and then it was heated in a vacuum oven at 65 ℃ to make the melt completely enter the pinhole groove of the mold. The mold was taken out after 24 h. After it became cooling at room temperature, the microneedle was gently taken out of the mold and put in a desiccator for later use.

3.3 Preparation of curcumin microneedles by the solvent methodThe curcumin drug powder and PVP were first dissolved in ethanol and then completely dissolved by ultrasonic waves. After the solution was magnetically stirred for 30 min to be mixed thoroughly, and it was transferred to a rotary evaporator to fully volatilize the solvent. After the obtained solid was dried in a vacuum oven for 24 h, it was taken out to obtain a solid dispersion. About 0.5 g of the solid dispersion powder was swelled in 2 mL of purified water, and transferred to a microneedle mold with a pipette gun. It was pressurized to make the solution completely enter the groove of the mold and dried at room temperature for 24 h. The solution was supplemented three times, and the volume was about 50 μL each time. The completely dried microneedles was gently taken out of the mold[9-10]and put in a desiccator for later use.

4 Object identification

4.1 Microneedles

4.1.1Microneedle morphology. Fig.1 shows the shape of blank microneedles under an optical microscope. They were quadrangular pyramid in shape, and the appearance was good. The height was about 600 μm.

Note: A. Microneedle front; B. Microneedle side; C. A single microneedle.

Fig.1 Photo of blank microneedles

4.1.2The skin pierced with the microneedles. After a mouse was sacrificed by removing the abdomen from the neck, the abdominal skin was cut out and placed in physiological saline. After it was stored in a refrigerator at about 0 ℃ for 24 h, it was taken out, and the hair was removed from the skin. The skin was rinsed with physiological saline, and dried with filter paper. The prepared microneedles were pierced into the skin and hold for 5 min. From the pictures of the skin pierced with the microneedles, it can be seen that PVP K30 had the best puncturing effect, and could form complete microneedle array holes. F68 microneedles were not sufficiently hard and partially adhere to the skin.

Note: A. Blank microneedles; B. PVP K30; C. PEG; D. F-68.

Fig.2 The skin pierced with the microneedles

4.2 Identification of solid dispersions

4.2.1Preparation of samples. The microneedles of curcumin solid dispersions were mashed into powder in a mortar, and passed through a 80-mesh sieve to make curcumin solid dispersion powder for later use. Afterwards, the curcumin drug powder and matrix powder were mashed into powder in a mortar, and passed through a 80-mesh sieve to make curcumin physical mixture for later use.

4.2.2Appearance analysis by scanning electron microscope (SEM). The microscopic morphology of the prepared curcumin solid dispersion powder and the physical mixture was observed by SEM. The photographs at different magnifications were observed under SEM. The scanning electron microscope results show that the curcumin drug existed in the form of crystals, but it already existed in the carrier matrix in an amorphous form in the solid dispersion (Fig.3).

Note: A. Curcumin crystals; B. PVP solid dispersion; C. PVP physical mixture; D. PEG solid dispersion; E. PEG physical mixture; F. F68 solid dispersion; G. F-68 physical mixture.

Fig.3 Scanning electron microscope images

4.2.3Differential scanning calorimetry (DSC). At first, 10 mg of curcumin, physical mixture and solid dispersion were taken, and an empty aluminum crucible was as a reference. The heating rate was 10 ℃/min, and the scanning range was 25-250 ℃. As shown in Fig.4, the curcumin drug had a sharp downward endothermic peak at 174 ℃, which was the characteristic peak of melting point of curcumin. F-68 and PEG had a sharp downward endothermic peak at 54.5 and 61.5 ℃ respectively, and PVP had a broad endothermic peak at 127 ℃. For the physical mixtures, F-68, PEG, and PVP had characteristic peaks of melting point at the corresponding positions, and an endothermic peak still existed at around 174 ℃, which was the melting point peak of curcumin. It indicates that curcumin still existed in the form of drug crystals in the physical mixtures.

In the DSC curve of the solid dispersion, the original endothermic peak of the drug had completely disappeared, indicating that the curcumin drug crystals did not exist in the solid dispersion, but were dispersed in the matrix in an amorphous state.

Note: a. F68; b. F68-Cur; c. F68 SD; d. PEG; e. PEG-Cur; f. PEG SD; g.PVP; h. PVP-Cur; i. PVP SD; j. Cur.

Fig.4 Curves of differential scanning calorimetry

4.2.4Fourier transform infrared spectroscopy (FTIR) analysis. At first, 2-3 mg of curcumin, physical mixture and solid dispersion were taken respectively, and mixed with dry potassium bromide powder. After they were ground until the color was consistent, they were pressed into tablets and detected, and finally the infrared spectra were obtained. Seen from Fig.5, no new characteristic peaks were found in the solid dispersion, indicating that no new compounds were produced. Compared with the physical mixture, the absorption peak of the solid dispersion became smaller, possibly due to a change in the crystal form.

Note: a. F68; b. F68-Cur; c. F68 SD; d. PEG; e. PEG-Cur; f. PEG SD; g.PVP; h. PVP-Cur; i. PVP SD; j. Cur.

Fig.5 Fourier transform infrared spectra

5 Establishment of analytical method

5.1 Determination of the maximum absorption wavelength and specific experimentsAn appropriate amount of curcumin reference substance was dissolved in ethanol. Ethanol solvent was used as the blank control, and the curcumin solution was scanned for ultraviolet spectra in the wavelength range of 200-600 nm. The scanning results show that there was a maximum absorption at 426 nm. Appropriate amounts of PVP K30, PEG 6000, and poloxamer 188 were weighed, and purified water was used as the solvent. They were diluted by a certain multiple and scanned for ultraviolet spectra in the wavelength range of 200-600 nm. The scanning results show that all the excipients had no absorption peak near 426 nm, and had no interference to the measurement, so 426 nm was selected as the detection wavelength.

5.2 Establishment of the standard curve10.6 mg of curcumin was accurately weighed, placed in a 100 mL volumetric flask, dissolved in ethanol, diluted to the mark, and shaken to obtain the curcumin reference stock solution with the mass concentration of 106.0 μg/mL, which was stored in the dark. Afterwards, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL of the stock solution was precisely taken into six 10 mL measuring flasks, to which 0.2% sodium lauryl sulfate solution was added. They were diluted to the mark and shaken well to be used as the test solution.

0.2% SDS solution was used as the blank, and the absorbance (A) was measured at 426 nm. Linear regression was performed with the mass concentration (C) and absorbance, and the regression equation was obtained:A=0.219 2C-0.011 9, showing that curcumin had good linearity in the range of 1.06-6.36 μg/mL (R2=0.999 3).

Fig.6 Standard curve

6 Dissolution experiment

6.1 Solubility testExcess curcumin powder and excess solid dispersion containing 10% curcumin were placed in a test tube with 10 mL of ultrapure water, and they were treated with ultrasonic waves until supersaturation. They were fully shaken in a 25 ℃ constant temperature shaker for 24 h in the dark and then centrifuged. The supernatant was appropriately diluted, and the absorbance was measured with an ultraviolet spectrophotometer at 426 nm to calculate the drug solubility. The results show that the solubility of the curcumin drug substance was less than 1.06 μg/mL; the solubility of the solid dispersion based on PVP K30 was 634.07 μg/mL, which was 743.34 times that of the drug; that of the solid dispersion based on poloxamer 188 was 452.16 μg/mL, which was 530.08 times that of the drug; that of the solid dispersion based on PEG 6000 was 415.09 μg/mL, which was 486.63 times that of the drug. It can be seen that the preparation of curcumin in the form of a solid dispersion can significantly enhance the solubility of the drug.

6.2 Test of dissolution rateThis determination was carried out according to the second method (paddle method) of the "Measurement Method for Dissolution Rate and Release Rate" stipulated in the 2015 edition ofChinesePharmacopoeia[11]. 900 mL of 0.2% SDS solution was used as the dissolution medium, and the rotation speed was 100 r/min, while the temperature was (37 ± 0.5) ℃. The curcumin microneedles were put into the medium, and then 5 mL of a sample was collected after 5, 10, 20, 30, 45, 60, 90, 120 min. The same amount of dissolution medium at the same temperatur was added. After the samples were immediately centrifuged at a speed of 4 000 r/min for 10 min, the absorbance of the supernatant A was measured at 426 nm. The cumulative dissolution rate of the drug was calculated according to the regression equation, and the curve of cumulative dissolution rate was drawn.

As shown in Fig.7, the cumulative dissolution rate of Cur SD was significantly higher than that of the Cur drug. As the drug content decreased, the cumulative dissolution rate of the drug became higher.

Fig.7 Dissolution curve of Cur SD within 120 min

7 Discussion

Most of soluble microneedles use water-soluble polymer materials as the matrix, but when the conditions for making curcumin into a solid dispersion are met, the selection range of the matrix is limited. During the experiment, it was found that the poloxamer 188 is not hard enough, and the needle was easily broken when being taken out of the mold. Moreover, it stuck to the skin when the skin was pierced. This problem was alleviated by freezing it in a refrigerator at -20 ℃ for about 30 min before being taken out of the mold. The molding degree of PVP was the best, but because the water content could not be measured, the drug content could not be further accurately determined. In the dissolution experiment, as the drug content increased, the cumulative dissolution rate reduced. A smaller amount of the drug was more easily dispersed in the matrix, so its solubility increased in the dissolution solution, and the cumulative dissolution rate only reach 60%. Because the microneedles were prepared by the melting method, and the drug could not be completely dispersed in the matrix. Although the prepared microneedles could penetrate the skin of mice well, the transdermal performance of transdermal administration needs to be verified by further transdermal experiments.