Colloids and Surfaces B: Biointerfaces 90 (2012) 28–35
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Self-assembled nanoparticles of hyaluronic acid/poly(dl-lactide-co-glycolide) block copolymer Young-Il Jeong, Do Hyung Kim 1 , Chung-Wook Chung, Jin Ju Yoo, Kyung Ha Choi, Cy Hyun Kim, Seung Hee Ha, Dae Hwan Kang ∗ National Research and Development Center for Hepatibiliary Disease, Pusan National University YangSan Hospital, Beomeo-ri, Mulgeum-eup, Yangsan, Gyeongnam 626-770, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 August 2011 Received in revised form 20 September 2011 Accepted 20 September 2011 Available online 2 October 2011 Keywords: Block copolymer Nanoparticles Hyaluronic acid PLGA CD44 Receptor-mediated endocytosis
a b s t r a c t We synthesized block copolymer composed of hyaluronic acid (HA) and poly(dl-lactide-co-glycolide) (PLGA) (HAbLG) for antitumor targeting. 1 H NMR was employed to confirm synthesis of block copolymer. At 1 H NMR study, HabLG nanoparticles showed HA intrinsic peaks only at D2 O, indicating that they contained HA as a hydrophilic outer-shell and PLGA as a inner-core. Anti-tumor activity was studied using CD44-overexpressing HCT-116 human colon carcinoma cells. Addition of doxorubicin (DOX)incorporated nanoparticles to tumor cells resulted in the expression of a strong red fluorescence color while they expressed very weak fluorescence when CD44 receptor was blocked with free HA. Flow cytometry data also showed similar results, indicating that the fluorescence intensity of tumor cells treated with nanoparticles was significantly decreased when CD44 receptor was blocked. These results indicate that HAbLG nanoparticles were able to target CD44-overexpressing tumor cells via receptor-mediated endocytosis. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hyaluronic acid (HA), a linear polysaccharide, has a unique structure of repeating disaccharide units composed of alternating d-glucuronic acid and N-acetyl-d-glucosamine units [1,2], and it is one of the glycosaminoglycan components of the extracellular matrix (ECM). Especially, receptors of HA such as CD44 and RHAMM are also overexpressed at sites of tumor attachment to the mesentery, which suggests HA acts as a matrix for spreading, migration, invasion, and metastasis of tumor cells [3–6]. Especially, expression of HA receptors such as CD44 and RHAMM in tumor cells is also known to be positively correlated with cell migration and invasion; specifically, malignant tumor cells invade HA-rich environments [3–10]. Based on these points of view, many scientists use HA as a targeting molecule for anti-tumor drug delivery, indicating that invasive or metastatic tumor cells excessively express CD44 and that polymer conjugates containing HA as well as anti-cancer agents can target this receptor via receptor-mediated uptake [11–14]. Furthermore, HA is regarded as an excellent bioinert material based on its characterization as an immunoneutral,
∗ Corresponding author. Tel.: +82 55 360 3870; fax: +82 55 360 3879. E-mail address:
[email protected] (D.H. Kang). 1 This author equally contributed to this work. 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.09.043
biocompatible, and biodegradable material [12,15–17]. Therefore, HA is considered to be a superior candidate for anti-tumor drug delivery. Amphiphilic macromolecules such as hydrophobic polysaccharides, block copolymers, and graft copolymers have been extensively investigated for use as a targeted drug delivery system [11,18–22]. These polymers are frequently used as nanostructured medicine via self-assembling properties, i.e., liphophilic domains of polymers can form a drug-containing solid-core based on hydrophobic interactions with polymers and anti-cancer agents, whereas the hydrophilic domain forms an outer-shell of the nano-assemblies [18–22]. Among them, block copolymers are primarily considered to be applicable as a nanomedicine for anticancer drug targeting due to their unique structures and ability to conjugate their targeting moiety to a hydrophilic segment [21–24]. Jeong et al. [21] and Suo et al. [24] reported the cellular recognition of galactose-tagged poly(ethylene glycol)-based block copolymers using hepatocellular carcinoma cells. Therefore, nanoparticles composed of block copolymers are considered to be ideal vehicles for solubilizing hydrophobic drugs, site-specific drug delivery via active or passive targeting mechanisms, reducing the amount of drug administered, and avoiding unwanted side effects [21–23,25,26]. In this study, we synthesized HA-based block copolymer using low molecular weight HA and poly(dl-lactic acid-co-glycolic acid)
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(PLGA) (abbreviated as HAbLG), followed by the preparation of core–shell type nanoparticles containing an anti-cancer agent, DOX. Furthermore, HA in HAbLG block copolymer may have formed a hydrophilic outer segment, which acted as an ideal moiety for tumor cell targeting, whereas PLGA acted as a drug incorporation part. We also investigated the physicochemical properties of nanoparticles composed of HAbLG copolymer as well as their in vitro target ability against tumor cells using HCT116 cells, a CD44-overexpressing human colon carcinoma cell type.
2. Materials and methods 2.1. Materials HA (molecular weight: 7460 Da) was purchased from Lifecore Biomedical (Chaska, MN, USA). Triethylamine (TEA), doxorubicin HCl, sodium cyanoborohydride, hexamethylene diamine (HMDA), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma Chem. Co. (St. Louis, USA). N,N -Dicyclohexyl carbodiimide (DCC) and N-hydroxysuccimide (NHS) were purchased from Aldrich Chemical Co. USA. The dialysis membranes with molecular weight cutoffs (MWCO) of 2000, 8000, and 12,000 g/mol were purchased from Spectra/ProTM Membranes. Dichloromethane (DCM) and dimethyl sulfoxide (DMSO) were of HPLC grade or extra-pure grade. Poly(dl-lactic acid-co-glycolic acid) (PLGA-5005, Mw = 5000 g/mol) was purchased from Wako Pure Chemicals Co. (Osaka, Japan). The molecular weight (Mw ) of PLGA was measured by GPC, as described previously [12]. The weight average Mw , number average Mw , and polydispersity of PLGA were 4920, 4780, and 1.029, respectively.
2.2. Synthesis of HAbLG block copolymer Aminated HA was prepared as described by Maruyama et al. [27] with brief modification. HA (400 mg) was dissolved in 10 ml of H2 O/DMSO (3/7, v/v), and an excess amount of sodium cyanoborohydride was then added. This mixture was stirred for 24 h at room temperature. Next, 10 equiv. of hexamethylene diamine was added, and the mixture was stirred for 24 h at room temperature. Reactants were introduced into the dialysis membrane (MWCO, 2000 g/mol) and were dialyzed against deionized water for 2 days. The dialyzed solution was lyophilized for 3 days. The conjugation yield of amine was analyzed by 1 H NMR spectroscopy. N-Hydroxysuccimide PLGA (PLGA–NHS) was prepared as follows: 500 mg of PLGA was dissolved in dichloromethane, after which 1.5 equiv. of DCC and NHS were added. This solution was stirred for 6 h, and then reactants were filtered to remove any byproducts. Then, the solvent was removed under reduced pressure, and the solid was washed with methanol three times and dried under a vacuum for 1 day. For synthesis of HAbLG copolymer, 150 mg of aminated HA and 100 mg of PLGA were dissolved in dry DMSO. This solution was stirred in nitrogen atmosphere for 2 days at room temperature. Then, reactants were precipitated in deionized water, and this solution was introduced into a dialysis membrane (MWCO: 12,000 g/mol). To remove unreacted HA and organic solvent, solution was dialyzed for 2 days against deionized water and then lyophilized for 3 days. Following this, a white solid was obtained. This product was added to dichloromethane to remove any unreacted PLGA, after which precipitates were harvested by filtration. This procedure was repeated three times. The resulting white solid was dried under a vacuum for 3 days. The yield of the final product was 78% (w/w). Yield was calculated by the following
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equation: Yield of product (%, w/w) =
weight of final product weight of aminated HA + PLGA–NHS
× 100.
2.3. Preparation of DOX-incorporated nanoparticles of HAbLG DOX-incorporated nanoparticles composed of HAbLG block copolymer were prepared as follows: 40 mg of HAbLG block copolymer was dissolved in 4 ml of H2 O/DMSO (1/3, v/v). Then, 5 or 10 mg of DOX was separately dissolved in 1 ml of DMSO containing a 2 equiv. molar ratio of TEA, and this solution was added to the above HAbLG solution. This mixture was further stirred for 1 h and then dropped into 15 ml of deionized water over 10 min to form the nanoparticles. The solvent was removed by dialysis against deionized water using a dialysis membrane (8000 g/mol) for at least 1 day. Deionized water was exchanged at 1–2 h intervals during the dialysis procedure. Subsequently, the resulting solution was used for analysis or lyophilized. Empty nanoparticles of HAbLG copolymer were prepared by the same procedure in the absence of DOX. For evaluation of drug loading contents, 5 mg of DOXincorporated nanoparticles were dissolved in 10 ml of H2 O/DMSO (2/8, v/v) and then diluted 100 times with DMSO. The DOX concentration was measured using a UV-spectrophotometer (UV spectrophotometer 1201, Shimadzu Co. Japan) at 479 nm. Empty nanoparticles of HAbLG copolymer were prepared as the blank. Drug content =
drug weight in the nanoparticle
Loading efficiency =
weight of nanoparticle
× 100
weight of remained drug in nanoparticles feeding weight of drug ×100
2.4. Analysis of HAbLG nanoparticles Characterization of the nanoparticles was performed in DMSO or D2 O by 500 MHz 1 H NMR spectroscopy (500 MHz Superconducting FT-NMR Spectrometer, Unity-Inova 500). The morphology of nanoparticles was observed using a transmission electron microscope (TEM, JEOL JEM-2000 FX II, Japan). One drop of nanoparticle solution was placed onto a carbon film coated on a copper grid. Phosphotungstic acid (0.05% (w/w)) was used for negative staining. Observation of nanoparticles was performed at 80 kV. The size of the nanoparticles was measured with dynamic laser scattering (DLS-7000, Otsuka Electonics Co. Japan). A sample solution prepared via dialysis was used to determine the particle size (concentration: 1 mg/ml). Fluorescence spectroscopy (Shimadzu RF-5301 PC spectrofluorophotometer, Shimadzu Co. Ltd., Japan) was performed to prove the potential of HAbLG self-assembly. To determine the critical association concentration (CAC) of HAbLG copolymers, empty nanoparticles were prepared as described above. The resultant suspension was adjusted to contain various concentrations of nanoparticles. The CAC of the HAbLG copolymers was estimated using pyrene as a hydrophobic probe [24]. Sample solutions were prepared as follows: a known amount of pyrene in acetone was added to each of the vials, after which acetone was evaporated. The final concentration of pyrene was 6.0 × 10−7 M. Then, 10 ml each of various concentrations of the nanoparticle solutions were added,
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followed by arming for 3 h at 65 ◦ C. Equilibration of pyrene and the HAbLG nanoparticles was achieved by cooling the solutions for 3 h at room temperature. The fluorescence excitation spectra were measured at an emission wavelength of 390 nm. Excitation and emission bandwidths were 1.5 nm and 1.5 nm, respectively. 2.5. Drug release study in vitro Release of DOX from HAbLG nanoparticles was performed in vitro. Five milligrams of DOX-incorporated nanoparticles was reconstituted in 5 ml of phosphate-buffered saline (PBS, 0.1 M, pH 7.4), and this solution was introduced into the dialysis membrane (MWCO: 8000 g/mol). This dialysis membrane was placed in a 200 ml bottle containing 95 ml of PBS. Following this, the bottle was placed in a shaking incubator at a stirring speed of 100 rpm and 37 ◦ C. Whole media were removed at specific time intervals in order to measure the drug concentration and replaced with fresh PBS to avoid drug saturation. The concentration of DOX released into PBS was measured using a UV-spectrophotometer (UV spectrophotometer 1201, Shimadzu Co. Japan) at 479 nm. 2.6. In vitro cell cytotoxicity test HCT-116 cells (human colon carcinoma cell line) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (5% CO2 at 37 ◦ C) to evaluate the anti-tumor activity of DOXincorporated nanoparticles. Growth inhibition of tumor cells was evaluated by MTT cell proliferation assay. HCT-116 cells at a density of 1 × 104 cells were seeded in 96-well plates with 100 l of medium and then incubated overnight in a CO2 incubator (5% CO2 at 37 ◦ C). One hour before the addition of nanoparticles, HA receptor (CD44) of tumor cells was blocked with 10 equiv. of HA. Following this, free DOX, DOX-incorporated nanoparticles, and empty nanoparticles were added to 96-well plates. Controls were treated with 0.5% (v/v) of DMSO. After 1 day of incubation, 30 l of MTT (5 mg/ml) was added to 96-well plates, followed by incubation for 4 h. The formazan crystals formed were solubilized with DMSO, and the absorbance (560 nm-test/630 nm-reference) was determined using an automated computer-linked microplate reader (Molecular Device Co., USA). Each drug concentration measurement was obtained as the mean value of eight wells. The amount of formazan present was proportional to the number of viable cells, as only living cells could reduce MTT to blue formazan. The results were expressed as a percentage of absorbance present in the drugtreated cells compared to that in the control cells. 2.7. Receptor-mediated endocytosis of nanoparticles into HCT-116 cells To observe tumor cells, HCT-116 cells were seeded onto a coverglass and incubated overnight in a CO2 incubator (5% CO2 at 37 ◦ C). One hour before the addition of nanoparticles, HA receptor (CD44) of tumor cells was blocked with 10 equiv. of HA. Following this, nanoparticles were added to tumor cells and incubated for 1 h in a CO2 incubator. Then, cells were washed with PBS (pH 7.4, 0.1 M) and treated with 4% paraformaldehyde, followed by washing again with PBS and fixing with immobilization solution (IMMU-MOUNT, Thermo Electron Corporation, Pittsburgh, PA 15275, USA). These cells were then observed with a confocal laser scanning microscope (CLSM, TCS-SP2; Leica, Wetzlar, Germany). To evaluate receptor-mediated endocytosis of the nanoparticles, tumor cells were seeded in 6-well plates at a density of 1 × 106 cells/well. Before the addition of nanoparticles, HA receptor (CD44) of tumor cells was blocked with 10 equiv. of HA. Following this, nanoparticles were added to the tumor cells and incubated for 1 h in a CO2 incubator. Then, cells were washed with PBS (pH
7.4, 0.1 M) and harvested by trypsinization, followed by washing again with PBS and flow cytometry analysis (FACScan). An excitation wavelength of 488 nm and emission wavelength of 522 nm were used to observe DOX fluorescence intensity. 3. Results 3.1. Characterization of HAbLG block copolymer For block copolymer synthesis, aminated HA and PLGA–NHS were synthesized as shown in Fig. 1. Since polysaccharides have one reductive end, the reductive end of HA was treated with sodium cyanoborohydride. To construct the aminated end of HA, an excess amount of HMDA was added, as shown in Fig. 1(a), after which aminated HA was purified by dialysis. Aminated HA (HA-NH2 ) was analyzed by 1 H NMR as shown in Fig. 1(b). Specific peaks of HA were observed at 1.9, 3.0–3.8, and 4.2–4.8 ppm, whereas peaks of HMDA were observed at 1.3, 1.6, and 2.8–2.9 ppm (data not shown). Coupling yield of HMDA was calculated based on the ratio between the methylene peaks of HA (1.9 ppm) and ethylene peaks of HMDA (1.3 ppm). Coupling yield of HMDA was at least higher than 91% (data not shown). For coupling of PLGA with aminated HA, NHS-activated PLGA (PLGA–NHS) was prepared by treatment of PLGA with DCC and NHS. PLGA–NHS was mixed with aminated HA to synthesize HAbLG copolymer, as shown in Fig. 1(a). To remove unreacted HA, the reactants were purified by dialysis against deionized water using a dialysis membrane (MWCO: 12,000 g/mol). Following this, unreacted PLGA was removed by precipitation of copolymer in DCM, since copolymer was insoluble in DCM while PLGA itself was soluble in DCM. 1 H NMR spectra of the resulting block copolymer were shown in Fig. 1(b). The coupling yield of PLGA to aminated HA was evaluated by 1 H NMR. Since the peak of HA at 1.9 ppm (position 2 in HA) was similar before and after conjugation with PLGA, the peak ratio between 1.9 ppm (methylene of position 2) of HA and 1.18 ppm of PLGA (methylene at position 1) was compared to estimate conjugation yield and Mw of copolymer. The Mw of HA in the block copolymer was estimated by 1 H NMR spectroscopy based on the Mw of PLGA (weight average Mw 4920 g/mol). The estimated Mw of HA in the block copolymer was about 6800 g/mol. The estimated Mw of PLGA was slightly smaller than that of the manufacturer’s data. These results indicate that HAbLG block copolymer was successfully synthesized. 3.2. Characterization of nanoparticles composed of HAbLG copolymer Since HAbLG has amphiphilic properties, nanoparticles composed of HAbLG copolymer might be formed by self-assembling in an aqueous solution. To clarify the self-assembling behavior of HAbLG copolymer in aqueous solution, a fluorescence probe technique was employed using pyrene. The fluorescence excitation spectra of pyrene at various concentrations of HAbLG copolymer are shown in Fig. 2(a). The intensity of pyrene was increased according to the increased concentrations of HAbLG copolymer (Fig. 2(a)), indicating that self-assembled HAbLG copolymer was formed in water. Furthermore, a red shift was observed in the excitation spectra based on the increased concentration of HAbLG copolymer. This result suggests that pyrene was preferentially solubilized into the hydrophobic core of the HAbLG nanoparticles. To estimate the CAC, the intensity ratios of I336 /I333 vs. log c of HAbLG copolymer for the pyrene excitation spectra were plotted as shown in Fig. 2(b). A flat region and sigmoidal change in the crossover region were observed at low concentrations of HAbLG copolymer, indicating that the signal change in the crossover region could have been related to the CAC of HAbLG copolymer. The CAC was
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Table 1 Characterization of DOX-incorporated self-assembled nanoparticles of HAbLG. Polymer/DOX (mg/mg)
40/0 40/5 40/10
Drug contents (%, w/w)
– 7.8 11.7
Loading efficiency (%, w/w)
– 67.7 53.0
estimated from the fluorescence excitation spectra and was found to be 0.0089 g/l. Consequently, nanoparticles composed of HAbLG copolymer were formed by self-association in an aqueous environment. Fig. 2(c) shows the morphology of nanoparticles of HAbLG copolymer. To examine nanoparticles, HAbLG copolymer dissolved in DMSO was dialyzed against water and observed with TEM. As shown in Fig. 2(c), spherical nanoparticles of HAbLG copolymer were observed, and their sizes were mostly less than 100 nm, even though some particles were larger than 100 nm. The size of the nanoparticles as determined by TEM was almost similar to the results of the particle size measurement shown in Table 1. These results indicate that HAbLG copolymer formed nanoparticles in an aqueous environment. Due to their amphiphilic properties, HAbLG nanoparticles may have a core–shell structure, i.e., a hydrophilic HA domain forms the outer segment of the nanoparticles while a hydrophobic PLGA domain forms the inner-core of the nanoparticles. Furthermore, a
Particle size (nm)
Zeta potential (mV)
Intensity average
Weight average
Number average
59.5 ± 27.2 80.5 ± 16.8 116.1 ± 18.9
56.2 ± 24.3 71.8 ± 20.7 103.2 ± 14.4
45.3 ± 20.2 68.8 ± 22.7 95.6 ± 16.0
−28.01 −7.89 −0.87
hydrophobic anti-cancer drug, DOX, can be incorporated into the core of the nanoparticles via hydrophobic interaction. To determine the core–shell structure of HAbLG nanoparticles, 1 H NMR spectroscopy was employed. Fig. 2(d) shows 1 H NMR spectra of DOX-incorporated HAbLG nanoparticles. As shown in Fig. 2(d), specific HA and PLGA peaks were observed from 1 ppm to 5 ppm when nanoparticles were introduced into DMSO. Furthermore, specific DOX peaks were observed from 7.6 ppm to 8.0 ppm. However, the specific PLGA and DOX peaks disappeared when the nanoparticles were distributed in D2 O, although HA peaks were still observed. These results indicated that HAbLG nanoparticles were composed of a hydrophilic outer-shell of HA and hydrophobic inner-core of PLGA. Furthermore, DOX was incorporated into the core of the nanoparticles. Table 1 shows the drug content and particle size of DOXincorporated HAbLG nanoparticles. Drug content increased with increased drug feeding ratio, whereas loading efficiency decreased.
Fig. 1. Synthesis scheme (a) and 1 H NMR spectra (b) of HAbLG copolymer. DMSO was used as a solvent for NMR measurement.
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Fig. 2. Self-association of HAbLG nanoparticles. Fluorescence excitation of pyrene (6.0 × 10−7 M) vs. the concentration of HAbLG copolymer in distilled water (em = 390 nm) (a). Plots of the intensity ratios I339 /I336 from the pyrene excitation spectra vs. log c of the HAbLG copolymers in distilled water (b). TEM photograph nanoparticles of HAbLG block copolymer (c). 1 H NMR spectra of HAbLG nanoparticles in D2 O (b) and DMSO (c).
Both particle size and zeta potential gradually increased according to the drug content. Fig. 3 shows the drug release from HAbLG nanoparticles in vitro. As shown in Fig. 3, initial burst release was observed until 12 h, after which DOX was continuously released
over 4 days. Furthermore, the higher the drug content, the slower the drug release, confirming that a high content of hydrophobic drug causes crystallization of the solid core of the core–shell type nanoparticles and that the drug release rate is slower compared to that at lower drug contents.
3.3. In vitro cell cytotoxicity
Fig. 3. Drug release from DOX-incorporated HAbLG nanoparticles according to the drug contents.
To investigate the anti-tumor activity of DOX-incorporated HAbLG nanoparticles, HCT-116 cells, which are CD44overexpressing human colon carcinoma cells, were used. For this test, CD44 receptor of HCT-116 cells was blocked with 10 equiv. of free HA to determine whether or not HAbLG nanoparticles enter tumor cells via receptor-mediated endocytosis and inhibit tumor cell growth. As shown in Fig. 4, HAbLG nanoparticles showed almost similar cytotoxic effect compared to DOX against HCT-116 cells. However, when CD44 receptor of tumor cells was blocked with HA, the survivability of the tumor cells was higher than DOX or nanoparticles without blocking. This result indicates that nanoparticles entered the tumor cells via receptor-mediated endocytosis and inhibited cell growth. To prove this hypothesis, HCT-116 cells were exposed to DOX-incorporated HAbLG nanoparticles with or without blocking of CD44 receptor and then observed by CLSM as shown in Fig. 5. When tumor cells were
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4. Discussion
Fig. 4. Growth inhibition of HCT-116 colon carcinoma cells by treatment of DOX-incorporated HAbLG nanoparticles (Drug contents: 7.8%, w/w in Table 1). 1 × 104 cells were exposed to DOX or DOX-incorporated HAbLG nanoparticles (a). One hour before start of nanoparticle treatment, 10 times higher amount of HA was added to block CD44 receptor of HCT-116 cells.
exposed to DOX-incorporated HAbLG nanoparticles, tumor cells expressed a strong red color, indicating that DOX-incorporated nanoparticles successively entered the cells. However, tumor cells expressed a very weak red color when CD44 receptor of tumor cells was blocked with free HA. These results therefore prove that DOX-incorporated HAbLG nanoparticles entered tumor cells via receptor-mediated endocytosis. To quantitatively investigate the receptor-mediated endocytosis of HAbLG nanoparticles, HCT-116 cells were exposed to DOX-incorporated HAbLG nanoparticles and then analyzed by flow cytometry as shown in Fig. 6. When HCT-116 cells were exposed to HAbLG nanoparticles (Fig. 6(d)), fluorescence intensity obviously increased. On the other hand, tumor cells with blocked CD44 displayed decreased fluorescence intensity (Fig. 6(e) and (f)). When DOX was treated to tumor cells, however, the extent of fluorescence intensity was not practically changed with or without CD44 blocking as shown in Fig. 6(b) and (c). These results indicated that HAbLG nanoparticles entered HCT-116 cells via CD44 receptor-mediated endocytosis.
Since HA receptors such as CD44 and RHAMM are excessively expressed in tumor cells [13], HA has been used as a targeting moiety and drug carrier for drug targeting against humor [11,12,14,17]. Luo et al. [28] reported that HA-paclitaxel conjugates enter tumor cells via receptor-mediated endocytosis. Practically, they proved that the selective cytotoxicity of HA-paclitaxel against tumor cells could be blocked by either excess HA or an anti-CD44 antibody. Auzenne et al. [29] reported that HA-paclitaxel prodrug significantly enhances the survivability of tumor xenograft-induced mice. Furthermore, Yin et al. [30] reported that a paclitaxel/HA combination is effective for inhibiting tumor metastasis and migration in an animal tumor model. Amphiphilic polymers such as block or graft copolymers and hydrophobized polysaccharides can normally form self-aggregated nanoparticles or polymeric micelles [18–20]. Especially, due to their unique structures, block or graft copolymer generally forms a core–shell structure, i.e., the hydrophilic domain forms an outershell and the hydrophobic domain forms a hydrophobic inner-core via self-assembly. Furthermore, the advantages of this structure are that hydrophobic anti-cancer agents can be incorporated into the core of the nanoparticles; the hydrophilic domain acts as a defense membrane against attack by the reticuloendotheial system and protein absorption [18–20]. In this study, we designated block copolymer composed of HA as a hydrophilic block and PLGA as a hydrophobic one. As far as we know, this is the first report using HA and PLGA to synthesize block copolymer for drug targeting against cancer cells. In particular, one of the main advantages of our HAbLG copolymer nanoparticles is that HA segment can be freely directed toward the outer-environment while the PLGA domain is in charge of drug incorporation. The fact that HA is exposed on the surface of nanoparticles may be helpful in targeting CD44 receptor of tumor cells. Practically, we showed that HA was exposed on the surface of nanoparticles in an aqueous environment, whereas PLGA and DOX composed the solid inner-core as shown in Fig. 2(d). Furthermore, TEM photograph also showed the core–shell structure of HAbLG nanoparticles (arrows in Fig. 2(c)); the relatively white section is the PLGA inner-core and the grey-colored section is the HA segment. Most reports on the HA-anti-cancer agent delivery system are based on the direct conjugation of the anticancer agent with HA [1,28,29], simple mixtures between HA and
Fig. 5. Fluorescence images of DOX-incorporated nanoparticle-treated HCT-116 tumor cells with or without blocking of CD44 receptor. HCT-116 cells were exposed to DOX-incorporated HAbLG nanoparticles at 10 g/ml of DOX concentration for 1 h. Fluorescence images of cells were observed with CLSM.
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Fig. 6. Flow cytometric analysis of DOX-incorporated HAbLG nanoparticle-treated HCT-116 tumor cells with or without blocking of CD44 receptor. Prior to DOX or nanoparticle treatment, 1 mg or 5 mg of free HA was pretreated to HCT-116 cells for 3 h for blocking of CD44 receptor. HCT-116 cells were exposed to DOX or DOX-incorporated HAbLG nanoparticles at 10 g/ml of DOX concentration for 1 h. (a) Control; (b) DOX without HA; (c) DOX with CD44 blocking (HA concentration: 1 mg/ml); (d) DOX-incorporated HAbLG nanoparticles without blocking of CD44; (e) DOX-incorporated HAbLG nanoparticles with blocking of CD44 (HA concentration: 1 mg/ml); (f) DOX-incorporated HAbLG nanoparticles with blocking of CD44 (HA concentration: 5 mg/ml); (g) comparison of relative fluorescence intensity between each samples. The experiment was triplicated and expressed as average with S.D. *, **P < 0.001.
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drugs [11,30], or HA-coated nanoparticles [31,32]. In addition to its potential for tumor targeting, HA is regarded as a superior candidate for the enhancement of solubility of hydrophobic anti-cancer drugs [12,29]. We incorporated DOX as a model drug into HAbLG nanoparticles. We choose DOX because it has hydrophobic properties and exhibits strong red fluorescence intensity in a biological environment. Loading efficiency of DOX was higher than 50% at any formulation. Additionally, particle size was less than 200 nm in all formulations. The small particle size of HAbLG nanoparticles is also favorable for drug targeting since particles smaller than 200 nm are regarded as essential candidates for drug targeting [19,33]. The decreased anti-tumor activity of DOX-incorporated HAbLG nanoparticles with blocked CD44 receptor was due to the receptormediated uptake of HAbLG nanoparticles; uptake of nanoparticles by tumor cells was blocked when CD44 receptor was blocked. Results of tumor cell observation by CLSM (Fig. 5) and analysis by flow cytometry (Fig. 6) supported this result, indicating that the red fluorescence intensity of DOX-incorporated nanoparticles without blocked CD44 was higher than those with CD44 blocking. Luo et al. [34] also reported that HA-modified and DOXconjugated N-(2-hydroxypropyl)methacrylamide (HPMA) polymer (HA-HPMA-DOX) is able to selectively target CD44-overexpressing tumor cells compared to non-targeted HPMA-DOX conjugates. They also observed that HA-modified HPMA has enhanced cytotoxicity and targeting efficiency against HCT-116 cells. They showed that the uptake of HA-HPMA-DOX conjugates into CD44 overexpressed tumor cells was increased with time course while tumor cell uptake of non-targeted HPMA-DOX was slowly increased with incubation time. We also have no beneficial effect with nontargeted DOX-loaded dextran-PLGA nanoparticles against tumor cells [35]. Even though the differences were not significantly changed at cell viability (Fig. 4), proliferation inhibition effect of DOX-incorporated HAbLG nanoparticles against CD44 overexpressed tumor cells was controlled receptor-mediated pathway. Based on these results, we suggest that nanoparticles composed of HAbLG copolymer are a promising candidate for an anti-tumor drug delivery system. 5. Conclusion We have synthesized HabLG block copolymer for antitumor drug targeting system. 1 H NMR spectra confirmed successful synthesis of HabLG copolymer. Core–shell type nanoparticles prepared by dialysis have spherical shape with size less than 200 nm. When HabLG nanoparticles were reconstituted in D2 O, intrinsic peaks of HA segment was only observed at 1 H NMR study while specific peaks both of HA and PLGA was observed at DMSO, indicating that HabLG nanoparticles have HA as a hydrophilic outer-shell and PLGA as a solid inner-core. DOX was incorporated in the HAbLG nanoparticles with loading efficiency higher than 67%. Antitumor activity was studied using CD44-overexpressing HCT-116 cells. HCT-116 cells with nanoparticle treatment expressed strong red fluorescence color while fluorescence intensity was significantly decreased when CD44 receptor was blocked with HA. We suggest
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that HAbLG nanoparticles are superior candidate for targeted drug delivery for cancer. Acknowledgement This study was supported by a grant of the Korean Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (Project No. A091047). References [1] [2] [3] [4]
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