An epidemiological study shows that boron (B) intake can reduce the risk of prostate cancer11 , 12 for human by up to 54% (ref. 13). Boric acid (BA), the dominant form of B in plasma, has been tested as a preventative and therapeutic agent against prostate cancer.
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Physicochemical characterization of hollow BN spheres
Hollow BN spheres were synthesized via the chemical vapour deposition (CVD) reaction of trimethoxyborane (B(OMe) 3 ) using modified method developed previously39, in which the second-stage annealing process was conducted in Ar rather than in NH 3 atmosphere. Transmission electron microscopic images prove the successful synthesis of BN nanospheres (Fig. 1a). The BNs-a sample shows a solid sphere structure with an approximate diameter of 200 nm and low crystallinity characterized by a long-range disorder. The BNs-b shows a hollow sphere structure with the same diameter and wall thickness of 50–60 nm. For BNs-c, a hollow nanostructure with a wall thickness of about 20 nm and high crystallinity characterized by the state of long-range order is observed.
Figure 1: Physicochemical characterization of BN spheres. (a) Transmission electron microscopic images of BN nanospheres: BNs-a (a,b), BNs-b (c,d), and BNs-c (e,f). Scale bar: a,c,e, 200 nm; b,d,f, 5 nm. (b) Wide-angle X-ray diffraction patterns of the BN nanospheres. (c) FTIR patterns of the BN nanospheres. (d) Particle size distribution of the BN spheres in water. (e) Suspension of BN spheres in culture medium at 100 μg ml−1. (f) Zeta potential of BNs-a, -b and -c in PBS buffer. (g) Boron release for BNs-a, -b and -c in acetate buffer pH=4.6 at different time. (h) The residual particles after immersing same amount of BNs-a, -b and -c samples in acetate buffer for 1 month. Full size image
The BN spheres are adjustable with respect to crystallinity by means of posttreatment temperature variations (Fig. 1b). As a whole, with an increase in posttreatment temperature, the crystallinity increased and the full width at half-maximum for the peaks in the wide-angle X-ray diffraction patterns becomes narrower. The BNs-a spheres exhibit an amorphous nature with a broad peak at around 26°. The BNs-b spheres are higher in crystallinity than BNs-a, exhibiting two crystalline peaks at around 26.4° and 42° that correspond to (002) and (100), respectively. The BNs-c spheres are the highest with regard to crystallinity, exhibiting a much narrower peak width for (002) and (100) reflections than those for BNs-b.
All the BN spheres show typical fourier transform infrared spectroscopy (FTIR) absorption bands of B-N stretching at 1,382 cm−1 and B-N-B bending at 795 cm−1 (Fig. 1c). The presence of hydroxyl groups is confirmed by the O-H stretching band at 3,421 cm−1 for BNs-a. A shoulder at 3,227 cm−1 indicates the asymmetric stretching of the N-H group. A weak band at around 1,200–1,250 cm−1 is attributed to B-N-O stretching. With an increase in calcination temperature from 900 to 1,025 °C for BNs-b, the O-H stretching band becomes weaker while the N-H stretching band becomes stronger. For BNs-c, calcined at 1,400 °C, both the O-H and N-H bands become very weak. X-ray photoelectron spectroscopy reveals the high content of oxygen (O) in BN spheres (Supplementary Fig. 1). As shown in Fig. 1d, the BNs-a, -b and -c nanospheres exhibit hydrodynamic diameters of 263.5±66.5, 334.1±87.8 and 351.8±56.3, respectively, which are slightly higher than those obtained from the transmission electron microscopic observations. In addition, unlike other BN materials such as BN nanotubes, all the BN spheres show good dispersibility in cell culture medium (Fig. 1e). The presence of large amount of hydroxyl groups (Fig. 1c) and a high content of oxygen (Supplementary Fig. 1) result in their high hydrophilicity and good dispersibility in an aqueous solution. At pH=7.4, the BNs-a, -b and -c spheres all show zeta potentials centred at around −21 to −25 mV (Fig. 1f).
B release from hollow BN spheres
Release of B from BN spheres with controlled crystallinity kept in dialysis membrane bags was analysed in an acetate buffer at pH 4.6. BN spheres with high crystallinity show slow B release (Fig. 1g). BNs-a spheres with the lowest crystallinity show the highest B release speed, which is about 20 times that of BNs-c spheres and 2 times that of BNs-b spheres. Figure 1h displays the BN nanospheres remain in the dialysis membrane bags after immersing the same amount of BNs-a, -b and -c samples in the acetate buffer for 1 month. It can be seen that the BNs-a sample almost totally degrades, while the BNs-c sample remains in a large amount. Supplementary Fig. 2 shows the B release rate for different BN spheres under various conditions of pH, temperature and concentration. For all the BN spheres, the B release rate increases with the increase in temperature. In addition, the B release rate nearly linearly increases with the increase in the initial BN materials concentration. For BNs-b, pH value has a negligible effect on B release at a low temperature, whereas, at a high temperature, high pH value results in the increase in the B release. The dynamic studies of structural evolution for BN spheres are presented in Supplementary Fig. 3. For BNs-a sample, the spherical particle, around 200 nm in diameter, gradually degrades from the edge area at the initial stage and then transforms into smaller clusters, about 5–20 nm in diameter, 3 days later. For BNs-b sample, the hollow spherical particle degrades in a different way compared with the BNs-a sample. The defect sites in the wall gradually degrade to form a porous structure and the hollow spherical wall structures are still preserved after 3 days. After 10 days, the partial degradation in the edge area and a marginal amount of small clusters are observed. Supplementary Fig. 4 reveals FTIR spectra of the hydrolysed products of BN nanospheres. New absorption bands at 1,228 and 1,185 cm−1 are attributed to the B-O group for the BA and B-N-O groups, respectively. In addition, the obtained products possess new absorption bands at 1,096, 1,023, 916 and 689 cm−1, which are identified as ammonium borate hydrates. In contrast with Fig. 1b, wide-angle X-ray diffraction patterns of the hydrolysed products of BN spheres suggest the presence of BA and ammonium borate hydrates (Supplementary Fig. 5), which is consistent with the FTIR results.
Cytotoxicity assay
Inhibitory effects of hollow BN spheres on proliferation and viability of androgen-sensitive LNCap and androgen-independent DU145 prostate cancer cell lines were assessed by the WST-8 method (Cell Counting Kit-8). Both BA and the BN spheres reduce cell viability in a dose-dependent manner (Supplementary Fig. 6). For LNCap prostate cancer cells, all the BN spheres decrease cell viability greater than BA. Among them, BNs-b with a moderate crystallinity and B release best inhibits LNCap cell viability. For example, after 3 days’ exposure at 5 μg ml−1, BNs-b induces much lower cell viability than BA, BNs-a and BNs-c. In addition, at a higher concentration or longer exposure time, BNs-b as well as BNs-c induces much higher inhibition for LNCap cells than BA and BNs-a. There is also a tendency for BNs-b to inhibit best DU145 cell viability after 3-day exposure. Moreover, after 6 days’ exposure at a concentration of up to 5 μg ml−1, BNs-b still induces the lowest DU145 cell viability. After long incubation for 6 days at a high concentration of 25 μg ml−1, BA, BNs-a and BNs-b decrease DU145 cell viability greatly, compared with BNs-c. As a whole, LNCap prostate cancer cells are more sensitive to BNs-b and -c with relatively higher crystallinity and slower B release, while DU145 prostate cancer cells are more sensitive to BA, BNs-a and BNs-b with the lower crystallinity and faster B release.
Light microscopic observation revealed significant morphological alterations between the cells treated with BN spheres and BA as shown in Fig. 2. After 6-day exposure of LNCap cancer cells to BA and BNs-a, there are no obvious changes in morphology. However, the presence of BNs-b results in a significant decrease in cell number and obvious aggregation. The presence of BNs-c also results in a decrease in cell number, although the trend is not obvious as for BNs-b. DU145 cells treated with BA, BNs-a and BNs-b for 6 days overall shrink and become smaller, showing a typical apoptosis process. Especially for BNs-b, the obvious aggregation of particles in the cytoplasm can be observed. However, for BNs-c, a large number of DU145 cells remain attached to the plate and only a small number of cells become smaller.
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Figure 2: BN spheres alter cell morphology. (a,b) Optical microscopy images of (a) LNCap and (b) DU145 prostate cancer cells after exposure to original culture mediums (a) and culture medium containing different samples at 5 μg ml−1 (b: BA; d: BNs-a; f: BNs-b; h: BNs-c) and 25 μg ml−1 (c: BA; e: BNs-a; g: BNs-b; i: BNs-c) for 6 days. BA at the equivalent B concentration was used as control. Full size image
Cell death mechanism induced by hollow BN spheres
LNCap and DU145 prostate cancer cells treated with BA or BN spheres were evaluated and compared by a spectral cell analyser (Figs 3 and 4). Cells stained annexin-V-FITC+/PI− are considered as early apoptotic; cells stained annexin-V-FITC+/PI+ are considered as late apoptotic; cells stained annexin-V-FITC−/PI+ are considered as necrotic. Compared with blank control, all BN spheres exhibit obvious cytotoxicity against LNCap cells in a dose-dependent manner (Fig. 3c,d). In contrast, BA shows only weak cytotoxicity to LNCap cells regardless of dose. BNs-a enhances the fraction of early apoptosis, late apoptosis and necrosis, compared with BA (Fig. 3a,b). BNs-b best enhances the fraction of early apoptosis, late apoptosis and necrosis regardless of incubation time. For example, 3 days’ exposure to 5 μg ml−1 of BNs-b is obviously cytotoxic, being characterized by 11.82% in early apoptosis, 29.04% in late apoptosis and 26.80% in cellular necrosis fractions (Fig. 3a(f)). When BNs-b dose is increased to 25 μg ml−1, the cytotoxicity increases to a level of 6.42% in early apoptosis, 47.01% in late apoptosis and 37.47% in necrosis (Fig. 3a(g)). However, exposure to BNs-c with high crystallinity and a very low rate of B release results in lower level of early apoptosis and late apoptosis than exposure to BNs-b. For example, 3 days’ exposure to 25 μg ml−1 of BNs-c shows 0.69% in early apoptosis and 9.68% in late apoptosis (Fig. 3a(i)), which is much lower than exposure to BNs-b under the same conditions (6.42% and 47.01%, respectively). In addition, a decrease in solubility, thus an increase in crystallinity, clearly correlates with an increase in necrosis of LNCap cells: for example, the necrosis ratios for BA, BNs-a, -b and -c are about 14.48, 18.71, 37.47 and 43.76%, respectively, at 25 μg ml−1 for 3 days.
Figure 3: BN spheres induce apoptosis and necrosis in LNCap prostate cancer cells. (a,b) Evaluation of the death pathways of LNCap cells treated with BA or BN nanoparticles supplemented culture medium at BN concentration of 5 μg ml−1 (b: BA; d: BNs-a; f: BNs-b; h: BNs-c) and 25 μg ml−1 (c: BA; e: BNs-a; g: BNs-b; i: BNs-c) for 3 days (a) or 6 days (b). BA containing equivalent B was used for comparison. Culture medium was used as control (a). Q1, Q2, Q3 and Q4 zones represent necrosis, late apoptosis, normality and early apoptosis, respectively. (c,d) Statistical analysis of the percentage of apoptosis and necrosis in LNCap cells treated with BA or BN nanoparticles for 3 days (c) and 6 days (d). Blank: 5 μg ml−1; Slash: 25 μg ml−1. Data in c,d are shown as mean±s.d., ANOVA, *P<0.05, n=3. Full size image
Figure 4: BN spheres induce apoptosis and necrosis in DU145 prostate cancer cells. (a,b) Evaluation of the death pathways of DU145 cells treated with BA or BN nanoparticles supplemented culture medium at BN concentration of 5 μg ml−1 (b: BA; d: BNs-a; f: BNs-b; h: BNs-c) and 25 μg ml−1 (c: BA; e: BNs-a; g: BNs-b; i: BNs-c) for 3 days (a) or 6 days (b). BA containing equivalent B was used for comparison. Culture medium was used as control (a). Q1, Q2, Q3 and Q4 zones represent necrosis, late apoptosis, normality and early apoptosis, respectively. (c,d) Statistical analysis of the percentage of apoptosis and necrosis in DU145 cells treated with BA or BN nanoparticles for 3 days (c) and 6 days (d). Blank: 5 μg ml−1; Slash: 25 μg ml−1. Data in c,d are shown as mean±s.d., ANOVA, *P<0.05, n=3. Full size image
Similarly, BA and BN spheres exhibit obvious cytotoxicity against DU145 cells in a dose-dependent manner (Fig. 4c,d). For BA at a low concentration of 5 μg ml−1 or after a short period of 3 days, cytotoxicity against DU145 cells is weak. However, 6-days exposure to 25 μg ml−1 of BA is obviously cytotoxic, being characterized by 9.60% in cellular necrosis, 22.57% in late apoptosis and 5.48% in early apoptosis fractions (Fig. 4b(c)), which suggests that the cytotoxicity of BA is related to both the apoptosis and necrosis. Three days’ exposure to 25 μg ml−1 of BNs-a enhances fractions of early (16.96%) and late (14.42%) apoptosis (Fig. 4a(e)), compared with that of BA (3.57% and 4.43%, respectively) (Fig. 4a(c)). Three days’ exposure to BNs-b considerably enhances fractions of early apoptosis (40.05% at 5 μg ml−1; 32.68% at 25 μg ml−1) and late apoptosis (8.62% at 5 μg ml−1; 52.41% at 25 μg ml−1) (Fig. 4a(f,g)). After 6 days’ incubation, BNs-b demonstrates similar results to those after 3 days. However, BNs-c shows much weaker effects on DU145 cells compared with BA and other two BN spheres regardless of incubation time.
Furthermore, the levels of two key damage-associated molecular pattern protein biomarkers, capase-3/7 and lactate dehydrogenase (LDH) release, were examined to evaluate apoptosis and necrosis, respectively (Fig. 5; Supplementary Figs 7 and 8). The results indicate that the apoptosis (capase-3/7) and the necrosis (LDH) are enhanced remarkably by BN spheres, compared with BA, further confirming the flow cytometric results. Both LNCap and DU145 prostate cancer cells show an increase in LDH release with the increase in crystallinity and decrease in solubility. It can be seen that the LDH release shows the following sequence: BNs-c>BNs-b>BNs-a>BA>control. As a whole, LNCap cells exhibit much higher LDH release than DU145 cells. With the addition of BA or BN spheres, caspase-3/7 contents increase. Among all the samples, BNs-b shows the highest caspase-3/7 activity both for LNCap and DU145 cells. Moreover, totally, DU145 cells exhibit higher caspase-3/7 activity than LNCap cells.
Figure 5: The levels of two key damage-associated molecular pattern protein biomarkers. LDH cytotoxicity for (a) LNCap and (b) DU145 prostate cancer cells after 16 h (n=4); Caspase-3/7 activity for (c) LNCap and (d) DU145 prostate cancer cells after 16 h (n=2). Data in a–d are shown as mean±s.d., ANOVA, *P<0.05. Full size image
In vivo anticancer effects of hollow BN spheres
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We investigated in vivo anticancer efficacy of BA and hollow BN spheres to suppress the tumour growth in mice with prostate cancers induced by the injection of LNCap prostate cancer cells through subcutaneously injected models first (Fig. 6). Saline group was used as the control. BN spheres and BA significantly suppress the prostate tumour growth compared with the control (Fig. 6c). Among all the groups, BNs-b is the most effective for controlling tumour progression. Thirty-three days after inoculation of LNCap cells, 50% of mice in the saline groups are free from tumour formation by visual inspection, compared with 75% in the BA, BNs-a and BNs-c groups and 100% in the BNs-b group. After 61 days, ratios of tumour-free mice for the saline, BA, BNs-a, BNs-b and BNs-c groups are 0, 50, 75, 100 and 25%, respectively. Finally, after 96 days, the ratios of tumour-free mice for the saline, BA, BNs-a, BNs-b and BNs-c groups are 0, 25, 50, 75 and 25%, respectively. Over the 3-month period, the average tumour volume increases to 827 mm3 in the saline group, compared with 2 mm3 in the BNs-b group and 287 mm3 in the BA group (Fig. 6d). It can be seen that BNs-b can inhibit tumour volume by about 99.75% compared with the control. The fact that the tumour growth is significantly suppressed in the BA group over the control group indicates that B can inhibit prostate cancer. The inhibitory effects on prostate tumour growth are further enhanced by the alternative use of BNs-a or -b sphere as a novel B carrier to realize the sustained release of B. However, BNs-c spheres exhibit much weaker inhibitory effects on prostate tumour growth owing to too high crystallinity associated with too low B release.
Figure 6: Effects of BN spheres on cellular and in vivo subcutaneously injected prostate cancer models. (a) BA or hollow BN spheres with controlled B release resulting in different LDH release and caspase-3/7 activity in LNCap prostate cancer, which is responsible for necrosis and apoptosis, respectively; (b) Effects of saline, BA and hollow BNs-b spheres on mice preinjected with LNCap prostate cancer cells, respectively. (c) Percentage of mice without development of tumour over time after LNCap cancer cell injection (data are shown as mean±s.d., Kaplan–Meier log rank test, *P<0.05 vs saline group, n=4); (d) Quantitative analysis of the effects of different samples on tumour size (data are shown as mean±s.d., t-test, *P<0.05, n=4). Full size image
Then orthotopical tumour growth models in mouse injected with low dose of LNCap cells were used to further confirm in vivo anticancer efficacy of BA and hollow BN spheres to suppress the LNCap tumour occurrence and growth (Fig. 7). At the end point, 12 weeks later, the average mouse weight for the saline, BA and BNs-b groups is 17, 24 and 27 g, respectively, while the average tumour volume is in the following sequence: control group (494 mm3)>BA group (224 mm3)>BNs-b group (39 mm3). The orthotopical tumour growth models exhibit the same trends with subcutaneously injected models.
Figure 7: In vivo anticancer effects of BN spheres by using orthotopically injected models. (a) Mouse weight for different groups 12 weeks after LNCap cancer cell injection at the dose of 2 × 106 cells per mouse (n=4); (b) quantitative analysis of the effects of different samples on tumour size at the end point (data are shown as mean±s.d., t-test, *P<0.05, n=4). Full size image
Cancer therapy efficacies using hollow BN spheres, chemotherapy drug paclitaxel (PTX) and the PTX–hollow BN spheres complex were further evaluated in mice orthotopically injected with high dose of LNCap cells (Fig. 8). Both hollow BNs-b spheres and PTX drugs significantly inhibit the tumour growth compared with the control of saline group. Most importantly, the combination of hollow BNs-b spheres with PTX drugs demonstrates the cooperative and synergetic effects on the tumour suppression, as the PTX–hollow BN spheres complex group exhibits the minimum tumour volume among all the groups.
Figure 8: Comparison of cancer therapy efficacies using different combinations of BN spheres and PTX. Quantitative analysis of the tumour size for different groups 8 weeks after LNCap cancer cell injection at the dose of 5 × 106 cells per mouse (data are shown as mean±s.d., t-test, *P<0.05, n=4). Full size image
In vivo safety and systemic biodistribution
Healthy C57/BL6 mice were intravenously administered via tail vein with 50 μg of BNs-b and then the blood biochemistry, haematology and biodistribution analysis were carried out. Various biochemistry parameters, such as blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase were tested (Supplementary Fig. 9A–E). The liver function markers (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase) and kidney function markers (creatinine, blood urea nitrogen) are only slightly varied and are still within the normal range, compared with the control. No obvious hepatic or renal toxicity is observed in treated mice. For the haematological analysis, the following important parameters, such as white blood cells, red blood cells, haemoglobin, haematocrit, mean cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet, were tested (Supplementary Fig. 10). All of the above parameters in the BNs-b treated groups appear to be normal compared with the control groups. As a whole, no obvious toxicity of BNs-b is noted from the blood biochemistry and haematological data.
Moreover, the biodistribution of BNs-b after intravenous injection into C57/BL6 mice was monitored after 1 h, 1 day, 3 days and 15 days (Supplementary Fig. 9F). During the whole period of time, no obvious signs of abnormal behaviour in eating, drinking and activity were documented. At a certain time, the mouse was killed, various organs and tissues were collected and the boron contents were measured by ICP. The results show that BNs-b distributes in many different organs and mainly accumulates in the reticuloendothelial system such as the liver and spleen. The distributed amounts in the different organs and tissues decrease with time owing to gradual degradation and clearance.
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