Therapy of cervical cancer using 131 I-labeled nanoparticles

Materials

Liposome RGD-bovine serum albumin (BSA)-polycaprolactone (PCL) was synthesized and provided by Professor Chang Jin, Tianjin University. ¹⁰ Tyrosine peptide chain (TPC) and a peptide with a random sequence (random peptide chain [RPC]) were purchased from the Chinese Peptide Company (Hangzhou, China). The TPC and RPC sequences were YYYHYYKYYRYHYYYRYYHYYKY and HPLGSPGSASDLETSGLEEQR, respectively. ¹³¹I-Na was purchased from the China Institute of Atomic Energy, Beijing, China.

Cell lines

The HeLa cervical cancer cell line was cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (GIBCO Cell Culture [subsidiary of Invitrogen Corp.], Carlsbad, CA, USA), and 1% penicillin/streptomycin (Beijing Dongsheng Tebo Technology Company, Beijing, China). Cells were stored in a humidified atmosphere containing 5% CO₂ buffered with ambient air at 37 °C. The RGD peptide was overexpressed in HeLa cells.^11,12

¹³¹I labeling

The liposomes and TPC were labeled with ¹³¹I using the chloramine-T method. ¹³ The ¹³¹I-liposomes and ¹³¹I-TPC were purified using centrifugal filtration (Amicon Pro Purification System, Merck Millipore, Billerica, MA, USA) to remove the remaining ¹³¹I-Na. The dose of radioactivity, radiochemical purity, and the rates of incorporation of radioactivity into the products were determined using paper chromatography. ¹⁴ The developer was prepared using a 3:2:5 ratio of butyl alcohol:2 ethyl alcohol:5 ammonium hydroxide.

Preparation of ¹³¹I-labeled liposomes

¹³¹I-labeled liposomes were prepared as follows: ¹⁵ The RGD-targeting liposome encapsulating an RPC was directly labelled with ¹³¹I on the surface and named ¹³¹I-RGD-L. The RGD-targeted liposomes that encapsulated ¹³¹I-TPC were named RGD-¹³¹I-TPC-L. The structures of radionuclide nanoparticles are shown in Figure 1a-1b. The emulsion solvent evaporation method was performed to encapsulate TPC or RPC into liposomes. The procedure was as follows: 2 mg of liposomes were dissolved in 2 mL of deionized water and then 300 µL of trioxymethylene solution was added. Immediately after the trioxymethylene and liposomes were sonicated (SCIENTZ-IID; Xin Zhi Biotechnology Co., Ltd., Zhejiang, China), the mixture was vibrated and centrifuged. The quantity of bound polypeptide was measured using a UV-visible spectrometer (UV-2450; Shimadzu Corporation, Beijing, China) at 220 nm.

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Figure 1.

Characteristics of ¹³¹I-labeled nanoparticles

Cellular uptake of ¹³¹I

The RGD-BSA-PCL liposome was labeled with fluorescein isothiocyanate (FITC), ¹⁰ which can be easily imaged using confocal laser scanning microscopy (CLSM). The cellular uptake, targeting, and therapeutic effects of RGD-BSA-PCL are published. ¹⁶ CLSM was used to evaluate the uptake by HeLa cells of FITC-labeled liposomes that encapsulated TPC or RPC.

Cellular uptake of ¹³¹I-RGD-L and RGD-¹³¹I-TPC-L

Cervical cancer-derived HeLa cells were seeded in 6-well plates and cultured with 1.85 MBq/mL of radioactive nanoliposomes. ¹⁶ Cells were washed, lysed, centrifuged, and counted at different times. Radioactivity was measured using a γ-counter (LKB Gamma Counter 1261; LKB Instruments, Mount Waverley, Australia). All of experiments were performed in triplicate.

Apoptosis assays

The MTS assay was conducted according to a published procedure ¹⁷ to calculate the half-maximal inhibitory concentration (IC₅₀) of 131-labelled liposomes after 24 hours. ¹⁸ Flow cytometry was performed according to the IC₅₀ value. HeLa cells were seeded into a 6-well plate and incubated with 18.7 MBq/mL of RGD-¹³¹I-L, RGD-¹³¹I-TPC-L, or Na¹³¹I for 24 hours, washed twice, lysed, and centrifuged. The cells were then incubated with FITC-Annexin-V and propidium iodide. Flow cytometric analysis (BD Biosciences, San Jose, CA, USA) was performed.

Mouse model

BALB/c mice (female, aged 4–5 weeks, 15 to 20 g) were purchased from the Beijing Experimental Animal Center of Peking Union Medical, China. Mice were kept under specific pathogen-free conditions in the Laboratory Animal Center of Tianjin Medical University, China. All animal studies were conducted in accordance with a protocol approved by the Tianjin Medical University General Hospital Ethics Committee. The animal experiment guidelines were followed according to the regulations of Swiss veterinary law.

HeLa cells were subcutaneously injected into the right flank. When the tumor volume reached 0.7 cm in diameter, the mice were randomly divided into three groups of five mice each. According to the principles of the human thyroid perchlorate discharge test, 0.05 mg/mL sodium perchlorate was added to the drinking water of all mice 1 day before injection of the radionuclide. ¹⁹ The mice were killed when neurological symptoms appeared or a loss of 20% of original body weight.

Distribution of ¹³¹I in mice

Mice were sacrificed at 24, 48, and 72 hours postinjection. Heart, spleen, liver, kidney, and tumor samples were collected for weighing, and radioactivity was measured using a γ-counter. The percentage injected dose (ID) per gram of tissue was calculated. ²⁰

Radioiodine therapy

When a tumor’s diameter reached 0.7 cm, 74 MBq of ¹³¹I-labeled liposomes, ¹³¹I-Na, or an equivalent volume of normal saline was injected into the tumor. During treatment, the animal’s body weight and in vivo tumor growth were measured. Body weights and tumor volumes (volume = 1/6 × π × length [cm] × width [cm] × height [cm]) were measured. Antitumor activity was evaluated by determining the relative tumor increase rate (T/C) as follows: T/C (%) = TRTV/CRTV x 100. Therapeutic efficiency was evaluated as follows: T/C >40% indicated no therapeutic effect, whereas T/C ≤40% (P<0.05) indicated a positive therapeutic effect. The tumor inhibition rate (TIR) was calculated by comparing the weights of the transplanted tumors of the treatment group with those of the negative control group as follows: TIR (%) = (1-mean weight of the transplanted tumor of the treatment group per mean weight of the transplanted tumor of the negative control group) × 100. ²¹ The therapy groups were followed for 30 days after injection and then killed.

SPECT/CT whole-body imaging of mice

To assess the organ localization of ¹³¹I, single-photon computed tomography/computed tomography (SPECT/CT) (Discovery VH 670; GE, Chicago, IL, USA) was performed. Mice from the three treatment groups, except for the normal saline group, received an intratumor injection of 74 MBq (740 MBq/mL) of radioliposomes and Na¹³¹I, respectively. ²²

Histopathology studies

When the radioiodine therapy experiment concluded, normal tissues including the heart, liver, spleen, and kidney were isolated. Sections were stained with hematoxylin and eosin for histopathological analyses. The histopathological changes of the tissue were examined using light microscopy, ×40 magnification (Olympus Th4-200; Olympus Optical Company, Beijing, China ).

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics, version 22.0 (IBM Corp., Armonk, NY, USA). Data were evaluated using the Student t test or one-way ANOVA. The difference between the two groups was considered significant when the P<0.05 indicated a significant difference between two groups.

Characteristics of nanoparticles

Dynamic light scattering measurements showed that the diameters of RGD-TPC-L and RGD-L were not significantly different (264.7 ± 17.6 nm and 275.4 ± 18.7 nm, respectively) (Figure 1c). The polydispersity index was 0.17, and the zeta potential was −41.30 mV.

Encapsulation of TPC and RPC into liposomes

The amounts of TPC or RPC encapsulated into liposomes using 100 µg, 200 µg, 300 µg, 500 µg, and 1000 µg of starting material were not significantly different. Their respective amounts (µg) encapsulated into liposomes were as follows: 75.9 ± 10.91 vs 69.1 ± 12.88, 123.7 ± 25.1 vs 133.2 ± 34.5, 158.9 ± 33.94 vs 146.7 ± 36.2, 192.8 ± 31.3 vs 189.0 ± 41.26, and 219.6 ± 51.4 µg vs 234.7 ± 45.87, respectively. UV spectroscopy showed that the amount of each encapsulated polypeptide was 500 µg.

¹³¹I-labeling

The maximum yields of radioactivities of 1 mg of liposomes labeled with ¹³¹I-RGD-L and RGD-¹³¹I-TPC-L were 170.2 ± 50.3 MBq and 699.3 ± 79.6 MBq, respectively (P<0.05). The efficiencies of labeling RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L were 85.7 ± 7.4% vs 72.5 ± 9.8%, respectively (P<0.05). The radiochemical purities of ¹³¹I-TPC, RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L were not significantly different (96.5 ± 1.9%, 92.0 ± 2.6%, and 94.8 ± 1.7%, respectively (Figure 1d).

Internalization of liposome-encapsulated polypeptides

Confocal microscopy was used to evaluate the internalization of liposomes in HeLa cells. Liposomes encapsulating TPC and RPC were significantly internalized in HeLa cells and exhibited strong green fluorescence (Figure 1e–h). The fluorescence intensities of FITC-L-TPC and FITC-L-RPC were similar after incubation with HeLa cells for 2 hours and 6 hours.

Intracellular retention of ¹³¹I

The retention times of ¹³¹I by nuclear liposomes in HeLa cells are shown in Figure 2a. RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L exhibited increased intracellular retention, which reached maximum levels at 6 hours. The CPM/10⁵ cells of RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L were 138 763.6 ± 7421.9 vs 125 692.1 ± 9 430.3, respectively. However, the radioactivity of the Na¹³¹I group was relatively low.

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Figure 2.

The characteristics and treatment of ¹³¹I-labeled nanoparticles

Apoptosis assay

The MTS assay determined that the IC₅₀ values of ¹³¹I- RGD-L and RGD-¹³¹I-TPC-L were approximately 1.85 MBq/mL. The rates of late apoptosis measured using flow cytometry (Annexin V+/PI+) of HeLa cells incubated with ¹³¹I-RGD-L, RGD-¹³¹I-TPC-L, and Na¹³¹I were 10.3 ± 0.67%, 11.9 ± 0.46%, and 5.1 ± 0.38%, respectively (P<0.05) (Figure 2d–2f). RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L were more cytotoxic than Na¹³¹I and normal saline. There was not a significant difference between the cytotoxicities of RGD-¹³¹I-TPC- L and ¹³¹I-RGD-L.

Biodistribution analysis

Comparisons of the biodistribution data of RGD-¹³¹I-TPC-L, ¹³¹I-RGD-L and Na¹³¹I in the tumor and normal tissues of mice were measured according to γ-counts. The uptake values of RGD-¹³¹I-TPC-L, ¹³¹I-RGD-L 24, 48, and 72 hours postinjection were 25.7 ± 5.13% ID/g, 17.4 ± 3.43% ID/g, 8.7 ± 2.64% ID/g versus 19.7 ± 4.66% ID/g, 9.9 ± 2.11% ID/g, and 3.6 ± 1.03% ID/g, respectively, and were significantly higher compared with those of the Na¹³¹I group (0.41 ± 0.12% ID/g, 0.31 ± 0.08% ID/g, 0.29 ± 0.10% ID/g (Figure 2g–2j). However, the uptake of all radionuclides was low and differed slightly among organs such as the heart, liver, spleen, and kidneys at all times.

Radiotherapy

The differences between in vivo and in vitro experiments required further evaluation of radioactive iodine treatment of cervical cancer using two types of nuclear liposomes. Therefore, a mouse xenograft model of cervical cancer was established. The average tumor volumes of the RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L groups were smaller compared with those of the Na¹³¹I and normal saline groups (Figure 2b, 2c). The tumor volumes of the RGD-¹³¹I-TPC-L, ¹³¹I-RGD-L groups were significantly reduced compared with that of the Na¹³¹I or normal saline groups. On day 15, compared with the other groups, RGD-¹³¹I-TPC-L exhibited improved tumor inhibition, and the differences in the declining values of the T/C were significant (P < 0.01) (Figure 2l). The TIR values of the RGD-¹³¹I-TPC-L group were higher compared with those of the other groups, and the differences among these groups were significant (P < 0.01) (Figure 2k). These results show that RGD-¹³¹I-TPC-L had the best therapeutic effects in our mouse model of cervical cancer.

Histopathological analysis

To test the potential toxicity of liposomes in experimental mice, hematoxylin and eosin–stained sections of the heart, liver, spleen, and kidneys were examined after radiotherapy. The histopathological images of the major organs are shown in Figure 2g–2j. Significant pathological changes in vital organs were not observed in nude mice following treatment using RGD-¹³¹I-TPC-L, ¹³¹I-RGD-L or Na¹³¹I, indicating the limited toxicity of ¹³¹I-labeled liposomes. Pathological examination revealed degeneration and necrosis of tumor cells in groups injected with RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L.

SPECT/CT imaging

RGD-¹³¹I-TPC-L, ¹³¹I-RGD-L, and Na¹³¹I were injected into the tumors. Representative SPECT/CT images are shown in Figure 2l–2n. The Na¹³¹I group emitted a weak signal from the xenografted tumor on the day of injection. There was no significant subsequent uptake of ¹³¹I uptake into the xenografted tumor (Figure 2l). In contrast, the RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L groups had higher accumulations of ¹³¹I in the region of the xenografted tumors compared with the Na¹³¹I group. The levels of radioactivity emitted by RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L were detectable 20 days after injection, indicating that the RGD-¹³¹I-TPC-L and ¹³¹I-RGD-L groups retained radioactivity significantly longer than the Na¹³¹I group.