Co-encapsulation of borneol and paclitaxel by liprosomes improved anti-tumor effect in a xenografted glioma model

Abstract

In this study, a borneol (BOR) and paclitaxel (PTX) co-encapsulated lipid–protein nanocomplex (BP–liprosome) was developed for the treatment of brain glioma. The prepared BP–liprosome had small particle size (107.5 ± 3.2 nm), high entrapment efficiency for BOR (85.89 ± 0.78%) and PTX (90.40 ± 1.2%), and exhibited sustained release profiles in vitro. Raman spectra showed that both PTX and BOR existed in the BP–liprosome in a noncrystalline state. In vivo imaging showed that the BP–liprosomes exhibited stronger fluorescence intensity in the brains of mice than PTX encapsulated lipid–protein nanocomplexes (P–liprosome) without BOR. Furthermore, the results of the in vivo distribution study indicated that the BP–liprosome significantly increased the accumulation of PTX in the brain compared to the P–liprosome after i.v. administration in mice. Most importantly, the BP–liprosome exhibited a stronger anti-tumor effect in C6 tumor-bearing mice than the P–liprosome. The tumor inhibition rates of BP–liprosome, P–liprosome and PTX solution were 85.71%, 62.39% and 49.00%, respectively. Overall, the combination of BOR and PTX with the lipid–protein nanocomplex appears to be a promising approach for the treatment of brain glioma.

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1. Introduction

Despite the many efforts that have been made, many serious brain diseases affecting millions of people world-wide cannot be treated. For example, brain tumor, the most common primary central nervous system tumor, is a life-threatening disease characterized by a low survival rate.¹ Glioblastoma, known as a grade IV glioma, is the most common and malignant form of brain tumor, which accounts for more than 60% of new primary brain cancer diagnoses.^2,3 Glioblastoma can rapidly diffuse and invade the surrounding normal tissue, which makes it impossible to remove the tumor mass completely by conventional surgical methods, and tumor recurrence from the residual tumor is very possible.⁴ In addition, the therapeutic effect for glioblastoma produced by systemic delivery therapeutic drugs is very limited, due to their poor permeability across the blood-brain barrier (BBB) and insufficient drug concentration in the brain.⁵ Therefore, it is important to improve the transport of therapeutic drugs across the BBB for the treatment of brain glioma.

Paclitaxel (PTX) is in the first class of microtubule stabilizing agents, and has been widely used in clinics for the treatment of a variety of solid tumors, including breast cancer, ovarian cancer, lung cancer, head and neck malignancies and other tumors.⁶ However, PTX is not able to transport across the BBB and reach therapeutic concentrations in the brain tissue.⁷

Borneol (BOR) is an indispensible constituent for traditional Chinese medicine, such as “An Gong Niu Huang” pills. The pills have been used clinically in the treatment of strokes for hundreds of years. Recent studies have indicated that BOR could improve the permeation of drugs across the BBB and enhance their distribution in the brain.^8,9 On the basis of that, the co-administration of PTX and BOR may increase the transport of PTX across the BBB. However, when co-administering free drugs, the different physico-chemical properties of the free drugs may lead to issues with pharmacokinetic interactions and toxic effects. Hence, it is essential to explore a safe and effective method for the co-administration of PTX and BOR.

Nano-technology based drug delivery systems have attracted much attention for the co-delivery of multiple drugs. This strategy is based on the co-encapsulation of free drugs in a single carrier, which protects drugs from degradation in biological environments and increases drug accumulation in the target site.^10–12 Various nanocarriers have been developed for co-encapsulation of free drugs, such as human serum albumin nanoparticles co-delivery of pirarubicin and paclitaxel,¹³ liposomes co-loaded with elacridar and tariquidar,¹⁴ and polymeric nanoparticles co-encapsulation of tamoxifen and quercetin.¹⁵ Moreover, Ren et al. reported that co-encapsulation of BOR and ganciclovir by solid lipid nanoparticles markedly enhanced the transport of ganciclovir to the brain in mice after i.v. administration.¹⁶

In our previous work, a lipid–protein nanocomplex (liprosome) was successfully prepared from egg yolk lecithin (PL 100M) and bovine serum albumin (BSA). To some extent, the liprosome enhanced PTX transport across the BBB while reduced its systemic toxicity as compared to that of PTX solution.¹⁷ However, the brain tissue accumulation of PTX was still relatively low, and hence its potential as a drug carrier will be limited. Encouraged by the high potential of BOR as a promoter for drug transport across the BBB, we further sought to co-encapsulate BOR and PTX in a liprosome, so as to transport more PTX across the BBB and improve the anti-glioma effect of PTX in vivo.

In the present study, a BOR and PTX co-encapsulated lipid–protein nanocomplex (BP–liprosome) was prepared. Its physicochemical properties and drug release profile were characterized. In vivo distribution in mice was evaluated quantitatively and qualitatively. Moreover, the anti-tumor effect was also evaluated in xenografted C6 glioma mice. It is speculated that the BP–liprosome might be a suitable drug delivery system for the treatment of brain glioma.

2. Materials and methods

2.1. Materials and cells

Paclitaxel and docetaxel were obtained from Tianfeng Bioengineering Technology Co., Ltd. (Liaoning, China). BOR was obtained from Yunnan Linyuan spicery Co., Ltd. (Yunnan, China). Egg yolk lecithin PL 100M was obtained from the Q.P. Corporation (Tokyo, Japan). Bovine serum albumin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cremophor EL was kindly supplied by the BASF Corporation (Ludwigshafen, Germany). Penicillin–streptomycin, fetal bovine serum, DMEM, and 0.25% (w/v) trypsin–0.03% (w/v) EDTA were purchased from Gibco BRL (Gaithersberg, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (Dir) was obtained from Biotium (Hayward, Canada).

The C6 cells were generously provided by Prof. Jingyu Yang (Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China). The cells were cultured in DMEM medium, supplemented with 10% fetal bovine serum, penicillin and streptomycin sulfate at 37 °C with 5% CO₂ under fully humidified conditions.

2.2. Preparation of the BP–liprosome

The BP–liprosome was prepared by a desolvation-ultrasonication technique as detailed in our previous report.¹⁷ In brief, 0.20 g BSA was dissolved in 5 mL deionized water. 0.3 mL anhydrous ethanol solution containing 0.01 g BOR and 0.01 g PTX was added dropwise to the BSA solution, and then 0.3 mL anhydrous ethanol anhydrous solution containing 0.10 g PL 100M was also added with magnetic stirring. Then, the mixtures were introduced by probe sonication (JY92-II, Ningbo Scientz Biotechnology Co., Ltd., China). After that, anhydrous ethanol was removed by a rotary evaporator (Ya Rong Biochemical Instrument Factory, Shanghai, China). Eventually, the samples were centrifuged and passed through a 0.22 μm filter membrane. The preparation of P–liprosome was the same as that of the BP–liprosome, but excluded BOR from the formulation.

PTX solution was prepared in accordance with the commercially available prescription. In brief, 0.012 g of PTX was dissolved in a mixture of 1 mL anhydrous ethanol and 1 mL cremophor EL. A proper concentration of PTX solution was obtained in the experiment by diluting the stock solution with saline.

BOR solution was prepared as follows. In brief, 0.01 g of BOR was dissolved in 5 mL anhydrous ethanol under magnetic stirring. The BOR solution was diluted properly before testing.

2.3. Characterization of the BP–liprosome

The mean particle size, polydispersity index (P.I.) and zeta potential were determined by dynamic light scattering (DLS) (PSS NICOMP 380, USA). The shape and size of BP–liprosome were observed using a transmission electron microscope (TEM) (JEOL, Japan). The Raman spectra were determined by a confocal Raman spectrometer (NTEGRA SPECTRA, NT-MDT, Russia).

2.4. Analysis of drug entrapment efficiency and loading capacity

The entrapment efficiency (EE) and loading capacity (LC) of PTX in BP–liprosome were determined as depicted in our previous work.¹⁷ Acetonitrile was added to precipitate BSA in order to release PTX from the BP–liprosome. After 12 000 rpm centrifugation for 10 min, the PTX content in the supernatant was determined by high performance liquid chromatography (HPLC). The HPLC system (LC-10Avp, SHIMAZU, Japan) involved a UV-Vis detector (SPD-10Avp, SHIMAZU, Japan) and a Diamonsil C18 reversed phase column (particle size 5 μm, 4.6 × 250 mm). UV detection was performed at 227 nm with a column temperature of 30 °C. The mobile phase consisted of water–methanol (25 : 75, v/v), and the flow rate was 1.0 mL min⁻¹. The EE and LC of PTX were calculated as follows.

where W_{total PTX}, W_{feeding PTX} and W_{feeding drugs and excipients} represents the weight of PTX in the BP–liprosome, the weight of feeding PTX, and the weight of feeding drugs and excipients, respectively.

The EE and LC of BOR in BP–liprosome were determined by separation of the free BOR in aqueous BP–liprosome suspensions using ultrafiltration. In brief, 0.5 mL BP–liprosome suspensions were added to ultrafiltration tubes (Millipore, MWCO: 3 kDa). After centrifugation at 3000 rpm for 10 min, the BOR in the ultrafiltrate was determined by gas chromatography (GC) (Agilent 7890B, USA) using a flame ionization detector and a PEG-20 M capillary column (30 m × 0.25 mm, 0.4 μm). Nitrogen was used as the carrier gas and the flow rate was 17 mL min⁻¹. The temperature of the injection port was set at 200 °C, the hydrogen flow rate was 30 mL min⁻¹, the oven temperature was 150 °C, and the detector temperature was 250 °C. A split ratio of 10 : 1 was used in this study. The sample of 5 μL was injected using an autosampler (Agilent 7693, USA). The EE and LC of BOR were calculated according to the following formulas.

where W_{total BOR}, W_{filtrate BOR}, W_{feeding BOR} and W_{feeding drugs and excipients} represents the weight of BOR in the BP–liprosome, the weight of free BOR in the aqueous phase, the weight of feeding BOR, and the weight of feeding drugs and excipients, respectively.

2.5. In vitro drug release

The release profiles of BOR and PTX in BP–liprosome were evaluated using a dialysis method.¹⁸ In brief, a sample of 1.0 mL was added into a dialysis bag at the PTX concentration of 0.09 mg mL⁻¹ (BOR concentration, 0.086 mg mL⁻¹). After that, the dialysis bag was suspended in 30 mL phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 0.5% w/v Tween 80 in an Erlenmeyer flask. Then the flask was put in an orbital shaker and vibrated horizontally at 120 rpm under 37 °C. Samples of 0.2 mL were withdrawn from the release medium at predetermined time intervals. The concentrations of BOR and PTX were determined as described in the analysis of drug encapsulation efficiency and loading capacity.

2.6. In vivo imaging

In order to observe the in vivo real-time distribution of BP–liprosomes, the liprosome was labeled with a hydrophobic dye Dir. The Dir encapsulated liprosome (D–liprosome) was prepared with the same procedure as the P–liprosome, except the PTX was replaced with Dir. BOR and Dir co-encapsulated liprosome (BD–liprosome) was prepared with the same procedure as the BP–liprosome, except the PTX was replaced with Dir. Mice were treated with D–liprosome and BD–liprosome suspensions via i.v. administration at 5 mg kg⁻¹. At 1, 2, 4, 8, 12, 24 h, the mice were scanned using an Fx Pro multimodal imaging system (Bruker Corp., USA). After 24 h, the mice were sacrificed, and the brain, heart, liver, spleen, lung and kidney were removed. Then the fluorescence images of these tissues were visualized.

2.7. In vivo distribution

The in vivo distribution study was conducted to further quantitatively explore the PTX distribution in major organs. Male Kunming strain mice (18–22 g) were purchased from the Experimental Animal Center (Shenyang Pharmaceutical University, China). All of the animal experiments were approved by the Shenyang Pharmaceutical University Ethics Committee and conformed to the Guidelines for the Use of Laboratory Animals. Mice were divided into two groups. Each group of twelve mice was given P–liprosome or BP–liprosome by i.v. administration at 20 mg kg⁻¹. Then the mice were sacrificed at previously determined times (1, 4, 8 h), and the tissues of interest were excised. The tissues were washed with physiological saline and dried on filter paper. And then, pre-weighed tissue (0.1 g) was homogenized with saline (0.5 mL). Next, the tissue homogenate was vortex-mixed with 10 μL of docetaxel (100 μg mL⁻¹), the internal standard and 4 mL of methyl tert-butyl ether. After centrifugation (4000 rpm, 10 min), the organic layer was taken out and dried under nitrogen. The residue was dissolved in 50 μL mobile phase (water–acetonitrile, 50 : 50, v/v) and centrifuged at 12 000 rpm for 10 min. The supernatant of 20 μL was subjected to HPLC analysis.

2.8. In vivo anti-tumor effect of the BP–liprosome

The anti-tumor effect was investigated in Kunming mice bearing a C6 brain glioma xenograft.¹⁹ C6 cells were injected subcutaneously at 3 × 10⁷ cells in the armpit of the right anterior limb. When the tumor volume reached about 100 mm³, the mice were divided into four groups (n = 6), and the formulations of PTX solution, P–liprosome and BP–liprosome were given at a dose of 10 mg kg⁻¹ every other day for a total of 5 times. Saline was used as a control. Tumor volumes and body weights were measured with a caliper every other day. Tumor volume (V) was determined by the following formula.
where a and b represent the long and short axis of the tumor, respectively.

At the end of experiments the mice were sacrificed and the tumors were removed and weighed. The tumor inhibition rate (TIR) was calculated as follows.

where W₀ and W_t represent the mean tumor weight in the saline group and test formulations group, respectively.

In order to examine the tumor tissue toxicity of the above-mentioned formulations, pathological examination was also conducted. The removed tumor tissues were fixed with 10% formaline and then embedded with paraffin. Tumor tissue sections of 4 μm thickness were prepared and stained by hematoxylin and eosin (H&E). Photographs were captured with a light microscope.

2.9. Statistical analysis

Analysis of statistical significance was performed with the SPSS statistics software 16.0. The data are presented as the mean ± SD. Student’s t-test was used to analyze the differences.

3. Results and discussion

3.1. Characterization of the BP–liprosomes

The particle size has a crucial impact on the in vivo fate of a nano-drug delivery system. DLS analysis showed that the BP–liprosome had a small particle size (107.5 ± 3.2 nm) with a narrow size distribution (P.I. = 0.171 ± 0.02). Such small particles could accumulate more readily in tumor tissues due to the EPR effect.²⁰ TEM imaging showed that the BP–liprosome had a spherical core–shell structure (Fig. 1). The zeta potential of the BP–liprosome was found to be −21.36 ± 0.3 mV. The negative charge was ascribed to the phospholipids located on the surface of the BP–liprosome. The loading capacity of BOR and PTX in BP–liprosome was 2.68 ± 0.02% and 2.83 ± 0.15%, respectively. The entrapment efficiency of BOR and PTX in BP–liprosome was 85.89 ± 0.78% and 90.40 ± 1.2%, respectively, demonstrating the strong affinity between BOR or PTX and BSA through hydrophobic interactions.^21,22

Fig. 1 TEM image of BP–liprosome.

The Raman spectrum of PTX showed several peaks at 180, 429, 618, 849, 893, 946, 1004, 1027, 1601 and 1714 cm⁻¹ (Fig. 2e). The Raman spectrum of BOR displayed several peaks at 258, 394, 501, 533, 597, 655, 773, 836, 858, 1082 and 1460 cm⁻¹ (Fig. 2d). However, these peaks disappeared in the spectrum of BP–liprosome (Fig. 2c), indicating that the PTX and BOR molecules inside the BP–liprosome were in a noncrystalline state. No obvious peaks appeared in the spectra of PL 100M and BSA (Fig. 2a and b), implying that the state of PL 100M and BSA in the BP–liprosome cannot be speculated.

Fig. 2 Raman spectra of PL 100M (a), BSA (b), BP–liprosome (c), BOR (d), PTX (e).

3.2. In vitro drug release of the BP–liprosomes

The release of BOR and PTX from BP–liprosome was performed in pH 7.4 PBS to simulate blood release conditions. The release profiles are presented in Fig. 3. Almost 100% cumulative release was observed for BOR solution and PTX solution within 8 h. Both the BOR and PTX in BP–liprosome exhibited a sustained release profile compared with that of BOR solution and PTX solution. BP–liprosome showed 78.5% release of BOR and 64.4% release of PTX. The cumulative release of PTX was lower than that of BOR from BP–liprosome at all time points, which could be due to the higher affinity of PTX with BSA in comparision with BOR. The release characteristics of PTX and BOR are beneficial for PTX transport across the BBB, because the earlier release of BOR could improve the permeability of the BBB, and subsequently increase the transport of PTX to the brain.

Fig. 3 In vitro release profile of BOR and PTX from BP–liprosome in phosphate buffered saline (0.5% Tween 80 in PBS, pH 7.4) at 37 ± 0.5 °C (n = 3).

3.3. In vivo imaging of the liprosomes

Real-time fluorescence imaging can be used to qualitatively investigate the in vivo distribution of nanoparticles. As shown in Fig. 4, the fluorescence intensity of BD–liprosome in the brain was stronger than that of D–liprosome from 1 h to 24 h after i.v. administration, indicating that BOR could improve the permeability of the BBB. After 24 h, the different tissues of mice were removed and captured, the fluorescence intensity of BD–liprosome was still higher than D–liprosome in the brain. The result further confirmed that the coencapsulation of BOR in liprosomes could promote Dir transport across the BBB, and thus increased the accumulation of Dir in the brain.

Fig. 4 In vivo image. Fluorescence imaging of mice after i.v. administration with Dir encapsulated liprosome (D–liprosome) and BOR and Dir coencapsulated liprosome (BD–liprosome) at 5 mg kg⁻¹ (a); ex vivo imaging at 24 h (b).

3.4. In vivo distribution of the BP–liprosome

The tissue distribution of BP–liprosome and P–liprosome were investigated in vivo after i.v. administration in mice. As shown in Fig. 5, almost no obvious change was observed in the accumulation of PTX in the heart, liver, spleen and kidney between BP–liprosome and P–liprosome. The results demonstrated that BOR encapsulated in the P–liprosome did not influence the distribution characteristics of P–liprosomes in the corresponding tissues. However, the BP–liprosome had an increased accumulation of PTX in the brain and lung in comparision with the P–liprosome. Compared to the P–liprosome, the PTX concentration in the brain from BP–liprosome was significantly increased by 1.42-fold, 1.79-fold and 11.41-fold, respectively after 1, 4 and 8 h administration.

Fig. 5 Mean PTX concentration in different tissues after i.v. administration of P–liprosome and BP–liprosome in mice at 1 h (a), 4 h (b) and 8 h (c) (n = 4). *p < 0.05, significant difference compared with P–liprosome, #p < 0.01, significant difference compared with P–liprosome.

In a word, the BP–liprosome clearly increased the accumulation of PTX in the brain compared to the P–liprosome. This is probably due to the particle characteristics of the BP–liprosome and the synergistic effect of BOR (Fig. 6). First, the BP–liprosome had a smaller particle size, which could improve the interaction between BP–liprosome and brain microvascular endothelial cells (BMECs), promoting the liprosome transport across BMECs via transcytosis. Previous studies have shown that BOR could increase the number and volume of pinocytosis vesicles in BMECs and then enhance the transport of drugs by cell pinocytosis.²³ Second, BOR could significantly loosen the intercellular tight junctions in the BBB and accelerate the transport of drugs through the intercellular passages by decreasing the levels of the tight junction proteins localized in the cell–cell junctions.^23,24 Third, it has been found that BOR could bind to a site on the cellular membrane or be adsorbed to the membrane surface to promote the membrane fluidity of the epithelium, thereby increasing the orderly arrangement of the membrane phospholipid molecule chains and reducing the drug collisions with phospholipid molecules, which would facilitate drug permeation across the cell membrane.^25,26 Finally, BOR could overcome P–glycoprotein (P–gp) mediated drug efflux at BMECs, which enhanced the transport of drugs across the BBB.²⁷ In addition, the opening of the BBB combined with BOR is physiological, which has a protective effect on brain tissue and cannot damage the BBB.²⁸

Fig. 6 Schematic illustration of PTX transport across the brain-blood barrier (BMECs, brain microvascular endothelia cells).

3.5. In vivo anti-tumor effect

The subcutaneous xenografted model provides convenience in tumor separation and visualization, and deciding the time for drug administration. It has been used widely in the assessment of the anti-tumor effect for various cancers, including breast cancer,²⁹ colon cancer,³⁰ and brian glioma.^31–33 Consequently, the anti-glioma effect of BP–liprosome was evaluated in the subcutaneous xenografted model in mice. The changes of tumor volume after i.v. injection of different formulations are shown in Fig. 7a, the tumor proliferation rate of the different groups in decreasing order is BP–liprosome, P–liprosome, PTX solution and saline group, respectively. The result showed that all the PTX formulations could significantly inhibit the growth of tumor compared with the saline group. BP–liprosome exhibited the greatest advantage in inhibiting the tumor growth. At the end of the test, the excised tumor masses from different treatment groups were captured and shown in Fig. 7b. The significantly lowest tumor weight was obtained from BP–liprosome (0.132 ± 0.046 g) in comparison with PTX solution (0.470 ± 0.146 g) and P–liprosome (0.347 ± 0.152 g) (Fig. 7c). The calculated tumor inhibition rates of PTX solution, P–liprosome and BP–liprosome were 49.00%, 62.39% and 85.71%, respectively. The results further demonstrated that BP–liprosome showed an obviously better anti-tumor efficacy than P–liprosome.

Fig. 7 Anti-tumor effect of BP–liprosomes on C6 glioma tumor-bearing Kunming mice. Mice were injected i.v. with saline, PTX solution, P–liprosome and BP–liprosome at a dosage of 10 mg kg⁻¹. The changes in tumor volume after administration (a). The tumor photos of different treatment groups after tumors were excised at the end of the test (b). The weight of excised tumors at the end of the test (c). The changes in body weight after administration (d) (n = 6). *p < 0.05, significant difference compared with saline, #p < 0.01, significant difference compared with PTX solution and P–liprosome. Histological (H&E) analysis of tumor tissue sections from different treatment groups (e). Magnification, ×100.

Moreover, the anti-tumor effect of different treatment groups was evaluated by pathological examination. As shown in Fig. 7e, negligible necrosis of tumor tissues was observed in the saline group. The PTX solution group showed more obvious spotty necrosis than the saline group. The P–liprosome group exhibited focal necrosis and nucleus pycnosis. Larger areas of necrosis and nucleus pycnosis were observed in the BP–liprosome group than for PTX solution and P–liprosome. It was concluded that BP–liprosomes significantly induced the apoptosis of tumor cells and thus inhibited the tumor growth, which was in agreement with the results of anti-tumor effect described above. The mechanism of the enhanced anti-tumor effect by BP–liprosome may be ascribed to two aspects. First, the nanoparticles can preferentially accumulate at tumor tissues by passive targeting due to EPR effects. Second, the BOR could inhibit the action of P–gp on the cell membrane, and thus increase the PTX accumulation in the tumor cells.³⁴ As a consequence, the increased intracellular PTX concentration would mediate the tumor cell death.

In addition, the body weight of mice was evaluated for the toxicity of different PTX formulations. As shown in Fig. 7d, there was no body weight loss in any group, demonstrating the low systemic toxicity in the treated animals. Accordingly, BP–liprosome is a safe and effective drug delivery system for the treatment of brain glioma.

4. Conclusions

The present study developed BP–liprosomes for improving the anti-glioma effect of PTX. The prepared BP–liprosome was spherical with a suitable particle size and a narrow size distribution. The in vitro drug release profiles confirmed the sustained release of BOR and PTX from the BP–liprosome. Compared with P–liprosome, BP–liprosome exhibited more PTX accumulation in the brain after i.v. administration in mice. Finally, BP–liprosome remarkably enhanced antitumor effect in xenograft C6 glioma bearing mice models. Therefore, the BP–liprosome could be a useful candidate for the treatment of brain tumors.

Declaration of interest

The authors declare that there is no conflict of interest in this work.

Acknowledgements

The authors thank Prof. Jingyu Yang of Shenyang Pharmaceutical University for gifting the cells, and Wuxiyar Otkur, Dr Mingyu Xia and Prof. Takashi Ikejima of Shenyang Pharmaceutical University for their help in the cell experiments. Dr David B. Jack is thanked for polishing the language.

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