Optimization of O/W Emulsion Solvent Evaporation Method for Itraconazole Sustained Release Microspheres

Abstract

Itraconazole, a commonly used antifungal drug in the clinic approved by U.S. Food and Drug Administration (FDA), has been gradually found to have anti-tumor, angiogenesis inhibition and other pharmacological activities. However, its poor water solubility and potential toxicity limited its clinical application. In order to improve the water solubility and reduce the side effects caused by the high concentration of itraconazole, a novel preparation method of itraconazole sustained release microspheres was established in this study. Firstly, five kinds of polylactic acid-glycolic acid (PLGA) microspheres loaded with itraconazole were prepared by oil/water (O/W) emulsion solvent evaporation and then characterized by infrared spectroscopy. Then the particle size and morphology of the microspheres were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). After that, the particle size distribution, drug loading rate, entrapment efficiency, and drug release experiments were evaluated. Our results showed the microspheres prepared in this study had uniform particle size distribution and good integrity. Further study found that the average drug loading of the five kinds of microspheres prepared with PLGA 7505, PLGA 7510, PLGA 7520, PLGA 5020 and PLGA 0020 were 16.88, 17.72, 16.72, 16.57, and 16.64%, respectively, and the encapsulation rate all reached about 100%. More surprisingly, the release experimental results showed that the microspheres prepared with PLGA 7520 did not show sudden release, showing good sustained release performance and high drug release rate. To sum up, this study optimized the preparation method of sustained-release microspheres without sudden release, which provides a new solution for the delivery of itraconazole in the clinic.

Introduction

Diseases caused by fungal infections are increasing year by year because it spreads easily, leading to severe illnesses that can be life-threatening.¹⁾ Itraconazole (Fig. 1) is a traditional antifungal agent with a wide range of antifungal effects and is widely used in clinical practice.^2,3) Recently, the excellent antitumor activity of itraconazole has been discovered and caused extensive research.^4–6) My previous study also found that itraconazole had good anti-tumor effect on liver cancer cells, and further explored the anti-tumor mechanism.⁷⁾ As a novel anti-tumor drug candidate, itraconazole has been used in phase ii clinical trials in recent years.^8–11) However, itraconazole has poor oral water solubility, low gastrointestinal absorption rate, and systemic application is often accompanied by gastrointestinal discomfort, headache, and other adverse effects.^12,13) Therefore, itraconazole should not be used in patients with abnormal liver and kidney function.^14–16) By developing new dosage forms, drug efficacy can be improved and toxicity reduced, but the poor water solubility of itraconazole makes the development of sustained and controlled release dosage forms more challenging.¹⁷⁾

Fig. 1. Chemical Structure of Itraconazole

In order to improve the solubility and release performance of poorly soluble drugs, multiple dosage forms have been developed such as solid dispersions, nano suspensions, polylactic acid-glycolic acid (PLGA) microspheres and so on.^18–20) Besides, a series of long-term sustained release preparation have been reported,^21,22) among which the most common is drug-loaded PLGA microspheres, and their properties such as drug loading, encapsulation rate and drug release rate have been studied in detail.^23–25) PLGA, an aliphatic polyester, has been applied in clinical practice due to its good biocompatibility and biodegradability^26,27) and is one of the most successfully developed biodegradable polymers. Recently, PLGA microsphere delivery system is widely used in anti-tumor,²⁸⁾ anti-inflammatory²⁹⁾ and anti-bacterial³⁰⁾ treatment. As a continuous controlled release preparation, PLGA microspheres can release drugs slowly to achieve long-term effects,²⁷⁾ thereby improving the efficacy of drugs and reducing the side effects.^31,32)

Literature reports showed that PLGA microspheres can be prepared by various methods such as Leuprolide acetate-PLGA microsphere reported³³⁾ as industrial application and PLGA nanoparticles reported by Niwa et al.³⁴⁾ In this study, microspheres were prepared by oil/water (O/W) emulsion solvent evaporation method and the significance of this study lies in two aspects. Firstly, itraconazole was encapsulated in the microspheres to improve the solubility and bioavailability. Secondly, the innovation is that itraconazole microspheres were successfully prepared with high drug loading and encapsulation efficiency without burst release, showing a continuous slow release, which has never been reported in previous studies. In addition, our preparation of microspheres has potential applications in antifungal, antitumor and so on, and the synthesis method in our study also has reference significance for the preparation of other drug sustained-release microspheres in the future.

Experimental

Materials

Itraconazole (batch No. 092-05581) purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), PLGA copolymer (Lactic acid : Glycolic acid (LA : GA) = 75 : 25; PLGA 7505; average molecular weight 5000; batch No. 829-11946), PLGA copolymer LA : GA = 75 : 25; PLGA 7510; average molecular weight 10000; batch No. 826-11956), PLGA copolymer (LA : GA = 75 : 25; PLGA 7520; average molecular weight 20000; batch No. 820-11976), PLGA copolymer (LA : GA = 50 : 50; PLGA 5020; average molecular weight 20000; batch No. 822-11936), PLGA 0020 (PLA; average molecular weight 20000; batch No. 826-11836), were acquired from Wako Pure Chemical Corporation. Polyvinyl alcohol (PVA) (polymerization degree of 500, saponification degree of 86.5–89.0 mol%, batch No. 28311-25), phosphate buffer powder (1/15 mol/L, pH 7.0, batch No. 161-12191) was purchased from FUJIFILM Wako Pure Chemical Corporation and Tween 80 was obtained from Nacalai Tesque Ltd. (Kyoto, Japan). High-quality reagent-grade ethyl acetate and methanol (FUJIFILM Wako Pure Chemical Corporation) were used as good solvents for PLGA. Acetonitrile was chromatographic grade and the water used for the experiment was high purity water.

Experimental Instrument

Perista pump (SJ-1211, Japan ATTO Corporation, Tokyo, Japan), IKA T10 basic ULTRA-TURRAX^® Homogenizer (IKA company of Germany), EYELA FREEZE DRYER FD-1000 (Tokyo Physical and Chemical Equipment Co., Ltd., Tokyo, Japan), HPLC (LC-2010C, SHIMADZU, Kyoto, Japan), Japanese electron field emission scanning electron microscope (Instrument no. A14000001), Transmission electron microscope (JEM-1230 (HC), Japan Electronics Corporation).

Preparation of PLGA Microspheres of Itraconazole

Five kinds of PLGA microspheres of itraconazole were prepared by O/W emulsion solvent evaporation method. The preparative procedures can be summarized as follows: 2% PVA solution was prepared and 10 mL of PVA solution was accurately measured as the aqueous phase. One hundred milligram PLGA and 20 mg itraconazole were dissolved in a certain proportion of methanol and ethyl acetate as oil phase. And then the organic phase solution dropped into the aqueous phase through the peristaltic pump at a speed of 4 mL/min and stirred to form an O/W emulsion. The solution was then stirred on a magnetic stirrer for 2.5 h, followed by homogenization at 27800 rpm for 10 min, after which it was stirred for another 3 h, and the organic solvent was volatilized. Finally, the microspheres were centrifuged at 12000 rpm for 10 min at 4 °C, washed 3 times with high pure water, and then freeze-dried for 2 d to obtain dry solid microspheres.

In this experiment, five kinds of PLGA microspheres with different polymerization ratios and molecular weights were selected to prepare PLGA microspheres, including the PLGA 7505, PLGA 7510, PLGA 7520, PLGA 5020, PLGA 0020. By comparing the differences of the five kinds of microspheres, the best experimental scheme for preparing itraconazole microspheres was optimized. In addition, the toxicity of ethyl acetate and methanol used in this study is lower than that of dichloromethane and trichloromethane, which are relatively safe.

Calculation of the Yield of Microspheres

The higher the yield of microspheres, the more drug-loaded microspheres can be obtained under the condition of adding the same amount of drugs, indicating a lower preparation cost. Five kinds of freeze-dried microspheres were weighed accurately and the yield was calculated.

  

Drug Loading and Encapsulation Rate

One milligram microspheres of itraconazole was weighed accurately and dissolved with 5 mL mixed solvent (acetonitrile : water as 3 : 2). Next, the solution was homogenized by ultrasonic apparatus and the concentration of itraconazole was determined by HPLC. The mobile phase was acetonitrile: water 3 : 2, the flow rate was 1 mL/min, the column temperature was 30 °C, and the injection volume was 10 µL. And then the absorbance of itraconazole was determined at 260 nm. Drug loading and encapsulation efficiency were defined as Ref. 35:

  

Characterization of Microspheres

Fourier transform (FT)-IR spectra were obtained using FT-IR spectrophotometer (Thermo Scientific Nicolet iS10 FT-IR Spectrometer). In this experiment, 10 mg samples were taken and pressed by potassium bromide tablet method and followed by the acquisition of FT-IR spectra over the scanning range 500–4000 cm⁻¹.

Scanning Electron Microscope (SEM)

The surface morphology of microspheres was observed by taking SEM images using Japanese electron field emission scanning electron microscope (Instrument No. A14000001). Briefly, the microspheres were attached to brass stubs with double-sided tape and then coated in a thin layer of foil in a vacuum to conduct electricity. And then the image of the microsphere surface was taken at the excitation voltage of 10 KV.

Transmission Electron Microscope (TEM)

In our study, the morphology of the microspheres was investigated by TEM. Firstly, a drop of the pellet suspension was placed on a 400-mesh copper mesh coated with a carbon-containing polymer, excess samples were removed with filter paper, and analyzed by TEM. Besides, transmission images of the microspheres were taken at an excitation voltage of 10 KV.

Release of Itraconazole from PLGA Microspheres

Drug release of microspheres (1 mg) in vitro was performed using 500 mL phosphate buffer containing 0.05% Tween-80 (pH 7.0) at 37 °C and 100 rpm by paddle method.^36,37) At various time intervals (5, 30 min, 1, 2, 3, 4, 5, 6, 8, 10, 24, 48, 72, 96, 120, 144, 168 h), 1 mL aliquots were withdrawn and replaced with the same volume of fresh medium. Afterwards, the samples centrifuged for 10 min at 1200 rpm and 800 µL supernatant was transferred as the sample to be measured. Next, the sample was quantified at 260 nm by HPLC, which is the same as the liquid phase method used for the determination of encapsulation rate. Besides, 1 mg itraconazole was dissolved in 1 mL methanol and added to 500 mL phosphate buffer containing a certain concentration of Tween-80 for full dissolution. And then the standard solution was obtained by gradient dilution method and the concentration of itraconazole in the sample solution was further calculated by the standard curve method.

Results

Yield of Five Kinds of Microspheres

In our study, five kinds of microspheres were dried thoroughly and then weighed accurately to calculate the yield of microspheres. Our results showed (Table 1) that the yield of the five kinds of microspheres was high, especially the yield of itraconazole microspheres prepared by PLGA 7510, 7520, 5020 was higher than 85%.

Table 1. The Quantity and Yield of Five Kinds of Microspheres

PLGA	Quantity (mg)	Yield (%)
7505	93.48	77.9
7510	109.18	90.98
7520	107.50	89.58
5020	104.80	87.33
0020	100.41	83.68

Drug Loading and Encapsulation Efficiency of Microspheres

Through the calculation of experimental data, the drug loading and encapsulation rates of five kinds of microspheres were obtained. As showed in the Table 2, the loading efficiency of the five kinds of microspheres were all between 16 and 18%, with high loading efficiency and no significant difference. In addition, the encapsulation efficiency is an important parameter to evaluate the performance of microspheres. As we can see in the Table 2, the encapsulation efficiency of the five kinds of microspheres prepared in this study was very high, all around 100%, indicating that the preparation method adopted in this study was very successful.

Table 2. Drug Loading Rate and Encapsulation Rate of the Five Kinds of Microspheres

PLGA	LE (%)	Average (%)	EE (%)	Average (%)
7505	14.99	16.88	89.94	101.26
	18.48		110.88
	17.16		102.97
7510	17.02	17.72	102.12	106.28
	18.06		108.36
	18.06		108.35
7520	17.61	16.72	105.63	100.31
	15.60		93.60
	16.95		101.70
5020	17.33	16.57	103.99	99.38
	15.30		91.77
	17.07		102.37
0020	15.70	16.64	94.15	99.79
	17.76		106.53
	16.45		98.69

IR Spectroscopy

We characterized itraconazole, PLGA 7520, physical mixture and five kinds of PLGA microspheres by IR spectroscopy. As we can see in Fig. 2, the characteristic peaks at 1700 cm⁻¹ (3) (ν _{C=O, C=O stretching vibration of aromatic ketones}), 1550 cm⁻¹ (4) (ν _{C=C, C=C skeleton stretching vibration of benzene ring}) and 1250 cm⁻¹ (5) (ν _{as(C-O-C), Asymmetric stretching vibration of ether bond C-O-C}) of itraconazole disappeared in the PLGA 7520 microspheres, indicating that our preparation method loaded itraconazole into PLGA 7520 and the microspheres was prepared successfully.³⁸⁾ Furthermore, we detected another four kinds of microspheres using IR spectrum scanning and found the IR spectrum of four kinds of microspheres prepared by PLGA 7505, PLGA 7510, PLGA 5020 and PLGA 0020 are very consistent with PLGA 7520 microspheres in the peaks of 1650 cm⁻¹ (1) and 1350 cm⁻¹ (2). More importantly, the IR spectra of the five kinds of microspheres were significantly different from those of itraconazole and the physical mixture, showing the disappearance of multiple peaks. The above results indicated that five different itraconazole microspheres were successfully prepared in this study.

Fig. 2. IR Spectra of Itraconazole, PLGA 7520, Physical Mixtures and Five Kinds of PLGA Microspheres Prepared in This Study

SEM

The morphology of PLGA 7505, PLGA 7510, PLGA 7520, PLGA 5020 and PLGA 0020 microspheres loaded with itraconazole were observed by SEM. The results showed (Fig. 3) that most of the PLGA 7505 microspheres were around 1–5 μm with a few fragments and the particle size of PLGA 7510 microspheres was also around 1–5 μm. Meanwhile, PLGA 5020 microspheres had more fragments and the particle size was 1–5 μm while the surface of PLGA 0020 microspheres is smooth and the particle size is also about 1–5 μm. Different from the above, itraconazole microspheres prepared with PLGA 7520 had significantly smaller particle size, uniform particle size distribution and almost no fragmentation.

Fig. 3. The Morphology of PLGA 7505 (A), PLGA 7510 (B), PLGA 5020 (C), PLGA 7520 (D) and PLGA 0020 (E) Loaded Itraconazole Microspheres Was Observed by SEM

TEM

The morphology of PLGA 7505, PLGA 7510, PLGA 7520, PLGA 5020 and PLGA 0020 microspheres loaded with itraconazole was observed by TEM. As shown in the Fig. 4, we found that the microspheres above were all spherical and the PLGA 7505, PLGA 7510, PLGA 5020 and PLGA 0020 microspheres prepared in our study have similar particle sizes, while PLGA 7520 microspheres was significantly smaller, about 1 μm. Besides, we also observed some visible fragments in the PLGA 5020 microspheres.

Fig. 4. Five Kinds of PLGA Microspheres Loaded with Itraconazole Were Observed by TEM

(A) PLGA 7505 microspheres. (B) PLGA 7510 microspheres. (C) PLGA 7520 microspheres. (D) PLGA 5020 microspheres. (E) PLGA 0020 microspheres.

Release Experiment of Microspheres

In order to determine the sustained release ability of the prepared microspheres, a series of release experiments in vitro were carried out. The experimental results showed that the five kinds of microspheres prepared all had certain sustained release ability while the sustained-release effect was different (Table 3 and Fig. 5). Firstly, the release rate of PLGA 7505 microspheres was 17.5% within 5 min, and the release rate was basically completed at 144 h, with the maximum release rate was 42.3%. Secondly, PLGA 7510 microspheres were suddenly released within 5 min, and the release rates at 5 min and 168 h were 25.4 and 62.8%, respectively.

Table 3. Drug Release of PLGA 7505, PLGA 7510, PLGA 7520, PLGA 5020 and PLGA 0020 Microspheres Loaded with Itraconazole in 168 h

Release Rate (%)	PLGA 7505	PLGA 7510	PLGA 7520	PLGA 5020	PLGA 0020
Time(h)
0.08	17.5	25.4	9.0	7.2	5.2
0.5	25.3	19.8	12.5	6.7	5.7
1	28.3	25.3	18.8	10.6	10.1
2	29.6	27.9	27.6	12.3	12.5
3	29.9	28.6	34.2	15.1	14.3
4	30.3	32.6	38.0	22.4	16.6
5	33.2	35.5	42.1	18.6	18.2
6	34.0	32.6	44.8	20.1	19.9
8	31.7	36.6	49.6	22.6	21.6
10	30.3	37.9	51.5	22.6	30.3
24	32.3	43.0	63.8	30.0	45.5
48	35.9	48.0	66.7	33.1	44.7
72	×	×	73.9	42.8	×
96	34.5	47.2	70.7	41.9	45.4
120	37.7	51.8	83.5	62.4	45.3
144	41.2	59.8	66.83	47.4	31.5
168	42.3	62.8	65.05	47.4	27.2

Fig. 5. Line Diagram of Five Kinds of Microspheres Released in Vitro

(A) PLGA 7505 microspheres. (B) PLGA 7510 microspheres. (C) PLGA 7520 microspheres. (D) PLGA 5020 microspheres. (E) PLGA 0020 microspheres.

Surprisingly, PLGA 7520 microspheres had good release performance, which was reflected in that there was no sudden release within 5 min, and the release peak reached 83.5% at 120 h, indicating that PLGA 7520 microspheres had good sustained release effect. Meanwhile, the release rate of PLGA 5020 microspheres was 7.2% at 5 min, and the highest release rate was 62.4% at 120 h. Besides, the release rate of PLGA 0020 microspheres was only 5.2% at 5 min without sudden release, and the highest release rate was 45.3%. By comparing, the final release rate of PLGA 7520 microspheres was about 20% higher than that of PLGA 5020 microspheres, and about 40% higher than that of PLGA 0020 microspheres. Overall, the most successful part of this study was the preparation of PLGA 7520 microspheres loaded with itraconazole, which not only had the highest final release rate but also showed good sustained release properties.

Discussion

For clinical drugs with low safety factor, sudden release is often controlled.³⁹⁾ As an antifungal drug, itraconazole has been used in clinical practice for many years.⁴⁰⁾ However, it has been found that itraconazole will produce certain hepatotoxicity and other toxicity with the increase of concentration.^41,42) Besides, the poor water solubility of itraconazole also limits its clinical application.^43,44) Therefore, itraconazole was loaded into PLGA microspheres to prepare sustained-release microspheres in this study, which can improve its water solubility and reduced adverse reactions after oral administration. Firstly, as we can see in Fig. 6, we prepared five kinds of microspheres and the yield, drug loading and encapsulation rate were measured. Furthermore, we studied the particle size and release behavior in vitro of microspheres to determine the optimal process. Our data showed that the yield of the five kinds of microspheres was high, while the PLGA 7510 microspheres and PLGA 7520 microspheres reached about 90%, indicating the superiority of the preparation method. In addition, the yield of PLGA 7505 microspheres was less than 80%, which was significantly lower than that of the other four microspheres, probably due to its relatively low molecular weight. Further study showed that all five kinds of microspheres showed very good drug encapsulation rate and excellent drug loading.

Fig. 6. Preparation of Sustained Release Microspheres of Itraconazole

As the molecular weight of PLGA increases, the chain length becomes longer and the viscosity increases, which will reduce the amount of drug remaining on the surface of the microspheres and then reduce the burst release. It was found that the molecular weight of PLGA increased from 28000 to 90000, and the burst release rate of risperidone microspheres decreased from 13 to 0.8%.⁴⁵⁾ In our study, the release rate of PLGA 7520, PLGA 5020 and PLGA 0020 microspheres of itraconazole was less than 10% in 5 min, while PLGA 7505 and PLGA 7510 microspheres showed obvious sudden release. Therefore, we hypothesize that the molecular weight of itraconazole microspheres determines the occurrence of sudden release in our study, and no sudden release occurs at the molecular weight of 20000, which is important for the future study.⁴⁶⁾ Besides, by comparing the release rate of PLGA 7505, PLGA 7510 and PLGA 7520 microspheres, we found that the higher the molecular weight, the slower the release rate in the first two hours, and the drug release rate increased significantly from the third hour. Therefore, to prepare microspheres with low sudden release and high total release rate, copolymers with high molecular weight should be selected. Therefore, itraconazole microspheres with good productivity can be produced when the copolymer ratio (L/G) is 75/25 and the molecular weight is 20000.

PLGA is composed of lactic acid (LA) and glycolic acid (GA), and overall, with the increase of GA ratio, the hydrophilicity of PLGA is enhanced, and the burst release is relatively increased.⁴⁷⁾ But not all studies showed the same regular changes, and some even showed opposite trends. This is because the particle size, embedding efficiency, and release curve of microspheres depend not only on the polymer support, but also on the physicochemical properties of the encapsulated drug, the solvent used to dissolve the organic phase support, and the preparation method.²⁶⁾ By comparing PLGA 7520, PLGA 5020 and PLGA 0020 microspheres, we found that the higher of the copolymer ratio (L/G) in itraconazole microspheres, the higher the release rate within 5 min, while the final total release rate was also higher. Meanwhile, PLGA 7520, PLGA 5020 and PLGA 0020 microspheres all reached the release peak at 120 h and then showed a downward trend, indicating that itraconazole may produces crystal precipitation. As shown in Fig. 6, in addition to the excellent drug release performance, PLGA 7520 microspheres also showed a higher yield and the smallest particle size compared to other microspheres.

In conclusion, in this study, sustained-release microspheres of itraconazole with high release rate and no sudden release were developed for the first time, which has not been reported previously. The preparation method adopted in this study can provide reference for the preparation of sustained-release microspheres of other drugs. In addition, through literature research and my previous research, I found that itraconazole has the potential to develop anticancer drugs such as anti-liver cancer. Therefore, the sustained-release microspheres prepared in this study can be further studied as anti-cancer drugs in our future research.

Acknowledgments

Part of this research was completed during my study at the Mukogawa Women’s University in Japan, and the other part was completed at Beijing University of Traditional Chinese Medicine and China-Japan Friendship Hospital. I am very grateful to Mukogawa Women’s University of Japan for the support of this study.

Funding

This work was financially supported by the National Natural Science Foundation of China (82204676) and Fundamental Research Funds for the Central Universities (Project’s number: 3332022080).

Author Contributions

WWP conducted the experiments and wrote the manuscript. KH designed the experiments and helped complete the experiments. MG, UT, XY, and NJ designed the study and provided financial support for the study. All authors discussed and commented on the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Data availability

Data supporting the findings of this study are available from the first author or corresponding author upon reasonable request.

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