Sorafenib and 5-Fluorouracil Loaded Dual Drug Nanodelivery Systems for Hepatocellular Carcinoma and Colorectal Adenocarcinoma


 Purpose: Here, we reported the sysnthesis of two clinically used drugs, 5-fluorouracil (5FU) and Sorafenib (SF)-loaded in chitosan nanoparticles and their priliminary study of therapeutics effect on hepatocellular carcinoma and colorectal adenocarcinoma cell lines. We have formulated chitosan nanoparticles (CS NPs) loaded dual (SF and 5-FU) drugs nanodelivery system for SF/5FU-CS NPs and their coating version with folic acid (FA) for SF/5FU-CS-FA NPS. Human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (HT29) cell lines were selected for in vitro cytotoxicity studies to evaluate the preliminary anticancer efficacy of both nanoparticles.Characterization: The physiochemical characterization of SF/5FU-CS NPs and SF/5FU-CS-FA NPs were investigated by DLS, FESEM, HRTEM, EDX, XRD, TGA, FTIR, and HPLC methods.Results: DLS study has shown the size of SF/5FU-CS and SF/5FU-CS-FA nanoparticles were about 78±14 nm and 142±25 nm, respectively. HRTEM and FESEM studies confirmed the spherical shape with size of 60-70nm for SF/5FU-CS and 90-150 nm for SF/5FU-CS-FA NPs. The XRD results indicated the drug loading and folate-coating comfirmation. FTIR peaks confirmed the presence of drugs in the nanoparticles, as well as folate-coating on the surface of the nanoparticles. TGA results demonstrated the thermostability of both nanoparticles. The release profiles of SF and 5FU from the two designed NPs were found to be in a sustained manner according to the pseudo-second-order kinetics model indicating a good delivery system for tumor cells. The cytotoxicity studies confirmed the better anti-cancer activity of the nanoparticles compared to the free 5-fluorouracil and sorafenib against liver cancer cells, HepG2 and colon cancer cells, HT29. Conversely, both NPs were found not toxic towards normal human dermal fibroblast cells (HDF) cells.

6 therapeutic system for HepG2 cells. 28 FA conjugated polyethylene glycol-based polymer NPs have also 160 shown a good load of 5-FU drug with effective HT29 cell inhibition. 29 A novel active targeted nanoparticle-161 based oral drug delivery system comprised of chitosan and gelatin integrated with 5FU has been developed. 162 The nanoparticle has exhibited a superior anti-cancer activity as well as pro-apoptotic efficacy compared 163 to free drugs and nanoparticles alone. 30 Chitosan-based 5-fluorouracil nanoparticles of around 169.2 nm to 164 259.8 nm in size and the tumor inhibition rates of high, medium, low concentration group of drug-loaded 165 nanoparticles was found to be 62.05%, 48.79% and 36.14%, respectively. 31 166 In this study, we simultaneously incorporated Sorafenib and 5-fluorouracil drugs using chitosan 167 nanoparticles as the host to form chitosan sorafenib/5-fluorouracil drug nanoparticles (SF/5FU-CS NPs) 168 and their conjugation with FA, folate-chitosan conjugated sorafenib/5-fluorouracil drug nanoparticles 169 (SF/5FU-CS-FA NPs). Particle size, shape, structure, and drug release kinetic from the nanoparticles were 170 characterized. The human liver cancer cells (HepG2) and human colon cancer (HT29) cells were used to 171 study the anticancer effect of these nanoparticles. Besides, FA was used on colon/liver cancer cells targeted 172 drug delivery of SF and 5-FU. Furthermore, the cytotoxic study of these NPs was investigated in the normal 173 healthy cell to test the toxic level in normal human dermal fibroblast cells (HDFa) cells.

Preparation of Sorafenib/5-Fluorouracil-loaded Chitosan nanoparticles 182
The ionic gelation method has been applied to prepare SF/5FU-CS NPs. LMW chitosan powder (0.5 183 mg/mL) of was totally dissolved in 1.0 % (v/v) acetic acid solution. 1M of 0.15 g of SF and 1M of 0.15g 184 of 5-FU drugs were dissolved in DMSO. The drug solutions and 2% (v/v) of TWEEN-80 as a surfactant 185 were added to the dissolved chitosan solution and the pH was fixed at 5 by NaOH (1M) aqueous solution 186 maintaining vigorous stirring. 7 mg/mL sodium tripolyphosphate (TPP) solution was prepared in dH 2 O 187 separately and fixed at pH 2 using HCl (1M). The TPP solution was slowly added dropwise using a burette 188 into the chitosan-drugs solution. The resulting suspension was subsequently centrifuged at 4000 rpm for 10 7 min, followed by washed 2 times with dH 2 O. Finally, the sample was freeze-dried for further analysis and 190 cytotoxicity study. Figure 1 showed the schematic diagram of preparation steps of SF/5FU-CS NPs 191

Preparation of Folate-conjugated Sorafenib/ 5-Fluorouracil loaded Chitosan nanoparticles 192
SF/5FU-CS-FA NPs were also prepared by ionic gelation method by dissolving 0.5 mg/mL of CS powder 193 in 1.0 % (v/v) acetic acid solution. 0.15g of SF and 0.15g of 5-FU were dissolved in DMSO and added into 194 the chitosan solution followed by adding 2% v/v of TWEEN-80 and fixing to pH 5 by NaOH aqueous 195 solution. 7 mg/mL TPP solution of pH 2 was prepared in dH 2 O water and added dropwise into chitosan-196 drugs solution with vigorous stirring. After that, a solution of 1g FA and 0.25g of EDC (1-ethyl-3-197 (dimethylamino propyl) were prepared and added to chitosan drug solution and stirred for 48 hours at room 198 temperature in the dark to let folic acid conjugate onto chitosan molecules. The resulting suspension was 199 subsequently centrifuged and washed with dH 2 O then freeze-dried for further analysis and cytotoxicity 200 study. Figure 1 showed the schematic diagram of preparation steps of SF/5FU-CS-FA NPs.

Instrumentation/Characterization 209
The information of particle size distribution and polydispersity index (PDI) of SF/5FU-CS and SF/5FU-210 CS-FA NPs were achieved by a high-performance dynamic light scattering (DLS) nanosizer (Malvern, 211 UK). A Lambda 35 ultraviolet-visible spectrophotometer (Perkin Elmer) apparatus was used to determine 212 the drug release of the samples. The structures, different crystallinity patterns of both drug SF and 5FU, 213 CS-NPs, FA, SF/5-FU-CS, SF/5-FU-CS-FA nanoparticles were investigated through an X-ray diffraction 214 machine from SHIMADZU XRD 6000, JAPAN in the range of 2-40° using λ =1.5406 Å radiation of CuKα 215 at 40 kV and 30 mA. The morphological analysis of particle's shape, size distribution, particle size in nano 216 dimensions was determined by a high-resolution transmission electron microscope from HITACHI H-7100, 217 TOKYO, at 100 kV voltage. A drop of sonicated SF/5FU-CS, SF/5FU-CS-FA nanoparticle's solution was 218 placed on a carbon film 300 mesh copper grid and air-dried before observation by HRTEM. A field emission 219 scanning electron microscope (FESEM) and Energy Dispersive X-Ray Spectroscopy (EDX) from NOVA 220 NANOSEM 230, CALIFORNIA, USA was used to investigate the surface, shape, morphology, and 221 elemental composition of atomic and weight percentage of existing elements of SF/5FU-CS and SF/5FU-222 CS-FA NPs. The drop of nanoparticles was put separately on top of a stub and oven-dried before analyzed. 223 The thermal decomposition of both drug SF and 5FU, CS-NPs, FA, SF/5-FU-CS, SF/5-FU-CS-FA 224 nanoparticles was determined by the thermogravimetric and differential thermogravimetric (TGA/DTG) 225 analyses using a Mettler-Toledo Instrument from GREIFENSEE, SWITZERLAND. This is to determine 226 the mass reduction and thermal behavior of the nanoparticles, the temperature was used in the range of 25-227 1000 °C at a heating rate of 10 °C min −1 . The Fourier transform infrared spectroscopy (FTIR) analysis of 228 CS NPs, SF, 5-FU, SF/5FU-CS, SF/5FU-CS-FA nanoparticles, and raw chitosan samples was recorded 229 using a PerkinElmer FTIR spectrometer (SPECTRUM 1000). 6.0-8.0 mg of the samples were subjected to 230 the infrared spectrum with a resolution of 4 cm −1 at 500 -4000 cm −1 wavenumbers. 231 232

Encapsulation Efficiency and Loading Capacity 233
The encapsulation efficiency and loading capacity measurement of SF and 5-FU drugs was performed by 234 the high-performance liquid chromatography (HPLC) using Alliance e2695, USA. The absorbance was 235 measured using the HPLC at wavelengths of 265 nm for SF and 264 nm for 5-FU. In brief, 10 mg of 236 SF/5FU-CS and SF/5FU-CS-FA NPs powder samples were put separately in 10 mL buffer solution and 237 shook for around 3 hours using an orbital shaker and centrifuged at 40,000 rpm. 2 mL of supernatant was 238 used for the absorbance measurement at 265 nm for SF and 264 nm for 5-FU against blank. The mobile 239 phase of the HPLC was methanol/water with a flow rate of 1.0 mL min −1 at 25 °C. The encapsulation 240 efficiency (EE%) and loading content (LC%) of SF/5FU-CS, SF/5FU-CS-FA nanoparticles were calculated 241 using the following formula.

In Vitro Drug Release 247
The release percentage was observed over 6 days. 10 mg of each separate sample of SF/5FU-CS, SF/5FU-248 CS-FA NPs were suspended in 10 mL of pH 7.4 and 4.8 PBS buffer solutions with continuous shaking 249 using an orbital shaker at 37 ⁰C with 400 rpm. From 1 until 12 hours, and 2, 3, 5, 6 days incubation, 3 mL 250 of the sample was collected from each tube to analyze the drug concentration using the HPLC. The 251 withdrawn solution was replaced with fresh PBS solution. 252

Encapsulation Efficiency and Loading Content 269
The drug encapsulation and loading into a nanoparticle depend on the nature of the drug molecule and the 270 carrier materials, as well as drug and carrier's concentration. The drug loading for SF and 5-FU loaded in 271 the CS nanoparticles was determined by the HPLC technique. In this study, we found that the ratio of 272 chitosan should be higher than the drugs so the interactions between chitosan and drugs increase and thus 273 result in a higher concentration of drug loading. For folate-coated SF/5FU-CS-FA nanoparticles, the ratio 274 of folic acid should be higher than chitosan/drug ratio in order to reach the optimum drug loading. Loading 275 content and encapsulation efficiency of SF, as well as 5-FU, is slightly higher in SF/5FU-CS-FA than 276 SF/5FU-CS nanoparticles which does not significantly affect the % EE and %LC. Table 1

Particle Size Analysis 285
Dynamic light scattering (DLS) is a non-imaging method to investigate the particle size distribution. The 286 nanoparticles size distribution, polydispersity index (PDI) was determined by a DLS nanosizer. Using the 287 DLS, the hydrodynamic particle size of SF/5FU-CS, SF/5FU-CS-FA NPs were found as peaks 78±14 nm 288 and 142±25 nm respectively with excellent dispersity index (PDI ~0.30) and monomodal narrow 289 distribution of particle size ( Figure-2A,2B). Henceforward, the DLS analysis confirmed the nanoscale level 290 of particles and the larger particle size is due to SF and 5-FU loading into the chitosan polymer chains. The

Morphological studies using the High-resolution Transmission Electron Microscopy 298
The morphology of the nanoparticles was revealed by performing image analysis through high-resolution 299 transmission electron microscopy (HRTEM) by observing the actual size of the nanoparticles, mean core 300 size of the particles, size distributions, shape, inner structure, interface image of particle's dispersion in the 301 solution and morphology of nanoparticles after coating. The particle images of both SF/5FU-CS and 302 SF/5FU-CS-FA nanoparticles were found to be spherical with a uniformly dispersed and smooth surface. 303 SF/5FU-CS nanoparticles have sizes in between 60-100 nm whereas folic acid-conjugated SF/5FU-CS-FA 304 NPs have a larger spherical shape in between 90-150 nm with uniformly dispersed. The larger particle size 305 of both NPs is responsible for two different drug incorporation. Due to folate conjugation, SF/5FU-CS-FA 306 NPs showed relatively higher in particle size. Apparently, no debris and no aggregation were observed from 307 the TEM image which indicates the good synthesis of the nanoparticles. Figure

Surface properties using the Field Emission Scanning Electron Microscopy 319
The field emission scanning electron microscopy (FESEM) was used for observing the external 320 morphology of surface properties of NPs as shown in Figure 4.

Qualitative Elemental Analysis using Energy Dispersive X-Ray 340
The compositional analysis of SF/5FU-CS and SF/5FU-CS-FA nanoparticles are necessary to confirm the 341 NPs synthesized in this work. Energy Dispersive X-Ray Analysis (EDX) in conjunction with FESEM study 342 was conducted to investigate the compositional elements that are present in the nanoparticles (Figure 4). 343 The EDX elemental components of weight and atomic percentages of all the samples are shown in Figure  344 4. Different areas were focused during the EDX measurement to get their corresponding elemental contents. 345 Table 2 represents the elemental composition of weight and atomic percentages of all the samples. The 346 weight % and atomic % of carbon and oxygen were found to be the highest in both NPs and the presence 347 of nitrogen, fluorine and chlorine is due to the presence of drugs in the molecular structure. The higher 348 percentages of nitrogen in SF/5FU-CS-FA nanoparticles is due to the FA coating. In weight % of carbon, 349 nitrogen, oxygen, florin, phosphorus, chlorine, and sulfur were present in the synthesized SF/5FU-CS and 350 SF/5FU-CS-FA nanoparticles. Phosphorus and sodium were found to be existed in large amounts in the 351 CS-nanoparticles due to the high sodium-tripolyphosphate crosslinking used in the synthesis process.

X-Ray Diffraction 358
The X-ray diffraction patterns of SF, 5-FU, FA, CS NPs, SF/5FU-CS NPs, SF/5FU-CS-FA NPs are shown 359 in Figure 5.  carboxylic acid, respectively. The free SF (Fig. 6-B)  In pH 7.4 PBS solution, 5FU took 30 hours for 70% release and the remaining release was sustained 466 compared to 30 hours for 85% release for SF. In pH 4.8, both the drugs took 30 hours for the initial release 467 of approximately 85% followed by the sustained release which lasted for 100 hours. At 4.8 pH, the drug 468 SF release was approximately 95%, 97% and 100% while for 5FU, the release was approximately 90%, 469 95% and 100% in the period of 60, 80 and 120 hours, respectively. However, at pH 7.4, the SF release was 470 approximately 90%, 95% and 100% while for 5FU release was approximately 82%, 89% and 100% in the 471 period of 60, 80 and 120 hours, respectively. The release profiles in Figure 8 indicates that the maximum 472 percentages of drug release in pH 4.8 solution are higher than that at pH 7.4. The resulted release showed 473 that both SF/5FU-CS and SF/5FU-CS-FA nanoparticles are more responsive at pH 4.8 than pH 7.4 which 474 made both NPs are suitable for slow delivery purposes. The drug transport or release from nanocarriers involves various physicochemical factors, which require 485 the mathematical models to explain. To develop an effective nanodrug delivery system, it is important to 486 determine their drug release profiles to identify the process of drug release from the system designed. An 487 ideal delivery system should follow the kinetics model to identify nanocarrier's drug release behavior. 488 Different kinetics models namely the pseudo-first-order, pseudo-second-order and parabolic diffusion were 489 applied to determine the kinetics release of the drugs. where M o and M t are the drugs that remained in the NPs at release time 0 and t, respectively. The above 506 three models were applied to determine the kinetics of drug release and it was found that the release of both 507 drugs, SF and 5FU follows the pseudo-second-order kinetics as evident by the plots shown in Figure 9-12. 508 The higher correlation coefficient values for the release kinetics of 5-FU and SF from SF/5FU-CS NPs 509 before Folic acid (FA) coating and SF/5FU-CS-FA NPs after Folic acid coating are shown in Tables 3 and  510 4. 511 512    The anticancer activity of the nanoparticles was studied by treating the HepG2 cell line (Figure- According to the cytotoxicity assay shown in Figure 15, the results suggest that both nanoparticles have 615 better anticancer activity compared to pristine drugs 5-FU and SF.  profile was favourable for nanoparticles treatment. Cytotoxicity studies revealed that the anticancer action 646 of synthesized nanoparticles was higher than the drugs at the same timepoint. Based on this study, it seems 647 that dual drug loaded chitosan nanoparticles and folate coated dual drug loaded chitosan NPs are preferable 648 to deliver the chemotherapy drug-sorafenib, and 5-flurouracil in the application of liver and colon cancer 649 treatment with minimal side effects and superior inhibitory effect on HepG2 and HT-29. 650

Future Aspects 651
The future aspects of study will be included by LDH assays, Caspase activity assays, cellular uptake, 652 western blots and in vivo for the SF/5FU-CS and SF/5FU-CS-FA nanoparticles.