http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2015.1012311
RESEARCH ARTICLE
A nanoparticulate drug-delivery system for glaucocalyxin A: formulation, characterization, increased in vitro, and vivo antitumor activity Meihua Han1, Zhitao Li2, Yifei Guo1, Jian Zhang3, and Xiangtao Wang1
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1
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China, 2School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, PR China, and 3College of Pharmaceutical Sciences, Soochow University, Suzhou, PR China Abstract
Keywords
Glaucocalyxin A (GLA) is a phytochemical component with multiple pharmacological activities; however, glaucocalyxin A’s wider use has been restricted by its poor solubility. In this study, GLA nanosuspensions were prepared with precipitation-combined ultrasonication and were characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and differential scanning calorimetry (DSC). The GLA nanosuspensions were spherical with a smooth surface and a small size of 143 nm, the drug payload achieved 8.95%, and the maximum GLA concentration reached 1 mg/mL. The lyophilized powders for the GLA nanosuspensions were amorphous and displayed a biphasic drug release pattern with an initial burst release and a consequent sustained release. In contrast to the free drug solution, GLA nanosuspensions showed higher in vitro antitumor activity against HepG2 cells (IC50 value of 1.793 versus 2.884 mg/mL at 24 h, p50.01). Meanwhile, nanosuspensions displayed better anticancer efficacy than free GLA on H22 bearing mice (54.11% versus 36.02% tumor inhibition rate). These results indicate that GLA nanosuspensions have great potential for the treatment of hepatic cancer.
Characterization, Glaucocalyxin A nanosuspensions, in vitro antitumor efficacy, in vivo antitumor efficacy, preparation
Introduction Glaucocalyxin A (GLA, Figure 1) is a biologically active entkauranoid (Sun et al., 2006) diterpenoid that is isolated from the ethanol extract of the leaves of Rabdosia japonica (Burm.f.) Hara var. Glaucocalyx (Maxim.) Hara, a traditional Chinese medicinal herb that grows in northeastern China (Zhang & Long, 1993). Glaucocalyx has been used in folk medicine as an anti-bacterial, anti-inflammatory, stomachic, and anthelmintic agent in China (Amit & Ben-Neriah, 2003). Moreover, its anti-cancer activities were proved early in the 1960s (Arai et al., 1963). Many phytochemical studies on this plant have revealed the presence of more than 30 entkauranoids (Xu et al., 1996; Zhang & Ren, 2003; Xiang et al., 2008), including glaucocalyxin A. Glaucocalyxin A is known to have various biological activities, such as inhibition of platelet aggregation induced by platelet-activating factor, immunosuppressive activity (Chen et al., 2006), antioxidative, and cytotoxic activity (Zhang et al., 2008). Address for correspondence: Jian Zhang, College of Pharmaceutical Sciences, Soochow University, No. 199 Ren0 ai Road, Suzhou Industrial Park, Suzhou 215123, PR China. E-mail: [email protected]. Mr. Xiangtao Wang, Professor, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel: +86 10 57833266. E-mail: [email protected]
History Received 17 December 2014 Revised 21 January 2015 Accepted 22 January 2015
However, the poor solubility of GLA in water has presented a serious obstacle for its practical use as a therapeutic agent. The usual approach for overcoming the problem of solubility is to prepare the formulation using hydroxyproply-b-cyclodextrin, DMSO, and methanol (Shang et al., 2011; Li et al., 2013; Wei et al., 2013). However, adverse effects, such as inflammation of the blood vessels and topical pain caused by delivery of miscible solvents by injection, negate its clinical utility (Zhang et al., 2003). As a consequence, there is a growing need for developing a suitable formulation of GLA that can overcome this problem. Recently, the carrier-free drug nanosuspensions have become an extensively utilized protocol to tackle formulation problems for insoluble compounds (Mu¨ller et al., 2001; Patravale et al., 2004). The most outstanding feature of drug nanosuspensions is the significantly increased drug solubility and dissolution velocity that can be achieved due to the small particle size. Nanosuspensions with a small particle size can be delivered to specific sites by size-dependent, ‘‘passive’’ targeting. It has been reported that nanosuspensions injected intravenously were taken up by the reticuloendothelial system in the liver after only a few minutes as a result of the opsonization process. When the diameter is less than 200 nm, these nanosuspensions can be captured easily by the reticuloendothelial system, especially by the Kupffer cells in the liver. In addition, the aqueous nanosuspensions, which are
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Preparation of GLA nanosuspensions GLA nanosuspensions were prepared using the precipitationultrasonication method. Briefly, GLA and lecithin were dispersed into 1 mL of alcohol to form an organic solution. Afterwards, 1 mL of the organic solution was added into 5 mL of an aqueous solution containing fetal calf serum (20%) drop by drop under continuous ultrasonication (250 W). The resultant GLA nanosuspensions were evaporated under vacuum at 40 C until no residual alcohol remained. Figure 1. The chemical structure of glaucocalyxin A.
Characterization of GLA nanosuspensions
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Particle size and zeta potential of GLA nanosuspensions dispersed in water, can mitigate the side effects caused by miscible solvents (Wang et al., 2010). There are two reports about nanoparticulates of GLA (Cao et al., 2008; Yang et al., 2012); however, the drug payload mentioned in one report was 5.36% and there was no research on the in vitro and in vivo antitumor activity of the resultant GLA nanoparticles in the two reports. In order to obtain higher drug-loaded formulation, in this study, GLA nanosuspensions with good stability were firstly successfully prepared, in which the drug-loading content achieved 8.95%, and GLA nanoparticles demonstrated improved in vitro and in vivo antitumor activity than free GLA, thus have great potential for cancer treatment.
Materials and methods Materials Glaucocalyxin A (98% pure) was kindly supplied by the School of Pharmaceutical Sciences, Soochow University; lecithin (Injection grade) was purchased from Guangzhou Hanfang Modern Chinese Medicine Research and Development Co., Ltd (Guangzhou, China); and fetal calf serum was obtained from Yuanhengjinma Biotechnology Development Co., Ltd. (Beijing, China). Hydroxycamptothecin (HCPT) injection (used as the sodium carboxylate in China) was obtained from Shenghe Pharmaceutical Ltd (Sichuan, China). The water used in the experiments was deionized, and all other organic solvents were of the highest commercially available grade. Animals and cell line Female ICR mice (20 ± 2 g) were supplied by Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the animals were acclimatized at a temperature of 25 ± 2 C and a relative humidity of 70 ± 5% under natural light/dark conditions for 1 week and provided food and water ad libitum. All experimental procedures were performed in accordance with the Guidelines for Ethical and Regulatory for Animal Experiments as defined by The Institute of Medicinal Plant Development (IMPLAD), China. The human hepatoma cell line HepG2 was supplied by the Department of Pharmacology, Beijing Normal University; The H22 cell line (murine sarcoma) was obtained from the Xiyuan Hospital, China Academy of Traditional Chinese Medicine.
The average particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were determined using the dynamic light scattering (DLS) (Zetasizer Nano ZS 90, Malvern Instruments, Malvern, UK). All measurements were made at least in triplicates. Morphology observation by transmission electron microscope (TEM) The morphologies of GLA nanosuspensions were observed using a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan). A drop of nanosuspensions was spread on a 200-mesh copper grid and negatively stained with 2% (w/v) phosphotungstic acid for 30 s. The grid was allowed to dry further for 10 min and was then examined with the electron microscope. Differential scanning calorimetry (DSC) measurement The thermal properties of the freeze-dried powder of GLA nanosuspensions, GLA, lecithin, and fetal calf serum were investigated using a differential scanning calorimeter (DSC Q200, TA Co., Cambridge, MA). The scanning temperature for each sample was set from 0 to 400 C with a heating rate of 10 C/min. Five to eight milligrams of each sample were placed in perforated aluminum-sealed pans, and a blank pan was used as the reference. Entrapment efficiency (EE) and drug loading (DL) The drug EE and DL amount of GLA nanosuspensions were determined according to the method of centrifugation ultrafiltration (Dai et al., 2010). Generally, GLA nanosuspensions were ultracentrifuged by OptimaÔ L-80 XP Ultracentrifuge (Beckman Coulter, Fullerton, CA) at 50 000 rpm for 60 min. The supernatant was sampled, and the concentration of GLA in the supernatant was determined by a reversed-phase high-performance liquid chromatography (HPLC) method. A 20-mL sample solution was injected at least three times into the chromatographic column. The mobile phase was a mixture of water and acetonitrile in the volume ratio of 10:90. The elution rate was 0.3 mL/min, and the GLA detection wavelength was set at 231 nm. The standard curve for the quantification of GLA was linear over the range of 0.1–100 mg/mL with a correlation coefficient of 0.9999. In vitro release behavior To measure the dissolution kinetics, 1 mL (1 mg/mL) of the GLA nanosuspensions, GLA coarse suspensions (made by
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suspending GLA bulk powder in an aqueous solution), and GLA solutions (GLA solutions were prepared by dissolution of GLA in DMSO and then sequential additions of polyoxyethylene hydrogenated castor oil, Tween 80 and physiological saline (DMSO:polyoxyethylene hydrogenated castor oil:Tween 80:physiological saline 0.5:0.5:0.5:8.5, v/v/ v/v)) was placed in a preswelled dialysis bag (molecular cutoff ¼ 8000–14 000). The dialysis bag was then immersed fully in 30 mL of purified water as the release medium and kept in an incubator at 37 ± 0.5 C with stirring at 100 rpm. One milliliter of the release medium was withdrawn at specific time intervals and replaced with an equal volume of fresh release medium. The samples were centrifuged, and the resulting supernatant was analyzed by HPLC. The release medium was replaced every 12 h. All the experiments were performed in triplicate. In vitro cytotoxicity of GLA nanosuspensions against HepG2 cells Cell culture The human hepatoma cell line HepG2 was cultured in RPMI1640 containing 10% fetal bovine serum, 100 U/ penicillin and 100 mg/mL streptomycin at 37 C in a humidified atmosphere containing 5% CO2. MTT assay The cell viability was assessed using a MTT assay. Cells at a density of 5 103 cells/well were seeded in 96-well culture plates with RPMI1640 supplemented with 10% fetal bovine serum. After a 24-h incubation, the medium was replaced by GLA nanosuspensions and a free GLA control solution at various concentration for indicated time periods. The crude drug control group was treated with the same volume of culture media with 0.2% DMSO (v/v). Subsequently, to each well was added 20 mL of MTT (5 mg/mL), and the cells were then incubated for 4 h at 37 C. The medium was discarded, and 150 mL of DMSO was added to each well. Light absorbance of each well was measured on a ThermoMax Microplate Reader (Molecular Devices, Oceanside, CA) at a test wavelength of 570 nm. The cell inhibitory rate was calculated as follows: inhibitory rate ¼ (Abs570control cells Abs570treated cells)/Abs570control cells100%. All the assays were performed with three parallel samples. The IC50 value was defined as the drug concentration required to inhibit growth by 50% relative to controls.
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Statistical analysis Values are expressed as the mean ± SD for all treatments. Statistical analysis of the inhibitory effect on tumor growth was performed using one-way analysis of variance. For p values that were 0.05 or less, the difference was considered significant.
Results and discussion Preparation of the nanosuspensions Preparation method In preliminary experiments, several methods were examined for the preparation of GLA nanosuspensions, but the precipitation–ultrasonication method proved to be the most successful due to its reproducibility and minimal residual solvent. Ideal nanosuspensions should meet the following requirements: (1) small size (5200 nm) with a narrow size distribution; (2) stability in normal saline, PBS, rat plasma, or gastrointestinal fluids; (3) the absence of hemolysis; and (4) facile reconstitution after lyophilization (reconstituted nanosuspensions showing no significant change in size or shape). For these purposes, a suitable stabilizer has to be selected from a series of excipients for the preparation of GLA nanosuspensions. The in vitro test demonstrated that GLA nanosuspensions prepared using lecithin and fetal calf serum met all the above requirements, which showed GLA nanosuspensions that were stable in rat plasma and were suitable for i.v. injection. The main stabilizing function of fetal calf serum was believed to be ascribed to the presence of a variety of serum proteins, including bovine serum albumin (BSA), which has been widely used to prepare drug nanoparticles. The way in which BSA or HAS (human serum albumin) stabilize nanoparticles has been reported and reviewed (Kratz, 2008; Elzoghby et al., 2012). Characterization of drug-loaded nanoparticles Particle size and zeta potential of GLA nanosuspensions Figure 2(A) displays the size and size distribution of drugloaded nanoparticles. The results exhibited that the particles were unimodal and had a relatively narrow particle size distribution. The mean particle diameter of GLA nanosuspensions was 143.3 ± 2.9 nm, and its polydispersity index (PDI) was 0.188 ± 0.01. The small PDI value of 0.188 ± 0.01 indicated a narrow size distribution. The zeta potential was 9.86 ± 1.59 mV (n ¼ 3).
In vivo antineoplastic activity of GLA nanosuspensions The in vivo antineoplastic activity of GLA nanosuspensions was assessed using H22-tumor-bearing mice as the animal model. ICR mice were inoculated subcutaneously with H22 liver tumor cells (5 106 cells per mouse) in the axillary region. At 24-h post-inoculation, the H22-bearing mice were randomly assigned to the following four groups (six per group): the HCPT injection group (the positive group, 10 mg/mL), the saline group (negative control), the GLA nanosuspensions group (10 mg/kg), and the free GLA solution group (10 mg/kg). The drugs were administered by injection through the tail vein once every 2 d for 10 d.
Morphology observation by TEM The morphologies of GLA nanosuspensions examined by TEM are shown in Figure 2(B). TEM observation revealed that GLA nanosuspensions were nearly spherical, had smooth surfaces, and were no more than 100 nm in size. No drug crystal or fragment could be seen. The size was smaller than that determined by dynamic light scattering (143.3 ± 2.9 nm) because of the shrinkage of the GLA nanoparticles during TEM sample preparation (Zhang et al., 2007). In other words, the dynamic light scattering determines the hydrodynamic diameter or the ‘‘equivalent sphere diameter’’ in solution,
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Figure 2. Characterization of GLA nanoparticles. (A) Particle size distribution of GLA nanosuspensions (n ¼ 3); (B) TEM micrograph of GLA nanosuspensions ( 50 000); (C) DSC thermograms for the freeze-dried GLA nanosuspensions, coarse drug powders, lecithin, and lyophilized powders of fetal calf serum (top to bottom).
while TEM images were obtained in the absence of the solvent (Morita et al., 2001; Ye et al., 2002). DSC measurement DSC is commonly used to determine the differences in the crystalline state for the physical or chemical changes during the preparation process. Therefore, the DSC of freeze-dried GLA nanosuspensions, GLA, lecithin, and lyophilized powders of fetal calf serum were studied. As shown in Figure 2(C), an endothermic peak corresponding to the melting point of GLA emerged at 223.2 C. The thermograms of lecithin showed an irregular peak near 250 C, which is due to the degradation process, while the melting peak 223.2 C disappeared in the thermogram of GLA nanosuspensions, indicating that there was substantial conversion from the crystalline state to an amorphous state in the GLA nanosuspensions during preparation (Zhao et al., 2010). EE and DL To further evaluate the preparation of the nanoparticles, the drug EE and DL were measured. The prepared GLA nanosuspensions were directly lyophilized for extended storage and were easily reconstituted by vortexing with water. The highest drug concentration in the nanosuspensions
Figure 3. Solubility profiles of the GLA coarse suspensions, GLA nanosuspensions, and GLA solution (n ¼ 3).
was increased to 1 mg/mL. The average EE and DL of the GLA nanosuspensions were 84.57% and 8.95%, respectively. In vitro release behavior The dissolution profiles of the GLA coarse suspensions, GLA nanosuspensions, and GLA solution are shown in Figure 3.
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DOI: 10.3109/10717544.2015.1012311
The drug’s release from the GLA nanosuspensions was fitted to a two-stage exponential kinetic model. The fast release was normally attributed to the non-encapsulated drugs and drugs located at the surface of the particles. A slower and sustained release of GLA followed. For the GLA coarse suspensions, only 42.19% of the applied GLA was dissolved at 8 h, whereas the GLA nanosuspensions showed a significantly enhanced solubility, with 80.36% of the drug dissolved within 8 h, indicting an enormous effect of the nanosuspensions in promoting the solubility of the insoluble drugs. This enhanced solubility was attributed to the higher surface area of the nanoscale drug particles and the decreased diffusion layer thickness (Hintz & Johnson, 1989). In addition, the amorphous or metastable state of GLA in nanosuspensions had a higher internal energy and a greater molecular motion, thus accelerating the GLA solubility. Therefore, the enhancement of the GLA nanosuspension’s solubility may further improve the bioavailability of GLA when oral administration is necessary. In vitro cytotoxicity of GLA nanosuspensions The in vitro anti-cancer cytotoxic activity of GLA nanosuspensions and the free GLA solution against hepatocellular carcinoma cells HepG2 was experimentally determined with an MTT assay at different concentrations of GLA. As shown in Figure 4, both nanosuspensions and free solution inhibited the growth of HepG2 cells in a dose-dependent manner. In comparison with a GLA solution, the IC50 value of GLA nanosuspensions was apparently lower after an identical incubation time, accounting for 1.793 versus 2.884 mg/mL at 24 h, respectively. These data suggested that GLA nanosuspensions were much more effective at inhibiting the growth of the HepG2 cells compared with the GLA solution. It is generally considered that nanoparticles can be nonspecifically internalized into cells via endocytosis or phagocytosis (Lou et al., 2011). In addition, it is possible that the nanoparticles were not actively internalized by cells, but
Figure 4. The cytotoxicity of free GLA solution and GLA nanosuspensions on HepG2 cell lines after a 12-h exposure. The results are expressed as the mean ± SD (n ¼ 6). *p50.05 (nanosuspensions versus solution).
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adsorbed non-specifically by pinocytosis after accumulating on the surface of the cells (Des Rieux et al., 2006). These non-specific internalizations can enhance the interaction between the drug and the cells. In addition, GLA nanosuspensions possessing a markedly increased solubility and induced a sufficient molecular concentration around the cells. Therefore, for GLA nanosuspensions, the increased cytotoxic effect may be a result of the presence of dissolved free GLA, nanoparticles, or the combination of both. In vivo anti-tumor effects in H22-bearing mice The in vivo antitumor effect of GLA nanosuspensions and the GLA solution was assessed using H22 tumor-bearing mice as the animal model. The tumor growth curve measured with Vernier calipers is shown in Figure 5(A). On the 8th day, the average tumor volume in GLA nanosuspension-treated mice had increased relatively slowly and attained only 500 mm3 at 10 mg/kg. However, the average tumor volume in saline-treated mice grew rapidly and reached 1210 mm3. Within 6 d, the free GLA solution at 10 mg/kg did not significantly affect the tumor size compared to the GLA nanosuspensions at the same concentration. However, from the 8th day onwards, the tumor volume of the GLA solution group was obviously larger than that of the GLA nanosuspension group. These findings suggest that the treatment with a GLA nanosuspension displayed stronger tumor inhibition than treatment with the GLA solution at the same dosage. Piloerection and diminished vigor were also observed in the GLA solution treated mice. In stark contrast, mice treated with the GLA nanosuspensions remained vigorous, had a healthy appearance, and were groomed well throughout the entire experiment. Both GLA nanosuspensions and GLA solution yielded significantly enhanced tumor inhibition compared with the negative control group. As shown in Table 1, the inhibition rate of 10 mg/kg of GLA nanosuspensions (54.11%) against the tumor was close to approximately 70% of the HCPT injection (78.46 %) at the same dose, whereas the same dose of the GLA solution also had significant antitumor effects (the inhibition rates were 36.02%). There was no significant difference among the spleen coefficients of various formulations. It was apparent that the GLA nanosuspensions and GLA solution had no effect of immunological suppression on the spleen. However, the body weights of mice (Figure 5B) show that all the mice gained weight during the experiment, but the GLA solution resulted in less weight gain due to the toxicity of the solution. These findings provide strong evidence that GLA is a potent antitumor drug, while GLA nanosuspensions were a suitable drug delivery system for antitumor therapy. This behavior could be explained by the following observations: it has been acknowledged that the nanoparticles could escape from the vasculature through the leaky endothelial tissue that surrounds the tumor and then accumulate in certain solid tumors by the so-called enhanced permeation and retention (EPR) effect (Brannon-Peppas & Blanchette, 2004). Enhanced EPR effects led to the accumulation of GLA nanosuspensions in tumor tissue and might also sustain the therapeutic concentration over time. Additionally, relatively slower drug release made drug in GLA nanoparticles had
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Figure 5. (A) Tumor growth in H22 tumor-bearing mice at 10.0 mg/kg of GLA (n ¼ 6). (B) Relative body weight changes in mice after i.v. administration. The results are expressed as the mean ± SD (n ¼ 6).
Table 1. The in vivo antitumor effects of different formulations of GLA on H22 tumor-bearing mice.
Group Saline HCPT injection GLA nanosuspensions GLA solution
Dose (mg/kg)
Tumor weight (g)
– 10 10 10
1.95 ± 0.11 0.42 ± 0.04** 0.89 ± 0.04**# 1.25 ± 0.18**
Spleen Inhibition coefficient (103) rate (%) N. A. 78.46 54.11 36.02
7.94 ± 0.43 7.36 ± 0.41 7.11 ± 0.42 7.57 ± 0.60
**p50.01 versus the normal saline group. #p50.05 versus the GLA solution group. Each value represents the mean ± SD (n ¼ 6).
system is an effective strategy for achieving the improved therapeutic efficacy of relatively insoluble anticancer drugs.
Declaration of interest The authors are thankful to the National Natural Science Foundation of China for financial support (Project no. 81102813), Open Project for Key Laboratory, Heilongjiang University of Traditional Chinese Medicine (2013kf05) and Beijing Natural Science Foundation (7152099). The authors report no declarations of interest.
References longer circulation time and then more opportunity to reach the tumor target, leading to the prolonged exposure of the tumor to GLA. Many investigations have shown that nanoparticulate drug delivery systems can increase antitumor efficacy while reducing systemic side effects (Brigger et al., 2002; Lou et al., 2009; Mattheolabakis et al., 2009). Because H22 is a hepatic cancer cell line, it is now believed that GLA nanosuspensions will perform well in the treatment of hepatic cancer due to their liver targeting and EPR effects. Nanosuspensions seem to be a feasible strategy for developing the therapeutic potential of poorly soluble drugs, such as GLA.
Conclusion To our knowledge, this is the first report of the preparation of glaucocalyxin A nanosuspensions, including in vitro and in vivo studies. Glaucocalyxin A nanosuspensions formed smooth spheres with a hydrodynamic diameter of approximately 143 nm. Glaucocalyxin A existed in nanosuspensions in the amorphous state. The present study demonstrates that when formulated as nanosuspensions, GLA showed stronger antitumor activity compared with the free drug. Cytotoxicity tests using HepG2 cells showed that GLA nanosuspensions were more cytotoxic than the GLA solution. Similarly, in mice, GLA nanosuspensions were better tolerated and induced noticeable antitumor effects compared with the GLA solution. Therefore, a nanosuspension drug delivery
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Xiang ZB, Xu YX, Shen Y, et al. (2008). Two new diterpenoids from Rabdosia japonica var. Glaucocalyx. Zhongguo Hua Xue Kuai Bao 19:852–4. Xu FM, Hu HP, Wang ZQ. (1996). Studies on diterpenoid constituents in Rabdosia japonica (Burm.f.) Hara. Zhongguo Zhong Yao Za Zhi 21: 678–9. Yang LJ, Zhang J, Shi JW, et al. (2012). Preparation and properties of glaucocalyxin A-loaded self-assemble nanomicelles. Yiyao Dao Bao 31:775–8. Ye Q, Zhang Z, Jia H, et al. (2002). Formation of monodisperse polyacrylamide particles by radiation-induced dispersion polymerization: particle size and size distribution. J Colloid Interface Sci 253: 279–84. Zhang B, Long K. (1993). Effects of glaucocalyxin A on aggregation and cAMP levels of rabbit platelets in vitro. Zhongguo Yao Li Xue Bao 14:347–50. Zhang DR, Ren TC. (2003). The advance in pharmaceutical research on oridonin. Zhongguo Yao Xue Za Zhi 11:817–20. Zhang DY, Li ZY, Shi LY, et al. (2008). Advances in research on cytotoxinic activity of ent-Kaurane diterpenoids. Chin J Org Chem 11: 1911–17. Zhang JX, Han QB, Zhao AH, et al. (2003). Diterpenoids from Isodon japonica. Fitoterapia 74:435–8. Zhang L, Yang M, Wang Q, et al. (2007). 10-Hydroxycamptothecin loaded nanoparticles: preparation and antitumor activity in mice. J Control Release 119:153–62. Zhao YX, Hua HY, Chang M, et al. (2010). Preparation and cytotoxic activity of hydroxycamptothecin nanosuspensions. Int J Pharm 392: 64–71.
RESEARCH ARTICLE
A nanoparticulate drug-delivery system for glaucocalyxin A: formulation, characterization, increased in vitro, and vivo antitumor activity Meihua Han1, Zhitao Li2, Yifei Guo1, Jian Zhang3, and Xiangtao Wang1
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1
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China, 2School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, PR China, and 3College of Pharmaceutical Sciences, Soochow University, Suzhou, PR China Abstract
Keywords
Glaucocalyxin A (GLA) is a phytochemical component with multiple pharmacological activities; however, glaucocalyxin A’s wider use has been restricted by its poor solubility. In this study, GLA nanosuspensions were prepared with precipitation-combined ultrasonication and were characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), and differential scanning calorimetry (DSC). The GLA nanosuspensions were spherical with a smooth surface and a small size of 143 nm, the drug payload achieved 8.95%, and the maximum GLA concentration reached 1 mg/mL. The lyophilized powders for the GLA nanosuspensions were amorphous and displayed a biphasic drug release pattern with an initial burst release and a consequent sustained release. In contrast to the free drug solution, GLA nanosuspensions showed higher in vitro antitumor activity against HepG2 cells (IC50 value of 1.793 versus 2.884 mg/mL at 24 h, p50.01). Meanwhile, nanosuspensions displayed better anticancer efficacy than free GLA on H22 bearing mice (54.11% versus 36.02% tumor inhibition rate). These results indicate that GLA nanosuspensions have great potential for the treatment of hepatic cancer.
Characterization, Glaucocalyxin A nanosuspensions, in vitro antitumor efficacy, in vivo antitumor efficacy, preparation
Introduction Glaucocalyxin A (GLA, Figure 1) is a biologically active entkauranoid (Sun et al., 2006) diterpenoid that is isolated from the ethanol extract of the leaves of Rabdosia japonica (Burm.f.) Hara var. Glaucocalyx (Maxim.) Hara, a traditional Chinese medicinal herb that grows in northeastern China (Zhang & Long, 1993). Glaucocalyx has been used in folk medicine as an anti-bacterial, anti-inflammatory, stomachic, and anthelmintic agent in China (Amit & Ben-Neriah, 2003). Moreover, its anti-cancer activities were proved early in the 1960s (Arai et al., 1963). Many phytochemical studies on this plant have revealed the presence of more than 30 entkauranoids (Xu et al., 1996; Zhang & Ren, 2003; Xiang et al., 2008), including glaucocalyxin A. Glaucocalyxin A is known to have various biological activities, such as inhibition of platelet aggregation induced by platelet-activating factor, immunosuppressive activity (Chen et al., 2006), antioxidative, and cytotoxic activity (Zhang et al., 2008). Address for correspondence: Jian Zhang, College of Pharmaceutical Sciences, Soochow University, No. 199 Ren0 ai Road, Suzhou Industrial Park, Suzhou 215123, PR China. E-mail: [email protected]. Mr. Xiangtao Wang, Professor, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, PR China. Tel: +86 10 57833266. E-mail: [email protected]
History Received 17 December 2014 Revised 21 January 2015 Accepted 22 January 2015
However, the poor solubility of GLA in water has presented a serious obstacle for its practical use as a therapeutic agent. The usual approach for overcoming the problem of solubility is to prepare the formulation using hydroxyproply-b-cyclodextrin, DMSO, and methanol (Shang et al., 2011; Li et al., 2013; Wei et al., 2013). However, adverse effects, such as inflammation of the blood vessels and topical pain caused by delivery of miscible solvents by injection, negate its clinical utility (Zhang et al., 2003). As a consequence, there is a growing need for developing a suitable formulation of GLA that can overcome this problem. Recently, the carrier-free drug nanosuspensions have become an extensively utilized protocol to tackle formulation problems for insoluble compounds (Mu¨ller et al., 2001; Patravale et al., 2004). The most outstanding feature of drug nanosuspensions is the significantly increased drug solubility and dissolution velocity that can be achieved due to the small particle size. Nanosuspensions with a small particle size can be delivered to specific sites by size-dependent, ‘‘passive’’ targeting. It has been reported that nanosuspensions injected intravenously were taken up by the reticuloendothelial system in the liver after only a few minutes as a result of the opsonization process. When the diameter is less than 200 nm, these nanosuspensions can be captured easily by the reticuloendothelial system, especially by the Kupffer cells in the liver. In addition, the aqueous nanosuspensions, which are
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Preparation of GLA nanosuspensions GLA nanosuspensions were prepared using the precipitationultrasonication method. Briefly, GLA and lecithin were dispersed into 1 mL of alcohol to form an organic solution. Afterwards, 1 mL of the organic solution was added into 5 mL of an aqueous solution containing fetal calf serum (20%) drop by drop under continuous ultrasonication (250 W). The resultant GLA nanosuspensions were evaporated under vacuum at 40 C until no residual alcohol remained. Figure 1. The chemical structure of glaucocalyxin A.
Characterization of GLA nanosuspensions
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Particle size and zeta potential of GLA nanosuspensions dispersed in water, can mitigate the side effects caused by miscible solvents (Wang et al., 2010). There are two reports about nanoparticulates of GLA (Cao et al., 2008; Yang et al., 2012); however, the drug payload mentioned in one report was 5.36% and there was no research on the in vitro and in vivo antitumor activity of the resultant GLA nanoparticles in the two reports. In order to obtain higher drug-loaded formulation, in this study, GLA nanosuspensions with good stability were firstly successfully prepared, in which the drug-loading content achieved 8.95%, and GLA nanoparticles demonstrated improved in vitro and in vivo antitumor activity than free GLA, thus have great potential for cancer treatment.
Materials and methods Materials Glaucocalyxin A (98% pure) was kindly supplied by the School of Pharmaceutical Sciences, Soochow University; lecithin (Injection grade) was purchased from Guangzhou Hanfang Modern Chinese Medicine Research and Development Co., Ltd (Guangzhou, China); and fetal calf serum was obtained from Yuanhengjinma Biotechnology Development Co., Ltd. (Beijing, China). Hydroxycamptothecin (HCPT) injection (used as the sodium carboxylate in China) was obtained from Shenghe Pharmaceutical Ltd (Sichuan, China). The water used in the experiments was deionized, and all other organic solvents were of the highest commercially available grade. Animals and cell line Female ICR mice (20 ± 2 g) were supplied by Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All the animals were acclimatized at a temperature of 25 ± 2 C and a relative humidity of 70 ± 5% under natural light/dark conditions for 1 week and provided food and water ad libitum. All experimental procedures were performed in accordance with the Guidelines for Ethical and Regulatory for Animal Experiments as defined by The Institute of Medicinal Plant Development (IMPLAD), China. The human hepatoma cell line HepG2 was supplied by the Department of Pharmacology, Beijing Normal University; The H22 cell line (murine sarcoma) was obtained from the Xiyuan Hospital, China Academy of Traditional Chinese Medicine.
The average particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were determined using the dynamic light scattering (DLS) (Zetasizer Nano ZS 90, Malvern Instruments, Malvern, UK). All measurements were made at least in triplicates. Morphology observation by transmission electron microscope (TEM) The morphologies of GLA nanosuspensions were observed using a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan). A drop of nanosuspensions was spread on a 200-mesh copper grid and negatively stained with 2% (w/v) phosphotungstic acid for 30 s. The grid was allowed to dry further for 10 min and was then examined with the electron microscope. Differential scanning calorimetry (DSC) measurement The thermal properties of the freeze-dried powder of GLA nanosuspensions, GLA, lecithin, and fetal calf serum were investigated using a differential scanning calorimeter (DSC Q200, TA Co., Cambridge, MA). The scanning temperature for each sample was set from 0 to 400 C with a heating rate of 10 C/min. Five to eight milligrams of each sample were placed in perforated aluminum-sealed pans, and a blank pan was used as the reference. Entrapment efficiency (EE) and drug loading (DL) The drug EE and DL amount of GLA nanosuspensions were determined according to the method of centrifugation ultrafiltration (Dai et al., 2010). Generally, GLA nanosuspensions were ultracentrifuged by OptimaÔ L-80 XP Ultracentrifuge (Beckman Coulter, Fullerton, CA) at 50 000 rpm for 60 min. The supernatant was sampled, and the concentration of GLA in the supernatant was determined by a reversed-phase high-performance liquid chromatography (HPLC) method. A 20-mL sample solution was injected at least three times into the chromatographic column. The mobile phase was a mixture of water and acetonitrile in the volume ratio of 10:90. The elution rate was 0.3 mL/min, and the GLA detection wavelength was set at 231 nm. The standard curve for the quantification of GLA was linear over the range of 0.1–100 mg/mL with a correlation coefficient of 0.9999. In vitro release behavior To measure the dissolution kinetics, 1 mL (1 mg/mL) of the GLA nanosuspensions, GLA coarse suspensions (made by
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suspending GLA bulk powder in an aqueous solution), and GLA solutions (GLA solutions were prepared by dissolution of GLA in DMSO and then sequential additions of polyoxyethylene hydrogenated castor oil, Tween 80 and physiological saline (DMSO:polyoxyethylene hydrogenated castor oil:Tween 80:physiological saline 0.5:0.5:0.5:8.5, v/v/ v/v)) was placed in a preswelled dialysis bag (molecular cutoff ¼ 8000–14 000). The dialysis bag was then immersed fully in 30 mL of purified water as the release medium and kept in an incubator at 37 ± 0.5 C with stirring at 100 rpm. One milliliter of the release medium was withdrawn at specific time intervals and replaced with an equal volume of fresh release medium. The samples were centrifuged, and the resulting supernatant was analyzed by HPLC. The release medium was replaced every 12 h. All the experiments were performed in triplicate. In vitro cytotoxicity of GLA nanosuspensions against HepG2 cells Cell culture The human hepatoma cell line HepG2 was cultured in RPMI1640 containing 10% fetal bovine serum, 100 U/ penicillin and 100 mg/mL streptomycin at 37 C in a humidified atmosphere containing 5% CO2. MTT assay The cell viability was assessed using a MTT assay. Cells at a density of 5 103 cells/well were seeded in 96-well culture plates with RPMI1640 supplemented with 10% fetal bovine serum. After a 24-h incubation, the medium was replaced by GLA nanosuspensions and a free GLA control solution at various concentration for indicated time periods. The crude drug control group was treated with the same volume of culture media with 0.2% DMSO (v/v). Subsequently, to each well was added 20 mL of MTT (5 mg/mL), and the cells were then incubated for 4 h at 37 C. The medium was discarded, and 150 mL of DMSO was added to each well. Light absorbance of each well was measured on a ThermoMax Microplate Reader (Molecular Devices, Oceanside, CA) at a test wavelength of 570 nm. The cell inhibitory rate was calculated as follows: inhibitory rate ¼ (Abs570control cells Abs570treated cells)/Abs570control cells100%. All the assays were performed with three parallel samples. The IC50 value was defined as the drug concentration required to inhibit growth by 50% relative to controls.
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Statistical analysis Values are expressed as the mean ± SD for all treatments. Statistical analysis of the inhibitory effect on tumor growth was performed using one-way analysis of variance. For p values that were 0.05 or less, the difference was considered significant.
Results and discussion Preparation of the nanosuspensions Preparation method In preliminary experiments, several methods were examined for the preparation of GLA nanosuspensions, but the precipitation–ultrasonication method proved to be the most successful due to its reproducibility and minimal residual solvent. Ideal nanosuspensions should meet the following requirements: (1) small size (5200 nm) with a narrow size distribution; (2) stability in normal saline, PBS, rat plasma, or gastrointestinal fluids; (3) the absence of hemolysis; and (4) facile reconstitution after lyophilization (reconstituted nanosuspensions showing no significant change in size or shape). For these purposes, a suitable stabilizer has to be selected from a series of excipients for the preparation of GLA nanosuspensions. The in vitro test demonstrated that GLA nanosuspensions prepared using lecithin and fetal calf serum met all the above requirements, which showed GLA nanosuspensions that were stable in rat plasma and were suitable for i.v. injection. The main stabilizing function of fetal calf serum was believed to be ascribed to the presence of a variety of serum proteins, including bovine serum albumin (BSA), which has been widely used to prepare drug nanoparticles. The way in which BSA or HAS (human serum albumin) stabilize nanoparticles has been reported and reviewed (Kratz, 2008; Elzoghby et al., 2012). Characterization of drug-loaded nanoparticles Particle size and zeta potential of GLA nanosuspensions Figure 2(A) displays the size and size distribution of drugloaded nanoparticles. The results exhibited that the particles were unimodal and had a relatively narrow particle size distribution. The mean particle diameter of GLA nanosuspensions was 143.3 ± 2.9 nm, and its polydispersity index (PDI) was 0.188 ± 0.01. The small PDI value of 0.188 ± 0.01 indicated a narrow size distribution. The zeta potential was 9.86 ± 1.59 mV (n ¼ 3).
In vivo antineoplastic activity of GLA nanosuspensions The in vivo antineoplastic activity of GLA nanosuspensions was assessed using H22-tumor-bearing mice as the animal model. ICR mice were inoculated subcutaneously with H22 liver tumor cells (5 106 cells per mouse) in the axillary region. At 24-h post-inoculation, the H22-bearing mice were randomly assigned to the following four groups (six per group): the HCPT injection group (the positive group, 10 mg/mL), the saline group (negative control), the GLA nanosuspensions group (10 mg/kg), and the free GLA solution group (10 mg/kg). The drugs were administered by injection through the tail vein once every 2 d for 10 d.
Morphology observation by TEM The morphologies of GLA nanosuspensions examined by TEM are shown in Figure 2(B). TEM observation revealed that GLA nanosuspensions were nearly spherical, had smooth surfaces, and were no more than 100 nm in size. No drug crystal or fragment could be seen. The size was smaller than that determined by dynamic light scattering (143.3 ± 2.9 nm) because of the shrinkage of the GLA nanoparticles during TEM sample preparation (Zhang et al., 2007). In other words, the dynamic light scattering determines the hydrodynamic diameter or the ‘‘equivalent sphere diameter’’ in solution,
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Figure 2. Characterization of GLA nanoparticles. (A) Particle size distribution of GLA nanosuspensions (n ¼ 3); (B) TEM micrograph of GLA nanosuspensions ( 50 000); (C) DSC thermograms for the freeze-dried GLA nanosuspensions, coarse drug powders, lecithin, and lyophilized powders of fetal calf serum (top to bottom).
while TEM images were obtained in the absence of the solvent (Morita et al., 2001; Ye et al., 2002). DSC measurement DSC is commonly used to determine the differences in the crystalline state for the physical or chemical changes during the preparation process. Therefore, the DSC of freeze-dried GLA nanosuspensions, GLA, lecithin, and lyophilized powders of fetal calf serum were studied. As shown in Figure 2(C), an endothermic peak corresponding to the melting point of GLA emerged at 223.2 C. The thermograms of lecithin showed an irregular peak near 250 C, which is due to the degradation process, while the melting peak 223.2 C disappeared in the thermogram of GLA nanosuspensions, indicating that there was substantial conversion from the crystalline state to an amorphous state in the GLA nanosuspensions during preparation (Zhao et al., 2010). EE and DL To further evaluate the preparation of the nanoparticles, the drug EE and DL were measured. The prepared GLA nanosuspensions were directly lyophilized for extended storage and were easily reconstituted by vortexing with water. The highest drug concentration in the nanosuspensions
Figure 3. Solubility profiles of the GLA coarse suspensions, GLA nanosuspensions, and GLA solution (n ¼ 3).
was increased to 1 mg/mL. The average EE and DL of the GLA nanosuspensions were 84.57% and 8.95%, respectively. In vitro release behavior The dissolution profiles of the GLA coarse suspensions, GLA nanosuspensions, and GLA solution are shown in Figure 3.
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The drug’s release from the GLA nanosuspensions was fitted to a two-stage exponential kinetic model. The fast release was normally attributed to the non-encapsulated drugs and drugs located at the surface of the particles. A slower and sustained release of GLA followed. For the GLA coarse suspensions, only 42.19% of the applied GLA was dissolved at 8 h, whereas the GLA nanosuspensions showed a significantly enhanced solubility, with 80.36% of the drug dissolved within 8 h, indicting an enormous effect of the nanosuspensions in promoting the solubility of the insoluble drugs. This enhanced solubility was attributed to the higher surface area of the nanoscale drug particles and the decreased diffusion layer thickness (Hintz & Johnson, 1989). In addition, the amorphous or metastable state of GLA in nanosuspensions had a higher internal energy and a greater molecular motion, thus accelerating the GLA solubility. Therefore, the enhancement of the GLA nanosuspension’s solubility may further improve the bioavailability of GLA when oral administration is necessary. In vitro cytotoxicity of GLA nanosuspensions The in vitro anti-cancer cytotoxic activity of GLA nanosuspensions and the free GLA solution against hepatocellular carcinoma cells HepG2 was experimentally determined with an MTT assay at different concentrations of GLA. As shown in Figure 4, both nanosuspensions and free solution inhibited the growth of HepG2 cells in a dose-dependent manner. In comparison with a GLA solution, the IC50 value of GLA nanosuspensions was apparently lower after an identical incubation time, accounting for 1.793 versus 2.884 mg/mL at 24 h, respectively. These data suggested that GLA nanosuspensions were much more effective at inhibiting the growth of the HepG2 cells compared with the GLA solution. It is generally considered that nanoparticles can be nonspecifically internalized into cells via endocytosis or phagocytosis (Lou et al., 2011). In addition, it is possible that the nanoparticles were not actively internalized by cells, but
Figure 4. The cytotoxicity of free GLA solution and GLA nanosuspensions on HepG2 cell lines after a 12-h exposure. The results are expressed as the mean ± SD (n ¼ 6). *p50.05 (nanosuspensions versus solution).
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adsorbed non-specifically by pinocytosis after accumulating on the surface of the cells (Des Rieux et al., 2006). These non-specific internalizations can enhance the interaction between the drug and the cells. In addition, GLA nanosuspensions possessing a markedly increased solubility and induced a sufficient molecular concentration around the cells. Therefore, for GLA nanosuspensions, the increased cytotoxic effect may be a result of the presence of dissolved free GLA, nanoparticles, or the combination of both. In vivo anti-tumor effects in H22-bearing mice The in vivo antitumor effect of GLA nanosuspensions and the GLA solution was assessed using H22 tumor-bearing mice as the animal model. The tumor growth curve measured with Vernier calipers is shown in Figure 5(A). On the 8th day, the average tumor volume in GLA nanosuspension-treated mice had increased relatively slowly and attained only 500 mm3 at 10 mg/kg. However, the average tumor volume in saline-treated mice grew rapidly and reached 1210 mm3. Within 6 d, the free GLA solution at 10 mg/kg did not significantly affect the tumor size compared to the GLA nanosuspensions at the same concentration. However, from the 8th day onwards, the tumor volume of the GLA solution group was obviously larger than that of the GLA nanosuspension group. These findings suggest that the treatment with a GLA nanosuspension displayed stronger tumor inhibition than treatment with the GLA solution at the same dosage. Piloerection and diminished vigor were also observed in the GLA solution treated mice. In stark contrast, mice treated with the GLA nanosuspensions remained vigorous, had a healthy appearance, and were groomed well throughout the entire experiment. Both GLA nanosuspensions and GLA solution yielded significantly enhanced tumor inhibition compared with the negative control group. As shown in Table 1, the inhibition rate of 10 mg/kg of GLA nanosuspensions (54.11%) against the tumor was close to approximately 70% of the HCPT injection (78.46 %) at the same dose, whereas the same dose of the GLA solution also had significant antitumor effects (the inhibition rates were 36.02%). There was no significant difference among the spleen coefficients of various formulations. It was apparent that the GLA nanosuspensions and GLA solution had no effect of immunological suppression on the spleen. However, the body weights of mice (Figure 5B) show that all the mice gained weight during the experiment, but the GLA solution resulted in less weight gain due to the toxicity of the solution. These findings provide strong evidence that GLA is a potent antitumor drug, while GLA nanosuspensions were a suitable drug delivery system for antitumor therapy. This behavior could be explained by the following observations: it has been acknowledged that the nanoparticles could escape from the vasculature through the leaky endothelial tissue that surrounds the tumor and then accumulate in certain solid tumors by the so-called enhanced permeation and retention (EPR) effect (Brannon-Peppas & Blanchette, 2004). Enhanced EPR effects led to the accumulation of GLA nanosuspensions in tumor tissue and might also sustain the therapeutic concentration over time. Additionally, relatively slower drug release made drug in GLA nanoparticles had
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Figure 5. (A) Tumor growth in H22 tumor-bearing mice at 10.0 mg/kg of GLA (n ¼ 6). (B) Relative body weight changes in mice after i.v. administration. The results are expressed as the mean ± SD (n ¼ 6).
Table 1. The in vivo antitumor effects of different formulations of GLA on H22 tumor-bearing mice.
Group Saline HCPT injection GLA nanosuspensions GLA solution
Dose (mg/kg)
Tumor weight (g)
– 10 10 10
1.95 ± 0.11 0.42 ± 0.04** 0.89 ± 0.04**# 1.25 ± 0.18**
Spleen Inhibition coefficient (103) rate (%) N. A. 78.46 54.11 36.02
7.94 ± 0.43 7.36 ± 0.41 7.11 ± 0.42 7.57 ± 0.60
**p50.01 versus the normal saline group. #p50.05 versus the GLA solution group. Each value represents the mean ± SD (n ¼ 6).
system is an effective strategy for achieving the improved therapeutic efficacy of relatively insoluble anticancer drugs.
Declaration of interest The authors are thankful to the National Natural Science Foundation of China for financial support (Project no. 81102813), Open Project for Key Laboratory, Heilongjiang University of Traditional Chinese Medicine (2013kf05) and Beijing Natural Science Foundation (7152099). The authors report no declarations of interest.
References longer circulation time and then more opportunity to reach the tumor target, leading to the prolonged exposure of the tumor to GLA. Many investigations have shown that nanoparticulate drug delivery systems can increase antitumor efficacy while reducing systemic side effects (Brigger et al., 2002; Lou et al., 2009; Mattheolabakis et al., 2009). Because H22 is a hepatic cancer cell line, it is now believed that GLA nanosuspensions will perform well in the treatment of hepatic cancer due to their liver targeting and EPR effects. Nanosuspensions seem to be a feasible strategy for developing the therapeutic potential of poorly soluble drugs, such as GLA.
Conclusion To our knowledge, this is the first report of the preparation of glaucocalyxin A nanosuspensions, including in vitro and in vivo studies. Glaucocalyxin A nanosuspensions formed smooth spheres with a hydrodynamic diameter of approximately 143 nm. Glaucocalyxin A existed in nanosuspensions in the amorphous state. The present study demonstrates that when formulated as nanosuspensions, GLA showed stronger antitumor activity compared with the free drug. Cytotoxicity tests using HepG2 cells showed that GLA nanosuspensions were more cytotoxic than the GLA solution. Similarly, in mice, GLA nanosuspensions were better tolerated and induced noticeable antitumor effects compared with the GLA solution. Therefore, a nanosuspension drug delivery
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