Development of a Liposomal Formulation of Acetyltanshinone IIA for

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Development of a Liposomal Formulation of Acetyltanshinone IIA for Breast Cancer Therapy Qi Wang,† Man Luo,† Na Wei,† Alex Chang,‡ and Kathy Qian Luo*,§ †

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 Department of Oncology, Johns Hopkins Singapore, Singapore 308433 § Faculty of Health Sciences, University of Macau, Taipa, Macau, China ‡

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ABSTRACT: Acetyltanshinone IIA (ATA), synthesized in our group exhibiting good anti-breast cancer effects, is expected to replace the commonly used anti-ER+ breast cancer (breast cancer cells overexpressing the estrogen receptor) drug tamoxifen. To promote the clinical progress of ATA, polyethylene glycol (PEG)modified liposomes were used to encapsulate ATA along with improving its bioavailability and in vivo anticancer efficiency. The resulting liposomal ATA exhibited a spherical shape with an average size of 188.5 nm. In vitro evaluations showed that liposomal ATA retained the anti-breast cancer efficacy of ATA while exerting much less cytotoxicity toward noncancerous cells. Significantly, pharmacokinetics analysis showed that the AUC0−24h of liposomal ATA was 59 times higher than that of free ATA, demonstrating increased bioavailability of ATA. Preclinical experiments demonstrated that liposomal ATA reduced the growth of ER-positive human breast tumor xenografts by 73% in nude mice, and the liposomal ATA exhibited a much lower level of toxicity than that of free ATA with respect to zebrafish larval mortality, body formation, and heart function during development. Moreover, 7-day and 21-day tissue toxicity levels were determined in mice by intravenous administration of a maximum dosage of liposomal ATA (120 mg/kg). The results showed no obvious tissue damage in major organs, including the heart, liver, spleen, kidney, and brain. In summary, we have developed a clinical formulation of liposomal ATA with the high bioavailability and potent efficacy for the treatment of ER-positive breast cancer. KEYWORDS: anti-breast cancer drugs, acetyltanshinone IIA (ATA), mPEG-liposomes, bioavailability, pharmacokinetics, toxicity study

1. INTRODUCTION Breast cancer has become a major public health problem in the current society and suffered from the resistance of frequently used anti-breast cancer drugs including tamoxifen. Therefore, a new chemotherapeutic agent for more effective breast cancer treatment has been urgently needed recently. Tanshinone IIA (TIIA), one of the major hydrophobic components of Danshen,1−3 has received major attention from the scientists for the high anticancer activity.4 Deriving from TIIA, acetyltanshinone IIA (ATA) was synthesized in our group and was further approved to have high anti-breast cancer efficacy.5 Specifically, ATA displayed a strong growthinhibitory effect on estrogen receptor-positive (ER+) breast cancer cells through degrading the ERα protein.6 More significantly, ATA produced a more potent growth inhibitory activity toward ER+ breast cancer cells than that of tamoxifen, which is a commonly used anti-ER+ breast cancer drug.6 In light of improved therapeutic effects, it is of great potential to develop ATA into a new anti-breast cancer drug. However, our initial pharmacokinetic study showed that ATA was quickly removed from the plasma in both mouse and rats. © XXXX American Chemical Society

Encapsulated by poly(ethylene glycol) methyl ether-blockpoly(lactide-co-glycolide) (mPEG-PLGA), the 24 h bloodcirculation time of ATA was extended to 10-fold that of unbound ATA.7 However, the majority of the ATA was trapped in red blood cells rather than in plasma. Herein, it is urgent to develop ATA into a clinical formulation to promote its clinical process. In this report, Federal Drug Administration-approved liposomes have attracted our attention that the liposome-based formulations represented excellent biocompatibility and improved bioavailability in vivo.8−12 Also, a conventional liposome is a mature and widely used intravenous dosage form among all the contemporary formulations,8−12 which ensures the further clinical evaluation of liposomal ATA. In this report, the main constituents of liposomal ATA are phospholipids and cholesterol, which are components of the mammalian cell Received: May 6, 2019 Revised: July 24, 2019 Accepted: July 26, 2019

A

DOI: 10.1021/acs.molpharmaceut.9b00493 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

Figure 1. (A) Structural features of liposomal ATA. ATA molecules were encapsulated in the lipid bilayer of the liposome. The PEG chain is located on the surface of the liposome. (B) SEM images. The liposomal ATA was uniformly distributed. (C) Size distributions. The average size of liposomal ATA was 188.5 nm based on intensity. (D) Zeta potential. The zeta potential of liposomal ATA was −31.0 mV.

membrane, making them compatible with biological systems. On the other hand, poly(ethylene glycol) methyl ether (PEG)modified phospholipids were utilized to prevent the engulfment of liposomes by blood cells (Figure 1A). Also, PEG chains are expected to increase the circulation time of liposomal ATA in vivo, thus improving its bioavailability. Furthermore, based on previous reports, we hypothesized that the manufacture of liposomal ATA with a smaller particle size might facilitate its ability to reach tumor tissues through an enhanced permeability and retention (EPR) effect, resulting in an enhanced antitumor effect.13−16 The liposomal ATA was prepared using a reverseevaporation method and was optimized through an orthogonal array test generating the drug loading efficiency of 12.35% with the size of 188.5 nm. The liposomal ATA formulation could be stably stored at 4 °C and diluted in 5% glucose in clinical storage and use. Specifically, important preclinical experiments were conducted in this report including pharmacokinetic profiles, anti-breast cancer efficacy, cytotoxicity, and smallanimal toxicity to pave the way for clinical use of liposomal ATA. The information obtained from this study can be used to

guide the future manufacturing and development of liposomal ATA into a new anti-breast cancer drug.

2. MATERIALS AND METHODS 2.1. Materials. Pure phosphatidylcholine (PC3) and 1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG) were obtained from LIPOID Pte. Ltd. (Singapore). Cholesterol, sodium dodecyl sulfonate (SDS), Tween 80, dialysis sacks (Mw cutoff of 12,000 kDa), and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Chloroform (CHCl3) and methanol (MeOH) were obtained from Merck Pte. Ltd. (Singapore). Dulbecco’s Minimum Essential Medium (DMEM), penicillin/streptomycin (PS), and trypsin were purchased from GIBCO. Fetal bovine serum (FBS) was purchased from HyClone. Human umbilical vein endothelial cells (HUVECs) and MCF-7 breast cancer cells were obtained from the ATCC (Manassas, VA). 2.2. Preparation and Characterization of ATA-Loaded PEG-Liposomes (Liposomal ATA). A reverse-evaporation B


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buffered saline (PBS, pH 7.4) and incubated at 4 or 37 °C for 1−7 days. Under all conditions, 1 mL of the suspension was collected at designated intervals; the size and zeta potential were detected using a laser electrophoretic light scatter analyzer (Malvern Nano-ZS Particle Sizer), and the EE% was measured using HPLC. 2.6. Drug Release from Liposomes. The release of ATA from liposomes was investigated using dialysis and compared with the release of free ATA in the release medium (ultrapure water with 1% SDS). Six milliliters of the release medium containing an equivalent amount of liposomal ATA or free ATA was placed in a dialysis sack (MWCO 12,000, Sigma). The sack was then immersed in 50 mL of the release medium with gentle stirring at 37 °C. The samples (2 mL) were removed from the sack at designated time intervals, and an equal volume of the release medium was replenished at the same time.22−25 The amount of released drug was determined by RP-HPLC as described in Section 2.3: “Determination of Encapsulation Efficiency and the Drug Loading Rate”. The similarity of the release profiles between the free ATA and liposomal ATA was evaluated by the similarity factor (f 2) shown below

method was used to prepare the liposomal ATA. First, PC3 (120.8 mg), ATA (29.9 mg), DSPE-PEG (39.9 mg), and cholesterol (52.8 mg) were dissolved in chloroform (CHCl3, 5 mL). Distilled (DI) H2O (1.5 mL) was then added to the solution and treated with ultrasound (37% strength) for 3 min. CHCl3 was then removed by rotary evaporation. Finally, DI H2O (10 mL) was added to rehydrate it to obtain liposomal ATA, and the liposomal ATA was ultrasonicated for 8 min to produce a smaller particle size. Blank liposomes were prepared using the same method but without the addition of ATA to the formulation. The size distribution and zeta potential of the liposomes were determined using a laser electrophoretic light scatter analyzer (Malvern Nano-ZS Particle Sizer). The morphology was determined by scanning electron microscopy (SEM) (JSM6700-FESEM, JEOL). Briefly, liposome samples were spread onto the surface of a carbon tape and then sputtercoated with gold under a vacuum. Afterward, the prepared samples were observed under a scanning electron microscope using magnification ranges of 5000−20000 with an electron beam energy of 20 kV. 2.3. Determination of Encapsulation Efficiency and the Drug Loading Rate. A solution of liposomal ATA (1 mL) was centrifuged at a low speed of 500 rpm at 28 °C for 5 min to remove large particles. A fixed amount of the supernatant containing liposomal ATA was diluted with methanol, which will dissolve the liposome structure to release ATA to the desired concentration, and the concentration of ATA in the supernatant was referred to as the concentration of the encapsulated drug. The concentration of ATA was measured simultaneously by RP-HPLC analysis (Shimadzu LC-20AT pump liquid chromatograph; chromatographic column: Kromasil 100-5C8, 250 mm × 4.6 mm, 5 μm). A mobile phase system consisting of methanol−H2O (80:20, v:v) was pumped at a flow rate of 1 mL/min with a column temperature of 30 °C, and the column eluents were monitored at a wavelength of 254 nm.7,17,18 The drug loading rate (DL%) and percentage of encapsulation efficiency (EE%) were calculated as follows:

ÄÅ l o Å o 1 oÅÅÅ ÅÅ1 + f 2 = 50logm o Å o n Å oÅÇ n

ÉÑ−0.5 | o ÑÑ o o Ñ ( R T ) 100 − × } ∑ t t ÑÑ o o Ñ o ÑÖ t=1 ~ n

2Ñ Ñ

where n is the number of time points, Rt is the release value of liposomal ATA at time t, and Tt is the release value of free ATA.26,27 2.7. Determination of the Pharmacokinetic Profile of Liposomal ATA in Rats. Female SD rats (INVIVOS, Singapore) were randomly divided into two groups (n = 6): free ATA and liposomal ATA. Free ATA was suspended in a solution of PEG300:ethanol:Tween 80 (60:25:15, v/v/v). The free ATA suspension or liposomal ATA was administered at an equivalent dose of ATA at 20 mg/kg body weight via a single intravenous injection in the tail vain. At different time points (15 min, 30 min, and 1, 2, 3, 4, 5, 6, 8, and 24 h), blood samples (0.3 mL) were collected from the suborbital vein and placed in heparinized tubes. The blood samples were centrifuged at 3000 rpm at 4 °C for 10 min to collect plasma. The samples were then pretreated and injected into RP-HPLC for detection of ATA as described in a previous work.6 Microsoft Excel PKsolver was used to analyze the pharmacokinetic data. 2.8. Cell Culture. HUVECs and MCF-7 cells were cultured in a DMEM-based culture medium supplemented with 1% penicillin/streptomycin and 10% FBS. Cell cultures were maintained at 37 °C in a humidified incubator supplemented with 5% CO2. 2.9. Determination of the Anticancer Efficacy and Cytotoxicity of Liposomal ATA in Vitro. The MTT reduction ability was determined as an index of the metabolic activity of the mitochondria, which can indicate cell viability. Briefly, MCF-7 cells or HUVECs were seeded in 96-well plates and cultured overnight. Free ATA (dissolved in DMSO diluted in DMEM), liposomal ATA, and blank liposomes were added at different concentrations, and the cells were incubated for 24 h. MTT solution (10 μL; 5 mg/mL) was then added into each well, and they were incubated for 4 h at 37 °C in the CO2 incubator. One hundred microliters of the solubilization solution containing 1% SDS and 0.1% hydrochloric acid was

DL% = amount of drug in liposomes /amount of feeding materials EE% = amount of drug in liposomes /amount of feeding drug

2.4. Thermal Analyses of Liposomal ATA. Thermal analyses that include thermogravimetric (TG) investigation and differential thermal analysis (DTA) were performed using Diamond TG/DTA (PerkinElmer instrument).19−21 The thermal decomposition was conducted on free ATA, PC3, cholesterol, DSPE-PEG, a physical mixture of the four components, and freeze-dried liposomal ATA. Approximately 2 mg of a vacuum-dried coal sample with an average particle diameter less than 200 μm was placed in the sample crucible. The sample was heated in pure N2 from 30 to 400 °C at a heating rate of 10 °C/min. The melting points of the samples were determined from thermal graphs. 2.5. Storage Stability of Liposomal ATA. The storage stability of liposomal ATA was investigated by measuring the size, zeta potential, and EE% of liposomal ATA during the experiment. Liposomal ATA was diluted using phosphateC


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zebrafish were used for each group in each experiment, and the zebrafish experiments were carried out at least four times. 2.11.3. Malformation and Mortality Rates of Zebrafish Embryos Exposed to Liposomal ATA. To examine the effects of free ATA and liposomal ATA on embryos during development, several exposure durations were chosen. These durations included 6−10 hpf (gastrula period), 14−24 h (important organ formation stage), 10−24 hpf (somite stage), 24−48 hpf (pharyngeal phase), and 48−72 hpf (the incubation period). During each period, embryos were exposed to free ATA, liposomal ATA, or blank liposomes at the equivalent concentration of 5 μM. At 96 hpf, larvae or embryos were microscopically examined to determine the malformation and mortality rates. The heart rate was also recorded at 24, 72, and 96 hpf.28−31 N = 20 fish/group. 2.12. Toxicity Evaluation of Liposomal ATA on Mice. BALB/C mice purchased from (INVIVOS, Singapore) were used to evaluate the toxicity of ATA and liposomal ATA in vivo. The maximum dosage of liposomal ATA was determined to be 120 mg/kg body weight as this is the maximum amount of liposomal ATA that could be prepared in a 0.2 mL volume for intravenous injection. Twenty mice (10 female and 10 male) were used in this study. A solution of 0.2 mL liposomal ATA at a dose of 120 mg/kg body weight was intravenously injected into the tail vain of each mouse. The behaviors of the mice were observed, and the body weight was recorded after administration every other day. The mice were then sacrificed after 7 days (5 female and 5 male) or 21 days (5 female and 5 male), and different organs such as the heart, liver, spleen, lung, kidney, and brain were collected, fixed in 4% paraformaldehyde, and processed for H&E staining. The damage in each organ was assessed and imaged to record any pathological changes. The maximal dosage of free ATA [dissolved in PEG300:ethanol:tween 80 = 60:25:15 (v/v/v) as a stock solution then diluted using 0.9% NaCl at a volume ratio of 1:9 before injection] at 30 mg/kg body weight was also injected into BALB/C mice to evaluate the toxicity of free ATA as a control.32 2.13. Statistical Analysis. Statistical analysis was performed using SPSS 10.0 software. Descriptive data were expressed as the arithmetic mean value plus or minus the standard deviation. Statistics were performed for all comparisons. All quantitative results were obtained from at least triplicate samples. A t-test was applied to detect differences between groups. In all evaluations, *p < 0.05 was considered statistically significant.

added to each well to dissolve the formazan crystals. After 8 h, the absorbance of the solubilized MTT formazan product was measured using a spectrophotometer at an absorbance wavelength of 595 nm, and the cell viability was calculated from the optical density (OD) value of the tested sample and the vehicle control (untreated cells) using the following equation: cell viability (%) = OD tested sample/OD vehicle control × 100. Nine sets of data obtained in triplicate from three independent experiments were averaged to generate the final data with the standard deviation, which are presented as the mean ± SD. 2.10. Determination of the Antitumor Effect of Liposomal ATA in Vivo. This mouse experiment was conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee, Nanyang Technological University, Singapore. To ensure that the estrogen receptor-positive breast cancer MCF-7 cells could grow into solid tumors in female mice, a tablet of 1.5 mg βestradiol 17-acetate was implanted subcutaneously seven days prior to tumor inoculation into female nude mice (18−22 g, 6−8 weeks of age). MCF-7 cells (5 × 106) were mixed with Matrigel in a final volume of 0.2 mL and subcutaneously injected into the armpit of the mouse. After the tumor reached a minimum volume of 25 mm3, an aliquot (200 μL) of PBS, blank liposomes, free ATA, or liposomal ATA (20 mg ATA/kg) was intravenously injected into the tumor-bearing nude mice (n = 5 per group) every other day. The tumor size and the weight of the nude mouse were measured and recorded during the treatment period. The tumor volume was determined by measuring its length and width and calculated using the following formula: V = (L × W2)/2 where L is the longest dimension parallel to the skin surface, and W is the dimension perpendicular to L and parallel to the surface. After the treatment period, the tumors and major organs were harvested. 2.11. Toxicity Evaluation of Liposomal ATA in Zebrafish. 2.11.1. Zebrafish Maintenance and Fish Embryo Generation. Male and female zebrafish were bred in separate tanks. The temperature of the water in the tanks was maintained at 28.5 °C, and the pH value was within 6.8. Adult zebrafish were placed in the upper level of a spawning tank the evening prior to mating at a ratio of female/male = 2:1. The next morning after mating, the embryos were collected from the lower level of the spawning tank, washed, and kept in fresh E3 medium (13.7 mmol/L NaCl, 0.54 mmol/ L KCl, 0.025 mmol/L Na2HPO4, 0.044 mmol/L KH2PO4, 0.42 mmol/L NaHCO3, 1.3 mmol/L CaCl2, and 1.0 mmol/L MgSO4, and the pH was adjusted to 7.2 before usage). 2.11.2. Toxicity Evaluation of Liposomal ATA in Zebrafish Larvae. The chorion surrounding the embryo was removed enzymatically at 4 h post fertilization (hpf) following the procedures described in the literature.28,29 At 6 hpf, the embryos were transferred to 96-well plates at one embryo per well with 500 μL of E3 medium containing different compositions: E3 medium alone (negative control), E3 medium containing blank liposomes, various concentrations of liposomal ATA, or free ATA dissolved in DMSO. Unexposed embryos (embryos raised in E3 medium) were also incubated to monitor the natural quality of the embryos. Throughout the experiment, incubation solutions with freshly added ATA were replaced daily. Images of the zebrafish were captured using a dissecting microscope. The heart rate was also recorded as described previously.30,31 Twenty

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Liposomal ATA. To increase the water solubility and bioavailability of hydrophobic ATA, liposomes were utilized to encapsulate ATA. Three components were utilized to make liposomes: PC3, DSPE-PEG, and cholesterol. PC3 and cholesterol possess excellent biocompatibility in vivo. DSPE-PEG allows a longer circulation in vivo for this drug delivery system, protects ATA from degradation within the circulation system, and enhances the delivery of ATA to tumor tissues (Figure 1A). To determine an optimal condition to formulate liposomal ATA, an orthogonal array test was designed with nine experiments, [L9(34)]. This array test allowed us to examine four critical experimental factors (A: ratio of PC3:cholesterol, D


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Molecular Pharmaceutics B: ratio of DSPE-PEG:ATA, C: ultrasonic time, and D: homogeneous ultrasonic time) at three levels (Table S1).33,34 The reverse-phase evaporation method was employed to prepare liposomal ATA. HPLC was used to measure the concentration of ATA in each experiment from which the percentage of encapsulation efficiency (EE%) and rate of drug loading (DL%) were calculated (Table S2). The results of array tests were further analyzed, and the optimal conditions to formulate liposomal ATA were determined after comparing the calculated parameters (Table S3): PC3:cholesterol = 9:4, DSPE-PEG:ATA = 7, ultrasonic time = 3 min, and homogeneous ultrasonic time w = 8 min. Under these conditions, the EE% and DL% of liposomal ATA were found to be 98.94% and 12.35%, respectively. This high level of EE should prevent a burst release of encapsulated ATA and, thus, is expected to produce a smooth and steady drug release profile. In contrast, a moderate 12% drug loading rate would satisfy the requirement for decreased liposome usage, reduced costs, and improved compliance of patients. The functional performance of nanoparticle-based delivery systems depends on the physicochemical properties of the particles, such as size, morphology, charge, and physical state. SEM analysis showed that the liposomal ATA was spherical and not aggregated (Figure 1B). The average particle size of liposomal ATA was found to be 188.5 nm with a relatively narrow size distribution (Figure 1C). The nonaggregative dispersion and relatively narrow size distribution of liposomal ATA may ensure its smooth and steady drug release properties. In addition, a small size is supposed to allow the easy dissemination of liposomal ATA through the tumor neovascularization. Consequently, the passive targeting ability of liposomal ATA for tumors may be achieved. The zeta potential was −31.0 mV, which suggests the stability of the liposomal ATA (Figure 1D). Likewise, due to the PEG chain on the surface of liposomes, an enhanced circulation period could be obtained. Greater accumulation in the tumor tissue may be anticipated for liposomal ATA, potentially further facilitating its therapeutic effects and reducing the off-target effects on normal tissues. 3.2. Using Thermal Analysis (TG/DTA) to Study the Properties of Liposomal ATA. The thermodynamic study of liposomal ATA was conducted to achieve three objectives: (1) to confirm the existence of ATA in liposomes, (2) to evaluate the stability of drug-loaded liposomes, and (3) to validate whether the drug-loaded liposomes were successfully prepared. The thermal analysis of liposomal ATA was investigated using TG/DTA. The results of pure and formulated components are shown in Figure 2. The TG/DTA curve of pure ATA showed that the mass loss of ATA occurred in just one step in the temperature range of 220−310 °C. This step consisted of an accelerated mass loss reaching ∼100% that was associated with a small DTA endothermic peak at 175.72 °C, which was presumably caused by the phase transition of ATA. The materials used to prepare liposomal ATA showed different TG/DTA profiles compared with ATA. For example, the mass loss of cholesterol occurred in the temperature range from 220 to 330 °C that was associated with a DTA endothermic peak after 300 °C. The phase transition peak of cholesterol was 148.96 °C. The mass loss of DSPE-PEG occurred in the temperature range of 300− 400 °C associated with the DTA endothermic peak after 350 °C. The phase transition peak of DSPE-PEG was 54.90 °C.

Figure 2. TG/DTA profiles of liposomal ATA and the materials used to formulate ATA in an individual or mixed form. Four characteristic absorption peaks appeared in the DTA curve when ATA, cholesterol, DSPE-PEG, and PC3 were physically mixed together, while only one absorption peak appeared in the DTA curve in the formulated liposomal ATA.

The mass loss of PC3 was complicated, occurring in the range from 250 to 350 °C. The phase transition peak of PC3 was 109.16 °C (Figure 2). The individual phase transition peak of cholesterol, DSPEPEG, PC3, and ATA can be found by analyzing the TG/DTA curves obtained from the four physically mixed components. These four DTA peaks were found at 143.84 °C, 54.72 °C, 105.24 °C, and 165.23 °C, which corresponded to phase transition temperatures of cholesterol (148.96 °C in the cholesterol curve), DSPE-PEG (54.90 °C in the DSPE-PEG curve), PC3 (109.16 °C in the PC3 curve), and ATA (175.72 °C in the ATA curve). In opposition to the physical mixture, the curve for liposomal ATA showed only one phase transition temperature (65.04 °C), which suggested that ATA was molecularly dispersed within liposomes (Figure 2). The blank liposomes and the physical mixture of liposomal materials of cholesterol, DSPE-PEG, and PC3 were also analyzed by TG/DTA. From the enlarged DTA curve, three DTA peaks were detected from the components of liposome materials. In contrast, only one DTA peak at 72.03 °C was detected from the blank liposomes (Figure S1), which is quite different from the DTA peak at 65.04 °C from liposomal ATA (Figure 2). The thermodynamic results indicated that the physical mixture of four components of liposomal ATA would not affect E


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stability features were observed at 37 °C. The size and zeta potential did not significantly change in the first 5 days (Figure 3A,B). However, smaller liposomes started to aggregate to form larger liposomes after 5 days, resulting in a significant increase in average particle size from ∼190 to ∼380 nm and a significant reduction of the average zeta potential from −42 mV to nearly −68 mV. We do not expect that this aggregation problem will affect the in vivo efficacy of liposomal ATA as they should be metabolized in the body in less than 3 days. The EE% of liposomal ATA did not decrease at 37 °C in the first 4 days and was slightly reduced to ∼80% after 7 days (Figure 3C). In conclusion, liposomal ATA should be stored at 4 °C to maintain its small particle size and high encapsulation efficiency. The compatibility and stability of liposomal ATA with two commonly used intravenous dosing media, saline (0.9% NaCl) and isotonic glucose solution (5% glucose), were evaluated. After being incubated with 5% glucose at room temperature for 24 h, the particle size of the liposomal ATA was reduced by 15 nm, and the zeta potential of liposomal ATA was reduced by 6 mV. In contrast, incubation with 0.9% NaCl resulted in a much greater variation of the particle size and zeta potential of liposomal ATA at 50 nm and 38 mV, respectively (Figure S2A,B). More importantly, these changes occurred within the first several hours of incubation. A significant reduction of EE% was also detected when liposomal ATA was incubated with 0.9% NaCl for 12 h, while no reduction was observed when liposomal ATA was incubated with 5% glucose for 12 h (Figure S2C). These results indicated that liposomal ATA was more stable in 5% glucose than in 0.9% NaCl, and therefore liposomal ATA should be diluted with 5% glucose for intravenous injection in future clinical applications. The release profiles of ATA were compared between free ATA and liposomal ATA in aqueous medium. The graph in Figure 3D shows that most free ATA could not be released from the dialysis bag into the surrounding medium, likely because of its poor solubility, which could not produce a sufficient concentration gradient between the two sides of the dialysis membrane for the diffusion of ATA. However, ATA exhibited a better release profile when it was encapsulated with liposomes. For example, ∼78% of ATA was released from liposomal ATA, while only ∼38% was released from free ATA after 72 h. More interestingly, unlike the flat release profile of free ATA, a steady increase in ATA release was observed over the 72 h. This result not only supported the conclusion that ATA molecules were successfully encapsulated within liposomes, but it also demonstrated that encapsulating ATA within liposomes could prolong its release. Other important information obtained from this study included the finding that the liposomal ATA formulation could be more stably stored at 4 °C and diluted in 5% glucose for intravenous injection or diffusion. 3.4. Determination of the Pharmacokinetic Profile of Liposomal ATA in Rats. The most important objective of this study was to develop a clinical formulation of ATA with improved bioavailability. To assess whether the formulated ATA could meet this goal, free and liposomal ATA at 20 mg/ kg were administered to rats via a single intravenous injection. At different time points, blood samples were obtained from rats, and the concentrations of ATA in plasma and whole blood were determined using a well-validated HPLC method.6 The pharmacokinetic profiles shown in Figure 4 revealed that

the state of their existence because each melting point of these components could be easily found in the thermodynamic curve of the physical mixture. In contrast, when these components were molecularly integrated in the liposomal ATA, only one melting point was detected, suggesting that the ATA molecules were dissolved in the lipid bilayer of liposomes. This result confirmed that ATA was successfully and stably carried by the PEG-liposomes. 3.3. Evaluating the Storage Stability of Liposomal ATA. The size distribution, surface charge, and EE% of liposomal ATA were measured during a storage period of 7 days to assess their storage stability. This information is important for developing liposomal ATA into a clinical formulation. Figure 3A,B shows that the size and zeta potential of the liposomal ATA remained constant at 4 °C during the 7 days of storage. In addition, the EE% remained at a very high level (>95%) in the first 4 days at 4 °C and was slightly reduced to >80% from day 5 to 7 (Figure 3C). In contrast, different

Figure 3. Storage stability of liposomal ATA. Liposomal ATA was stored in PBS at 4 or 37 °C for 7 days. (A) Size changes in liposomal ATA. The size of liposomal ATA was more stable at 4 °C. (B) Zeta potential of liposomal ATA. The zeta potential of liposomal ATA was more stable at 4 °C. (C) EE% change in liposomal ATA. *p < 0.05, ***p < 0.001 compared with day 0. (D) Drug release profile. Liposomal ATA displayed a gradual and prominent release profile compared with free ATA. f 2 < 50 between free ATA and the liposomal ATA group. F


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Figure 4. Pharmacokinetic (PK) profiles of free and liposomal ATA from plasma and blood of rats after intravenous injection. N = 6 rats/group.

Table 1. Pharmacokinetic Parameters of Free ATA and Liposomal ATA in Rat Plasma and Whole Blooda free ATA

liposomal ATA

ratio (lipo/free-ATA)

parameter

plasma

blood

plasma

blood

plasma

blood

CL{[(mg/kg)/μM]/min} AUC0−inf (μM·min)

0.24 85

25 882

0.004 5009

2672 27,912

0.02 59

106 32

a

CL: clearance; AUC0−inf: area under the plasma concentration-time curve from time zero to infinity.

Figure 5. The cytotoxicity of free and liposomal ATA was evaluated by the MTT assay. (A) Liposomal ATA displayed a good growth inhibitory effect in breast cancer MCF-7 cells. (B) Liposomal ATA was not toxic to normal endothelial HUVECs at concentrations ≤5 μM. *p < 0.05, **p < 0.01, ***p < 0.001 between tested samples and the control group. Data were collected from triplicate wells in each experiment repeated three times.

parameters. Compared with free ATA, the 24 h area under the curve (AUC0−24h) of liposomal ATA was increased 59-fold in plasma and 32-fold in whole blood, whereas the clearance rate was reduced 50-fold in plasma and increased 106-fold in whole blood. These results demonstrated that liposomal ATA significantly extended the retention time and enhanced bioavailability of ATA in vivo. 3.5. In Vitro Determination of the Anticancer Efficacy and General Cytotoxicity of Liposomal ATA. As liposomal ATA was designed for intravenous injection, we wanted to investigate whether liposomal ATA could retain its anticancer effect without damaging endothelial cells. To assess this possibility, the viabilities of human breast cancer MCF-7 cells and human umbilical vein endothelial cells (HUVECs) were determined by the MTT assay after the cells were incubated with various concentrations of free ATA, liposomal ATA, or blank liposomes for 24 h. The MTT results showed that, at 0.5 μM, liposomal ATA significantly reduced the viability of MCF-7 cells to ∼60% compared with the control group, while free ATA did not affect the viability of MCF-7 cells (Figure 5A), demonstrating that formulating ATA with liposomes has increased its cytotoxicity on breast cancer cells. Importantly, 0.5 μM liposomal ATA did not reduce the viability of endothelial HUVECs (Figure 5B), suggesting that it has little toxicity

the concentrations of ATA in plasma and whole blood from the free ATA-treated group were at a very low level at 15 min. Compared with free ATA, liposomal ATA had significantly higher concentrations of ATA in both plasma and whole blood. We noticed that the concentration of ATA is higher in the whole blood group than in the plasma group, which may be due to the higher internalization of liposomal ATA by the blood cells. This distribution of liposomal ATA in the blood cells may increase its storage ability and contribute to a longer circulation time of ATA in the blood stream. The pharmacokinetic parameters of ATA were then calculated using Excel PKsolver. According to the requirements for selecting the pharmacokinetic calculation model, the values of Akaike’s information criterion and residual sum of squares (Re) were minimized, and the value of the determinate coefficient (r) was maximized. The free ATA and liposomal ATA belonged to the two-chamber model in whole blood. Free ATA belonged to the one-compartment model in plasma, and liposomal ATA belonged to the two-chamber model in plasma. The mean retention time was calculated using the statistical moment. The main pharmacokinetic parameters of ATA in rat plasma and whole blood are listed in Table 1 and Tables S4 and S5. As shown in Table 1, the free ATA and liposomal ATAtreated groups exhibited significantly different pharmacokinetic G


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After the MCF-7 cells had grown into solid tumors, liposomal ATA (20 mg/kg) was injected into the tumorbearing nude mice through the tail vein every other day for 3 weeks. During the treatment period, both the tumor size and body weight were measured. As shown in Figure 6A, tumors grew rapidly in the control group, whereas in the free ATAtreated group, tumor growth was significantly reduced. More importantly, liposomal ATA exerted an even better tumor growth inhibitory effect (73%) than that of free ATA (61%) (Table 3).

toward noncancerous cells. At elevated concentration ranges of 1−20 μM, both liposomal and free ATA produced a much stronger growth inhibitory effect on MCF-7 cells than that of the control group (Figure 5A). We also calculated the concentration that could reduce 50% of the cell viability after a 24 h incubation. The 24 h IC50 values showed that liposomal ATA had a lower (0.4-fold) IC50 value than that of free ATA in breast cancer MCF-7 cells and a higher (1.5-fold) IC50 value in endothelial cells (Table 2). In summary, these results suggested that liposomal ATA had a higher anticancer efficacy than that of free ATA and was less toxic to HUVECs in comparison to its free form.

Table 3. Tumor Growth Inhibition Ratio sample

Table 2. IC50 Values of Liposomal ATA vs Free ATA 24 h IC50 values liposomal ATA free ATA

breast cancer MCF-7 cells

ratio of IC50 (lipo/free ATA)

endothelial HUVECs

ratio of IC50 (lipo/free ATA)

1.0 μM

0.4-fold

22 μM

1.5-fold

2.3 μM

control blank liposomes free ATA liposomal ATA

14 μM

tumor size (mm3) 1,611 897 625 432

± ± ± ±

612 360 301 428

inhibition ratio (%)

p value

0 44 61 73

0.023 (*) 0.009 (**) 0.007 (**)

No significant body weight differences were found between the treatment groups (free ATA, blank liposomes, and liposomal ATA) and the control group injected with PBS during the experiment, which suggested that liposomal ATA did not cause serious side effects in nude mice (Figure 6B). At the end of the animal experiment, tumor tissues were removed and photographed. The images in Figure 6C revealed that three out of four tumors from the liposomal ATA-treated mice had a much smaller size than the tumors from free ATAtreated mice. We propose that the following reasons contributed to a higher antitumor efficacy of liposomal ATA. First, the encapsulation of ATA with liposomes significantly

3.6. Evaluating the in Vivo Antitumor Effect of Liposomal ATA. As ATA has been previously shown to effectively inhibit the growth of ER+ breast cancer MCF-7 cells, in this study, MCF-7 cells were utilized to establish human xenograft tumors in nude mice. A tablet of 1.5 mg βestradiol 17-acetate was first implanted dermatologically into female mice, and seven days later, MCF-7 cells (5 × 106) were mixed with Matrigel and subcutaneously injected into the armpit of the mice.

Figure 6. Determination of the antitumor effect of liposomal ATA in a human breast tumor xenograft model. (A) Graphs of the tumor size during the treatment period. Tumor growth was effectively suppressed after the treatment of liposomal ATA. (B) Body weight of nude mice during the treatment. No apparent body weight loss was found in all four groups. (C) Pictures of harvested tumors at the end of the experiment. The liposomal ATA treatment group had the smallest tumor size. N = 5 mice/group. *p < 0.05, **p < 0.01 between samples and the control group. H


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Figure 7. Toxicity evaluation of liposomal ATA in zebrafish embryos. (A) Types of induced malformations in zebrafish embryos at 96 hpf: (a) normal development, (b) yolk sac absorption delay, (c) pericardial edema, and a (d, e) bent body axis. (B) Deformity and mortality rates of zebrafish at 96 hpf. Less deformity and mortality were observed in zebrafish in the liposomal ATA-treated group compared with the free ATAtreated group under the same treatment concentration. (C) Heartbeat of zebrafish. *p < 0.05, **p < 0.01 between samples and the control group. N = 20 fish/group, and each experiment was performed at least four times.

3.7. Evaluation of the Potential Toxicity of Liposomal ATA on Zebrafish. To date, our data have shown that the newly formulated liposomal ATA displayed good antitumor effects with little toxicity to human endothelial cells at ≤5 μM and did not reduce the body weight of tumor-bearing mice at a dose of 20 mg/kg. As our ultimate goal for liposomal ATA is to use it to treat cancer, it is necessary to evaluate its potential toxicity in vivo. We first utilized zebrafish because they are cheap and easy to acquire in large quantities and because of the similarities of the zebrafish genome to the human genome and statuses of both fish and humans as vertebrates. In this experiment, zebrafish larvae at the age of 6 h post fertilization (hpf) were exposed to different concentrations of free or liposomal ATA, and the morphological changes were observed at 96 hpf. Moreover, the heart rates were measured at 48, 72, and 96 hpf. During the treatment with free or liposomal ATA, various kinds and degrees of toxicity were observed over

increased their plasma concentration. Second, the average particle size of liposomal ATA was 188.5 nm, which should facilitate its exit from the leaky tumor vascular system to reach tumor cells and enhance its therapeutic effect. On a separate note, we found that blank liposomes exhibited a minor tumor growth inhibitory effect (Figure 6A). The results of the MTT assay also showed that the blank liposome reduced the viability of MCF-7 cells (Figure 5A). These two observations suggested that internalized liposomes might affect the membrane integrity of tumor cells, which reduced cell viability and tumor growth. Further studies will be conducted to understand why blank liposomes produced a minor tumor growth inhibitory effect, which can also help demonstrate that the enhanced anticancer activity is due to the encapsulation of ATA with liposomes but not due to the additive effect of blank liposomes plus free ATA. I


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Figure 8. Toxicity study of 5 μM free or liposomal ATA during zebrafish development. (A) Zebrafish embryos at different stages. (B) Deformity and mortality rate of zebrafish at different periods during development. (C) Heartbeat of zebrafish at different developmental stages. *p < 0.05, **p < 0.01 between samples and the control group.

90% at 5 μM. The combined rate of deformity and mortality was 40% at 2.5 μM. These results suggested that free ATA at 5 μM was toxic to zebrafish development. In contrast, liposomal ATA showed much lower toxicity toward zebrafish than that of free ATA. The sum percentage of mortality and deformity caused by liposomal ATA was lower than that of free ATA. Importantly, no toxicity was observed at a low concentration of 1 μM, and 10% mortality was detected at 2.5 μM. In addition, the sum percentage of mortality and deformity did not exceed 50% at high concentrations of 10−20 μM. Additionally, the heart rate (heartbeats per minute) of zebrafish was measured to determine heart function (Figure 7C). In the control group, the heartbeat gradually increased

the course of zebrafish development. At high concentrations, ATA resulted in death or malformations in the early developmental period. Images of malformations observed in this experiment are shown in Figure 7A, including pericardial edema, yolk sac absorption delay, and curvature of the spine. The overall percentages of deformity and mortality are shown in Figure 7B. A 5% mortality and no deformity were observed in the control group. Blank liposomes did not cause significant deformities or mortalities under the tested concentrations. The data indicated that free ATA caused extensive death or body deformation in zebrafish at high concentrations (5−20 μM). In particular, 100% mortality was recorded in zebrafish treated with 10 μM and 20 μM free ATA, and the total amount of deformity and death rate exceeded J


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8C, blank liposomes and liposomal ATA did not cause obvious changes in the heartbeat compared with the control group in all treatment periods. Free ATA did not cause notable changes in the heartbeat in the 6−10 hpf and 14−24 hpf exposure periods but significantly increased the heartbeat in the periods at 10−24, 24−48, and 48−72 hpf compared with the control group. These results further confirmed that liposomal ATA was safer than free ATA. Although encapsulating ATA with liposomes reduced its toxicity, liposomal ATA still showed a negative impact during early embryonic development. This finding suggested that liposomal ATA should not be administered to breast cancer patients who are pregnant. 3.9. Evaluating the Toxicity of Liposomal ATA in Mice. The results of the toxicity study in zebrafish showed that liposomal ATA had much lower toxicity than that of free ATA. In this study, the acute toxicity evaluation was conducted in mice, which is a required process for the development of liposomal ATA into a therapeutic agent. Acute toxicity refers to the adverse effects of a substance caused by either a single exposure or multiple exposures in a short period of time (usually less than 24 h). Because liposomal ATA did not cause any mice to die at the dose of 120 mg/kg, which is the highest dose that could be prepared, this maximum dose was used in this study. Equal numbers of female and male mice (n = 10) were administered with 120 mg/kg liposomal ATA via an intravenous injection. The saline group and blank liposomes were used as controls. After injection, the mice were monitored for 21 days in terms of behavioral changes related to the following: respiratory system, motor system, convulsive reaction, reflex system, eyes, cardiovascular system, excretory system, and muscle tension (Table S6). The results revealed no death in either female or male mice after dosing, and the following abnormal behaviors were observed. First, all control, liposomal ATA, and blank liposome-treated groups produced reflex irritation and were sensitive to noise and touch, suggesting neuromuscular toxicity. This response quickly recovered, implying that the injection process induced a transient stress response. Second, liposomal ATA and blank liposomes increased spontaneous activities in mice, which could be quickly recovered, indicating the occurrence of a minor recoverable motor malfunction. Third, liposomal ATA resulted in transient salivation, which was related to disturbed autonomic functions. In summary, liposomal ATA may have toxic effects on motor function and autonomic function in mice. Liposomal ATA did not cause strong acute toxicity in male or female mice, and all the aforementioned symptoms could be rapidly recovered (i.e., in 1 day), which suggested that liposomal ATA was quite safe in mice. As the highest dose that could be prepared for free ATA is 30 mg/kg, we used this dose to conduct the toxicity study of free ATA. Mice appeared to have inspiratory difficulties and produced a wheezing sound when they breathed. These disorders involved malfunctions in pulmonary edema, respiratory secretion accumulation, and enhanced cholinergic function. However, at 120 mg/kg, which is 4 times higher than free ATA, liposomal ATA did not cause such problems. These results further confirmed our previous findings in zebrafish: liposomal ATA is safer than free ATA. In addition to observing behavioral changes, the body weights of the injected mice were measured at multiple time points over 21 days. The graphs in Figure S3 show that the

from 48 hpf to 96 hpf, which resulted from normal heart growth during zebrafish development. Similar to the control group, blank liposomes also formed this normal pattern at most liposome concentrations. Free ATA at 1−5 μM caused significant changes in the heartbeat compared with the control group. No heartbeats could be detected in the zebrafish treated with high concentrations of free ATA (10−20 μM) as they had all been killed. Compared with free ATA, lower toxicity was observed in the liposomal-ATA treated group. No signs of cardiotoxicity were observed in fish larvae treated with liposomal ATA at 1−10 μM at 96 hpf. Although the heartbeat of 20 μM at 48 hpf was significantly increased compared with the control group, this elevation was not observed at 72 hpf and 96 hpf, which suggested that this side effect could be overcome during fish development. 3.8. Determining the Effects of 5 μM Liposomal ATA on the Malformation and Mortality of Zebrafish Embryos during Early Development. The above zebrafish toxicity evaluation showed that 5 μM liposomal ATA did not affect heart function and had much less toxicity than that of free ATA. In this experiment, we further compared the differential toxicity between free and liposomal ATA (5 μM) during five major periods of zebrafish development (Figure 8A). These five time periods are the (1) gastrula period (6−10 hpf, morphogenetic movements of involution, convergence, and extension form the epiblast, hypoblast, and embryonic axis; through the end of epiboly), (2) somite stage (10−24 hpf, somite, pharyngeal arch primordium, and neuromere development; primary organogenesis; earliest movement; tail appearance), (3) important organ formation stage (14−24 h, primary organogenesis; earliest movement; the tail appears), (4) pharyngeal phase (24−48 hpf, phylotypic-stage embryo; body axis straightens from its early curvature around the yolk sac; circulation, pigmentation, and fins begin to develop), and (5) hatching period (48−72 hpf, completion of rapid morphogenesis of primary organ systems; cartilage development in the head and pectoral fin; hatching occurs asynchronously) (Figure 8A). Mortality, abnormality, and heart function were also assessed in each developmental period of zebrafish. Figure 8B shows the overall deformity and mortality rates of zebrafish after the treatment of various samples in different exposure periods. The sensitivity window was determined according to the statistical results. No deformity or mortality was observed in the control group during all developmental stages. The blank liposomes did not produce significant toxicity in zebrafish, and less than 18% mortality or deformity was observed in all tested periods. Free ATA caused the highest mortality (100%) during early development at 6−10 hpf and resulted in lower mortality (53.85%) in the later developmental periods at 10−24 hpf. Even lower mortality rates of 11.76% and 14.29% were detected in other periods of 14−24 hpf and 24−48 hpf, respectively. These results suggested that free ATA mainly affected the gastrula period and early somite stage of zebrafish development. In contrast, liposomal ATA significantly decreased the toxicity of ATA in zebrafish. For example, much lower mortality rates (29−31%) were recorded at exposure times of 6−10 and 10−24 hpf in comparison to free ATA. In addition to evaluating larval mortality and abnormality, zebrafish heart function was also examined. As shown in Figure K


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Molecular Pharmaceutics weights of male and female mice increased slightly and that no significant changes were observed between the liposomal ATA and saline control group. These results showed that a single intravenous dose of liposomal ATA at 120 mg/kg would not affect the normal increment of mouse body weight. Histopathology analysis was conducted for important organs, including the heart, liver, spleen, lung, kidney, and brain at 7 and 21 days post injection by H&E staining. The images in Figures 9 and 10 showed no obvious necrosis or

Figure 10. Long effect of liposomal ATA on various organs in BALB/ C mice (female) 21 days after injection. Images of H&E staining show that liposomal ATA caused light congestion in the lung (50×). N = 5 mice/group.

encapsulate a promising anticancer agent ATA into nanoparticles with a suitable size and further evaluate the preclinical properties. Encapsulating ATA into liposomes significantly increased its release capacity in vitro and increased the ATA plasma concentration 59-fold in rats. The formulation of liposomal ATA was stable for storage at 4 °C for at least 4 days and after being diluted with 5% glucose for intravenous administration for 12 h. Liposomal ATA displayed strong potency toward ER+ breast cancer cells by inhibiting their proliferation in vitro and by reducing tumor growth by 73% in nude mice. At the maximum dose of 120 mg/kg, in 21 days, liposomal ATA did not show obvious toxicity, such as death, reduced body weight or damage to major organs. However, liposomal ATA showed minor toxicity in zebrafish larvae during their early development and resulted in a slight abnormality in the lung tissue in mice. Thus, liposomal ATA should not be given to cancer patients who are pregnant, and more clinical studies are needed to determine whether ATA should be given to cancer patients with lung disease. The successful development of ATA into a clinical formulation with improved bioavailability and better anticancer efficacy provided an example for drug formulation. Furthermore, the information obtained from this study can be used to guide the future manufacturing and development of liposomal ATA into a new anti-breast cancer drug.

Figure 9. Short effect of liposomal ATA on various organs of BALB/ C mice (female). Blank liposomes and liposomal ATA (at an equivalent dosage of 120 mg/kg ATA) were injected into mice via a single intravenous injection. Images of H&E staining show no obvious necrosis in the heart, liver, spleen, kidney, and brain 7 days after injection (50×). More red blood cells were found in lung tissues of the liposomal ATA group. N = 5 mice/group.

significant inflammation in all tissues excluding the lung, indicating the relatively low toxicity of liposomal ATA. The lung tissue of the liposomal ATA-treated group appeared firmer and showed slight congestive phenomena compared with the control group, indicating weak toxicity to the lung tissue (Figures 9 and 10). A similar phenomenon was observed in the tissue sections of male mice (Figures S4 and S5).

4. CONCLUSIONS How to construct a clinical formulation of anti-breast cancer chemotherapeutic agent ATA with improved bioavailability is a difficult challenge to meet during drug development. In this study, by using PEG-modified liposomes, we were able to L


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.9b00493. Orthogonal array test design with four critical factors at three variable levels, results of the orthogonal array tests, analysis of orthogonal array tests, pharmacokinetic parameters of free and liposomal ATA in rat whole blood after iv administration, pharmacokinetic parameters of free and liposomal ATA in rat plasma after iv administration, behavioral changes in mice after injection of the maximum dose of liposomal ATA and blank liposomes, TG/DTA profiles of the mixture of ATA, cholesterol, and DSPE-PEG, blank liposomes, compatibility and stability of liposomal ATA, weight changes in female and male mice after injection of the maximum dose of liposomal ATA and blank liposomes, toxicity of liposomal ATA in male mice after injection of the maximum dose of liposomal ATA and blank liposomes, recovery of male mice after injection of the maximum dose of liposomal ATA and blank liposomes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 853-8822-4233. Fax: 8538822-2314. ORCID

Qi Wang: 0000-0003-3694-5826 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants awarded to K.Q.L. from the Johns Hopkins Singapore Research Fund, the Start-Up Fund from the Faculty of Health Sciences, and the Start-Up Research Grant (no. SRG2016-00068-FHS) from the University of Macau. We would like to thank Prof. Sierin Lim for her kind help in managing the JHS Research Fund.

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Development of a Liposomal Formulation of Acetyltanshinone IIA for

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