Thyroid-stimulating hormone (TSH)-armed polymer–lipid nanoparticles for the targeted delivery of cisplatin in thyroid cancers: therapeutic efficacy evaluation

Abstract

Thyroid-stimulating hormone (TSH)-conjugated polymer–lipid hybrid nanoparticles (TPLHC) were developed for the targeted delivery of cisplatin (CDDP) in thyroid cancers. In the present study, polymer–lipid hybrid nanoparticles were conjugated to TSH, which will bind to the TSH receptor (TSHr) on the surface of thyrocytes. This delivery system was mainly designed to achieve high concentrations in thyroid carcinomas. The TPLHC exhibited excellent properties, which were attributed to their nanoscaled size of 160 nm with a narrow distribution to benefit EPR-based passive targeting. The polymer–lipid nanoparticle efficiently controlled the release of drugs in physiological conditions. The TSH-conjugated nanoparticles displayed higher cellular uptake in cells, which overexpress TSHr. TPLHC consistently showed high intracellular levels of CDDP in FTC-133 cancer cells. Specifically, the TSH-conjugated nanoparticles showed a significantly enhanced anticancer effect compared to the other groups across all time points and concentrations tested. In addition, a 4-fold higher accumulation of the TSH-conjugated NP was observed in tumors compared to the non-targeted NP in xenograft mice. Importantly, the TPLHC inhibited the growth of tumors more efficiently than the other formulations. This enhanced tumor inhibition might be attributed to the specific binding of TSH to the TSHr overexpressed in FTC-133 tumors. Taken together, the TSH-conjugated nanoparticles hold great potential to be an effective and safe nanoscale delivery system for the targeted therapy of thyroid cancers.

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Introduction

Thyroid cancer is a disease in which malignant cells arise from the thyroid gland. Thyroid cancer is a rare type of cancer that affects the thyroid gland.¹ It most commonly occurs in age groups between 35 and 40 years, and specifically women are more prone to develop thyroid cancer than men. In UK, thyroid cancers account for less than 1% of the total cancer cases registered. At present, either surgical removal of the thyroid gland or radiotherapy or a combination of surgery and radiotherapy is performed to treat thyroid cancers.² Chemotherapy is not a very common choice of treatment in the case of thyroid cancer; however, it has been reported that if cancer in the thyroid gland has been detected in the early stages then chemotherapy can be induced. One of the main obstacles in chemotherapy of the thyroid gland is the anatomical location of this gland in the body.^3,4 Therefore, a smart approach has to be adopted for the successful delivery of anticancer agents in this specific location.

Cisplatin (CDDP), which is a potent anticancer drug, is often indicated for the treatment of thyroid cancer.⁵ CDDP binds to DNA and causes DNA cross linking, which triggers cell apoptosis when repair proves unsuccessful. Despite its potent anticancer effect, the therapeutic effect of CDDP has been limited due to serious side effects such as nephrological and neurological toxicities.^6,7 Nephrotoxicity is a major cause of CDDP associated acute and chronic morbidity, whereas neurotoxicity is cumulative-dose dependent. Despite the associated side effects, one of the major concerns is the rapid inactivation of the drug through non-specific protein binding in the blood stream, which results in less therapeutic efficacy and undesirable side effects.^8,9 With regard to thyroid cancer, CDDP will be effective if it can be delivered directly into the thyroid tissue. Due to the fact that the administration of CDDP directly to the thyroid is difficult, intravenous administration is preferred wherein the drug will accumulate in the cancer over a period of time. One of the potential disadvantages of IV administration is the rapid inactivation of free drugs in the systemic circulation. Therefore, a novel concept revolving around increasing the therapeutic window, increasing the therapeutic efficacy, and reducing side effects should be adopted.

In this regard, the use of biodegradable polymeric nanoparticles such as poly(lactic-co-glycolic acid), PLGA NP is gaining importance due to its excellent features such as high loading capacity and controlled drug release pattern.^10,11 In particular, PEG-substituted polymers offer stearic protection to the carrier system in the systemic circulation.¹² However, polymeric NP often show limited systemic stability and circulation half-lives.¹³ Therefore, we have combined the benefits of polymers and lipids and made a fusion NP.¹⁴ The fusion NP is a simple and single step based efficient formulation strategy. In this case, PLGA polymeric NP form the core, which is surrounded by a monolayer of soybean phosphatidylcholine and 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-carboxy (polyethylene glycol)2000. Particularly, the PEG shell provides electrostatic and steric stabilization and exhibits a longer circulation half-life. To increase the specificity to cancer, an active targeting moiety could be attached to the surface of NP.^14,15 For example, antibody fragments against human epidermal growth factor receptor 2 (HER2), which is overexpressed in breast cancer, could be tagged to the NP.^16,17 There are several reports wherein folate residues are attached on the NP surface to increase the selectivity towards folate receptor overexpressed cancer cells. Although there are several antibody tagged nanocarriers, there are limited studies on organ targeted delivery systems.^18–20 Specifically, there is no report on targeted drug delivery to the thyroid gland.

In this study, we attempted to increase the concentration of an anticancer agent in the thyroid gland to increase its therapeutic efficacy in thyroid cancer. Towards this goal, we have conjugated thyroid-stimulating hormone (TSH) to the surface of polymer–lipid nanoparticles. DSPE-mPEG2000-3-(2-pyridyldithio)propionate (PDP) was employed to conjugate TSH. The PDP moieties present on the NP surface was reduced by 1,4-dithiothreitol (DTT) to express the free –SH group, which was then conjugated with the TSH. The TSH-receptor (TSHr) is a glycoprotein G-protein-coupled receptor, which is expressed in the plasma membrane of thyrocytes. The presence of TSH on the NP surface will increase its accumulation in cancer cells via a ligand-mediated biological effect.^21,22 In vivo accumulation of the NP was monitored using an animal model and antitumor efficacy was demonstrated.

Result and discussion

In the present study, TSH-conjugated nanoparticles were prepared to increase the specificity towards thyroid cancers. TSH was attached to the PDP-conjugated nanoparticle surface via a thiol-activated functional group and formed a disulphide bond (Fig. 1). The TSH-receptor (TSHr) is a glycoprotein G-protein-coupled receptor, which is expressed in the plasma membrane of thyrocytes. The presence of TSH on the NP surface will increase its accumulation in cancer cells via a ligand-mediated biological effect. In vivo accumulation of the NP was monitored using an animal model and their antitumor efficacy was demonstrated.

Fig. 1 Schematic of preparation of the cisplatin-loaded polymer–lipid hybrid nanoparticles. Schematic shows that polymer and lipid self-assemble to form the polymer–lipid NP, which was then reduced with DTT to expose the free-SH group on the surface. TSH was then conjugated with the free-SH group on the surface of the nanoparticles. The core is composed of a hydrophobic PLGA core, whereas the shell consists of lipid materials.

Dynamic light scattering analysis

The particle size and size distribution of the nanoparticles were observed using the dynamic light scattering method (DLS). The average particle size of the CDDP-loaded TSH-conjugated polymer–lipid hybrid nanoparticles (TPLHC) was observed to be 185.8 nm with an excellent polydispersity index of 0.12 (Fig. 2a). A slight increase in particle size from 152.5 to 185.8 nm for TSH conjugation was attributed to the presence of additional molecular mass from TSH itself. Nevertheless, the final particle size was <200 nm, thus indicating the possibility of passive targeting via a unique enhanced permeability and retention (EPR) effect. The nanoparticles upon long circulation will accumulate in tumor tissues.^23,24 The TSH conjugation on the nanoparticle surface was further confirmed via zeta potential evaluation. The surface of the bare lipid nanoparticle was −28 mV, whereas it decreased to −19.2 mV for the conjugation of TSH. We observed that the particle size of the TSH-conjugated nanoparticles slightly increased compared to that of the non-conjugated nanoparticles. The colloidal stability of the TSH-NP was monitored for 12 h, and the particle sizes remained unchanged (DLS analysis). The ELISA assay was performed to evaluate the stability of TSH in biological fluid. For this purpose, TSH was incubated for 3 h in growth media supplemented with 10% FBS as well as in phosphate buffered saline (pH 7.4) and acetate buffered saline (pH 5.5). The results reveal that TSH maintains good stability in all the tested conditions. Based on these results, it can be expected that the TSH surface-conjugated nanoparticles would remain stable in in vivo conditions and would be available for the selective targeting of therapeutic systems.

Fig. 2 (a) Size distribution of TSH-conjugated polymer–lipid nanoparticles (b) representative morphology characteristic of TPLHC observed by transmission electron microscopy (TEM).

Particle morphology

TEM was used to investigate the morphology of the nanoparticles. As observed in Fig. 2b, the TSH-conjugated nanoparticles were present as individual spherical objects on the copper grid and no sign of aggregation was observed. This well-defined morphology and spherical shape is expected to increase their systemic performance. The greyish shell on the outer surface and darker core clearly reveals the lipidic shell and polymer core structure. The smaller size of the micelles observed by TEM compared to that determined by DLS was due to the dehydration of the micelles during drying and staining of the TEM specimen.

Drug loading and drug release kinetics

Knowledge of drug entrapment efficiency is very important for drug delivery applications. The TPLHC exhibited a high entrapment efficiency of >95% with a high drug loading of ∼22.6%. The drug release study was evaluated using the dialysis method in phosphate buffered saline (pH 7.4 conditions) at 37 °C. The release study was performed in PBS medium to simulate the blood environment. As observed in Fig. 3, both the CDDP-loaded polymer–lipid hybrid nanoparticles (PLHC) and TPLHC exhibited a controlled release profile for 48 h. Specifically, no burst release phenomenon was observed, which suggests that the entire drug was incorporated in the nanoparticles and is present in the core of the PLGA NP but not present on the lipid shell. As expected, the presence of TSH relatively limited the rate of drug release over a period of time. Approximately, 50% of CDDP was released from TPLHC compared to that of 65% from PLHC at the end of 24 h. Such a low or controlled release of drug in the pH 7.4 conditions was highly advantageous for targeted cancer therapy because the amount of drug released prematurely might be minimized in the blood circulation. On the other hand, one could expect that intracellular levels of drug could be increased once internalized via endocytosis.

Fig. 3 In vitro release profile of the PLHC and TPLHC formulation in phosphate buffered saline (PBS, pH 7.4) at 37 °C. The release study was performed up to 48 h.

Intracellular accumulation of nanoparticles

The selectivity of the PLHC and TPLHC towards wild-type Chinese hamster ovary (CHO) cells lacking TSHr (CHOw) and in TSHr overexpressed CHO cells was determined. As observed in Fig. 4a–c, wild-type CHO cells, which do not express TSHr, did not have any influence on the cellular uptake of either nanoparticle. The cellular uptake efficiency of both the nanoparticles was same regardless of the presence of TSH on the nanoparticles. The trend was different in the case of the CHO-t cells that express the receptor for TSH. The TPLHC exhibited an enhanced cellular uptake on CHO-t cells compared to that of PLHC. Simultaneously, the competitive binding nature of the PLHC and TPLHC towards CHO-t cells was evaluated. As observed, the TSH-conjugated nanoparticles showed a decrease in cellular uptake with an increase in the concentration of TSH in the incubated medium, whereas the cellular uptake of the non-conjugated nanoparticles was independent of the TSH level in the medium. All this evidence collectively suggests the preferable interaction of the TSH nanoparticles with the receptor expressed cells.

Fig. 4 Evaluation of the selectivity of the TSH-conjugated nanoparticles over the non-conjugated nanoparticles. The cellular uptake was studied in Chinese hamster ovary (CHO) cells without (CHO-w) (a) and with (CHO-t) the TSHr (b). Competitive binding effect of the TSH-conjugated and non-conjugated nanoparticles in the presence of free TSH.

Intracellular uptake of PLHC and TPLHC

The intracellular uptake of cisplatin loaded PLHC and TPLHC in FTC-133 thyroid cancer cells was studied as a function of time. As observed in Fig. 5a, the intracellular uptake of CDDP significantly increased in the cell group treated with the TSH-conjugated NP. Approximately, 10 ng mL⁻¹ of CDDP was internalized from PLHC compared to that of TPLHC, which exhibited 25 ng mL⁻¹ at the end of 8 h. These results clearly depict the ability of the TSH-conjugated nanoparticles as a potent delivery vehicle. The higher cellular uptake was further confirmed by means of confocal imaging. It can be clearly observed that TPLHC exhibited prominent red fluorescence compared to that of the non-targeted delivery system (Fig. 5b). It can be expected that the higher intracellular uptake of CDDP would result in a greater anticancer effect in cancer cells.

Fig. 5 (a–c) In vivo biodistribution of TSH-conjugated and non-conjugated nanoparticles in Wistar rats. The formulations were administered through tail vein injection and different organs were collected at specified time points.

In vitro anticancer effect

Subsequently, the anticancer effect of free CDDP, PLHC and TPLHC in FTC-133 thyroid cancer cells was investigated using the MTT assay. The cells were incubated for 24 and 48 h and the cytotoxic potential of the individual formulations was studied. As observed, the drug-loaded nanoparticles exhibited a significantly higher cytotoxic effect in thyroid cancer cells than compared to the free CDDP (Fig. 5c and d). Specifically, the TSH-conjugated nanoparticles showed a significantly enhanced anticancer effect than any of the other groups across all time points and concentrations tested. The IC50 value was calculated to quantify the effect of the individual formulation on cancer cells. The IC50 value of free CDDP, PLHC and TPLHC were 4.26 μg mL⁻¹, 2.31 μg mL⁻¹, and 0.52 μg mL⁻¹, respectively, after 24 h incubation. The superior anticancer effect of TPLHC was attributed to the high intracellular concentration of CDDP due to the receptor mediated uptake of the nanoparticles in the cancer cells. This is more evident from the fact that the uptake of the NP decreased when the concentration of TSH was increased in CHO cells. Overall, these results suggest that the presence of TSH on the surface of the nanoparticles could markedly improve the delivery of drugs in cancer cells expressing TSHr.^25,26

Biodistribution of the nanoparticles

Experiments evaluating the distribution of the targeted and non-targeted nanoparticles were performed in Wistar rats (Fig. 6). For cancer targeting, enhanced accumulation of nanoparticles in the tumor tissue is of great importance. As observed from Fig. 3a–c, a 4-fold higher accumulation of the TSH-conjugated NP was observed compared to that of the non-targeted NP. This could be due the interaction of the TSH-NP with the TSHr overexpressed in the thyroid cancer cells. It is worth noting that the TPLHC did not accumulate in other organs, such as the heart, kidney, spleen, and lungs, in a higher proportion. The biodistribution study therefore clearly depicted the in vivo targeting ability of the TPLHC to the thyroid gland.

Fig. 6 (a) Intracellular uptake of CDDP loaded PLHC and TPLHC formulation in FTC-133 thyroid cancer cells. (b) Confocal laser scanning microscopy images of PPLHC and TPLHC in cancer cells. In vitro cytotoxicity profile of different formulations in FTC-133 cancer cells after incubation of 24, and 48 h (c–d). The cytotoxicity assay was evaluated by means of MTT assay.

In vivo anticancer efficacy

The in vivo anticancer efficacy of the various CDDP formulations was examined in an FTC-133-bearing tumor xenograft animal model. The antitumor efficacy of the individual formulations was evaluated by measuring the tumor volume at a specified time interval to assess the tumor inhibition effect. All mice were alive during the course of the entire study period. The growth curves of the tumor treated with the different formulations are presented in Fig. 7a. The blank nanoparticle did not have any effect on the growth of the tumor, whereas all the other formulations effectively retarded or delayed the growth of the tumor compared to that of the untreated control group. In particular, TPLHC, which has TSH conjugated on its surface, inhibited tumor growth more efficiently compared with the other mice groups, which indicates that its targetability and pH-sensitivity could favor increased tumor inhibition.^27,28 The final tumor volumes remained at ∼2500 mm³, ∼1600 mm³, ∼1400 mm³, and ∼700 mm³ for the control, free CDDP, PLHC, and TPLHC, respectively. The enhanced tumor growth might be attributed to the specific binding of TSH to the TSHr overexpressed in FTC-133 tumors, which facilitates intracellular uptake through receptor-mediated endocytosis and enhances the suppression effect of CDDP on cancer cell proliferation.

Fig. 7 Tumor suppression at the whole-body level. (a) Changes of tumor volume after intravenous injection of the different formulations in a tumor xenograft animal model. (b) Body weight of mice after treatments. (c) Images of tumor sections.

The body weight of the mice was monitored throughout the study period, which is an important indicator for systemic toxic and side effects. As observed in Fig. 7b, none of the formulations induced any loss of body weight, which implies that the free drug as well as the delivery system is safe and has a good biocompatibility profile. Overall, TPLHC is an effective and safe drug formulation for the treatment of thyroid cancers.

Conclusion

In conclusion, TSH-conjugated polymer–lipid hybrid nanoparticles were successfully developed based on a PLGA/DSPE polymer–lipid mixture and TSH conjugation for the treatment of thyroid cancer. The delivery system was mainly designed to target the thyroid gland to achieve a high concentration in thyroid carcinomas. The TPLHC exhibited excellent properties, which were attributed to their nanoscaled size of 160 nm with a narrow distribution to benefit the EPR effect. The polymer–lipid nanoparticles efficiently controlled the release of the drug in physiological conditions. The TSH-conjugated nanoparticles displayed higher cellular uptake in cells, which express TSHr. Consistently, the TPLHC showed high intracellular levels of CDDP in FTC-133 cancer cells. Specifically, the TSH-conjugated nanoparticles showed a significantly enhanced anticancer effect compared to any of the other groups across all time points and concentrations tested. In addition, a 4-fold higher accumulation of TSH-conjugated NP was observed in tumors compared to the non-targeted NP in xenograft mice. Importantly, the TPLHC inhibited the growth of tumors more efficiently than the other mice groups. This enhanced tumor inhibition might be attributed to the specific binding of TSH to the TSHr overexpressed in FTC-133 tumors. Taken together, the TSH-conjugated nanoparticles hold great potential to be an effective and safe nanoscale delivery system for the targeted therapy of thyroid cancers.

Materials and methods

Materials

PLGA (poly(D,L-lactide-co-glycolide), (MW ∼ 12 000) with a 50 : 50 monomer ratio with a viscosity of 0.72–0.92 dL g⁻¹ and lecithin were purchased from Sigma-Aldrich, China. DSPE–PEG2000 (MW ∼ 2790.5) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(methoxypolyethylene glycol-2000)] (DSPE-mPEG2000-PDP (MW ∼ 2987.8)) were purchased from Avanti Polar Lipids, China. TSH (from human pituitary), N-(fluorescein-5-tiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (fluorescein-DHPE) was purchased from Sigma-Aldrich, China). All other chemicals were of reagent grade.

Preparation of lipid-assembled polymeric nanoparticles

The fusion nanoparticles were prepared using the nanoprecipitation method. Briefly, PLGA was dissolved in dichloromethane at a concentration of 20 mg mL⁻¹. Subsequently, lecithin, DSPE-PEG2000, DSPE-mPEG2000-PDP, and cisplatin (CDDP, 10%w/w) were dissolved in 4% ethanol solution and heated to 60 °C. The polymer solution was added drop-by-drop on the lipid solution and vortexed. The organic mixtures were stirred for 2 h until the organic solvents evaporated. The formed nanoparticles were obtained by centrifuging at high speed.

The TSH-conjugated polymer–lipid nanoparticles were prepared by the reduction of disulfide bonds on the surface. For this purpose, the NP were treated with 50 mM DTT solution for 30 min. Subsequently, free DTT was removed by centrifugation. The TSH (in nanomolar concentration) was added to the abovementioned NP and incubated overnight. The unconjugated TSH was removed by gel permeation chromatography.

Nanoparticle characterization

The particle size of the NP was determined by Quasi-elastic laser light scattering using a ZetaPALS dynamic light scattering (DLS) detector (15 mW laser, incident beam = 676 nm) (Brookhaven Instruments, Holtsville, NY). The zeta potential of nanoparticles was determined using a Zetasizer ZS Nano (Malvern Instruments, UK).

Transmission electron microscopy

The morphology of the NP was observed using a JEOL JEM-200CX instrument at an acceleration voltage of 200 kV. A 300-mesh carbon-coated copper grid was used to load the samples. An aqueous solution of NP was placed on the TEM grid and blotted away after 20 min. The samples were negatively stained with 2% phosphotungistic acid. The grids were then dried and observed under a TEM instrument.

Drug release study

The release profile of CDDP from the PLHC and TPLHC was observed in phosphate buffered saline (PBS pH 7.4) at 37 °C. For this purpose, 1 mL of nanoparticle dispersion was sealed in a dialysis bag and incubated in 100 mL of PBS. At specified time intervals, 1 mL of release medium was withdrawn and replaced with an equal amount of fresh release medium. The amount of CDDP released in the medium was determined via the ICP-MS method. A Quadrupole ICP-MS Thermo X-series (Thermo Electron, Windsford, Cheshire, U.K.) equipped with a Meinhard nebulizer, a Fassel torch, and an Impact Bead Quartz spray chamber cooled by a Peltier system was employed for total Pt determination. The ICP-MS operating conditions were as follows: forward power 1250 W, plasma gas 15 L min⁻¹, auxiliary gas 0.73 L min⁻¹, nebulizer gas 0.85 L min⁻¹, channels per AMU 10, and integration time 0.6 ms.

Cell culture

The selectivity of the non-conjugated and TSH-conjugated NP was tested in TSHr expressing CHO cells and CHO wild type cells (without TSHr). The cells were grown in F12 medium containing a 10% FBS and 1% penicillin-streptomycin combination. Moreover, FTC-133 thyroid cancer cells were incubated in DMEM/F12 (1 : 1) supplemented with glutamate, D-glucose, pyruvate, and other regular ingredients of normal growth media. The cells were grown in ambient conditions and the media was changed and cells were sub-cultured from time to time.

Intracellular accumulation of PLHC and TPLHC

Fluorescent labelled nanoparticles were used to observe the nanoparticle uptake and affinity towards cancer cells and TSHr overexpressed cells. For this study, TSHr non-expressing CHO-w and TSHr expressing CHO-t cells were used. The cells were seeded in 6-well culture plates, incubated overnight and then treated with the respective formulations. As a control experiment, free TSH of different concentrations was used to investigate the competitive binding of nanoparticles to the cells. The formulations were incubated for 3 h, cells were obtained, centrifuged, and media was removed. The cells were then dissolved in quaternary ammonium hydroxide solution and shaken for 1 h at 60 °C. After incubation for 1 h, the cells were treated with a liquid scintillation cocktail and the samples were vigorously mixed and analyzed using a Wallac Win Spectral 1414 liquid scintillation counter.

Intracellular uptake of PLHC and TPLHC

CHO-w and CHO-t cells were seeded in a 12-well culture plate and incubated overnight for the cells to become attached. In brief, the cells were exposed with the respective formulations and incubated for predetermined time intervals. At specific time intervals, the cells were harvested, washed, and resuspended in PBS. The cells were ruptured using a probe sonicator. The ruptured cell suspensions were centrifuged and the supernatant was collected. The internalized drugs were present in the supernatant solution. The CDDP signals were determined using an Agilent 7700x ICP-MS.

Cytotoxicity assay

The MTT assay was performed to evaluate the cytotoxicity potential of free CDDP, PLHC and TPLHC and dose–response curves were plotted. FTC-133 thyroid cancer cells were used in the present study and cell survival was noted after the assay protocols. In brief, 1 × 10⁴ cells were seeded in a 96-well plate in 100 μL of DMEM/F12 medium and incubated for 24 h. When the cells reached 80% confluence, they were treated with various concentrations of blank, free CDDP, PLHC and TPLHC formulations and further incubated for 24 h, 48 h, and 72 h, respectively. After each time point, the derivatives in the medium were removed and 20 μL of MTT (5 mg mL⁻¹ in MEM) was added and incubated for 4 h under normal growing conditions in an incubator. After 4 h, 100 μL of DMSO was added and incubated for 30 min. After 30 min, the optical density (OD) at 570 nm was measured using a plate reader. Cells without polymers were taken as the control.

In vivo biodistribution study

The studies on experimental animals were carried out strictly according to the guidance framed by the ‘Institutional Animal Care and Ethics Committee’, Qingdao University, China. All ethics protocols were followed while handling the animals. Female Wistar rats (4–5 months) were selected to perform the biodistribution study. [3H]CHE-radiolabeled PLHC and TPLHC were injected via the tail vein into the rats. After 4 h, the rats were sacrificed and individual organs were collected. The organs were mixed with quaternary ammonium hydroxide solution and shaken for 1 h at 60 °C in an incubator shaker. The tissue samples were decolorized with 2 mL of 24% (v/v) H₂O₂ at room temperature for 1 h. After incubation for 1 h, the cells were treated with a liquid scintillation cocktail and the samples were vigorously mixed and analyzed using a Wallac Win Spectral 1414 liquid scintillation counter.

Anticancer efficacy study

The anticancer efficacy was studied in NOD SCID mice. FTC-133 cancer cells (1 × 10⁶) cells were subcutaneously injected into the mice and the tumor was allowed to grow up to a volume of ∼400–500 mm³. Subsequently, the mice were randomly divided into 5 groups with 8 mice in each group. The 5 groups were control, blank NP, free CDDP, PLHC, and TPLHC. The formulations were injected 3 times into the mice via the tail vein at a fixed dose of 5 mg kg⁻¹. The tumor volume was measured at a specific point of time. The tumor volume was calculated from the equation; V = 0.5 × D × d², where V is the tumor volume and D and d were longest and shortest diameter of the tumor, respectively. The body weight of the mice was subsequently monitored on a regular basis. The animal experiments were carried out in accordance with the guidelines framed by the Affiliated Hospital of Qingdao University, China.

Statistical analysis

Results were statistically analyzed using the Student's t-test with the level of significance set at p < 0.05. The significance was tested using the GraphPad Prism 5 software (GraphPad Software Inc., USA). The results are expressed as mean ± standard deviation.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (No. 81171816) and the Science and Technology Research Foundation of Shandong Province (No. 2008GG10202045).

References

1. A. Mehta, L. Zhang, M. Boufraqech, Y. Zhang, D. Patel, M. Shen and E. Kebebew, Endocr.-Relat. Cancer, 2015, 22, 319.
2. H. Luo, S. Tulpule, M. Alam, R. Patel, S. Sen and A. Yousif, Case Rep. Oncol., 2015, 13, 233.
3. L. Lorusso and K. Newbold, Future Oncol., 2015, 11, 1719.
4. P. Valderrabano, V. E. Zota, B. McIver, D. Coppola and M. E. Leon, Cancer Control, 2015, 22, 152.
5. A. Longhi, C. Errani, M. de Paolis, M. Mercuri and G. Bacci, Cancer Treat. Rev., 2006, 32, 423.
6. X. Yanand and R. A. Gemeinhart, J. Controlled Release, 2005, 106, 198–208.
7. E. Cvitkovic, Cancer Treat. Rev., 1998, 24, 265–281.
8. B. L. van Leeuwen, W. A. Kamps, H. W. B. Jansen and H. J. Hoekstra, Cancer Treat. Rev., 2000, 26, 363.
9. S. Sevenson and D. A. Tomalia, Adv. Drug Delivery Rev., 2005, 57, 2106.
10. F. Gu, L. Zhang, B. A. Teply, N. Mann, A. Wang and A. F. Radovic-Moreno, et al., Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 2586.
11. L. Zhang, A. F. Radovic-Moreno, F. Alexis, F. X. Gu, P. A. Basto and V. Bagalkot, et al., ChemMedChem, 2007, 2, 1268–1271.
12. O. C. Farokhzad, J. Cheng, B. A. Teply, I. Sherifi, S. Jon and P. W. Kantoff, et al., Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 6315.
13. Y. Li, Y. Pei, X. Zhang, Z. Gu, Z. Zhou and W. Yuan, et al., J. Controlled Release, 2001, 71, 203.
14. L. Zhang, J. M. Chan, F. X. Gu, J. W. Rhee, A. Z. Wang and A. F. Radovic-Moreno, et al., ACS Nano, 2008, 2, 1696.
15. J. Thevenot, A. L. Troutier, L. David, T. Delair and C. Ladaviere, Biomacromolecules, 2007, 8, 3651.
16. H. Shmeeda, D. Tzemach, L. Mak and A. Gabizon, J. Controlled Release, 2009, 136, 155.
17. W. Punfa, S. Suzuki, P. Pitchakarn, S. Yodkeeree, T. Naiki, S. Takahashi and P. Limtrakul, Asian Pac. J. Cancer Prev., 2014, 15, 9249.
18. S. Mazzucchelli, M. Truffi, L. Fiandra, L. Sorrentino and F. Corsi, World J. Pharmacol., 2014, 3, 72.
19. M. Arruebo, M. Valladares and Á. González-Fernández, J. Nanomater., 2009, 439389.
20. J. L. Arias, J. D. Unciti-Broceta and J. Maceira, et al., J. Controlled Release, 2015, 197, 190.
21. E. Koren, A. Apte, A. Jani and V. P. Torchilin, J. Controlled Release, 2012, 160, 264.
22. D. Paolino, M. Licciardi, C. Celia, G. Giammona, M. Fresta and G. Cavallaro, Eur. J. Pharm. Biopharm., 2012, 82, 94.
23. D. Paolino, D. Cosco, R. Molinaro, C. Celia and M. Fresta, Drug Discovery Today, 2011, 16, 311.
24. C. Celia, D. Cosco, D. Paolino and M. Fresta, Med. Res. Rev., 2011, 31, 716.
25. M. Celano, M. G. Calvagno, S. Bulotta, D. Paolino, F. Arturi and D. Rotiroti, et al., BMC Cancer, 2004, 4, 63.
26. C. Celia, M. G. Calvagno, D. Paolino, S. Bulotta, C. A. Ventura and D. Russo, et al., J. Nanosci. Nanotechnol., 2008, 8, 2102.
27. V. P. Torchilin, Nat. Rev. Drug Discovery, 2005, 4, 145.
28. H. Wu, L. Zhu and V. P. Torchilin, Biomaterials, 2013, 34, 1213–1222.

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