Self-organized nanoparticle drug delivery systems from a folate-targeted dextran–doxorubicin conjugate loaded with doxorubicin against multidrug resistance

Abstract

A folate-targeted dextran–doxorubicin conjugate (folate–dextran–DOX) for drug delivery systems (DDSs) was synthesized by grafting DOX onto dextran through cleavable hydrazone bonds and a pH-sensitive spacer for controlling the drug release. Folate was coupled onto dextran as an ideal ligand for targeting hepatocytes. The conjugate was formulated into nanoparticles with excessive deprotonated DOX (DOX nano-DDSs) under aqueous conditions, which exhibited nanoparticles with larger size of 147.9 nm in diameter and improved drug entrapment to the level of 25.2%. DOX nano-DDSs delivered higher cytotoxicity and a greater extent of intracellular uptake in vitro against drug resistant HepG2 (HepG2/DOX) cells; moreover, they displayed equivalent effects with folate–dextran–DOX micelles in terms of inhibiting tumor volume and decreasing toxicity. In addition, DOX nano-DDSs achieved significantly greater effects than free DOX. The results indicated that these targeted self-organized DOX nano-DDSs have superior reversal efficacy to free DOX and serve as a highly promising nano-platform for future cancer therapy.

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1. Introduction

Polymer conjugates for anticancer drug delivery systems (DDSs) have been extensively developed due to their advantages. However, their drug efficacy was limited by their rapid clearance from the body by the reticuloendothelial system (RES).^1–3 Recently, nano-scaled DDSs (nano-DDSs) utilizing polysaccharides as drug carriers exhibited dramatic decreasing impact from RES. Their hydrophobic core and hydrophilic shells assist them to spontaneously form stable nanoparticles that result in prolonged systemic circulation and sustained release of drugs into the blood stream.^2,4 They were also appreciated for several natural characteristics such as biodegradability, water-solubility and non-antigenicity.^5,6 In addition, the enhanced permeable retention (EPR) effect resulting from the hypervascular permeability and impaired lymphatic drainages allowed the nano-DDSs to accumulate in tumors by the “filtration” mechanism.^7–10

Currently, a pH-sensitive nano-DDS based on polymeric chains for the delivery of drugs has attracted interest.^11–15 Compared with traditional spacers that are less sensitive to the environment, hydrazone bonds, in particular, are cleavable under mildly acidic conditions and stable under neutral pH conditions, which are facile to control the drug release.^16–19 Therefore, the combination of a drug and a polymer through a hydrazone bond contributes to the relatively larger amount of drug release in tumor tissue by preventing premature cleaving in the plasma.

Drugs of peripheral toxicity such as doxorubicin (DOX) produce a broad spectrum of antitumor behaviors and are easily extruded from the cell due to the emergence of multidrug resistance (MDR).²⁰ Their low drug efficiencies, as well as notorious side effects, serve to drastically decrease the therapeutic efficacy. To overcome these obstacles, nano-DDSs should be manipulated with tumor-targeting ligands. One of the best candidates is folate acid, whose receptor has been known to be vastly overexpressed in several human tumor cell lines.^21–28 By conjugating folate, the micelles can be directed to cancer cells and subsequently internalized by folate-mediated endocytosis.^{4,22,24,29,30} Therefore, the folate-targeted nano-DDSs could achieve both decreases in side effects by selectively locating in targeted cells and increase the uptake in tumor cells.

A pH-sensitive nano-DDS based on pullulan for a drug delivery system was synthesized, and in vitro cytotoxicity confirmed its improved drug release behavior when compared with a previous study.³¹ In order to increase the cellular uptake and decrease the toxicity, we took further steps to develop targeted nano-DDSs and implement more comprehensive tests through in vivo experiments. In our study, DOX molecules were chemically conjugated to dextran by hydrazone bonds. Folate acid was also grafted onto the dextran. It was desirable to incorporate the advantages of hydrazone spacers, targeted ligand as well as the natural biodegradable polysaccharide carrier.³² Larger drug content was achieved when combined with excessive DOX under aqueous conditions. The cytotoxicity of nano-DDSs against HepG2/DOX cells and the therapeutic effect on tumor cells implanted in mice were also observed.

2. Materials and methods

2.1. Materials

Dextran (MW 100 000) was purchased from Huzhou Langshexi Biotech. Co. in Zhejiang Province, China. Doxorubicin (DOX), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), folic acid, N-hydroxysuccinimide (HOSu) and hydrazine hydrate were obtained from Aldrich Chemical Co. China. Organic solvents were used without further purification. All other chemicals were commercially available analytical grade reagents unless otherwise stated.

2.2. 4-Nitrophenyl chloroformate activation of dextran

Dextran (2 g, 12.3 mmol) and 4-dimethylaminopyridine (DMAP) (0.15 g, 1.2 mmol) were added in 20 mL of DMSO/pyridine solution (vol. ratio 1/1). Then, 4-nitrophenyl chloroformate (2 g, 10 mmol) was dissolved at 0 °C. The reaction was carried out for 24 h at 0 °C. At the end of the reaction, the mixture was precipitated in anhydrous ethanol. The white precipitate obtained was rinsed repeatedly three times with the same solvent. Dextran 4-nitrophenyl carbonate was finally dried in vacuo and was identified by FTIR. The carbonate content was determined by UV analysis after activated dextran hydrolysis in NaOH. The degree of activation of dextran was determined by the hydrolysis of activated dextran in a NaOH solution. Activated dextran (100 mg) was dissolved into 10 mL of 0.1 N sodium hydroxide solution and the absorption of the carbonate residue was monitored by UV-visible spectroscopy at 402 nm for the p-nitroaniline group. The content of carbonate was determined using Beer's law (DS = 34% (mol%)).

2.3. Synthesis of dextran–hydrazide

Dextran–COO(C₆H₄)NO₂ (4.68 g) was dissolved in 50 mL dry DMF and hydrazine hydrate (80%, 15.7 mL, 30 equiv.) was added at room temperature. The mixture was keep at room temperature for 48 h with gentle oscillation. After removing majority of the solvent by vacuum distillation, the mixture was precipitated in anhydrous ether. The obtained dextran–hydrazide was freeze-dried and identified by FTIR. The DS was determined by oxidation reduction titration. During the process of titration, caprylic hydrazide could be oxidized to carboxylic acid, whereas potassium bromate was reduced to potassium bromide. The endpoint of the reaction could be indicated via the fading of pink color of methyl orange (http://www.med126.com/pharm/2009/20090109143304_72296.shtml) (DS = 28% (mol%)).

2.4. Synthesis of dextran–DOX

17.3 g dextran hydrazide was dissolved in 50 mL of DMF and excessive DOX·HCl (1.74 g) was added. The mixture was stirred at room temperature for 24 h and protected from the light. The product was dialyzed (MWCO 8000 Da, Spectra/Por membrane RC) against water and then freeze-dried to obtain the dextran–DOX conjugate. The percentages of DOX in conjugation were measured by a UV-spectrophotometer in DMSO at 480 nm. The content of DOX in conjugates was determined using absorbance at 480 nm by UV-visible spectroscopy.³³ A calibration curve was made by detecting different concentrations of DOX solution at 480 nm. The absorbance by the conjugate was measured at 480 nm and the DOX content was found by comparison with the calibration curve of DOX (DOX = 7.1% (wt)).

2.5. Synthesis of folate–dextran

4.4 g of folic acid was dissolved in 200 mL DMSO. 4.2 g HOSu and 2.2 g dicyclohexylcarbodiimide (DCC) were then added. The mixture was stirred at room temperature. After 6 h of reaction in the dark, the by-product dicyclohexylurea was removed by filtration. 17.3 g dextran hydrazide was then added. The reaction was performed for 24 h and 200 mL deionized water was added to the mixture. The precipitate was filtrated and lyophilized to obtain dextran hydrazide folate. The conjugation percentages of folic acid were calculated by determining the amount of folate conjugated to folate–dextran–DOX in DMSO at 365 nm, which was 2.3% (wt).

2.6. Conjugation of DOX to the folate–dextran

1.83 g dextran hydrazide folate was dissolved in 50 mL of DMF, and an excessive amount of DOX (1.74 g) was added. The mixed solution was oscillated in the dark at room temperature for 24 h. The precipitate was dialyzed (MWCO 8000 Da, Spectra/Por membrane RC) against water and then freeze-dried to obtain the folated dextran–DOX conjugate. The conjugation was identified by FTIR and its percentages of DOX were calculated by a UV-spectrophotometer in DMSO at 480 nm (DOX = 6.5% (wt)). The content of DOX in conjugates was determined using absorbance at 480 nm by UV-visible spectroscopy.³³ A calibration curve was made by detecting different concentration of DOX solution at 480 nm. The absorption of conjugate was measured at 480 nm and the DOX content of conjugate was found by comparison with the calibration curve of DOX.

2.7. Preparation and characterization of nano DDSs

The conjugated product (100 mg) was dissolved in 10 mL DMSO and dialyzed (MWCO 8000 Da) against excessive deionized water at 4 °C for 3–4 days with water changes every 8 h. The dialysate was obtained and filtrated through a 0.45 μm membrane and then freeze-dried for 3 days to shape the nanoparticles.

In order to acquire nano-DDSs loaded with DOX, 15 mg DOX was dissolved in anhydrous DMSO (10 mL) containing triethylamine. The molar ratio of triethylamine to DOX was 2 : 1. The conjugated product (50 mg) was added into the solution with mixing overnight in a dark and cold environment. At the end of the reaction, the solution was dialyzed against excessive deionized water at 4 °C for 3–4 days with water changes at 8 h intervals. By directly scattering the organic phase into the aqueous phase, the conjugation spontaneously aggregated into nanoparticles. Extensive dialysis against deionized water proceeded to remove unencapsulated DOX and triethylamine. The obtained novel nano-DDS was filtrated through a 0.45 μm membrane, centrifuged, separated and lyophilized.

2.7.1 Particle size and zeta potential measurement. Nanoparticle sizes and distribution were measured by dynamic light scattering (DLS; Zetasizer 3000, Malvern Instruments LTD, UK). Transmission electron microscopy (H-600, Hitachi LTD, Japan) was also conducted at the accelerating voltage of 200 keV to observe the nanoparticles.

2.7.2 Drug entrapment efficiency. The entrapment percentages of DOX were calculated by determining the amount of DOX in the nano-DDS in DMSO at 480 nm using a UV-spectrophotometer.

The DOX entrapment efficiency was calculated as follows:

where A is the amount of DOX added to the system and B is the amount of DOX in the supernatant.

2.8. In vitro release of DOX from the nano-DDS

The release study was conducted in serum, phosphate buffered saline (PBS, pH 7.4) and PBS at pH 5.0 (pH of endosomes or lysosomes) at 37 °C with moderate stirring. DOX nano-DDSs, folate–dextran–DOX and dextran–DOX (1 mg mL⁻¹) were transferred to 10 mL of PBS in a dialysis tube. At selected time intervals, the whole medium was removed and replaced with fresh PBS. The drug content was detected by UV at 480 nm.³³ A calibration curve was made by detecting different concentrations of DOX solution (0.5, 1, 2, 4, 8, 16 μg mL⁻¹ respectively) at 480 nm.

2.9. In vitro cytotoxicity assay

2.9.1 Cell culture. HepG2 cells (Yanyu Biotech Co., LTD, Shanghai) were cultured and preserved in an RPMI-1640 medium and supplemented with 10% fetal bovine serum. The drug resistant HepG2 (HepG2/DOX) cell line was developed from HepG2 cells incubated with DOX in a stepwise increasing concentration (from 0.01 to 2 μg mL⁻¹) for several months. The drug resistant cells were obtained by removing the dead cells. The drug resistance was maintained by culturing the cells at 1 μg mL⁻¹ DOX.

2.9.2 Cytotoxicity assay in vitro. The 3-(4,5-diemethylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT) assay (Sigma Co. USA) was implemented to evaluate the in vitro cytotoxicity of the 4 formulations (dextran–DOX, folate–dextran–DOX, DOX nano-DDSs and free DOX). HepG2/DOX cells were incubated with the three formulations at a dose equivalent to free DOX. Statistic was determined by cell viability. The reversal of MDR was evaluated by the IC₅₀ value. Control groups were treated with physiological saline. The cell viability was calculated as follow:
where A is the absorbance of the control group and B is the absorbance of the treated group.

2.9.3 Cellular uptake of drug. HepG2/DOX cells were pre-incubated with the 4 formulations (dextran–DOX, folate–dextran–DOX, DOX nano-DDSs and free DOX) for 2 h at a dose equivalent to free DOX (100 μg mL⁻¹), whereas LO2 normal hepatocyte cells were pre-incubated with the folate–dextran–DOX for 2 h at the same dosage. 1 mL of cell suspension (10⁷ HepG2/DOX cells) was mixed with 300 mL TM-2 buffer solution (10 mmol L⁻¹ Tris–HCl, pH 7.4, 2 mmol L⁻¹ MgCl₂, 0.5 mmol L⁻¹ PMSF) in an ice bath for 5 min. 300 μL 1.0% Triton X-100 was added to the mixture and cultured in an ice bath for 5 min. The mixture was filtered through the membrane (0.22 μm) 6 times. 1 mL of cell suspension (10⁷ HepG2/DOX cells) was added with DOX standard solution with different concentrations. 1 mL cell suspension (10⁷ HepG2/DOX cells) was mixed with 300 mL TM-2 buffer solution (10 mmol L⁻¹ Tris–HCl, pH 7.4, 2 mmol L⁻¹ MgCl₂, 0.5 mmol L⁻¹ PMSF) in an ice bath for 5 min. Furthermore, 300 μL of 1.0% Triton X-100 was added and cultured in the ice bath for 5 min. The mixture was then filtered using a membrane (0.22 μm) 6 times. The formulations were analyzed by HPLC with a Shimadzu HPLC system composed of and a SPD-10Avp ultraviolet detector (Shimadzu Corporation, Japan) and two pumps (LC-10Avp and LC-10AS) in reverse phase mode at different points of time. An extend-C18 column (4.6 × 250 mm I.D., 5 μm) was used and the mobile phase for the analysis was methanol–acetonitrile–phosphate buffer (pH 5.0, 0.2 M) (50 : 20 : 30, v/v/v) with a flow rate of 0.5 mL min⁻¹. The drug content was detected by UV at 480 nm.³³ A calibration curve was made by detecting different concentrations of DOX solution (0.014, 0.028, 0.056, 0.112, 0.168, 0.224 μg mL⁻¹) at 480 nm. The absorption of conjugated product was measured and the DOX content was found by comparison with the calibration curve of DOX.

2.10. In vivo studies

All study performed on the animals was in accordance with the “Guidelines for the Care and Use of Laboratory Animals” published by the National Institute of Health (NIH Publication No. 85–23, revised 1985). This study was supported by the Ethics Committee of Central China Normal University. Informed written consent was obtained from all the subjects prior to the study.

2.10.1 In vivo pharmacokinetic study. Every specific pathogen-free grade male BALB/c nude mouse (provided by the Experimental Animal Center of Wuhan Institute of Biological Products) was inoculated with 0.2 mL HepG2/DOX cell suspension (5 × 10⁶ cells per mL) in the right auxiliary region. After being incubated for three weeks, a solid tumor growth was increasing noticeable in the nude mice.

144 tumor-bearing mice were randomly divided into 4 groups of 36 mice each and injected with dextran–DOX, folate–dextran–DOX, DOX nano-DDSs and free DOX at a single dose (equivalent dose of DOX = 10 mg kg⁻¹). At different time intervals, 0.5 mL blood sample was withdrawn by a retro-orbital venous plexus puncture from the tumor-bearing mice (from all 4 groups, 6 mice from each group). The livers, hearts, tumors, and kidneys of all the mice were immediately separated and washed with Na₂HPO₄ buffer, followed by homogenization with ethyl acetate as the solvent. DOX was extracted after being incubated with acidic isopropanol for 12 h at 4 °C. The mixture was centrifuged at 1200 rpm for 15 min. The DOX concentration in the supernatant solution was detected by HPLC quantitatively. Free drug or released drug was extracted and determined without incubation by HPLC as described previously.³⁴

2.10.2 In vivo cytotoxicity of nano-DDS against drug resistant HepG2/DOX cells in mice. Specific pathogen-free grade male BALB/c naked mice (provided by the Experimental Animal Center of the Wuhan Institute of Biological Products, 4 weeks old, 20–22 g) were inoculated subcutaneously with HepG2/DOX cells (1 × 10⁷ cells per animal). After 3 weeks, solid tumor growth was established noticeably in most mice, dextran–DOX, folate–dextran–DOX, DOX nano-DDSs and free DOX (equivalent dose of DOX = 4 mg kg⁻¹) suspended in PBS were injected into the tail veins of the animals every week for four doses (days 0, 7, 14, and 21). A major axis and a minor axis of tumors were measured using calipers. Tumor volume was then measured. The survival time and number of long-term survivors (LTS) until day 50 were monitored.

2.11. Statistical analyses

Data are described as means ± standard deviations (SD) of multi-replicated determinations. Results were analyzed by a one-way evaluated of variance (ANOVA) with the Student–Newman–Keuls multiple comparisons or t-test when comparing the differences between the means of two groups at the same time point. Diversities at P < 0.05 were considered statistically significant.

3. Results

3.1. Synthetic routes of folate–dextran–DOX conjugate

3.1.1 Preparation and characterization of the folate–dextran–DOX conjugate. The attachment of DOX to the dextran was accomplished by a hydrazone bond spacer, as identified in Fig. 1. The peak around 1655 cm⁻¹ might be the carboxylation of dextran, forming dextran–COO(C₆H₄)NO_2. After amidation of the carboxylic group, the peak shifted to 1630 cm⁻¹, which indicated the incorporation of hydrazide to the activated dextran. The shifted peak at about 1613 cm⁻¹ might be attributed to the conjugating of DOX and folate. The content percentage of DOX, determined by the calculation and detection using UV-spectrophotometer, was approximately 6.5% (wt).

Fig. 1 Synthetic route of folate–dextran–DOX conjugate.

Fig. 2 FTIR (left) spectra of DOX (A), dextran (B), dextran hydrazide (D), 4-nitrophenyl-chloroformate–dextran (C) and folate–dextran–DOX conjugate (E) and ¹H-NMR (right) spectra of (B)–(E).

In the ¹H NMR spectrum of dextran–COO(C₆H₄)NO₂, the new peaks at 8.31 ppm and 7.50 ppm compared with dextran was attributed to the protons on the benzene rings, which indicated the loss of –COO(C₆H₄)NO₂ groups. The signals at 7.98 ppm and 1.97 ppm are the two types of protons in –NH–NH₂; moreover, the signals of 8.31 ppm and 7.50 ppm disappeared, which indicated the successful formation of dextran–hydrazide. In the spectrum of folate–dextran–DOX, the peak at 1.10 ppm was the result of the methyl group on DOX, which confirmed the conjugation of DOX. The peak at 8.72 ppm attributed to the proton on pyrazine was observed, indicating the conjugation of folate group.

(A): δ 5.46–5.27 (m), 5.05–4.63 (m), 4.29–4.02 (m), 3.94–3.80 (m).

(B): δ 8.31 (t, J = 5.6 Hz), 7.50 (d, J = 4.2 Hz), 5.74–5.46 (m), 4.81 (d, J = 17.6 Hz), 4.47–4.20 (m), 4.20–3.94 (m).

(C): δ 7.98 (s), 7.98 (s), 5.55 (d, J = 20.8 Hz), 5.55 (d, J = 20.8 Hz), 4.79 (s), 4.29 (ddd, J = 26.8, 21.2, 14.4 Hz), 4.20–3.87 (m), 1.97 (s).

(D): δ 8.72 (d, J = 10.2 Hz), 8.09 (s), 7.95 (d, J = 15.5 Hz), 7.70 (s), 6.81 (s), 5.52–5.30 (m), 5.19–4.50 (m), 4.25–4.03 (m), 3.90 (dd, J = 11.2, 8.6 Hz), 3.87–3.80 (m), 2.70 (s), 2.19 (d, J = 26.0 Hz), 1.10 (q, J = 3.5 Hz).

(E): δ 8.72 (d, J = 10.2 Hz), 8.09 (s), 7.95 (d, J = 15.5 Hz), 7.70 (s), 6.81 (s), 5.52–5.30 (m), 5.19–4.50 (m), 4.25–4.03 (m), 3.90 (dd, J = 11.2, 8.6 Hz), 3.87–3.80 (m), 2.70 (s), 2.19 (d, J = 26.0 Hz), 1.10 (d, J = 5.1 Hz).

3.2. Characterization of DOX nano-DDSs

The morphology of these novel nano-DDSs loaded with DOX is shown by transmission electron microscopy (Fig. 3). The mean diameter of the novel nano-aggregates is 147.9 nm with a PDI of 0.264 as measured by the DLS technique. The content of entrapped doxorubicin reached up to 25.2% and was threefold higher than that of the folate–dextran–DOX nanoparticles. The DOX entrapment efficiency was 81%.

Fig. 3 Dynamic laser scattering (DLS) results (left) and TEM micrograph (right) of DOX nano-DDSs.

3.3. DOX and folate released

The in vitro release behavior of the four formulations was examined under different conditions. The results are shown in Fig. 4. In both PBS and serum at pH 7.4, the amount of DOX released was negligible. In the buffer at pH 5.0, the drug was released with a noticeable accumulation as time proceeded. The release percentage of DOX nano-DDSs, folate–dextran–DOX and dextran–DOX after 48 h was 92.5%, 44.7% and 46.1%, respectively. Compared with other administrations, the release from nano-DDSs loaded with DOX was faster and more thorough. There were no significant differences in the release profile between folate–dextran–DOX and dextran–DOX conjugate. The amount of drug release from DOX nano-DDSs in pH 5.0 was drastically greater than that in pH 7.4, which indicated that the hydrazone bond is the appropriate spacer controlling drug release. The release percentages of folate was 15.3% in pH 6.0 buffer, which is 26.1% lower than that of DOX, indicating that folate was more stable than DOX when conjugated on dextran in mildly acidic environments and therefore facilitated nano-DDSs to realize folate-mediated internalization in tumor cells.

Fig. 4 Release profiles of DOX (left) and folate (right). (DOX release from DOX nano-DDSs (▼) (pH 5.0 buffer), DOX release from folate–dextran–DOX (▲) (pH 5.0 buffer), DOX release from dextran–DOX (◆), serum (●) and PBS (◄) (pH 7.4), DOX release from DOX nano-DDSs (►) (pH 6.5 buffer), folate release from folate–dextran–DOX (★) (pH 7.4 PBS), folate release from folate–dextran–DOX (☆) (pH 6.0 buffer).) Data were given as mean ± SD (n = 6) (P < 0.05).

3.4. In vitro cytotoxicity of the nano-DDSs against tumor cells

The MTT-based in vitro cytotoxicity assay determined by the cell growth inhibition assay of the HepG2/DOX cells was performed to compare the therapeutic effect of DOX nano-DDSs and other three formulations. According to the result shown in Fig. 5, the DOX nano-DDSs nanoparticles showed better anti-cancer effects against tumor cells than that of free DOX. The calculated IC₅₀ was 1.14 μg mL⁻¹, 1.09 μg mL⁻¹ and 0.49 μg mL⁻¹ for dextran–DOX, folate–dextran–DOX and DOX nano-DDSs, respectively, which indicated that nano-DDSs loaded with DOX demonstrated better inhibition effects than that of folate–dextran–DOX and dextran–DOX.

Fig. 5 In vitro cytotoxicity of free DOX (■), dextran–DOX (●), folate–dextran–DOX (▲) and DOX nano-DDSs (▼) against drug resistant HepG2/DOX cells. Data given as mean ± SD (n = 6) (P < 0.05).

3.5. Cellular uptake of DOX

HepG2/DOX cells were incubated in free DOX solution, dextran–DOX, folate–dextran–DOX and DOX nano-DDSs with equivalent doses of DOX for 2 h. The results are displayed in Fig. 6. DOX nano-DDSs and folate–dextran–DOX micelles, both containing the folate ligand, reached higher uptake amounts of 135.9 ng and 135.2 ng, respectively, after 2 h. In contrast, dextran–DOX without the targeting group showed a relatively lower uptake amount of 70.5 ng and free DOX was slightly internalized by tumor cells. The cellular uptake of DOX by LO2 normal hepatocytes at 2 h was 22.3 ng, which was significantly lower than that by HepG2/DOX cells (136.2 ng), indicating that the folate ligand on the prodrugs have selective targeting ability towards cells that express the folate receptor.

Fig. 6 Uptake of drug by MDR cells after incubation with free DOX (■), dextran–DOX (◆), folate–dextran–DOX (▲) and DOX nano-DDSs (▼) (left) and uptake of DOX by folate-expressing cells versus LO2 hepatocyte cells (that do not express the folate receptor) (right). Values are means ± SD (n = 3) (P < 0.05).

3.6. In vivo pharmacokinetic study in tumor-bearing mice

Bio-distribution profiles of DOX in blood together with other tissues were measured after administrating the four formulations DOX. The results are shown in Fig. 7 and Table 1.

Fig. 7 Drug concentration–time profiles in different tissues after a single dose of 10 mg kg⁻¹ of free DOX (■), dextran–DOX (◆) folate–dextran–DOX (▲) and DOX nano-DDSs (▼) in tumor-bearing mice (n = 12 per group). Values are means ± SD (n = 3) (P < 0.05).

Table 1 Pharmacokinetic parameters of doxorubicin in tumor-bearing mice after i.v. administration of the four formulations at a single dose of 10 mg kg⁻¹ (P < 0.05)

Tissue	Free DOX			Dextran–DOX			Folate–dextran–DOX			DOX–nano-DDSs
	AUC	MRT	T1/2	AUC	MRT	T1/2	AUC	MRT	T1/2	AUC	MRT	T1/2
	μg g^−1 h	h	h	μg g^−1 h	h	h	μg g^−1 h	h	h	μg g^−1 h	h	h
Blood	5677	3.509	2.417	96 130	7.234	5.001	90 090	7.507	5.185	84 170	7.834	5.411
Tumor	12.04	3.326	2.287	656.3	35.92	23.09	2552	67.82	46.41	2559	57.54	39.08
Liver	181.5	12.10	8.369	263.9	16.97	11.00	131.3	22.74	14.71	122.3	20.76	13.48
Kidney	21.17	2.33	1.597	45.41	4.355	2.655	78.33	19.39	12.68	81.08	18.56	12.21
Heart	159.9	12.73	8.805	28.18	21.05	14.57	18.88	21.06	14.58	8.698	7.370	5.092

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In the blood, the concentration of free DOX was 1.693 mg L⁻¹ in 0.5 h, 7-fold lower than DOX nano-DDSs, which was attributed to the rapid elimination from the circulation system by passive convection. DOX nano-DDSs and folate–dextran–DOX demonstrated nearly 20 times higher value of area under curve (AUC) than that of free DOX. Their mean residence time (MRT) reached up to 7.834 μg g⁻¹ h and 7.507 μg g⁻¹ h, which was twofold greater than free DOX. These nano-DDSs showed the excellent characteristic of prolonged circulation time before arriving at the tumor cell.

Significantly higher drug concentration, as we expected, was selectively distributed in the tumor when compared with other RES organs, including liver, heart and kidney. The AUC of DOX nano-DDSs in the tumor was 21-fold higher than in the liver, 31-fold higher than in the kidney and nearly 300-fold higher than in the heart. The drug concentration of DOX nano-DDSs reached up to a maximum of 74.59 μg g⁻¹ after 8 h and then decreased with relatively slower speed. The AUC values as well as the MRT of DOX–nano-DDSs had no obvious differences with those of folate–dextran–DOX, while the AUC of DOX nano DDSs were 200 fold higher than that of free DOX and the MRT of DOX nano DDSs were 20 fold higher than that of free DOX.

In the heart, negligible amounts of DOX were detected in dextran–DOX, folate–dextran–DOX and DOX nano-DDSs when compared with the concentration of free DOX. The AUC of DOX nano-DDSs was 18 times lower than that of free DOX, indicating the reduced cardiac toxicity of DOX nano-DDSs.

3.7. Antitumor activity in vivo

In vivo cytotoxicity experiments of DOX nano-DDSs, folate–dextran–DOX dextran–DOX and free DOX against tumor cells was implemented in order to analyze the inhibition effect on the growth of HepG2/DOX cells in mice. Consequently, nano-DDSs conjugated with the folate ligand demonstrated better therapeutic efficacy in suppressing the tumor cells as compared with that of non-targeted DOX. 20 days later, the volume of tumors treated with DOX nano-DDSs was about 47% less than those treated with free DOX. There was no significant different between the two targeted nano-DDSs in terms of inhibiting the tumor volume (Fig. 8). In addition, targeted nano-DDSs displayed longer life spans of tumor-bearing mice than for free DOX. Mice treated with DOX nano-DDS and folate–dextran–DOX showed longer life spans (47.2 days and 46.8 days) when compared with the life spans of mice treated with dextran–DOX and free DOX (40.8 days and 33.8 days) (Fig. 9).

Fig. 8 Tumor volume changes in vivo of the xenograft nude mice bearing the HepG2/DOX tumors. (PBS (◄), free DOX (■), dextran–DOX (◆) folate–dextran–DOX (▲) and DOX nano-DDSs (▼)). The tumor-bearing mice were treated with equivalent drug (4 mg kg⁻¹ DOX) by tail injections every week for four doses (days 0, 7, 14, and 21).

Fig. 9 Surviving profile of tumor-bearing mice treated with PBS (◄), free DOX (■), dextran–DOX (◆) folate–dextran–DOX (▲) and DOX nano-DDSs (▼) after injection of the HepG2/DOX cells. The tumor-bearing mice were treated with equivalent drug (4 mg kg⁻¹ DOX) by tail injections every week for four doses (days 0, 7, 14, and 21). The survival time and number of long-term survivors (LTS) until the 50^th day were monitored (P < 0.05).

4. Discussion

As a natural polysaccharide, dextran is an excellent polymeric carrier in drug delivery system due to the requisite properties of biodegradability, water-solubility and non-antigenicity. The amphipathic nano-DDSs (hydrophilic dextran and hydrophobic DOX) could stabilize in the aqueous environment, forming nano aggregates spontaneously. The decreased elimination impact by RES in circulation contribute noticeably to the prolonged circulation time, which could be explained statistically by higher MRT and AUC values of DOX nano-DDSs and folate–dextran–DOX over free DOX in blood (Table 1).

The folate–dextran–DOX conjugation was synthesized and its structure was identified by FTIR and ¹H-NMR spectra (Fig. 2). The content of DOX in the aggregates is 6.5%, which is probably restricted by the low drug loading efficiency and the instability of high attachment. When combined with excessive free DOX, these self-assembled DOX nano-DDSs exhibited higher drug content, higher drug entrapment, greater size and higher entrapment efficiency (Fig. 3). The DOX nano-DDSs with a mean diameter of 147.9 nm (Fig. 3) could access the solid tumor tissue in a more facile way by the EPR effect.

Drug release from the DOX nano-DDSs is controllable by the pH-sensitive spacer. The hydrazone bond could achieve high degrees of hydrolysis in acidic conditions of pH 5.0, a typical environment in tumor cells. This property can well be explained by the negligible drug release in PBS and serum at pH 7.4 when compared with the prompt release in acidic conditions (Fig. 4). The in vitro cytotoxicity study demonstrated that HepG2/DOX cells bear higher chemosensitivity toward nano-DDSs than free DOX (Fig. 5), which could partly be explained by their higher drug release rates (Fig. 4). Although there is no significant difference between folate–dextran–DOX and DOX nano-DDSs in terms of cytotoxicity after long treatment times and because of the ample time for release drug, the DOX nano-DDSs with higher drug content released drugs with faster speed and demonstrated a lower IC₅₀ value (Fig. 5).

DOX nano-DDSs not only have the advantages in controlling drug release but also their targeted conjugate gives rise to enhanced drug uptake, decreased side effects and a reversal of MDR. The efficacy of DOX was restricted by peripheral toxicity; in addition, its systemic injection had negligible effects on tumor regression and overall survival. Folate, an ideal targeting ligand, was covalently attached to the dextran carrier. The folate receptor on the tumor cells assists the internalization of nano-DDSs by receptor-mediated endocytosis, which results in the significant effect of increasing intracellular uptake compared to micelles without the folate ligand (Fig. 6). Based on the folate-receptor on the HepG2/DOX cells, nano-DDSs conjugated with folate could accomplish selective distribution in the tumor cells, locating sparsely on RES organs such as the heart, kidney and liver (Fig. 7).

For the in vivo anti-cancer activities, targeted nano-DDSs showed superior effects in terms of delayed tumor volume growth, which was probably responsible for the synergetic impact of passive and active targeting. Passive targeting of nano-DDSs was achieved through ‘filtration’ by EPR effect, whereas active targeting allowed them to be readily internalized by mediated receptors. On the other hand, the sustained release of DOX in nano-DDSs would contribute to the striking decrease in tumor sizes (Fig. 8).

5. Conclusion

The objective of this study was to develop self-organized nano-DDSs with the function of both controlling drug release and targeting tumor cells. The folate–dextran–DOX conjugate could form nanoparticles spontaneously in the aqueous phase. Larger amounts of drug content could be achieved by adding free DOX into the micelles, reaching up to 25.2%. Studies demonstrated the superior therapeutic effect of folate–dextran–DOX and DOX nano-DDSs as they exhibited excellent drug controlling, considerable drug release, improved cellular uptake and decreased side toxicity. Although the DOX nano-DDSs featured superior sizes and larger drug contents, they demonstrated negligible advantages over folate–dextran–DOX, which allows for further optimization studies.

Acknowledgements

This study was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry, 2010-1174).

References

1. K. H. Min, K. Park, Y. S. Kim, S. M. Bae and S. Lee, et al., J. Controlled Release, 2008, 127, 208–218.
2. D. Sutton, N. Nasongkla, E. Blanco and J. Gao, Pharm. Res., 2007, 24, 1029–1046.
3. J. A. S. Fatma and B. Cheng, Clin. Physiol. Funct. Imaging, 2013, 33, 293–301.
4. N. Nasongkla, E. Bey, H. A. Jimin Ren, C. Khemtong and J. Setti Guthi, et al., Nano Lett., 2006, 6, 2427–2430.
5. Y. Cao, D. Chen, P. Zhao, L. Liu and X. Huang, et al., Ann. Biomed. Eng., 2011, 39, 2456–2465.
6. C. Zheng, X. Liu, J. Zhu and Y. Zhao, Drug Dev. Ind. Pharm., 2012, 38, 653–658.
7. A. J. D'Souza and E. M. Topp, J. Pharm. Sci., 2004, 93, 1962–1979.
8. R. J. Christie and D. W. Grainger, Adv. Drug Delivery Rev., 2003, 55, 421–437.
9. K. Miyata, R. J. Christie and K. Kataoka, React. Funct. Polym., 2011, 71, 227–234.
10. H. Nehoff, N. N. Parayath, L. Domanovitch, S. Taurin and K. Greish, Int. J. Nanomed., 2014, 9, 2539–2555.
11. Y. Song, Q. Tian, Z. Huang, D. Fan and Z. She, et al., Int. J. Nanomed., 2014, 9, 2307–2317.
12. L. Y. Qiu and Y. H. Bae, Pharm. Res., 2006, 23, 1–30.
13. V. Pillay, A. Seedat, Y. E. Choonara, L. C. du Toit, P. Kumar and V. M. K. Ndesendo, AAPS PharmSciTech, 2013, 14, 692–711.
14. E. Marin, M. I. Briceno and C. Caballero-George, Int. J. Nanomed., 2013, 8, 3071–3090.
15. G. Doerdelmann, D. Kozlova and M. Epple, J. Mater. Chem. B, 2014, 2, 7123–7131.
16. M. D. Howard, A. Ponta, A. Eckman, M. Jay and Y. Bae, Pharm. Res., 2011, 28, 2435–2446.
17. T. Jiang, Y. M. Li, Y. Lv, Y. Jia Cheng, F. He and R. Xi Zhuo, Colloids Surf., B, 2013, 111, 542–548.
18. G. Mo, X. Hu, S. Liu, J. Yue, R. Wang, Y. Huang and X. Jing, Eur. J. Pharm. Sci., 2012, 46, 329–335.
19. H. Nakamura, T. Etrych, P. Chytil, M. Ohkubo, J. Fang, K. Ulbrich and H. Maeda, J. Controlled Release, 2014, 174, 81–87.
20. Y. Cao Y. Gu, L. Liu, Y. Yang, P. Zhao, P. Su, L. Liu C. Qi, X. Lei and C. Yang, Lett. Drug Des. Discovery, 2010, 7, 500–506.
21. J. M. Saul, A. V. Annapragada and R. V. Bellamkonda, J. Controlled Release, 2006, 114, 277–287.
22. H. S. Yoo and T. G. Park, J. Controlled Release, 2004, 96, 273–283.
23. J. M. Saul, A. Annapragada, J. V. Natarajan and R. V. Bellamkonda, J. Controlled Release, 2003, 92, 49–67.
24. H. Zhao and L. Y. Yung, Int. J. Pharm., 2008, 349, 256–268.
25. A. Gabizon, H. Shmeeda, A. T. Horowitz and S. Zalipsky, Adv. Drug Delivery Rev., 2004, 56, 1177–1192.
26. J. Varshosaz, F. Hassanzadeh, H. S. Aliabadi, M. Banitalebi, M. Rostami and M. Nayebsadrian, Colloid Polym. Sci., 2014, 292, 2647–2662.
27. A. Mehdizadeh, S. Pandesh, A. Shakeri-Zadeh, S. Kamran Kamrava, M. Habib-Agahi, M. Farhadi, M. Pishghadam, A. Ahmadi, S. Arami and Y. Fedutik, Lasers Med Sci, 2014, 29, 939–948.
28. E. Moradi, D. Vllasaliu, M. Garnett, F. Falcone and S. Stolnik, RSC Adv., 2012, 2, 3025–3033.
29. M. Prabaharan, J. J. Grailer, S. Pilla, D. A. Steeber and S. Gong, Biomaterials, 2009, 30, 5757–5766.
30. L. Xu Hua, Y. Sun, L. Lu Sha, Y. Yong Zhao, X. Yun Hu and J. Fan, Chin. Chem. Lett., 2012, 23, 1133–1136.
31. D. Lu, X. Wen, J. Liang, Z. Gu, X. Zhang and Y. Fan, J. Biomed. Mater. Res., Part B, 2009, 89, 177–183.
32. H. Li, S. Bian, Y. Huang, J. Liang, Y. Fan and X. Zhang, J. Biomed. Mater. Res., Part A, 2014, 102, 150–159.
33. H. S. Yoo and T. G. Park, J. Controlled Release, 2004, 100, 247–256.
34. L. W. Seymour, K. Ulbrich, J. Strohalm, J. Kopeček and R. Duncan, Biochem. Pharmacol., 1990, 39, 1125–1131.

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Footnote

† Equal contributors to the work.

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