Introduction

Glioma is the most common intracranial malignant tumor, and its prognosis is poor due to the existence of the blood-brain barrier (BBB), which hinders the effective targeting of chemotherapeutic agents to glioma [1]. To improve therapy for glioma, it is essential for chemotherapeutics to pass BBB and enter into the brain to target tumor cells with little side effects to surrounding normal cells [2]. Doxorubicin (Dox) is widely used for the treatment of breast, cervix, prostate, lung, and ovarian cancers. Although intratumoral injection of Dox improved glioma survival ratio [3], Dox can hardly pass the BBB, which limits its clinical utility on glioma treatment. The binding of adriamycin to polybutylcyanoacrylate nanoparticles can increase the ability to penetrate the cell membrane. Furthermore, nanoparticles with the surface modified by bioactive ligands could deliver a large amount of agents into the tumor [4, 5].

To improve targeted chemotherapy of glioma, in this study, we prepared doxorubicin-polybutylcyanoacrylate nanoparticles (Dox-PBCA-NP) and assessed its anti-glioma effects both in vitro and in vivo.

Materials and methods

Preparation of NP and Dox-encapsulated NP

The Dox-PBCA-NP was prepared using the emulsion polymerization method [6]. Dextran70 (Sigma) was dissolved in double-distilled water with pH 3.0 to get aqueous phase, and butylcyanoacrylate (BCA, Guangzhou Baiyun Med Co.) was dissolved in dichloromethane with the ratio of BCA to CH2C12 1:3 to get the oil phase. The oil phase was slowly dropped into water phase at a speed of 20 ml/h and stirred at 600 rpm at room temperature for 2 h. The blank polybutylcyanoacrylate nanoparticle (PBCA-NP) was obtained when colloidal solution was filtered with 0.45-um vertical containing glass funnel. To get Dox-encapsulated NP, Dox was added to PBCA-NP and stirred for 2 h.

Characterization of NP and Dox-encapsulated NP

NP or Dox-NP was diluted with distilled water and measured by Laser diffraction particle size analyzer (Mastersizer 3000HS) to assess the size and size distribution of the nanoparticles. NP or Dox-NP suspension was diluted appropriately and dropped on the carbon film-coated copper net, stained by 2 % phosphate tungsten, dried for 30 min, and then observed under transmission electron microscope (TEM; JEM-100CX, Japan). The freeze-dried Dox-NP powder was kept for 3 months at room temperature and then was dissolved in distilled water to detect the stability.

Drug loading and entrapment efficiency of doxorubicin in the nanoparticles were measured by UV spectra at 481 nm (Unicoam UV500 ultraviolet spectrophotometer, British Thereto electronic). Drug loading ratio (DL) and encapsulation efficiency (EE) were calculated as follows [7]:

DL% = Doxtotal − Doxsupernatant/BCA × 100 %; EE = Doxtotal − Doxsupernatant/Doxtotal × 100 %. Finally, NP-Dox was suspended in a dialysis system and Dox released into 0.01 M PBS (pH 4 and 7) was periodically taken out, and the supernatant was analyzed for the released Dox by UV spectrophotometry.

In vitro cytotoxicity assay

C6 glioma cells were seeded in 96-well plates at 1 × 104 cells/well and cultured for 24 h, then treated with different concentrations of Dox or Dox-PBCA-NP for 24 h at 37 °C in atmosphere with 5 % CO2. The cytotoxicity of Dox or Dox-PBCA-NP was evaluated by using MTT assay (Sigma). The experiments were performed in triplicate, dose-effect curves were made, and IC50 values were calculated.

Cell cycle analysis by flow cytometry

C6 cells were seeded in 6-well dishes at 1 × 10,000 cells/well and cultured for 24 h, then treated with Dox, Dox-PBCA-NP, or PBCA-NP for 24 h at 37 °C in atmosphere with 5 % CO2. The cells were collected and fixed overnight in 70 % (v/v) ethanol at −20 °C. The cells were washed twice with PBS and incubated for 30 min at room temperature with DNA-staining solution (MultiScience, China) containing 10 μg/ml RNase and 50 μg/ml propidium iodide (PI, Sigma). All samples were assessed by a FACS Calibur flow cytometer (Becton-Dickinson, USA) and the data were analyzed by Multicycles software.

Immunocytochemical staining

The expression of telomerase was measured by immunocytochemical method. Briefly, poly-l-lysine covered coverslips was placed in 24-well dishes, and then C6 cells were seeded in each well at a density of 105 cells/well and cultured for 24 h. Then cells were treated with Dox, Dox-PBCA-NP, or PBCA-NP for 24 h at 37 °C in atmosphere with 5 % CO2. The cells were fixed for 30 min at room temperature by paraformaldehyde, then washed twice with PBS and assessed by EnVision immunohistochemical staining kit (Shanghai, Dako Denmark A/S). The percentage of positive cells was calculated according to the instruction.

Biodistribution of Dox in the brain of glioma-bearing rats

Animal studies were approved by the Institutional Animal Care and Use Committee of Zhejiang University. The rats were anesthetized with 10 % chloral hydrate (0.4 g/kg), and 1 × 106 C6 cells were injected into right caudate nucleus on a stereotactic apparatus [8, 9]. Ten days after xenograft, ten rats were randomly divided into two groups in which free Dox or Dox-PBCA-Dox (dose of Dox 3 mg/kg) was injected through tail vain, respectively. Three hours later, the rats were sacrificed and the brain tissues were collected and washed by cold PBS to remove the surface blood. The left caudate nucleus (normal brain tissue) and right caudate nucleus (tumor tissue) were resected from the surrounding tissue under the operating microscope. After the homogenization of the tissues, the concentration of Dox in the tissues was analyzed by HPLC (Ex, 480 nm and Em, 580 nm) as described previously [10].

Survival curves

Forty glioma-bearing rats were randomly divided into four groups (n = 10), on the 7th day post xenograft. Animals in control group were administered with physiological saline. Animals in PBCA-NP group were administered with PBCA-NP at dose of 0.5 mg/kg. Animals in other two groups were treated with free Dox or Dox-PBCA-NP via tail vein at a dose of 1.5 mg/kg Dox, respectively. Administration was made every 2 days with a total of three doses per rat. All rats were maintained to monitor the survival curves. Kaplan-Meier survival curves were plotted for each group.

Serum assay

On the 5th day after the last administration, 0.5 ml of blood was collected from the rat’s tail. Serum level of aspartate aminotransferase (AST) was measured via automated chemistry analyzer with reagent kits (Micro lab 300, Merck).

Statistical analysis

Each experiment was performed in triplicate with results expressed as mean ± standard error (SE). The Student’s test was used. Survival data were presented using Kaplan-Meier plots and analyzed using a log-rank test. Statistical significance was set at P < 0.05.

Results

Characterization of NPs

TEM micrographs demonstrated that NP and Dox-NP vesicles were generally round with a diameter 105.3 ± 25.1 and 120.5 ± 30.8 nm, respectively (Fig. 1a, b). No obvious adhesion was found. DL of Dox in the Dox-PBCA-NP was about 10.58 % with EE above 87.43 %. Zeta potential of NP or Dox-PBCA-NP ranged from −8 to −10 mV. We took freeze-dried powder of Dox-PBCA-NP kept at room temperature for 3 months and dissolved it in distilled water; no obvious sediment was found and there were no significant changes in the size, shape, DL, and EE. The in vitro release analysis demonstrated that releasing rate of Dox-PBCA-NP was higher under acidic condition at 37 °C (Fig. 1c). Similarly, the degradation rate of PBCA-NP was higher under acidic condition (Fig. 1d).

Fig. 1
figure 1

Characterization of Dox-PBCA-NP. a TEM image of PBCA-NP. b TEM image of Dox-PBCA-NP. Scale bar, 100 nm. c Cumulative release (%) of Dox from Dox-PBCA-NP in medium with different pH at 37 °C. Dox concentration was calculated as described in “Materials and methods.” d The curve of concentration of urea released by PBCA-NP. Urea is one of PBCA-NP degradation products, and the concentrations of urea in PBCA-NP solution reflect the degradation of PBCA-NP

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In vitro cytotoxicity of NPs

The cytotoxicity of Dox, PBCA-NP, and Dox-PBCA-NP in C6 cells was evaluated by MTT assay. Even at the highest concentration (1 mg/mL) of NPs, the viability of C6 cells was over 95 %. The cytotoxicity of Dox or Dox-PBCA-NP was concentration dependent. IC50 (Dox concentration of 50 % inhibition) of Dox and Dox-PBCA-NP was 52.45 and 64.21 g/mL, respectively (Fig. 2). Moreover, there was a significant difference in the viability between C6 cells treated with Dox and C6 cells treated with Dox-PBCA-NP (P < 0.05).

Fig. 2
figure 2

In vitro cytotoxicity of Dox-PBCA-NP. The cytotoxicity of PBCA-NP and Dox on C6 glioma cells was evaluated by MTT assay. *P < 0.05 Dox versus Dox-PBCA-NP

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The effects of NPs on cell cycle progression of C6 cells

In the control and PBCA-NP-treated groups, there was no significant difference in cell cycle progression between the two groups (P > 0.05, Table 1). In Dox and Dox-PBCA-NP-treated groups, the number of cells in G1/G0 phase increased while the number of cells in S phase decreased significantly, indicating the inhibition of cell proliferation. At the same concentration of Dox, Dox had stronger inhibition on C6 cell proliferation compared to Dox-PBCA-NP (P < 0.05).

Table 1 Effects of Dox, Dox-PBCA-NP on cell cycle progression of C6 cells
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The effects of NPs on telomerase expression in C6 cells

Immunocytochemical analysis showed that the a positive rate of telomerase staining in blank control group and PBCA-NP group was 93.5 ± 2.4 and 95.1 ± 1.8 %, respectively. There was no significant difference between the two groups (P > 0.05). In Dox and Dox-PBCA-NP groups with Dox concentration of 10 μg/ml, the positive rate of telomerase staining was 55.2 ± 0.8 and 69.7 ± 1.3 %, with a significant difference between these two groups (P < 0.05, Table 2).

Table 2 Effects of Dox, Dox-PBCA-NP on telomerase expression in C6 cells
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Biodistribution of Dox in the brain of glioma-bearing rats

To evaluate the anti-glioma efficacy of Dox-PBCA-NP in vivo, the glioma-bearing rats were injected with the different agents via tail vain on the 10th day post intracranial implantation. Dox concentration in normal brain tissue (left caudate nucleus) and tumor tissue (right caudate nucleus) were detected by HPLC. The results showed that Dox concentration was much higher in tumor tissue than normal brain tissue in Dox-PBCA-NP group (P < 0.01, Fig. 3). Furthermore, Dox concentration of tumor tissue in Dox-PBCA-NP group was much higher than that of Dox groups (P < 0.05, Fig. 3).

Fig. 3
figure 3

Biodistribution of Dox in the brain of glioma-bearing rats. The C6 glioma cells-bearing rats received an i.v. of Dox or Dox-PBCA-Dox, Dox concentration in the tumor tissue or left caudate nucleus was detected by HPLC analysis (n = 5). *P < 0.01, **P < 0.05

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The effects of NPs on the survival of glioma-bearing rats

The survival of glioma-bearing rats from saline, PBCA-NP, free Dox, and Dox-PBCA-NP group was 13–27, 14–28, 14–25, and 17–30 days, respectively. The rank of median survival time was Dox-PBCA-NP (24 days) > NP (20 days) > saline (19.5 days) > Dox (19 days). By log-rank test, the median survival time of Dox-PBCA-NP group was significantly prolonged compared to the other three groups, while there was no significant difference between Dox, NP group, and saline group (P > 0.05, Fig. 4).

Fig. 4
figure 4

Kaplan-Meier plots showing the survival of glioma-bearing rats (n = 10)

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The cardiotoxicity of NPs

The administration of Dox (1.5 mg/kg/time, 3 times/rat) increased serum AST levels compared to saline control or blank NP group, but the administration of Dox-PBCA-NP decreased serum AST levels significantly compared to Dox group (Fig. 5). These results showed that Dox induced cardiotoxicity manifested by significant increases in serum AST levels [11]. However, serum AST levels decreased significantly in Dox-PBCA-NP group, suggesting less cardiotoxicity after entrapping Dox into nanoparticles.

Fig. 5
figure 5

Serum level of AST in glioma-bearing rats of different groups. *P < 0.05 versus PBCA-NP; **P < 0.05 versus Dox

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Discussion

The anti-glioma effects mainly depend on the transport of chemotherapeutic agent across BBB to enter the glioma site [12]. Traditional strategies to overcome BBB include intracerebral injection or infusion of hyperosmotic solutions such as mannitol [13]. These approaches can increase intracranial drug levels but cause a higher risk for patients such as electrolyte disturbances, intracranial hemorrhage, or infection. With the development of materials science, nanoparticles have been prepared to absorb or encapsulate drugs. PBCA is a widely used nano-material as drug carrier with little toxicity and good biocompatible characters [14, 15]. In this study, we developed a biodegradable NP with anti-tumor agent encapsulated inside. PBCA nanoparticles polymerized butylacrylate monomer with emulsion polymerization induced by -OH group. The reaction rate and degree of polymerization are influenced by many factors. During the polymerization reaction, Dextran70 acted as the stabilizer [16]. The average particle size was around 120 nm, with little polydispersity coefficient, which indicated that the nanoparticle size distribution is narrow. TEM showed that the nanoparticles had uniform particle size, smooth round surface, and little aggregation. Furthermore, after the nanoparticles were kept at room temperature for a long time, there were no obvious changes in solubility, DL, or EE.

Dox is widely used for tumor chemotherapy, but seldom used for glioma chemotherapy because it could not overcome the BBB. Moreover, the cardiotoxicity limits its administration [17, 18]. In this study, we encapsulated Dox into PBCA-NP to overcome BBB. After being encapsulated into PBCA-NP, the release of Dox is time dependent. To exclude the possible effects of blank PBCA-NP, the cytotoxicity of PBCA-NP was evaluated and no significant toxicity was found, and it had no significant effects on the proliferation of C6 cells. Cytotoxicity of Dox-PBCA-NP was lower than Dox in vitro at the same concentration of Dox, maybe due to the slow-releasing aspect of Dox-PBCA-NP. After the injection of Dox-PBCA-NP into the vein of glioma-bearing rats, the rats showed a longer survival, in agreement with Dox biodistribution in the glioma tissue. Dox is anthracycline anti-tumor agent of low molecular weight and could not pass the BBB. After encapsulation into PBCA-NP, the solubility of Dox-PBCA-NP improved significantly and the residence time in circulation system was prolonged. Furthermore, the slow-releasing effects contributed to its long circulation.

Dox-induced cardiotoxicity is the most serious side effect of the chemotherapeutic agent. AST is an enzyme that leaks from the hearts upon tissue damage and has been used as the marker of cardiotoxicity due to the tissue specificity and serum catalytic activity. To evaluate the cardiotoxicity of Dox-PBCA-NP, we collected serum samples from glioma-bearing rats and examined serum AST levels. We found that serum AST levels were significantly lower in Dox-PBCA-NP-treated group compared with Dox-treated group. These data suggest that entrapping of Dox in PBCA-NP significantly reduces the cardiotoxicity of Dox.

In summary, we prepared Dox-PBCA-NP via emulsion polymerization method. Dox-PBCA-NP demonstrated less effective anti-tumor effect on C6 glioma cells in vitro compared to Dox, which may be due to its slow-releasing aspect. However, Dox-PBCA-NP demonstrated better therapeutic efficacy on glioma in vivo manifested by prolonged survival of glioma-bearing rats. In addition, Dox-PBCA-NP alleviated Dox-induced cardiotoxicity in vivo. Therefore, Dox-PBCA-NP is a potential nano-scale drug delivery system for glioma chemotherapy.