Transferrin receptor-targeted HMSN for sorafenib delivery in refractory differentiated thyroid cancer therapy

Abstract

Background: Thyroid cancer becomes the most common endocrine cancer with the greatest growing incidence in this decade. Sorafenib is a multikinase inhibitor for the treatment of progressive radioactive iodine-refractory differentiated thyroid cancer (DTC), while the off-target toxicity effect is usually inconvenient for patients taking.

Methods: In this study, hollow mesoporous silica nanoparticles (HMSNs) with transferrin modification (Tf-HMSNs) were loaded with sorafenib (sora@Tf-HMSNs) to help targeted delivery of sorafenib. Due to the biocompatible Tf shell, Tf-HMSNs exhibited excellent bio-compatibility and increased intracellular accumulation, which improved the targeting capability to cancer cells in vitro and in vivo.

Results: Sora@Tf-HMSNs treatment exhibited the strongest inhibition effect of res-TPC-1 cells and res-BCPAP cells compared with sora@HMSNs and sorafenib groups and induced more cancer cell apoptosis. Finally, Western blot analysis was conducted to check the expression of RAF/MEK/ERK signaling pathway after sorafenib encapsulated Tf-HMSNs treatment.

Conclusion: Overall, sora@Tf-HMSNs can significantly increase the effective drug concentration in cancer cells and thus enhance the anticancer effect, which are expected to be promising nanocarriers to deliver anticancer drugs for effective and safe therapy for RAI-refractory DTC.

Keywords: RAF/MEK/ERK; RAI-refractory DTC; hollow mesoporous silica nanoparticles; sorafenib; transferrin.

Figure 1

Physicochemical characterization of Tf-HMSNs. Notes: (A) SEM images of HMSNs (scale bar=1 µm). (B) TEM images of HMSNs (scale bar=200 nm) (C) Coomassie Blue analysis of particles after transferrin modification with UV–vis spectroscopy. This spectrum is apparent in the different color; Tf modification particles are stained blue while the HMSNs maintain the dye as green. (D) The average size of HMSNs with dynamic light-scattering analysis. Abbreviations: HMSNs, hollow mesoporous silica nanoparticles; SEM, scanning electron microscope; TEM, transmission electron microscope; Tf-HMSNs, transferrin-conjugated HMSNs; UV–vis spectroscopy, ultraviolet–visible spectroscopy.

Figure 2

In vitro release profile of sorafenib from drug-loaded HMSNs and Tf-HMSNs at 37°C, respectively. Notes: The release study was performed in the PBS (pH 7.4) and performed until 72 hours. Twelve time points were included. Abbreviations: HMSNs, hollow mesoporous silica nanoparticles; Tf-HMSNs, transferrin-conjugated HMSNs.

Figure 3

(A, B) CLSM images of FITC-labeled sora@HMSNs and sora@Tf-HMSNs uptaken by res-TPC-1 and res-BCPAP cells incubated for 24 hours. Notes: The HMSNs and nuclei were stained with FITC (green) and DAPI (blue), respectively. All experiments were performed in triplicate. Abbreviations: CLSM, confocal laser scanning microscope; Ctrl, control; FITC, fluorescein isothiocyanate; HMSNs, hollow mesoporous silica nanoparticles; sora@HMSNs, HMSNs loaded with sorafenib; sora@Tf-HMSNs, transferrin-conjugated HMSNs loaded with sorafenib.

Figure 4

Sorafenib-loaded Tf-HMSNs (sora@Tf-HMSNs) increase cell cytotoxicity (A) res-TPC-1 cells and (B) res-BCPAP cells viability after incubation with different concentrations of sorafenib (sora), sorafenib-loaded HMSNs (sora@HMSNs), and Tf-HMSNs for 24 hours, respectively. Notes: The cell viability was assessed by CCK-8 assay. All the experiments were conducted for three times (Results were given as mean ± SD). Abbreviations: CCK-8, cell counting kit-8; ctrl, control; HMSNs, hollow mesoporous silica nanoparticles; Tf-HMSNs, transferrin-conjugated HMSNs.

Figure 5

Annexin V/FITC staining indicating enhanced apoptosis and cell death by sora@Tf-HMSNs compared to sora@HMSNs and sorafenib group using flow cytometry in TPC-1 cell line. Note: Each experiments were replicated three times and representative pictures are shown. Abbreviations: Ctrl, control; FITC, fluorescein isothiocyanate; HMSNs, hollow mesoporous silica nanoparticles; sora@HMSNs, HMSNs loaded with sorafenib; sora@Tf-HMSNs, transferrin-conjugated HMSNs loaded with sorafenib; Tf-HMSNs, transferrin-conjugated HMSNs; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Figure 6

Representative pictures of Western blots displaying the inhibition of RAF/MEK/ERK pathway after sora@Tf-HMSNs, sora@HMSNs, and sorafenib (sora) treatment. Abbreviations: Ctrl, control; HMSNs, hollow mesoporous silica nanoparticles; sora@ HMSNs, HMSNs loaded with sorafenib; sora@Tf-HMSNs, transferrin-conjugated HMSNs loaded with sorafenib; Tf-HMSNs, transferrin-conjugated HMSNs.

Figure 7

Sora@Tf-HMSNs could enhance drug accumulation and inhibit tumor growth of xenograft in nude mice. Notes: (A) TPC-1 thyroid cancer cells were injected subcutaneously in right flank of 21-day-old male BALB/c nude mice (6 mice each group). After 23 days feeding, the mice received DiR-labeled sora@Tf-HMSNs, DiR-labeled sora@HMSNs, and then anesthetized to obtain the tumors and major organs. Then the tumors and other major organs were collected for further analysis by in vivo fluorescence imaging. (B) The tumor volumes were measured every 3 days, and the growth rate was recorded as reproduction curve (tumor volume = length × width²/2). *P<0.05, **P<0.01. Abbreviations: Ctrl, control; HMSNs, hollow mesoporous silica nanoparticles; sora@HMSNs, HMSNs loaded with sorafenib; sora@Tf-HMSNs, transferrin-conjugated HMSNs loaded with sorafenib; Tf-HMSNs, transferrin-conjugated HMSNs.

Figure 7

Sora@Tf-HMSNs could enhance drug accumulation and inhibit tumor growth of xenograft in nude mice. Notes: (A) TPC-1 thyroid cancer cells were injected subcutaneously in right flank of 21-day-old male BALB/c nude mice (6 mice each group). After 23 days feeding, the mice received DiR-labeled sora@Tf-HMSNs, DiR-labeled sora@HMSNs, and then anesthetized to obtain the tumors and major organs. Then the tumors and other major organs were collected for further analysis by in vivo fluorescence imaging. (B) The tumor volumes were measured every 3 days, and the growth rate was recorded as reproduction curve (tumor volume = length × width²/2). *P<0.05, **P<0.01. Abbreviations: Ctrl, control; HMSNs, hollow mesoporous silica nanoparticles; sora@HMSNs, HMSNs loaded with sorafenib; sora@Tf-HMSNs, transferrin-conjugated HMSNs loaded with sorafenib; Tf-HMSNs, transferrin-conjugated HMSNs.

Scheme 1

Schematic representation of the construction of hollow mesoporous silica nanoparticle (HMSN) based cancer cell-targeted nanocarriers. Notes: Specifically, for protein transferrin attachment, HMSN is first modified with 3-GPTMS, loaded with sorafenib in DMSO. After which, HMSN was reacted with the transferrin (Tf) to construct particle–cell signaling and enhance the uptake. Abbreviations: CTAB, cetyltrimethyl ammonium bromide; DMSO, dimethyl sulfoxide; 3-GPTMS, 3-glycidoxypropyltrimethoxysilane.