Preparation and characterization of nanoparticles based on histidine–hyaluronic acid conjugates as doxorubicin carriers
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- Wu, J., Liu, C., Wang, X. et al. J Mater Sci: Mater Med (2012) 23: 1921. doi:10.1007/s10856-012-4665-8
Histidine–hyaluronic acid (His–HA) conjugates were synthesized using hyaluronic acid (HA) as a hydrophilic segment and histidine (His) as hydrophobic segment by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) mediated coupling reactions. The structural characteristics of the His–HA conjugates were investigated using 1H NMR. His–HA nanoparticles (HH-NPs) were prepared based on His–HA conjugates, and the characteristics of HH-NPs were investigated using dynamic light scattering, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and fluorescence spectroscopy. The particles were between 342 and 732 nm in size, depending on the degree of substitution (DS) of the His. TEM and SEM images indicated that the morphology of HH-NPs was spherical in shape. The critical aggregation concentrations of HH-NPs ranged from 0.034 to 0.125 mg/ml, which decreased with an increase in the DS of the His. Images of fluorescence microscopy indicate that HH-NPs were taken up by the cancer cell line (MCF-7), and significantly decreased by competition inhibition of free HA. From the cytotoxicity test, it was found that DOX-loaded HH-NPs exhibited similar dose and time-dependent cytotoxicity against MCF-7 cells with free DOX.
Cancer is a multifaceted disease that represents one of the leading causes of mortality in the world. Due to lack of selectivity against cancer cells, traditional chemotherapy produces serious systemic toxicity and adverse effects. For minimizing the side effects, self-assembled polymeric nanoparticles which were composed of hydrophilic shell and hydrophobic core have intensively investigated in recent years [1, 2]. Because hydrophilic shells contributes to prolong circulation in the bloodstream, and the hydrophobic core can encapsulate various drugs, and release them in a sustained manner passively at tumor site due to enhanced permeability and retention (EPR) effects , these self-assembled nanoparticles have displayed potent prosperity in cancer therapy. However, this passive targeting strategy of nanoparticles is limited in its ability to eradicate the tumor because the nanoparticles may be taken up by normal cells and release a considerable portion of drugs before arrival at target site .
To improve selectivity and efficacy towards tumor cells, the first strategy is to design active targeting nanoparticles modified by targeting moieties such as nucleic acids [5, 6], antibodies [7, 8] and various ligands [9–11]. As drug-delivery agents, these multifunctional nano-carriers are capable of targeting cancer cells, delivering and releasing drugs in a regulated manner, and detecting cancer cells with enormous specificity and sensitivity. Another strategy is to design nanoparticles which can be triggered to release encapsulated drugs when environmental conditions, such as pH [12–14], temperature [15, 16], light  and magnetic fields , change in vivo. These parameters can be actively controlled in time to navigate drug-loaded nanoparticles through the biological hurdles.
Hyaluronic acid (HA) is a linear, negatively charged polysaccharide, consisting of two alternating units of d-glucuronic acid and N-acetyl-d-glucosamine. It is biocompatible, biodegradable and present in the extracellular matrix and synovial fluids. HA can bind to CD44 receptor, which is overexpressed in various kinds of cancer cells. Thus HA, as a targeting moiety, is potent in pharmaceutical applications for anti-cancer therapeutics. In the recent studies, HA conjugates encapsulating anticancer agents such as paclitaxel [19–21], doxorubicin , and siRNA , have exhibited enhanced targeting ability to the tumor and higher therapeutic efficacy compared with free anti-cancer agents.
Owing to weakly acidic extracellular pH (pH 6.5–7.2) in the solid tumors, pH-responded nanoparticles have been investigated for their potential use in controlled release . His, an essential amino acid, has a positively charged imidazole functional group (pKb ∼ 6.5). The hydrophobic imidazole group in the His becomes hydrophilic as a result of protonation of the amine group at lower pH. The property of His contributes to the development of potent pH-responsive nanoparticles which can cumulate anticancer drugs in tumor cells [25, 26].
The objective of this study was to synthesize His–HA conjugates by chemical modification of His to the backbone of HA. These HH-NPs with different degrees of substitution of His were prepared and characterized. Their physicochemical characteristics were studied using 1H NMR, dynamic light scattering (DLS), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Cytotoxicity assay was evaluated by MTT assay. In addition, cellular uptake of HH-NPs in vitro was monitored to evaluate internalization via receptor-mediated endocytosis.
2 Materials and methods
Sodium hyaluronate (Mw: 100k Dr) was purchased from Shandong Freda Biopharm Co., Ltd. (China). l-Histidine (His) was purchased from Sinopharm Chemical reagent Co., Ltd. Doxorubicin HCl (DOX·HCl) was purchased from ShanXi powerdone pharmaceutical Co., Ltd. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from ShangHai Medpep Co., Ltd, Pyrene was purchased from Sigma (St. Louis, MO). Fluorescein isothiocyanate (FITC) was purchased from Huasheng Co., Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Beijing Solarbio Co., Ltd, human breast-cancer cell line cells (MCF-7) was purchased from QingDao University. All other chemicals were analytical grade.
2.2 Synthesis of His–HA conjugates
HA was dissolved in distilled water. In the presence of EDC and NHS, the His (different molar ratio) was added to the HA solution under stirring. The reaction was then allowed to proceed for 24 h at room temperature. The resulting solution was dialyzed against distilled water for 5 days. After freeze-drying, the chemical structure of His–HA conjugates was determined by 1H NMR (JNM ECP-600, JEOL, Japan), for which the sample was prepared by dissolving the conjugate in D2O.
2.3 The preparation of HH-NPs
His–HA conjugates were suspended in phosphate buffered saline (PBS, pH 7.4) under gentle shaking. The solution was sonicated three times using a probe-type sonifier (VCX-750, Sonics & Materials, CT, USA) at 90 W, and the pulse was turned on for 2 s with 3 s interval under ice bath.
2.4 Critical aggregation concentration
The critical aggregation concentration (CAC) of HH-NPs was evaluated using fluorescence spectroscopy in the presence of pyrene molecules . In brief, a pyrene solution (12 × 10−7 M in distilled water) was mixed with HH-NPs to obtain polymer concentrations from 1.0 × 10−4 to 1.0 mg/ml. The final concentration of pyrene in each sample was 6.0 × 10−7 M. Pyrene fluorescence spectra was obtained by RF-5301PC fluorescence spectrophotometer (Shimadzu Co., Kyoto, Japan).
2.5 DLS and zeta potential
The particle size of the HH-NPs was measured by Malvern Zetasizer 3000HSA (Malvern Instruments Ltd., Malvern, UK). All measurements were done at a wave-length of 635 nm at 25° with an detection angle of 90°. The concentration of nanoparticles was kept 1 mg/ml, and each batch was analyzed in triplicate.
Zeta potential of the nanoparticles was measured by Malvern Zetasizer 3000HSA. Each sample was measured three times at 25°. Each experimental result is an average of three independent measurements.
2.6 TEM and SEM
The morphology of the HH-NPs was observed by electron microscopy. One drop of the nanoparticles suspension was placed on a copper grid. The grid was allowed to dry at room temperature, and was examined with the TEM (Philips EM 400, Netherlands). The vacuum freeze-dried powder of nanoparticles were distributed evenly on the conductive adhesive, slightly pressed, sprayed gold by ion sputtering, and examined in the SEM (S-3400N Hitachi, Japan).
2.7 DOX loading efficiency and encapsulation efficiency
The DOX-loaded HH-NPs were prepared using a dialysis method . In brief, 100 mg of HH9 conjugate was dissolved in 5 ml of formamide by gently heating. And, 10 mg of DOX·HCl were dissolved in 2 ml of DMF containing TEA (1.3 mol ratio of DOX·HCl). The two solutions were well mixed by vortexing, dialyzed against distilled water to remove the unloaded drugs, DMF, TEA, formamide and triethylammonium chloride (TEA·HCl), followed by sonicating for 180 s at 90 W, and lyophilized.
Then 3 mg of DOX-loaded HH9 nanoparticles was dissolved in 9 ml of formamide by gently heating. The absorbance of the solution at 480 nm was measured by UV–Vis spectrophotometer, and the concentration of DOX in solution was obtained using the standard curve.
2.8 In vitro drug release study
In vitro the release of DOX was investigated using a dialysis method in PBS (pH 7.4 and 6.0). Briefly, 5 ml of DOX-loaded nanoparticle solution was placed in a dialysis bag, and dialyzed against 50 ml of the release medium at 100 rpm under 37°. At predetermined time intervals, 4 ml medium was removed and replaced with the same amount of fresh release medium. The amount of DOX released was determined by UV spectrophotometer at 480 nm, the amount of DOX released was calculated according to the standard curve . Each experimental result is an average of at least three independent measurements.
2.9 In vitro cytotoxicity of DOX-loaded HH-NPs
MCF-7 cells were cultured in DMEM medium containing 10 % (v/v) fetal bovine serum and 1 % (w/v) penicillin–streptomycin in a humidified 5 % CO2 at 37°.
The cytotoxicity of HH-NPs was evaluated using MTT assay according to the previously established method . The MTT assay is a colorimetric assay that measures activity of mitochondrial succinate dehydrogenase that reduces MTT to insoluble purple formazan crystals. Crystals are solubilized by the addition of a detergent so the absorbance can be read using a spectrophotometer. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells. In brief, MCF-7 cells were seeded in 96-well plates at a density of 5 × 103 cells/well at 37°. After 24 h, DOX-loading HH-NPs, free DOX-HCl were added to the cells, and followed by incubating for 24 h. MTT(20 μl) solution was added to each well, and the cells were incubated for an additional 4 h at 37°. Subsequently, the medium was removed and the cells were dissolved in DMSO. The cell viability was measured by absorbance at 490 nm in a microplate reader (FLx800B, Bio-Tek, USA). Each data point represents the average result of four wells and three independent experiments.
2.10 In vitro cellular uptake behavior of HH-NPs
MCF-7 cells were seeded in 6-well plates at a density of 5 × 104 cells/ml at 37°. After cell attachment, the medium was replaced with 2 ml of serum-free culture medium containing FITC-labeled HH-NPs loaded with DOX, followed by incubation for 2 h.
A competition study was designed to investigate whether HH-NPs were specifically taken up by MCF-7 cells through HA receptor (CD44) mediated endocytosis [30, 31]. DOX-loaded HH-NPs (DOX ~ 2 μg/ml) was added to 6-well culture plates containing MCF-7 cells, and free HA solution in D-Hanks (2 mg/ml) was treated simultaneously, followed by incubation for 2 h. The cells were then washed twice with D-Hanks (pH 7.4) and fixed with a 4 % paraformaldehyde solution. The intracellular localization of HH-NPs was observed by Fluorescence microscopy (excitation 479 nm, emission 587 nm) and images were taken.
3 Results and discussions
3.1 Synthesis of His–HA conjugates
3.2 Characteristics of HH-NPs
Mean diameters and zeta potential measurements of HA nanoparticles in different DS
Zeta potential (mV)
732 ± 47.1
−19.3 ± 1.6
351 ± 30.1
−17.3 ± 1.1
342 ± 29.6
−13.5 ± 0.7
3.3 In vitro DOX loading and release studies
When the initial feed ratio of DOX to HH-NPs was 10 wt%, EE of HH9 was 87.23 % and the LE of HH9 was 7.02 %. It showed that HH-NPs could solubilize and stabilize hydrophobic DOX molecules in aqueous solution due to its hydrophobic core and hydrophilic shell structures.
3.4 In vitro cellular uptake of HH-NPs
3.5 In vitro cytotoxicity of DOX-loaded HH-NPs
In this study, amphiphilic His–HA conjugates can form stable nano-sized particles composed of hydrophilic shell and hydrophobic core. Doxorubicin was successfully entrapped to form DOX-loaded HA nanoparticles. The study demonstrated that the HH-NPs can be taken up by cancer cells over-expressive CD44 through receptor-mediated endocytosis and that drug-loaded nanoparticles show dose and time-dependent cytotoxicity against cancer cells. These results imply that self-assembled HH-NPs have potential as a carrier for hydrophobic drugs in cancer therapy.
This work was supported by the Natural Science Foundation of Shandong Province of China (Grant ZR2009CM071).