Research on CT Imaging and Radiotherapy of Breast Cancer with Bi-DTPA

doi:10.21203/rs.3.rs-1888610/v1

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Research on CT Imaging and Radiotherapy of Breast Cancer with Bi-DTPA

https://doi.org/10.21203/rs.3.rs-1888610/v1

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Background

High z number element is advanced in Computed Tomography (CT) imaging and the sensibilization of radiotherapy because of higher mass energy absorption coefficients. Bi-Diethylene triamine pentaacetic acid (Bi-DTPA), structured on Bi element, is advanced in CT imaging ability and advantages of strong water solubility, good biocompatibility, low cost and mass production. So in this study, Bi-DTPA was used in CT imaging and radiotherapy of breast cancer to explore its feasibility and effect in CT imaging of mouse breast cancer model, and whether it had sensitization radiation characteristics through radiotherapy.

Methods

The characterization of Bi-DTPA was detected by FT-IR, ¹H-NMR and HR-MS. Compared the tumor CT imaging effect of Bi-DTPA with I 370 on 4T1 tumor bearing mouse model. The radiosensitization mechanism of Bi-DTPA was investigated by CCK-8 assay, clonogenic assay, ROS detection, DNA double strand break detection, flow cytometry for the detection of cell apoptosis, and treatment of 4T1 tumor bearing mouse model.

Result

The Bi-DTPA’s CT imaging signal was higher than I 370 at a safe concentration which synthesized by the one-pot method. During radiotherapy with Bi-DTPA, the viability of cells was reduced and the sensitization rate was 1.51. The level of ROS generated, DNA damage sites and the proportion of cell apoptosis was significantly higher than that in the radiotherapy group. The results of in vivo treatment showed that Bi-DTPA had better inhibitory effect on tumors.

Conclusion

The synthesized Bi-DTPA was advanced and applicable in CT imaging and possessed sound capacity in radiation therapy (RT) sensitization which can effectively kill tumor cells by ROS and inhibit tumor proliferation. Therefore, it has great potential in the future application of tumor integrated diagnosis and treatments.

Bi-DTPA

ROS

Radiosensitization

High z number metallic elements are considered as sound radiation attenuation media, such as Au, W, Bi, Ta etc(1–4), which has been widely used in Computed Tomography (CT) imaging. Materials containing these elements tend to have higher Mass Energy Absorption Coefficients, which causes increased dose attenuation in target region and decreased side-effect of irradiation in non-target region, thus these materials are also widely used in contrast-enhanced CT imaging and sensibilization of radiotherapy(5, 6), so they have a good application prospect in the integration of diagnosis and treat. Liao's team(7) synthesized Bi-DTPA with Bi and diethylene triamine pentaacetic acid (DTPA) through a simple one-pot method, and confirmed its excellent CT imaging ability and advantages of strong water solubility, good biocompatibility, low cost and mass production.

In this study, Bi-Diethylene triamine pentaacetic acid (Bi-DTPA) was used in CT imaging and radiotherapy of breast cancer to explore its feasibility and effect in CT imaging of mouse breast cancer model, and whether it had sensitization radiation characteristics through radiotherapy at the cellular level and in mouse breast cancer model as it contains the characteristics of the plateau z element, which would provide a more safe and effective way for CT imaging and radiotherapy of tumors, and lay a foundation for the follow-up research on integrated nanomaterials of diagnosis and treatment.

Synthesis and characterization of Bi-DTPA

Bi-DTPA was synthesized by one-pot method. In details, Bi₂O₃（0.5mmol）and DTPA（1mmol）were mixed and dissolved in 20mL of double distilled water for 2-hour heating at 85℃, followed by adjusting pH to 7 with NaOH and lyophilization. The verification of successful synthesis was completed by Fourier transform infrared (FT-IR), ¹H-Nuclear magnetic resonance (¹H-NMR) and High resolution mass spectrometry (HR-MS).

Establishment of 4T1 tumor bearing mouse model of breast cancer

4T1 cells were cultured with 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution in an incubator based on 5% CO₂ at 37 °C (the latter culture conditions were the same), and resuspended in Phosphate buffered saline (PBS) after digestion with trypsin, after the mice skin was disinfected with 75% alcohol, 100 μL of 4T1 cells (1×10⁶ cells) in logarithmic growth phase were injected subcutaneously on the back of nude mice, then the tumor grew to a size of 75 mm³ after 2-3 weeks.

CT imaging of breast cancer tumor with Bi-DTPA

The 4T1 tumor-bearing mice were randomly divided into two groups, including Bi-DTPA group and I 370 group. Mice were anesthetized by chloral hydrate (4%, 40mg/kg), and respectively accepted intratumor injection (25μL 0.5mol/L, 299mg/ml). CT scanning was conducted at different time point after injection (pre、0.25、0.5、1、3、6h). CT values of tumor and bladder were measured.

In vitro safety of Bi-DTPA

100 μL of Bi-DTPA solutions of different concentrations (0, 0.2, 0.4, 0.5, 0.6, 0.8, 1 mg/mL) were incubated with 4T1 cells (5 × 10³ per well) in a 96-well plate for 24 h. Then discard the supernatant and washed 3 times with PBS, and 100 μL of Cell Counting Kit-8 (CCK-8) solution (The ratio of CCK-8 and serum-free medium is 1:10, the same CCK-8 solution in the following) were added and incubated for 1 h, and the microplate reader was used to measure the wavelength of 450 nm (Optical density; OD value), calculated the cell viability according to the following formula, and found its safe maximum concentration (the cell viability remains above 90%).

Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

In vitro treatment

4T1 tumor cells were randomly divided into 4 groups, including Bi-DTPA+X-ray, Bi-DTPA, X-ray and control group. The concentration of Bi-DTPA applied in this experiment was set at 0.5 mg/ml, and irradiation dosing was 6Gy.

Colony survival analysis

4T1 cells were seeded in 6-well plate with different amount (100, 200, 400, 600, 800, 1000 cells) and incubated overnight for attachment. After that, Bi-DTPA (0.5mg/ml) was added into cells in Bi-DTPA+X-ray group for 24-hour co-incubation, while no treatment was done to cells in X-ray group. Then cells in both groups were irradiated at different dose (0, 2, 4, 6, 8Gy). Then cells were washed and added with fresh cell medium for another 10-day incubation. Finally, cells were washed, fixed and stained, and samples including at least 50 cell colonies were counted. Three parallel experiments were carried out, and survival fraction (SF) was calculated according to following formulas.

Planting efficiency (PE)= (the number of cell colonies)/ (the number of seeded cells) * 100%

SF= (the number of cell colonies formed after treatment)/ (the number of seeded cells * PE)

Three replicate data of each sample in the plotted survival curve were standardized by standard linear quadratic model. Referring to the survival curve, Sensitivity enhancement rate (SER) was calculated by Sigma Plot 14.0 software.

According to the equation: SF= exp [-(αD+βD²)]

D represents the dose of X-ray, and 1/α represents mean lethal dose (D_O).

SER=D_O (w/o RT sensitizer)/ (D_O with RT sensitizer)

CCK8 assay

4T1 cells were seeded in 96-well plate at a concentration of 5*10³ per well and divided into 4 groups in which 5 wells were set as replicates. After being treated correspondingly, cells were added with CCK8 agent and detected by microplate reader to gain OD value at a wavelength of 450 nm for the evaluation of cell viability.

The detection of Reactive oxygen species (ROS)

4T1 cells were seeded in confocal dishes at the concentration of 3*10⁴ /dish and incubated overnight for attachment. Then 1mL diluted 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (0.1ug/ml) was added and incubated away from light for 20 min. After 4 groups were all correspondingly treated, another 10-minute incubation was done. The production of ROS in each group was observed by Confocal laser scanning microscope (CLSM) (excitation/emission=502nm/530nm).

The detection of DNA double strand break

4T1 cells were seeded in confocal dishes at the concentration of 3*10⁴ /dish and incubated overnight for attachment. Two hours after 4 groups were all correspondingly treated, cells were fixed by 4% of paraformaldehyde for 10 min and washed by PBS, then incubated with 0.5% of Triton X-100 (dissolved in PBS) for 20-minute permeation at room temperature. Then cells were again washed by PBS for three times followed by one-hour block with 1% of BSA solution at room temperature and overnight incubation with primary antibody (anti-phosphohistone γ-H2AX mice monoclonal antibody, diluted by PBS, 1:500) at 4℃. Then cells were washed by Phosphate buffered saline tween-20 (PBST) for three times and incubated with secondary antibody (goat anti-rabbit IgG H&L, ab150077) away from light for 1 hour at room temperature. Then cells were washed by PBST for 3 times and stained by 4',6-diamidino-2-phenylindole (DAPI). Finally, cells were observed under CLSM (excitation/emission=495nm/519nm).

Flow cytometry for the detection of cell apoptosis

4T1 cells were seeded in 6-well plate at the concentration of 2*10⁵ /well and incubated overnight for attachment. After 4 groups were all correspondingly treated, cells were digested, collected and centrifugated at 1000 rpm for 5 min to remove medium. Then they were washed by PBS and centrifugated again to remove PBS, and pre-cooled 70% of ethanol was added to fix cells for 1-2 hours at 4 ℃. Cells were centrifugated to remove ethanol, resuspended by PBS for 5 min, filtered by 400-mesh filter, and centrifugated at 1000 rpm for 5 min to remove PBS. Then cells were stained by 1 ml of PI for 30 min away from light at 4℃. Finally, cell apoptosis in the prepared samples were analyzed by flow cytometry (excitation/emission=488nm/630nm).

In vivo treatment

4T1 tumor-bearing nude mice models were established and divided into 4 groups when tumor size reached 75mm³, including Bi-DTPA+X-ray, Bi-DTPA, X-ray and control group, 5 nude mice per group. Intratumor injection of Bi-DTPA (25μL 0.5mol/L, 299mg/ml) was adopted, and X-ray dose was 6Gy. After 4 groups were all correspondingly treated, tumors size were measured every two days, and weight of mice was also measured and recorded by photos during the 14-day treating process.

Tumor volume was calculated according to the following equation: V(cm³) = (length* width²)/2.

The second day after treatment, one mouse in each group was randomly selected and sacrificed for the tumor Hematoxylin-eosin (H&E) staining, Proliferating Cell Nuclear Antigen (PCNA) & TdT-mediated dUTP Nick-End Labeling (TUNEL) test, in order to examine the status of major organs by H&E staining.

Statistical analysis

All results are given as mean ± standard deviation, and were analyzed using SPSS 22.0 software (IBM, Armonk, NY, USA). Mann-Whitney U test and the students’-test were utilized for statistical evaluation. Differences were considered significant at P＜0.05.

Preparation and characterization of Bi-DTPA

As shown in Fig.1a, the synthetic product was a kind of white powder after lyophilization, with a productive rate of 93.01%±1.76. The result of FT-IR detection showed no absorption peak at 1733 cm^-1, 1700 cm^-1 and 1634 cm^-1, moreover the characteristic absorption peak of O-C-O (carboxylate radical) at 1597.8 cm^-1 disappeared, as shown in Fig.1b. Data of ¹H-NMR demonstrated as 4.23(s, 2H), 3.89-4.04(m,4H), 3.77(d, 6H), 3.27-3.33(m, 4H), 2.99-3.03(m, 2H) (Fig.1c). The result of HR-MS depicted that the peak at m/z 600.0716 represented Bi-DTPA, which indicated molecular weight of Bi-DTPA (Fig.1d). Data mentioned above together have proved the successful synthesis of Bi-DTPA.

CT-imaging of breast cancer tumor with Bi-DTPA

After the drug was injected intratumorally, the tumor area enhanced rapidly and then the CT value gradually decreased with time. While the CT value at the bladder gradually increased at first and then decreased gradually, reaching a peak at 1 hour, which was delayed compared with the peak value in the tumor area. The whole process The CT values of tumor and bladder in the Bi-DTPA group were higher than those in the I 370 group (Fig.2). There were no adverse reactions in the whole mice.

In vitro safety of Bi-DTPA

The results of CCK-8 assay showed that when the concentration of Bi-DTPA was 0.5 mg/mL and below, the cell viability could be maintained at more than 90%, and at 1 mg/mL, it could be maintained at more than 80% (compared with the 0 mg/mL group, P<0.05) (Fig.3).

In vitro treatment

Colony survival analysis

The results of clonal survival analysis showed that under the exposure of 6Gy irradiation dose or even more, cell proliferation was dramatically decreased (*P<0.05). After simulated calculation on software, the sensitivity enhancement rate turned out to be SER=1.51, as shown in Fig.4 (the fitting degrees of both survival curve were satisfying, with the values of R² in X-ray group and Bi-DTPA+X-ray group being 0.9979 and 0.9975, respectively), which illustrated the significant ability of Bi-DTPA in sensibilization of radiotherapy.

Therefore, the following experiments selected Bi-DTPA concentration of 0.5mg/mL and X-ray dose of 6Gy.

CCK8 assay

The efficacy of in vitro treatment was detected by CCK8 assay. Data in Fig.5 demonstrated that cell viability decreased from 88.28% to 53.88% when radiotherapy was accompanied by Bi-DTPA(P<0.05), while cells in control group or treated with equal radiotherapy only or Bi-DTPA only showed better viability. (*P<0.05, compared with the control group)

The detection of ROS

We then detected the change in ROS production by DCFH-DA. The results showed that cells in Bi-DTPA+X-ray group presented the brightest fluorescence which indicated the production of obviously increased ROS; although ROS was also detected in cells in X-ray group, it appeared to be weaker than that in Bi-DTPA+X-ray group (P<0.05); in Bi-DTPA group and the control group, no obvious production of ROS was detected (Fig.6, *p<0.05, compared with the control group). These results proved that Bi-DTPA itself could not cause the production of ROS in cells, but more ROS was produced when cells were treated with both Bi-DTPA and X-ray, so Bi-DTPA was safe and effective in killing tumor cells.

The detection of DNA double strand break

The detection of DNA double strand break was carried out using was carried out by using antibody labeled by fluorophores to bind to the break site. Observed by laser scanning confocal microscope, DNA damage in Bi-DTPA +X-ray group was severe, according to the increased green fluorescent spot number and signal intensity; cells treated by X-ray group only showed less fluorescent spots and weaker signal intensity(P<0.05); no obvious damage was observed in Bi-DTPA group and the control group (Fig.7, *P< 0.05, compared with the control group).

Flow cytometry for the detection of cell apoptosis

Apoptotic cells were detected by flow cytometry. The results demonstrated that cell viability in Bi-DTPA+X-ray group was only 51.29%, while it was 85.64% and 89.41% in X-ray group and Bi-DTPA group, respectively (Fig.8). More cells in Bi-DTPA+X-ray group suffered from apoptosis than in other groups, indicating promoted tumor-killing ability by the collaboration of Bi-DTPA and X-ray.

In vivo treatment

Referring to the growth curve of tumor volume after treatment, Bi-DTPA+X-ray exhibited the most apparent tumor inhibition effect; though X-ray suppressed tumor growth to some extent, it was less effective; tumors in Bi-DTPA group and the control group were not effectively suppressed and showed vigorous growth, as shown in Fig.9a, 9b (*P<0.05, **P<0.001), indicating the promoted tumor-inhibiting capacity of Bi-DTPA+X-ray treatment. Data in mice weight curve showed that no significant difference was observed in body weight of mice in each group during treatment (Fig.9c).

Mice in each group were randomly chosen and sacrificed on the second day after treatment, from which the tumor tissues were collected and detected by H&E staining. The results showed that more nucleus shrinkage was observed in Bi-DTPA+X-ray group than other groups (with more pink area was observed under microscope), showing obvious damages in tumor tissues; PCNA detection was performed on these slices, and the results indicated that the proliferation of cells in Bi-DTPA+X-ray group was significantly depressed compared to other groups; there were more apoptotic cells in Bi-DTPA+X-ray group than that in other groups, as presented by the results of TUNEL test (Fig.10). H&E staining of other normal tissues in all groups showed no obvious damage (Fig.11).

With their applaudable irradiation attenuation capacity, high z number metallic elements can be utilized to enhance CT imaging. Meanwhile, their ability of strengthening ionizing radiation allows them to produce more ROS and thus realize the sensitization of radiotherapy. Nanomaterials constructed on the basis of these high z number elements have been thriving, but the corresponding applications are confined due to their shortcomings of complicated synthesis, high cost, long-term retention in vivo and degradation resistance(8–10). To address these shortcomings, in this study, we synthesized Bi-DTPA with low-cost and fine biocompatibility by a simple one-pot reaction. By FT-IR detection and ¹H-NMR detection, we have observed the disappearance of original functional groups and structural changes in raw ingredients, and the new corresponding structures formed in the final product, along with the estimation of molecular weight by HR-MS, the successful synthesis of Bi-DTPA was verified.

The synthesized Bi-DTPA possesses great potential in CT imaging as a result of sound X-ray attenuation capacity(7). We have observed satisfying outcome of CT imaging of breast cancer after mice accepted intratumor injection of Bi-DTPA. No adverse effect was observed during the whole treatment process, and the drug was gradually metabolized out through bladder over time, with a higher signal intensity than that in I 370 group. Altogether, these phenomenons indicated that it was safe and feasible to apply Bi-DTPA in CT imaging of breast cancer, moreover, Bi-DTPA could be a better choice since it showed better imaging effect than the traditional CT imaging contrast agent.

The therapeutic mechanism of radiotherapy’s effectiveness is based on inducing DNA damage in tumor cells by ROS generated from ionizing radiation, thereby inhibiting tumor growth(11). However, the hypoxia tumor microenvironment constrains its effect, which can not be addressed by uplifting dose since the side effects do severe harm to normal tissues(12, 13). So we tried to break this dilemma by enhancing the sensitivity of tumor region towards radiotherapy so as to lowering down dosing. In vitro experiments, we found that with Bi-DTPA, the proliferation ability and cell viability of 4T1 cells after radiotherapy were significantly decreased, the level of ROS generated in the cells was significantly increased, the degree of DNA damage was more serious, the cell survival rate was lower, and the cell viability was significantly reduced. More apoptosis, indicating that Bi-DTPA can promote the generation of more ROS under the action of X-rays, more damage to cell DNA, and achieve the effect of sensitizing radiotherapy.

In the meantime, we further assessed its radiotherapy sensitization ability in vivo by establishing tumor-bearing mouse model. The results demonstrated that, collaborating with Bi-DTPA, radiotherapy achieved efficient inhibition of tumor growth, destruction of tumor tissues, decrease in tumor proliferation, severer apoptosis and undamaged normal tissue, indicating that Bi-DTPA promoted tumor-killing effect and inhibited tumor growth, while normal tissues were unharmed, which proved that safe and effective therapeutic effect was accomplished by the addition of Bi-DTPA.

According to results mentioned above, Bi-DTPA, which had the characteristics of CT imaging and radiotherapy sensitization, was expected to play a great potential in the application of integrated diagnosis and treatment. However, tumors in this experiment were not completely eliminated, which might be due to insufficient one dose treatment or the limitation of single Radiotherapy, and this problem needed to be further investigated.

To sum up, in this study, we synthesized Bi-DTPA by the simple one-pot method. The synthesized Bi-DTPA was advanced and applicable in CT imaging and possessed sound capacity in RT sensitization which can effectively kill tumor cells and inhibit tumor proliferation. Therefore, it has great potential in the future application of tumor integrated diagnosis and treatments.

CT: Computed Tomography; Bi-DTPA: Bi-Diethylene triamine pentaacetic acid; FT-IR: Fourier transform infrared; 1H-NMR: 1H-Nuclear magnetic resonance; HR-MS: High resolution mass spectrometry; CCK-8: Cell Counting Kit-8; ROS: Reactive oxygen species; DNA: Deoxyribonucleic acid; RT: Radiation therapy; PBS: Phosphate buffered saline; OD: Optical density; SER: Sensitivity enhancement rate; DCFH-DA: 2',7'-Dichlorodihydrofluorescein diacetate; CLSM: Confocal laser scanning microscope; PBST: Phosphate buffered saline tween-20; DAPI: 4',6-diamidino-2-phenylindole; PCNA: Proliferating Cell Nuclear Antigen; TUNEL: TdT-mediated dUTP Nick-End Labeling; H&E: Hematoxylin-eosin staining.

Ethics approval and consent to participate

The animal experiments in this study have been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was funded by the National Natural Science Foundation of China (Grant Numbers 82071926, 81630047).

Author Contributions

Yi Zhang: Conceptualization, Data curation, Investigation, Validation, Visualization, Writing – original draft; Dang Liu: Data curation, Formal analysis, Investigation, Writing – original draft; Bin Qiao: Conceptualization, Investigation, Writing – review & editing; Yuanli Luo: Methodology, Investigation, Validation; Chao Yang: Project administration, Resources; Liang Zhang: Conceptualization, Writing – review & editing; Yang Cao: Project administration, Writing – review & editing; Haitao Ran: Funding acquisition, Supervision, Writing – review & editing.

Acknowledgements

Authors are thankful to the National Natural Science Foundation of China support of this study. We are also grateful to Oncology Department of the First Affiliated Hospital of Chongqing Medical University for their technical supports

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No competing interests reported.

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Research on CT Imaging and Radiotherapy of Breast Cancer with Bi-DTPA

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Abstract

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Background

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