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Hydrophobically modified polysaccharide-based on polysialic acid nanoparticles as carriers for anticancer drugs
International Journal of Pharmaceutics 520 (2017) 111–118
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Hydrophobically modified polysaccharide-based on polysialic acid nanoparticles as carriers for anticancer drugs Bom Junga,1, Man-Kyu Shima,b,1, Min-Ju Parka , Eun Hyang Janga , Hong Yeol Yoonb , Kwangmeyung Kimb , Jong-Ho Kima,* a
Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea b
A R T I C L E I N F O
Article history: Received 19 October 2016 Received in revised form 28 December 2016 Accepted 27 January 2017 Available online 4 February 2017 Keywords: Polysialic acid Nanoparticles Targeted cancer therapy EPR effect Doxorubicin
A B S T R A C T
This study presented the development of hydrophobically modified polysialic acid (HPSA) nanoparticles, a novel anticancer drug nanocarrier that increases therapeutic efficacy without causing nonspecific toxicity towards normal cells. HPSA nanoparticles were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling between N-deacetylated polysialic acid (PSA) and 5b-cholanic acid. The physicochemical characteristics of HPSA nanoparticles (zetapotential, morphology and size) were measured, and in vitro cytotoxicity and cellular uptake of PSA and HPSA nanoparticles were tested in A549 cells. In vivo cancer targeting of HPSA nanoparticles was evaluated by labeling PSA and HPSA nanoparticles with Cy5.5, a near-infrared fluorescent dye, for imaging. HPSA nanoparticles showed improved cancer-targeting ability compared with PSA. Doxorubicin-loaded HPSA (DOX-HPSA) nanoparticles were prepared using a simple dialysis method. An analysis of the in vitro drug-release profile and drug-delivery behavior showed that DOX was effectively released from DOX-HPSA nanoparticles. In vivo cancer therapy with DOX-HPSA nanoparticles in mice showed antitumor effects that resembled those of free DOX. Moreover, DOX-HPSA nanoparticles had low toxicity toward other organs, reflecting their tumor-targeting property. Hence, HPSA nanoparticles are considered a potential nanocarrier for anticancer agents. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In rapidly growing cancers, vascularization is disorganized and the vascular architecture is defective (Kim et al., 2006). Because of the pathophysiological dissimilarity of tumor areas from normal tissues, nanoparticles ranging from 10 to 500 nm in size can escape from the vascular capillary bed and accumulate in the interstitial space of tumor areas, where they ultimately accumulate owing to the insufficient lymphatic drainage of tumor interstitial fluid (Biswas and Torchilin, 2014; Torchilin, 2011). This phenomenon of selective extravasation and retention of macromolecules or nanoparticles in tumor areas is known as the enhanced permeability and retention (EPR) effect (Biswas and Torchilin, 2014; Fang
* Corresponding author at: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. E-mail address: [email protected] (J.-H. Kim). 1 These authors equally contributed. http://dx.doi.org/10.1016/j.ijpharm.2017.01.055 0378-5173/© 2017 Elsevier B.V. All rights reserved.
et al., 2011; Maeda, 2001a; Maeda et al., 2009, 2006; Seki et al., 2009; Torchilin, 2011). Since passive drug delivery by the EPR effect occurs only in tumor areas, penetration of drug-loaded nanoparticles into normal tissues is limited, thereby reducing the side effects of chemotherapy (Biswas and Torchilin, 2014; Fang et al., 2011; Iyer et al., 2006; Maeda, 2001a,b; Seki et al., 2009). Moreover, intravenously injected, drug-loaded nanoparticles have a comparatively increased plasma half-life because uptake by the spleen and liver is limited (Kim et al., 2006; Koshkaryev et al., 2012, 2011). This prolonged circulation facilitates accumulation of nanoparticles in tumor areas through the EPR effect. Polysialic acid (PSA), a negatively charged homopolymer of either a-2,8- or a-2,9-linked sialic acid residues, was first discovered in Escherichia coli K-1 and K-235 strains by Barry and colleagues (Barry and Goebel, 1957). PSA has great potential for use as a biomaterial owing to its biocompatibility, biodegradability, and non-immunogenicity (Gregoriadis et al., 2000). In addition, PSA can be produced easily in large scale by fermentation of E. coli (Zheng et al., 2013). PSA has received attention as a drug-delivery agent because it evokes the production of only small amounts of
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antibody capable of clearing drug-loaded PSA from the bloodstream, and thus allows PSA to exhibit a long circulation time in vivo (Fernandes and Gregoriadis, 1997, 2001). PSA has several functional groups, including carboxyl, hydroxyl and amine groups, which can react with macromolecules to produce nanoparticles for drug delivery. For example, Bader et al. employed PSA and decylamine to prepare self-assembled micelles that could load the hydrophobic drug, cyclosporine (Bader et al., 2011). Zhang et al. produced methotrexate-loaded PSA-N-trimethyl chitosan nanoparticles that exhibited fine, slow-release of methotrexate (Wu et al., 2015; Zhang and Bader, 2012). Wilson et al. used PSA and polycaprolactone to produce a self-assembling cyclosporineencapsulated micelle (Wilson et al., 2014; Wu et al., 2015). Clearly, PSA exhibits characteristics appropriate for an effective drugdelivery agent in cancer therapy, and may overcome limitations in the pharmacokinetics, stability, and anti-immunogenicity of therapeutic agents. In this study, we prepared hydrophobically modified PSA (HPSA) that formed nano-sized particles in aqueous solutions owing to their amphilicity. We previously developed hydrophobically modified polysaccharides, including glycol chitosan and hyaluronic acid with bile acids, deoxycholic acid, or 5b-cholanic acid. Bile acids introduce hydrophobicity to hydrophilic polysaccharides, enabling them to incorporate hydrophobic anticancer drugs with a high loading efficiency and release them in a sustained manner. We found that 5b-cholanic acid-modified nanoparticles were more stable and had higher drug loading efficiency than deoxycholate-modified nanoparticles. Therefore, we prepared HPSA with 5b-cholanic acid as a new anticancer drug carrier for tumor-targeted therapy (Fig. 1). We characterized HPSA nanoparticles with respect to size, cytotoxicity, cellular uptake and tumor targeting, and evaluated the therapeutic efficacy of drugloaded HPSA nanoparticle in a tumor-bearing mouse model.
2. Experimental 2.1. Materials Polysialic acid (PSA, colominic acid sodium salt, Mw = approx. 30 kD) was purchased from Nacalai Tesque (Kyoto, Japan). 5bCholanic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) were obtained from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Doxorubicin (DOX) was obtained from Sigma Aldrich. Cellulose membrane dialysis tube (molecular weight cutoff = 3.5 kD) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). All other chemicals were analytical grade and used without further purification. 2.2. Preparation of HPSA and Dox-HPSA nanoparticles N-deacetylation of PSA was performed as described elsewhere (Erbing et al., 1976). Briefly, PSA (100 mg) dissolved in water (6 mL), 10 M NaOH (2 mL), thiophenol (200 mL), and DMSO (30 mL) were added. The mixture was heated at 80 C for 3 h, dialyzed against 0.01 M ammonium carbonate at 4 C, and lyophilized. N-deactylated PSA was hydrophobically modified with 5bcholanic acid as described in previous study (Kim et al., 2006). In brief, N-deactylated PSA was hydrophobically modified with various 5b-cholanic acid (molar ratio of PSA:5b-cholanic acid; 5% = 1:20, 10% = 1:10, and 20% = 1:5) in water/methanol mixture (1:1, v/v) for 24 h at room temperature. To activate carboxylic acid pertaining to 5b-cholanic acid, equal amounts (1.2 equiv/[5bcholanic acid]) of EDC and NHS were added into the polymer solution. These react with the primary amino groups in PSA to form an amide linkage. The reaction mixture was dialyzed for 24 h against the excess amount of water/methanol mixture (1:4, v/v), and lyophilized to obtain HPSA nanoparticles. For in vitro cellular uptake and in vivo tumor targeting, cy5.5labeled HPSAs were prepared with simple conjugation as follows; hydroxysuccinimide ester cy5.5 was chemically coupled to HPSA in
Fig. 1. Schematic illustration of HPSA nanoparticles for cancer targeting.
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DMSO. The reaction was performed at 25 C in the dark for 6 h. Byproducts and unreacted cy5.5 molecules were removed by dialysis for two days and the product was lyophilized. Doxorubicin loaded HPSA (Dox-HPSA) nanoparticles were prepared by a dialysis method. Briefly, HPSA10 nanoparticles (10 mg) were dissolved in methanol (10 mL) and Dox (1 mg) in DMSO (1 mL) were added to the HPSA nanoparticles solution for 24 h at room temperature. Then, the mixture was dialyzed against water using the cellulose membrane dialysis tube for 24 h and lyophilized to obtain Dox-HPSA nanoparticles. 2.3. Characterization of HPSA nanoparticles The chemical structure of the HPSA nanoparticles was characterized (dissolved in D2O) by 600 MHz proton nuclear magnetic resonance (1H NMR) spectroscopy (DD2 600 MHz FT NMR, Agilent Technologies, Santa Clara, CA, USA). The sizes of the HPSA nanoparticles were determined at 25 C using dynamic light scattering (DLS) (90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation, Holtsville, NY, USA). The zeta potentials of the particles were measured at 25 C using 90 Plus Particle Size Analyzer. The concentration of HPSA nanoparticles was 1 mg/mL. The morphology of the particles was observed by transmission electron microscopy (TEM) (Tecnai F20 G2, FEI, Hillsboro, OR, USA). 2.4. Cytotoxicity A549 cells were seeded at 1 104 cells/well in 96-well plate and stabilized for 24 h at 37 C CO2 incubator. After stabilizing, cells were washed with DPBS and cultured for 24 h with various concentrations (0, 5, 10, 20, 50, and 100 mg/mL) of PSA and HPSA nanoparticles. Then, 20 mL of the 3-[4,5-dimethythiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) solution (0.83 mg/mL in RPMI 1640 media) was added to each well, and cells were incubated for an additional 2 h at 37 C. MTT-containing media was removed, and cells were dissolved in 100 mL of DMSO. The absorbance of each well was measured at 540 nm using a microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Ortenberg, Germany). 2.5. In vitro cellular uptake Observation of cellular uptake activity of HPSA nanoparticles was performed on A549 cells. In brief, 2 104 cells of A549 were seeded into 35 mm cover glass bottom dishes. After 24 h culture, 50 mg/mL of PSA, HPSA5, HPSA10 or HPSA20 containing RPMI 1640 media were added to the cells for 24 h at 37 C CO2 incubator. Then, the cells were washed twice with DPBS and fixed with formaldehyde-glutaraldehyde combined fixative for 10 min in dark condition. After fixation, the cells were washed twice with DPBS and stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) with 405 diode (405 nm), Ar (458, 488,514 nm) and HeNe (633 nm) lasers.
Table 1 Characterization of HPSA nanoparticles including zeta-potential, size and polydispersity index (PDI). Materials PSA HPSA5 HPSA10 HPSA20
Zeta-potential (mV) 1.25 0.37 3.16 0.25 3.90 0.26 5.28 0.39
Size (nm)
PDI (polydispersity index)
– 611.05 79.05 821.81 90.08 645.30 155.70
– 0.287 0.170 0.193
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2.6. Tumor targeting of HPSA nanoparticles All experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Korea Institute of Science and Technology (KIST), and institutional committees approved the experiment. Athymic nude mice (5 weeks old, 20–25 g, male) were purchased from Orient Bio Inc. (Gyeonggi-do, Korea). To prepare tumor-bearing mice models, a suspension of 1 107 A549 cells in RPMI 1640 media (100 mL) was injected into left flanks of mice. When the tumors grew to approximately 200 mm3 in volume, 1 mg/mL Cy5.5-labeled PSA and HPSA nanoparticles (5%, 10% and 20% respectively) were administered (200 mL) by intravenous injection (4 mise per group). After injection, all time point fluorescence in tumors was measured by IVIS Lumina Series III (PerkinElmer, Waltham, MA, USA). To observe the tumor targeting effect in ex vivo, each group of mice was sacrificed 48 h post-injection. Then, major organs and the tumors were excised and observed using the IVIS Lumina Series III. Fluorescence intensities were calculated using the Living Image1 software (PerkinElmer, Waltham, MA, USA). 2.7. In vitro drug release and intracellular delivery Dox-HPSA nanoparticles (1, 2 mg/mL) were dispersed in PBS (pH 7.4). Each of the Dox-HPSA nanoparticles solution (2 mL) was placed into the cellulose membrane dialysis tube. The dialysis tube was placed in 18 mL of PBS buffer and gently shaken at 37 C in a water bath at 100 rpm. At various time points, the medium was refreshed. Samples were taken predetermined times from the medium outside of the dialysis tube, and their Dox concentrations were determined by fluorescence excitation at 470 nm and emission at 590 nm using a microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Ortenberg, Germany). Observation of Dox cellular delivery of Dox-HPSA nanoparticles into cancer cells was conducted on A549 cells. Briefly, 2 104 cells of A549 were grown in 4-chamber slides. After 24 h incubation, the cells were treated with free Dox at 30 mg/mL, Dox-HPSA nanoparticles at 300 mg/mL or Dox-HPSA nanoparticles at 150 mg/mL in RPMI 1640 media for 24 h culture. The cells were washed twice with DPBS and fixed with 4% paraformaldehyde for 15 min at room temperature, then removed the fixative and added sufficient volume of the permeabilization reagent (0.25% Triton1 X-100 in PBS) for 20 min at room temperature. After fixation and permeabilization, the cells were treated with terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay, with a commercial apoptosis detection kit; Click-iT1 Plus TUNEL assay for in situ apoptosis detection with Alexa Fluor1 488 dye (Thermo Fisher Scientific, Waltham, MA, USA). The cells were stained with DAPI and observed using a confocal laser microscope (Eclipse Ti-S, Nikon Instruments Inc., Melville, NY, USA). 2.8. In vivo anti-tumor effect of Dox-HPSA Nanoparticles To evaluate the anti-tumor effect of Dox-HPSA nanoparticles, A549 tumor-bearing mice were prepared, as previously described in biodistribution test. Mice were divided in four Groups: (i) normal saline, (ii) HPSA at 50 mg/kg, (iii) free Dox at 5 mg/kg, and (iv) Dox-HPSA nanoparticles at 5 mg/kg (6 mice per group). When tumors reached 200 mm3 in volume, each sample was injected once every three days. Tumor size were calculated as a b2/2, were a was the largest and b the smallest diameter. To analysis tumor tissues apoptosis by Dox-HPSA nanoparticles, tumors were dissected from A549 tumor-bearing mice at 24 h after intravenous injection of Dox-HPSA nanoparticles. Then, fixed with 3% paraformaldehyde solution, and embedded in paraffin. Embedded tumors were sectionalized for 8 um using a Cycle Type Paraffin
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Fig. 2. Characterization of HPSA nanoparticles. (A) Size, as determined by DLS, (B) cytotoxicity in A549 cells, and (C) cellular uptake of Cy5.5-labeled PSA and HPSA. Results are expressed as means SD of three replicate measurements from a single experiment, and are representative of three separate experiments (*P < 0.05, **P < 0.01). In fluorescent images, red (Cy5.5) corresponds to nanoparticles and blue (DAPI) represents nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Section Machine (QP3268, Changzhou Haosilin Medical Insrument Co., Ltd., Changzhou, Jiangsu, China). After section of tumors, the tumors were treated with TUNEL assay as formerly described in in vitro doxorubicin delivery into A549 cells. The tumors were treated with DAPI staining and observed by a confocal laser microscope (Eclipse Ti-S, Nikon Instruments Inc., Melville, NY, USA). 2.9. Statistical analysis The results are presented as mean SD or SE, and statistical comparisons between groups were carried out using one-way ANOVA followed by the Student’s t-test using SigmaPlot version 10.0. 3. Results and discussion 3.1. Characterization of HPSA nanoparticles To produce HPSA nanoparticles, we N-deacetylated PSA to form amide bonds between PSA and 5b-cholanic acid, eliminating the acetyl groups. The structure of N-deactylated PSA was determined by 600-MHz 1H NMR spectroscopy. The peak of PSA at 1.95 ppm was attributed to the protons of the acetyl group in PSA. As expected, the peak at 1.95 ppm disappeared in N-deacetylated PSA (Supplementary data, Fig. S1), indicating successful N-deacetylation. The amphiphilic character of HPSA nanoparticles, composed
of hydrophilic PSA and hydrophobic 5b-cholanic acid at different molar ratios of PSA:5b-cholanic acid (5%, 1:20 [HPSA5]; 10%, 1:10 [HPSA10]; and 20%, 1:5 [HPSA20]), induces self-assembly in an aqueous solution. As the amount of 5b-cholanic acid increased, the zeta potential value of PSA and HPSA nanoparticles decreased (Table 1). The mean diameters of HPSA nanoparticles, measured by dynamic light scattering (DLS), were in the 600–800 nm range (Table 1, Fig. 2A), a size somewhat larger than that of other reported nanoparticles. However, they showed good deformability, with most HPSA nanoparticles easily passing through smaller-size filter membranes (Supplementary data, Fig. S2). This indicates that these nanoparticles are appropriate for cellular uptake and in vivo experiments. The various-size HPSA nanoparticles showed differences in zeta potential value; at 821.81 90.08 nm, HPSA10 nanoparticles were the largest. A transmission electron microscopy (TEM) analyses demonstrated that HPSA nanoparticles were nearly spherical in shape (Fig. 2A, inset images). Except for nanoparticles with a 5b-cholanic acid composition of 20% (HPSA20), HPSA nanoparticles were less cytotoxic toward A549 cells than PSA at concentrations higher than 50 mg/mL (Fig. 2B). We did not observe any difference among any treated groups at lower concentrations. Notably, HPSA5 and HPSA10 nanoparticles had no significant impact on cell viability at the highest concentration tested (100 mg/mL). As illustrated in Fig. 2C, Cy5.5-labeled HPSA nanoparticles showed greater accumulation within A549 cells than Cy5.5-labeled PSA.
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Fig. 3. Cancer targeting of HPSA nanoparticles in A549-inoculated athymic nude mice. (A) In vivo biodistribution of Cy5.5-labeled PSA and HPSA nanoparticles. (B) Ex vivo NIRF images of dissected major organs. (C) Quantification of ex vivo results (n = 4). Results are expressed as means SE of three replicate measurements (**P < 0.01; ND, no significant difference).
3.2. In vivo cancer targeting of HPSA nanoparticles Athymic nude mice bearing A549 tumors were intravenously injected with Cy5.5-labeled PSA and HPSA nanoparticles, and the in vivo biodistribution of PSA and nanoparticles was monitored
over time (Fig. 3A). A strong near-infrared fluorescence (NIRF) signal was detected throughout the body of mice within 1 h of intravenous injection of either PSA or HPSA nanoparticles, demonstrating that PSA and HPSA nanoparticles promptly circulate in the blood. A maximum NIRF signal was detected in tumors
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Fig. 4. In vitro release of Dox. (a) Release profiles of Dox from Dox-HPSA at 37 C. (B) Distribution of Dox (red fluorescence) in A549 cells. (C) TUNEL assays of A549 cells treated with free Dox or Dox-HPSA. A549 cells were immunostained with TUNEL (green), and nuclei were counterstained with DAPI (blue). Results are expressed as means SD of three replicate measurements from a single experiment, and are representative of three separate experiments (**P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
almost immediately after injection, and was maintained for up to 2 days before progressively decreasing. To evaluate the tumor targeting effect ex vivo, we assessed Cy5.5-labeled PSA and HPSA nanoparticle fluorescence in the major organs (liver, lung, spleen, kidney, and heart) excised from mice. PSA and HPSA nanoparticle uptake was chiefly observed in tumors and not in normal tissues (Fig. 3B). Moreover, the NIRF intensity of PSA and HPSA nanoparticles was similar in each organ, but not in tumors, where the NIRF intensity of HPSA nanoparticles was 2.3–2.9-fold higher than that of PSA. These observations provide crucial evidence that the cancer-targeting ability of HPSA nanoparticles is much greater than that of PSA, a conclusion supported by statistical analyses (Fig. 3C). 3.3. In vitro drug delivery into cancer cells Dox, an effective anticancer drug, was employed as a representative chemotherapeutic agent in this study. Dox was loaded into HPSA nanoparticles using a simple dialysis method, as previously reported (Kim et al., 2006). We prepared two Doxloaded HPSA nanoparticles: Dox-HPSA, containing 10% Dox (relative to HPSA), and 1/2-Dox-HPSA, containing 5% Dox. Both preparations showed no significant size change after drug loading and exhibited encapsulation efficiencies greater than 90%
(Supplementary data, Table S1). To evaluate the stability of Doxloaded HPSA, we measured changes in the size of Dox-HPSA nanoparticles. These analyses showed that Dox-HPSA nanoparticles maintained their stability for 7 days in phosphatebuffered saline (PBS; Supplementary data, Fig. S3). To assess the potential of HPSA nanoparticles as a drug carrier, we evaluated the release profile of Dox from HPSA nanoparticles at 37 C in PBS (pH 7.4). For the duration of these experiments, sink conditions were preserved by regularly replacing the dialysis medium. The in vitro drug-release profile of Dox-HPSA nanoparticles is illustrated in Fig. 4A, which shows an initial burst of Dox release within 2 days and a sustained release until day 7. Dox-HPSA nanoparticles released more Dox than 1/2-Dox-HPSA nanoparticles, owing to the relative difference in Dox content. As noted above, HPSA nanoparticles accumulate in tumors through the EPR effect. At 48 h after intravenous injection, at least 2.5-times more HPSA nanoparticles accumulated in the tumor than in the liver. Therefore, we conclude that Dox is released from HPSA nanoparticles and accumulates in tumor tissue. Because the cytotoxic action of Dox occurs in the nucleus of cancer cells (Gewirtz, 1999), we measured Dox delivery to the nucleus by Dox-HPSA nanoparticles compared with that of free Dox. As shown in Fig. 4B, free Dox and Dox-HPSA nanoparticles exhibited similar Dox delivery. This effect was concentration
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Fig. 5. In vivo antitumor activity of DOX-HPSA nanoparticles in A549 tumor-bearing mice. (A) Changes in tumor volume and photographs of dissected tumors. (B) Tumor weights in treatment groups after 16 days. (C) TUNEL assay of dissected tumor tissues. Tumor tissues were immunostained with TUNEL (green), and nuclei were counterstained with DAPI (blue). (D) H&E staining of major organs (liver, lung, spleen, kidney, and heart) after 16 days. Results are expressed as means SE of three replicate measurements from one mouse, and are representative of six mice (*P < 0.05 and **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
dependent, as evidenced by the fact that the 1/2-Dox-HPSA nanoparticles group showed relatively less delivery of Dox. To evaluate the cytotoxicity of Dox released from HPSA nanoparticles, we assessed apoptosis in Dox-HPSA–treated A549 cells using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays (Fig. 4C). Cells in all groups exhibited apoptosis signals, except cells in the control group. Notably, treatment with free Dox as well as Dox-HPSA and 1/2-Dox-HPSA nanoparticles caused a significant, concentration-dependent reduction in the number of A549 cells compared with untreated controls, confirming the cytotoxicity of Dox. Taken together, our HPSA biodistribution experiments, analyses of Dox release from HPSA, and evaluation of the cytotoxicity of Dox-HPSA, which collectively show that Dox HPSA nanoparticles specifically accumulate in tumors through the EPR effect, release Dox in a sustained manner and effectively kill cancer cells, suggest the potential of HPSA nanoparticles as drug carriers.
3.4. In vivo cancer therapy of Dox-HPSA nanoparticles The antitumor effects of Dox-HPSA nanoparticles were evaluated in A549 tumor-bearing athymic nude mice. After tumors reached a volume of 200 mm3, mice were injected with one of the following four formulations every 3 days: (i) normal saline, (ii) HPSA nanoparticles only at 50 mg/kg, (iii) free Dox at 5 mg/kg, and (iv) Dox-HPSA nanoparticles at 5 mg/kg. The therapeutic efficacy of Dox-HPSA nanoparticles was investigated by measuring tumor volumes for 16 days (Fig. 5A). At the end of this period, mean tumor volume in the group treated with HPSA alone was 5029.9 mm3, a volume similar to that observed in the normal saline group (5110.7 mm3), indicating that HPSA nanoparticles alone had no effect on tumor volume. By contrast, treatment with Dox-HPSA nanoparticles caused a substantial decrease in tumor volume, reducing mean tumor volume to 774.5 mm3 on day 16. Notably, this decrease was
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considerably greater than that in the free-Dox group, where mean tumor volume was 3044.4 mm3. The effects of these treatments on tumor volumes were also reflected in final tumor weights, which were decreased to the greatest extent in mice treated with DoxHPSA nanoparticles (748.7 mg). By comparison tumor weights in mice treated with free Dox, HPSA only, and normal saline were 2335.7, 4524.1 and 4602.0 mg respectively (Fig. 5B). Excised tumors were stained for histological analysis, and slides were evaluated by an independent pathologist. TUNEL assays revealed increased apoptosis in median tumors excised from mice treated with Dox-HPSA nanoparticles (Fig. 5C). By comparison, tumors in mice treated with normal saline, HPSA, or free Dox showed higher numbers of tumor cells and lower apoptosis signals. To evaluate the toxicity of HPSA nanoparticles towards non-target organs, we performed H&E staining on liver, lung, spleen, kidney, and heart tissue after therapy. As shown in Fig. 5D, none of these major organs exhibited any histomorphological differences. In the Dox-HPSA-treated group, histomorphology was similar to that in the saline-treated group, despite the significantly enhanced therapeutic efficacy of Dox-HPSA. These results suggest that Dox-HPSA nanoparticles enhance the cancer-targeted delivery and therapeutic efficacy of anticancer drugs while reducing associated toxicity. 4. Conclusion PSA is one of the most biodegradable and biocompatible polysaccharides. HPSA nanoparticles containing 5b-cholanic acid self-assembled in aqueous solutions. They also specifically accumulated in tumors and exhibited appropriate Dox loading content, sustained Dox release, and good anticancer therapy without causing tissue toxicity. However, it remains possible that the initial burst release of Dox within 2 days during which HPSA accumulates in tumors leads to unexpected effects. Despite this limitation, our results, taken together, suggest that HPSA nanoparticles have outstanding potential as a novel nanocarrier system for therapeutic agents owing to their low toxicity, long systemic retention, and specific accumulation in tumor cells. Acknowledgements This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2057861). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2017.01.055.
References Bader, R.A., Silvers, A.L., Zhang, N., 2011. Polysialic acid-based micelles for encapsulation of hydrophobic drugs. Biomacromolecules 12, 314–320. Barry, G.T., Goebel, W.F., 1957. Colominic acid, a substance of bacterial origin related to sialic acid. Nature 179, 206. Biswas, S., Torchilin, V.P., 2014. Nanopreparations for organelle-specific delivery in cancer. Adv. Drug Deliv. Rev. 66, 26–41. Erbing, C., Granath, K., Kenne, L., Lindberg, B., 1976. A new method for the Ndeacetylation of carbohydrates. Carbohydr. Res. 47. Fang, J., Nakamura, H., Maeda, H., 2011. The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Deliv. Rev. 63, 136–151. Fernandes, A.I., Gregoriadis, G., 1997. Polysialylated asparaginase: preparation, activity and pharmacokinetics. Biochim. Biophys. Acta 1341, 26–34. Fernandes, A.I., Gregoriadis, G., 2001. The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics. Int. J. Pharm. 217, 215–224. Gewirtz, D.A., 1999. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57, 727–741. Gregoriadis, G., Fernandes, A., Mital, M., McCormack, B., 2000. Polysialic acids: potential in improving the stability and pharmacokinetics of proteins and other therapeutics. Cell. Mol. Life Sci.: CMLS 57, 1964–1969. Iyer, A.K., Khaled, G., Fang, J., Maeda, H., 2006. Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov. Today 11, 812–818. Kim, J.H., Kim, Y.S., Kim, S., Park, J.H., Kim, K., Choi, K., Chung, H., Jeong, S.Y., Park, R. W., Kim, I.S., Kwon, I.C., 2006. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. J. Controll. Release 111, 228–234. Koshkaryev, A., Thekkedath, R., Pagano, C., Meerovich, I., Torchilin, V.P., 2011. Targeting of lysosomes by liposomes modified with octadecyl-rhodamine B. J. Drug Target. 19, 606–614. Koshkaryev, A., Piroyan, A., Torchilin, V.P., 2012. Increased apoptosis in cancer cells in vitro and in vivo by ceramides in transferrin-modified liposomes. Cancer. Biol. Ther. 13, 50–60. Maeda, H., Greish, K., Fang, J., 2006. The EPR effect and polymeric drugs: a paradigm shift for cancer chemotherapy in the 21st century. Adv. Polym. Sci. 193, 103–121. Maeda, H., Bharate, G.Y., Daruwalla, J., 2009. Polymeric drugs for efficient tumortargeted drug delivery based on EPR-effect. Eur. J. Pharm. Biopharm. 71, 409– 419. Maeda, H., 2001a. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv. Enzyme Regul. 41, 189–207. Maeda, H., 2001b. SMANCS and polymer-conjugated macromolecular drugs: advantages in cancer chemotherapy. Adv. Drug Deliv. Rev. 46, 169–185. Seki, T., Fang, J., Maeda, H., 2009. Tumor-targeted macromolecular drug delivery based on the enhanced permeability and retention effect in solid tumor. Pharm. Perspect. Cancer Ther. 93–120. Torchilin, V., 2011. Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Deliv. Rev. 63, 131–135. Wilson, D.R., Zhang, N., Silvers, A.L., Forstner, M.B., Bader, R.A., 2014. Synthesis and evaluation of cyclosporine A-loaded polysialic acid-polycaprolactone micelles for rheumatoid arthritis. Eur. J. Pharm. Sci. 51, 146–156. Wu, J.R., Zhan, X.B., Zheng, Z.Y., Zhang, H.T., 2015. Synthesis and characterization of polysialic Acid/carboxymethyl chitosan hydrogel with potential for drug delivery. Bioorg. Khim. 41, 627–632. Zhang, N., Bader, R.A., 2012. Synthesis and characterization of polysialic acid-Ntrimethyl chitosan nanoparticles for drug delivery. Nano LIFE 2, 1–11. Zheng, Z.Y., Wang, S.Z., Li, G.S., Zhan, X.B., Lin, C.C., Wu, J.R., Zhu, L., 2013. A new polysialic acid production process based on dual-stage pH control and fed-batch fermentation for higher yield and resulting high molecular weight product. Appl. Microbiol. Biotechnol. 97, 2405–2412.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Hydrophobically modified polysaccharide-based on polysialic acid nanoparticles as carriers for anticancer drugs Bom Junga,1, Man-Kyu Shima,b,1, Min-Ju Parka , Eun Hyang Janga , Hong Yeol Yoonb , Kwangmeyung Kimb , Jong-Ho Kima,* a
Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, 5 Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea b
A R T I C L E I N F O
Article history: Received 19 October 2016 Received in revised form 28 December 2016 Accepted 27 January 2017 Available online 4 February 2017 Keywords: Polysialic acid Nanoparticles Targeted cancer therapy EPR effect Doxorubicin
A B S T R A C T
This study presented the development of hydrophobically modified polysialic acid (HPSA) nanoparticles, a novel anticancer drug nanocarrier that increases therapeutic efficacy without causing nonspecific toxicity towards normal cells. HPSA nanoparticles were prepared by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling between N-deacetylated polysialic acid (PSA) and 5b-cholanic acid. The physicochemical characteristics of HPSA nanoparticles (zetapotential, morphology and size) were measured, and in vitro cytotoxicity and cellular uptake of PSA and HPSA nanoparticles were tested in A549 cells. In vivo cancer targeting of HPSA nanoparticles was evaluated by labeling PSA and HPSA nanoparticles with Cy5.5, a near-infrared fluorescent dye, for imaging. HPSA nanoparticles showed improved cancer-targeting ability compared with PSA. Doxorubicin-loaded HPSA (DOX-HPSA) nanoparticles were prepared using a simple dialysis method. An analysis of the in vitro drug-release profile and drug-delivery behavior showed that DOX was effectively released from DOX-HPSA nanoparticles. In vivo cancer therapy with DOX-HPSA nanoparticles in mice showed antitumor effects that resembled those of free DOX. Moreover, DOX-HPSA nanoparticles had low toxicity toward other organs, reflecting their tumor-targeting property. Hence, HPSA nanoparticles are considered a potential nanocarrier for anticancer agents. © 2017 Elsevier B.V. All rights reserved.
1. Introduction In rapidly growing cancers, vascularization is disorganized and the vascular architecture is defective (Kim et al., 2006). Because of the pathophysiological dissimilarity of tumor areas from normal tissues, nanoparticles ranging from 10 to 500 nm in size can escape from the vascular capillary bed and accumulate in the interstitial space of tumor areas, where they ultimately accumulate owing to the insufficient lymphatic drainage of tumor interstitial fluid (Biswas and Torchilin, 2014; Torchilin, 2011). This phenomenon of selective extravasation and retention of macromolecules or nanoparticles in tumor areas is known as the enhanced permeability and retention (EPR) effect (Biswas and Torchilin, 2014; Fang
* Corresponding author at: Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea. E-mail address: [email protected] (J.-H. Kim). 1 These authors equally contributed. http://dx.doi.org/10.1016/j.ijpharm.2017.01.055 0378-5173/© 2017 Elsevier B.V. All rights reserved.
et al., 2011; Maeda, 2001a; Maeda et al., 2009, 2006; Seki et al., 2009; Torchilin, 2011). Since passive drug delivery by the EPR effect occurs only in tumor areas, penetration of drug-loaded nanoparticles into normal tissues is limited, thereby reducing the side effects of chemotherapy (Biswas and Torchilin, 2014; Fang et al., 2011; Iyer et al., 2006; Maeda, 2001a,b; Seki et al., 2009). Moreover, intravenously injected, drug-loaded nanoparticles have a comparatively increased plasma half-life because uptake by the spleen and liver is limited (Kim et al., 2006; Koshkaryev et al., 2012, 2011). This prolonged circulation facilitates accumulation of nanoparticles in tumor areas through the EPR effect. Polysialic acid (PSA), a negatively charged homopolymer of either a-2,8- or a-2,9-linked sialic acid residues, was first discovered in Escherichia coli K-1 and K-235 strains by Barry and colleagues (Barry and Goebel, 1957). PSA has great potential for use as a biomaterial owing to its biocompatibility, biodegradability, and non-immunogenicity (Gregoriadis et al., 2000). In addition, PSA can be produced easily in large scale by fermentation of E. coli (Zheng et al., 2013). PSA has received attention as a drug-delivery agent because it evokes the production of only small amounts of
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antibody capable of clearing drug-loaded PSA from the bloodstream, and thus allows PSA to exhibit a long circulation time in vivo (Fernandes and Gregoriadis, 1997, 2001). PSA has several functional groups, including carboxyl, hydroxyl and amine groups, which can react with macromolecules to produce nanoparticles for drug delivery. For example, Bader et al. employed PSA and decylamine to prepare self-assembled micelles that could load the hydrophobic drug, cyclosporine (Bader et al., 2011). Zhang et al. produced methotrexate-loaded PSA-N-trimethyl chitosan nanoparticles that exhibited fine, slow-release of methotrexate (Wu et al., 2015; Zhang and Bader, 2012). Wilson et al. used PSA and polycaprolactone to produce a self-assembling cyclosporineencapsulated micelle (Wilson et al., 2014; Wu et al., 2015). Clearly, PSA exhibits characteristics appropriate for an effective drugdelivery agent in cancer therapy, and may overcome limitations in the pharmacokinetics, stability, and anti-immunogenicity of therapeutic agents. In this study, we prepared hydrophobically modified PSA (HPSA) that formed nano-sized particles in aqueous solutions owing to their amphilicity. We previously developed hydrophobically modified polysaccharides, including glycol chitosan and hyaluronic acid with bile acids, deoxycholic acid, or 5b-cholanic acid. Bile acids introduce hydrophobicity to hydrophilic polysaccharides, enabling them to incorporate hydrophobic anticancer drugs with a high loading efficiency and release them in a sustained manner. We found that 5b-cholanic acid-modified nanoparticles were more stable and had higher drug loading efficiency than deoxycholate-modified nanoparticles. Therefore, we prepared HPSA with 5b-cholanic acid as a new anticancer drug carrier for tumor-targeted therapy (Fig. 1). We characterized HPSA nanoparticles with respect to size, cytotoxicity, cellular uptake and tumor targeting, and evaluated the therapeutic efficacy of drugloaded HPSA nanoparticle in a tumor-bearing mouse model.
2. Experimental 2.1. Materials Polysialic acid (PSA, colominic acid sodium salt, Mw = approx. 30 kD) was purchased from Nacalai Tesque (Kyoto, Japan). 5bCholanic acid, N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) were obtained from Sigma Aldrich (St. Louis, MO, USA) and used without further purification. Doxorubicin (DOX) was obtained from Sigma Aldrich. Cellulose membrane dialysis tube (molecular weight cutoff = 3.5 kD) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). All other chemicals were analytical grade and used without further purification. 2.2. Preparation of HPSA and Dox-HPSA nanoparticles N-deacetylation of PSA was performed as described elsewhere (Erbing et al., 1976). Briefly, PSA (100 mg) dissolved in water (6 mL), 10 M NaOH (2 mL), thiophenol (200 mL), and DMSO (30 mL) were added. The mixture was heated at 80 C for 3 h, dialyzed against 0.01 M ammonium carbonate at 4 C, and lyophilized. N-deactylated PSA was hydrophobically modified with 5bcholanic acid as described in previous study (Kim et al., 2006). In brief, N-deactylated PSA was hydrophobically modified with various 5b-cholanic acid (molar ratio of PSA:5b-cholanic acid; 5% = 1:20, 10% = 1:10, and 20% = 1:5) in water/methanol mixture (1:1, v/v) for 24 h at room temperature. To activate carboxylic acid pertaining to 5b-cholanic acid, equal amounts (1.2 equiv/[5bcholanic acid]) of EDC and NHS were added into the polymer solution. These react with the primary amino groups in PSA to form an amide linkage. The reaction mixture was dialyzed for 24 h against the excess amount of water/methanol mixture (1:4, v/v), and lyophilized to obtain HPSA nanoparticles. For in vitro cellular uptake and in vivo tumor targeting, cy5.5labeled HPSAs were prepared with simple conjugation as follows; hydroxysuccinimide ester cy5.5 was chemically coupled to HPSA in
Fig. 1. Schematic illustration of HPSA nanoparticles for cancer targeting.
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DMSO. The reaction was performed at 25 C in the dark for 6 h. Byproducts and unreacted cy5.5 molecules were removed by dialysis for two days and the product was lyophilized. Doxorubicin loaded HPSA (Dox-HPSA) nanoparticles were prepared by a dialysis method. Briefly, HPSA10 nanoparticles (10 mg) were dissolved in methanol (10 mL) and Dox (1 mg) in DMSO (1 mL) were added to the HPSA nanoparticles solution for 24 h at room temperature. Then, the mixture was dialyzed against water using the cellulose membrane dialysis tube for 24 h and lyophilized to obtain Dox-HPSA nanoparticles. 2.3. Characterization of HPSA nanoparticles The chemical structure of the HPSA nanoparticles was characterized (dissolved in D2O) by 600 MHz proton nuclear magnetic resonance (1H NMR) spectroscopy (DD2 600 MHz FT NMR, Agilent Technologies, Santa Clara, CA, USA). The sizes of the HPSA nanoparticles were determined at 25 C using dynamic light scattering (DLS) (90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation, Holtsville, NY, USA). The zeta potentials of the particles were measured at 25 C using 90 Plus Particle Size Analyzer. The concentration of HPSA nanoparticles was 1 mg/mL. The morphology of the particles was observed by transmission electron microscopy (TEM) (Tecnai F20 G2, FEI, Hillsboro, OR, USA). 2.4. Cytotoxicity A549 cells were seeded at 1 104 cells/well in 96-well plate and stabilized for 24 h at 37 C CO2 incubator. After stabilizing, cells were washed with DPBS and cultured for 24 h with various concentrations (0, 5, 10, 20, 50, and 100 mg/mL) of PSA and HPSA nanoparticles. Then, 20 mL of the 3-[4,5-dimethythiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) solution (0.83 mg/mL in RPMI 1640 media) was added to each well, and cells were incubated for an additional 2 h at 37 C. MTT-containing media was removed, and cells were dissolved in 100 mL of DMSO. The absorbance of each well was measured at 540 nm using a microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Ortenberg, Germany). 2.5. In vitro cellular uptake Observation of cellular uptake activity of HPSA nanoparticles was performed on A549 cells. In brief, 2 104 cells of A549 were seeded into 35 mm cover glass bottom dishes. After 24 h culture, 50 mg/mL of PSA, HPSA5, HPSA10 or HPSA20 containing RPMI 1640 media were added to the cells for 24 h at 37 C CO2 incubator. Then, the cells were washed twice with DPBS and fixed with formaldehyde-glutaraldehyde combined fixative for 10 min in dark condition. After fixation, the cells were washed twice with DPBS and stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA). The cells were observed using a confocal laser microscope (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) with 405 diode (405 nm), Ar (458, 488,514 nm) and HeNe (633 nm) lasers.
Table 1 Characterization of HPSA nanoparticles including zeta-potential, size and polydispersity index (PDI). Materials PSA HPSA5 HPSA10 HPSA20
Zeta-potential (mV) 1.25 0.37 3.16 0.25 3.90 0.26 5.28 0.39
Size (nm)
PDI (polydispersity index)
– 611.05 79.05 821.81 90.08 645.30 155.70
– 0.287 0.170 0.193
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2.6. Tumor targeting of HPSA nanoparticles All experiments with live animals were performed in compliance with the relevant laws and institutional guidelines of Korea Institute of Science and Technology (KIST), and institutional committees approved the experiment. Athymic nude mice (5 weeks old, 20–25 g, male) were purchased from Orient Bio Inc. (Gyeonggi-do, Korea). To prepare tumor-bearing mice models, a suspension of 1 107 A549 cells in RPMI 1640 media (100 mL) was injected into left flanks of mice. When the tumors grew to approximately 200 mm3 in volume, 1 mg/mL Cy5.5-labeled PSA and HPSA nanoparticles (5%, 10% and 20% respectively) were administered (200 mL) by intravenous injection (4 mise per group). After injection, all time point fluorescence in tumors was measured by IVIS Lumina Series III (PerkinElmer, Waltham, MA, USA). To observe the tumor targeting effect in ex vivo, each group of mice was sacrificed 48 h post-injection. Then, major organs and the tumors were excised and observed using the IVIS Lumina Series III. Fluorescence intensities were calculated using the Living Image1 software (PerkinElmer, Waltham, MA, USA). 2.7. In vitro drug release and intracellular delivery Dox-HPSA nanoparticles (1, 2 mg/mL) were dispersed in PBS (pH 7.4). Each of the Dox-HPSA nanoparticles solution (2 mL) was placed into the cellulose membrane dialysis tube. The dialysis tube was placed in 18 mL of PBS buffer and gently shaken at 37 C in a water bath at 100 rpm. At various time points, the medium was refreshed. Samples were taken predetermined times from the medium outside of the dialysis tube, and their Dox concentrations were determined by fluorescence excitation at 470 nm and emission at 590 nm using a microplate reader (FLUOstar Omega, BMG LABTECH GmbH, Ortenberg, Germany). Observation of Dox cellular delivery of Dox-HPSA nanoparticles into cancer cells was conducted on A549 cells. Briefly, 2 104 cells of A549 were grown in 4-chamber slides. After 24 h incubation, the cells were treated with free Dox at 30 mg/mL, Dox-HPSA nanoparticles at 300 mg/mL or Dox-HPSA nanoparticles at 150 mg/mL in RPMI 1640 media for 24 h culture. The cells were washed twice with DPBS and fixed with 4% paraformaldehyde for 15 min at room temperature, then removed the fixative and added sufficient volume of the permeabilization reagent (0.25% Triton1 X-100 in PBS) for 20 min at room temperature. After fixation and permeabilization, the cells were treated with terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay, with a commercial apoptosis detection kit; Click-iT1 Plus TUNEL assay for in situ apoptosis detection with Alexa Fluor1 488 dye (Thermo Fisher Scientific, Waltham, MA, USA). The cells were stained with DAPI and observed using a confocal laser microscope (Eclipse Ti-S, Nikon Instruments Inc., Melville, NY, USA). 2.8. In vivo anti-tumor effect of Dox-HPSA Nanoparticles To evaluate the anti-tumor effect of Dox-HPSA nanoparticles, A549 tumor-bearing mice were prepared, as previously described in biodistribution test. Mice were divided in four Groups: (i) normal saline, (ii) HPSA at 50 mg/kg, (iii) free Dox at 5 mg/kg, and (iv) Dox-HPSA nanoparticles at 5 mg/kg (6 mice per group). When tumors reached 200 mm3 in volume, each sample was injected once every three days. Tumor size were calculated as a b2/2, were a was the largest and b the smallest diameter. To analysis tumor tissues apoptosis by Dox-HPSA nanoparticles, tumors were dissected from A549 tumor-bearing mice at 24 h after intravenous injection of Dox-HPSA nanoparticles. Then, fixed with 3% paraformaldehyde solution, and embedded in paraffin. Embedded tumors were sectionalized for 8 um using a Cycle Type Paraffin
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Fig. 2. Characterization of HPSA nanoparticles. (A) Size, as determined by DLS, (B) cytotoxicity in A549 cells, and (C) cellular uptake of Cy5.5-labeled PSA and HPSA. Results are expressed as means SD of three replicate measurements from a single experiment, and are representative of three separate experiments (*P < 0.05, **P < 0.01). In fluorescent images, red (Cy5.5) corresponds to nanoparticles and blue (DAPI) represents nuclei. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Section Machine (QP3268, Changzhou Haosilin Medical Insrument Co., Ltd., Changzhou, Jiangsu, China). After section of tumors, the tumors were treated with TUNEL assay as formerly described in in vitro doxorubicin delivery into A549 cells. The tumors were treated with DAPI staining and observed by a confocal laser microscope (Eclipse Ti-S, Nikon Instruments Inc., Melville, NY, USA). 2.9. Statistical analysis The results are presented as mean SD or SE, and statistical comparisons between groups were carried out using one-way ANOVA followed by the Student’s t-test using SigmaPlot version 10.0. 3. Results and discussion 3.1. Characterization of HPSA nanoparticles To produce HPSA nanoparticles, we N-deacetylated PSA to form amide bonds between PSA and 5b-cholanic acid, eliminating the acetyl groups. The structure of N-deactylated PSA was determined by 600-MHz 1H NMR spectroscopy. The peak of PSA at 1.95 ppm was attributed to the protons of the acetyl group in PSA. As expected, the peak at 1.95 ppm disappeared in N-deacetylated PSA (Supplementary data, Fig. S1), indicating successful N-deacetylation. The amphiphilic character of HPSA nanoparticles, composed
of hydrophilic PSA and hydrophobic 5b-cholanic acid at different molar ratios of PSA:5b-cholanic acid (5%, 1:20 [HPSA5]; 10%, 1:10 [HPSA10]; and 20%, 1:5 [HPSA20]), induces self-assembly in an aqueous solution. As the amount of 5b-cholanic acid increased, the zeta potential value of PSA and HPSA nanoparticles decreased (Table 1). The mean diameters of HPSA nanoparticles, measured by dynamic light scattering (DLS), were in the 600–800 nm range (Table 1, Fig. 2A), a size somewhat larger than that of other reported nanoparticles. However, they showed good deformability, with most HPSA nanoparticles easily passing through smaller-size filter membranes (Supplementary data, Fig. S2). This indicates that these nanoparticles are appropriate for cellular uptake and in vivo experiments. The various-size HPSA nanoparticles showed differences in zeta potential value; at 821.81 90.08 nm, HPSA10 nanoparticles were the largest. A transmission electron microscopy (TEM) analyses demonstrated that HPSA nanoparticles were nearly spherical in shape (Fig. 2A, inset images). Except for nanoparticles with a 5b-cholanic acid composition of 20% (HPSA20), HPSA nanoparticles were less cytotoxic toward A549 cells than PSA at concentrations higher than 50 mg/mL (Fig. 2B). We did not observe any difference among any treated groups at lower concentrations. Notably, HPSA5 and HPSA10 nanoparticles had no significant impact on cell viability at the highest concentration tested (100 mg/mL). As illustrated in Fig. 2C, Cy5.5-labeled HPSA nanoparticles showed greater accumulation within A549 cells than Cy5.5-labeled PSA.
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Fig. 3. Cancer targeting of HPSA nanoparticles in A549-inoculated athymic nude mice. (A) In vivo biodistribution of Cy5.5-labeled PSA and HPSA nanoparticles. (B) Ex vivo NIRF images of dissected major organs. (C) Quantification of ex vivo results (n = 4). Results are expressed as means SE of three replicate measurements (**P < 0.01; ND, no significant difference).
3.2. In vivo cancer targeting of HPSA nanoparticles Athymic nude mice bearing A549 tumors were intravenously injected with Cy5.5-labeled PSA and HPSA nanoparticles, and the in vivo biodistribution of PSA and nanoparticles was monitored
over time (Fig. 3A). A strong near-infrared fluorescence (NIRF) signal was detected throughout the body of mice within 1 h of intravenous injection of either PSA or HPSA nanoparticles, demonstrating that PSA and HPSA nanoparticles promptly circulate in the blood. A maximum NIRF signal was detected in tumors
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Fig. 4. In vitro release of Dox. (a) Release profiles of Dox from Dox-HPSA at 37 C. (B) Distribution of Dox (red fluorescence) in A549 cells. (C) TUNEL assays of A549 cells treated with free Dox or Dox-HPSA. A549 cells were immunostained with TUNEL (green), and nuclei were counterstained with DAPI (blue). Results are expressed as means SD of three replicate measurements from a single experiment, and are representative of three separate experiments (**P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
almost immediately after injection, and was maintained for up to 2 days before progressively decreasing. To evaluate the tumor targeting effect ex vivo, we assessed Cy5.5-labeled PSA and HPSA nanoparticle fluorescence in the major organs (liver, lung, spleen, kidney, and heart) excised from mice. PSA and HPSA nanoparticle uptake was chiefly observed in tumors and not in normal tissues (Fig. 3B). Moreover, the NIRF intensity of PSA and HPSA nanoparticles was similar in each organ, but not in tumors, where the NIRF intensity of HPSA nanoparticles was 2.3–2.9-fold higher than that of PSA. These observations provide crucial evidence that the cancer-targeting ability of HPSA nanoparticles is much greater than that of PSA, a conclusion supported by statistical analyses (Fig. 3C). 3.3. In vitro drug delivery into cancer cells Dox, an effective anticancer drug, was employed as a representative chemotherapeutic agent in this study. Dox was loaded into HPSA nanoparticles using a simple dialysis method, as previously reported (Kim et al., 2006). We prepared two Doxloaded HPSA nanoparticles: Dox-HPSA, containing 10% Dox (relative to HPSA), and 1/2-Dox-HPSA, containing 5% Dox. Both preparations showed no significant size change after drug loading and exhibited encapsulation efficiencies greater than 90%
(Supplementary data, Table S1). To evaluate the stability of Doxloaded HPSA, we measured changes in the size of Dox-HPSA nanoparticles. These analyses showed that Dox-HPSA nanoparticles maintained their stability for 7 days in phosphatebuffered saline (PBS; Supplementary data, Fig. S3). To assess the potential of HPSA nanoparticles as a drug carrier, we evaluated the release profile of Dox from HPSA nanoparticles at 37 C in PBS (pH 7.4). For the duration of these experiments, sink conditions were preserved by regularly replacing the dialysis medium. The in vitro drug-release profile of Dox-HPSA nanoparticles is illustrated in Fig. 4A, which shows an initial burst of Dox release within 2 days and a sustained release until day 7. Dox-HPSA nanoparticles released more Dox than 1/2-Dox-HPSA nanoparticles, owing to the relative difference in Dox content. As noted above, HPSA nanoparticles accumulate in tumors through the EPR effect. At 48 h after intravenous injection, at least 2.5-times more HPSA nanoparticles accumulated in the tumor than in the liver. Therefore, we conclude that Dox is released from HPSA nanoparticles and accumulates in tumor tissue. Because the cytotoxic action of Dox occurs in the nucleus of cancer cells (Gewirtz, 1999), we measured Dox delivery to the nucleus by Dox-HPSA nanoparticles compared with that of free Dox. As shown in Fig. 4B, free Dox and Dox-HPSA nanoparticles exhibited similar Dox delivery. This effect was concentration
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Fig. 5. In vivo antitumor activity of DOX-HPSA nanoparticles in A549 tumor-bearing mice. (A) Changes in tumor volume and photographs of dissected tumors. (B) Tumor weights in treatment groups after 16 days. (C) TUNEL assay of dissected tumor tissues. Tumor tissues were immunostained with TUNEL (green), and nuclei were counterstained with DAPI (blue). (D) H&E staining of major organs (liver, lung, spleen, kidney, and heart) after 16 days. Results are expressed as means SE of three replicate measurements from one mouse, and are representative of six mice (*P < 0.05 and **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
dependent, as evidenced by the fact that the 1/2-Dox-HPSA nanoparticles group showed relatively less delivery of Dox. To evaluate the cytotoxicity of Dox released from HPSA nanoparticles, we assessed apoptosis in Dox-HPSA–treated A549 cells using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays (Fig. 4C). Cells in all groups exhibited apoptosis signals, except cells in the control group. Notably, treatment with free Dox as well as Dox-HPSA and 1/2-Dox-HPSA nanoparticles caused a significant, concentration-dependent reduction in the number of A549 cells compared with untreated controls, confirming the cytotoxicity of Dox. Taken together, our HPSA biodistribution experiments, analyses of Dox release from HPSA, and evaluation of the cytotoxicity of Dox-HPSA, which collectively show that Dox HPSA nanoparticles specifically accumulate in tumors through the EPR effect, release Dox in a sustained manner and effectively kill cancer cells, suggest the potential of HPSA nanoparticles as drug carriers.
3.4. In vivo cancer therapy of Dox-HPSA nanoparticles The antitumor effects of Dox-HPSA nanoparticles were evaluated in A549 tumor-bearing athymic nude mice. After tumors reached a volume of 200 mm3, mice were injected with one of the following four formulations every 3 days: (i) normal saline, (ii) HPSA nanoparticles only at 50 mg/kg, (iii) free Dox at 5 mg/kg, and (iv) Dox-HPSA nanoparticles at 5 mg/kg. The therapeutic efficacy of Dox-HPSA nanoparticles was investigated by measuring tumor volumes for 16 days (Fig. 5A). At the end of this period, mean tumor volume in the group treated with HPSA alone was 5029.9 mm3, a volume similar to that observed in the normal saline group (5110.7 mm3), indicating that HPSA nanoparticles alone had no effect on tumor volume. By contrast, treatment with Dox-HPSA nanoparticles caused a substantial decrease in tumor volume, reducing mean tumor volume to 774.5 mm3 on day 16. Notably, this decrease was
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considerably greater than that in the free-Dox group, where mean tumor volume was 3044.4 mm3. The effects of these treatments on tumor volumes were also reflected in final tumor weights, which were decreased to the greatest extent in mice treated with DoxHPSA nanoparticles (748.7 mg). By comparison tumor weights in mice treated with free Dox, HPSA only, and normal saline were 2335.7, 4524.1 and 4602.0 mg respectively (Fig. 5B). Excised tumors were stained for histological analysis, and slides were evaluated by an independent pathologist. TUNEL assays revealed increased apoptosis in median tumors excised from mice treated with Dox-HPSA nanoparticles (Fig. 5C). By comparison, tumors in mice treated with normal saline, HPSA, or free Dox showed higher numbers of tumor cells and lower apoptosis signals. To evaluate the toxicity of HPSA nanoparticles towards non-target organs, we performed H&E staining on liver, lung, spleen, kidney, and heart tissue after therapy. As shown in Fig. 5D, none of these major organs exhibited any histomorphological differences. In the Dox-HPSA-treated group, histomorphology was similar to that in the saline-treated group, despite the significantly enhanced therapeutic efficacy of Dox-HPSA. These results suggest that Dox-HPSA nanoparticles enhance the cancer-targeted delivery and therapeutic efficacy of anticancer drugs while reducing associated toxicity. 4. Conclusion PSA is one of the most biodegradable and biocompatible polysaccharides. HPSA nanoparticles containing 5b-cholanic acid self-assembled in aqueous solutions. They also specifically accumulated in tumors and exhibited appropriate Dox loading content, sustained Dox release, and good anticancer therapy without causing tissue toxicity. However, it remains possible that the initial burst release of Dox within 2 days during which HPSA accumulates in tumors leads to unexpected effects. Despite this limitation, our results, taken together, suggest that HPSA nanoparticles have outstanding potential as a novel nanocarrier system for therapeutic agents owing to their low toxicity, long systemic retention, and specific accumulation in tumor cells. Acknowledgements This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2057861). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2017.01.055.
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