Octreotide acetate-templated self-assembly Pt nanoparticles and their anti-tumor efficacy

Abstract

Platinum nanoparticles (PtNPs) were assembled in a chain-like structure by activating chemical groups of the octreotide acetate (AOC) template. Tumor-bearing mice were inoculated with cervical carcinoma cells, and then treated with a low dose of AOC-PtNPs (AOC-PtNPs-L), a high dose of AOC-PtNPs (AOC-PtNPs-H), sterile physiological saline and cyclophosphamide. The results suggested that tumor inhibition rates of cyclophosphamide, AOC-PtNPs-L and AOC-PtNPs-H were 87.0%, 38.3% and 42.5%; and the apoptosis rates of the tumor-bearing mice were 30.95%, 23.41% and 26.64%, respectively. More importantly, the histopathological study results implied that AOC-PtNPs had no toxicity or side-effects on liver and kidney tissues, but obvious inhibitory effects on tumors. In addition, MTT assay results showed that the as-prepared AOC-PtNPs had a higher inhibition rate on Hela cells than that of AOC or PtNPs alone. Therefore, AOC-PtNPs have great potential as anti-tumor drugs for cancer therapy in the future.

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Introduction

Cervical carcinoma, which is one of the main causes of cancer-related deaths in females, arouses great concern, and it is also the second most common cancer among women. Approximately 83% of cases occur in developing countries, especially in South America, Asia and Africa, accounting for 15% of all cancers in women.¹ So far, most treatments of cervical cancer derive from studies including surgery, radiation and chemotherapy. Among the above therapeutic treatments, chemotherapy is the foremost remedial approach for the treatment of localized and metastasized tumors.² For chemotherapy, agents such as doxorubicin, cyclophosphamide or combination drugs have been widely used in cervical carcinoma treatment and they ultimately improve quality of life.³ However, these agents also show unexpected toxicity and serious side-effects. A main obstacle for successful chemotherapy is the resistance of cancer cells to therapeutic drugs and the destructive actions of these drugs to normal cells, tissues, and organs.⁴

Platinum compounds such as cisplatin and carboplatin have been widely applied in cancer chemotherapy.^5,6 Also other platinum compounds are under investigation for anti-cancer treatment, such as ormaplatin and oxaliplatin.⁷ In addition to platinum compounds, derivatives of other metals are being investigated for their anti-tumor properties e.g. titanocene.⁸ Cisplatin is used in the treatment of a number of cancers, but its applicability is still limited to a relatively narrow range of tumors. In comparison to cisplatin, carboplatin has lower toxicity which has been the advantage that enabled it to achieve worldwide approval and use. Unfortunately, carboplatin is still only active in the same range of tumors as cisplatin and is still administered intravenously.

Currently, cancer therapy is a multidisciplinary challenge requiring close collaboration among clinicians, biologists, materials scientists and chemists. The ultimate goal of cancer therapeutics is to increase the survival time and the quality of life of the patient by reducing the systemic toxicity of chemotherapy.⁹ At present, octreotide acetate is used as an anti-tumor drug for clinical treatment.^10–12 Octreotide, as a hydrosoluble spheroprotein, is composed of eight amino acids, containing amines amidogen, and a hydroxyl disulfide bond, which is liable to combine with ions. Hence, an octreotide template is prone to combine with platinum to form nanoparticles.¹³ Moreover, nano-sized platinum particles are used in multiple biomedical applications such as thermotherapy, radiation and catalysis. In our previous study, the combination of octreotide acetate with platinum nanoparticles is available.¹⁴ In this study, we mainly investigate the anti-tumor activity and cytotoxicity of a complex of octreotide acetate-templated Pt nanoparticles.

Materials and methods

Materials and animals

U14 cervical carcinoma cells were purchased from the Institute of Meteria Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College. Kunming mice were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (China).

Thirty-two healthy female Kunming mice (7–8 weeks old), which were in a good state and at similar weights (about 20 g), were fed for 7 days in a temperature-controlled mouse room. The mice were maintained under standard conditions of temperature (22–25 °C), a humidity of 60 ± 10% and a 12 h light/dark cycle. The animals were fed with a standard diet of mouse chow and water was allowed ad libitum. All experimental animal protocols were approved and in accordance with the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care).

Preparation of AOC-PtNPs

The procedures for preparing AOC-PtNPs were the same as in our previous work. 0.5 mg of AOC was dissolved in 1 mL of acidic solution (pH 2.0) to obtain 0.5 mM of AOC acidic solution. Then, 200 µL of the AOC acidic solution was mixed with a prepared aqueous solution of PtCl₄ (5 mM, 200 µL) and co-incubated in a shaking bath (13 °C, 130 rpm) for 24 h. Finally, the incubation solution was reduced by adding fresh DMAB solution (25 mM, 150 µL) drop by drop to prepare AOC-PtNPs. In the reduction process, the color of the solution changed from faint yellow to grayish-black, and there was no precipitation in the solution.

Characterization of AOC-PtNPs

Transmission electron microscopy (TEM). The morphologies and sizes of the samples were observed using TEM operated at 80 kV. TEM samples were prepared by placing drops of the AOC-PtNP suspension onto carbon-coated copper grids. To investigate the stability of the as-prepared AOC-PtNPs, the AOC-PtNP suspension was centrifuged at 10 000 rpm for 5 min and washed with double-distilled water three times. Finally, the black precipitate was dissolved with double-distilled water and the TEM test was carried out.

Particle size and zeta potential. Malvern Zetasizer ZS (Malvern Instruments, UK) was used to measure the sizes and surface zeta potentials of the prepared AOC-PtNPs. The mean diameters and zeta potentials of the AOC-PtNPs were determined by dynamic light scattering (DLS) and electrophoretic mobility measurements, respectively. The zeta potential of an AOC solution at pH 2 was also measured. All characterization measurements were repeated three times at 25 °C.

AOC loading on platinum nanoparticles. To investigate the loading efficiency and loading capacity of AOC on Pt nanoparticles, the prepared AOC-PtNP solution was centrifuged at 12 000 rpm for 10 min and washed with double-distilled water two times in order to remove free AOC molecules. After that, the loading capacity and efficiency were evaluated by measuring the relative intensity of the UV-vis absorption of the standard AOC solution and supernatant at 277 nm, respectively.

U14 mouse cervical carcinoma-bearing solid tumor model. To investigate the anti-tumor efficacy of AOC-PtNPs in vivo, we established the cervical carcinoma xenograft animal model through transplantation of the U14 cells into female Kunming mice. Before the solid tumor model was established, all the tested mice were fasted and given free access to water for 12 h. The imbibed ascites from celiac tumor-bearing mice were diluted with sterile physiological saline (1 : 3, v/v). The density of the cell suspension was approximately adjusted to 2 × 10⁶ mL⁻¹. All these procedures were performed in sterile conditions. Then U14 mouse cervical carcinoma cells were transplanted subcutaneously into the oxter of the right forelimb of the mice. The successful ratio of cell inoculation in the experiment was up to 100%. Various drugs in different doses were offered to the mice after 24 h of inoculating the tumor cells.

Groups and treatment scheme of experimental animals. The mice were randomly divided into 4 groups with each group containing 8 mice. The mice in each group were marked after being weighed. The treatment methods for each group were as the following description: mice in the negative control (NC) group were injected intraperitoneally with sterile physiological saline; the mice in the positive control (PC) group were injected intraperitoneally with 5 mg mL⁻¹ cyclophosphamide solution once a day for a total of 10 days; and mice in the AOC-PtNPs-L and AOC-PtNPs-H groups were injected intraperitoneally with 0.3 and 0.6 mg mL⁻¹ AOC-PtNP solution once a day for a total of 10 days, respectively.

Effects of AOC-PtNPs on the body weight of the mice. The body weight of the mice was monitored every day after drug treatment. Ten days later, all of the mice were treated with aether anesthesia and sacrificed. The tissues (liver, kidney and tumor) were removed and flushed with 0.9% NaCl (w/v) three times to thoroughly remove the remaining blood.¹⁵ Afterwards, all of the tissues were weighed separately and stored in formalin.

Determination of tumor inhibition rate. The tumor tissues were removed and weighed after visual inspection. The tumor inhibition rate was calculated accordingly: tumor inhibition rate = (mean tumor weight of NC − mean tumor weight of treatment group)/mean tumor weight of NC × 100%.

Annexin V-FITC/PI double staining assay. To understand the specific mechanism underlying the enhanced anti-tumor efficacy of AOC-PtNPs, cell apoptosis was further analyzed using FCM. Apoptosis was detected by staining with annexin V and PI labeling, since annexin V could identify the externalization of phosphatidylserine during the apoptosis process.¹⁶ A series of morphology changes can be observed in the apoptosis procedure, and transformation of the cell membrane is one of the characteristics in the earlier stages. An annexin V-FITC/PI kit was chosen to determine the percentage of apoptosis. The cells were collected by centrifugation and stained for 5 min at room temperature with annexin V-FITC/PI double staining. The cells were then analyzed by the flow cytometer (Beckman Coulter) using the manufacturer’s analysis software. Approximately 10 000 counts were made for each sample.

Histopathological study. The liver, kidney and tumor tissues were carefully removed and fixed in 10% neutral buffered formalin for 24 h. All tissues were dehydrated in a series of ethanol, cleared in xylene and embedded in paraffin wax. Sections of 3–5 µm thickness were obtained using a rotator microtome (Leica Company, Germany). The sections attained were stretched on slides, subjected to paraffin removal and rehydrated in a regressive series of ethanol and stained with hematoxylin eosin.¹⁷ It is important to point out that all glass slides used in experiments were pretreated with poly-L-lysine solution or APES so that sections could adhere to the slides tightly. All sections were observed with a light microscope and photographed for histological examination.

MTT assay. The classical MTT method was utilized to detect the inhibition effects of the prepared AOC-PtNPs on Hela cells in vitro. For comparison, separate AOC and PtNPs served as the control groups. In this study, the PtNPs were obtained by ultracentrifugation of AOC-PtNPs at 41 000 rpm for 1 h, not by simply reducing a PtCl₄ solution with NaBH₄. The above samples were filtered through a sterile 0.22 µm filter membrane, and then they were diluted with phosphate-buffered saline to obtain various drug concentrations (10 µg mL⁻¹, 50 µg mL⁻¹ and 100 µg mL⁻¹).

Briefly, Hela cells were grown in DMEM with 10% fetal bovine serum under sterile conditions with 5% of CO₂ at 37 °C, and according to the growth conditions of the cells, subcultures were carried out at intervals of 2–3 days. Ultimately, Hela cells in the logarithmic phase were used for the following MTT assay. Adequate numbers of cells were plated in each well of a 96-well plate in 0.1 mL of complete culture medium and allowed to attach for 24 h. After that, each sample was transferred in triplicate (100 µL per well) into the appropriate well of the culture dish. The drug-treated cells were incubated for 24 hours under the conditions mentioned above, and the inhibition effect of each sample on Hela cells was analyzed using the MTT agent. 24 hours later, the medium was discarded and the cells were incubated with 200 µL of fresh medium containing 0.5 mg mL⁻¹ MTT for 4 hours. After removing the unreduced MTT, 150 µL of DMSO was added to each well to dissolve the formazan crystals. Finally, the absorbance of samples was measured by an ELISA reader (MK3, Thermo Co., USA) at 570 nm.

Statistical analysis. All the obtained data are expressed as mean ± standard error unless particularly outlined. Origin software (version 7.0) was utilized to deal with numeric plots. SPSS statistical software (version 13.0) was performed for statistical analysis. Statistical significance was assessed by a one-way analysis of variance test. Differences were considered statistically significant if P was <0.05.

Results and discussion

Morphological analysis of the prepared AOC-PtNPs

From Fig. 1, the chain-like structure of the as-prepared AOC-PtNPs can be clearly seen. There is a uniform distribution of the particles along the Pt nanochains. Almost all of the AOC was combined with the Pt particles, and no loose particles could be found, which indicates that the AOC molecules were used effectively to mostly form spheroidal Pt nanochains. In addition, the morphology of the sample washed by double-distilled water was still a chain-like structure, and there was no separation between Pt particles and AOC. Therefore, it can be concluded that the prepared AOC-PtNPs were stable in neutral conditions.

Fig. 1 TEM images of platinum nanochains produced using AOC as a bio-template at pH 2.0 ((A) unwashed; (B) washed).

Particle size and zeta potential

In this study, the zeta potentials were determined by electrophoretic mobility measurements. The surface zeta potentials of the single AOC solution (pH 2) and the prepared AOC-PtNPs can be seen in Fig. 2. The single AOC solution at pH 2 had a marked positive charge (7.64 ± 0.34 mV), which contributed to combining the Pt complex ion which had negative charges. The surface zeta potential of the prepared AOC-PtNPs was 21.63 ± 0.85 mV, indicating that the AOC molecules aggregated during the process of forming AOC-PtNPs. Positively charged AOC-PtNPs can be combined with the negative charges on the surface of the tumor cells. Hence, the prepared AOC-PtNPs tend to aggregate in the tumor cells and cause cell uptake, which can effectively inhibit the growth of tumor cells.

Fig. 2 Surface zeta potentials of AOC and AOC-PtNPs. Data represent the mean ± SE (n = 3).

The particle sizes were detected by DLS. The size distribution with the log-normal fitting curve of the prepared AOC-PtNPs is shown in Fig. 3. The average diameter of the AOC-PtNPs was around 174.18 nm. The result also indicated that the particle sizes of the prepared AOC-PtNPs were relatively uniform.

Fig. 3 Size distribution of the as-prepared AOC-PtNPs with the log-normal fitting curve.

Loading efficiency and loading capacity of AOC on PtNPs

As shown in Fig. 4, there is a significant reduction in the absorption intensity, indicating that the AOC has a good ability in loading on the platinum nanoparticles. The loading efficiency of the AOC on platinum particles is 79.7%, and the loading capacity is 408.7 mg of AOC per g of platinum nanoparticles.

Fig. 4 UV-vis absorption spectra of the standard AOC solution before and after loading on Pt nanoparticles.

Effects of AOC-PtNPs on the body weight of the mice

The body weight of the mice was monitored throughout the test period as an indication of the effects of the drugs. In the whole experimental process, all the mice were in a favorable condition; body weight increased gradually, and there were no deaths.

The results shown in Fig. 5 suggest that the body weight of the mice in the NC group was significantly lower than that in the other groups. Thus it can be seen that physiological saline had no inhibiting effect on the tumor, and the tumor had a great influence on the health status. However, the mice in the PC group grew normally and the increase in body weight was obvious.

Fig. 5 Trends in body weight of mice during the therapy process (values are expressed as the mean ± SE of 8 mice per group; * indicates P < 0.05 compared with the NC group).

Though the body weights of the mice in the drug-treated groups decreased on the second day, the overall trend was for their body weights to increase. Therefore cyclophosphamide, AOC-PtNPs-L and AOC-PtNPs-H all had significant inhibiting effects on tumors.

Inhibition effect of AOC-PtNPs on U14 cervical cancer solid tumors

After 10 days of drug treatment, it was observed that cyclophosphamide, AOC-PtNPs-L and AOC-PtNPs-H all exhibited pronounced inhibitory effects on transplanted tumors. The results are shown in Table 1.

Table 1 Inhibition effect of AOC-PtNPs on U14 cervical cancer solid tumors of mice^a

Groups	Tumor weight (g)	Inhibition rate (%)
a Note: values are expressed as the mean ± SE of 8 mice per group. NC, negative control group; PC, positive control group; AOC-PtNPs-L, mice treated with a low dose of AOC-PtNPs; and AOC-PtNPs-H, mice treated with a high dose of AOC-PtNPs. * indicates P < 0.05 compared with the NC group; # indicates P < 0.01 compared with the PC group.
NC	1.48 ± 0.11	—
PC	0.19 ± 0.01*	87.0*
AOC-PtNPs-L	0.91 ± 0.02*#	38.3*#
AOC-PtNPs-H	0.84 ± 0.06*#	42.5*#

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All data obtained were analyzed by one-way ANOVA; the results showed that the mean tumor weights of the treatment groups were notably lower than that of the NC group. The mean tumor weight of the NC group was 1.48 ± 0.11 g, whereas the values for AOC-PtNPs-L, AOC-PtNPs-H and PC groups were only 0.91 ± 0.02 g, 0.84 ± 0.06 and 0.19 ± 0.01 g, and the inhibition rates were 38.3%, 42.5% and 87.0% respectively (Table 1). The mean tumor weights of the AOC-PtNPs-L and AOC-PtNPs-H groups showed significant differences (P < 0.05) compared with the NC group. The results demonstrated that AOC-PtNPs suppressed U14 tumor growth prominently, and there were significant dose-dependent effects on tumor weights by AOC-PtNP treatment.

Measurement of apoptosis using FCM

Flow cytometry (FCM) can be used to analyze quantitatively and screen unicellular creatures and other biological particles at a functional level. There are many advantages in applications of FCM, such as rapid speed precision, great accuracy and so on; especially when it is used in the process of apoptosis, the detection accuracy of FCM is remarkable.¹⁸ In the measurement with annexin V, the kit can distinguish between annexin and PI: cell necrosis (cell death independent of apoptosis), located upper left (annexin V/PI = −/+); necrotic and late apoptotic cells, upper right (annexin V/PI = +/+); viable cells, lower left (annexin V/PI = −/−); and apoptotic cells in the early stage, lower right (annexin V/PI = +/−) (Fig. 6).

Fig. 6 Flow cytometry studies of apoptosis for each sample: (A) negative control (NC); (B) positive control (PC); (C) AOC-PtNPs-L; (D) AOC-PtNPs-H.

In the experiment with annexin V-FITC, there were significant differences (P < 0.05) in viability, apoptosis and necrosis with cyclophosphamide, AOC-PtNPs-L and AOC-PtNPs-H (Fig. 6).

Apoptosis rates of tumor cells of the tested mice in the treatment groups were 30.95%, 23.41% and 26.64%, respectively. The result suggested that apoptosis rate of the PC group was obviously higher than that of the AOC-PtNP groups, and it was presumable that both cyclophosphamide and AOC-PtNPs presented inhibitory effects on tumor growth; meanwhile, the effects of inducing apoptosis were distinct. Furthermore, a prominent increase was observed in the AOC-PtNPs-H group compared with the AOC-PtNPs-L group. Therefore, it was indicated that the anti-tumor activity of AOC-PtNPs was remarkable.

Effects of AOC-PtNPs on histopathology of the tumor-bearing mice

Effects of AOC-PtNPs on the kidney tissue structure of mice. The kidney tissues were immediately removed after the mice were sacrificed. It was observed that the color, gloss and texture of the kidney tissues were normal in the AOC-PtNPs-L and AOC-PtNPs-H groups. No histopathological changes were discovered in the sections of the kidneys. As shown in Fig. 7, the structure of the kidney tubules was very clear, and the shape of the glomerulus was quite regular. Moreover, nephrocytes were arranged closely. It was indicated that AOC-PtNPs caused no kidney injuries.

Fig. 7 Histological sections of the kidney tissue for: (A) negative control; (B) positive control; (C) AOC-PtNPs-L; and (D) AOC-PtNPs-H.

Effects of AOC-PtNPs on the liver tissue structure of mice. Histopathologically, the same normal phenomena were observed in the color and texture of the liver from the AOC-PtNPs-L and AOC-PtNPs-H groups. No histopathological alterations of liver tissue were observed. As shown in Fig. 8, hepatocytes were arranged closely and regularly. The volume of hepatic cells was normal and no dropsy was noted. Furthermore, the nucleolus was clear, and the central veins and hepatic lobule were obviously seen as well. It was indicated that both AOC-PtNPs-L and AOC-PtNPs-H didn’t cause damage to the livers of mice.

Fig. 8 Histological sections of the liver tissue for: (A) negative control; (B) positive control; (C) AOC-PtNPs-L; and (D) AOC-PtNPs-H.

Effects of AOC-PtNPs on the tumor tissue structure of mice. The tumor tissues dissected in the NC mice were fluidified severely, and were adjacent to pleura. Some tumor tissues even invaded the skeletal muscles, and represented diffusible growth and abundant blood vessels. Based on the comparison of those in the NC mice, tumor tissues in the other groups showed a clear borderline with other body parts. No invasion of the skeletal muscle was observed; tumor tissues could be easily dissected. The tumor body was tight and firm, the color was white and the blood vessels were not rich. In addition, the tumor weight in the treatment groups was lower than that of the NC group, according to the tumor weight of experimental mice.

Histological sections of the tumor tissue are shown in Fig. 9, and the tumor cells in the NC group were round or polygonal. The nucleolus was round or elliptical and mostly very large, and the nuclear envelope was clear. Rough chromatin and little cytoplasm were shown to be prominent. The number of tumor cells in the AOC-PtNPs-H group decreased obviously compared with that of the NC group. Malignant tumor cells presented heteromorphosis; apoptosis of many single dispersive tumor cells and tumor necrosis were seen. The adhesion of tumor cells was poor. It was also found that monolayer and necrotic cells appeared in tumor tissues of mice treated with AOC-PtNPs and cyclophosphamide. The results demonstrated that AOC-PtNPs indeed had anti-tumor activity.

Fig. 9 Histological sections of the tumor tissue for: (A) negative control; (B) positive control; (C) AOC-PtNPs-L; and (D) AOC-PtNPs-H.

Anti-tumor efficacy of AOC-PtNPs on Hela cells in vitro

From Fig. 10, it can be seen that all the samples show significant inhibition effects on Hela cells when their concentrations increase. At 10 µg mL⁻¹, inhibition rates were not significantly different between AOC and PtNPs, which were 27.23% and 31.63%, respectively. However, when the concentrations increased, inhibition rates of the PtNPs were significantly higher than those of AOC, though the inhibition rate of AOC also increased obviously. To sum up, inhibition rates of the AOC-PtNPs on Hela cells were higher than those of the separate AOC and PtNPs at any concentration. The results implied that the anti-tumor efficacy of separate AOC and PtNPs can be enhanced when they are combined into AOC-PtNPs.

Fig. 10 Inhibition effects of AOC, PtNPs and AOC-PtNPs on Hela cells. All values are expressed as the mean ± SE (n = 3) (* indicates P < 0.05 compared with AOC; # indicates P < 0.05 compared with PtNPs).

Discussion

Platinum, as a kind of noble metal, exhibits special efficacy particularly in cancer therapy when its size reaches the nanometer grade.¹⁹ In the case of the anti-tumor mechanism of PtNPs, earlier reports have emphasized that PtNPs inside the cell can be hydrolyzed to release Pt²⁺, which could block cell division by binding to DNA.²⁰ We speculate the involvement of an analogous mechanism in PtNP toxicity wherein released Pt²⁺ ions might halt DNA synthesis. In this sense, PtNPs can interact with DNA to produce Pt–DNA adducts through hydrolyzation.

The synthetic somatostatin analogue octreotide has been tested for efficacy in various solid tumours, including breast, prostate, colon, pancreas, and small cell lung carcinoma.²¹ Though the exact mechanism of the anti-tumor effect of octreotide on carcinoma described above has not been elucidated, octreotide has been suggested to be mediated by inhibiting endothelial cell proliferation, invasion and differentiation, and the production of vascular endothelial growth factor in tumor cells.^22–24

According to the findings of our experiment, the combination of octreotide with PtNPs has been shown to induce apoptosis in Hela cells, indicating that the linkage of PtNPs to octreotide does not change the active center of the octreotide. In relation to the anti-tumor mechanism of the AOC-PtNPs on cervical carcinoma, at the same time as octreotide inhibits the growth of Hela cells, Pt(0) on the surface of octreotide can be hydrolyzed to release Pt²⁺, which interacts with DNA to form Pt–DNA adducts, thereby blocking fundamental cellular processes.

However, the tumor inhibition rate of the AOC-PtNPs is lower than that of the PC group, which is consistent with the dose-dependent property of AOC-PtNPs. By comparing the treatment scheme of experimental animals, it can be seen that the dosage of the PC group is 5 mg mL⁻¹, more than 8 times as many as that of the AOC-PtNPs-H group and 16 times as many as that of the AOC-PtNPs-L group, on the premise that the total volume of each group is equal. Nonetheless, the tumor inhibition rate of the PC group is approximately only twice as high as that of the AOC-PtNP group. Comparing the AOC-PtNPs-H group with the AOC-PtNPs-L group, the dosage of the former is twice as much as that of the latter, and the tumor inhibition rate is increased by 4.2%. Therefore, it was indicated that the anti-tumor activity of the AOC-PtNPs was remarkable.

Histopathological studies have been utilized to help establish causal relationships between contaminant exposure and various biological responses.²⁵ The paraffin methodology requires less specialised equipment and could supply high quality slides adequate for many purposes.²⁶ Our histopathological results indicate that no changes of liver or kidney tissues take place after treatment with the AOC-PtNPs. The histopathological sections of tumor tissues in the tested groups demonstrate that the AOC-PtNPs have strong inhibitory effects on transplanted U14 cells. Our present investigations clearly reveal that AOC-PtNPs would serve as an anti-tumor drug for future tumor therapy.

Conclusions

According to the results, the AOC-templated Pt nanoparticles administered via injection significantly suppressed tumor growth compared with the NC group. In particular, a high dose of AOC-PtNPs remarkably enhanced the inhibitory ratio of tumor cells. Flow cytometry analysis revealed that a high dose and low dose of AOC-PtNPs suppressed tumor growth through induction of apoptosis. Furthermore, a high dose of AOC-PtNPs showed a higher apoptosis ratio. In addition, the findings from the histopathological observations implied that the AOC-PtNPs had no toxicity or side-effects on liver and kidney tissues, which also indicated that the AOC-PtNPs had an inhibitory effect on tumors, and the inhibition action of a high dose of AOC-PtNPs was particularly evident. MTT assay results suggested that the cell inhibition effects of the AOC-PtNPs on Hela cells were significantly stronger than those of the AOC or PtNPs alone. Therefore, this study can provide a basis for further investigation on the anti-tumor activity of AOC-PtNP formulation.

Acknowledgements

This work was financially supported by a research fund from the National Natural Science Foundation (no. 21476190), Hebei Province Key Basic Research Foundation (no. 15961301D) and Qinhuangdao Science and Technology Research and Development Project (no. 201202B029).

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Footnote

† Weili Xue and Xiaoning Zhao contributed equally to the paper.

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