Journal of Materials Science: Materials in Medicine

, Volume 23, Issue 8, pp 1921–1929

Preparation and characterization of nanoparticles based on histidine–hyaluronic acid conjugates as doxorubicin carriers

Authors

  • Jing-liang Wu
    • College of Marine Life ScienceOcean University of China
    • College of Pharmaceutical and Biological ScienceWeiFang Medical University
    • College of Marine Life ScienceOcean University of China
  • Xiao-lei Wang
    • College of Marine Life ScienceOcean University of China
  • Zhen-hua Huang
    • College of Marine Life ScienceOcean University of China
Article

DOI: 10.1007/s10856-012-4665-8

Cite this article as:
Wu, J., Liu, C., Wang, X. et al. J Mater Sci: Mater Med (2012) 23: 1921. doi:10.1007/s10856-012-4665-8

Abstract

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.

1 Introduction

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 [3], 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 [4].

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 [911]. 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 [1214], temperature [15, 16], light [17] and magnetic fields [18], 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 [1921], doxorubicin [22], and siRNA [23], 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 [24]. 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

2.1 Materials

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 [27]. 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 [28]. 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.

The loading efficiency (LE) of nanoparticles and the encapsulation efficiency (EE) were calculated by using following equation:
$$ {\text{LE}} = {\text{Ws}}/{\text{Wc}} \times 100\% \quad {\text{EE}} = {\text{Ws}}/{\text{Wa}} \times 100\% $$
Wa = total amount of DOX in added solution; Ws = total amount of DOX in freeze-drying nanoparticles; and Wc = weight of the nanoparticles measured after freeze-drying.

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 [21]. 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 [29]. 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

His–HA conjugates were synthesized using HA as a hydrophilic segment and His as hydrophobic segment by EDC mediated coupling reactions (Fig. 1) [32]. The chemical structure of HA, His and His–HA conjugates were characterized by 1H NMR. The peaks at 7.11 and 8.44 ppm (Fig. 2A) showed the methylene protons (a, –N–CH=C–; b ,–N=CH–) located in imidazole group of His. The peak at 2.0 ppm (Fig. 2B) was attributed to the methyl protons of HA (c, NHCO–CH3). Successful conjugation was supported by the presence of the peaks of His at 7.11 and 8.44 ppm (Fig. 2C). The degree of substitution (DS) was determined by comparing the integral ratio between the methyl protons of HA and the methylene protons of His. By varying the free ratio of His to HA polymers, the DS was controlled in the range of 4–10.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-012-4665-8/MediaObjects/10856_2012_4665_Fig1_HTML.gif
Fig. 1

Synthesis scheme of HA–His conjugates

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Fig. 2

1H-NMR spectra of A His, B HA, and C HA–His conjugates in D2O

3.2 Characteristics of HH-NPs

When His–HA conjugates were dispersed in PBS (pH 7.4) and sonicated, the conjugates formed nano-sized particles. As shown in Table 1, the mean diameters of HH-NPs (HH5, HH9, and HH10) were in the range of 342–732 nm, depending on the DS value. The size of HH-NPs decreased as the DS value increased, indicating that His as hydrophobic segment in the conjugate allowed for formation of compact hydrophobic cores.
Table 1

Mean diameters and zeta potential measurements of HA nanoparticles in different DS

HH-NPs

DLS (nm)

Zeta potential (mV)

HH5

732 ± 47.1

−19.3 ± 1.6

HH9

351 ± 30.1

−17.3 ± 1.1

HH10

342 ± 29.6

−13.5 ± 0.7

The CAC of HH-NPs was determined using fluorescence spectroscopy in the presence of pyrene, which had been widely utilized to monitor self-aggregation behavior of various surfactants and amphiphilic polymers [30]. The variation in the ratio of intensity of third (383 nm) to first (372 nm) vibronic peaks (I383/I372) was quite sensitive to the polarity of microenvironments where pyrene was located. The formation of the aggregates with a hydrophobic inner core could be detected by means of plotting I383/I372 versus polymer concentration. Figure 3 showed the intensity ratio (I383/I372) of the pyrene excitation spectra versus the logarithm of the HH-NPs concentration. The CAC values of the HH-NPs were 0.098, 0.045, 0.039 mg/ml, respectively as the DS increased, showing a strong hydrophobic interaction in the inner core of HH-NPs. The CAC values of the HH-NPs was significantly lower than those of low molecular surfactants such as sodium dodecyl sulfate in water (2.3 mg/ml). The low CAC value was one of the essential parameters for the use of self-assembled nanoparticles as a drug carrier, since conjugates with low CAC values might have resistance to dissociation at highly diluted conditions in the body [21].
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Fig. 3

The intensity ratios (I383/I372) from pyrene emission spectra as a function of logarithm of HA–His conjugate concentration in distilled water

The zeta potential of HH-NPs ranged from −19.3 to −13.5 mV as the DS value increased, this is due to that carboxylic acid groups of HA and amine groups of His reacted to form more amide bonds. The TEM (Fig. 4a) and SEM (Fig. 4b) images of HH-NPs were shown in Fig. 4, which revealed intact and well-separated, spherical nanoparticle structures.
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Fig. 4

TEM and SEM images of HH-NPs. a TEM image, b SEM image

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.

The release profiles of DOX were shown in Fig. 5. It exhibited an initial burst of 15.8 ± 1.24 % (pH 7.4) and 21.4 ± 1.56 % (pH 6.0) in 4 h, and an accumulative release of 27.98 ± 1.07 % (pH 7.4) and 50.97 ± 2.23 % (pH 6.0) after 48 h, respectively. It showed that the HH-NPs could release large amount of drug at acidic environments, suggesting that HH-NPs form a loose sphere due to low hydrophobicity caused by the protonation of imidazole groups at lower pH. The loose sphere results in more drug release from the hydrophobic core [25]. Owing to weakly acidic extracellular pH in the solid tumors, this pH sensitivity may make the nanoparticles more effective for targeting solid tumors to release anti-cancer drugs entrapped.
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Fig. 5

Release behavior of DOX from HH-NPs in different pH mediums in vitro

3.4 In vitro cellular uptake of HH-NPs

Cellular uptake of FITC-labeled nanoparticles and DOX-loaded nanoparticles was examined by fluorescent microscopy. As shown in Fig. 6a–c strong green fluorescent signals were detected in the cytoplasm for FITC-labeled His–HA nanoparticles tested in MCF-7 cells, indicating that nanoparticles were readily internalized into MCF-7 cells. On the other hand, the auto-fluorescence of DOX was observed in the cytoplasm as well as the nucleus when the cells were incubated with the DOX-loaded nanoparticles (Fig. 6d–f), which were attributed to the DOX molecules released from the nanoparticles. Therefore, it is reasonable to conclude that DOX encapsulated into the nanoparticles was transferred into the cells by the nanoparticles. Figure 6 shows the fluorescent microscopic images of cells incubated with FITC-labeled nanoparticles or DOX-loaded nanoparticles.
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-012-4665-8/MediaObjects/10856_2012_4665_Fig6_HTML.jpg
Fig. 6

Fluorescence microscopy images of MCF-7 tumor cells treated with FITC-labeled particles or DOX-loaded particles. FITC channel for FITC-labeled nanoparticles (green), TRITC channel for DOX (red) and DAPI channel for nucleus (blue) are simultaneously presented: a FITC, b DAPI, c merged panels of (a)–(b), d DOX, e DAPI, and f merged panels of (d)–(e) (Color figure online)

HA can bind to CD44 receptor, which is overexpressed in various kinds of cancer cells such as MCF-7 cells lines [21, 22]. To confirm receptor-mediated endocytosis of HH-NPs, the competition study was carried out. The strong fluorescence from DOX was observed in the cells (Fig. 7a), while it was significantly reduced by treatment with 2 mg/ml HA (Fig. 7b). These results suggest that the His–HA nanoparticles prepared in this study selectively bind to CD44 and internalize into the cancer cells via receptor-mediated endocytosis [4, 22, 33].
https://static-content.springer.com/image/art%3A10.1007%2Fs10856-012-4665-8/MediaObjects/10856_2012_4665_Fig7_HTML.jpg
Fig. 7

Fluorescence microscopy analyses of competition between free HA and DOX-loaded HH-NPs. a DOX-loaded nanoparticles, b DOX-loaded nanoparticles with free HA

3.5 In vitro cytotoxicity of DOX-loaded HH-NPs

The cytotoxicity of DOX-loaded His–HA nanoparticles and free DOX on MCF-7 was compared in Fig. 8. The results showed that DOX-loaded His–HA nanoparticles exhibited similar dose and time-dependent cytotoxicity against MCF-7 cells with free DOX. Detailed observation of the cell viability in Fig. 8a indicated that DOX-loaded His–HA nanoparticles induce more cell death than free DOX after 24 h of incubation, implying that DOX-loaded His–HA nanoparticles show higher cytotoxicity than free DOX, the possible explanation is that the His–HA nanoparticles are internalized into cells by HA receptor-mediated endocytosis which inhibits P-glycoprotein-mediated drug efflux [7]. As the treatment time went on, however, free DOX showed better in vitro therapeutic effects than DOX-loaded nanoparticles after 72 h of incubation (Fig. 8c). This result is consistent with previous studies where DOX-loaded micelles show lower cytotoxicity than free DOX at 72 h. The somewhat lower toxicity of the nanoparticles system may result from the gradual release of DOX within the cell and further studies are needed to elucidate exact mechanism [34].
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Fig. 8

Viability of MCF-7 cells after treatment with free DOX or DOX-loaded nanoparticles for 24 h (a), 48 h (b), and 72 h (c)

4 Conclusion

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.

Acknowledgments

This work was supported by the Natural Science Foundation of Shandong Province of China (Grant ZR2009CM071).

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© Springer Science+Business Media, LLC 2012