International Journal of Biological Macromolecules 72 (2015) 510–518

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Paclitaxel loaded hyaluronic acid nanoparticles for targeted cancer therapy: In vitro and in vivo analysis Reju G. Thomas a , MyeongJu Moon b , SeJy Lee a , Yong Yeon Jeong a,∗ a b

Department of Radiology, Chonnam National University Hwasun Hospital, Chonnam National University Medical School, Gwangju 501-746, Korea DKC corporation (BioActs), Incheon, South Korea

a r t i c l e

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Article history: Received 25 July 2014 Received in revised form 14 August 2014 Accepted 21 August 2014 Available online 16 September 2014 Keywords: Nanoparticle Cancer Hyaluronic acid

a b s t r a c t The main aim of this work was to evaluate a nanoconjugate system of paclitaxel loaded self-assembling, biodegradable micelles for targeting CD44 overexpression in cancer cells. The shape and size, zeta potential, encapsulation efficiency and cell uptake of these drug-loaded micelles were evaluated. To understand their bio distribution profile, the hyaluronate (HA) micelles were labeled with FlammaTM -774 NIR dye and injected into SCC7 tumor induced mice. Cell viability in response to drug loaded and unloaded micelles was studied in SCC7 cancer cells using the MTS assay. An in vivo tumor inhibition study was conducted by intravenous injection of paclitaxel-loaded HA micelle nanoparticles as well as control nanoparticles without paclitaxel. The shape of the nanomicelles was evaluated by loading them with hydrophobic superparamagnetic iron oxide nanoparticle and then visualizing them by TEM. In conclusion, paclitaxelloaded HA nanoparticulate micelles might be found to be a specific and efficient chemotherapeutic treatment for CD44 overexpressing cancer cells. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The solubility of hydrophobic anti-cancer drugs in the human body is a major problem facing drug developers [1]. Paclitaxel is an established naturally derived anti-cancer drug that is widely commercialized in the form of hydrophilic formulations, such as Taxol® or Abraxane® , developed for the treatment of different types of cancers [2]. Apart from facing a few biocompatibility issues [3], some of these paclitaxel formulations rely on achieving tumor accumulation by the enhanced permeation and retention (EPR) effect, which is not an ideal solution for tumor targeting [4–6]. A main aim of researchers is to develop a formulation that is biocompatible, actively targets cancer and has slow releasing properties, and this work begins with finding a good drug delivery vehicle. Head and neck cancer is still the sixth most common type of cancer worldwide. Ninety percent of the tumors in the head and neck are squamous cell carcinomas (SCC7), which present as aggressive and recurrent malignancies [7]. CD44 is recognized as a cell surface

Abbreviations: F772, FlammaTM -552; F774, FlammaTM -774; CA, 5ß-cholanic acid; NIR, Near infra red; HA, Hyaluronic acid; EtCA, aminoethyl 5ß-cholanomide; SPION, Superparamagnetic Iron Oxide Nanoparticle. ∗ Corresponding author. Tel.: +82 61 379 7102; fax: +82 91 379 7133. E-mail addresses: [email protected] (R.G. Thomas), [email protected] (Y.Y. Jeong). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.054 0141-8130/© 2014 Elsevier B.V. All rights reserved.

marker for cancer stem-like cells in epithelial tumors, such as head and neck cancer [8]. CD44 expression may have a prognostic value in SCC7 of the head and neck [9]. Hyaluronic acid (HA) is associated with malignant tumor as a prognostic agent and plays a crucial role in the progression of cancer. High levels of hyaluronan are related to the abundance of CD44 receptors found in epithelial cell cancers, such as head and neck cancer [10–12]. HA is readily available, biocompatible, non-toxic and has many functional groups for chemical modification [13,14]. HA is widely regarded as a polymer of choice for developing drug delivery vehicles against CD44 overexpressing tumors [15–19]. Amphiphilic conjugates of HA are capable of forming hydrophilic drug carriers in aqueous conditions. Additionally, they can carry hydrophobic drugs, such as paclitaxel, to the tumor area without affecting the payload [20]. Several biomolecules have been tested as the hydrophobic moiety of these conjugates, including cholesterol, octadecylamine (C18 ), ICG (Indo Cyanine green), 5ßcholanic acid and so on [21–25]. HA carries the ligand for CD44, which facilitates receptor mediated endocytosis of drug loaded micelles by these cells. Hyaluronidase present in the cytoplasm of cell cleaves the HA backbone to release paclitaxel, which inhibits mitosis by stabilizing the microtubules, thereby interfering with normal cell division [26]. In this study, the hydrophilic polymer HA was chemically conjugated to modified 5ß-cholanic acid [13] and used to prepare a nano-micelle formulation, encapsulating the anti-neoplastic drug

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paclitaxel, targeted against SCC7 of the head and neck. The main aim of this work was to evaluate the nanoconjugate system of paclitaxel loaded self-assembling, biodegradable micelles for targeting CD44 overexpressing SCC7.

2. Materials & methods 2.1. Materials Sodium hyaluronate (0.48 MDa) was purchased from Bioland, Korea. Formamide, Pyrene and 5ß-cholanic acid (CA) was purchased from Sigma–Aldrich, USA. Fluorescent probes FlammaTM FCR-552 (F552) and FlammaTM FCI-774 (F774) were obtained from BioActs, Korea. N-N dimethyl formamide was purchased from Merck, Germany. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were purchased from Sigma–Aldrich, USA. 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium) (MTS) was purchased from Promega, USA. Paclitaxel was purchased from Genebio, Korea. Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 were purchased from Thermo Scientific, USA. All other reagents were of analytical or chromatographic grade. 2.2. Preparation of the hyaluronate-cholanic acid (HA-CA) conjugate The HA-CA conjugate was synthesized as described elsewhere [13]. Briefly, 500 mg of 5ß-cholanic acid (CA) was dissolved in 5 ml of methanol, 1 ml of 37% HCl was added and the reaction was refluxed at 60 ◦ C for 6 h. The mixture was cooled to 0 ◦ C to obtain white precipitate, which was filtered out using a membrane filter (pore size: 0.45 ␮m, Millipore). In the next step, the vacuum dried filtrate was dissolved in 5 ml of ethylene diamine (EDA) and refluxed at 130 ◦ C for 6 h. The mixture was cooled to room temperature to obtain a white precipitate, which was aminoethyl 5ß-cholanomide (EtCA). Next, 120 mg of HA (0.48 MDa) was dissolved in 70 ml of formamide by overnight stirring. 48.5 mg of EDC was then added and stirred for one hour, after which 29.1 mg of NHS was added. Subsequently, 0.26 mg of EtCA dissolved in 28 ml of dimethyl formamide was added drop-wise to the HA solution and stirred for one day. The resultant product was dialyzed against a water/methanol mixture for 2 days, followed by 2 additional days against water alone, using a cellulose ester dialysis membrane bag (MWCO = 3500) to remove unreacted chemicals. To obtain the micelles in powder form, the sample was cooled in liquid nitrogen and lyophilized at 0.01 mBar and −81 ◦ C for 5 days (Labconco Free Zone, Kansas, USA). 2.3. Characterization of the HA-CA conjugate The lyophilized HA-CA conjugate was subjected to 1 H-NMR analysis to study the level of conjugation between HA and EtCA. To characterize the morphology of micelles, hydrophobic oleic acid coated superparamagnetic iron oxide (SPION) was loaded into the HA-CA micelles, which were then visualized by transmission electron microscopy (TEM) and subjected to thermogravimetric (TGA) analysis (S1) to quantify the HA-CA conjugate content in the SPION loaded micellar system. The size of the HA-CA micelles and HA-CA-Paclitaxel micelles was analyzed using DLS, and the charge of the particles was analyzed on a Zetasizer instrument (Nano-Z590, Malvern Instruments, Worcestershire, UK).

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2.4. Conjugation of HA-CA micelles with fluorescent probes HA-CA micelles were conjugated with FlammaTM -552 and FlammaTM -774 probes using DCC/NHS chemistry. More specifically, the free carboxylic acid groups of HA were reacted with the amine activated florescent probes in the presence of DCC and NHS. Briefly, HA-CA was dissolved in DMSO and FlammaTM -552 (abs -551 nm, em -570 nm) and FlammaTM -774 (abs -778 nm, em -808 nm) were added in a fixed molar ratio of 1:100 (HA:probe). Subsequently, DCC/NHS was added at a 5 and 10 times molar value to the fluorescent probe present in the solution. The reaction mixture was stirred for one day in the dark and dialyzed using a membrane of MWCO = 3500 to remove DMSO and unreacted materials. The lyophilized sample was analyzed for conjugation efficiency by the absorbance method using a multimode microplate reader (TECAN, Infinite M200 PRO, Männedorf, Switzerland). The final conjugation efficiency was calculated as the ratio of the concentration of the HA-CA conjugated fluorescent probes, obtained from a standard curve of free dye, compared to the theoretical concentration.

2.4.1. In vitro bioimaging of HA-CA micelles labeled with FlammaTM -552 Dye SCC7 (Squamous cell carcinoma) and NIH3T3 cells (mouse embryonic fibroblasts) were seeded at a density of 5 × 104 cells in an 8 well chamber slide (Lab-Tek2, USA) and incubated in a humidified environment in CO2 at 37 ◦ C for one day. RPMI medium containing 10 vol% FBS and 1 wt% of antibiotic was added to culture the cells. Prior to adding dye conjugated HA-CA micelles, 1 mg of HA (0.48 MDa) was added to one set of wells to study the effect of competitive inhibition. HA-CA micelles labeled with FlammaTM 552 dye were added to the next set of wells at a concentration of 30 ␮g/ml. Similarly, free dye at concentration equivalent to that in the HACA micelles was added to the third set of wells. Following these treatments, the cells were incubated for another 4 h. After the incubation period, the cells were washed with PBS and fixed with a 4% formaldehyde solution. The cells were then observed using a confocal microscope (Zeiss LSM 510, Oberkochen, Germany) equipped with HeNe (543 nm) and Diode (405 nm) lasers for fluorescence at a magnification of 40x.

2.4.2. In vivo and ex vivo bioimaging of HA-CA conjugates labeled with FlammaTM -774 fluorescent dye Athymic (nu/nu-ncr, Balb/c mice) (5–6 weeks old, 20–25 g) were obtained from Jungang Lab Animal, Inc., Korea. The Chonnam National University Medical School Research Institutional Animal Care and Use Committee approved the experimental protocol (CNU IACUC-H-2011-5). For developing tumors in the mice, SCC7 cells cultured in RPMI (37 ◦ C, 5%CO2 ) were injected subcutaneously at a density of at least 1 × 106 cells per injection. When the tumor size reached 100–150 mm3 , HA-CA-FlammaTM 774 dissolved in PBS at a concentration of 5 mg/kg was injected into each mouse. For in vivo imaging, mice (n = 3) were analyzed at predetermined time interval using an IVIS Lumina (Xenogen, Toronto, USA) imaging system with ICG (Indo Cyanine Green) excitation and emission filters at an exposure time of one second. For the ex vivo organ distribution study, 16 Balb/C mice in randomized groups of 4 were injected with HA-CA-FlammaTM 774 (5 mg/kg). Their organs were harvested at 3 h, 1 day, 2 days and 6 days (n = 4). The fluorescence of HA-CA-FlammaTM 774 was captured using an IVIS Lumina with ICG excitation and emission filters at an exposure time of one second.

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2.5. Preparation of HA-CA-Paclitaxel micelles 10 mg of lyophilized HA-CA micelles was dissolved in distilled water. 1 mg of paclitaxel dissolved in 100 ␮l of 100% ethanol was added drop-wise to the HA-CA micelles in water and stirred for 24 h. Next, the HA-CA-Paclitaxel micellar solution was centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the obtained pellet was dissolved in acetonitrile (500 ␮l) and probe sonicated for 10 min at a 2:3 pulse rate to disrupt the micellar structure. Paclitaxel that was freely soluble in acetonitrile was analyzed by HPLC using a mobile phase of acetonitrile (50:50, v/v). The encapsulation efficiency was calculated based on the ratio of the amount of paclitaxel in the micelles to the total amount of paclitaxel added in the solution, and the drug loading capacity was determined by the total amount of paclitaxel in a specific amount of micelles [27]. 2.5.1. Cytotoxicity tests of HA-CA-Paclitaxel micelles The cytotoxicity of free paclitaxel and HA-CA-Paclitaxel against SCC7 cells was evaluated using the MTS assay. The cells were seeded into a 96-well plate at a density of 104 cells/well. The cells were cultured in a CO2 incubator at 37◦ C in a humidified environment for one day. Free paclitaxel or HA-CA-Paclitaxel was added to the cells in triplicate to analyze the cytotoxicity over the concentration range from 0.01 ␮g/ml to 100 ␮g/ml. Triton was added at 5 ␮g/ml as a positive control. The cells were incubated for 24 h after treatment. Then, 20 ␮l of MTS reagent was added to each of the treated wells and incubated for 4 h. Finally, the absorbance at 490 nm was measured using a microplate reader. 2.5.2. In vivo tumor inhibition study with HA-CA-Paclitaxel micelle The mice injected subcutaneously with SCC7 cells (1 × 106 cells) were divided into four groups (n = 3) when tumor size reached 9 mm or 100 mm3 in volume. First group was used as a control group. The second group was injected with paclitaxel in a 1% ethanol PBS solution (5 mg/kg). The third and fourth groups were injected with 2 mg/kg and 5 mg/kg, respectively, of HA-CAPaclitaxel dissolved in PBS at pH 7.4. This dosage was repeated twice, with three days between injections. Tumor growth was analyzed so that the upper limit of tumor size was 1000 mm3 . The tumor volume was measured using a digital vernier caliper (Mitutoyo Corp., Kawasaki, Japan) and calculated using the following equation [28]: Volume = length × width2 /2.

2.6. Data analysis The results are expressed as the mean ± SD and the mean ± SEM. Statistical significance was taken as P < 0.05, using an unpaired Student’s t-test. A repeated measures ANOVA test was used to analyze the data from the tumor inhibition study. 3. Results 3.1. Characterization of HA-CA micelles HA-CA micelles were prepared by amide bond formation between the carboxyl groups of HA and the amine group of EtCA using EDC/NHS chemistry (Fig. 1A). The 1 H-NMR spectrum of the HA-CA conjugate, in which the N-acetyl peak of HA appears at 2 ppm and the methyl group of EtCA is at 0.68 ppm, is shown (Fig. 1B).

The size and charge of the HA-CA micelles were 298 ± 102 nm and −26.3 ± 11.8 mV, respectively, whereas the HA-CApaclitaxel micelles had a size of 258 ± 57.85 nm and a charge of −31.8 ± 6.1 mV (Fig. 2). A decrease in the size of the particles was observed after encapsulating paclitaxel. This result may have been due to strong interactions between the drug and the EtCA moiety by hydrophobic effects, as these interactions would bring the two molecular species together and compact the micellar structure. There was not much change in the zeta potential between the HA-CA and HA-CA-paclitaxel micelles, as the outer negatively charged carboxylic acid groups of the HA backbone were left unaffected. A critical micellar concentration of 40 ␮g/ml was measured (S2) by fluorescence spectroscopy using pyrene molecules as the hydrophobic agent for encapsulation inside HA micelles. To verify the morphology of micelles after encapsulating paclitaxel, hydrophobic SPION were used to mimic the encapsulation process and enable visualization by TEM. Fig. 2B shows the circular morphology of the micelles, with SPION tightly packed into compact structures. In contrast, fluorescently labeled HA-CA micelles exhibited larger particle sizes and less negative charge (Table 1), which can be explained by the reduced number of free carboxyl groups due to fluorescent probe conjugation. 3.1.1. In vitro bioimaging of HA-CA micelles labeled with FlammaTM -552 dye Confocal fluorescent images of SCC7 cells treated with free dye at a concentration equivalent to the conjugated dye clearly showed that cellular uptake of the dye by passive diffusion was minimal (Fig. 3A). NIH3T3 cells showed some uptake of HA-CAFlammaTM 552, as these cells emitted slightly higher fluorescence than was observed with the free dye (Fig. 3B). This result can be attributed to the low number of CD44 receptors on these cells. To further confirm the role of HA in increased uptake of the particles by SCC7 cells, 10 mg of sodium HA was added in one group and HA-CA-FlammaTM 552 was added 1 h later. Fig. 3C shows reduced fluorescence in this group, which can be explained by the free HA occupying the CD44 receptors, thereby blocking the HA-CA-F552 micelles from binding to the cells. In contrast, the SCC7 cells treated with HA-CA-FlammaTM 552 showed high intensity fluorescence, indicating much greater uptake of the micelles (Fig. 3D). These findings verified the role of CD44 receptor mediated endocytosis in CD44 overexpressing tumors. 3.1.2. In vivo bioimaging of HA-CA conjugates labeled with FlammaTM 774 NIR dye HA-CA micelles were labeled with FlammaTM -774dye to study in vivo biodistribution in mice. Based on the in vitro cell uptake study, SCC7 cells were injected into the subcutaneous region of Balb/C nude mice to induce tumor formation. After the tumor size reached approximately 150 mm3 , HA-CA-FlammaTM 774 dissolved in PBS was injected at a dose of 5 mg/kg. Accumulation was evident in the liver 15 min after injection and increased in the tumor region at later time points. After 24 h, HA-CA-F774 was cleared from liver and was observed to be concentrated in the tumors until day 4. The shift in the accumulation pattern can be attributed to the active targeting of CD44 by HA, as well as the EPR effect due to increased vascularization in the tumor region. From day 5 to day 7, clearance of the micelles from the tumors and overall clearance from the mice was observed (Fig. 4). The action of hyaluronidase enzymes, overproduced in the tumor microenvironment, may have degraded the HA conjugates and led to rapid clearance [29]. The tumor/liver average fluorescence intensity clearly shows that the maximum accumulation of particles occurred from day 2 to day 4, after which the intensity

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Fig. 1. Preparation of HA-CA-Paclitaxel micelles. (A) Synthesis of paclitaxel loaded HA-CA micelles. (B) 1 H-NMR spectrum of HA-CA-paclitaxel micelles.

Fig. 2. (A) DLS and Zeta potential of HA-CA and HA-CA-paclitaxel in PBS at 7.4 pH. (B) TEM image of a SPION loaded HA-CA conjugate micelle.

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Table 1 The physiochemical characterization of FlammaTM -552 and FlammaTM -774 conjugated HA micelles. Sample

Molar ratio (HA to dye)

Size (nm)

Zeta potential (mV)

Conjugation efficiency (%)

HA-CA-F552 HA-CA- F774

1/100 1/100

386 ± 81.31 443.6 ± 119.9

−17.6 ± 6.8 −23.6 ± 5.31

7.7 8.9

decreased (Fig. 4D). To further confirm active targeting property by the HA micelles, HA-CA-FlammaTM 774 was injected into another set of mice where the tumor size was ≤100 mm3 . The results showed accumulation and retention up to day 6, closely following the pattern observed for larger tumors (S3). The biodistribution profile of HA-CA micelles in organs was analyzed ex vivo at various predetermined time points (Fig. 5). The liver was found to have a reasonably high accumulation due to HARE over abundance, and the kidney also showed accumulation, owing to the presence of CD44 receptors [11]. The spleen, lung and heart initially showed little accumulation, which subsided as time progressed. Tumor accumulation remained high for up to 2 days due to the combined effects of CD44 receptor targeting and EPR at the tumor site (Fig. 5E). These results correlated with the in vivo biodistribution profile.

3.2. Paclitaxel encapsulation efficiency of HA-CA conjugates HA-CA micelles and paclitaxel prepared in a 10:1 ratio (10% w/w) were shown to have an encapsulation efficiency (EE) of 77.3% and a drug loading of 7.7%, as analyzed by HPLC. As we increased the drug/carrier ratio to 10:3 (30% w/w) the EE dropped down to 29.1% with only a 1% increase in drug loading (Table 2). This result

showed that the optimum paclitaxel to HA-CA micelle ratio was 10% w/w, which was used for our further experiments. 3.2.1. Cytotoxicity tests of HA-CA-paclitaxel The cell toxicity of HA-CA-paclitaxel was analyzed using the SCC7 cell line, which expresses the CD44 receptor. Paclitaxel dissolved in 1% ethanol in PBS was used as a control. Both free paclitaxel and paclitaxel loaded HA micelles were dissolved in optiMEM® reduced serum medium (Gibco, USA). HA-CA-paclitaxel treatment resulted in a visible decrease in cell viability as the concentration increased compared to free paclitaxel (Fig. 6A). At concentrations of 1 ␮g/ml up to 100 ␮g/ml, the toxicity difference was found to be statistically significant for paclitaxel loaded micelles compared to free paclitaxel (* P < 0.05 and ** P < 0.01). To check the cell viability profile dependency on incubation time, we treated free paclitaxel and HA-CA-Paclitaxel on SCC7 cells for 4 h and kept for another 48 h after treatment period. There was a marked decrease in cell viability in both groups, with 88% and 75% cell death at 100 ␮g/ml of paclitaxel concentration and 72% and 60% cell death at 10 ␮g/ml concentration, in HA-CAPaclitaxel and free Paclitaxel group respectively (S4). Lastly, we verified cytocompatibility of HA-CA-paclitaxel in normal mouse embryonic fibroblast cells (NIH3T3) in comparison with free paclitaxel. After 4 h treatment and 24 h incubation, both free and

Fig. 3. Confocal imaging of HA-CA micelles labeled with FlammaTM 552 dye. (A) SCC7 cells incubated with free FlammaTM 552 dye. (B) NIH3T3 cells incubated with 30 ␮g/ml of HA-CA-FlammaTM 552. (C) 10 mg of HA added 1 h prior to incubating SCC7 cells in 30 ␮g/ml of HA-CA-F552. (D) 30 ␮g/ml of HA-CA-FlammaTM 552 incubated with SCC7 cells. The incubation period was 2 h for all experiments.

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Fig. 4. In vivo biodistribution profile of HA-CA-FlammaTM 774 in Balb/c nude mice (n = 3). (A) Red color indicates accumulation of FlammaTM 774 dye conjugated HA micelles, after injecting 5 mg/kg, from 5 min to 2 h, (B) 4 h to 4 days and (C) 5 days to 14 days after injection. (D) Tumor-to-liver ratio up to day 6. Mean ± SD. Fluorescence intensity was quantified by the region-of-interest method.

Fig. 5. Ex vivo biodistribution profile of HA-CA-FlammaTM 774 in the liver, kidney, spleen, lung, heart and tumor (n = 4). (A) Balb/c nude mice were sacrificed after injecting 5 mg/kg of FlammaTM 774 dye conjugated HA micelles at (A) 3 h, (B) 1 day, (C) 2 days and (D) 6 days after injection, and IVIS images were taken. (E) Fluorescence intensity was quantified by the region-of-interest (ROI) method. The data are presented as the mean ± SEM. (F) Tumor-to-liver ratio. *P < 0.05 relative to the liver ROI.

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Table 2 Physicochemical characterization of paclitaxel loaded HA micelles. Sample

Drug/carrier (w/w)%

Size (nm)

Zeta potential (mV)

CMC (␮g/ml)

EE (%)

DL (%)

HA-CA-Paclitaxel

10%

256.8 ± 57.8

−31.8 ± 6.1

40 ± 10

77.3

7.7

CMC: Critical micellar concentration, E.E: Encapsulation efficiency, DL: Drug loading.

Fig. 6. In vitro cytotoxicity of paclitaxel (in 1% ethanol–PBS solution) and HA-CA-paclitaxel (in PBS, pH 7.4). (A) SCC7 cell line; (B) NIH3T3 cell line. MTS assay data show the mean cell viability of quadruplicate samples ± SD. *P < 0.05 and ** P < 0.01 relative to the free paclitaxel treated group.

HA-CA micelle encapsulated paclitaxel showed minimal toxicity, towards NIH3T3 cell line at 0.01–100 ␮g/ml concentration range (Fig. 6B).

3.2.2. In vivo tumor reduction with HA-CA-paclitaxel Balb/C nude mice bearing subcutaneous tumors and treated with 5 mg/kg of HA-CA-paclitaxel showed significantly lower tumor growth compared to the other groups (Fig. 7). A dose of 2 mg/kg also resulted in a comparatively smaller tumor size (540 mm3 ) than free paclitaxel (1071 mm3 ) and the control (883 mm3 ) at day 8 (Table 3). Statistical analysis by repeated measures ANOVA showed that the differences for HA-CA-paclitaxel (5 mg/kg) from the control and HA-CA-paclitaxel (2 mg/kg) at days 6 and 8 were highly significant (P < 0.05). Free paclitaxel and the control group were not significantly different at all times points. Observation was continued until day 14 for all groups and their tumor volume was measured and recorded. The size of the tumors increased from day 8, after the paclitaxel-loaded micelle treatment was stopped, indicating that the paclitaxel activity in the tumors had worn off. There was a significant decrease in weight loss of the mice in the free paclitaxel dosage group, with the minimum average weight being reached at day 4. The HA-CA-paclitaxel groups did not show significant weight loss until day 8, possibly due to paclitaxel being released from micelles circulating in the bodies of the mice and causing general toxicity [30] (S5).

Table 3 Average tumor volume of the mice at the end of study. Group (n = 3)

Drug dose (mg/kg)

Tumor volume (mm3 )

Control Paclitaxel HA-CA-Paclitaxel HA-CA-Paclitaxel

0 5 2 5

894 1066 575 334

4. Discussion HA-CA micelles were labeled with FlammaTM -552 dye for the purpose of studying cellular uptake by CD44 receptor mediated endocytosis [31,32]. The SCC7 cell line was used to study free dye accumulation, competitive inhibition and particle uptake, while the NIH3T3 cell line was used as a control. In a similar study using CA conjugated HA, Cy5.5 dye was used to observe uptake by SCC7 cells, and intense fluorescence was observed at concentration of 50 ␮g/ml of Cy5.5 conjugated HA polymer [13]. In our study, 30 ␮g/ml of HA-FlammaTM -552 showed a significant difference in uptake between SCC7 cells and NIH3T3 cells. Furthermore, SCC7 cells pretreated with HA showed significant decreased uptake rather than HA-CA treated SCC7 cells. These results demonstrate that cellular uptake was due to CD44 receptor mediated endocytosis of the HA-CA micelles. Preferential micelle uptake by the liver is a major challenge facing the tumor targeted HA-CA micelle system [33]. Previous studies have shown that PEGylation of the HA polymer backbone considerably increased tumor accumulation by up to 1.6-fold and decreased uptake by the liver [34,35]. Our study showed that HA-CA had accumulated in the liver at day 1 after injection. After 24 h, HA-CA was cleared from the liver and was concentrated in the tumor, which lasted until day 4. The in vivo and ex vivo biodistribution profiles both indicated a high tumor-to-liver ratio from days 2 to 6, which clearly showed the ability of the HA-CA micelles to target CD 44 expressing tumors. Our study showed that paclitaxel was loaded with an optimal EE of 77.3%. EE and DL are two important factors that determine the drug entrapment capability of the polymer. They mainly depend on the degree of substitution of the carboxyl groups of the HA-CA micelles by EtCA units [27]. However, for CD44 specific targeting with HA, it is important to maintain a comparatively low degree of substitution to protect its carboxyl groups, which are mainly involved in CD44 receptor binding. The results from the MTS assay demonstrated that HACA-Paclitaxel had significantly higher cell toxicity profile

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Fig. 7. In vivo tumor inhibition study. Balb/c nude mice were injected with PBS, free paclitaxel and HA-CA-Paclitaxel at Day 0 (n = 3). Comparison of tumor size on Day 8 of (A) Control (B) Paclitaxel (C) HA-CA-Paclitaxel (2 mg/kg) (D) HA-CA-Paclitaxel (5 mg/kg). Tumor inhibition data show mean tumor volume, of triplicate samples ± SD. *P < 0.05 relative to HA-CA-paclitaxel (2 mg/kg) and **P < 0.05 relative to control group.

(1–100 ␮g/ml) for drug encapsulating micelles compared to free drug. The in vivo tumor study conducted with 5 mg/kg of paclitaxel in HA-CA micelles showed significant inhibition of tumor growth compared to the 2 mg/kg HA-CA-paclitaxel and free paclitaxel groups, as well as the control, on days 6 and 8. The tumor inhibition effect of 5 mg/kg HA-CA-paclitaxel was evidently more effective than 2 mg/kg due to the delivery of a higher concentration paclitaxel.

Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (MEST) (2012-0008258) and BK 21 PLUS Program funding through Center for Creative Biomedical Scientists at Chonnam National University. Appendix A. Supplementary data

5. Conclusion Our results demonstrate the effective targeting property of HA micelles to CD44 overexpressing tumors, as shown by higher fluorescence in vitro cell uptake by HA-CA-F552 and in vivo and ex vivo biodistribution studies which was based on high tumor/liver ratio by HA-CA-F774. The accumulation and toxicity of HA-CA-paclitaxel in SCC7 cells was quantified by cytotoxicity assay and in vivo tumor inhibition study validated inhibition of tumor growth with minimal side effects compared with free paclitaxel group. Thus, polymeric nanoparticulate micelles of HA-CA-paclitaxel were found to be a specific and efficient chemotherapeutic treatment for CD44 overexpressing cancer cells.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally (match statement to author names with a symbol).

Conflicts of Interest The authors declare no competing financial interest.

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