Accepted Manuscript

Title: Hyaluronic acid-coated Solid Lipid Nanoparticles for

Targeted Delivery of Vorinostat to CD44 Overexpressing
Cancer Cells

Author: Tuan Hiep Tran Ju Yeon Choi Thiruganesh

Ramasamy Duy Hieu Truong Chien Ngoc Nguyen Han-Gon
Choi Chul Soon Yong Jong Oh Kim

PII: S0144-8617(14)00795-4
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2014.08.026
Reference: CARP 9163

To appear in:

Received date: 18-6-2014

Revised date: 4-8-2014
Accepted date: 5-8-2014

Please cite this article as: Tran, T. H., Choi, J. Y., Ramasamy, T., Truong, D. H., Nguyen,
C. N., Choi, H.-G., Yong, C. S., and Kim, J. O.,Hyaluronic acid-coated Solid Lipid
Nanoparticles for Targeted Delivery of Vorinostat to CD44 Overexpressing Cancer
Cells, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.08.026

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1 Hyaluronic  acid‐coated  Solid  Lipid  Nanoparticles  for  Targeted  Delivery  of  Vorinostat  to  CD44 

2 Overexpressing Cancer Cells 

3  

4 Tuan Hiep Tran1, Ju Yeon Choi1, Thiruganesh Ramasamy1, Duy Hieu Truong1,  

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5 Chien Ngoc Nguyen2, Han‐Gon Choi3, Chul Soon Yong1**, Jong Oh Kim1* 

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6  

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1
7 College  of  Pharmacy,  Yeungnam  University,  214‐1,  Dae‐Dong,  Gyeongsan  712‐749, 

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8 South Korea. 
2
9 National  Institute  of Pharmaceutical Technology, Hanoi University  of Pharmacy,  13‐15 

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10 Le Thanh Tong, Hoan Kiem, Ha Noi, Viet Nam. 
3
11 College  of  Pharmacy,  Hanyang  University,  55,  Hanyangdaehak‐ro,  Sangnok‐gu,  Ansan 
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12 426‐791, South Korea 

13  
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14  
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15 Authors to whom correspondence should be addressed: 
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16  
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*
17 Corresponding author:           Jong Oh Kim, Prof. Ph.D. 

18  Tel: +82‐53‐810‐2813 
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19  Fax: +82‐53‐810‐4654 

20  E‐mail: jongohkim@yu.ac.kr 
**
21  Co‐corresponding author:   Chul Soon Yong, Prof. Ph.D. 

22  Tel: +82‐53‐810‐2812 

23  Fax: +82‐53‐810‐4654 

24  E‐mail: csyong@yu.ac.kr  

Page 1 of 34
25  

26 Abstract 

27 Hyaluronic  acid  (HA)‐decorated  solid  lipid  nanoparticles  (SLNs)  were  developed  for 

28 tumor‐targeted  delivery  of  vorinostat  (VRS),  a  histone  deacetylase  inhibitor.  HA,  a 

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29

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naturally  occurring  polysaccharide,  which  specifically  binds  to  the  CD44  receptor,  was 

30 coated on a cationic lipid core through electrostatic interaction. After the optimization 

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31 process,  HA‐coated  VRS‐loaded  SLNs  (HA‐VRS‐SLNs)  were  spherical,  core‐shell 

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32 nanoparticles,  with  small  size  (~100  nm),  negative  charge  (~  ‐9  mV),  and  narrow  size 

33 distribution. In vitro release profile of HA‐VRS‐SLNs showed a typical bi‐phasic pattern. 

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34 In addition, the intracellular uptake of HA‐VRS‐SLNs was significantly enhanced in CD44 

35 overexpressing  cells,  A549  and  SCC‐7  cells,  but  reduced  when  HA‐VRS‐SLNs  were 
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36 incubated  with SCC‐7  cells pretreated with HA  or MCF‐7 cells with low over‐expressed 

37 CD44. Of particular importance, HA‐VRS‐SLNs were more cytotoxic than the free drug and

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38 VRS-SLNs in A549 and SCC-7 cells. In  addition,  HA  shell  provided  longer  blood 
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39 circulation and reduced VRS clearance rate in rats, resulting in enhanced higher plasma 
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40 concentration and bioavailability. These results clearly indicated the potential of the HA‐
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41 functionalized lipid nanoparticle as a nano‐sized drug formulation for chemotherapy. 

42  
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43 Keywords: vorinostat, solid lipid nanoparticles, hyaluronic acid, chemotherapy, targeting 

44  

45

46

47

48
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48

49 1. Introduction

50 Vorinostat (VRS), a histone deacetylase inhibitor, has been approved by the FDA for

51 treatment of cutaneous T-cell lymphoma (CTCL) (Cai et al., 2010; Choo, Ho & Lin, 2008).

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52 In clinical trials, it was used for a variety of hematopoietic and solid tumor indications

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53 (Bolden, Peart & Johnstone, 2006; Kelly et al., 2005). It can effectively induce cell cycle

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54 arrest, cell differentiation, and apoptosis (Marks & Breslow, 2007). Despite showing such

55 chemotherapeutic promise, the clinical application of VRS has been restricted due to its poor

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56 aqueous solubility and low membrane permeability, belonging to class IV of the

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57 Biopharmaceutics Classification System (BCS) (Mohamed et al., 2012). In addition, VRS

58 exhibited a short half-life of 40 min following intravenous (IV) administration. To overcome

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59 these problems, VRS has been incorporated into micelle nanocarriers (Mohamed et al., 2012),

60 inclusion cyclodextrins (Cai et al., 2010), and silicon microstructures (Strobl, Nikkhah &
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61 Agah, 2010), however, the efficiency of therapy is still a major question.

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62 Solid lipid nanoparticles (SLNs) are a suitable candidate for delivery of hydrophobic and

63 hydrophilic anti-cancer drugs. Owing to their lipid core, SLNs are expected to show high
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64 drug loading capacity, overcome multidrug resistance (MDR), modulate release kinetics,
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65 improve the blood circulation time, and increase the overall therapeutic efficacy of anti-

66 cancer drugs (Bhushan et al., 2012; Lee, Lim & Kim, 2007; Luan et al., 2014). In addition,
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67 surface modification of SLNs with targeting moiety enables these SLNs to increase their

68 selectivity for cellular binding and internalization (Venishetty, Komuravelli, Kuncha, Sistla

69 & Diwan, 2013). In particular, the frequent overexpression of the hyaluronic acid (HA)

70 receptors CD44 and CD168 (RHAMM) on many types of tumors opens new avenues for

71 targeting by the naturally-occurring high-molecular weight HA (Rivkin, Cohen, Koffler,

72 Melikhov, Peer & Margalit, 2010; Sun, Benjaminsen, Almdal & Andresen, 2012).
3

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73 Therefore, in this study, we developed HA-coated VRS-loaded SLNs (HA-VRS-SLNs)

74 for tumor-targeted delivery. We hypothesize that this system will demonstrate the combined

75 advantages of high drug loading capacity by using lipid core, long blood circulation resulting

76 from the hydrophilic surface and improved delivery by selective target of HA. To validate

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77 that, we investigated at the molecular, cellular, and whole animal levels of organization. The

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78 optimized HA-VRS-SLNs were evaluated in terms of physicochemical, thermal analyses, and

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79 ultrastructure characterization.  The targeting efficacy of the HA-VRS-SLNs to tumor cells

80 was estimated by intracellular uptake through confocal fluorescence image as well as flow-

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81 cytometric analysis in different cancer cell lines; the activity of VRS of HA-VRS-SLNs was

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82 then confirmed via cytotoxicity study. VRS plasma concentration, retention time and AUC0-∞

83 in blood circulation were compared among free drug, VRS-SLNs, and HA-VRS-SLNs for the
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84 in vivo study.

85
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86
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87 2. Materials and methods

88 2.1. Materials
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89 VRS was purchased from LC Laboratories (MA, USA). Compritol 888 ATO (Compritol)
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90 (powder state; melting point, 70°C) was obtained from Gattefosse (Cedex, France). 3-(4,5-

91 Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and didecyldimethyl

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92 ammonium bromide (DDAB) were purchased from Sigma (St. Louis, MO, USA). Soybean

93 lecithin was purchased from Junsei Co. Ltd (Tokyo, Japan). Hyaluronic acid (3kDa) was

94 supplied by B&K Technology Group Co. Ltd (China). NBD-PC was supplied by Avanti

95 Polar Lipids, Inc., and Lyso TrackerRed was purchased from Thermo Fisher Scientific Inc.

96 The squamous cell carcinoma (SCC-7), human breast adenocarcinoma (MCF-7), human lung

97 adenocarcinoma epithelial (A549) cells were originally obtained from the Korean Cell Line
4

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 98 Bank (Seoul, South Korea). All cell lines were cultured in RPMI 1640 containing 10% fetal

99 bovine serum (FBS), 100 U/mL penicillin G sodium, and 100μg/mL streptomycin sulfate

100 (complete 1640 medium) and incubated at 37 °C in 5% CO2 atmosphere. All the cells threats

101 such as fungi, bacteria, mycoplasma were checked before carrying out experiments. All other

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102 chemicals were of reagent grade and were used without further purification.

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103

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104 2.2. Preparation of HA-VRS-SLNs

105 HA-VRS-SLNs were prepared by the hot homogenization method using an emulsification-

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106 sonication technique (Tran et al., 2014a). Based on a preliminary analysis of potential lipids

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107 and surfactants (data not shown), Compritol 888 ATO, DDAB, lecithin, and Tween 80 were

108 selected for preparation of SLNs. Briefly, the lipid phase was prepared by melting Compritol
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109 888 ATO, DDAB, lecithin, and VRS at 10ºC above the lipid melting point to obtain a clear

110 transparent solution. The aqueous phase was prepared by dissolving Tween 80 in distilled
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111 water and heating to the final temperature of the lipid phase. Next, hot aqueous phase was
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112 gently added drop-wise into the lipid phase with constant stirring at 13,500 rpm in an Ultra

113 Turrax® T-25 homogenizer (IKA®-Werke, Staufen, Germany) for 3 min. The resulting coarse
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114 emulsion was immediately sonicated using a high-intensity probe sonicator (Vibracell
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115 VCX130; Sonics, USA) at 80% amplitude for 10 min. The resulting suspension was then

116 cooled in an ice bath. For preparation of HA-VRS-SLNs, 1 mL of VRS-SLNs dispersion was
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117 slowly added under gentle stirring to a HA solution. The various concentrations of HA were

118 investigated in order to obtain the optimum formulation.

119 Trehalose (5%, w/v) was used as a cryoprotectant in the freeze-drying process. The

120 resulting nanoparticle dispersions were poured into glass bottles and pre-frozen at −80°C for

121 24 h; later, the samples were freeze-dried using a lyophilizer (FDA5518, IlShin, South Korea)

Page 5 of 34
122 at a temperature of −25°C for 24 h. The lyophilized powders were collected for further

123 experiments.

124

125 2.3. Characterization of HA-VRS-SLNs

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126 2.3.1. Measurement of particle size and zeta potential

127 Particle sizes of VRS-SLNs and HA-VRS-SLNs were analyzed by the dynamic light

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128 scattering (DLS) technique using a Zetasizer Nano–Z (Malvern Instruments, Worcestershire,

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129 UK) at a fixed scattering angle of 90º and a temperature of 25 ºC. Prior to the measurement,

130 colloidal dispersions were adequately diluted with distilled water. DLS results are presented

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131 as z-average diameter, polydispersity index (PDI), and ζ-potential. The data were determined

132 using the Nano DTS software (version 6.34) provided by the manufacturers. All
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133 measurements were performed in triplicate.

134
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135 2.3.2. Morphological analysis

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136 The VRS-SLNs and HA-VRS-SLNs were deposited onto the surface of a copper grid (mesh

137 size of 300) coated with carbon. Using 2% phosphotungstic acid (w/v), negative staining was
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138 performed on the samples, followed by air-drying for 15 minutes. The stained grids were
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139 subsequently probed using transmission electron microscopy (TEM) (Hitachi H-7100, Japan)
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140 to visualize the morphology of nanoparticles.

141

142 2.3.3. Physical characterization

143 Differential scanning calorimetry (DSC) analysis were performed using a differential

144 scanning calorimeter DSC-Q200 (TA Instruments, New Castle, DE, USA) for lipid, drug and

145 optimized formulation. Briefly, 2-3 mg lyophilized sample was loaded in an aluminum pan

Page 6 of 34
146 and then heated over the temperature range of 40–200ºC with the heating rate of 10ºC/min

147 under nitrogen gas flow of 50 mL/min. An empty pan was used as a reference. The XRD

148 patterns of the above mentioned samples were obtained using an X-ray diffractometer (X’Pert

149 PRO MPD diffractometer, Almelo, The Netherlands) using Cu Kα radiation under voltage of

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150 40 kV and current of 30 mA with a scan step size of 0.02.

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151

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152 2.4. Drug entrapment efficiency and drug loading capacity

153 The drug entrapment efficiency (EE) and drug loading capacity (LC) of VRS-SLNs and HA-

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154 VRS-SLNs were determined by measuring the amount of free drug in the dispersion medium.

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155 Ultrafiltration was performed on the VRS-SLNs and HA-VRS-SLNs using centrifugal 10-

156 kDa molecular weight cut-off devices (Amicon Ultra, Millipore, USA). 2 mL of formulations
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157 was dispensed onto the top compartment of the devices, followed by centrifuging at 5000

158 rpm for 10 min. The filtrate was collected and diluted with acetonitrile at a ratio of 1:1. The
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159 amount of free VRS in the aqueous phase was determined using high-performance liquid
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160 chromatography (HPLC). Formic acid (0.1%)/acetonitrile (70/30) was used as a mobile phase

161 at a flow rate of 1.0 mL/min. VRS was detected at 241 nm. The drug entrapment efficiency
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162 and drug loading capacity were calculated using the following equations (Tran et al., 2014a).
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163 EE(%) = (Winitial drug− Wunbound drug)/Winitial drug× 100

164 LC(%) = (Winitial drug− Wunbound drug)/Wlipid × 100

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165 where EE is the entrapment efficiency; LC is the drug loading capacity; W is the weight (in

166 mg)

167

168 2.5. In vitro release studies

169 The in vitro release behavior of VRS-SLNs and HA-VRS-SLNs was determined in

170 dissolution medium composed of phosphate buffered saline (pH 7.4) using a dialysis method
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171 (Luan et al., 2014; Tran et al., 2014b). 1 mL of formulation was applied in the dialysis bag

172 with a 3500 Da cut-off (Spectra/Por®, CA, USA). The dialysis bag was soaked in a Falcon

173 tube containing 40 mL of dissolution medium. The tube was capped and placed on a shaking

174 water bath (HST – 205 SW, Hanbaek ST Co., Korea) at 37°C with a shaking speed of 100

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175 rpm. The VRS released into the medium at each time point was taken and determined using

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176 the HPLC system described above.

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177

178

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2.6. In vitro cytotoxicity studies and cellular uptake

179 2.6.1. Flow cytometric analysis

Cells (2×105 cells/mL) were seeded in each well of a 6-well plate and incubated for 24h using

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180

181 RPMI 1640 as media. 200μL of RPMI 1640 (with or without HA at concentration 1mg/mL)
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182 was added for 2h, and the medium was then discarded and the cells were washed twice with

183 phosphate buffered saline (PBS, pH 7.4). The cells were then treated with NBD-PC-loaded
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184 HA-SLNs at a concentration of 1μg/mL in a humidified incubator with 5% CO2 atmosphere

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185 at 37°C. After incubation for 30 min, the medium was removed and washed twice with 2 mL

186 of PBS. Cells were then harvested using 0.25% (w/v) trypsin solution then dispersed in
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187 1.5mL of PBS for flow cytometric measurements using a FACS Calibur flow cytometer (BD
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188 Biosciences, San Jose, CA, USA).

189
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190 2.6.2. Qualitative cellular uptake study by fluorescent microscopy

191 Cellular uptake of VRS-SLNs and HA-VRS-SLNs was evaluated by confocal microscopy in

192 SCC-7 and MCF-7 cells. In detail, 2x105 cells per well were cultured in 12-well plates

193 containing an antiseptic glass coverslip at the bottom of each well and incubated to grow

194 overnight. Cells were continuously treated with 1 µg/mL NBD-PC loaded VRS-SLNs and

195 HA-VRS-SLNs for 30 min, followed by continuous treatment with Lyso-Tracker Red and
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196 incubation for 10 min. Cells were then fixed with 4% paraformaldehyde for 15 min after

197 washing three times with PBS. The glass coverslip with the cells was then taken out of the

198 well and placed on a regular glass slide in the presence of mounting agent. Confocal analysis

199 was performed on a Leica TCS SP2 Confocal Microscope (Leica Co., Wetzlar, Germany).

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200

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201 2.6.3. In vitro cytotoxicity studies

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202 Cell-killing activity of VRS-SLNs and HA-VRS-SLNs was measured using the MTT assay

203 with some adjustments (Tran et al., 2014b). 100 µL of cell suspension with 5x103 cells per

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204 mL were cultured into wells of 96-well test plates and incubated for 24 h at 37ºC in 5% CO2.

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205 A concentration range of blank SLNs, blank HA-SLNs, free VRS, VRS-SLNs, and HA-VRS-

206 SLNs were added to each well-plate, followed by incubation for 24 hours. The medium with
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207 sample was removed and then washed twice with phosphate-buffered saline. 100 µL MTT

208 solution (1.25 mg/mL MTT in supplemented RPMI medium) was added to the cultures,
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209 followed by incubation for 4 h at 37ºC. The obtained formazan crystals were dissolved in 100
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210 µL DMSO. Subsequently, an incubation of 15 min under light protection and at room

211 temperature was performed. The absorbance was measured at 570 nm using a microplate
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212 reader (Multiskan EX, Thermo Scientific, Waltham, MA, USA). Cell viability was calculated
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213 using the following formula:

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214

215 The half-maximal inhibitory concentration (IC50) of each group was calculated using

216 GraphPad Prism 5 software.

217

218 2.7. Pharmacokinetics study

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219 2.7.1. Intravenous administration of VRS formulations to rats

220 Male Sprague-Dawley rats weighing 250 ± 10 g were divided into three groups of four rats.

221 The animals were quarantined in an animal house maintained at 25 ± 2°C and 50-60% RH,

222 and fasted for 12 h prior to the experiments. The protocols for the animal studies were

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223 approved by the Institutional Animal Ethical Committee, Yeungnam University, South

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224 Korea.

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225 Three groups of rats received free VRS, VRS-SLNs, and HA-VRS-SLNs by

226 intravenous administration (Tran et al., 2014b). For IV injection of free VRS, VRS was

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227 dissolved in PEG 400 and administered at a dose of 10 mg/kg. In case of HA-VRS-SLNs, the

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228 lyophilized powder was dissolved in physiological saline (0.9 % NaCl) at VRS concentration

229 of 3 mg/mL, accordingly. The pH of solution is 6.4 before injection. Blood samples (300 µL)
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230 were collected from the right femoral artery at pre-determined times (0.25, 0.5, 1, 2, 3, 4, 6,

231 8, 12 and 24 h) after administration of these formulations. The samples were collected in
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232 heparin-containing tubes (100 IU/mL) and then immediately centrifuged (Eppendorf,
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233 Hauppauge, NY, USA) at 14,000 rpm for 10 min. The plasma supernatant was collected and

234 stored at -20 ºC until further analysis.

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235
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236 2.7.2. Plasma sample processing

237 For extraction of VRS and for precipitation of unwanted protein, 150 μL of plasma was
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238 mixed with 150 μL of acetonitrile for 15 min. The samples were then centrifuged at 13,000

239 rpm for 10 min and 20 µL of the supernatant was injected into the HPLC system for VRS

240 analysis, as described above.

241

242 2.7.3. Analysis of pharmacokinetic data

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243 The pharmacokinetic profiles of free VRS, VRS-SLNs, and HA-VRS-SLNs were calculated

244 using the Win-NonLin pharmacokinetic software (v4.0, Pharsight Software, Mountain View,

245 CA, USA). The pharmacokinetic data measured consisting of the area under curves of the

246 plasma drug concentration–time from time zero to infinity (AUC0–∞), the half-life of

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247 elimination (t1/2), and the mean residence time (MRT) were obtained by summation of the

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248 central and tissue compartments. Analysis of variance (ANOVA) was performed to

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249 investigate differences between the experimental treatments. A p-value < 0.05 was

250 considered statistically significant in all cases, and all data were expressed as mean ±

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251 standard deviation.

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252

253
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254 3. Results and discussion

255 3.1. Preparation of HA-VRS-SLNs

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256 HA-VRS-SLNs were developed using a cationic solid lipid core and then shedding by
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257 anionic polymer, HA (Fig. 1), in order to selectively and preferentially interact with tumor

258 cell surface for effective cancer treatment. As shown in Fig. 1, the core of cationic SLNs was
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259 prepared using Compritol as a solid lipid element. Lecithin, DDAB, and Tween 80 were used
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260 as helper lipid, cationic lipid, and surfactant, respectively. Fig. 2A shows that SLNs were

261 fabricated in particles with average sizes in the range of 50 and 150 nm, and polydispersity
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262 index (PDI) of 0.2 and 0.6. Cationic lipid concentration had significant effects on the system,

263 especially PDI and surface charge. Zeta potential measurements determined the surface

264 charge of all particles in aqueous medium in the range between -20.6 and +29.3 mV. In the

265 presence of 8% DDAB as a cationic lipid, VRS was incorporated into SLNs in order to find

266 the optimal formulation. VRS-loaded SLNs were maintained in terms of size, PDI, and

267 surface charge (Fig. 2B). The resulting cationic SLNs were covered with HA via electrostatic
11

Page 11 of 34
268 interaction. The outer layer of HA dramatically increased particle size and converted the

269 surface charge to a negative charge. When the HA concentration increased, the surface

270 charge also increased, but beyond 1 mg/mL the charge remained constant (Fig. 2C). The drug

271 entrapment efficiency and loading capacity demonstrated compatible encapsulation of

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272 hydrophobic compound into the lipid core. Upon increase of drug amount, the drug loading

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273 capacity was augmented whereas the drug entrapment efficiency was reduced (Fig. 2D). This

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274 phenomenon is consisted with previous reports (Martins, Sarmento, Nunes, Lúcio, Reis &

275 Ferreira, 2013; Raza, Singh, Singal, Wadhwa & Katare, 2013).

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276 Finally, the formulation, which is composed of 40 mg DDAB as a cationic lipid, 460 mg

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277 Compritol, 100 mg Lecithin, 500 mg Tween 80, 15 mg VRS and then coating by 1 mg/mL

278 HA solution was selected for further studies with small size (102.3 ± 1.6 nm), narrow
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279 distribution (0.293 ± 0.057), negative charge (-9.0 ± 0.7 mV) and high drug loading (1.86 ±

280 0.01%). The particles were stored and checked over at least three months, indicating long
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281 term physical stability (data not shown).

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282

283 3.2. Physical characterization

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284 TEM photomicrographs of the optimized VRS-SLNs and HA-VRS-SLNs are shown in Fig.
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285 3A and 3B. Morphology of both formulations is spherical in shape and well supported by

286 DLS characterization results. Nanoparticles appeared in the range of 60 nm and 110 nm,
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287 respectively. In particular, the structure of HA-VRS-SLNs was clearly observed with a black

288 core and transparent shell indicating the HA layer.

289 DSC and X-ray diffraction were performed for VRS, lipid, HA, VRS-SLNs and HA-

290 VRS-SLNs for characterization of the drug–lipid interactions and the crystallinity of drug

291 before and after formulation in lipid matrices. As shown in Fig. 3C, DSC thermogram of bulk

292 compritol, DDAB showed sharp melting peaks at 73°C and 88°C, respectively, whereas VRS
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Page 12 of 34
293 exhibited a melting peak at 163°C, corresponding to their natural crystal forms. In contrast,

294 VRS-SLNs and HA-VRS-SLNs did not show the endothermic peak for the VRS around

295 163°C. This finding indicates that VRS was in amorphous state or incorporated inside the

296 lipid core (Ramasamy et al., 2014). In addition, sharp peaks were observed for VRS at 2θ-

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297 scattered angles of 16.3, 17.2, 19.2, 19.8, 22.2, and 23.7, corresponding to the crystalline

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298 nature of the drug, whereas the crystalline state of Compritol was still presented at 2θ-

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299 scattered angles of 21.3 and 23.5 (Fig. 3D). The intensity of VRS and Compritol peaks was

300 decreased in VRS-SLNs and HA-VRS-SLNs. Less ordered crystal lattices of lipid suggested

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301 the successful drug-lipid interaction. In addition, less ordered lipids prevent expulsion of the

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302 drug from the particles. Thus, Compritol provides more space for accommodation of drug

303 molecules and limits drug expulsion (Peer, Karp, Hong, Farokhzad, Margalit & Langer,
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304 2007).

305
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306 3.3. In vitro drug release

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307 In vitro release profiles of VRS from VRS-SLNs and HA-VRS-SLNs formulation were

308 evaluated in pH 7.4 (Fig. 4). The release follows a biphasic profile, characterized by a rapid
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309 initial burst (releasing 40% of the drug in 10 h), and followed by a second stage in which few
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310 changes were recorded. This two-phase release behavior has been reported for solid lipid

311 nanoparticles (Nabi-Meibodi et al., 2013; Sun, Bi, Chan, Sun, Zhang & Zheng, 2013). The
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312 attached drug in the exterior shell and on the particle surface is related to burst release, while

313 the drug that has been encapsulated into the lipid core is released in a sustained manner. This

314 phenomenon was consistent with supposition of drug loading and physical characteristics. On

315 the other hand, HA-VRS-SLNs exhibited a slightly lower release pattern in whole time

316 points. HA layer plays a role as a barrier that could restrict the dispersion of drug to media.

317
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318 3.4. Intracellular uptake study

319 Three cell lines present different expression of CD44 receptors (Ghosh, Neslihan Alpay &

320 Klostergaard, 2012; Qhattal & Liu, 2011). To evaluate the intracellular uptake of

321 formulations and the role of CD44 expression on cell surface, the intensity of incorporated

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322 dye was visualized and quantified using fluorescence microscopy and flow cytometry (NBD-

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323 PC labeled SLNs and HA-SLNs), respectively. Flow cytometry analysis was used to study

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324 the cellular uptake efficacy of HA-SLNs with or without pretreated HA. HA-SLNs showed a

325 higher fluorescence shift compared to the controlled group and SLNs in SCC-7 cells, while a

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326 few right shifts were observed in the HA-SLNs after pretreatment with HA, indicating less

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327 cellular uptake into the SCC-7 cell line (Fig. 5A). This phenomenon clearly demonstrates the

328 role of HA-CD44 interaction in terms of enhancing cellular uptake. In contrast, degree of
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329 cellular internalization of SLNs into MCF-7 cells after incubation for 30 min was slightly

330 higher than that of HA-SLNs due to possible interaction between positive charge of SLN and
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331 negative charge of cell membrane (Fig. 5B). This result demonstrates the capacity of HA-
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332 SLNs to penetrate high overexpressed CD44 cells (SCC-7) through a process called receptor-

333 mediated endocytosis; meanwhile, there is no significant difference for that system into low
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334 overexpressed CD44 cells (MCF-7) (Qhattal & Liu, 2011).

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335 The internalization of SLNs and HA-SLNs was further visualized by confocal laser

336 scanning microscope (CLSM). As shown in Fig. 6, similar results were obtained and agreed
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337 with flow cytometry data. In SCC-7 cells, the fluorescence intensity of HA-SLNs was higher

338 than that of SLNs in SCC-7 cells, however, it was reduced by pretreatment with HA, which

339 indicated that surface decoration with HA enhanced cellular uptake of SLNs based on CD44-

340 mediated internalization. In addition, cellular uptake of HA-SLNs in MCF-7 cells was

341 slightly lower than that of SLNs, suggesting that an affinity for CD44 may influence the

342 efficient delivery of payloads.

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343

344 3.5. In vitro cytotoxicity

345 The efficacy of the formulations against cancer cells is indicated by their cytotoxicity. Fig. 7

346 shows cytotoxicity results for cancer cells incubated with five different formulations,

t
347 including blank HA-SLNs, blank SLNs, free VRS, VRS-SLNs, and HA-VRS-SLNs at 0.5,

ip
348 1.0, 2.5, 5.0, 12.5, 25.0, and 50.0 μg/ml drug concentrations. Blank HA-SLNs did not show

cr
349 any appreciable cytotoxicity throughout the entire range of concentrations and the cell

350 viability remained at more than 80% in all cell lines following 24 hour exposure,

us
351 demonstrating its biocompatible nature and tolerance (Petersen, Steiniger, Fischer, Fahr &

an
352 Bunjes, 2011; Tran et al., 2014a). However, blank SLNs showed a slight cytotoxicity

353 compared with blank HA-SLNs in all cell lines, which could be attributed by cationic DDAB
M
354 on the surface of SLNs, leading to cell membrane destruction (Yang, Li, Li, Zhang, Feng &

355 Zhang, 2013). In the case of blank HA-SLNs, the outer surface of the lipid was coated with
d

356 HA so that HA can protect against disruption of cellular membranes, thereby reducing
te

357 cytotoxicity versus the plain SLN itself. In addition, Fig. 7 showed that the cell-killing effects

358 of VRS formulations were concentration-dependent. In all cell lines, in vitro antitumor
p

359 activity of HA-VRS-SLNs was significantly higher than that of free VRS. The enhanced
ce

360 cytotoxic effect of HA-VRS-SLNs compared to free VRS may reflect the lipophilic nature of

361 the carrier and enhanced intracellular uptake, by a rapid CD44 receptor-mediated endocytosis
Ac

362 process (Ramasamy et al., 2013; Tran et al., 2014b). In particular, higher toxicity of HA-

363 VRS-SLNs compared with VRS-SLNs was observed in A549 and SCC-7 cells with a high

364 level of CD44 expression, while HA-VRS-SLNs did not show a better effect than VRS-SLNs

365 MCF-7 cells with low CD44 levels. The good achievement of HA-VRS-SLNs could also be

366 supposed by charge interaction between positive charge of SLNs and negative surface cell

367 membrane after degradation of HA by HAase (Jiang et al., 2012). This result is consistent
15

Page 15 of 34
368 with in vitro cellular uptake studies by flow cytometric analyses and CLSM. Hence,

369 development of the formulation could improve the therapeutic effect of VRS (Qhattal & Liu,

370 2011; Wei, Senanayake, Warren & Vinogradov, 2013).

371

t
372 3.6. Pharmacokinetic study

ip
373 Fig. 8 shows the plasma concentration-time curves of VRS after intravenous administration

cr
374 of free drug suspension, VRS-SLNs and HA-VRS-SLNs. The mean plasma concentrations of

375 VRS after IV injection of HA-VRS-SLNs were higher than those after injection of free VRS

us
376 and VRS-SLNs. Owing to its hydrophobic property, free VRS was rapidly cleared from the

an
377 blood stream. Half of VRS was eliminated after 0.65 ± 0.07 hour, leading to mean residence

378 time of only 0.77 ± 0.12 hour, whereas t1/2 and MRT of VRS-SLNs were 2.02 ± 0.52 and 3.20
M
379 ± 0.39 hour, respectively (Table 1). However, among them, HA-VRS-SLNs showed the best

380 performance with higher plasma concentration, longer half-life and MRT (4.55 ± 0.50, 6.26 ±
d

381 0.96 hour). With the decrease in in vivo clearance, AUC0-∞ values of HA-VRS-SLNs (58.74 ±
te

382 6.19 μg.h/mL) were increased significantly (P<0.05), compared with those of free VRS

383 (16.62 ± 1.80 μg.h/mL) and VRS-SLNs (29.81 ± 3.84 μg.h/mL). As shown Fig. 8, VRS-
p

384 SLNs improved drug state in vivo, but it faced clearance by the reticuloendothelial system
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385 (RES) due to a positive surface (Dobrovolskaia, Aggarwal, Hall & McNeil, 2008). Coating

386 particles with long circulating agents such as PEG or HA are proposed as a solution to steric
Ac

387 stabilization of vesicles and provide protection from opsonization, which is an essential

388 desirable property in drug delivery (Peer, Karp, Hong, Farokhzad, Margalit & Langer, 2007).

389 These results clearly indicated that the HA-coated VRS-SLNs would be successful in causing

390 a reduction in dose and improvement in efficacy of VRS.

391

392
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393 4. Conclusions

394 In this study, hyaluronic acid was successfully attached onto the surface of cationic SLNs by

395 electrostatic attraction. The resultant particles were spherical in shape, uniform in size with a

396 visually clear outer shell. HA-VRS-SLNs exhibited favorable characteristics that suggested

t
397 their capability for efficient drug delivery and targeting. First, VRS was released slowly in

ip
398 order to maintain drug concentration in control, leading to reduced toxicity on normal cells.

cr
399 The HA-VRS-SLNs penetrated rapidly into cancer cells, especially on high level CD44 over-

400 expression cells. In addition, the better performance of HA-VRS-SLNs clarified the role of

us
401 HA in selective targeting to tumor cells. As expected, the HA-VRS-SLNs showed more

an
402 potency in inhibiting the growth of all cancer cells: A549, SCC7, and MCF-7, compared to

403 that for the VRS and VRS-SLNs. Finally, due to a hydrophilic and negative charge surface,
M
404 HA-VRS-SLNs stayed longer in blood circulation and prolonged therapeutic drug

405 concentration, which gave the drug a greater chance of reaching the tumor area. Taken
d

406 together, our data indicated that HA-VRS-SLNs could be successfully developed as a
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407 targetable drug delivery system with high bioavailability.

408
p

409
ce

410 Acknowledgements

411 This research was supported by the National Research Foundation of Korea (NRF) grant
Ac

412 funded by the Ministry of Education, Science and Technology (No. 2012R1A2A2A02044997

413 and No. 2012R1A1A1039059).

414

415

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415

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420 pharmaceutics, 10(1), 225-235.

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496 Kim, J. O. (2014a). Formulation and Optimization of Raloxifene-Loaded Solid Lipid

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502 Venishetty, V. K., Komuravelli, R., Kuncha, M., Sistla, R., & Diwan, P. V. (2013). Increased

503 brain uptake of docetaxel and ketoconazole loaded folate-grafted solid lipid nanoparticles.

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508 Yang, X.-y., Li, Y.-x., Li, M., Zhang, L., Feng, L.-x., & Zhang, N. (2013). Hyaluronic acid-

509 coated nanostructured lipid carriers for targeting paclitaxel to cancer. Cancer letters, 334(2),

510 338-345.

511
512

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513

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513

514 Figure legends

515 Fig. 1 Diagram illustrating the preparation process for HA-VRS-SLNs.

516 Fig. 2 Effect of (A) Cationic lipid concentration, (B) Drug concentration, (C) HA

t
ip
517 concentration on formulation parameters: particle size, polydispersity index (PDI), zeta

518 potential (ZP). Effect of (D) drug concentration and (E) HA coating on drug entrapment

cr
519 efficiency and loading capacity. Data are expressed as the mean ± S.D. (n = 3).

us
520 Fig. 3 TEM images of (A) VRS-SLNs and (B) HA-VRS-SLNs. (C) Differential scanning

521 calorimetric thermograms and (D) X-ray diffraction patterns of DDAB, Compritol, free VRS,

an
522 VRS-SLNs, and HA-VRS-SLNs.

523 Fig. 4 In vitro drug release of VRS from VRS-SLNs (○) and HA-VRS-SLNs (♦) in PBS
M
524 buffer (pH 7.4, 0.14 M NaCl) at 37°C. Data are expressed as mean ± S.D. (n = 3).

525 Fig.5 Quantitative uptake of analysis by flow cytometry of SLNs, HA-SLNs and HA-SLNs
d

526 pretreated with HA on (A) SCC-7 cells, and (B) MCF-7 cells.
te

527 Fig. 6 Intracellular uptake of SLNs and HA-SLNs by confocal laser scan microscope images
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528 in (A) SCC-7 cells and (B) MCF-7 cells. SLNs and HA-SLNs containing NBD-PC (green)
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529 and Lysotracker Red (red) staining lysosome were used for this experiment.

530 Fig. 7 In vitro cytotoxicity of blank SLNs, blank HA-SLNs, free VRS, VRS-SLNs and HA-
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531 VRS-SLNs after 24 h exposure in (A) A549, (B) SCC-7, and (C) MCF-7 cells. Data are

532 expressed as the mean ± S.D. (n = 8).

533 Fig. 8 Plasma concentration-time profile of VRS after intravenous administration at a dose of

534 10 mg/kg of free VRS (◊), VRS-SLNs (■) and HA-VRS-SLNs (●). Each value represents the

535 mean ± S.D. (n = 4).

536

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536

537

538 Table 1 Pharmacokinetic parameters of VRS in rats after intravenous administration of free

539 VRS, VRS-SLNs, and HA-VRS-SLNs at a dose of 10mg/kg.

t
ip
Parameter Free VRS VRS-SLNs HA-VRS-SLNs

Cmax (µg/mL) 11.17 ± 0.83 11.49 ± 0.11 11.67 ± 0.53

cr
t1/2 (h) 0.65 ± 0.07 2.02 ± 0.52a 4.55 ± 0.50a,b

AUC0-∞ (µg.h/mL) 16.62 ± 1.80 29.81 ± 3.84a 58.74 ± 6.19 a,b

us
MRT (h) 0.77 ± 0.12 3.20 ± 0.39a 6.26 ± 0.96 a,b
540 Data are expressed as the mean ± standard deviation (n=4).

541

542
a, p<0.05, compared to the free drug

b, p<0.05, compared to VRS-SLNs an

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543

544
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545
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545 Highlights

546 • Hyaluronic acid-coated solid lipid nanoparticles for tumor-targeted delivery of

547 vorinostat (VRS-SLN) were formulated and characterized.

548 • Vorinostat loaded HA-SLNs showed high drug loading capacity and a pattern of

t
549 sustained release.

ip
550 • Selectivity of decorated nanoparticles is promising to ensure effective chemotherapy

cr
551 on the tumor.

us
552 • The obtained nanoparticles maintained drug concentration for a long period of time in

553 blood circulation and improved bioavailability.

an
554

555
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Figure(s)

Fig. 1

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Fig. 2

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Fig. 3

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(C)

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Fig. 4

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Fig. 5

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 HA-SLN
HA-SLN SLN
Fig. 6A

(Pretreated with HA)

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NBD-PC

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LysoTracker Red

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Fig. 6B

NBD-PC LysoTracker Red Merged

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HA-SLN

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Fig. 7

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Fig. 8

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