Self-assembled reduced albumin and glycol chitosan nanoparticles for

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Self-assembled reduced albumin and glycol chitosan nanoparticles for paclitaxel delivery Muhamad Alif Razi, Rie Wakabayashi, Masahiro Goto, and Noriho Kamiya Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02809 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Self-assembled reduced albumin and glycol chitosan nanoparticles for paclitaxel delivery Muhamad Alif Razi1, Rie Wakabayashi1, Masahiro Goto1,2, and Noriho Kamiya1,2, * 1

Department of Applied Chemistry, Graduate School of Engineering, Kyushu University,

Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan 2 Division

of Biotechnology, Center for Future Chemistry, Kyushu University, Motooka 744,

Nishi-ku, Fukuoka 819-0395, Japan

ACS Paragon Plus Environment

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ABSTRACT

Cancer continues to pose health problems for people all over the world. Nanoparticles (NPs) have emerged as a promising platform for effective cancer chemotherapy. NPs formed by the assembly of proteins and chitosan (CH) through non-covalent interactions are attracting great interest. However, the poor water solubility of CH and low stability of this kind of NP limit their practical application. Herein, the formation of reduced bovine serum albumin (rBSA) and glycol chitosan (GC) nanoparticles (rBG-NPs) stabilized by hydrophobic interactions and disulfide bonds, was demonstrated for paclitaxel (PTX) delivery. The effects of a rBSA:GC mass ratio and pH on the particle size, polydispersity index (PDI), number of particles, and surface charge were evaluated. The formation mechanism and stability of the NPs were determined by compositional analysis and dynamic light scattering. Hydrophobic and electrostatic interactions were the driving forces for the formation of the rBG-NPs and the NPs were stable in physiological conditions. PTX was successfully encapsulated into rBG-NPs with high encapsulation efficiency (~90%). PTX-loaded rBG-NPs had a particle size of 400 nm with low PDI (0.2) and positive charge. rBG-NPs could be internalized by HeLa cells, possibly via endocytosis. An in vitro cytotoxicity study revealed that PTX-loaded rBG-NPs had lower anti-cancer activity compared with a Taxol-like formulation at 24 h, but had similar activity at 48 h, possibly owing to the slow release of PTX into the cells. Our study suggests that rBG-NPs could be used as a potential nanocarrier for hydrophobic drugs.

Keywords: Bovine serum albumin, glycol chitosan, complexation, nanoparticles, reduction, paclitaxel


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INTRODUCTION Cancer remains a leading cause of death across the globe, accounting for 13% of all deaths.1 The current treatments for cancer are surgery, radiation therapy, and chemotherapy, which involves the systemic administration of anti-cancer drugs to the body. However, most of the anti-cancer drugs used in chemotherapy are poorly soluble in water, which leads to low drug bioavailability.2 The conventional and commercial approach for solubilizing hydrophobic anticancer drugs is by using surfactants and organic solvents, as exemplified by the paclitaxel (PTX) injection formulation (Taxol) that uses considerable amounts of cremophor EL and dehydrated ethanol. However, the use of Taxol in cancer treatment has been associated with hypersensitivity reactions, hyperlipidemia, and peripheral neuropathy.3 In the recent years, nano-sized drug delivery systems (DDS) have gained tremendous attention as potentially safer and more effective cancer treatments owing to their ability to solubilize or encapsulate hydrophobic drugs, reduce non-specific toxicity, control the pharmacokinetics and pharmacodynamics of the drugs, and enhance drug penetration into tumors via the enhanced permeability and retention (EPR) effect.1,2,4 Various nano-size DDS have been explored, including polymeric-based nanoparticles (NPs), liposomes, nanoemulsions, and metallic and inorganic-based nanocarriers. 5–9 Organic-based nano-size DDS are often preferable to their inorganic counterparts because they are less toxic and exhibit better biocompatibility and biodegradability.10 Proteins and polysaccharides are attractive components for the development of NPs for cancer treatment owing to their excellent biocompatibility.11,12 NPs fabricated from proteins and polysaccharides have emerged as a promising NPs for improving the water solubility, stability, controlled release, and bioactivity of the drugs and/or therapeutic


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molecules.13–18 NPs can be formed by the facile complexation of proteins and polysaccharides in aqueous solution via non-covalent interactions, for example electrostatics.19 Albumin is an important abundant protein found in the blood (3550 g/L in human serum) that transports many biomolecules, including fatty acids, hormones, and nutrients; it is synthesized in the liver and has a globular structure.20 Albumin has been widely explored for use in nano-size DDS owing to its non-toxic properties, biodegradability, biocompatibility, low immunogenicity, highly water solubility, and because it is well-characterized.21,22 In fact, human serum albumin (HSA)-based NPs (Abraxane) have been commercially used for cancer treatment. Bovine serum albumin (BSA) (Figure 1a), albumin obtained from bovine serum, has been extensively used as a drug nanocarrier owing to its similarity to HSA, relative cost-effectiveness, and being well-received by the pharmaceutical industry.21 BSA has a molecular weight of 69.3 kDa with 583 amino acid residues and contains 17 disulfide bonds and one free thiol (Cys34). BSA has an isoelectric point (pI) of 4.7 and possesses two tryptophan (Trp) residues at position 212 (Trp212) present in a hydrophobic pocket at sub domain IIA and at position 134 (Trp134) at sub domain IB, which is exposed to solvent.20–22 BSA is negatively charged in solution, meaning it can interact with cationic polymers/polysaccharides. Among known polycations, chitosan (CH) has been recognized as a promising material for development of nano-size DDS owing to its cationic nature, non-toxic profile, biodegradability, and biocompatibility.23 CH is amino polysaccharides obtained from deacetylation of chitin and is composed of 1,4-linked 2-amino-2deoxy--D-glucan and 1,4-linked 2-acetamido-2-deoxy--D-glucan.24 NPs formed from the assembly of CH and albumins have previously been reported for the encapsulation and delivery of anti-cancer drugs such as doxorubicin (DOX)18, alprazolam25, paclitaxel (PTX)26, and ibuprofen27 and as a gene carrier28. However, one limitation in utilizing CH as a drug nano


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carrier is its poor water solubility and possible aggregation in physiological conditions (neutral pH and high salt concentrations) due to its pKa of 6.5.23,24 In this study, we fabricated NPs by assembling reduced BSA (rBSA) and glycol chitosan (GC) (Figure 1b), a water soluble derivative of CH, for encapsulation and delivery of hydrophobic anti-cancer drug, PTX (Figure 1c). We hypothesized that reducing BSA would increase the complexation with GC and stable NPs could be formed via hydrophobic interactions and/or disulfide bonds. This simple and energy-efficient approach would eliminate the use of external crosslinkers and high temperatures, which have commonly been used to form stable NPs in the previous reports.13,16.18 The use of GC would also overcome the insolubility issue and poor cellular penetration of CH at the neutral pH, as previously reported.14 The formation of nanocomplexes from rBSA and GC (rBG-NPs) was studied as a function of rBSA:GC mass ratio and pH. The physical characteristics and stability of the NPs were evaluated by dynamic light scattering (DLS) and compositional analysis. Finally, the ability of rBG-NPs as nano-sized PTX DDS was demonstrated.

Figure 1. The crystal structure of (a) BSA (PDB-ID:3v03) and the chemical structures of (b) GC and (c) PTX.


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MATERIALS AND METHODS Materials. Bovine serum albumin (free fatty acid, 98% purity), glycol chitosan (free amine group  60%, degree of polymerization of minimum 400), methanol, acetonitrile (HPLC grade), ethanol, urea, and tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Paclitaxel and L-cysteine were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The Quick Start Bradford Protein Assay was purchased from Bio-Rad, Inc. (Hercules, CA, USA). Tween 80, rhodamine B isothiocyanate (RBITC), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Inc (St. Louis, MO, USA). 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). HeLa and NIH-3T3 cell lines were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The cell counting kit, WST-8, and Hoechst 33342 solution were purchased from Dojindo Laboratories, Inc. (Kumamoto, Japan). Minimum essential medium (MEM), fetal bovine serum (FBS), penicillin, streptomycin, and OPTI-MEM were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Dulbecco’s phosphate-buffered saline (D-PBS) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Cremophor EL was obtained from Fluka Biochemika, Germany. All other chemicals were analytical grade. Reduction of BSA. The reduction of BSA—to give rBSA—was carried out using TCEP.HCl under denaturing conditions.29 Briefly, BSA (100 mg) and TCEP.HCl (86 mg) were separately dissolved in 5 mL of reducing buffer (25 mM Tris-HCl, 8 M urea, and 0.1 mM EDTA). The BSA solution was then mixed with the TCEP.HCl solution and incubated at 25 ºC for 4 h. The resulting solution was then dialyzed against 500 mL of 1 mM HCl using a 1214 kDa molecular


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weight cut-off (MWCO) dialysis membrane (Spectra/Por, Repligen Corp., Waltham, MA, USA) for 3 days at 4 ºC with 4-6 changes of release medium to remove excess urea and TCEP.HCl. The protein concentration of the rBSA solution was measured using the Bradford assay. The number of free thiols per BSA molecule was determined using the Ellman’s reagent. L-cysteine was used to obtain a standard calibration curve for quantifying the number of free thiols. Under these experimental conditions, the number of free thiols per BSA molecule generated after the reduction process was about 21. The rBSA solution was stored at 4 ºC before use. Purification of glycol chitosan. GC was purified by dialysis and filtration methods.30 Briefly, GC (10 mg/mL) was dissolved in Milli-Q water with gentle magnetic stirring, and the pH was adjusted to 5.5. The GC solution was then dialyzed against Milli-Q water using a 1214 kDa MWCO membrane overnight at room temperature and filtered using a 0.4 m membrane filter. Finally, the GC solution was lyophilized for 48 h and stored at 4 ºC before use. Formation of rBG-NPs. The formation of rBG-NPs was achieved by mixing rBSA and GC solutions to yield rBG-NPs. Briefly, a GC solution (2 mg/ml) was prepared by dissolving lyophilized GC in 10 mM acetate buffer (pH 5.5), with stirring until completely dissolved and stored at 4 ºC overnight for complete hydration. An rBSA solution (2 mg/ml) was prepared from the stock solution by diluting with 1 mM HCl. rBG-NPs were formed by slowly adding rBSA solution to an equal volume of GC solution with mild stirring at room temperature (25 ºC), and the final pH was adjusted to the desired values using 0.1 M HCl or NaOH. The composition of rBSA and GC in the NPs. The composition of rBSA and GC in the NPs was determined using a centrifugation method. In general, 1 mL of rBG-NPs solution was centrifuged (18,000 g, 20 ºC, 30 min), and the supernatant was collected. The amount of rBSA in


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the rBG-NPs was measured using the Bradford assay, whereas the amount of GC in rBG-NPs was estimated using RBITC-labeled GC. The fluorescent labeling protocol of GC was carried out based on a previously reported method31 with a slight modification. Briefly, 0.1 g of GC was dissolved in 10 mL of Milli-Q water, an equal volume of methanol was added and the solution was stirred for 2 h. 6.5 mL of a methanolic solution of RBITC (0.2 mg/mL) was then added to the GC solution and the mixture was stirred overnight under light protection. Purification was then carried out by centrifugal filtration (Amicon Ultra; MWCO, 30 kDa; Millipore Corp., Billerica, MA, USA) and dialysis against 1 L of Milli-Q water (1214 kDa MWCO, Spectra/Por, Repligen Corp., Waltham, MA, USA). Finally, the pH of the RBITC-GC solution was adjusted to 5.5 and the solution was lyophilized. The degree of labeling was calculated to be 0.08 (% mol/mol). The concentration of GC was estimated using a standard curve generated from the absorbance of RBITC-GC at 556 nm. Stability of rBG-NPs in various buffers. Briefly, 1 mL of rBG-NPs solution was purified using a centrifugation method (9,000 g, 20 ºC, 30 min) under a 10 L glycerol bed. The supernatant was removed, and the precipitated rBG-NPs were washed twice with Milli-Q water. The purified rBG-NPs were then redispersed in PBS or the desired buffer solutions and stored at 37 ºC for 1 day. The particle size and polydispersity index (PDI) of rBG-NPs in various buffers were noted. PTX-loaded rBG-NPs. PTX was loaded into rBG-NPs using both adsorption and a mixed method. In the adsorption method, a small amount of ethanolic PTX solution (4 mg/mL) was added to 2 mL of rBG-NPs solution and gently stirred for 10 min. In the mixed method, PTX was first added to an rBSA solution, and then the mixture was added slowly to the GC solution with gentle magnetic stirring. In some cases, PTX-loaded rBG-NPs were freeze-dried using 5% trehalose as a cryoprotectant.


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Characterization of rBG-NPs and PTX-loaded rBG-NPs. The Z-average particle size, PDI, and derived count rate were measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, United Kingdom). Zeta potential was measured using the same instrument. The shape and morphology of the NPs were observed by transmission electron microscopy (TEM) (JEM 2010, JEOL) at an accelerating voltage of 120 kV. The NPs were stained with 2% uranyl acetate before imaging. The structural characteristics of rBSA were analyzed using a circular dichroism (CD) spectropolarimeter (J-725G, JASCO, Tokyo, Japan) and a fluorescence spectrophotometer (LS55C, Perkin Elmer, Waltham, MA, USA). The encapsulation efficiency (EE) was determined using an extraction method. Briefly, an aliquot of PTX-loaded rBG-NPs was mixed with an ethanol at a 1:4 volume ratio and filtered (0.2 m; Milex-LG; Millipore Corp., Billerica, MA, USA) before injection into a reversed-phase high performance liquid chromatograph equipped with an auto sampler (Jasco LC-4000, Jasco Inc., Easton, MD, USA). A C-18 column (InertSustain; GL Sciences, Inc., Tokyo, Japan) was used with a mobile phase of acetonitrile (60% by vol) and Milli-Q water at a flow rate of 0.8 mL/min. The EE (%) of PTX-loaded rBG-NPs was calculated using the following equation:

EE (%) =

Total PTX loaded × 100% Total PTX added

In vitro PTX release from rBG-NPs. The release profile of PTX from rBG-NPs was established using a dialysis method at pH 7.4. Briefly, 1.5 mL of lyophilized PTX-loaded rBG-NPs (25 g/mL) in PBS pH 7.4 was added to a dialysis membrane (Spectra/Por, 1214 kDa MWCO). The dialysis bags were then immersed in 25 mL of release medium (10 mM PBS pH 7.4 containing 5% ethanol and 1% Tween 80) and placed in an incubator shaker at 37 ºC and 60 rpm for 10 h. 5% ethanol and 1% Tween 80 was used to provide sink conditions.32,33 At a


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predetermined time, 500 L of release medium containing PTX was collected and replaced with the same amount of pre-warmed release medium. The amount of PTX was determined using HPLC analysis, as described above. Cellular uptake of rBG-NPs. Cellular uptake of rBG-NPs in HeLa cells was evaluated by CLSM. RBITC-GC was used to detect the fluorescent signal of rBG-NPs. Briefly, HeLa cells were seeded in a multi-well glass bottom dish (Matsunami Glass Industries, Ltd., Osaka, Japan) at a density of 10,000 cells per well and incubated for 24 h at 37 ºC in an atmosphere of CO2. The medium was then removed and 200 L of RBITC-labeled rBG-NPs, dispersed in OPTIMEM, was added to the dish. After 2 h of incubation, the samples were removed and washed three times with D-PBS. The cells were then fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.) at 25 ºC for 10 min. Nuclear staining of HeLa cells was carried out using Hoechst 33342, according to the manufacturer’s instructions. Finally, the cells were viewed using an LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a diode laser. A rhodamine filter was used to observe the red fluorescence of RBITC-labeled rBG-NPs. In vitro cytotoxicity studies. The cytotoxicity of rBG-NPs and PTX-loaded rBG-NPs against HeLa cells was evaluated by WST assay. The cytotoxicity of a Taxol-like formulation was also evaluated as a positive control. HeLa cells were grown in MEM medium containing 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific, Inc.). HeLa cells were seeded at a density of 5,000 cells per well in a 96-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated for 24 h in an atmosphere of 5% CO2. The medium was then removed and the cells were washed with D-PBS. Subsequently, the cells were exposed to a Taxol-like formulation, rBG-NPs, or PTX-loaded rBG-NPs in a reduced serum medium (OPTI-MEM,


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Thermo Fisher Scientific, Inc.) at varying concentrations. After 24 and 48 h of incubation, the samples were removed and 100 L of WST-8 cell counting solution in OPTI-MEM was added to each well. After a 3 h of incubation, the A450 was read using a microplate reader (Bio-Tek Instrument, Inc., Winooski, VT, USA) and cell viabilities were expressed as the percentage of living cells over untreated cells. Statistical analysis. The statistical analysis was performed using ANOVA and Student’s t-test with Microsoft Excel (Version 2017 for Macintosh, Microsoft Corp, Redmond, WA, USA). P values of < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION Reduction of BSA. The formation of rBG-NPs involved the initial reduction of BSA and the complexation of rBSA and GC under certain conditions. The reduction of BSA was expected to increase its hydrophobicity as the hidden hydrophobic domain should be exposed as a result of the alteration of the BSA structure.34 The number of free thiols per BSA molecule should also increase owing to the breakdown of disulfide bridges. An increase in the hydrophobicity and the number of free thiols of BSA would benefit the stability of rBG-NPs by allowing for a stronger hydrophobic interaction with GC, and BSA self-crosslinking through thiol-disulfide exchange reactions and/or oxidation of the free thiols.35 In this study, TCEP.HCl was selected as a disulfide reductant owing to its lower sensitivity to oxygen and good activity in a broad pH range, compared with other commonly used reductants such as dithiothreitol (DTT)36. The number of free thiols per BSA was about 21, corresponding to ~60% of the maximum number of free thiols that can be generated. The structural characteristics of rBSA were evaluated by CD


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analysis and fluorescence spectroscopy. The CD spectrum of native BSA shows negative bands at 222 and 208 nm (Figure S1a), which are characteristics of an -helix structure.37 In contrast, the CD spectrum of rBSA shows a denatured-like form, indicating that the secondary structure of BSA was changed. BSA has two Trp residues that can be used to monitor the structural changes of BSA using fluorescence spectroscopic analysis. The fluorescence intensity of BSA decreased and a slight red-shift in the emission maxima was seen after reduction, suggesting that protein unfolding took place (Figure S1b).38 Transformation from the globular state (folding) to linear form (unfolding)35 would also be expected to enhance the electrostatic complexation between BSA and GC. Effect of rBSA/GC mass ratio. rBG-NPs were formed by mixing rBSA and GC in an aqueous solution with low ionic strength (5 mM acetate buffer). As the physical characteristics of the NPs were affected by the initial mass ratio of protein/polysaccharide, the effect of rBSA:GC mass ratio on the physical properties of the rBG-NPs (particle size, PDI, zeta potential, and derived count rate) was investigated by the Zetasizer Nano ZS. The electrostatic interactions were expected to occur at pH values above the isoelectric point (pI) of BSA (~4.9) and below neutral pH (~7) where GC was protonated. The experimental conditions were therefore set as, pH 5.7, total biopolymer concentration of 2 mg/mL (predetermined in a preliminary study) and varied rBSA:GC mass ratios (4:1, 2:1, 1:1, and 1:2). The Z-average diameter (particle size) remained the same—average diameter ~250 nm—as the rBSA:GC mass ratios were varied, except for the rBSA:GC mass ratio 1:2, which showed a markedly larger particle size of ~350 nm (Figure 2a). The PDI values for all mass ratios were consistent with an average of 0.2, indicating relatively narrow particle size distributions. Irrespective of the rBSA:GC mass ratios, the zeta potentials of the rBG-NPs were positive (+30 mV). The zeta potentials tended to increase with increasing GC


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concentration although a slight decrease in the zeta potential at the highest GC concentration (rBSA:GC mass ratio of 1:2) was observed (Figure 2b). The derived count rate for rBG-NPs decreased with increasing GC concentrations (Figure 2b). The derived count rate can be used to estimate the number of particles in the solution.39 These findings indicate that higher rBSA concentrations are required to form rBG-NPs with smaller particle size and higher number of particles. Based on the colloidal stability of rBG-NPs, an rBSA:GC mass ratio of 1:1 was selected for the subsequent experiments owing to the higher zeta potential value (+35 mV), small size, and sufficient number of particles and their better stability against aggregation during purification.

Figure 2. The effect of rBSA:GC mass ratio on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and pH was 5.7.


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Effect of pH. The effect of pH on the particle size, PDI, zeta potential, and the derived count rate of rBG-NPs was assessed by the Zetasizer Nano ZS (Figure 3). The Z-average diameter increased (from 70  2 nm to 367  36 nm) when the pH was increased from 4.9 to 6.5. Similarly, the derived count rate increased with increasing pH. In contrast, the zeta potential values decreased with increasing pH, with the lowest zeta potential value of + 24 mV being recorded at pH of 6.5. The increase in the particle size and a decrease in the zeta potential values might be due to bridging effects and charge neutralization of the complex40 as pH was increased from 4.9 to 6.5. The PDI values also decreased with increasing pH from 4.9 to 6.5, with the lowest PDI of ~0.2 at pH 5.7 and 6.5.

Figure 3. The effect of pH on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and the rBSA:GC mass ratio was 1:1.


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The composition of rBG-NPs. To get an insight into the nature of the interaction between rBSA and GC and to confirm the findings of the DLS analysis, we evaluated the composition of rBSA and GC in rBG-NPs using a centrifugation method, assuming that the centrifugal forces do not affect the original structure of the rBG-NPs. The amount of rBSA in the rBG-NPs was determined using the Bradford assay, while the amount of GC was estimated using RBITClabeled GC. It should be noted that RBITC conjugation into GC was optimized such that the formation of RBITC-GC NPs was prevented, as previously reported.41 The amount of rBSA and GC in rBG-NPs was studied as a function of rBSA:GC mass ratio and pH, with similar experimental conditions to those previously discussed. The compositional analysis showed that the amount of rBSA and GC was not greatly affected by rBSA:GC mass ratio, although the amount of GC incorporated into rBG-NPs tended to be higher at higher rBSA concentrations, particularly at an rBSA:GC mass ratio of 4:1 (Figure 4a). In contrast, the pH appeared to affect the amount of rBSA and GC in rBG-NPs (Figure 4b). Increasing the pH from 4.9 to 6.5 increased the percentage of rBSA (from 67  3% to 97  1%) and GC (from 3.6  1% to 15.1  3%) present in rBG-NPs, indicating that the complexation of rBSA and GC was promoted at slightly higher pH near the pKa of GC (~8). This result could be attributed to the neutralization of the complex at this pH, which increases the amount of rBSA and GC present in rBG-NPs. It has also been suggested that other non-covalent forces, such as hydrophobic interactions, play an important role in the complexation of BSA and native chitosan (CH)42, and this may also apply for GC and rBSA.


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Figure 4. Composition of rBSA and GC in rBG-NPs as a function of (a) rBSA:GC mass ratio at pH 5.7 and (b) pH at an rBSA:GC mass ratio of 1:1. The amount of rBSA/GC in the NPs was estimated by concentration difference of rBSA/GC in total added and in the supernatant before and after centrifugation (18,000 g). The concentration of rBSA and GC in the supernatant was determined using the Bradford assay and fluorescently labeled-GC.

Stability and morphology of rBG-NPs. To evaluate the stability of rBG-NPs in various buffers and to understand the interactions affecting the formation of rBG-NPs, rBG-NPs formed at a rBSA:GC mass ratio of 1:1 and pH 6.5 were purified using a centrifugation method and redispersed in PBS pH 7.4, PBS with 25 mM glutathione (GSH), PBS with 100 mM Tween 20, and PBS with 100 mM urea. The particle size and PDI were measured by DLS after a 1-day


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incubation at 37 ºC. A marked increase in the particle size was observed after rBG-NPs were incubated in PBS containing 100 mM Tween 20, suggesting that hydrophobic interactions play a dominant role in the formation and stability of NPs (Figure 5a). In contrast, urea, which is responsible for breaking strong hydrogen bonds43 was not effective in destabilizing rBG-NPs, indicating that hydrogen bonding is not a critical factor for rBG-NPs. A slight increase in PDI was observed when rBG-NPs were incubated in PBS with GSH, although GSH did not appear to affect the particle size. In addition, the number of free thiols in rBG-NPs decreased after incubation in PBS (Figure 5b). This suggests that the self-crosslinking of rBG-NPs through the free thiols of rBSA occurs. The morphology of rBG-NPs was examined using TEM. Figures 5c and 5d show a representative TEM image of rBG-NPs formed at a rBSA:GC mass ratio of 1:1 and pH 6.5. rBGNPs were nearly spherical in shape and had smaller particle size, compared with DLS measurements. Some agglomeration of particles was observed. The smaller size and agglomeration of particles were possibly due to the drying effect during sample preparation.


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Figure 5. (a) Effect of destabilizing compounds on the particle size and PDI of rBG-NPs. rBGNPs were incubated at 37 °C for 1 day. PDI value of rBG-NPs in PBS + Tween 20 was excluded (PDI > 0.5). * indicates significance at p < 0.05. (b) The number of free thiols in rBG-NPs before and after incubation in PBS pH 7.4 at 37 °C. The NPs were purified by a centrifugation, followed by redispersion and incubation in PBS. (c) Representative TEM image of rBG-NPs, and that of rBG-NPs with higher magnification (d). The NPs were prepared at a total biopolymer concentration of 2 mg/mL, rBSA:GC mass ratio of 1:1, and pH 6.5. Scale bars are 200 nm (c) and 100 nm (d).

The structure and formation mechanism of rBG-NPs. DLS, compositional analysis, and stability tests in various destabilizing agents suggest that rBG-NPs possess a core-shell structure and their formation is mainly promoted by both hydrophobic and electrostatic interactions. rBSA


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is prone to self-aggregation in aqueous solution due to its exposed hydrophobic domains, especially at pH values near its pI (~5). rBSA self-aggregation conceivably forms the core of rBG-NPs, whereas GC acts as a stabilizer to prevent strong rBSA self-aggregation via the electrostatic interactions at a pH where rBSA and GC are oppositely charged (~5 < pH < ~8). According to DLS and compositional analysis, the complexation of rBSA and GC is strengthened at a relatively higher pH (6.5). Beyond this pH value, either precipitation or complex dissociation was observed (data not shown), suggesting that the electrostatic interactions are also critical in stabilizing the complexation between rBSA and GC. In addition, the free thiols in BSA generated by the reduction process might also contribute to enhancing the stability of rBG-NPs via thiol crosslinking reactions. PTX-loaded rBG-NPs. To evaluate the ability of rBG-NPs to encapsulate and deliver a hydrophobic anti-cancer drug, PTX was selected as a representative model drug. rBG-NPs formed at a rBSA:GC mass ratio of 1:1 and pH 6.5 were used for encapsulation of PTX. PTX was encapsulated into rBG-NPs using mixed and adsorption methods at a final concentration of 100 g/mL. The properties of PTX-loaded rBG-NPs were evaluated by DLS (Table 1). The particle size of PTX-loaded rBG-NPs prepared using the adsorption method was 349  26 nm, which is similar to that of blank rBG-NPs. In contrast, after loading PTX using the mixed method, an increase in the particle size (421  29 nm) was observed, which might be due to the PTX-induced rBSA self-aggregation.44 No difference in PDI and the derived count rate was observed between the two methods. The encapsulation efficiencies (EE) of PTX in rBG-NPs using the mixed and adsorption method were 96  5% and 84  13%, respectively (Table 1). The high encapsulation efficiency was owing to effective PTX binding to rBG-NPs through hydrophobic interactions with the hydrophobic domains of rBSA. We also confirmed that PTX


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could be completely released from rBG-NPs in 10 mM PBS at pH 7.4 (containing 5% ethanol and 1% Tween 80 to prevent the precipitation of PTX), which simulates physiological conditions (Figure S2).

Table 1. Characteristics of PTX-loaded rBG-NPs

Samples

Size [nm]

PDI

Derived count rate EE [%] [103 kcps]

Mixed Method

421  29

0.212 0.015

1069  212

96  5

Adsorption Method

349  26

0.194 0.010

1170  128

84  13

Data, mean standard deviation, n = 3.

Cellular uptake of rBG-NPs. The cellular uptake of rBG-NPs was evaluated in HeLa cells using CLSM. GC was labeled with RBITC to yield a red fluorescence signal that could be used to follow the intracellular fate of rBG-NPs. HeLa cells were incubated with rBG-NPs for 2 h at 37 ºC. Hoechst 33342 (blue) was used to stain the nuclei. The z-stack mode of CLSM was used to precisely determine the spatial location of rBG-NPs within HeLa cells. CLSM analysis showed that a considerable red fluorescence signal could be found inside the cells (Figures S3a and S3c), which suggests that rBG-NPs were internalized. NPs can undergo internalization via endocytosis-related pathways such as macropinocytosis, caveola-mediated endocytosis, and clathrin-mediated endocytosis.45 To establish whether endocytosis is the pathway for rBG-NPs uptake, the cellular uptake experiments were performed for 2 h at 4 ºC, at which temperature the internalization via endocytosis decreases due to changes in the cell membrane fluidity.46 CLSM analysis revealed that most of the red fluorescence signals were not found inside the cells


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(Figures S3b and S3d), suggesting that the intracellular transport of rBG-NPs is via an endocyticrelated pathway. It has been reported that the particle size can determine the internalization pathways of NPs.47 Since rBG-NPs are relatively large in size (about 390 nm), macropinocytosis and/or caveola-mediated endocytosis could be the cellular internalization pathways. Further cellular experiments are required to confirm the intracellular fate of rBG-NPs in more details. Indeed, other researchers using GC-based NPs have demonstrated such a possibility.48,49 In vitro cytotoxicity of PTX-loaded rBG-NPs. The in vitro cytotoxicity of PTX-loaded rBGNPs was evaluated using HeLa cells. A Taxol-like formulation and blank rBG NPs served as a positive and negative control, respectively. HeLa cells were treated with samples containing various PTX concentrations for 24 and 48 h and cell viability was determined using a WST-8 assay. The in vitro cytotoxicity results for the 24 h of incubation are shown in Figure 6a. The cell viability of HeLa cells was not considerably affected by exposure to blank rBG-NPs. In addition, no marked decrease in the cell viability of normal mouse fibroblast NIH-3T3 cells was observed after incubation with blank rBG-NPs at a concentration of 1 mg/mL for 24 h (Figure S4), indicating that rBG-NPs were biocompatible and nontoxic. Cell viability decreased with increasing PTX concentrations after exposure to Taxol-like formulation and PTX-loaded rBG. However, the cytotoxicity of PTX-loaded rBG-NPs was lower than that of the Taxol-like formulation. After increasing the incubation time to 48 h, the cytotoxicity of PTX-loaded rBGNPs was comparable to that of the Taxol-like formulation (Figure 6b). These results suggest that PTX was slowly released from rBG-NPs. The slow release of PTX might be useful for prolonging the circulation of PTX and reducing the side effects. In addition, the slow release


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properties of PTX-loaded rBG-NPs might also indicate that rBG-NPs have good stability in the serum free medium.

Figure 6. In vitro cytotoxicity of Taxol-like formulation, PTX-loaded rBG-NPs, and rBG-NPs in HeLa cells by WST-8 assay after 24 h (a) and 48 h (b) incubation. Data, mean  standard deviation, n = 3.

CONCLUSIONS


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The formation of core-shell NPs based on the complexation of rBSA and GC (rBG-NPs) was demonstrated. Electrostatic and hydrophobic interactions played dominant roles in the formation and stability of rBG-NPs, as determined by DLS and compositional analysis. The particle size of rBG-NPs could be tuned by adjusting the rBSA:GC mass ratio and pH. rBG-NPs were spherical in shape and had excellent stability at neutral pH (10 mM PBS pH 7.4). rBG-NPs were able to encapsulate the hydrophobic anti-cancer drug PTX with high encapsulation efficiency through hydrophobic interactions. CLSM analysis revealed that rBG-NPs could be effectively internalized by cells, possibly via an endocytosis-pathway. WST-8 assay showed that PTX-loaded rBG-NPs had a comparable cytotoxicity in vitro in HeLa cells to a Taxol-like formulation after a 48 h of incubation, which might be due to the slow release of PTX from the NPs. Ultimately, rBG-NPs showed little toxicity, suggesting that the NPs hold great promise for use as a hydrophobic drug nanocarrier in the pharmaceutical and medical applications. Corresponding Author *Email: [email protected] Author Contributions The manuscript was written with contributions from all authors. All authors have approved to the final version of the manuscript. ACKNOWLEDGMENT This research was funded by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers of JP16H04581 and JP16H06369 from the Ministry of Education, Cultures, Sports, Science and Technology (MEXT). M. Alif Razi was supported by a scholarship


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from MEXT (scholarship ID No. 150043). We thank Sarah Dodds, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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FIGURE CAPTIONS

Figure 1. The crystal structure of (a) BSA (PDB-ID:3v03) and the chemical structures of (b) GC and (c) PTX. Figure 2. The effect of rBSA:GC mass ratio on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and pH was 5.7. Figure 3. The effect of pH on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and the rBSA:GC mass ratio was 1:1. Figure 4. Composition of rBSA and GC in rBG-NPs as a function of (a) rBSA:GC mass ratio at pH 5.7 and (b) pH at an rBSA:GC mass ratio of 1:1. The amount of rBSA/GC in the NPs was estimated by concentration difference of rBSA/GC in total added and in the supernatant before and after centrifugation (18,000 g). The concentration of rBSA and GC in the supernatant was determined using the Bradford assay and fluorescently labeled-GC. Figure 5. (a) Effect of destabilizing compounds on the particle size and PDI of rBG-NPs. rBGNPs were incubated at 37 °C for 1 day. PDI value of rBG-NPs in PBS + Tween 20 was excluded (PDI > 0.5). * indicates significance at p < 0.05. (b) The number of free thiols in rBG-NPs before and after incubation in PBS pH 7.4 at 37 °C. The NPs were purified by a centrifugation, followed by redispersion and incubation in PBS. (c) Representative TEM image of rBG-NPs, and that of rBG-NPs with higher magnification (d). The NPs were prepared at a total biopolymer concentration of 2 mg/mL, rBSA:GC mass ratio of 1:1, and pH 6.5. Scale bars are 200 nm (c) and 100 nm (d). Figure 6. In vitro cytotoxicity of Taxol-like formulation, PTX-loaded rBG-NPs, and rBG-NPs in HeLa cells by WST-8 assay after 24 h (a) and 48 h (b) incubation. Data, mean  standard deviation, n = 3.


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For Table of Contents Only


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Figure 1. The crystal structure of (a) BSA (PDB-ID:3v03). The chemical structure of (b) GC and (c) PTX. 339x190mm (133 x 133 DPI)


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Figure 2. The effect of rBSA:GC mass ratio on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and pH 5.7. 190x229mm (197 x 197 DPI)


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Figure 3. The effect of pH on (a) particle size and PDI, (b) zeta potential and derived count rate. Total rBSA + GC concentration was 2 mg/mL and rBSA:GC mass ratio of 1:1. 190x229mm (197 x 197 DPI)


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Figure 4. Composition of rBSA and GC in rBG-NPs as a function of (a) rBSA:GC mass ratio at pH 5.7 and (b) pH at a rBSA:GC mass ratio of 1:1. The amount of rBSA/GC in the NPs was estimated by concentration difference of rBSA/GC in total added and in supernatant before and after centrifugation (18000 g). The concentration of rBSA and GC in supernatant was determined by Bradford assay and fluorescent labelledGC. 338x228mm (111 x 111 DPI)


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Figure 5. (a) Effect of destabilizing compounds on the particle size and PDI of rBG-NPs. rBG-NPs were incubated at 37 oC for 1 day. PDI value of rBG-NPs in PBS + Tween 20 was excluded (PDI > 0.5). * indicates significant at p < 0.05. (b) The number of free thiols in rBG-NPs before and after incubation in PBS pH 7.4 at 37 oC. The NPs were purified by a centrifugation, followed by redispersion and incubation in PBS. (c) Representative TEM image of rBG-NPs, and that of rBG-NPs with higher magnification (d). The NPs were prepared at total biopolymer concentration of 2 mg/mL, rBSA:GC mass ratio of 1:1, and pH 6.5. Scale bars are 200 nm (c) and 100 nm (d). 329x253mm (114 x 114 DPI)


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Figure 6. Cytotoxicity in vitro of Taxol-like formulation, PTX-loaded rBG-NPs, and unloaded rBG-NPs in HeLa cells by WST-8 assay after 24 h (a) and 48 h (b) incubation. Data, mean ± standard deviation, n = 3 228x304mm (167 x 167 DPI)


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Table of Contents/Abstract Graphic 253x152mm (178 x 178 DPI)


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Self-assembled reduced albumin and glycol chitosan nanoparticles for

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