Gambogic Acid-Loaded Electrosprayed Particles for Site-Specific

Article pubs.acs.org/molecularpharmaceutics

Gambogic Acid-Loaded Electrosprayed Particles for Site-Specific Treatment of Hepatocellular Carcinoma Dengke Yin,†,‡,§ Ye Yang,*,†,‡,§ Hanxu Cai,† Fei Wang,† Daiyin Peng,†,§ and Liqing He† †

School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, P. R. China Key Laboratory of Xin’an Medicine, Ministry of Education, Hefei 230031, P. R. China § Anhui Province Key Laboratory of R&D of Chinese Medicine, Hefei 230031, P. R. China ‡

ABSTRACT: This study aims to assess the targeted effect and antitumor efficacy of Gambogic-acid-loaded particles (GA-Ps). GA-Ps with uniform particle sizes of 69.8 ± 17.8 nm (GA-P1), 185.6 ± 33.8 nm (GA-P2), 357.8 ± 81.5 nm (GA-P3), and 7.56 ± 0.95 μm (GA-P4) were prepared using an electrospray technique and exhibited extremely high entrapment efficiency. As the particle size increased from the nano- to microscale, the in vitro GA release rate sharply decreased. After tail-vein injection in mice, GA-P samples GA-P1, GA-P2, GA-P3, and GA-P4 improved the uptake of GA 1.67-times in the liver, 1.78-times in the liver, 2.18-times in the spleen, and 2.35-times in the lung, respectively, compared with GA solution (GA-S). The antitumor efficacy of GA-P2, with an 82.51% targeting efficiency (Te) for the liver, was examined in hepatocellular carcinoma (HCC) model mice. After 2 weeks of administration, HCC mice in the GA-P2 group exhibited a lower degree of tumor invasion and cell lesions in hepatic tissue, recovered liver function, and significantly prolonged survival time, compared with mice in the model, GA-S, and normal saline (NS) groups. Pharmacokinetic studies indicated that the superior antitumor efficacy of GA-P2 was attributed not only to tissue targeting but also to low clearance, extended retention, high bioavailability in plasma, and increased GA stability. KEYWORDS: gambogic acid (GA), electrospray, gambogic acid-loaded particle (GA-P), tissue targeting, antitumor efficacy

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However, the poor aqueous solubility (∼10 μg/mL) and highly toxic side effects in normal tissue impede its clinical application. Therefore, researches into structural modifications of GA derivatives5−7 and drug carrier designs such as polymeric micelles and magnetic Fe3O4 nanoparticles8−10 have been investigated. Because the 30-carboxy group of GA can tolerate a variety of modifications with little or no effect on its bioactivity, as speculated from the results of a structure−activity relationship assay,11 a series of novel GA derivatives were designed and synthesized by the coupling of various hydrophilic alkanolamines.5 Although the structural modifications improved the solubility of GA, the complex synthesis operations reduce the product yield. GA-loaded micelles and magnetic Fe3O 4 nanoparticles have also been prepared to improve the solubility

INTRODUCTION

Gambogic acid (GA; C38H44O8, Figure 1), the main active ingredient isolated from the dry gum-resin of Garcinia hanburyi, has recently been established as a potent anticancer agent that can inhibit the growth of a variety of tumor cells1−4 and currently is undergoing a phase II clinical trial in China.

Received: Revised: Accepted: Published:

Figure 1. Chemical structure of GA. © 2014 American Chemical Society

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April 21, 2014 July 13, 2014 September 24, 2014 September 24, 2014 dx.doi.org/10.1021/mp500214a | Mol. Pharmaceutics 2014, 11, 4107−4117

Molecular Pharmaceutics

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experimentation, fed with a standard diet and allowed water ad libitum. All animal experiments were performed in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (document number 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of our university. Cells and Cell Culture. H22 cells were preserved by our lab and used to establish the HCC mice model. The cells were cultured in RPMI-1640 medium (Gibco BRI, Rockville, MD) with 10% heat-inactivated fetal calf serum (FCS; Gibco BRI, Grand Island, NY) and placed in a 75 cm2 cell-culture flask at 37 °C with 5% CO2 in air. Preparation of GA-Loaded Particles. Four milligrams of GA and 100 mg of PDLLA were dissolved in 2.0 mL of chloroform. The mixed solution was electrosprayed using a previously described method. Briefly, the mixed solution was added into a 2 mL syringe attached to a metal capillary. The electrospray apparatus is equipped with a high-voltage statitron (Tianjing High Voltage Power Supply Co., Tianjing, China) and a microinject pump (Zhejiang University Medical Instrument Co., Hangzhou, China). The electrosprayed particles were collected using ultrapure water with a counter electrode located approximately 15 cm from the capillary tip, under magnetically stirring, separated by ultracentrifuge, and then the residual solvent was removed by vacuum drying. The resulting particles of GA-P1, GA-P2, GA-P3, and GA-P4 were respectively prepared with flow rates of 0.02, 0.02, 0.04, and 0.10 mL/min by the precision pump, capillary external diameters of 0.4, 0.4, 0.5, and 0.9 mm, and applied voltages of 20, 18, 15, and 13 kV. Preparation of Particles with APTS-Labeled GA. GA was labeled with APTS according to the method established by Liu et al.19 In brief, GA was darkly reacted with 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HCl; 5 mmol/L) and N-hydroxysuccinimide (NHS; 0.33 mmol/L) in dimethylformamide (DMF) for 4 h at room temperature. Then, APTS was added to the above reaction system at five times the concentration of GA, and the reaction was continued for 4 h. The reaction mixture was dialyzed to remove any residual APTS and freeze-dried. APTS-GA-loaded particles were prepared according to the above methods and parameters and were named as A-GA-P1, A-GA-P2, A-GA-P3, and A-GA-P4. Particle Characterization. The morphology of electrosprayed particles was determined using a scanning electron microscope (SEM; FEI Quanta 200, The Netherlands). In brief, the electrosprayed particles were mounted on metal stubs, sputtercoated with gold for a period up to 120 s, and then detected at an accelerating voltage of 20 kV. The mean particle size distributions were detected using dynamic light scattering (DLS) on a Malvern Nano-2S90 (Malvern instruments, Malvern, U.K.) at 25 °C after resuspension with distilled water, by ultrasonic dispersion. Drug Loading and Entrapment Efficiency. Drug loading and entrapment efficiency of GA into electrosprayed particles were determined by dissolving the electrosprayed particles in chloroform, and the GA content was detected using high performance liquid chromatography (HPLC). In brief, a known amount of electrosprayed particles (ca. 10 mg) was dissolved in 500 μL of chloroform, centrifuged at 15,000 × g for 10 min and filtered with a 0.22 μm microfiltration membrane. The GA content was detected using a HPLC system, consisting of a Waters Alliance 2695 Separations Module, a Waters 2487 Dual Absorbance Ultraviolet Detector, and a C18 Hypersil BDS 4.6 mm × 250 mm × 5 μm analytical column (Hypersil, Runcorn,

and inhibitory effect of GA in tumor cells; however, their entrapment efficiency is dissatisfactory.8−10 Electrospray is a well-known technique to prepare monodispersed particles with diameters ranging from nanometers to micrometers, which was first applied to mass spectrometry.12 The electrospray system includes a liquid delivery system (pump), a spray nozzle, a high voltage direct current power supply, and a grounded electrode that is a short distance away from the needle. The material for electrospray can be both a solution and liquation of inorganic, organic, or polymeric materials. In the absence of an electric field, a drop at the tip of a needle grows until its weight exceeds the surface tension of the liquid at the needle−drop interface. When a high electric field is applied, a conical meniscus (Taylor cone) forms at the tip of the needle, and then the jet emerging from the apex breaks into monodispersed droplets.13 As the droplets move through the air, evaporation, disintegration, or solidification ensues, simultaneously shrinking the droplet diameter and leading to solid particle formation. Because of the electrostatic repulsion between these highly charged droplets, the particles are monodispersed with no coalescence. The technique of electrospray has gradually attracted widespread attention in the field of drug delivery because of its advantages of simplicity, cost-effectiveness, monodispersed product, and fast production time. Proteins,14 genes,15 cells,16 and poorly water-soluble drugs17 have been encapsulated in particles, in solid/core− shell/liposome configuration,14−18 using electrospray. Furthermore, as the encapsulation of biologically active substances by electrospray is one-step; the entrapment efficiency and activity retention are extremely high. In the present study, a particle delivery system for GA (GAPs) was established using electrospray, resulting in particle sizes ranging from the nano- to microscale. After the detection of targeting efficiency, the liver targeting GA-P was administered to hepatocellular carcinoma (HCC) model mice through intravenous injection, and its antitumor efficacy was investigated. To analyze the different antitumor effects between GAP and GA solution (GA-S), the conventional dosage form, the pharmacokinetic characteristics of each drug were also investigated.

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MATERIALS AND METHODS Materials. Gambogic acid (GA; ≥ 99.0%) was extracted and isolated from gamboge, the resin from trees of the family Clusiaceae, in our lab. Poly-DL-lactide (PDLLA, Mw = 49 kDa, Mw/Mn = 1.08) was synthesized in our lab by ring-opening polymerization of lactide. The molecular weight was determined by gel permeation chromatography (GPC; Waters 2695 and 2414, Milford, MA), using polystyrene beads as the standard, Styragel HT 4 (7.8 × 300 mm) as the column, and tetrahydrofuran (THF) as the mobile phase, at a flow rate of 1.0 mL/min. The fluorescent marker, 1,3,6-pyrenetrisulfonicacid, 8-amino-, sodium salt (1:3) (APTS), was purchased from Aladdin Reagent Database Inc. (Shanghai, China). Ultrapure water used in the experiments was from a Milli-Q biocel purification system (UPI-IV-20, Shanghai UP Scientific Instrument Co., Shanghai, China). All other chemicals and solvents were of reagent grade or better. Animals. The female Kunming mice (weight, 18−22 g; age, 6−8 weeks) and Sprague−Dawley rats (SD rats; , 180−200 g; age, 5 months) that were supplied by the Experimental Animal Center of Anhui Medical University (Anhui, China) were allowed to acclimatize for at least 1−2 weeks before 4108

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particles of A-GA-Ps were injected into the tail vein using the methods described above. At the same defined time points as above, mice were sacrificed and the previously specified tissues were excised, fixed, paraffin-embedded, sectioned, and observed using a fluorescence microscope (Leica DMR HCS, Germany). Creation of the Hepatocellular Carcinoma (HCC) Mouse Model. The HCC mouse model was established as described previously.22 H22 cell suspension was triply rinsed with normal saline, centrifuged at 10,000 r/min for 1 min, and diluted with normal saline to a concentration of 1 × 107 tumor cells/mL. Healthy Kunming mice were anesthetized with pentobarbital (45 mg/mL), supinely fixed, sliced in the costal margin, scissored in layers to sufficiently expose the left hepatic lobe, obliquely and slowly injected with 0.1 mL of tumor cell suspension, gently pressed to stop bleeding, and then the peritoneum was closed in layers. Mice in the control group underwent the same treatment, but were injected with normal saline. Antitumor Test. Fifty-four HCC model mice were fed in the conventional manner for a week and were then randomly and evenly divided into 3 groups and respectively given injections of liver-targeting GA-loaded particle (2.5 mg/kg dose, GA-P group), GA solution (2.5 mg/kg dose, GA-S group), and normal saline (NS group) in the tail vein. The GA solution was prepared as mentioned above. HCC model mice without any administration (model group, n = 18) and normal mice (normal group, n = 6) were used as controls. All mice were weighed daily. At the first and second week after administration, one-third of mice in each group were sacrificed, and the left hepatic lobe was excised. Four tenths of a gram of liver was accurately weighed, homogenized, centrifuged at 7000 r/min for 15 min, and the supernatant was collected to evaluate the liver function indices of alanine transaminase (ALT), bile acid (TBA), prealbumin (PA), alkaline phosphatase (AKP), and glutamyltranspeptidase (GGT) using an automated biochemical analyzer (7600, Hitachi, Japan). The remaining sections of liver were excised, fixed, paraffin-embedded, sectioned, and stained to observe the tissue histomorphology. The last third of the HCC model mice in each group were fed in the conventional manner until natural death to evaluate the mean survival time of HCC mice in each group. Histological Examinations. Hematoxylin-eosin (HE) staining was used to observe the histomorphology of tissues. In brief, the paraffin sections were immersed in 10% formaldehyde, washed with distilled water; nuclei were stained with hematoxylin, rinsed in running tap water, differentiated with 0.3% acid alcohol, rinsed in running tap water, stained with eosin for 2 min, dehydrated, cleared and mounted, and observed using a light microscope (Nikon Eclipse E400, Japan). Sirius Red (SR) staining is one of the best understood techniques of collagen histochemistry and is used to observe the degree of liver fibrosis. In brief, paraffin sections were stained with hematoxylin for 8 min for nuclei staining, washed in running tap water for 10 min, stained in SR solution (SigmaAldrich, St. Louis, MO) for 1 h, washed in two changes of acidified water, shaken vigorously to remove water, then dehydrated, cleared and mounted, and observed using a polarized light microscope (Leica 12POLS, Germany). Pharmacokinetic Studies in Rats. SD rats were randomized into 2 groups (n = 6 for each group) and administered liver-targeting GA-loaded particle (GA-P group) and GA solution (GA-S group) at a 2.5 mg/kg dose by bolus injection into the tail vein. The GA solution was prepared as

U.K.) with a mixed mobile phase of methanol and 0.1% phosphoric acid (95:5, v/v) at a flow rate of 1 mL/min at a 25 °C column temperature and a detected wavelength of 360 nm. The concentration was obtained using a standard curve prepared from known concentrations of GA solution. In vitro GA Release. One hundred milligrams of GA-Ps were immersed in 10 mL of PBS with 0.5% of sodium dodecyl sulfate (SDS)8 and kept in a thermostated shaking water bath (Taichang Medical Apparatus Co., Jiangsu, China) that was maintained at 37 °C and 100 cycles/min. At predetermined time intervals, 0.5 mL of the release buffer was removed for analysis, and 0.5 mL of fresh PBS (with 0.5% SDS) was added for continuing incubation. The GA amount present in the release buffer was determined using the HPLC method described above. The amount of GA in the medium taken out for detection was also added into the accumulated release total. Blank particles of PDLLA with the same particle sizes as GA-Ps were used as a control for the GA amount determination. Tissue Targeting and Drug Distribution. Mice were randomized into 6 groups (n = 36 for each group) and administered the GA-Ps of GA-P1 (GA-P1 group), GA-P2 (GAP2 group), GA-P3 (GA-P3 group), GA-P4 (GA-P4 group), and GA solution (GA-S group) at a 2.5 mg/kg dose via tail-vein injection with normal saline injection as a control (NS group). As the GA has low solubility in water, it was dissolved in 20% trimethylene glycol aqueous solution, in order to administrate through vein injection, and the preparation was accomplished immediately before drug administration, according to the prescription presented by Wang et al.20 After dosing, mice were sacrificed at defined time points (5, 10, 15, 30, 60, and 90 min), and tissues from the heart, liver, spleen, lung, and kidney were excised. These tissues were weighed, grinded with liquid nitrogen, deproteinized using perchloric acid, extracted using ethyl acetate, blow-dried using nitrogen at 40 °C in a water bath, redissolved in methanol, and analyzed for GA content using HPLC. To eliminate tissue specific effects, a series of GA solutions with known concentrations were added into each type of tissue homogenate and processed in accordance with the methods described above to prepare GA standard curves for different tissues. Indices of relative tissue exposure (re), drug targeting index (DTI), targeting efficiency (Te), and relative targeting efficiency (RTe) were calculated to evaluate the targeting effects of electrosprayed GA-Ps to tissues, according to the following equations:21 re =

(AUCi )p (AUCi )s

DTI =

Te = RTe =

(1)

[GA concentration(t )i × (weight of tissue)i ]p [GA concentration(t )i × (weight of tissue)i ]s

AUCi × n ∑i = 1 [AUCi

(weight of tissue)i × 100% × (weight of tissue)i ]

(2)

(3)

(Te)p (Te)s n

=

[AUCi × (weight of tissue)i ]/∑i = 1 [AUCi (weight of tissue)i ]p n

[AUCi × (weight of tissue)i ]/∑i = 1 [AUCi (weight of tissue)i ]s

(4)

APTS-labeled GA was loaded into electrosprayed particles to observe the particle distribution in different tissues. In brief, 4109



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distributions of GA-P1, GA-P2, GA-P3, and GA-P4 are 69.8 ± 17.8 nm, 185.6 ± 33.8 nm, 357.8 ± 81.5 nm, and 7.56 ± 0.95 μm, respectively. The DLS results determined particle size distributions of electrosprayed particle samples (Figure 2a2− d2) that are in accordance with the SEM results. GA amounts in particle samples GA-P1, GA-P2, GA-P3, and GA-P4 are (3.3 ± 0.2)%, (3.3 ± 0.3)%, (3.6 ± 0.2)%, and (3.5 ± 0.2)%, respectively, which are similar to the theoretic value of 4.0% (w/w). The GA entrapment efficiencies in particle samples GAP1, GA-P2, GA-P3, and GA-P4 are (82.2 ± 5.8)%, (83.1 ± 6.5)%, (90.4 ± 5.5)%, and (88.7 ± 4.9)%, respectively. The variation in preparation parameters to synthesize different particle sizes does not influence the drug loading and entrapment efficiency significantly (p > 0.05). To observe the GA distribution in different tissues, GA was labeled with the fluorescence indicator, APTS, and the A-GA-Ps were prepared using the same preparation parameters as the corresponding particles mentioned above. As determined by the SEM, DLS, and HPLC analyses, there were no significant differences in morphology, particle size distributions, GA loading, and entrapment efficiency between GA-Ps and AGA-Ps. In Vitro GA Release. In the in vitro GA release experiments, the amounts of released media met sink conditions. Figure 3

mentioned above. Blood samples were obtained via the retinal vein plexus at 2, 5, 10, 20, 40, 60, 120, 240, 360, 480, and 600 min after drug administration. The blood samples were centrifuged, and the plasma was separated and stored at −70 °C until analysis. The plasma samples (100 μL) were deproteinized using acetonitrile (60 μL), vortexed, and vacuum filtered; then, 300 μL of 0.15% acetic acid was added, and 10 μL was injected into the HPLC system. The chromatographic conditions were the same as mentioned above. The concentration was obtained using a standard curve prepared from known concentrations of GA dissolved in plasma. The pharmacokinetic parameters of area under the concentration− time curve from zero to t (AUC0−t) and to infinity (AUC0−inf), mean residence time (MRT), maximum concentration (Cmax), clearance (CL), and terminal elimination half-life (t1/2) of GA in GA solution and GA-loaded particle were determined using Pksolver software. Statistical Analysis. Values were expressed as the mean ± standard deviation (SD). Whenever appropriate, a two-tailed Student’s t test was used to discern significant differences between groups. Differences were considered statistically significant for p < 0.05 or p < 0.01.

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RESULTS Particle Characterization. As the SEM images show in Figure 2a1−d1, the GA-Ps are uniform and smooth. After random selection and statistical calculation, the particle size

Figure 3. In vitro release profile for GA-P1 (□), GA-P2 (■), GA-P3 (△), and GA-P4 (▲).

shows the GA release profiles from electrosprayed particles. The ordinates are the amount of accumulated GA released from the particles. The release kinetics for all samples can be illustrated in three stages: the initial stage with a gentle release rate, the intermediate stage with a gradually accelerated release rate, and the last stage with an extremely slow release rate and small release amounts. There is only (8.66 ± 1.23)%, (5.93 ± 1.93)%, (5.23 ± 1.34)%, and (2.47 ± 0.59)% of the total GA released from particles GA-P1, GA-P2, GA-P3, and GA-P4 in the initial 40 h, respectively. After this initial slow release, each particle sample exhibits a gradually accelerated release rate for several days. As shown in Figure 3, the amount of GA released from particle samples GA-P1, GA-P2, GA-P3, and GA-P4 in this accelerated release phase are (78.04 ± 8.64)%, (81.47 ± 8.88)%, (78.42 ± 7.82)%, and (80.46 ± 5.39)%, respectively. The statistical analysis indicates that there are no significant differences between the release profiles of samples GA-P1, GAP2, and GA-P3 in this accelerative release phase (p > 0.05), although when the particle size increases from the nanoscale to the microscale (sample GA-P4), the release rate is remarkably decreased (p < 0.05). In the final stage, each particle sample exhibits an extremely slow release rate and low release quantity. Tissue Targeting and Drug Distribution. The GA tissue concentrations versus time after GA-Ps tail-vein injection (GAPs group) with different particle sizes, and equivalent GA

Figure 2. Representative SEM images and particle size distribution of GA-P1 (a), GA-P2 (b), GA-P3 (c), and GA-P4 (d). 4110



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However, when GA is entrapped into polymeric particles, the times of peak GA concentration (Tmax) in all tissues is delayed to 10 min postdose (Figure 4b−e). The GA concentrations in the liver in the GA-P1 (Figure 4b) and GA-P2 (Figure 4c) groups, in the spleen in the GA-P3 group (Figure 4e), and in the lung in the GA-P4 group (Figure 4e) are much higher than the other tissues (p < 0.05), and these significant differences exist throughout the whole animal experiment. Moreover, the Cmax of GA in the dominate tissue in the GA-Ps groups are significantly higher than the GA-S group. Table 1 summarizes the area under concentration−time curve (AUC) of GA in each tissue after administration of GA solution or GA-Ps and the AUC ratio, designated as re, between the GA-Ps group and GA-S group. As shown in Table 1, the re of the liver in the GA-P1 and GA-P2 groups, the spleen in the GA-P3 group, and the lung in the GA-P4 group is 1.67, 1.78, 2.18, and 2.35, respectively, indicating 1.67-, 1.78-, 2.18-, and 2.35-times higher GA AUC than that of GA-S group. The re of the heart in the GA-P1 and GA-P4 groups are 0.54 and 0.67, indicating a remarkable reduction of the GA distribution in the heart. Figure 5 shows the ratios of the GA-amount in tissues (GA concentrationi × weight of tissuei) in the GA-Ps group to the

solution (GA-S group) are shown in Figure 4a−e. As shown in Figure 4, after tail-vein injection, no matter the dosage form,

Figure 4. GA concentration in tissues of the heart, liver, spleen, lung, and kidney after tail-vein injection of GA solution (a), GA-P1 (b), GAP2 (c), GA-P3 (d), and GA-P4 (e) at a dose of 2.5 mg/kg.

GA is widely distributed in all of the main tissues; heart, liver, spleen, lung, and kidney tissue. In the GA-S group (Figure 4a), GA reaches its maximal concentration (Cmax) in all tissues at 5 min postdose and drops swiftly over time. At 5 min postdose, the GA concentration in the liver is higher than the other tissues, but this significant difference is subtle and transient.

Figure 5. Drug targeting index (DTI) for GA amount ratios between the tissues of the heart (□), liver (●), spleen (▲), lung (*), and kidney (◇) in mice administered GA-P1 (a), GA-P2 (b), GA-P3 (c), and GA-P4 (d) compared with mice administered GA solution.

Table 1. Mean Values of GA AUC (μg × min/g) and re in Tissues after Tail-Vein Injection of GA Solution or GA-Loaded Particles, with Various Particle Sizes, at a Dose Equivalent to GA 2.5 mg/kga tissue groups GA-S GA-P1 GA-P2 GA-P3 GA-P4

a

AUCi AUCi re AUCi re AUCi re AUCi re

heart

liver

spleen

lung

kidney

36.62 ± 4.66 19.84 ± 3.26 0.54 ± 0.11 29.37 ± 3.42 0.80 ± 0.06 31.03 ± 3.27 0.85 ± 0.08 24.47 ± 3.87 0.67 ± 0.07

58.48 ± 5.32 97.58 ± 7.83b 1.67 ± 0.17 103.83 ± 5.99b 1.78 ± 0.13 56.76 ± 5.69 0.97 ± 0.10 48.77 ± 3.28 0.83 ± 0.07

53.12 ± 5.28 48.53 ± 4.39 0.91 ± 0.07 52.69 ± 5.13 0.99 ± 0.07 115.93 ± 7.76b 2.18 ± 0.15 51.93 ± 4.32 0.98 ± 0.09

46.36 ± 4.76 48.30 ± 5.26 1.04 ± 0.17 45.12 ± 4.68 0.97 ± 0.07 47.89 ± 5.18 1.03 ± 0.09 108.89 ± 8.64b 2.35 ± 0.14

45.03 ± 5.19 49.37 ± 4.88 1.10 ± 0.11 47.94 ± 5.12 1.06 ± 0.11 50.51 ± 5.22 1.12 ± 0.11 44.92 ± 5.22 1.00 ± 0.06

Data are presented as the mean ± SD (n = 6). b*p < 0.05 vs GA-S group. 4111



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Table 2. Mean Value of Te (%) and RTe of GA in Tissues after Administration of GA Solution or GA-Loaded Particles with Various Particle Sizes at a Dose Equivalent to GA 2.5 mg/kga tissue groups GA-S GA-P1 GA-P2 GA-P3 GA-P4 a

Te (%) Te (%) RTe Te (%) RTe Te (%) RTe Te (%) RTe

heart

liver

± ± ± ± ± ± ± ± ±

72.96 ± 4.64 81.71 ± 5.22c 1.12 ± 0.07 82.51 ± 4.25b 1.13 ± 0.07 68.66 ± 3.33 0.94 ± 0.06 63.11 ± 3.68 0.86 ± 0.12

3.56 1.28 0.36 1.80 0.50 2.90 0.82 2.44 0.68

0.55 0.12b 0.06 0.21b 0.03 0.27 0.11 0.36 0.11

spleen

lung

kidney

± ± ± ± ± ± ± ± ±

6.91 ± 0.66 4.84 ± 0.22b 0.70 ± 0.06 4.29 ± 0.62b 0.62 ± 0.04 6.93 ± 0.76 1.00 ± 0.05 16.87 ± 2.02b 2.44 ± 0.16

14.04 ± 1.72 10.39 ± 1.12b 0.74 ± 0.06 9.58 ± 1.13b 0.68 ± 0.08 15.38 ± 2.12 1.09 ± 0.10 14.64 ± 1.27 1.04 ± 0.08

2.85 1.77 0.62 1.82 0.63 6.12 2.15 2.93 1.02

Data are presented as the mean ± SD (n = 6). b*p < 0.05 vs GA-S group.

0.13 0.15b 0.05 0.15b 0.04 0.13b 0.11 0.27 0.11

c#

p < 0.01 vs GA-S group.

Figure 6. Fluorescent microscopy images of tissues in mice administered ATPS-GA solution (a) at 5 min postdose and ATPS-GA-loaded particles AGA-P2 (b), A-GA-P3 (c), and A-GA-P4 (d) at 10 min postdose.

maximums of 2.81 ± 0.17, 3.66 ± 0.21, and 3.88 ± 0.22 at 15 min postdose. Table 2 summarizes the Te for the distribution ratio of GA in each tissue and the RTe for Te ratios between the GA-Ps groups and the GA-S group. In the GA-P1 group, the Te and RTe in the liver are 81.71% and 1.12. The GA-P2 group exhibits a similar Te and RTe in the liver as the GA-P1 group. The Te and RTe of

amount in the GA-S group, designated as DTI. At 5 min postdose, the DTI of tissues in all particle groups are close to zero. Over time, the DTI of the liver in the GA-P1 group reaches a maximum of 2.70 ± 0.26, indicating 2.7-times higher GA-amounts in the liver in the GA-P1 group compared with the GA-S group. The DTI of the liver in GA-P2 group, spleen in GA-P3 group, and lung in GA-P4 group, respectively, have 4112



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Table 3. Liver Function Indices of Normal Mice and HCC Model Mice at the 1st and 2nd Week after Being Injected with LiverTargeting GA-Loaded Particles (GA-P2 Group) or GA Solution (GA-S Group) by Tail-Vein Injection at a Dose of 2.5 mg/kg or after Injection of an Equal Volume of Normal Saline (NS Group)a liver function indices groups

ALT (U/L)

TBA (μmol/L)

PA (mg/L)

normal model

38.7 ± 3.2 366.1 ± 58.5b 426.1 ± 37.4b 347.5 ± 8.3b 382.8 ± 47.9b 286.9 ± 35.2b 205.4 ± 32.6b,c,d 177.7 ± 27.6b,c 148.9 ± 31.1b,c

1.6 ± 0.2 41.5 ± 3.8b 73.2 ± 5.5b 44.6 ± 5.6b 62.3 ± 4.8b 15.1 ± 2.2b,c 6.5 ± 1.7b,c,d 8.3 ± 1.6b,c 3.1 ± 0.6b,c,d

233.7 ± 35.6 126.6 ± 22.6b 97.6 ± 1.7b 121.2 ± 31.7b 121.7 ± 26.4b 171.1 ± 15.6b,c 177.5 ± 26.4c 179.2 ± 18.7c 220.5 ± 28.3c,d

NS GA-S GA-P2 a

1st week 2nd week 1st week 2nd week 1st week 2nd week 1st week 2nd week

Data are presented as the mean ± SD (n = 6). b*p < 0.05 vs normal group.

other tissues in the GA-P1 and GA-P2 groups are greatly reduced. In the GA-P3 group, the RTe of the spleen is 2.15, and the RTe of the heart is reduced to 0.82. In the GA-P4 group, the RTe of the lung is 2.44, and the RTe of the heart and liver are reduced to 0.68 and 0.86. To observe the particle distribution, GA was labeled with APTS, and the fluorescent particles, which were of various sizes, were administered to mice. Figure 6 shows the fluorescent microscope images of tissues at Tmax after administration. As shown in Figure 6a−d, the yellow−green fluorescence of APTS-labeled GA can be observed in all tissues. At 5 min postdose in the GA-S group, the fluorescence intensity in the liver (Figure 6a2) is higher than other tissues. Figure 6b−d shows that the fluorescence intensity in the livers of mice in the GA-P2 group (Figure 6b2), in the spleens of mice in the GA-P3 group (Figure 6c3), and in the lungs of mice in the GA-P4 group (Figure 6d4) are much higher than other tissues. The fluorescence intensity and distribution in the GA-P1 group are similar to the GA-P2 group and are not shown. The fluorescence distribution in different tissues in each group corresponds with the GA concentration data, as shown in Figure 4. However, the unique structures possessed by the liver, spleen, and lung allow discriminate fluorescence intensities between tissues with similar GA concentration. Antitumor Efficacy. As the GA distribution ratio in the liver in the GA-P2 group reaches 82.51%, the particle sample GA-P2 was used as a site-specific GA delivery system to the liver, and its antitumor efficacy in a HCC mice model was investigated in comparison with GA solution. Table 3 summarizes the liver function indices of normal mice and HCC model mice at the first and second week after administration. As shown in Table 3, the liver indices of ALT, TBA, AKP, and GGT of HCC model mice (model group) are significantly higher than those of normal mice (p < 0.05), while PA is significantly decreased (p < 0.05). The administration of normal saline (NS group) did not influence the liver function of HCC model mice. With administration of GA solution (GA-S group) or GA-P2 (GA-P2 group), liver function significantly recovered (p < 0.05, vs model group) and liver function recovery was positively correlated with the duration of administration time (p < 0.05, second week vs first week). Notably, the PA and GGT indices of mice in GA-P2 group return to normal after 2 wk administration (p > 0.05, vs normal group). Figure 7 shows the liver histomorphology of normal mice and HCC model mice in each experimental group after 2 wk

c#

AKP (U/L) 195.3 775.5 810.0 733.4 858.9 674.3 510.6 512.3 276.5

p < 0.05 vs model group.

± ± ± ± ± ± ± ± ±

22.1 117.2b 68.4b 43.2b 42.1b 145.2b 57.8b,c 107.3b,c 55.6b,c,d

GGT (U/L) 5.2 ± 1.7 46.3 ± 6.8b 106.6 ± 0.5b 42.3 ± 8.3b 91.0 ± 11.5b 28.3 ± 4.2b,c 16.6 ± 3.7b,c,d 26.4 ± 3.7b,c 8.3 ± 2.6c,d

d&

p < 0.05 vs 1st week.

Figure 7. (a−e) Liver histomorphology in normal mice (a) and HCC model mice at the 2nd wk after modeling (b1−b3) and after tertiary tail-vein injection of normal saline (c), GA solution (d), or liver targeting GA-P2 (e). Samples were stained with HE (a, b1, b2, c, and d) and SR (b3). (f) Survival time of HCC model mice with or without any drug administration.

administration using HE and SR staining. The micrograph of hepatic tissue in normal mice exhibits a typical lobular architecture with a centered terminal venule (Figure 7a, white arrow) and radiating hepatic cords, polygonous hepatic cells with a rounded nucleus in the center, and fat-storing cells with fat vacuoles and a tent-like nucleus (Figure 7a, blue arrow). The micrograph of hepatic tissue in the model group mice shows a banded nest (Figure 7b1) with filamentary collagen fiber accumulation (Figure 7b3), heteromorphic and irregularly arranged hepatic cells, basophilic cytoplasts, pleomorphic, hyperchromatic, and vesicular nuclei (Figure 7b2, black arrow), increased nucleo-cytoplasmic ratio, bile-plug deposition (Figure 7b2, yellow arrow), and eosinophilic and globular bodies in cytoplasm. In the NS group (Figure 7c), the hepatic tissue exhibits almost the same morphologic characteristics as the HCC model mice and lymphocyte infiltration (Figure 7c, red arrow). Figure 7d shows the liver histomorphology in GA-S group, exhibiting some vesicular nuclei (Figure 7d, black arrow) 4113



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toxicity or adverse drug reactions in noncancerous tissues. In the present study, a series of GA-Ps were prepared using electrospray techniques. The morphology, size distribution, drug loading, encapsulation efficiency, and in vitro GA release profiles of GA-Ps were investigated. After determining the GA concentration−time curves in each main tissue, the targeting indices of re, DTI, Te, and RTe for each GA-P sample were calculated. Additionally, APTS-labeled GA was synthesized, entrapped into polymeric particles, and tail-vein injected in the same manner as GA-Ps, to observe the GA distribution in tissues. Subsequently, the GA-P with the highest targeting efficiency to the liver was administered to HCC model mice, and its antitumor efficacy was investigated in comparison with GA solution. To explain the differences in antitumor efficacy between liver-targeting GA-P and GA-S, the pharmacokinetic parameters of AUC0−t, AUC0−inf, MRT0−inf, Cmax, CL, and t1/2 for these two formulations of GA in plasma were determined. SEM and DLS (Figure 2) analyses demonstrated that the particle size of GA-Ps is monotonically increased with an increase of capillary external diameter and flow rate and decreased applied voltage. A larger capillary external diameter, faster flow rate, and lower applied voltage are thought to offer larger droplets and result in a larger particle, which have been confirmed by early research.23 However, the morphology of GA-Ps, which was approximately spherical and display modest faceting, was not affected by parameter variation. In comparison with commonly used preparation methods for drug loading particles such as microsphere hardening, solvent evaporation, and phase separation, the electrospray technique has the prominent advantages of simplicity and narrow size distributions. In the present study, part of the particles exhibited sticky properties, as shown in SEM images. It is speculated because of the ultracentrifuge leading aggregation. However, because of the reliance on Coulomb repulsion in the electrospray process to disperse the liquid into charged droplets; the generated particles are easily redispersible.24 Entrapment efficiency is an important performance index to evaluate drug delivery systems. In early research, GA was embedded in micelles,8,9,25 Fe 3O 4 nanoparticles, 10 and lactoferrin nanoparticles.26 The optimal entrapment efficiency of GA in micelles is 72.7%, while the entrapment efficiency of all the GA-Ps prepared by electrospray is more than 80%. Although the entrapment efficiency of GA carrying lactoferrin nanoparticles is similar to that of electrosprayed GA-Ps, the heterogeneous protein used in the albumin-bound technique carries a remote risk of virus transmission. Besides these, as the technique of electrospray is a simple and straightforward process, the drug loss in the present study was speculated to be resulted from the collection using ultrapure water and the solubility of GA in water. In the in vitro GA release experiments, the amounts of released media met sink conditions. The cumulative in vitro GA release profiles from all the electrosprayed GA-Ps exhibit an

and globular bodies in cytoplasm (Figure 7d, green arrow), but insignificant cell proliferation. Figure 7e shows the liver histomorphology of mice in GA-P 2 group, exhibiting insignificant cell proliferation or a nucleo-cytoplasmic ratio change, and few pleomorphic or vesicular nuclei (Figure 7e, black arrow). Figure 7f is the HCC model mice survival time in each experimental group. It shows that the survival times of mice in the model group, NS group, GA-S group, and GA-P2 group are 24.0 ± 5.3, 22.2 ± 4.7, 29.8 ± 2.6, and 44.3 ± 3.8 days, respectively. There is no significant difference in survival times between the model, NC, and GA-S groups (p > 0.05), while the survival time of GA-P2 group is significantly longer than that of other groups (p < 0.05). Plasma Pharmacokinetic Parameters. Figure 8 exhibits the mean concentration−time profiles of GA in plasma in rats

Figure 8. Mean concentration−time profiles of GA in plasma after a single tail-vein injection in rats at a dose of 2.5 mg/kg with formulations of solution (○) or liver-targeting particles (●).

after a single 2.5 mg/kg tail-vein injection dose of solution or liver-targeting particle formulations. In comparison to the rapid and constant decrease of GA plasma concentration observed in rats in the GA-S group, the GA plasma concentration−time curve of rats in GA-P2 group exhibit a peak at 10 min after injection. Importantly, at each time point the GA plasma concentrations of rats in GA-P2 group are significantly higher than the GA-S group. A noncompartmental model was used to calculate the pharmacokinetic parameters. Table 4 shows the significant differences in pharmacokinetic parameters between the GA-S and GA-P2 groups. The liver targeting GA-P2 exhibits 4.01-, 3.56-, and 1.86-times higher AUC0−t, AUC0−inf, and Cmax values, 1.80- and 1.86-times longer MRT0‑inf and t1/2, and 3.54times lower CL than the GA solution.

■

DISCUSSION GA has been demonstrated to be an effective antitumor agent for variety of tumors; however, its poor water-solubility and toxic side effects impede preclinical study and further application. Entrapment with polymeric particles is a promising strategy to achieve the appropriate dosage for tumor growth inhibition, improve targeting effects to tumor tissue, and reduce

Table 4. Pharmacokinetic Parameters of Liver Targeting GA-Loaded Particles after Bolus Tail-Vein Injection at Dose of 2.5 mg/kg in Comparison with GA Solutiona pharmacokinetic parameters

a

formulations

AUC0−t (μg/mL·h)

AUC0−inf (μg/mL·h)

MRT0−inf (h)

Cmax (μg/mL)

CL (L/h·kg)

t1/2 (h)

GA solution liver targeting GA-loaded particle

2.74 ± 0.22 11.00 ± 0.42b

3.70 ± 0.27 13.16 ± 0.57b

2.79 ± 0.17 5.03 ± 0.16b

6.32 ± 0.55 11.78 ± 1.24b

4.32 ± 0.28 1.22 ± 0.12b

2.80 ± 0.17 5.20 ± 0.33b

Data are presented as the mean ± SD (n = 6). b*p < 0.05 vs GA solution. 4114



Article

1.0, indicating their liver targeting properties. This more selective GA localization in the liver is by virtue of reticular endothelial system (RES) uptake.30 Additionally, the GA RTe in other tissues in groups P1 and P2 were greatly reduced, indicating the reduced risk of potential toxicity in other tissues. Similarly, the outstanding GA concentration, DTI, re, and RTe of the spleen in the GA-P3 group and the lung in the GA-P4 group indicate the remarkable targeting effects of GA-P3 and GA-P4, on the spleen and lung, respectively. The reduction of RTe in the heart in groups GA-P3 and GA-P4 demonstrate the reduced risk of potential cardiac toxicity. Fluorescent images of APTS-labeled GA in tissues (Figure 6) provide direct visual evidence for the above results and speculations. In the present study, the antihepatoma effects of the liver targeting GA-loaded particle GA-P2 were evaluated in comparison with GA solution. As the utility of the tumorassociated antigens of alpha fetal protein (AFP) and carcinoembryonic antigen (CEA) on HCC diagnosis are uncertain, histological examination and liver function were chosen to evaluate the degree of tumor invasion and cell lesions. As the orthotopic injection of H22 cells into the liver led to a series of tumor-like lesions and biochemical abnormalities in liver in previous experiments, this method was chosen to establish the orthotopic xenograft mouse model of HCC. In antitumor experiments, the HCC model mice exhibited significantly higher ALT, TBA, AKP, and GGT and lower PA than normal mice (p < 0.05), as shown in Table 3. The value of ALT is one of the most commonly used liver function indices in all hepatic diseases, and ALT increases are positively correlated with the degree of liver damage, increased hepatocyte permeability, and necrosis. TBA is a cholesterol metabolite and inevitably increases in cases of hepatoma. A higher TBA value indicates a decreased metabolic function of the liver. PA is synthesized by hepatocytes, and lower PA values in the HCC model mice sensitively indicate the decreased synthetic function and reserve capacity of liver.31 GGT is synthesized in the liver and excreted by the bile. In hepatoma cases, the synthesis of GGT is sthenic, permeation is increased, and excretion is blocked by biliary obstruction. Thus, the elevation of GGT values in the HCC model mice indicates the occurrence of not only hepatoma but also biliary obstruction. AKP is another commonly used liver function index, and an elevated value also indicates dysfunctional bile excretion in the liver. All the abnormal biochemical indices in the HCC model group correspond with the histological changes of cell heteromorphism, inflammatory cell infiltration, fibrosis, and bile-plug deposition, as shown in Figures 7b1−b3. HCC model mice with normal saline administration acting as the negative control for GA-loaded particle suspensions in the antitumor experiment exhibited similar morphological changes and liver dysfunction compared with the HCC model mice (Figure 7c and Table 3). In the GA-S group, the hepatocytes exhibited partial heteromorphism and globular bodies in cytoplasm, indicating a degree of hepatocyte injury (Figure 7d). Although the HCC mice administered with GA solution still exhibited abnormal liver function; all liver function index values were significantly recovered compared with mice in HCC model group (Table 3). In the GA-P2 group, smaller morphological abnormalities and a higher degree of liver function was recovered compared with the GA-S group, indicating a stronger antitumor effect for liver-targeting GA-loaded particles versus GA solution. In particular, the PA and GGT values of HCC mice in the GA-P2 group were close to normal, indicating the

initial stage with a gentle release rate, an intermediate stage with a gradually accelerated release rate, and the final stage with an extremely slow release rate and small release amounts, as shown in Figure 3. As the particle matrix material of the PDLLA polymer used in the current study is relatively stable and requires long incubation periods for degradation, the possible GA release mechanism from particles is diffusion through the polymer matrix and/or diffusion through pores in the particles. In most polymer-based drug-loaded micro/nanoparticles, the fast dissolving of drug located on/near particle surface causes the initial burst release.27 However, in our study, the GA-loaded particles were collected by ultrapure water and separated by ultracentrifuge, so it showed rare GA on the particle surface. Moreover, because of the poor aqueous solubility of GA, the affinity of GA with PDLLA molecular chains is much higher than the aqueous release media, and the GA detachment from the particle matrix is very low, there is slight drug release from particles in the initial stage. However, with the releasing out of drug from particles, GA detachment leads increasing numbers of pores in the particles, resulting in faster water distribution into the particle matrix and faster GA solvation. The significant difference between the release rates in the accelerative release phase between nano-GA-Ps and micro-GA-P should result from the higher specific surface area of nanoscale particles. After this accelerative release phase, the release of the majority of GA leads to the shrinkage and collapse of the particle matrix, and the remaining GA may be released until the matrix polymer degrades. This speculation derives from previous research in electrospun drug-loaded fibers.28 Yu et al.8 and Zhang et al.26 have discussed the GA release profiles from albumin-bound particles and micelles, respectively, which exhibit rapid GA release and short release time. In the present study, the GA release from polymeric particles lasts for several days in vitro. However, given the higher solubility of lipophilic drugs and faster polymer degradation in vivo, GA release from polymeric particles in vivo should be much faster. Previous studies have demonstrated that GA in a solution formulation rapidly and widely distributes to all tissues after intravenous injection regardless of the dosage form.29 This has also been confirmed in our study, as shown in Figure 4. After concentration−time curves were calculated in each tissue, the liver in the GA-S group exhibits a much higher AUC, drug amount, and drug distribution than other tissues. Because the differences between GA concentrations in the liver and other tissues are subtle and transient, we propose that the primary reason for increased GA in the liver in the GA-S group may be attributed to the greater mass of the liver. While Figure 4 shows the differences in GA concentrations in each tissue between the GA-S group and GA-Ps groups, Figure 5 shows the drug amount ratio in each tissue between GA-Ps groups and GA-S group at each time point, referred to as DTI. The delayed Tmax and low DTI values of less than 1.0 at 5 min postdose in the GA-Ps groups indicate a gradual drug release from GA-Ps. The significantly elevated GA concentrations and high DTI values of the dominate tissue in the GA-Ps groups at subsequent time points indicate that the GA entrapment in polymeric particles can reduce the GA elimination rate. In the GA-S group, the (72.96 ± 4.64)% Te value of the liver indicates that the liver is the dominant organ for GA distribution; however, there is GA distribution to other organs, which may result in the highly toxic side effects in normal tissue. In the GA-P1 and GA-P2 groups, the Te values of the liver are significantly increased (p < 0.01), and the corresponding re and RTe are much greater than 4115


Molecular Pharmaceutics effective protection of liver-targeting GA-loaded particles on the synthetic function, reserve capacity, and excretory function of the liver. The increased survival time of HCC model mice in the GA-P2 group compared with other groups also confirms the increased antitumor effect of liver-targeting GA-loaded particles compared with GA solution. The plasma pharmacokinetic parameters of liver-targeting GA-loaded particles and GA solution were determined and calculated to discuss their different antitumor effects. The delayed Tmax, elevated GA concentration, prolonged MRT0−inf, and lower CL in the GA-P group indicate that the entrapment of GA into polymeric particles can significantly reduce the plasma clearance and extend the retention time. Additionally, the higher AUC and t1/2 values of the GA-P group indicate that entrapment can obviously improve GA bioavailability and stability. Thus, the increased antitumor efficacy of livertargeting GA-loaded particles in HCC is a result of the comprehensive effects of tissue targeting, lower clearance, extended retention time, and higher stability of GA.

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CONCLUSIONS

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AUTHOR INFORMATION

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ACKNOWLEDGMENTS

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REFERENCES

Article

This project was supported by the National Natural Science Foundation of China (81173556 and 81303239) and the Key Fund Project of Anhui Provincial Department of Education (KJ2012A181).

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A site-specific delivery system for GA was prepared using an electrospray technique and its morphologic, loading efficiency, in vitro drug release profile, in vivo target distribution, antitumor efficacy on HCC, and pharmacokinetic profile characteristics were investigated. By altering the preparation parameters, the sizes of obtained GA-Ps ranged from nano- to microscale. The particles exhibited high encapsulation efficiency, regardless of particle size. In vitro release studies demonstrated that the GA release rates from particles are closely related to the particle sizes. When the particle sizes increased from approximately 100 nm to 7 μm, the GA release time ranged from 6 to 10 days. In vivo studies of GA concentrations and tissue distributions demonstrated that the GA entrapment into polymeric particles can effectively alter GA tissue distribution. Particularly, the GAPs with diameters of 69.8 ± 17.8 (GA-P1) and 185.6 ± 33.8 nm (GA-P2) exhibited over 80% of Te to the liver. Histological examinations using HE and SR staining and liver function assessment using ALT, TBA, PA, AKP, and GGT demonstrated that the liver targeting GA-P2 exhibited superior antitumor efficacy displaying a lower degree of tumor invasion and cell lesion in hepatic tissue, greatly recovered liver function, and significantly prolonged survival time, in HCC model mice compared with GA solution administration. Pharmacokinetic studies indicated that the GA entrapped in particles exhibited lower clearance, longer retention time, and higher bioavailability and stability in plasma versus GA dissolved in NS. In summary, the GA-loaded particle formulation synthesized using an electrospray technique may be an ideal approach to achieve satisfactory antitumor efficacy and to overcome the limitations of GA, including its poor water-solubility and highly toxic side effects in normal tissue.

Corresponding Author

*Tel: +86 551 65169235. Fax: +86 551 65169222. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 4116



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Gambogic Acid-Loaded Electrosprayed Particles for Site-Specific

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