Transferrin-conjugated paclitaxel prodrugs for targeted cancer therapy

Abstract

Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs ever developed and is effective against a wide spectrum of tumors. The clinical application of PTX, however, is limited by its severe side effects. We developed new ligand-mediated prodrugs, namely, PTX conjugated with Fmoc-L-glutamic acid 5-tert-butyl ester (linker) and transferrin (Tf, ligand/carriers), to specifically target tumor cells/tissues. PTX was labeled with FITC or a near-infrared (NIR) dye ICG-Der-02, respectively, for in vitro and in vivo imaging studies. MTT assay and apoptosis studies demonstrated that the prodrug had efficient inhibition of tumor cell proliferation with low toxicity to normal cells. More importantly, the prodrug Tf–Glu–PTX displayed a potential to overcome PTX resistance in drug-resistant MDA-MB-231 cell lines. Our in vivo studies also demonstrated that Tf–Glu–PTX significantly decreased side effects and enhanced the antitumor efficiency compared to free PTX. Collectively, our study showed that Tf–Glu–PTX is a promising prodrug for targeted cancer therapy.

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Introduction

Chemotherapy drugs have been widely used in clinical cancer therapy over the past several decades. However, the serious side effects and drug resistance of much current chemotherapy present daunting hurdles against the efficacious applications of these drugs.¹ One example is paclitaxel (PTX), which is effective against many types of malignant tumors including breast, ovarian, lung and head and neck cancers.² PTX promotes the polymerization of microtubules, induces abnormal stabilization of cells, and causes nonfunctional microtubules to block the cell cycle at the G2-M phase, eventually leading to cancer cell apoptosis.^3,4 Therefore, PTX stops cell division as a mitotic inhibitor and chemotherapeutic agent, and it further can prevent tumor metastasis. Nevertheless, because of its intrinsic lack of targeting capability and poor water solubility, PTX induces serious side effects such as hypersensitivity reactions,⁵ which essentially prohibits PTX from wide clinical use. In addition, many types of tumors are resistant to PTX, which further diminishes its anticancer efficacy.^6,7

To overcome these limitations, it is thus highly desired to deliver drugs specifically to tumor tissues and cells, but not to healthy tissues or cells. One approach to this end is by loading drugs into nanocarriers, which, via the Enhanced Permeation and Retention (EPR) effect in many types of tumor, are able to efficiently deliver loaded drugs to tumor tissues, resulting in therapeutic benefits. Particularly for PTX, loading PTX into nanoparticles carriers, such as polylactide (PLA), chitosan, albumin, hyaluronic acid (HA), poly(butyl cyanoacrylate) (PBCA) and liposomes has received much attention.^8–14 The nanoparticle delivery system (NPs) has become an effective treatment method to enhance solubility and targeting ability, as well as decrease severe toxicities.^14–17 However, one drawback of these nanoparticles is the low drug loading efficiency. For example, the loading efficiency of human serum albumin and PEG–PLA are only about 10 and 16.7 wt% PTX, respectively.^18,19 Abraxane®, a PTX albumin-bound NP formulation with the particle size of ∼130 nm, was approved by the FDA in 2005 for the treatment of metastatic breast cancer.²⁰ This formulation was demonstrated to have reduced toxicity compared to Taxol. Nevertheless, whether Abraxane® could improve survival and address P-gp-mediated drug resistance is still unclear.²¹ Therefore, alternative PTX formulations are still in demand.

Transferrin (Tf), a 78 kDa-monomeric serum glycoprotein, acts as an important transporter to deliver iron into cells by binding to the transferrin receptor and subsequently being internalized via receptor-mediated endocytosis.²² The transferrin receptor is overexpressed in tumor tissues but not in healthy tissues, with high efficiency of internalization and fast recycling once transferrin is internalized.²³ It has been shown in rats that the cellular uptake of transferrin by tumors is correlated with the proliferation activity of the tumor cells (i.e., the faster the tumor growth, the higher the uptake of transferrin).²⁴ In addition, the uptake of transferrin by tumors results in a lack of transferrin from the blood circulation, which is one of the causes of the anemia observed in malignant diseases.^25,26 These finding have been extensively exploited preclinically for tumor diagnosis and targeted delivery of anticancer therapeutics to tumors. Tf (ligand)-conjugated NPs have thus been explored for targeted delivery.^27–30 To the best of our knowledge, however, Tf has not been developed as carriers to develop PTX prodrugs.

In this study, a novel ligand-mediated prodrug was developed for targeted delivery of PTX. Particularly, paclitaxel was conjugated at its 2′-hydroxy function by introducing Fmoc-Glu(OtBu)–OH linkers. The carboxyl groups of the linkers were covalently attached to transferrin through amide bond formation to produce Tf–Glu–PTX prodrugs. The release properties of the parent drug PTX from the ester bond of PTX-linker were investigated. The dynamic behavior and tumor targeting ability were investigated by Tf–Glu–PTX–FITC/ICG02 to carry out the optical imaging. Finally, the therapeutic efficacy of the prodrug was evaluated in vitro and in vivo.

Results

Characterization of Tf–Glu–PTX, Tf–Glu–PTX–FITC and Tf–Glu–PTX–ICG02 targeted prodrugs

Tf–Glu–PTX, Tf–Glu–PTX–FITC and Tf–Glu–PTX–ICG02 were synthesized by following the procedures described in the Experimental section, as shown in Fig. 1. As shown in Fig. 2A, the absorption peaks of Tf–Glu–PTX, Tf–Glu–PTX–FITC and Tf–Glu–PTX–ICG02 displayed almost the same as that of free PTX after the conjugated of Tf and FITC/ICG02, indicating the successful conjugation of Tf–Glu–PTX, Tf–Glu–PTX–FITC and Tf–Glu–PTX–ICG02. The successful conjugations of prodrugs were further evidenced by gel electrophoresis, as shown in Fig. 2B. In addition, the fluorescence of FITC/ICG02 was observed from the band of purified Tf–Glu–PTX–FITC/Tf–Glu–PTX–ICG02 under TLC/NIR imaging system, which further demonstrated that the FITC/ICG02 was attached to the Tf–Glu–PTX prodrugs (ESI,† Fig. 1B). Mass spectrum indicates that the molecular weight (MW) of FITC–Glu–PTX is 1389.43, whereas the calculated MW is 1390.43 based on its molecular structure. Glu–PTX: MS (ESI, m/z): 1283.4 ([M + Na]⁺) (ESI,† Fig. 1A and C).

Fig. 1 Synthesis scheme and structures of Tf–Glu–PTX and Tf–Glu–PTX–FITC/ICG02.

Fig. 2 Characterization of Tf-conjugated paclitaxel prodrugs. (A) The absorption peaks of Tf–Glu–PTX, Tf–Glu–PTX–FITC/Tf–Glu–PTX–ICG02 prodrug overlaps with the peaks of Tf, Glu, PTX, FITC and ICG-Der-02 at 280 nm, 220 nm, 227 nm, 347 nm and 778 nm, respectively, (B) gel electrophoresis further confirmed the successful synthesis of Tf, Tf–Glu–PTX, Tf–Glu–PTX–FITC/Tf–Glu–PTX–ICG02. The fluorescence of FITC/ICG-Der-02 can be observed from the band of purified Tf–Glu–PTX–FITC/Tf–Glu–PTX–ICG02 using fluorescent/NIR imaging system. (C) Size of Tf modified particles was measured by LPSA. (D) Morphology of Tf–Glu–PTX complex was measured by TEM. (E) The release of free PTX from Tf–Glu–PTX in water at different temperature. (F) The release of free PTX from –NH₂–Glu(OtBu)–PTX under different medium. (G) HPLC of incubation studies of –NH₂–Glu(OtBu)–PTX intermediate compounds in human plasma at 37 °C, the peak of free PTX at 4 h. (H) HPLC of incubation studies of Tf–Glu–PTX at 37 °C for 50 h.

To quantify the number of PTX molecules attached to the amino groups of Tf surface, a recovery method was strictly followed for the calculation of unreacted PTX based on the regression equation of the PTX standard curve Y(OD) = 0.05581X (μg mL⁻¹) + 0.1525 (R² = 0.9997). The linear range of the standard curve was from 0.0018–15.18 μg mL⁻¹. The modified PTX molecules were 67 ± 19 per Tf particle (n = 4). The size and morphology of Tf–Glu–PTX were measured by LPSA and TEM, as shown in Fig. 2C. The effective diameters of Tf–Glu–PTX (n = 4) were 4.3 ± 0.37 nm. Further, the morphologies of Tf–Glu–PTX was observed by TEM using negatively stained samples (Fig. 2D).

The release rate of free PTX from the conjugation is an important factor influencing the therapeutic efficacy. We investigated the cleavage of the linkers (Fmoc-Glu(OtBu)–OH) in the intermediate products (Glu–PTX: MS (ESI, m/z): 1283.4 ([M + Na]⁺). We measured the free PTX released from the compounds after incubating the product in different buffer and different temperature, respectively. The free PTX was measured by HPLC, as displayed in Fig. 2G and H. The release profiles are depicted in Fig. 2F. The intermediate product Glu–PTX displayed faster release of PTX in human plasma (∼32.81% at 4 h) and intermediate release in rat plasma (∼23.35%) and lower release in Tris (∼15.81%). Glu–PTX released faster in human plasma than in the Tris buffer, indicating that the faster release of the PTX is related to enzyme in plasma. Aqueous stability studies showed that the amount of PTX released from prodrug Tf–Glu–PTX was lower than 8% of drug when the aqueous solution of the prodrug was incubated at 37 °C for 50 h, which demonstrates the high stability of prodrugs.

In vitro tumor-targeting ability

To study the receptor mediated targeting of PTX-conjugated Tf carriers to tumor cell lines (MDA-MB-231, MCF-7, A549), the Tf-receptor (TfR) proteins expression levels were investigated by western blot, as shown in Fig. 3A. The TR protein expression on the different cancer cells was found to be in the following order: MDA-MB-231 > MCF-7 > A549 > 293T.

Fig. 3 Targeting capability of Tf-based PTX prodrugs in different tumor cell lines. (A) Protein levels of Tf receptors on the tumor and normal cell lines were determined by western blot. (B) Targeting ability of PTX prodrugs in MDA-MB-231, MCF-7, A549, and 293T cells. Tumor cells showed increased uptake of PTX prodrug compared to the normal cell lines. (C) Flow cytometric analysis of Tf-conjugated PTX prodrug in MDA-MB-231, MCF-7, A549, and 293T cells.

To investigate the tumor-targeting ability of Tf–Glu–PTX, fluorescence images were acquired on different cells incubated with free Glu–PTX–FITC or Tf–Glu–PTX–FITC, as shown in Fig. 3B, the fluorescence intensity from the cells incubated with Tf–Glu–PTX–FITC is much higher than that of free Glu–PTX–FITC. Flow cytometry analysis indicated Tf–Glu–PTX–FITC were taken in MDA-MB-231, MCF7 A549 and 293T cells at about 85.73%, 64.07%, 43.25% and 8.29%, respectively. The uptake levels of Tf–Glu–PTX–FITC in the three tumor cells are consistent with their protein expression levels.

In vitro cytotoxicity and antitumor activity

To evaluate the therapeutic efficacy of Tf–Glu–PTX and its potential cytotoxicity, cell viability assays were carried out in cancer cells (MCF7) and normal cells (293T). The results in Fig. 4A and B indicated that Tf–Glu–PTX effectively reduced the viability of cancer cells with little cytotoxicity in normal cells compared with free PTX. As shown in Fig. 4B, Tf–Glu–PTX had a higher therapeutic efficacy for cancer cells than free PTX. Fig. 4A displays that there is a significant decrease of the viability of PTX-treated 293T cells, due to the high cytotoxicity of non-targeting PTX. Further, Tf–Glu–PTX prodrug has negligible cytotoxicity in 293T normal cells. We used Tf–Glu–PTX and free PTX to treat PTX-sensitive MDA-MB-231 cells (PS) and PTX-resistant MDA-MB-231 cells (PR) to PTX have similar antitumor effect on the PTX sensitive MDA-MB-231 cells. Significant drug resistance was observed in MDA-MB-231 cells treated by free PTX, as shown in Fig. 4C–E. However, Tf–Glu–PTX showed high antitumor efficacy on MDA-MB-231 (PR) cells, indicating that Tf–Glu–PTX has the potential to overcome PTX-resistance in drug-resistant MDA-MB-231 cell lines. The mean concentration of paclitaxel that caused 50% cell inhibition (IC50) of Tf–Glu–PTX was decreased to 18.83 μg mL⁻¹, 21.49 μg mL⁻¹, 24.12 μg mL⁻¹ compared with 62.67 μg mL⁻¹, 64.46 μg mL⁻¹, 65.91 μg mL⁻¹ of free paclitaxel in MDA-MB-231, MCF-7 and A549, respectively. The IC50 of Tf–Glu–PTX was increased to 42.18 μg mL⁻¹ compared with 28.19 μg mL⁻¹ of free paclitaxel in 293T. This finding suggests that Tf-conjugated PTX delivery system displayed low cytotoxicity to normal cells.

Fig. 4 In vitro antitumor efficacy and cytotoxicity of Tf–Glu–PTX. Cell viability of 293T cells (A) and MCF-7 cells (B) incubated with either free PTX or Tf–Glu–PTX. Cell viability of Tf and Tf–Glu–PTX-treated PTX sensitive MDA-MB-231 cells (C) and PTX resistant MDA-MB-231 cells (D). Fluorescence images of MDA-MB-231/PS and MDA-MB-231/PR cells incubated with FITC–Glu–PTX or Tf–Glu–PTX–FITC (E) (green, V+/PI+, red). Late apoptosis/necrotic value of the three cell lines (MDA-MB-231, MCF-7 and A549) (F) data are given as mean ± SD (n = 6).

The morphological changes of MDA-MB-231 tumor cell lines after the treatments of Tf, PTX and Tf–Glu–PTX were observed under fluorescence microscopy. Annexin-V staining was performed to identify the cell apoptosis by using annexin-V-fluorescein isothiocyanate, which specifically binds phosphatidyl serine (PS) residues on the cell membrane. By staining cells with propidium iodide (PI), it is possible to distinguish and analyze non-apoptotic cells (V−/PI−), early apoptotic cells (green, V+/PI−), late apoptotic cells (green, V+/PI+, red) and necrotic cells (green, V+/PI+, red). Cells were also examined under fluorescence microscopy. The results of a representative experiment are shown in Fig. 4E. Flow cytometry quantitative analysis indicated the apoptosis cells to be 6.51% (Tf), 19.15% (PTX) and 28.49% (Tf–Glu–PTX) in MDA-MB-231 cell lines respectively. Fig. 4E showed that the late apoptosis/necrotic value of Tf–Glu–PTX was higher than other groups. The above qualitative and quantitative analysis demonstrate that the PTX conjugated Tf greatly enhanced the antitumor activity with low-dose PTX.

In vivo tumor targeting capability

To investigate the dynamics and tumor targeting ability of Glu–PTX–ICG02 and Tf–Glu–PTX–ICG02, the MDA-MB-231 cells that express high level of Tf receptors were used. Representative fluorescence images after administration of Glu–PTX–ICG02 and Tf–Glu–PTX–ICG02 are shown in Fig. 5. The fluorescent Glu–PTX–ICG02 initially spread in the whole body at about 30 min post-injection and gradually appeared in the tumor, as well as the excretion organs such as the liver, gastrointestinal system, and bladder (Fig. 5A). The Tf–Glu–PTX–ICG02 initially was distributed all over the body and was subsequently cleared by the hepatobiliary and renal pathways (Fig. 5B). However, the tumor sites were detectable within one hour post-injection of the prodrug. Over time, the drug increasingly accumulated in the tumors and the fluorescence intensity peaked at about 4 h. The bright fluorescence signal in tumor gradually disappeared after two days (data not shown).

Fig. 5 Dynamic behavior and targeting capability of PTX prodrugs in MDA-MB-231 tumor bearing mice. (A) Images of the tumor bearing mice after administration of ICG02–Glu–PTX within 24 h. (B) Images of the tumor bearing mice after administration of Tf–Glu–PTX–ICG02 within 24 h. (C) Tumor-to-normal tissue (T/N) ratio in MDA-MB-231 tumor tissues for ICG02–Glu–PTX and Tf–Glu–PTX–ICG02 prodrugs. Statistical analysis indicates that there is significant difference of T/N ratio in the MDA-MB-231 tumors between the two prodrugs (P < 0.05). The data are represented as mean ± standard deviation (SD), n = 5 per group.

To quantify the targeting ability of the drugs in tumor, fluorescence intensity was analyzed by using ROI. The signal dynamics of two drugs (Glu–PTX–ICG02 and Tf–Glu–PTX–ICG02) in tumors are shown in Fig. 5C. Fluorescence intensity ratio between MDA-MB-231 tumor and normal tissue (T/N) was 2.63 ± 0.325 at 1 h post injection of Tf–Glu–PTX–ICG02, reaching a peak at 4 h post injection (5.24 ± 0.34) and slowly decreasing to 3.02 ± 0.43 by 24 h (Fig. 5C). In contrast, MDA-MB-231 tumor-bearing mice intravenously injected with Glu–PTX–ICG02 showed a T/N ratio of 1.51 ± 0.46 after 0.5 h, and a peak uptake at 4 h reached 1.85 ± 0.23 and slowly reduced to 0.96 ± 0.15 by 8 h (Fig. 5C). Statistical analysis indicated a significant difference of T/N ratio in MDA-MB-231 tumors for the two PTX drug (P < 0.05). NIR images of mice with MDA-MB-231 tumor xenografts after injection of Tf–Glu–PTX–ICG02 drug showed good fluorescence intensity and fast tumor targeting in vivo from 0.5 to 48 h, and Tf–Glu–PTX–ICG02 could extend the residence time or concentration of drug in tumor.

In vivo therapeutic experiment

In vivo antitumor efficacy of Tf–Glu–PTX was evaluated in mice bearing MDA-MB-231 tumors by measuring the tumor growth rate and the body weight of the mice. As shown in Fig. 6B, the tumors of Tris-treated mice grew faster than that of the free PTX or Tf–Glu–PTX-treated mice. The inhibition rate of Tf–Glu–PTX-treated MDA-MB-231 tumor (55.79%) is about 13.21% higher than that of PTX-treated tumor (42.58%). In addition, the body weight of mice bearing MDA-MB-231 tumors in Tf–Glu–PTX and Tris-treated groups gradually increased during the treatment period (Fig. 6A). However, a significant decrease of body weight (5 g) in the free PTX-treated group was observed at the end of the treatment period. Body weight of PTX-treated mice bearing MDA-MB-231 tumors reduced dramatically and 80% mice in this group died within 16 days (Fig. 6C), whereas Tf–Glu–PTX-treated mice only slightly reduced. The 16 day survival rates of mice in the Tf–Glu–PTX groups were 85%, whereas the survival rate of the control group decreased to 50% (Fig. 6C).

Fig. 6 In vivo therapy. (A) Animal weights of MDA-MB-231-bearing mice as a function of time (d). (B) Tumor volumes of MDA-MB-231-bearing mice as a function of time (d). Data were given as mean (n = 6). (C) The 16 day survival rates of mice after administration of Tris, free PTX, or Tf–Glu–PTX. (D) Hematoxylin and eosin-stained livers and tumors of Tris pH 8.0 treated mice, PTX-treated mice, or Tf–Glu–PTX-treated mice. Data are given as mean ± SD, (n = 5), P < 0.05.

To further evaluate the antitumor effect of Tf, PTX and Tf–Glu–PTX on nude mice bearing MDA-MB-231 tumor, the tumors were excised for pathological analysis. Fig. 6D displays the representative tissue sections from different mice groups. Tumor tissue in the PTX, Tf–Glu–PTX groups exhibits spotty necrosis, spherical cells and intercellular blank. To evaluate the toxicity of Tf–Glu–PTX, liver from mice in each group were collected at the 5th day and histologic examination was conducted. Compared with the control group, no distinct pathologic changes were found in the liver of the mice injected with Tf–Glu–PTX. In marked contrast, More than 90% of mice in the PTX-treated group showed noticeable pathologic changes. These results indicate that although the survival rates of Tf–Glu–PTX and free PTX groups are the same, the safety and antitumor efficacy of Tf–Glu–PTX is far superior to free PTX.

Discussion

Despite the promise that PTX has engendered, serious drawbacks hamper PTX's clinical usefulness. In particular, PTX lacks selective cytotoxicity between cancer and normal cells, which frequently leads to deleterious side effects. In addition, PTX is a substrate of P-glycoprotein (P-gp), which actively pumps PTX out of the cells and induces drug resistance.^33,34 To overcome these obstacles, we developed a novel active targeting prodrug, Tf–Glu–PTX, which exhibited significantly stable PTX release rate, specific tumor-targeting capability, high anticancer efficiency, as well as reduced PTX resistance.

Tf has also been actively pursued as a drug delivery vehicle due to its unique receptor-mediated endocytosis pathway as well as its additional advantages of being biodegradable, nontoxic, and nonimmunogenic.³⁵ Previously, studies found that Tf can improve the targeting ability of Tf-conjugated NPs through a receptor-mediated endocytosis pathway.³⁶ Our study suggested that Tf carriers-based prodrugs had high selectivity as well as high accumulation and retention rates in tumors, with the targeting ability correlated with the TfR protein expression levels in cancer cells and normal cells (Fig. 3A). In vitro tumor targeting ability of Tf–Glu–PTX was studied by fluorescence imaging of MDA-MB-231, A549, MCF-7 cells and 293T cells incubated with free PTX or Tf–Glu–PTX (Fig. 3B and C). The results using four kinds of cell lines showed that Tf–Glu–PTX was translocated into the cytoplasm after incubation of 8 h, whereas only a very low level of free PTX was observed inside these cell lines. On the other hand, the uptake of PTX in 293T cells was higher than that of Tf–Glu–PTX due to the fact that the uptake of free PTX was mainly through molecular diffusion but the uptake of Tf–Glu–PTX was mostly mediated by TfR. The high tumor-targeting ability of the Tf-based prodrug has also been shown in tumor-bearing mice (Fig. 5B). In vitro and in vivo targeting experiments indicated that Tf-based prodrug had great potential for targeted cancer therapy. Tf–Glu–PTX initially was spread in the whole animal after 30 minutes. The tumor sites were identifiable at 2 hour post-injection and the probe was gradually accumulated in the tumor sites. Strong fluorescence emission from the tumors was observed even at 24 h post-injection. Tumor contrast as quantified by ROI analysis of optical imaging shown in Fig. 5C indicated that the tumor/normal tissue ratio was reduced from 5.24 ± 0.34 to 3.02 ± 0.43 and 1.85 ± 0.23 for Glu–PTX–ICG02 with free PTX, respectively.

In this study, MTT assay and IC50 were conducted to evaluate the therapeutic efficacy of PTX in the Tf-based prodrugs on cancer cells and its potential toxicity to normal cells. The results in Fig. 4 and ESI Table 1† indicated that Tf–Glu–PTX effectively reduced the viability of cancer cells while having little cytotoxicity in normal cells compared to free PTX. As shown in Fig. 4A and B, Tf–Glu–PTX had a higher therapeutic efficacy for cancer cells than free PTX. Fig. 4A shows that there is a significantly decrease in cell viability of PTX-treated 293T cells due to the high cytotoxicity of non-targeting PTX. IC50 value displayed that Tf–Glu–PTX has negligible cytotoxicity in 293T normal cells. Histologic studies on animal livers consistently showed that the toxicity of Tf–Glu–PTX was reduced, as shown in Fig. 6D.

We investigated the inhibition rates of PTX-resistant MDA-MB231/PR cells and PTX-sensitive MDA-MB-231/PS cells using Tf–Glu–PTX. As shown in Fig. 4E, Tf–Glu–PTX was identified in MDA-MB-231/PR, primarily due to the tumor-targeting capacity of the prodrug with the Tf ligand. The fluorescence emission from Tf–Glu–PTX–FITC treated cells (both PTX sensitive and PTX resistant) was much higher than that from FITC–Glu–PTX-treated cells (Fig. 4E), which suggests that Tf–Glu–PTX has the potential to prevent overcome PTX-resistance in treating multidrug-resistant tumor cells. After being modified with Tf, the prodrug has targeted the tumor cells and to a certain degree escaped the effluxing of P-glycoprotein, which is the molecular alteration found to be most consistently associated with the multidrug resistance phenotype and has been correlated with the degree of resistance.^37–40 Thus, special conjugation of PTX to Tf carries resulted in its increased anticancer efficiency.

The antitumor efficiency of Tf–Glu–PTX was studied both in vitro and in vivo. In vitro study showed that Tf–Glu–PTX had a higher tumor cell inhibition rate compared to free PTX. The cellular uptake and apoptosis-inducing effect of the PTX-conjugated Tf prodrugs, fluorescence microscopy and flow cytometric analysis were used for qualitative and quantitative analysis. The Tf–Glu–PTX prodrug induced a remarkable accumulation of necrotic cell population (28.49%), compared to that of the PTX monomer (19.15%) or Tf (6.51%). The impressive therapeutic efficacy of Tf–Glu–PTX may be because of that Tf system transported more prodrugs to the tumor site by receptor-mediate internalization. The in vivo study indicated that the anticancer effect of Tf–Glu–PTX was significantly greater than that of free PTX. Furthermore, mice treated with Tf–Glu–PTX showed a slight increase in body weight, whereas mice treated with free PTX exhibited a significant weight loss, as shown in (Fig. 6A). Fig. 6D displays the representative tissue sections from different groups of mice. Tumor tissues in the PTX and Tf–Glu–PTX groups exhibit spotty necrosis, spherical cells and intercellular blank. Finally, the tumor tissue in Tf–Glu–PTX group had intercellular blank space. These results suggested that the Tf–Glu–PTX greatly reduced the systemic toxicity and enhanced the antitumor activity of PTX.

Conclusions

Tf–Glu–PTX increased tumor-targeting capability and decreased toxicity to normal cells. It is also potential to circumvent paclitaxel resistance in cancer cells. In vitro and in vivo studies demonstrated that PTX-conjugated Tf carriers shown higher apoptosis and stronger inhibition rate in drug-resistant MDA-MB-231 tumor cells than the free drug. The toxicity study and treatment experiment indicated that PTX-conjugated Tf drug delivery system could significantly decrease organ toxicity compared to free PTX. Taken together, the PTX-conjugated Tf carrier is a prospective targeting drug delivery system for tumor therapy.

Experimental

Materials

Transferrin protein was purchased from Sino Geno Max Co (Beijing, China). Paclitaxel (PTX, MW 845.9) was purchased from Jiangsu Research Institute (WuXin, China). Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu(OtBu)–OH, MW 425.49), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, MW 191.7), N-hydroxysuccinimide (NHS, MW 115.09), 2-methylpiperidine and fluorescein isothiocyanate (FITC, MW 389.4). ICG-Der-02 (ICG02 MW 995) was prepared in State Key Laboratory of Natural Medicines of China Pharmaceutical University. All other solvents and reagents used in this study were certified analytical reagent grade, and were from Sigma Chemical (St. Louis, MO).

Synthesis of transferrin-conjugated paclitaxel prodrugs

Synthesis of glutamate–paclitaxel (Fmoc-Glu(OtBu)–PTX). The glutamate–paclitaxel compound was synthesized as shown in Fig. 1A. First, PTX (100 mg, 0.117 mmol, 1 equiv.) and Fmoc-Glu(OtBu)–OH (59.7 mg, 0.1404 mmol, 1.2 equiv.) were dissolved in CH₂Cl₂ (10 mL), and 4-dimethylaminopyridine (DMAP, 14.27 mg, 0.117 mmol 1 equiv.) was subsequently added. Cold EDC (5 mL CH₂Cl₂) was added dropwise over 20 min to the mixture and stirred at room temperature for 20 h. The organic layer was washed with water, saturated aqueous NaHCO₃, and dried over MgSO₄. The residue obtained after evaporation (vacuum) of the organic solvent was purified by recrystallization from diethyl ether. Secondly, Fmoc-Glu(OtBu)–PTX (45 mg, 0.0196 mmol) was dissolved in CH₃OH and CH₃OH–HCl was added dropwise (1 mL) over 5 min to mixture. The residue obtained after evaporation (vacuum) of the organic solvent was purified by recrystallization from diethyl ether. The purified Fmoc-Glu(COOH)–PTX was obtained as green solid in 95% yield. The molecular weight of purified Fmoc-Glu(COOH)–PTX was determined by LC-MS.

Synthesis of transferrin–glutamate–paclitaxel (Tf–Glu–PTX) prodrug. 352.48 mg Fmoc-Glu(COOH)–PTX in 3 mL DMSO was activated with EDC and NHS (molar ratio is 1 : 1.5 : 1.5) at room temperature in dark for 3 h. Then, the activated Fmoc-Glu(COOH)–PTX was added in 4 mL Tris buffer (10 mM, pH 8.0) containing Tf (100 mg, 1.25 μmol mL⁻¹). The mixture was stirred in dark for 12 h at room temperature, the reaction mixture was purified by filtration over a Sephadex G-10 column equilibrated with Tris buffer (10 mM, pH 8.0) to remove unconjugated Tf and Fmoc-Glu(COOH)–PTX fragments. The resulting product Tf–(Fmoc)Glu–PTX was stored at −20 °C.

Synthesis of FITC/ICG02-labelled transferrin-conjugated paclitaxel (Tf–Glu–PTX–FITC/ICG02) prodrugs. Fmoc-Glu(OtBu)–PTX (127.49 mg, 0.1 mmol) was dissolved in CH₂Cl₂ (5 mL), and 2-methylpiperidine (1 mL) was added to the solvent for the de-protection and the mixture was stirred at room temperature for 12 h. The residue obtained after evaporation (vacuum) of the organic solvent was purified by recrystallization from diethyl ether. The purified –NH₂–Glu(OtBu)–PTX was obtained as light yellow solid in 88% yield. The –NH₂–Glu(OtBu)–PTX (105.27 mg, 0.1 mmol) was dissolved in CH₃OH, and the activated FITC (38.93 mg, 0.1 mmol) was added to the mixture, which was stirred at room temperature in the dark for 15 h. The reaction mixture was purified by recrystallization from acetone. The purified FITC–Glu(OtBu)–PTX was obtained as brown solid in 80% yield. 99.5 mg ICG-Der-02 in 5 mL DMSO was activated with EDC and NHS (molar ratio of ICG-Der-02 : EDC : NHS is 1 : 1.5 : 1.5) at room temperature in the dark for 3 h. –NH₂–Glu(OtBu)–PTX (105.6 mg, 0.1 mmol) was dissolved in CH₃OH, and the activated ICG-Der-02 was added to the mixture, which was stirred at room temperature in dark for 20 h. The reaction mixture was concentrated in vacuum and purified by recrystallization from diethyl ether. The purified ICG02–Glu(OtBu)–PTX was obtained as green solid in 85% yield. FITC–Glu(OtBut)–PTX (144.21 mg, 0.1 mmol) or ICG02–Glu(OtBu)–PTX (243.71 mg, 0.1 mmol) was dissolved in CH₃OH and CH₃OH–HCl was added drop wise (1 mL) over 5 min to mixture. The residue obtained after evaporation (vacuum) of the organic solvent was purified by recrystallization from diethyl ether. The purified FITC–Glu(COOH)–PTX was obtained as brown solid in 87% yield. The purified ICG02–Glu(COOH)–PTX was obtained as green solid in 85% yield. 138.9 mg FITC–Glu–PTX or 198.81 mg of ICG02–Glu(COOH)–PTX in 3 mL DMSO was activated with EDC and NHS (molar ratio is 1 : 1.5 : 1.5) at room temperature in the dark for 3 h. The activated FITC–Glu(COOH)–PTX or ICG02–Glu(COOH)–PTX was added in 4 mL Tris buffer (10 nM, pH 8.0) containing Tf (100 mg, 1.25 μmol mL⁻¹). The mixture was stirred in the dark for 12 h at room temperature, the reaction mixture was purified by filtration over a Sephadex G-10 column equilibrated with Tris buffer (10 nM, pH 8.0) to remove un-conjugated Tf and FITC–Glu(COOH)–PTX or ICG02–Glu(COOH)–PTX fragments. The resulting product was stored at −20 °C.

Characterization of PTX targeted prodrugs. Q-TOF Micro Mass Spectrometer (Waters) was used to confirm synthesis of Glu–PTX and FITC–Glu–PTX (ESI†). The purified ICG02–Glu–PTX (MW: 1988.03) was evaluated by TLC under NIR imaging system. The above synthesized Tf–Glu–PTX–FITC and Tf–Glu–PTX–ICG02 were evaluated by spectroscopic measurements at 280 nm, 347 nm, 220 nm, 227 nm and 778 nm, which are the absorption peaks of Tf, FITC, Glu, PTX and ICG-Der-02, respectively. The successful conjugation of PTX to Tf was further confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The size and morphology of Tf, Tf–Glu–PTX were measured by Mastersizer 2000 Laser Particle Size Analyzer (LPSA) and JEM-2100 transmittance electron microscope (TEM, JEOL, Japan). The modified number of PTX molecules to the primary amino groups of Tf carrier (∼679 amino acid residue)²¹ was calculated by following the recovery method (n = 4). The regression equation of standard curve was obtained by dissolving different amounts of pure PTX in CH₃OH.

Stability of ester bond in PTX-conjugated compounds. To investigate the stability of ester bond, free PTX broken from the derivatives was measured under different medium, including Tris buffer (10 nM, pH 8.0), rat plasma and human plasma at 37 °C for up to 12 h. In addition, stability of Tf–Glu–PTX in water at 0 °C, 4 °C and 37 °C were evaluated by HPLC (ESI†). The PTX conjugated intermediate products (5 mg each of Glu–PTX) were dissolved in 2.5 mL each medium. Then, 100 μL each sample was taken at each designated time points. The purified of Tf–Glu–PTX was dissolved in distilled water with 1 m mL⁻¹ concentrate, and was stored at 0 °C, 4 °C and 37 °C, respectively. The free PTX was then extracted by using 1.5 mL ethyl acetate, and measured by reverse-phase high-performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) on a C18 column (300 mm × 3.9 mm Nova-Pak Waters) with acetonitrile/water gradient.

In vitro antitumor activity

Cell culture. The human breast cancer cell lines (MDA-MB-231, MCF7), human live cancer cell line (A549), PTX-sensitive (PS) MDA-MB-231 cell, PTX-resistant (PR) MDA-MB-231 cell (and 293T (human renal epithelial) cell lines were all purchased from ATCC. The cell line was cultured at 37 °C in a humidified atmosphere containing 5% CO₂ in DMEM and RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin.

Targeted-ability of Tf-conjugated PTX in tumor cells. Four cell lines (MDA-MB-231, MCF-7, A549 and 293T) were used for Tf receptor protein expression evaluation by western blot analysis were prepared as described previously.³¹ Cells were incubated in RIPA buffer (30 min, 4 °C with mild shaking) and centrifuged (30 min, 4 °C, 16 100g). 50 mg of protein samples were loaded in 4–12% gradient polyacrylamide pre-casted gels, ran (90 min, 100 mV) and transferred onto nitrocellulose membrane using iBlot transfer stacks.

To investigate the tumor cell targeting of Tf–Glu–PTX, the prodrug was labeled with visible fluorescent dye FITC for microscopy. Different cells were respectively seeded in the 6-well plates and incubated in culture medium overnight. Then, cells were incubated in a 200 μL solution of Tf–Glu–PTX–FITC (1 mg mL⁻¹) for 8 hours. After washing with PBS, the cells were directly visualized by fluorescence microscopy (40× objective magnification). Cell cytometry was used to further quantify the uptake of Tf–Glu–PTX–FITC into the cell lines. Briefly, 5 × 10⁵ cells were seeded in 6-well plates and incubated in culture medium overnight. Subsequently, 200 μL of 1 mg mL⁻¹ Tf–Glu–PTX–FITC was added into each well. After 8 h incubation at 37 °C, the cells were re-suspended in 500 μL PBS. The cell suspension was immediately analyzed by a flow cytometry.

In vitro therapeutic efficacy. To evaluate the antitumor activity and cytotoxicity of Tf–Glu–PTX, MTT assays were conducted on MCF-7, A549 and 293T cell lines by following standard protocol. Cells were plated at a density of 5 × 10³ cells per well in 96-well plates and subsequently infected for 24 h with PTX, Tf–Glu–PTX at the same concentration from 0.03125 to 1 mg mL⁻¹. Each drug was tested in 6 wells. Then MTT solution (20 μL, 5 mg mL⁻¹) was added into each well. The optical density (OD) was measured at 595 nm with a multi-well plate reader. PTX-resistance was evaluated in PTX-sensitive and PTX-resistant MDA-MB-231 cells. After 24 hour cultivation, Tf, Tf–Glu–PTX and free PTX of different concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 mg mL⁻¹) were added into the cells of the 96 wells and incubated for 24 hours (n = 6). Then MTT solution (20 μL, 5 mg mL⁻¹) was added into each well. The absorbance of the solution in each well was measured at 595 nm with a multi-well plate reader. The mean percentage of cell survival relative to that of control cells was determined from data of four individual experiments, and all the data were expressed as mean ± SD.

Apoptosis study. Annexin-V staining was performed to identify the cell apoptosis by using annexin-V-fluorescein isothiocyanate, which specifically binds phosphatidyl serine (PS) residues on the cell membrane. Propidium iodide (PI) was used to identify the cell apoptosis by binding to DNA once the cell membrane has become permeable.¹⁹ Briefly, cells were plated at a density of 5 × 10⁴ cells per well (MDA-MB-231) in 6-well plates and subsequently infected for 24 h with 100 μL Tris (control), Tf, PTX, Tf–Glu–PTX. Tf in these prodrugs was fixed at conjugated PTX was 0.1 mg mL⁻¹. Free PTX was added 0.1 μL (1 mg mL⁻¹). Each concentration was tested in 6 wells. After incubation for 24 h, the cells were washed twice with PBS and detached by trypsin (0.25%), followed by centrifugation at 1000 rpm for 5 min. The medium was discarded and the cells were re-suspended in 500 μL of binding buffer, 5 μL annexin V-FITC and 5 μL propidium iodide were subsequently added to mixture. After incubation at room temperature in the dark for another 10 min, the cell suspension was immediately analyzed by a flow cytometry.

In vivo study

Animal subjects and tumor model. Athymic nude mice were purchased from Charles River Laboratories (Shanghai, China) for prodrug investigations. All experiments (animals, human plasma and human cell) were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China (document no. 55, 2001) and approved by the Laboratory Animal Welfare & Ethics Committee of Suzhou University (SYXK: 2015-0182).

MDA-MB-231 tumor cell lines (6 × 10⁶) were implanted in the upper right axillary fossa in the nude mice (n = 5). As the tumors grew to a diameter of 0.2–0.5 cm, the mice were used to for therapy study.

Dynamics and targeting ability of Tf–Glu–PTX–ICG02 in tumor bearing mice. Nude mice bearing MDA-MB-231 tumor were used for the investigation of the dynamics and targeting ability of the prodrugs (n = 5, each group). ICG02–Glu–PTX (0.2 mL, 5 mg kg⁻¹) and Tf–Glu–PTX–ICG02 (0.2 mL, 5 mg kg⁻¹) were administered into the blood stream of the subject mice groups through tail vein injection. Fluorescence imaging was acquired with our NIR imaging system using the method reported previously.³² A series of images were collected from mice and the background images were taken for each mouse prior to injection.

To quantify the targeting ability of these drugs in MDA-MB-231 tumor, the average fluorescence intensity in tumor regions was acquired by selecting the region of interests (ROI) and compared with normal muscle tissue.³³ Tumor to normal tissue contrast ratios (T/N) were calculated by using the ROI functions of Living Image software.

In vivo antitumor activity. Nude mice-bearing MDA-MB-231 tumors were randomly assigned into 5 groups (n = 5 per group). The mice in each group were treated every 3 day for 15 days via tail vein injection with different solutions (0.2 mL): (A) Tris buffer (10 nM, pH 8.0); (B) pure PTX solution (5 mg kg⁻¹); (C) Tf–Glu–PTX (5 mg kg⁻¹). The therapeutic efficacies and systematic toxicities of Tf–Glu–PTX on these tumor bearing mice were assessed by measuring tumor volume and body weight of each mouse every day.

Histology examination. To further investigate the side effects of Tf-conjugated PTX on other organs, histology analysis of liver and tumor tissues of the treated mice was conducted. All sliced organs were stained with hematoxylin and eosin, and examined under a microscope.

Statistical analysis. All data are reported as the mean ± SD of n independent measurements. Statistical analysis was performed by using Student's t-test with statistical significance assigned for P value < 0.05.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are grateful to Natural Science Foundation Committee of China (NSFC81220108012, 661335007, 81171395 and 81328012), China Scholarship Council (201608340040), Program of Study Abroad for Young Scholar sponsored by Education Department of Anhui Province (gxfxZD2016266), and Key project of Anhui Educational Committee (KJ2014A249, KJ2015A220), and Quality Project of AnHui Education Department – The plan of “ChuangKe” laboratory (2015ckjh109). The Scientific Research Platform of Suzhou University (2015ykf15).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra15141h

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