Nanostructures, concentrations and energies: an ideal equation to extend therapeutic efficiency on radioresistant 9L tumor cells using ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ ceramic nanostructured particles

Tantalum pentoxide TNSPs were synthesised using a thermal oxidation reaction. This process involved solid state thermal oxidation of Ta metal foil with purity 99.99%. The foil was placed in an alumina crucible prior to heating at 800 °C for 1 h, which leads to complete oxidation and formation of ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ nano powder.

Tantalum pentoxide PNSPs were synthesised using a precipitation-based chemical reaction utilising ${\rm{Ta}}{({\rm{OEt}})}_{5}$ (Sigma-Alrich 99.99%) and an ethoxide decomposition reaction with water. This method was derived from the article written by Kominami et al [21]. Two drops (1 ml) of ${\rm{Ta}}{({\rm{OEt}})}_{5}$ was first added to a small amount of ethanol (15 ml) and mixed carefully to avoid disrupting the surface of the ethanol. Excess deionised water was added resulting in a white fine precipitate that was filtered out and washed, using a centrifuge (Eppendorf model 5702). The filtered tantalum hydroxide powder was placed in a furnace and heated for 8 h at 700 °C, where by thermal decomposition occurs and ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ nanocrystallites form. Drying of the resultant NSPs was necessary to avoid violent agglomeration. This was achieved by placing them on an aluminum crucible inside a furnace at 140 °C for a minimum of 2 h. Finally, sterilisation inside an autoclave at 121 °C for 45 min ensured the elimination of any contaminants that could jeopardise cell culture experiments. The NSPs were suspended in phosphate buffered saline (PBS) (without calcium and magnesium ions) to ensure homogeneity in concentration before being applied to the cells. Furthermore, sonication of the NSP solution was employed to reduce the degree of aggregation and create a more even distribution of the NSPs in solution. A 15 ml falcon tube containing the solution was sonicated with a fine tip probe in an ultrasonic bath environment for 30 min total in 10 min intervals.

Cellular experiments employed a culture of 9L gliosarcoma cancer cells derived from rodent brain cells [16]. The cells were maintained in a T75 cm² flask with complete Dulbecco's Modified Eagle Medium (c-DMEM from GIBCO, supplemented with 10% fetal bovine serum and 1% Penicillin and Streptomycin) and incubated at 37 °C and 5% (v/v) CO₂.

Characterisation of the NSP involved the use of XRD for determination of phase, structure, particle size and unit cell parameters. A sample of tantalum pentoxide NPs was placed in a GBC MMA XRD system (GBC Scientific Equipment Pty Ltd, Victoria, Australia) using Cu K-alpha radiation with wavelength ${\lambda }=1.5418\,\mathring{\rm A}$, accelerating voltage 40 kVp and cathode current 30 mA, in order to determine information regarding crystal structure and mean particle size (t). Mean crystallite size (t) was determined through utilisation of Scherrer's equation [22].

Morphological parameters—crystallinity, size, agglomeration and shape—were investigated using a 200 kVp (HRTEM, JEOL, 2011, USA).

9L cells were exposed to ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ TNSPs and PNSPs at concentrations of 0, 50 and 500 μg ml⁻¹ for 24 h. Trypsinisation was used to detach the 9L cells before being placed in a centrifuge at 1500 rpm and 4 °C for 5 min and rinsing twice with cold PBS (without calcium and magnesium). Flow cytometric measurements and analyses were performed using a Becton-Dickinson fluoresence-activated cell sorting (FACS) flow cytometer (BD LSR II; BD Biosciences, Franklin Lakes, USA). Each analyzed sample contained a minimum of 10 000 cells where cell doublets and aggregates were gated out using a two parameter histogram of FL2-Area versus FL2-Height. Data analysis was performed using the FACSDiva software, assessing both forward (FSC) and side scatter (SSC) intensities. The median cell SSC values were employed as a marker of cellular granularity, which is proportional to particle uptake and internalisation [23].

Clonogenic assays were used to assess cell survival. Cells were plated in triplicate for: a control (no NSP), 50 μg ml⁻¹ and 500 μg ml⁻¹ concentration of tantalum pentoxide NSPs. The 9L cells were brought to 90% confluence, tantalum pentoxide NSP solution prepared and added to the cell culture medium; then once exposure time had been reached the cells were split into 100 mm petri dishes with 10 ml c-DMEM and incubated for 21 days, equivalent to 15 doubling times. Following this, each dish was washed with 5 ml PBS (with calcium and magnesium ions) and stained with 5 ml of staining solution (25% crystal violet and 75% ethanol). Colonies were counted and considered viable if they consisted of approximately 50 cells or more (n), and compared with inital seeding values (I) to obtain the plating efficiency (PE).

$$\begin{eqnarray}&&{\rm{PE}}=\displaystyle \frac{n}{I}.\end{eqnarray} \tag{ 1 }$$

The surviving fraction (SF) could then be determined by comparing the control PE (${{\rm{PE}}}_{{\rm{c}}}$) with the PE of the group containing the NSP (${{\rm{PE}}}_{x}$).

$$\begin{eqnarray}&&{\rm{SF}}=\displaystyle \frac{{{\rm{PE}}}_{{x}}}{{{\rm{PE}}}_{{\rm{c}}}}.\end{eqnarray} \tag{ 2 }$$

To assess the toxicity of TNSPs and PNSPs to 9L cells, NSP concentrations of 0 (2 controls), 50, 100, 200 and 500 μg ml⁻¹ were exposed to 9L cells for a period of 24 h prior to plating. 9L cells were brought to 90 % confluence inside T12.5 ${{\rm{cm}}}^{2}$ flasks (BD Falcon^TM) before exposure to the NSP. Solutions of TNSPs and PNSPs were prepared according to the 'NSP Preparation' protocol, and the appropriate volumes corresponding to each concentration added to the cells. Once the desired exposure time had been reached, cells were plated in triplicate according to the 'clonogenic cell survival assay' technique. Following plate staining and colony counting, SFs were obtained for each NSP at each concentration, and normalised to the control group (0 μg ml⁻¹).

All radiation experiments were carried out at the radiation oncology department at the Prince of Wales Hospital (Randwick, NSW, Australia). Beam energies of 10 MV and 150 kVp were investigated using an Elekta Axesse^TM LINAC (Elekta AB, Kungstensgatan, Stockholm, Sweden) and a Nucletron Oldelft Therapax DXT 300 Series 3 Orthovoltage unit (Nucletron B.V., Veenendaal, The Netherlands), respectively. The 9L cell culture was exposed to the tantalum pentoxide NSPs for 24 h at concentrations of 50 and 500 μg ml⁻¹. For MV experiments, 9L cells were irradiated in T12.5 ${{\rm{cm}}}^{2}$ flasks (BD Falcon^TM) completely filled with Hank's balanced salt solution so no air bubbles were present inside the flask to ensure electronic equilibrium conditions were present at the depth of the cells. For kVp experiments, cells were contained and irradiated in 6 mm of medium to maximise the accuracy in delivering the prescribed dose to the cell monolayer. All doses (1, 2, 3, 5 and 8 Gy) were delivered in single fractions at room temperature with dose rates of 5 and 0.7 Gy min⁻¹ for 10 MV and 150 kVp x-ray photon beam energies, respectively. Tissue culture flasks were irradiated horizontally surrounded by a 30 × 30 × 10 ${{\rm{cm}}}^{3}$ solid water phantom to ensure backscattering effects were taken into account and human physiological conditions accurately modeled, with the cell monolayer situated at an SSD of 100 cm and 50 cm for 10 MV and 150 kVp beam energies, respectively. Unirradiated control samples (with and without tantalum pentoxide NSPs) were utilised and handled under the same conditions as the irradiated samples.

It is worth noting here that although different dose rates have been utilised at each energy, the dose rate effect has been tested on 9L cells at the dose rates utilised in this study and revealed an insignificant effect on the survival of 9L cells [24].

Survival curves were fit according to the linear quadratic model (LQM) using the associated error bars of each point as weighting factors. The error bars for each point represent 1 standard deviation from the mean. The LQM [25] describes cell SF mathematically as a function of absorbed dose (D).

$$\begin{eqnarray}&&{\rm{SF}}({D})={{\rm{e}}}^{-\alpha {D}-\beta {{D}}^{2}}.\end{eqnarray} \tag{ 3 }$$

The parameters α (${{\rm{Gy}}}^{-1}$) and β (${{\rm{Gy}}}^{-2}$) are indicative of cell radiosensitivity and repair effectiveness respectively. We were able to extrapolate these values based on the fit for each survival curve and compare experiments (with and without the NSP) to quantify the degree of dose enhancement by calculation of the SER (the ratio of doses giving 10% SF on the cell survival curves).

We measured the degree of dose enhancement by means of the SER, defined as the ratio of doses giving 10% SF on the cell survival curves. Given dose (D) as a function of SF on two curves, namely the control curve with no NSP (${D}_{{\rm{c}}}$(SF)) and the curve containing the NSP (${D}_{x}$(SF)), the SER can be mathematically expressed as:

$$\begin{eqnarray}&&{\rm{SER}}=\displaystyle \frac{{D}_{{\rm{c}}}(0.1)}{{D}_{x}(0.1)}.\end{eqnarray} \tag{ 4 }$$

Light and fluorescence microscopy images were obtained using a Leica confocal laser scanning microscope (Leica TCS SP5 Advcanced System UVVIS-IR and X1-Port Access with SMD FCS and CO₂ incubation chamber, Germany) and the accompanying image manager software Leica Application Suite Advanced Fluorescence (LAS AF, v.2.6.17314, Germany) located at the Illawarra Health and Medical Research Institute (IHMRI), Wollongong, Australia.

Cell survival experiments were repeated to ensure reliability and measured in triplicate for each sample. All values are expressed as the mean of three measurements with the experimental uncertainty given as one standard deviation. The LQM model was fitted to cell survival data using KaleidaGraph software. The fit of the data was weighted according to the error for each dose in the determination of the radiobiological constants, α and β. Statistical analysis of SF data was performed using a two-tailed Student's t-test under the assumption of equal variance. A P value less than, or equal to 0.05 was considered statistically significant.

Diffraction patterns were constructed for each NSP, shown in figure 1, with each curve containing peaks of similar shape, intensity and position, indicative that the NSP samples are of similar atomic composition, namely ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$. Closer inspection of the TNSP pattern revealed the existence of additional smaller peaks, compared to the PNSP pattern, signifying a structural difference between the samples despite their similar chemical composition. Peak analysis and database comparison of the diffraction patterns revealed the TNSPs to be orthorhombic beta phase (JCPDS:25-0922), while the PNSPs are hexagonal delta phase (JCPDS:19-1299). This structural difference is fundamentally linked to the production methods employed for each NSP, where by the annealing temperature of 700 °C produces hexagonal phase ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ for the PNSPs, as opposed to 800 °C for the TNSPs, which results in orthorhombic formation. This is supported in the literature, where the temperature threshold for orthorhombic transformation in ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$ NPs has been shown to be 750 °C [26]. The most notable feature of this transformation is the splitting of (1,1,0) hexagonal phase peak into (0,22,0) and (2,15,1) orthorhombic phase peaks [27], clearly evidenced in figure 1.

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Additionally, the unit cell parameters were derived for each sample (data summarised in table 1) with a = b characteristic of the hexagonal structure in the PNSPs [28]. Mean crystallite size (t) was calculated by means of Scherrer's equation [22] and analyis of an appropriate peak (0,0,1), proving the TNSPs (56 nm) to be larger than the PNSPs (45.5 nm). With regards to DERT, these sizes are quite large compared to Au NPs, which commonly employ 1.9 nm [20] and even 1.4 nm [29], suggesting their ability to be internalised by cells may be undermined. However, a study into NP size-dependency of internalisation has revealed 50 nm to be the optimal size for internalisation of Au NPs [30], suggesting damage to cells will be maximised and validating our choice to investigate NSPs of this size.

Table 1.  Mean crystallite size, unit cell parameters, crystal structure and phase summarised for the TNSPs and PNSPs.

Type of ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$	Size (nm)	a (Å)	b (Å)	c (Å)	Structure	Phase
TNSP	56	6.2	40.29	3.89	Orthorhombic	Beta
PNSP	45.5	3.62	3.62	3.88	Hexagonal	Delta

Table 2.  Alpha, beta and SER for the TNSPs and PNSPs at 0, 50 and 500 μg ml⁻¹, as well as 150 kVp and 10 MV. Errors are given ±1 standard deviation from the mean.

Beam energy and type of ${{\rm{Ta}}}_{2}{{\rm{O}}}_{5}$	NSP concentration($\mu$ g ml−1)	α(${{\rm{Gy}}}^{-1}$)	β(${{\rm{Gy}}}^{-2}$)	${{\rm{SER}}}_{0},{{\rm{SER}}}_{50},{{\rm{SER}}}_{500}$
150 kVp TNSP	0	0.30 ± 0.04	N/A	1
	50	0.32 ± 0.05	N/A	1.07 ± 0.03
	500	0.33 ± 0.02	N/A	1.10 ± 0.07
150 kVp PNSP	0	0.30 ± 0.04	N/A	1
	50	0.40 ± 0.01	N/A	1.33 ± 0.07
	500	0.43 ± 0.01	N/A	1.43 ± 0.08
10 MV TNSP	0	0.19 ± 0.05	0.012 ± 0.008	1
	50	0.38 ± 0.03	N/A	1.33 ± 0.07
	500	0.40 ± 0.03	N/A	1.40 ± 0.07
10 MV PNSP	0	0.19 ± 0.05	0.012 ± 0.008	1
	50	0.39 ± 0.03	N/A	1.36 ± 0.07
	500	0.43 ± 0.03	N/A	1.46 ± 0.07

High quality images acquired (figure 2) indicated that both NSPs are highly crystalline in nature, possessing mean crystallite sizes in agreement with that derived using Scherrer's formula. The tendency for agglomeration is seen to differ significantly between the two samples, with the TNSPs exhibiting larger aggregates approximately 400 μm in maximum size, while the PNSPs are globally much more dispersed with maximum aggregates of 100 μm. It must be noted that the sample preparation process causes the NSPs to agglomerate, so that the actual aggregate size in solution is much less. This difference in particle aggregation may be attributed to their respective production methods, where by the higher reaction temperature in thermal decomposition of the TNSP results in larger crystallites, with increased size distribution, and larger aggregates. Comparatively, the precipitation reaction utilises a lower reaction temperature, and involves dispersion treatment so that the crystallites are less likely to aggregate in an attempt to reduce the total surface energy of the material. These differences in the methods of synthesis directly affect the NSP (crystallite) morphology, there by influencing aggregate morphology as well. The TNSPs consist of larger crystallites, higher in aspect ratio, irregular in shape and less dispersed leading to aggregates which are larger and less dispersed than their PNSP counterparts.

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The two NSPs are also morphologically dissimilar in terms of shape. The TNSPs display an irregular shape while the PNSPs are distinctly spherical. Based on the smaller crystallite size and resistance to aggregation, we would naturally expect the PNSPs to be more easily internalised than the TNSPs. The less obvious fact, though, is that the spherical nature of the PNSPs also increases their degree of internalisation. It has been shown for Au that spherical NPs portray preferential uptake to other shapes, such as nanorods, in HeLa cells. In this particular study, endocytosis was identified as the mechanism of internalisation, with low aspect ratio spherical NPs being preferentially internalised due to greater availability of cell receptor bonding sites for serum proteins, which initiate endocytosis [30]. Evidently, we not only expect the PNSPs to be more easily internalised than their thermal counterparts, there by imparting greater damage to the cells, but we may also expect the internalisation mechanism to be endocytosis. This is confirmed with the results presented in the FACS flow cytometry and confocal microscopy sections.

The magnitude of forward (FSC) and side (SSC) scatter are representative of cell diameter (size) and granularity respectively, with changes in cellular granularity (mean SSC) proportional to the degree of internalisation [31]. Data was presented in the form of a histogram with SSC graphed as a function of FSC abscissa (figure 3).

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For all concentrations tested, we see no significant difference in the mean SSC values when comparing the two NSPs themselves [23], suggesting both NSPs are internalised to a similar degree. When examining each NSP on its own and comparing to the control, we see significant increases in the degree of internalisation as the concentration of the NSP increases. Although this dependency has been rarely tested explicitly, it is not unheard of, with bismuth ferrite (BiFeO₃) NPs [31] and superparamagnetic iron oxide NPs [32] exhibiting similar trends. For both NSPs, we see an approximate 10% increase in the mean SSC values at 50 μg ml, and at 500 μg ml⁻¹ we observe a substantial increase in the mean SSC of approximately 300%. These results suggest that at low concentrations the majority of the NSPs remain external to the cell membrane as aggregates, while at higher concentrations a greater portion of the NSPs are internalised by the 9L cells.

Maximum toxicity values of approximately 20% and 30% were measured (figure 4) for TNSPs and PNSPs respectively. Significant drops in cell colonies and saturation occured at 100 μg ml⁻¹ for both NSPs. Additionally, both NSPs exhibited 80% SF at 100 μg ml⁻¹, which is exactly the same SF as 1.9 nm Au NPs tested at the same concentration on MDA-231-MB (breast cancer) cells (more sensitive than 9L) [20]. This suggests they are, at a minimum, comparable to Au NPs in terms of cytotoxicity, which are already used clinically in DERT and widely considered non-toxic [33]. Moreover, there is considerable support in the literature to suggest toxicity values as high as 50% may be considered non-toxic [34], supporting the fact that both tantalum pentoxide NSPs may be considered relatively non-toxic at the highest concentrations tested. These results formed the basis for the concentrations utilised in radiation experiments.

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Radiation experiments were performed using the clonogenic assay technique to assess the influence of particle morphology, NSP concentration and beam energy on sensitisation of 9L cells by TNSPs and PNSPs, with resultant survival curves (figure 5) fit according to the LQM.

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Alpha and beta parameters were extrapolated based on SF data, and SERs calculated based on comparison of 10% survival doses (table Table 2). In all cases, cells containing either of the NSPs demonstrated lower SFs in comparison to the control, indicative of greater induced damage to the cells. Comparing results for each NSP, we see the PNSP exhibits significant sensitisation, far greater than that of the TNSP, for irradiation at 150 kVp and both concentrations examined (50 μg ml⁻¹, p = 0.0274 and 500 μg ml⁻¹, p = 0.0126), suggesting a morphological difference in the PNSPs gives them superior sensitisation ability at kVp beam energies. Similarly, we observe greater sensitisation by the PNSP at 10 MV for both concentrations tested, however the difference in SERs between the two NSPs proves to be statistically insignificant (50 μg ml⁻¹, p = 0.6274 and 500 μg ml⁻¹, p = 0.3531). At 10 MV, the effects of differing morphology, attributed to the TNSPs and PNSPs, on cell damage become less pronounced due to the long range (centimetres) of secondary Compton scattered electrons, as well as electrons and positrons originating from pair production, that are produced. These long range, low LET (0.2 keV μm⁻¹) particles produce uniform dose enhancement across the cell culture independent of NSP distribution, but rather dependent on the total NSP concentration.

Analyzing the effect of NSP concentration on sensitisation, we see insignificant increases in the SERs for the TNSP of approximately 3% at both beam energies tested (150 kVp, p = 0.5655 and 10 MV, p = 0.2879). The PNSP exhibits a slightly higher increase of 8% at both beam energies used (150 kVp, p = 0.2017 and 10 MV, p = 0.1551).

Finally, we observe significant beam energy dependence of sensitisation for the TNSPs, with approximate 25% increases in cell death from 150 kVp to 10 MV at both concentrations tested (50 μg ml⁻¹, p = 0.0274 and 500 μg ml⁻¹, p = 0.063). In contrast, the PNSPs exhibit a weaker dependence with an approximate increase of 3% from 150 kVp to 10 MV at both concentrations tested (50 μg ml⁻¹, p = 0.6274 and 500 μg ml⁻¹, p = 0.6585). This again supports our hypothesis that morphological differences in the two NSPs give rise to varying degrees of sensitisation for any given beam energy and concentration. Evidently, the results imply that the PNSPs are superior radiosensitisers to their TNSP counterparts, not only in terms of the magnitude of sensitisation, but also in consistency of delivering effective radiosensitisation across varying beam energies and nanocrystallite concentrations.

Confocal microscope images were captured detailing the interaction of the TNSPs and PNSPs with 9L cells at concentrations of 0 (control), 50 and 500 μg ml⁻¹.

Analysis of the resultant images (figure 6) revealed that both NSPs show a specificity to the 9L cells at all concentrations, forming aggregates and being attracted to the cellular membrane. This aggregation of the NSPs is more pronounced at the highest concentration tested (500 μg ml⁻¹). Comparing the two NSPs, we see the TNSPs are more prone to aggregation, forming much larger clusters around the 9L cells compared to the PNSPs, for all particle concentrations. On the other hand, the PNSPs show little to no aggregation and are of uniform size. Despite the lack of aggregation, clustering of the PNSPs is still observed around the cellular membrane and nucleus due to an intrinsic attraction of the PNSPs to the 9L cells. We also observe the presence of endosome-encapsulated NSPs (indicated by red arrows in figure 6), particularly in the 500 μg ml⁻¹ cases, suggesting the internalisation mechanism activated here is endocytosis. This is similar to Au NPs, which have been shown to exhibit clustering inside the cytoplasm of HeLa cells, and through the use of confocal imaging, were observed to localise in lysosomes [35]. Evidently, the images suggest the PNSPs are again superior to the TNSPs in terms of particle morphology (aggregation), specificity and internalisation. However, based on the significant increase in specificity and clustering from 50 to 500 μg ml⁻¹ for both NSPs, it would be logical to expect greater enhancement than what was observed in radiation experiments, although an explanation for this phenomenon is offered and supported by the results presented.

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