Novel folate-targeted docetaxel-loaded nanoparticles for tumour targeting: in vitro and in vivo evaluation

Abstract

Cholesterol-PEG₁₀₀₀ (Chol-PEG) has shown great potential in drug delivery. In our work, the synthesis of Chol-PEG₁₀₀₀-FA (folic acid) was firstly achieved via an amide reaction of NH₂ on the surface of Chol-PEG₁₀₀₀-NH₂, and then docetaxel-loaded Chol-PEG₁₀₀₀-FA nanoparticles were prepared by the precipitation–ultrasonication combined high-pressure homogenization (HPH) technique, and were characterized by particle size, zeta potential and morphology; meanwhile the average drug loading of the resulting DTX/Chol-PEG₁₀₀₀-FA nanoparticles reached up to 68%. The anti-tumor efficiency and targeting ability of docetaxel-loaded Chol-PEG₁₀₀₀-FA nanoparticles (DTX-FA-Nps) were demonstrated by their in vitro and in vivo anti-tumor activity against 4T1 cells. The docetaxel-loaded Chol-PEG₁₀₀₀ nanoparticles with and without FA conjugation proved to be of satisfactory size and distribution, with favorable drug release and high drug encapsulation efficiency. In vitro results showed the higher anti-tumor efficiency of the DTX-FA-Nps and DTX-Nps (docetaxel-loaded Chol-PEG₁₀₀₀ nanoparticles) than docetaxel solutions (DTX-Sol). The tumor-targeting effects of the FA conjugated Chol-PEG₁₀₀₀ are also studied. The IC₅₀ values of DTX-Sol, DTX-Nps and DTX-FA-Nps are 1.6260, 0.3772 and 0.0171 μg mL⁻¹, respectively, for 4T1 cancer cells after 48 h of treatment. The inhibition rate for the DTX-FA-Nps at 10 mg kg⁻¹ was 74.83% (P < 0.01). Based on these results, the DTX-loaded Chol-PEG₁₀₀₀-FA nanoparticles may be a promising targeted delivery system for breast cancer therapy.

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1. Introduction

Currently, the delivery of high doses of drugs to tumour sites in cancer therapy for maximum treatment efficacy and to minimize the side effects on normal organs has been extensively exploited.^1,2 Nanoparticulate drug delivery systems can accumulate in tumour tissues by escaping through abnormally leaky tumour blood vessels,^3–6 enabling them to be useful for drug delivery applications. There have been several reports about the use of nanoparticles to improve conventional cancer therapy in recent years.^7–11 The novel designed nanocarriers are expected to be non-cytotoxic, able to highly and efficiently load the drugs, enhance the circulation time in the bloodstream, and actively target the cancer cells.¹² Nanoparticles with amphiphilic block copolymers are considered to be effective delivery systems due to their unique properties such as stability, high drug loading capacity, appropriate size (20–200 nm), selective accumulation in the solid tumour, and ability to escape the reticuloendothelial system (RES).¹³

Cholesterol-PEG₁₀₀₀ (Chol-PEG₁₀₀₀) is a water-soluble derivative of cholesterol, namely PEGylated cholesterol, which has an amphiphilic structure comprising a lipophilic tail and hydrophilic polar head portion, whose bulky structure and large surface area make it an excellent solubilizer, emulsifier, and bioavailability enhancer of hydrophobic drugs, and long chain PEG can help the nanoparticles escape from being recognized and eliminated by the RES. To further increase the cancer specificity and intracellular uptake, targeted PEG carriers have been developed through synthetic methods, including folates, aptamers, antibodies and peptides. Folic acid (FA) is a promising targeting probe because it can be efficiently internalized into cells through receptor-mediated endocytosis and even conjugated with widely bioactive molecules. Moreover, folate receptors are over-expressed in many types of human cancer cells including breast, ovarian, and prostate cancers, while being little distributed in normal tissues.^14–16 Folic acid has been used widely as a molecule for targeted delivery.¹⁷ For example, it can been used widely in the targeted therapy of many cancers through receptor mediation including leukemic cells,¹⁸ PC3 cells^19,20 and ovarian tumours.²¹ Meanwhile in our work, FA was linked to Chol-PEG₁₀₀₀ in order to induce an active tumour targeting effect on 4T1 cells in mice models; it can be predicted that such newly designed nanocarriers should have a higher cellular uptake of the model drug, higher anti-tumor activity, and thus higher therapeutic effects and lower side effects.

In this study, docetaxel was used as a model anti-cancer drug. Docetaxel is a semi-synthetic analogue of paclitaxel. As reported, docetaxel was shown to be superior to paclitaxel in some preclinical models because of its improved cellular uptake and increased potential for promoting the assembly of microtubules used as an inhibition of the disassembly process of tubulin.²² However, its current clinical dosage form (Taxotere) includes the non-ionic surfactant Tween 80 (polysorbate 80) and ethanol, which were found to cause serious side effects including neurotoxicity, fluid retention and musculoskeletal toxicity.^23–25

Therefore, in this study, we firstly synthesized Chol-PEG₁₀₀₀-FA as a stabilizer to encapsulate DTX, which has not yet been reported.²⁶ The average drug loading of the resulting DTX/Chol-PEG₁₀₀₀-FA nanoparticles reached up to 68%, which was much higher than the reported nanoparticles (about 17.2%).²⁷ The results of in vitro and in vivo antitumor activity tests demonstrated the higher antitumor efficiency of the DTX-FA-Nps than that of DTX-Nps (docetaxel-loaded Chol-PEG₁₀₀₀ nanoparticles) and docetaxel solutions (DTX-Sol).

In short, our newly designed nanoparticles (docetaxel-loaded Chol-PEG₁₀₀₀-FA nanoparticles) can successfully reach target tumour cells and exhibit good anti-tumor activity, and can therefore be considered to be a promising targeted delivery system for anticancer agents.

2. Materials and methods

2.1 Materials

Docetaxel (99%) was provided by Dalian Meilun Biology Technology Co., Ltd, Chol-PEG₁₀₀₀ was purchased from Biomatrik Biology Technology Co., Ltd, folic acid, N,N-dicyclohexylcarbodiimide (DCC), and dimethyl sulfoxide (DMSO), were all provided by Sinopharm Chemical Reagent Beijing Co., Ltd. Fetal bovine serum (FBS) was provided by Zhejiang Tianhang Biological Technology Stock Co., Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM) and MTT was purchased from Sigma. 1,1′-Dioctadecyltetramethyl indotricarbocyanine iodide (DiR) and Cy5.5 (a fluorescent probe) were purchased from AAK (USA). The water used was deionized in the experiments, and all organic solvents were of the highest commercially available grade.

2.2 Cell line and animals

The murine breast cancer cell line 4T1 and normal cell line (HUVEC cells) were purchased from Beijing Acupuncture & Herb Clinic. The cells were cultured in RPMI1640 (contain 10% fetal bovine serum, 100 U per penicillin and 100 μg mL⁻¹ streptomycin) at 37 °C in a humidified atmosphere containing 5% CO₂.

Six- to eight-week-old BALB/c mice (female) and nude mice (female) were purchased from the Experimental Animal Center of Academy of Military Medical Sciences (Beijing, China) and kept under SPF conditions for 1 week before the study with free access to standard food and water. All of the studies complied with the principles of care and use of laboratory animals of the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences institute of medicinal plant animal laboratory.

2.3 Synthesis of Chol-PEG₁₀₀₀-FA

Folic acid (70.624 mg, 0.16 mmol) and DCC (70 mg, 0.33 mmol) were added to a solution of anhydrous DMSO (5 mL) and pyridine (1 mL); Chol-PEG₁₀₀₀-NH₂ (100 mg, average M_r1000) was then added to the mixture while stirring. Then, the mixture was maintained in the dark at 25 °C for 24 h, 10 mL of water was added and then the mixture was centrifuged at 9000 rpm for 30 min. The supernatant was removed and dried.²⁸ The crude product was further separated by preparative thin layer chromatography (developing agent: CHCl₃ : MeOH : H₂O 75 : 36 : 6) to obtain Chol-PEG₁₀₀₀-FA. The conjugate was characterized by ¹HNMR (Bruker AV300) and UV-vis (solvent; ethyl alcohol).

2.4 Preparation of DTX-FA-Nps

DTX-FA-Nps were prepared using the precipitation–ultrasonication combined high pressure homogenization method. Briefly, 15 mg DTX, 3.75 mg Chol-PEG₁₀₀₀ and 1.25 mg Chol-PEG₁₀₀₀-FA were dispersed into 0.5 mL of alcohol and 0.5 mL of acetone to form an organic solution. Afterwards, 1 mL of the organic solution was added into 5 mL of aqueous solution drop by drop under continuous ultrasonication (250 W). Acetone, alcohol and a portion of the volume of water were removed on a rotary evaporator under vacuum and then processed using a homogenizer at 1500 bar for 15 cycles. As a reference, nanoparticles without Chol-PEG₁₀₀₀-FA were prepared under an identical protocol as that used for Chol-PEG₁₀₀₀-FA, but without Chol-PEG₁₀₀₀-FA in the organic phase. The FA-coated and uncoated nanoparticles were called DTX-FA-Nps and DTX-Nps for short, respectively.

2.5 Characterization of DTX-Nps and DTX-FA-Nps

2.5.1 Particle and zeta potential characterization. The average particle size, polydispersity index (PDI), and zeta potential of DTX-Nps and DTX-FA-Nps were determined using dynamic light scattering (DLS) (Zetasizer Nano ZS 90, Malvern Instruments, UK). All measurements were made at least in triplicate.

2.5.2 Morphology observation by transmission electron microscope (TEM). The morphologies of DTX-Nps and DTX-FA-Nps were observed using a JEM-1400 electron microscope (JEOL Ltd., Tokyo, Japan). A drop of the Nps was spread on a 200 mesh copper grid and negatively stained with 2% (w/v) acetic acid glaze for 30 s. The grid was allowed to dry further for 10 min and was then examined with the electron microscope.

2.6 Entrapment efficiency and drug loading

The drug entrapment efficiency (EE) and drug-loading (DL) amount of DTX-Nps and DTX-FA-Nps were determined according to the method of centrifugation ultrafiltration.²⁹ Generally, DTX-Nps and DTX-FA-Nps were ultracentrifuged by an Optima™ L-80 XP Ultracentrifuge (Beckman Coulter, Fullerton, California) at 50 000 rpm for 60 min. The supernatant was sampled, and the concentration of DTX was determined by a reversed-phase high-performance liquid chromatography (HPLC) method. A 20 μL sample solution was injected at least three times at a volume of 20 μL into the chromatographic column. The mobile phase was a mixture of water and acetonitrile in a volume ratio of 10 : 90. The elution rate was 0.3 mL min⁻¹ and the DTX detection wavelength was set at 230 nm. The standard curve for the quantification of DTX was linear over the range of 1.5625–100 μg mL⁻¹ with a correlation coefficient of 0.9999. The EE and DL of the drug-loaded nanoparticles were calculated according to the following formula:

EE (%) = (weight of DTX in the nanoparticles/initial weight of DTX used) × 100%

DL (%) = (weight of DTX in the nanoparticles/total weight of nanoparticles) × 100%.

2.7 In vitro release behaviour

The drug release from the DTX-Nps and DTX-FA-Nps was analyzed using dialysis bag diffusion. DTX-Nps and DTX-FA-Nps (1 mL, 1 mg mL⁻¹ DTX) were placed in a preswelled dialysis bag (molecular cutoff = 8000–14 000). The dialysis bag was then incubated in 100 mL of phosphate buffer (pH 7.4) and cellular conditions (RPMI1640 containing 10% fetal bovine serum, 100 U per penicillin and 100 μg mL⁻¹ streptomycin) with gentle shaking and kept in an incubator at 37 ± 0.5 °C. The incubation medium was changed at every predetermined time point. The collected incubation medium (containing the released drug) was freeze-dried and dissolved in acetonitrile; the drug quantity was determined by the same HPLC procedure as mentioned above. All measurements were made in triplicate.

2.8 In vitro cytotoxicity of DTX against 4T1 cells

2.8.1 Cellular uptake studies. Cy5.5 (0.375 mg) was co-dissolved with mPEG₁₀₀₀-Chol in acetone according to section “2.4 Preparation of DTX-FA-Nps”. Cells were incubated with DTX-FA-Nps, DTX-Nps and DTX-Sol (DTX concentration: 10 μg mL⁻¹, Cy5.5 dose: 0.25 μg mL⁻¹) at 37 °C and 5% CO₂ for 4 h. The FA inhibition group was injected with 200 μL of a FA solution 0.5 h before adding the Cy5.5 DTX/Chol-PEG₁₀₀₀-FA nanoparticles. The cells were then thoroughly washed with PBS three times. Images were obtained using a Leica TCS SP2 microscope. DTX was observed using an Ar/ArKr 458/488 nm laser and the emission wavelength was read from 560 to 610 nm and expressed as red. Images were produced using the lasers sequentially with a 20 objective lens.

2.8.2 MTT assay. The cell viability against 4T1 cell and normal cell lines (HUVEC cells) were measured using the MTT calorimetric assay. Cells at a density of 1 × 10⁴ cells per well were seeded in 96-well culture plates. After 24 h of incubation, the medium was replaced by DTX-Nps, DTX-FA-Nps and DTX solution (DTX-Sol) at various concentrations for 24, 48 and 96 h (the above were discarded and changed to fresh drug solution at 48 h). Subsequently, to each well was added 20 μL of MTT (5 mg mL⁻¹) and the cells were then incubated for 4 h at 37 °C. The medium was discarded and 150 μL of DMSO was added to each well. The light absorbance of each well was measured on a ThermoMax Microplate Reader at a test wavelength of 570 nm. The cell inhibitory rate was calculated as follows: inhibitory rate = (Abs₅₇₀ control cells − Abs₅₇₀ treated cells)/Abs₅₇₀ control cells × 100%. All assays were done with three parallel samples. The IC₅₀ value was defined as the drug concentration required to inhibit growth by 50% relative to the controls.

2.9 In vivo antineoplastic activity of DTX-Nps and DTX-FA-Nps

The in vivo antineoplastic activity of DTX-Nps and DTX-FA-Nps was assessed using 4T1-tumor bearing mice as the animal model. BALB/c mice were inoculated subcutaneously with 4T1 breast tumour cells (1 × 10⁶ cells per mouse) in the axillary region. At 24 h post-inoculation the 4T1 bearing mice were randomly assigned to the following six groups (10 per group): negative control, DTX-Sol 10 mg kg⁻¹ group, DTX-Nps 10 mg kg⁻¹ group, DTX-Nps 20 mg kg⁻¹ group, DTX-FA-Nps 10 mg kg⁻¹ group and DTX-FA-Nps 20 mg kg⁻¹ group. The drugs were administered by injection through the tail vein, once every 3 days for 22 days. The tumour size was monitored every other day using a two-dimensional caliper (length and width). The tumour volume was estimated using the following equation: V = length × (width) 2/2 by measuring the tumour dimensions with a vernier caliper. Additionally, the body weight and survival rate of the mice in each group were also monitored for 22 days. On day 22, the animals were sacrificed.

2.10 Biodistribution by fluorescence imaging

For analysis of the in vivo tumour targeting of the nanoparticles, the near-infrared dye DiR was loaded into DTX-Nps and DTX-FA-Nps. The preparation method of DiR loaded nanoparticles was exactly the same as that of DTX-loaded nanoparticles, except that DTX was replaced by DTX and DiR. The amount of DiR in the nanoparticles was determined by a reversed-phase high-performance liquid chromatography (HPLC) method. BALB/c nude mice (female, 18–22 g) were inoculated via injection in the right side of the axillary region with 1 × 10⁶ 4T1 cells. When the volume of tumours reached 100 mm³, the mice were randomly assigned to four groups: (i) DiR loaded DTX-Sol 10 mg kg⁻¹ group, (ii) DiR loaded DTX-Nps 10 mg kg⁻¹ group, (iii) DiR loaded DTX-FA-Nps 10 mg kg⁻¹ group, and (iv) DiR loaded DTX-FA-Nps 10 mg kg⁻¹ group (FA inhibition). The FA inhibition group was injected with 200 μL of a FA solution 0.5 h before administering the Dir loaded DTX-FA-Nps. All mice were administrated with the formulations via i.v. injections (tail vein) and then anesthetized with isoflurane, and the in vivo fluorescence imaging was carried out with a MAESTRO in vivo imaging system (Hopkinton, USA) at predetermined times (0.5, 1, 2, 4, 8, 24, 48, 72 and 96 h). At 96 h, the mice were sacrificed and the tumours as well as organs were excised from the mice. Again, fluorescence images of the tumours and organs were taken under the same conditions mentioned above.

2.11 Statistical analysis

Values are expressed as mean ± SD for all treatments. Statistical analysis of the inhibitory effect on tumour growth was performed using one-way analysis of variance. For P-values that were 0.05 or less, the difference was considered significant.

3. Results and discussion

3.1 Synthesis and characterization of Chol-PEG₁₀₀₀-FA

The Chol-PEG₁₀₀₀-FA synthesis process is shown in Scheme 1. Chol-PEG₁₀₀₀-FA conjugates were synthesized by an acid amide reaction between the carboxyl group of folic acid and the amino group of Chol-PEG₁₀₀₀-NH₂ using pyridine as an activator and DCC as a coupling agent.²⁸ The γ-activated carboxyl group of FA was reportedly more accessible;^30–32 therefore, the acid amide reaction may have occurred at the γ-carboxyl group, resulting in the γ-activated derivative. Chol-PEG₁₀₀₀-FA showed a single point on the thin plate, but Chol-PEG₁₀₀₀-NH₂ was significantly different with the FA. Fig. 1A shows the ¹H NMR spectra of Chol-PEG₁₀₀₀-FA in CDCl₃; the peaks at 3.1–4.4 ppm and 5.3 ppm are attributed to the –CH₂– protons and PEG₁₀₀₀ protons of Chol-PEG₁₀₀₀-NH₂, respectively. From the UV-vis spectrum (Fig. 1B), an absorption peak at 280 nm and a broad shoulder peak at 370 nm were observed both in FA and Chol-PEG₁₀₀₀-FA, and their absorption peaks were not found on the Chol-PEG₁₀₀₀-NH₂ spectrum, implying that FA was linked to Chol-PEG₁₀₀₀-NH₂.³³

Scheme 1 Synthesis of Chol-PEG₁₀₀₀-FA. Reagents and conditions: DMSO and pyridine, DCC, 25 °C, 24 h. DCC = dicyclohexylcarbodiimide; DMSO = dimethyl sulfoxide.

Fig. 1 ¹H NMR spectra of Chol-PEG₁₀₀₀-FA (A). UV-vis analysis of Chol-PEG₁₀₀₀-NH₂, Chol-PEG₁₀₀₀-FA and folic acid (B).

3.2 Characterization of drug-loaded nanoparticles

3.2.1 Particle size and zeta potential of DTX-Nps and DTX-FA-Nps. The results of the size and size distribution of DTX-Nps and DTX-FA-Nps showed that the mean particle diameter of the DTX-Nps was 258.8 ± 3.4 nm with a PDI of 0.248 ± 0.015, while for DTX-FA-Nps, the mean particle size was 275.8 ± 2.6 nm with a PDI of 0.235 ± 0.116, suggesting that the nanoparticles still retained a uniform size after the conjugation reaction. The particle size results indicate that the incorporation of the targeting ligand on the nanoparticles’ surface (DTX-FA-Nps) led to a slight increase in size in comparison to the DTX-Nps.

Moreover, the zeta potential of DTX-FA-Nps (−14.6 ± 1.9) is slightly more positive than that of DTX-Nps (−17.0 ± 0.83). Surface charge is an important factor for the stability of the nanoparticle system. The repulsion of the same type of surface charge among the nanoparticles provides extra stability.³⁴ Besides, the surface charge of the nanoparticles has a significant effect on the cellular uptake. Usually a positive charge will promote the internalization rate of the nanoparticles and further increase their internalization amount, which is possibly due to electrostatic attraction between the negatively charged cell surface and the positively charged nanoparticles. In contrast, the negatively charged nanoparticles will have less cellular uptake due to their electrostatic repulsion.^35,36 However, in the in vivo study, positively charged nanoparticles are usually faster removed from the circulation system due to their quicker recognition as foreign objects.³⁷

3.2.2 Morphology observation. The morphologies of DTX-Nps and DTX-FA-Nps examined by TEM are shown in Fig. 2A and B, respectively. The sizes of both DTX-Nps and DTX-FA-Nps were about 100 nm and the nanoparticles presented a nearly spherical morphology. The mean diameters of the nanoparticles determined by TEM were less than the values measured by DLS. The diameter obtained from the DLS reflected the hydrodynamic diameter of the nanoparticles, while the diameter observed by TEM indicated dried nanoparticles.³⁸ Therefore, the size obtained from TEM was smaller than by DLS, probably resulted from the dehydration and shrinkage of high-density particles during sample preparation.

Fig. 2 TEM images of DTX-Nps (A) and DTX-FA-Nps (B) (n = 3). Scale bar: 500 nm.

3.3 Drug entrapment efficiency and loading capacity

Drug entrapment efficiency and loading capacity were measured to further evaluate the preparation of the nanoparticles. The entrapment efficiencies of the DTX-Nps and DTX-FA-Nps were 91.23% and 86.59%, respectively, higher than the reported nanoparticles (below 74%).^39,40 The average drug loading of the DTX-Nps and DTX-FA-Nps was 70.81% and 68.94%, respectively, higher than the reported nanoparticles (about 17%).²⁷ The results show the entrapment efficiency and drug loading of the DTX-FA-Nps were lower than that of the DTX-Nps, which might be because the Chol-PEG₁₀₀₀-FA polymers obstructed DTX from entering the lipid core of the nanoparticles in the course of the formation of DTX-FA-Nps.⁴¹

3.4 In vitro drug release

Fig. 3 shows the accumulated percentage release of DTX from the DTX-Sol, non-targeted DTX-Nps and targeted DTX-FA-Nps in the medium of PBS (pH 7.4) and cellular conditions (RPMI1640 containing 10% fetal bovine serum, 100 U per penicillin and 100 μg mL⁻¹ streptomycin) to simulate the sink condition. DTX-Sol, DTX-Nps and DTX-FA-Nps all exhibited a sustained release without any burst release (Fig. 3). The drug release profiles of DTX-Nps and DTX-FA-Nps were very close, while DTX-Sol released the drug a little faster. On the whole, the drug release of these three formulations was faster in the cell culture medium than in PBS, all reaching a plateau at 96 h in contrast to more than 160 h in PBS. The faster drug release in the cell culture medium may be partially ascribed to the presence of 10% fetal bovine serum.

Fig. 3 Release profiles of DTX from DTX-Sol, DTX-Nps and DTX-FA-Nps. The assay was dialyzed against phosphate buffer (A) and cellular conditions (RPMI1640 containing 10% fetal bovine serum, 100 U per penicillin and 100 μg mL⁻¹ streptomycin) (B) at 37 ± 0.5 °C. The results are presented as the mean ± SD (n = 3).

3.5 In vitro cytotoxicity

3.5.1 Cellular uptake studies. Cy5.5 was loaded into the DTX-FA-Nps, DTX-Nps, and DTX-Sol as a tracer to study their cell internalization into the cells using a Leica TCS SP2 microscope. As shown in Fig. 4, the cellular uptakes of DTX-FA-Nps and DTX-Nps were different from that of DTX-Sol. DTX-FA-Nps easily entered the cells, as evidenced by the strong fluorescence throughout the cells; only very weak Cy5.5 fluorescence was observed in DTX-Nps, especially in the DTX-Sol and DTX-FA-Nps (FA inhibition) group. These results imply that folate decoration could clearly increase the cellular uptake of the nanoparticles. The high expression of folate receptors on the 4T1 breast cancer cells caused a great proportion of targeted nanoparticles (DTX-FA-Nps) to be taken up and further internalized by the cells compared to the non-targeted nanoparticles (DTX-Nps). Therefore, we firmly believed that the DTX-FA-Nps with smaller hydrodynamic size would have a higher cellular uptake efficiency.^42,43

Fig. 4 Cellular uptake study of DTX-FA-Nps, DTX-Nps, DTX-Sol and DTX-FA-Nps (FA inhibition) using a Leica TCS SP2 microscope (from left to right). The Cy5.5-loaded nanoparticles and DTX-Sol (Cy5.5 dose: 0.25 μg mL⁻¹) were cultured individually with 4T1 cancer cells at 37 °C for 4 h.

3.5.2 MTT assay. In vitro cytotoxicity against the 4T1 cell lines was assessed after 24, 48 and 96 h incubation with the DTX-Nps and DTX-FA-Nps at 37 °C with DTX-Sol as a control at the equivalent drug concentration. 4T1 cells were used because of their folate receptor overexpression.⁴⁰ The cytotoxicity of DTX-Sol, DTX-Nps, and DTX-FA-Nps against 4T1 cells has been studied again and the MTT results for 24, 48 and 96 h are all obtained. It is true that DTX-Nps and DTX-FA-Nps showed little difference in cytotoxicity at 24 h (IC₅₀, 7.7000 vs. 0.7745 μg mL⁻¹, P > 0.05), and the cell inhibition rates of DTX-Sol were lower than 50% at all doses (0.001–10 μg mL⁻¹). At 48 h, DTX-Sol, DTX-Nps, and DTX-FA-Nps all inhibited the growth of 4T1 cells in a concentration-dependent manner (Fig. 5A), but DTX-Nps exhibited much higher cytotoxicity than DTX-Sol (IC₅₀, 0.3772 vs. 1.6269 μg mL⁻¹, P < 0.01) and DTX-FA-Nps exhibited much higher cytotoxicity than DTX-Nps (IC₅₀, 0.0171 vs. 1.6269 μg mL⁻¹, P < 0.01). It was possible that the small size of the NPs facilitated their adhesion to the cells and increased the contact area and time between the drug and the cells.^44,45 It is generally considered that nanoparticles can be non-specifically internalized into cells via endocytosis or phagocytosis.⁴⁶ In addition, it is possible that the nanoparticles were not actively internalized by cells, but adsorbed non-specifically by pinocytosis after accumulating on the surface of the cells.⁴⁷

Fig. 5 Inhibition efficiency of DTX-Sol, DTX-Nps, and DTX-FA-Nps on 4T1 cells (A), inhibition efficiency of DTX-Sol, DTX-Nps, and DTX-FA-Nps on normal cells (HUVEC cells) (B) at 48 h (n = 6) (*P < 0.01 vs. DTX-Sol; #P < 0.01 vs. DTX-Nps).

At 96 h, the cell inhibition rates of DTX-Sol, DTX-Nps and DTX-FA-Nps against the 4T1 cells were all above 80%.

The cytotoxicity of DTX-Sol, DTX-Nps and DTX-FA-Nps against normal cells (HUVEC cells) at different doses is also studied. All the three formulations showed cytotoxicity against HUVEC cells at 24 or 48 h, but the highest cell inhibition rate was less than 50% (Fig. 5B, at 10 μg mL⁻¹), suggesting that all of the three formulations had lower toxicity against the normal cells than 4T1 tumor cells.

3.6 In vivo anti-tumour effects in 4T1 bearing mice

The anti-tumor efficacy of DTX-Sol, DTX-Nps and DTX-FA-Nps was studied in mice bearing breast tumours. As shown in Fig. 6, the tumour size of the saline group, the DTX-Sol, the non-targeted (DTX-Nps) and targeted (DTX-FA-Nps) group were 3267 ± 322.3 mm³, 1861 ± 352.1 mm³, 1364 ± 204.0 mm³ and 1038 ± 204.6 mm³ at 10 mg kg⁻¹, while the tumour size of the non-targeted (DTX-Nps) and targeted (DTX-FA-Nps) group were 869 ± 162.3 mm³ and 673 ± 171.5 mm³ at 20 mg kg⁻¹, respectively. Table 1 shows the tumour inhibition rate compared to that of the DTX-Sol group. Significant tumour inhibition (inhibition rate > 47%) was achieved in all DTX-treated groups. It can be seen that the inhibition rates were 74.83% (P < 0.01) for the DTX-FA-Nps of 10 mg kg⁻¹, 66.19% (P < 0.05) for the DTX-Nps of 10 mg kg⁻¹, but much higher than the 10 mg kg⁻¹ DTX-Sol (47.22%). These data provided strong evidence that the DTX-Nps, and especially DTX-FA-Nps were more efficient for anti-tumor therapy than the DTX-Sol at a dose of 10 mg kg⁻¹ (P < 0.01); the enhanced efficiency may be attributed to the effect on the cancer cells of folate receptor targeting by the targeted nanoparticles (DTX-FA-Nps).⁴⁸ These results imply that folate decoration could clearly increase the cellular uptake of the nanoparticles. The high expression of folate receptors on the 4T1 breast cancer cells caused a great proportion of targeted nanoparticles (DTX-FA-Nps) to be taken up and further internalized by the cells compared to the non-targeted nanoparticles (DTX-Nps or DTX-Sol).⁴⁹ However, there is no significant difference between DTX-FA-Nps and DTX-Nps at 20 mg kg⁻¹.

Fig. 6 Tumour growth of 4T1-bearing mice given an i.v. injection of saline, DTX-Sol (10 mg kg⁻¹), DTX-Nps (10 mg kg⁻¹, 20 mg kg⁻¹), and DTX-FA-Nps (10 mg kg⁻¹, 20 mg kg⁻¹). The results represent the mean ± SD (n = 10).

Table 1 The in vivo antitumor effects in 4T1-bearing mice (x ± s, n = 10)^a

Group	Dose (mg kg^−1)	Tumor weight (g)	Inhibition rate (%)
a **P < 0.01 vs. normal saline group; *P < 0.05 vs. normal saline group. ##P < 0.01 vs. DTX-Sol at 10 mg kg^−1. #P < 0.05 vs. DTX-Sol at 10 mg kg^−1.
Saline	—	2.08 ± 0.25	—
DTX-Sol	10	1.10 ± 0.20**	47.22
DTX-Nps	10	0.70 ± 0.09**^#	66.19
DTX-FA-Nps	10	0.52 ± 0.06**^##	74.83
DTX-Nps	20	0.33 ± 0.12**^##	84.25
DTX-FA-Nps	20	0.22 ± 0.07**^##	89.63

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The body weight of the mice treated with DTX-Sol, DTX-Nps and DTX-FA-Nps was studied with saline as the control (after 22 days). No significant body weight loss in all groups was observed for all animals except the DTX-Sol group (Fig. 7), suggesting less harm from the Chol-PEG₁₀₀₀-FA nanoparticles than the DTX-Sol group.

Fig. 7 Body weight changes in mice treated with normal saline, DTX-Sol, DTX-Nps and DTX-FA-Nps at 10 mg kg⁻¹ or 20 mg kg⁻¹. The results represent the mean ± SD (n = 10).

3.7 Biodistribution by fluorescence imaging

We first marked the DTX-loaded nanoparticles with a DiR dye for near-infrared fluorescence imaging to visualize the biodistribution of DTX-loaded nanoparticles in 4T1 tumour bearing mice. The biodistribution of the non-targeted (DTX-Nps) and targeted (DTX-FA-Nps) nanoparticles was investigated with the DTX-Sol as a control. Fig. 8 shows that after intravenous administration of the formulations, the whole bodies of live mice were monitored at 0.5, 1, 2, 4, 8, 24, 48, 72 and 96 h. The living imaging test shows most of the DiR accumulated in the liver and tumour for DTX-Sol and both DTX nanoparticles. At 4 h, the fluorescence density of the liver decreased, meanwhile the DiR signals gradually increased in the tumour and the preferential accumulation of fluorescence was obvious in the tumour site over the liver or other normal tissues from 8 h after injection. At 48 h, the strong fluorescence signal could only be obtained in the tumour region, which suggested the enhanced anti-tumor efficacy of the DTX-FA-Nps. The nanoparticles that are not located in tumour zones can easily be cleared through the liver, inducing minimal side effects caused by the drugs.

Fig. 8 Time-dependent in vivo images of the mouse bearing 4T1 tumour after intravenous injection of DTX formulations (n = 10).

Besides, the DTX-FA-Nps proved to have a higher tumour-targeting efficiency, which meant that the fluorescence in the DTX-FA-Nps treated group was much higher than that of the DTX-Nps treated group in tumours at the same time points. The results provided decisive evidence that the DTX-FA-Nps were available for tumour-specific drug delivery. The high tumour-targeting efficiency of the nanoparticles might be attributed to a combination of an EPR effect and receptor-mediated uptake of nanoparticles.^50,51 Fig. 9 can further confirm the higher fluorescence accumulation in the tumours of the DTX-FA-Nps group compared with the DTX-Nps.

Fig. 9 Ex vivo fluorescent imaging of the tumour parts and other vital organs (from left to right: tumour, heart, liver, spleen, lung, kidney, and brain) (n = 5).

In Fig. 9, real-time images of DTX-FA-Nps with or without the pre-injection of FA are recorded. The fluorescence signal in the FA pre-injected animal group was lower than that in the non pre-injection group at the tumour region. This may be induced by the saturation of folate receptors being followed by a reduction in cellular nanoparticle uptake in the FA pre-treatment group. High doses of the FA were pre-injected intravenously to block folate receptors prior to DTX-FA-Nps binding to assess the target mechanism by a competitive inhibition assay.

In Fig. 9, ex vivo fluorescence images of the organs excised from the mice show that a strong fluorescence signal was observed mostly in the tumour tissue which is consistent with the in vivo whole body imaging; meanwhile only marginal fluorescence was detected in the liver and spleen. No fluorescence was found in the brain, kidney, lung and heart, suggesting that the injected DTX formulations can preferentially accumulate in the tumour region with no severe non-specific uptake by the normal tissues. The low accumulation of DTX-Sol and both DTX nanoparticles in the other organs could be caused by the high absorption and scattering characteristics of both MPS organs,⁵² and even when DiR was used, signal detection will be hindered by tissue attenuation, signal penetration and even autofluorescence, which is an intrinsic limitation of using ex vivo fluorescence imaging techniques to elevate the biodistribution of nanoparticles.⁴⁹ In future work, we will carry out further studies using isotopes, which would enable the assessment of organs that have up to now not been included in the evaluation, such as the intestines.

ROI analysis was performed by ex vivo fluorescence imaging to semi-quantitatively evaluate the biodistribution of the nanoparticles in each organ;⁵³ this was conducted by quantifying the amount of fluorescent intensity (average fluorescent signals per square centimetre i.e., radiant efficiency) of the DTX in the various organs including the heart, liver, spleen, brain, kidneys, lungs and tumour between the DTX-FA-Nps and DTX-Nps (Fig. 10). The results proved that the mean fluorescent intensity of both nanoparticles was significantly higher than for the DTX-Sol in the tumour after i.v. administration, which shows the higher tumour uptake of the DTX formulated in nanoparticles. Furthermore, the fluorescent intensity of the DTX-FA-Nps exhibited over 1.7-fold higher intensity at the tumour tissue than the DTX-Nps and shows obviously higher accumulation of DTX in comparison with the DTX-Nps and DTX-Sol in all the organs except the heart, brain and kidney. Besides, the higher accumulation of the DTX-FA-Nps confirms the folate receptor mediated codelivery.

Fig. 10 Column graph of luminous intensity for fluorescent probes measured in different organs in mice.

Only one paper has reported the bio-distribution in tumor-bearing mice by living imaging technology after the administration of targeted DTX-NPs (a novel FA conjugated poly(L-g-glutamyl glutamine) nanoparticle loaded with DTX).⁴⁰ The results showed high accumulation of targeting NPs in the tumor tissue, which is quite similar to the result obtained in our study.

The above results have demonstrated the advantages, by in vivo fluorescence imaging, of the DTX-FA-Nps over the DTX-Nps and DTX-Sol, indicating that DTX-FA-Nps are expected to be a highly efficient drug delivery system to achieve the targeted delivery of anti-tumour drugs.

4. Conclusions

In this study, Chol-PEG₁₀₀₀-FA was synthesized as a new type of targeting amphiphilic polymer, which was used as a stabilizer to encapsulate DTX. The constructed DTX nanoparticles can successfully target tumours and exhibit good anti-tumor activity. Thus the DTX-loaded Chol-PEG₁₀₀₀-FA nanoparticles may be a promising targeted delivery system for breast cancer therapy.

Acknowledgements

This study was financially supported by the Beijing Natural Science Foundation (7152099) and the NSFC-Guangdong Joint Foundation (U1401223).

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