Purpose: Quercetin is a potent chemotherapeutic drug. Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility. To overcome this limitation, this study is aimed to develop liposomal quercetin and investigate its distribution in vivo and antitumor efficacy in vivo and in vitro.
Experimental Design: Quercetin was encapsulated in polyethylene glycol 4000 liposomes. Biodistribution of liposomal quercetin i.v. at 50 mg/kg in tumor-bearing mice was detected by high-performance liquid chromatography. Induction of apoptosis by liposomal quercetin in vitro was tested. The antitumor activity of liposomal quercetin was evaluated in the immunocompetent C57BL/6N mice bearing LL/2 Lewis lung cancer and in BALB/c mice bearing CT26 colon adenocarcinoma and H22 hepatoma. Tumor volume and survival time were observed. The mechanisms underlying the antitumor effect of quercetin in vivo was investigated by detecting the microvessel density, apoptosis, and heat shock protein 70 expression in tumor tissues.
Results: Liposomal quercetin could be dissolved in i.v. injection and effectively accumulate in tumor tissues. The half-time of liposomal quercetin was 2 hours in plasma. The liposomal quercetin induced apoptosis in vitro and significantly inhibited tumor growth in vivo in a dose-dependent manner. The optimal dose of liposomal quercetin resulted in a 40-day survival rate of 40%. Quantitative real-time PCR showed that liposomal quercetin down-regulated the expression of heat shock protein 70 in tumor tissues. Immunohistochemistry analysis showed that liposomal quercetin inhibited tumor angiogenesis as assessed by CD31 and induced tumor cell apoptosis.
Conclusions: Our data indicated that pegylated liposomal quercetin can significantly improve the solubility and bioavailability of quercetin and can be a potential application in the treatment of tumor.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a naturally occurring flavone found in the plant kingdom. It is a component of most edible fruits and vegetables, with the highest concentrations being found in onions, apples, and red wine (1, 2). Quercetin has many biological activities, such as antitumor and antiproliferative effects on a wide range of human cancer cell lines and inhibition of glycolysis, macromolecule synthesis, and enzymes (3–7). Its inhibition of the heat shock protein 70 (HSP70) synthesis and expression in tumor cells was reported in our previous work (8). Clinical trials exploring different schedules of administration of quercetin have been hampered by its extreme water insolubility requiring dissolution in DMSO. There are concerns about using higher doses of DMSO, as it causes dose-dependent hemolysis, it is harmful to the liver and kidneys, and it has an unpleasant odor for 48 hours after administration (9–11). A potential solution to this problem is to improve its solubility by chemical modification (12), but the decreased activity of quercetin was observed in our laboratory.
Liposomes have been used previously as carriers for delivery of a variety of drugs, including antibiotic, antifungal, and cytotoxic agents (13). As carriers for anticancer drugs, they have been shown to reduce side effect, such as anthracycline-induced cardiomyopathy (14). Liposomes accumulate preferentially at tumor sites because of their ability to extravasate through “pores” or “defects” in the capillary endothelium. These “pores” seem to be a consequence of the rapid angiogenesis occurring in tumors and are generally not present in normal tissues or organs (15). After coating the liposome surface with inert, biocompatible polymers, such as polyethylene glycol (PEG), it forms a protective layer over the liposome surface and slows down liposome recognition by opsonins and therefore subsequent clearance of liposomes, so that a liposome has a long circulation time and provides slow release of an encapsulated drug, resulting in sustained exposure to tumor cells and enhanced efficacy (16).
In the present work, we encapsulated quercetin in the nonaqueous interior of the PEG liposome. The pharmacokinetic properties and biodistribution in tumor-bearing mice were tested, and anticancer efficacy of liposomal quercetin was observed in vivo and in vitro.
Materials and Methods
Materials. High-purity lecithin of soybean, PEG 4000, glucosidase, cholesterol, and quercetin were purchased from Sigma Chemical Co. (St. Louis, MO). Cisplatin (DDP) was purchased from Qilu Pharmaceutical Co. (Shandong, China). A rat antimouse CD31 monoclonal antibody was purchased from BD Biosciences Co. (PharMingen, San Diego, CA). In situ Cell Death Detection kit was purchased from Roche Co. (Promega, Madison, WI) Real-time One-Step RNA PCR kit was purchased from TaKaRa Co. (Dalian, China).
Cell culture and tumor model. Mouse colorectal carcinoma cell line CT26, Lewis lung cancer cell line LL/2, and hepatoma cell line H22 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). These cells were grown in RPMI 1640 (Life Technologies, Bedford, MA) or DMEM (Life Technologies) containing 10% heat-inactivated FCS, 100 units/mL penicillin, and 100 units/mL streptomycin in a humid chamber at 37°C under 5% CO2.
The CT26 and H22 tumor models were established in 8-week-old female BALB/c mice. LL/2 tumor model was established in C57BL/6N mice as described previously (17). Briefly, these BALB/c mice were inoculated with CT26 or H22 cells (5 × 105) and C57BL/6N mice with LL/2 cells (1 × 106) s.c. in the dorsal area. All these mice were purchased from Sichuan University Animal Center (Sichuan, Chengdu, China). All studies involving mice were approved by the Institute's Animal Care and Use Committee.
Preparation of pegylated liposomal quercetin and empty pegylated liposomes. PEG-modified liposomal quercetin (Q-PEGL) were prepared in our laboratory and described briefly as follows: the mixtures of lecithin/cholesterol/PEG 4000/quercetin in 13:4:1:6 weight ratio were dissolved in chloroform/methanol (3:1, v/v) and evaporated to dryness under reduced pressure in a rotary evaporator. The dried lipid films were sonicated in 5% glucose solution at constant container followed by concentrated and lyophilized. The preparation of empty pegylated liposomes (PEGL) was the same way as the Q-PEGL without quercetin in the mixtures. The final Q-PEGL and the PEGL were small unilamellar liposomes in our studies and in a size range of 130 ± 20 and 100 ± 20 nm, respectively. In following studies, the dose of Q-PEGL was on a quercetin basis, and Q-PEGL or PEGL was dissolved in 5% glucose water.
Pharmacokinetic studies in tumor-bearing BABL/c mice. Female BABL/c mice (n = 64) were inoculated with CT26 cells. On day 15, the Q-PEGL or the free quercetin dissolved in DMSO was given i.v. to these mice at a dose of 50 mg/kg. Mice were sacrificed at defined time points (5, 15, 30, 60, 120, 240, 720, and 1,440 minutes). At each time point, four mice were sacrificed and their blood was collected from the orbital cavity vein plexus, heparinized, and centrifuged to obtain the plasma. The tumor, kidney, liver, lung, intestine, heart, and spleen tissues were excised and weighed, and homogenized. Glucosidase was added into plasma sample and incubated in 37°C for 2 hours. Plasma and the homogenized organs were extracted with ethyl acetate, and supernatant fluid was collected and evaporated to dryness. The dry residues were dissolved in methanol for high-performance liquid chromatography analyses.
In vitro cytotoxicity. The growth-inhibitory activities of Q-PEGL on the CT26 and LL/2 cell lines were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The CT26 and LL/2 cells were cultured in DMEM containing 10% fetal bovine serum in a 96-well multiplate. Exponentially growing cells at 2 × 105/mL were exposed to each various doses of Q-PEGL or free quercetin with equivalent dose quercetin for various time intervals. The control culture was treated with PEGL, which diluted along with Q-PEGL dilutions. Then, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to these wells at different time point. Cell viability was measured by the absorbance at 570 nm.
To quantitative assessment of apoptosis and analysis of cell cycle specificity, flow cytometric analysis was done to identify sub-G1 cells/apoptotic cells and to measure the percentage of sub-G1 cells after propidium iodide staining in hypotonic buffer as described (8, 18). Briefly, cells were suspended in 1 mL hypotonic fluorochrome solution containing 50 μg propidium iodide/mL in 0.1% sodium citrate plus 0.1% Triton X-100 and the cells were analyzed by flow cytometer (ESP Elite, Beckman-Coulter, Miami, FL). Apoptotic cells appeared in the cell cycle distribution as cells with a DNA content of less than that of G1 cells and were estimated with Listmode software.
In vivo antitumor activity. In the first experiment, CT26-bearing BABL/c mice and LL/2-bearing C57BL/6N mice were coded and divided into seven groups (n = 10 for each group). Treatment was initiated when tumor volume was ∼90 mm3. These mice were given i.v. with PBS, PEGL (100 mg/kg), and Q-PEGL (12.5, 25, 50, 100, and 200 mg/kg), respectively. All these reagents were given every 3 days for 15 days. The mice were sacrificed on day 18 to ascertain the size of tumor lesions, and excised tumors were weighed.
In the second study, the tumor-bearing BABL/c mice inoculated with CT26 cells and C57BL/6N mice with LL/2 cells were coded and divided into four groups (n = 10 for each group). These mice were given i.v. with Q-PEGL (50 mg/kg), free quercetin (50 mg/kg), free quercetin (50 mg/kg) plus PEGL (100 mg/kg), and PBS, respectively. All these treatment were given every 3 days for 15 days. Then, these mice were sacrificed on day 18 to ascertain the size of tumor lesions, and excised tumors were weighed as above.
In the third study, tumor-bearing mice inoculated with CT26, H22, and LL/2 cells were classified into three groups (n = 10 for each group). In each tumor-bearing mice model, mice were i.v. given with Q-PEGL (50 mg/kg every 3 days for 21 days), DDP (5 mg/kg on days 1, 8, and 15) for positive control groups, and PBS (0.2 mL every 3 days for 21 days) for negative control groups. DDP was dissolved in 0.9% normal solution. Survival time and tumor volumes were observed. Tumor size was determined by caliper measurement of the largest and perpendicular diameters every other day. Tumor volumes were calculated according to the formula: V = a × b2 × 0.52, where a is the largest superficial diameter and b is the smallest superficial diameter. The mice were sacrificed when they became moribund. Then, the sacrificed date was recorded to calculate the survival time. To detect the microvessel density, apoptosis, and HSP70 expression, tumors tissues excised were fixed in 10% formalin and frozen in −80°C.
Detection of HSP70 mRNA expression in tumor tissues with quantitative real-time PCR. Quantitative real-time PCR was done to confirm HSP70 expression in tumor tissues. The HSP70-specific primers are as follows: forward primer 5V-CGAGAGCCGGTCGTTCTTC-3V and reverse primer 5V-CAGGTACGCCTCAGCGATCT-3V. The amplified fragment was 202 bp. The probe of HSP70 was 5V-FAM-CGTCCATGGTGCTGACGAAGATGAAG-TAMRA-3V. β-Actin mRNA expression was used to normalize the results.
Total RNA was extracted from tumor tissues (50 mg) by acid-guanidinium thiocyanate-phenol-chloroform (19). The RNA was resuspended in diethyl pyrocarbonate–treated water and quantitated spectrophotometrically for analysis. Reverse transcription reactions were carried out according to Wong et al. (20). Briefly, the percentage efficiency for reverse transcription reactions was estimated to be 69 ± 1.9 (mean ± SE for n = 7). The PCR profile was denaturing at 94.8°C for 30 seconds, annealing at 55.8°C for 30 seconds, and extension at 72.8°C for 30 seconds. In each case, the number of cycles was 35. PCR was carried out in a Perkin-Elmer (Cupertino, CA) 2400 Thermal Cycler. Analysis of PCR product capillary electrophoresis was carried out in a 47-cm silica capillary containing POP-4 polymer (PE Applied Biosystems, Branchburg, NJ). Each sample was run for 24 minutes at 60 kJ voltage with 5 seconds of injection time. We did LIFCE in an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
Detection CD31 by immunohistochemistry and apoptosis by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling in tumor tissues. The antiangiogenesis effects of liposomal quercetin were determined by CD31 immunostaining. The frozen tissue sections were fixed in acetone, incubated, and stained with an antibody reactive to CD31. The sections were then stained with labeled streptavidin biotin reagents. Vessel density was determined by counting the number of microvessels per high-power field in the sections as described (21). Tumor species embedded by paraffin were prepared as described above. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining was done following the manufacturer's protocol. Four equal-sized fields in tissue sections were randomly chosen and analyzed. The positive staining was evaluated in each field, yielding the density of apoptotic cells (apoptosis index).
Statistical analysis. Data were assayed by ANOVA and Student's t test. For the survival time of animals, Kaplan-Meier curves were established for each group, and the survivals were compared by the log-rank test. Differences between means or ranks as appropriate were considered significant when yielding a P < 0.05. Results are presented as means ± SD. Experiments were done at least in duplicate. The half-lives were calculated using a numerical module of the 3P97 computer program (Chinese Pharmacological Society) for kinetic analysis. The data were fitted into a biexponential equation with a bolus injection as an experimental model.
Pharmacokinetics and tissue distribution of Q-PEGL. After i.v. Q-PEGL, quercetin released from pegylated liposomes quickly conjuncted with glucuronide. Glucosidase was added into plasma sample to break the conjugation and detect free quercetin by high-performance liquid chromatography. Our results show that Q-PEGL prolonged blood circulation times in BALB/c tumor-bearing mice in comparison with free quercetin. The lifetime of Q-PEGL is 2 hours in the plasma, but the lifetime of free quercetin only 5 minutes. The maximum concentration of quercetin in the plasma was 105.6 ± 17.5 nmol/mL for the Q-PEGL at time point of 30 minutes and 113.5 ± 23.5 nmol/mL for the free quercetin at time point beginning to detected (Fig. 1A). The levels of quercetin could be still detected at 1,440 minutes for Q-PEGL in plasma.
In the mice treated by free quercetin, quercetin was widely distributed in normal organs and s.c. tumor tissues, and the concentrations of quercetin were relatively high in kidney and liver, moderate in spleen and tumor tissues, and low in lung. These results were consistent to other reports because free quercetin was rapidly cleared up in plasma (Fig. 1A and B). When encapsulated with PEG, quercetin was highly accumulated in liver, tumor tissues, and spleen but low in kidney and lung tissues (Fig. 1C) and had a long retention time in liver, spleen, and tumor tissues. These results show that Q-PEGL could selectively accumulate in s.c. tumor tissues and lengthen retention time at tumor site compared with free quercetin (Fig. 1B and C).
Inhibition of cell proliferation by Q-PEGL treatment. The Q-PEGL treatment resulted in inhibition of cell proliferation in vitro, which has similar antiproliferative effect compared with free quercetin. The inhibition effect was dependent on the dose of Q-PEGL and incubation time. For example, when CT26 cells were treated for 36 hours, the percentage inhibition of 1 and 10 μg/mL Q-PEGL was 21% and 71.5%, respectively (Fig. 2A). When CT26 cells were treated by 10 μg/mL Q-PEGL, the percentage inhibition was 42% for 24 hours and 81% for 96 hours (Fig. 2B). The PEGL could also inhibit CT26 cell proliferation but did not show such time- and dose-dependent effect.
By the use of flow cytometry, we could assess the number of sub-G1 cells (apoptotic cells). Results obtained with flow cytometry strongly show that the Q-PEGL lead to the CT26 death by inducing apoptosis. The quantitative assessment of sub-G1 cells by flow cytometry was used to estimate the number of apoptotic cells. The apoptosis-inducing effect of Q-PEGL was dose and time dependent, being observed at 1 μg/mL and reaching a maximum at 15 μg/mL when analyzed by flow cytometry. The increased number of apoptotic cells was detected after 12 hours of continuous Q-PEGL treatment, reaching a maximum by 60 hours (Fig. 3).
In vivo antitumor activity. In the first test, CT26-bearing BABL/c mice and LL/2-bearing C57BL/6N mice were treated with Q-PEGL at different dose. The mice treated with 12.5 and 25 mg/kg Q-PEGL showed moderate inhibition response to the tumor. The 50 and 100 mg/kg almost have no difference in inhibiting the tumor growth (P > 0.05), but the 100 mg/kg group significantly reduced the lung metastases in LL/2-bearing C57BL/6N mice. There was no difference between PBS and PEGL control (P > 0.05). The mice treated with 200 mg/kg resulted in death of 3 to 10 after the Q-PEGL administration. Therefore, we selected the dose of 50 mg/kg as effective dose.
In the second study, the free quercetin was found to have antitumor efficiency in inhibiting tumor progress compared with PBS (P < 0.05). Free quercetin plus PEGL did not enhance the antitumor efficiency compared with single free quercetin (P > 0.05). There was difference between free quercetin plus PEGL and Q-PEGL in inhibiting tumor progress although both with same dose based on quercetin (P = 0.0014). The antitumor efficiency of the Q-PEGL groups was better than that of the free quercetin groups.
In the third test, tumor volumes and life span of mice assay showed that the suppression of tumor growth in the Q-PEGL groups was more effective than the PBS control. The Q-PEGL groups resulted in a 40-day survival rate of 40% and decreased the tumor volume versus the PBS control. The inhibition rate of tumor volume treated with Q-PEGL was 45% compared with PBS. The Q-PEGL treatment had resulted in 10-, 11-, and 9-day (DDP 14-, 16-, and 15-day) delay of tumor growth to reach a volume of 900 mm3 compared with PBS control in CT26, H22, and LL/2 tumor-bearing mice, respectively (Fig. 4A-C). In the CT26, H22, and LL/2 tumor models, control animals that received PBS treatment survived 28.5, 28.5, and 29 days on average, respectively. In contrast, systemic therapy with Q-PEGL significantly resulted in improving the survival time versus PBS (P < 0.01, log-rank test; Fig. 4D-F). However, no complete reaction was found in these groups treated with Q-PEGL. Additionally, the appearance of lung metastases in the LL/2 tumor model was significantly delayed by treatment with Q-PEGL in comparison with PBS control.
Q-PEGL inhibited HSP70 expression in tumor tissues. Our studies showed that the antitumor efficacy of Q-PEGL was correlated with HSP70 expression in tumor tissues revealed by quantitative real-time PCR analysis. The relative levels of HSP70 expression were little detected in Q-PEGL group but highly found in the DDP and PBS groups in CT26, LL/2, and H22 tumor-bearing mice model (P < 0.01; Fig. 5).
Q-PEGL inhibited angiogenesis and induced apoptosis. Microvessel density was quantified to measure angiogenesis by immunolabeling of CD31 in frozen tumor tissue sections. The most highly vascularized area of each tumor was identified on low power, and five high-power fields were counted in this area of greatest vessel density. The Q-PEGL apparently reduced the number of vessels compared with PBS or DDP alone (P < 0.05). There was no difference between DDP and PBS in microvessel density (P < 0.05; Fig. 6A-C).
The tumor tissues were subjected to terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assays for respective determination of apoptotic index. Both the Q-PEGL group and the DDP group have a high apoptosis rate of tumor cells compared with the PBS control (P < 0.05). There was no difference between DDP and Q-PEGL in apoptotic index (P > 0.05; Fig. 6D-F).
High and selective accumulation of chemotherapeutic drugs at the tumor site is essential for the success of drug treatment in vivo. Previous studies have reported that quercetin in rats were widely distributed in tissues, with the highest concentrations in lungs and the lowest in brain, white fat, and spleen (22). Our studies showed that Q-PEGL can selectively accumulate in tumor site in comparison with free quercetin. Furthermore, Q-PEGL can significantly improve the water insolubility of quercetin, prolong the circulation times in the blood, and enhance antitumor efficacy.
Previous studies in vitro and in vivo identified that anticancer agents encapsulated by pegylated liposome can significantly prolong the plasma residence time of drugs that would otherwise be rapidly distributed or cleared (14, 23–26). Altered plasma pharmacokinetics is one mechanism by which liposomal drugs may show substantial changes in tissue distribution, efficacy, and toxicity from their free drugs. Increasing therapeutic efficacy was found in these liposomal drugs. These benefits derive from the altered pharmacokinetics and biodistribution afforded by the liposomal carrier. This may be especially important for water insolubility natural plant, such as quercetin. Increasing lifetime of Q-PEGL in the plasma compared with that of free quercetin was seen in our studies. Biodistribution studies showed that Q-PEGL was high concentration accumulated in tumor tissues. Thus, the increase in antitumor efficacy seen in the present experiments may be due to prolong exposure as determined by the increase in plasma half-time and to accumulation of the drug at the tumor site.
Defects in the capillary endothelium of tumor vasculature are typically in the size range of 400 to 600 nm; therefore, liposomes having diameters smaller than 400 nm can efficiently extravasate and accumulate within the tumor interstitial space (15). Otherwise, tumors have impaired lymphatic drainage (27), which makes extravasated liposomal drug have a long retention time in tumor tissues (13). An earlier study showed that large multilamellar vesicles have limited value as drug carriers because of their rapid clearance from the circulation (28). In addition, liposomes of small unilamellar vesicles are easily prone to endocytosis and are taken up by a large number of solid tumors due to the enhanced permeability and retention effect (29, 30). In present studies, the diameter of Q-PEGL was examined no more than 150 nm, which was favorable to increase quercetin concentration in tumor tissues.
Caltagirone et al. (31) have observed that quercetin given in vivo reduce the melanoma growth and metastasis in mice. In phase I and II clinical trials, Ferry et al. (32) found that i.v. quercetin could inhibit tumor progress in hepatocellular carcinoma and ovarian cancer, but the vehicle, DMSO, is unsuitable for further clinical development of quercetin. In our studies, the free quercetin could inhibit mouse colon carcinoma and Lewis lung cancer growth in vivo, but the antitumor efficiency of free quercetin was significantly less than that of Q-PEGL with equivalent dose quercetin.
It is known that members of the HSP70 families are often associated with cell cycle–related proteins, including p53, Cdk4, c-myc, pRb, and p27 (33–34). Compared with the corresponding normal tissues, increased amounts of cytoplasmic HSP70 have been found in lung, colorectum, and liver carcinomas (35–37). Recently, HSP70 overexpression has been identified as a marker of poor prognosis in many malignant tumors (38). Current results confirmed that HSP70 expression in tumor tissues was obviously down-modulated in mice treated by quercetin and apoptosis was increased. Such results were not found in DDP-treated mice group. There may be different antitumor mechanism between quercetin and DDP in inducing tumor cell apoptosis.
Although the present results show a significant suppression of tumor growth in vivo by Q-PEGL, it should be pointed out that the treatment of mice with Q-PEGL did not result in the complete eradication of the tumor cells with the dose and schedules used in this study. The tumor rapidly grew after cease the usage of Q-PEGL. Although Q-PEGL significantly prolonged the mean survival time of the mice, all of the survived animals died within 50 days in these models because of the big volume of tumor tissue and metastasis. This may be partly attributed to the fact that Q-PEGL was given every 3 days partly to a limited penetration of these long-circulating liposomes into the interior of established solid tumors in vivo (39). Clearly, additional development of these liposomal formations should be pursued to rectify these deficiencies. Detailed pharmacokinetic studies are in progress to improve antitumor efficacy and further antitumor mechanism of quercetin in vivo is being explored in our laboratory.
In conclusion, this study showed that the Q-PEGL could effectively accumulate in tumor tissues, lengthen the circulation time of quercetin in vivo, effectively inhibit multiple kinds of tumor growth, and prolong the survival time of tumor-bearing mice. Solid tumors belong to the most aggressive human cancers with short survival times. It is encouraged that the Q-PEGL could archive such good efficacy in the several solid tumor models. It is also important to note that Q-PEGL was unable to inhibit tumor growth fully. It is necessary to optimize liposomal formations and therapeutic schema to obtain better therapeutic efficacy.
Grant support: National Basic Research Program of China grants 2001CB510001 and 2004CB518800, National Natural Science Foundation of China, and National 863 Program.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.