Oligochitosan modified albumin as plasmid DNA delivery vector: Endocytic trafficking, polyplex fate, in vivo compatibility

Journal Pre-proofs Oligochitosan modified albumin as plasmid DNA delivery vector: endocytic trafficking, polyplex fate, in vivo compatibility Monika Kumari, Chi-Hsien Liu, Wei-Chi Wu PII: DOI: Reference:

S0141-8130(19)36095-7 https://doi.org/10.1016/j.ijbiomac.2019.09.121 BIOMAC 13366

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2 August 2019 15 September 2019 16 September 2019

Please cite this article as: M. Kumari, C-H. Liu, W-C. Wu, Oligochitosan modified albumin as plasmid DNA delivery vector: endocytic trafficking, polyplex fate, in vivo compatibility, (2019), doi: https://doi.org/10.1016/ j.ijbiomac.2019.09.121

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Oligochitosan modified albumin as plasmid DNA delivery vector: endocytic trafficking, polyplex fate, in vivo compatibility

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Monika Kumari a, Chi-Hsien Liu a,b,c,d*, Wei-Chi Wu d,e

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a

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First Road, Kwei-Shan, Tao-Yuan 333, Taiwan

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b

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Ecology, Chang Gung University of Science and Technology, 261, Wen-Hwa First Road, Taoyuan, Taiwan

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c

Department and Graduate Institute of Chemical and Materials Engineering, Chang Gung University, 259, Wen-Hwa

Research Center for Chinese Herbal Medicine and Research Center for Food and Cosmetic Safety, College of Human

Department of Chemical Engineering, Ming Chi University of Technology, 84, Gung-Juan Road, New Taipei City,

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Taiwan

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d

Department of Ophthalmology, Chang Gung Memorial Hospital, Linkou, 5, Fu-Hsing Street, Taoyuan, Taiwan

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e

College of Medicine, Chang Gung University, 259, Wen-Hwa First Road, Taoyuan, Taiwan

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*Corresponding author’s E mail: [email protected]

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Abstract

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Cationic macromolecules condense DNA into small nanoparticles and form polyplex. The composition of the

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polyplex determines the endocytic process, the intracellular routing and the fate of the polyplex. Previously,

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oligochitosan-modified vectors with different protein moieties are used as gene delivery vector and the types of

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protein moiety can influence the endosome escape ability and transfection efficiency. Among the modified vectors,

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oligochitosan-modified bovine serum albumin (BSA) showed 90% transfection efficeincy compared to the

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modified zein and ovalbumin. These data encouraged us to investigate the mechanism of internalization

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involved in the superior transfection efficiency of modified BSA/ plasmid polyplex. The effect of specific

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endocytic inhibitors was studied in two adherent cell lines. The caveolae-mediated and lipid-mediated pathways

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play a significant role in the polyplex internalization. Next, a colocation of polyplex with lysosome was

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investigated in the presence of LysoTracker using confocal microscopy. Up to 70% of polyplex successfully

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escaped the lysosome without degradation. Four non-adherent cell lines showed above than 60% transfection

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efficiency at an optimized vector/plasmid ratio. Moreover, no significant hemolytic effect was observed up to

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500 μg/mL of cationic BSA, indicating no detectable cell membrane disruption. Overall, the hybrid

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biomacromolecule showed good intracellular delivery and safety in a mice model.

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Keywords: albumin; oligochitosan; gene delivery; trafficking

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Introduction

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Gene therapy is gaining considerable interest for curing both acquired and inherited diseases. The design of

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vectors offers significant amelioration for the delivery of a desired sequence of DNA into the nucleus of the

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target cell without degradation [1]. Traditionally, there are two types of DNA delivery vectors: viral and non-

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viral. Recently, the synthesis of a non-viral vector was found to enhance gene delivery efficiency and cell

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viability by forming polyplex. Polyplexes are formed through the electrostatic interaction between the positively

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charged polymers with negatively charged plasmids into small, condensed nanoparticles. Therefore, a non-viral

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vector consisting of positive charges, such as cationic polymer and lipids, provides a potential tool to study the

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gene delivery approach into target cells [2]. However, the major limitation of a non-viral vector is its low

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transfection efficiency due to its multiple barriers, such as the binding of nanoparticles to the cell surface and its

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process (i.e., endosome escape ability and transport into the nucleus of the target cells) [3, 4]. Besides, the

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cellular uptake mechanism is poorly understood. The mechanism of the cellular uptake pathway requires a

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detailed investigation to improve the transfection efficiency of designed vectors. Previous studies demonstrate

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that endocytosis is the major pathway for the cellular uptake mechanism of the cationic macromolecule and thus

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enhances the gene delivery efficiency [5].

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Typically, the endocytic pathways are divided into two types: phagocytosis and pinocytosis. Further,

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pinocytosis is sub-divided into three classes: clathrin-mediated endocytosis (CME), clathrin-independent

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endocytosis (CIE), and macropinocytosis. The intracellular fate of polyplex followed by each endocytic

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pathway varies with the involvement of specific proteins [6]. The CME uptakes polyplex by forming clathrin-

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coated pits and then travels from early to late endosomes. Finally, the late endosome fuse with the lysosomes

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and eventually release the polyplex in endo-lysosomes compartment [7]. The CIE is subdivided into two types:

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caveolae-independent and caveolae-mediated endocytosis (CvME). The polyplex via caveolae-mediated

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pathway comes through the formation of small flask-shaped invaginations called caveolae in the plasma

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membrane [8]. To study the mechanisms, different endocytic inhibitors are used such as chlorpromazine (CPZ),

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genistein, and methyl-β-cyclodextrin related to CME, CvME, and lipid-mediated pathways. Each inhibitor

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utilizes distinct mechanism and has its own key feature to block the specific pathway. CPZ inhibition involves

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the loss of clathrin and AP2a adaptor complex from the cell surface and their artificial assembly on endosomal

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membranes [9]. The treatment with genistein inhibits the activity of Src kinase for caveolin 1 phosphorylation

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[10]. The role of the endosome-lysosome system is elucidated using lysosomotropic agents like monensin and

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PVP [11]. Monensin blocks the endocytosis pathway by disrupting the Golgi complex and lysosome function

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[12, 13]. PVP is an agent related to the intracellular vesicular swelling due to osmosis. It also decreases the

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function of lysosome enzymes and restricts the fusion of lysosomes with polyplex [14]. The intracellular 2

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delivery of polyplex also differs depending upon the physical nature of the complexes, such as their type of

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conjugation, size, and stability. The degree of successful transfection is influenced by cell metabolism and

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varies for different types of cells. Indeed, the recent literature has indicated that the internalization route taken

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by polyplex plays an important role in successful gene delivery [15]. Rejman et al. suggested that the CvME

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pathway shows a high endosome escape ability with good transfection efficiency compared with CME

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pathways [16]. An excess amount of glucosamine in a chitosan-based gene delivery system helps to internalized

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polyplex via endocytic pathways [17]. Peng et al. found that CvME has gained significant attention for gene

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delivery applications, as it prevents the degradation of nanoparticles by lysosomal enzyme [18]. Therefore,

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several authors have made numerous efforts to design a vector that can be internalized via CvME pathways and

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driven toward efficient transfection efficiency [3]. Recently we prepared a protein-OC cationic vector with

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different protein moieties like bovine serum albumin (BSA), zein, and ovalbumin (OVA) via Schiff base

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formation. The obtained transfection efficiency of BSA-OC (up to 95%) was higher than zein-OC (up to 84%)

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and OVA-OC (up to 70%) in a serum-free condition [19, 20]. These data generated a curiosity to investigate the

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cellular uptake pathway and intracellular distribution of BSA-OC/pDNA polyplex.

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Herein, the DNA complexation and releasing ability of three synthesized vectors was investigated. Meanwhile,

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the cell lines for recombinant protein production, i.e., CHO-K1 and HEK 293T, were used to understand the

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roles of endocytic pathways involved for the delivery of pDNA by BSA-OC vectors. In the presence of different

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endocytic inhibitors, the intracellular distribution of polyplex and the integrity of endo-lysosomal membrane

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after polyplex transfection were visualized by confocal microscopy. The gene transfection efficiency in three

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different suspended cell lines and their biocompatibility were discussed. Finally, the blood biocompatibility of

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polyplexes was observed and their gene delivery to the mice was also used to evaluate the potential applicability

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of the developed vector.

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Materials and methods

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2.1. Materials

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The oligochitosan (OC, average MW=662 g/mol, degree of deacetylation >90%) was obtained from Yaizu

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Suisan Kagaku Industry. Hoechst 33342, trypsin-EDTA, glutaraldehyde, chlorpromazine, sodium azide,

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genistein, monensin, FITC, ethidium bromide (EtBr), polyvinylpyrrolidone (PVP), and acridine orange were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Methyl-β-cyclodextrin was acquired from Wako Pure

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Chemical Industries, Ltd. (Osaka, Japan). Rhodamine B was obtained from Acros Organics (PA, USA). 3

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LysoTracker Red was purchased from Molecular Probes/Invitrogen (Eugene, Oregon, USA). Plasmid tdTomato

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was obtained from Addgene (plasmid 30530). Plasmid EGFP-C3 (size 4.7 kb) was procured from Takara Bio

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(Shiga, Japan). Both plasmids were amplified in Escherichia coli strain DH-5 and purified from the cell

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pellets using a purification kit (GeneMark, Taipei, Taiwan). Fetal bovine serum (FBS) was purchased from

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Biological Industries (Haemek, Israel). All reagents were used without further purification.

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2.2. Cell culture

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HEK 293T, CHO-K1, and the hybridoma cells (BCRC 60427 and BCRC 60252) were obtained from the

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Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). HEK 293T and CHO-K1 are adherent

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cell lines, were cultured in Dulbecco’s modified Eagle’s medium-high glucose supplemented with 10% FBS.

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BCRC 60252 was maintained in RPMI-1640 medium supplemented with 10% FBS. The hybridoma cell line

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(CRL-1754) was obtained from American Type Culture Collection (Manassas, VA, USA). BCRC 60427 and

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CRL-1754 were maintained in serum-free CD Hybridoma medium supplemented with 8 mM L-glutamine

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(Sigma). The subculture of adherent cell lines was performed when cells had 80% of confluence using trypsin-

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EDTA. Four non-adherent cell lines were passaged every four days to maintain an exponential growth phase.

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All cells were maintained in a humidified air atmosphere at 37°C with 5% CO2. The cell morphology and

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growth were monitored daily using a light microscope. The cell density and viability were determined using the

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Beckman Coulter counter (MS3 model) and the trypan blue staining.

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2.3. Synthesis of the protein-OC vector and formation of polyplex

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The protein (BSA, zein, or OVA) was cross-linked with OC in the presence of glutaraldehyde via the one-step

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process. The detail synthesis procedure was reported in our previous publication [19]. Briefly, the mixture of

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protein, OC, and glutaraldehyde was reacted for 2 hours at room temperature. Finally, the free aldehyde group

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was quenched by the addition of 0.1 mg/mL glycine and vortexed for 30 minutes. The unreacted reagents were

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removed by washing twice with deionized water using the 50 kDa cutoff Vivaspin tube through the

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centrifugation technique to obtain the purified vectors.

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2.4. DNA binding and stability test of the synthesized vector

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The DNA binding and stability of all three synthesized vectors was investigated using EtBr assay and agarose

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gel electrophoresis assay. The DNA binding affinity was studied by EtBr assay as follows. Briefly, EtBr (0.2

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μg) was mixed with pDNA (0.2 μg) and total volume was maintained to 50 μL by using the water with a 5-

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minute incubation. The vector (10 μg to 60 μg) was mixed with EtBr-DNA solution (total volume 100 μL) and

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incubated at different time duration to form complexes. EtBr displacement from DNA in the presence of a 4

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synthesized vector was recorded by fluorescence spectroscopy (Molecular Devices Spectramax i3x

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spectrophotometer (Sunnyvale, CA, USA)) into a 96 well black plate. The fluorescence intensity was measured

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at the excitation and emission of 516 nm and 605 nm. EtBr and DNA-EtBr fluorescence was also measured at

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the same condition as controls [21]. 𝐹𝑜𝑏𝑠 ― 𝐹𝑂

Percentage of EtBr and DNA binding = 𝐹𝐷𝑁𝐴 ― 𝐹𝑂 ∗ 100

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Where, FO, FDNA, and Fobs represent the fluorescence intensities of only EtBr, EtBr-DNA complex and EtBr-

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DNA complex with the vector respectively. The stability and releasing ability of DNA was investigated in the

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presence of anionic molecules i.e., heparin. Briefly, 10 μL of the prepared polyplex at a weight ratio of 60:1 was

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exposed to 8 μg/μl of heparin for 2 hours at 37°C. The disintegration of polyplexes was visualized by using the

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agarose electrophoresis.

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2.5. Quantitative measurement for the uptake of polyplexes

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For cellular uptake analysis, BSA-OC was labeled with rhodamine dye (Rh). CHO-K1 and HEK 293T cells

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were seeded into 48-well plate containing sterile coverslip at a density of 5×104. After 90% confluence, the cells

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were treated with polyplex containing the Rh-labeled BSA-OC vector for 3 hours. After completion of

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incubation times, the cells were washed with PBS and the nuclei of the cells were labeled with Hoechst 33342.

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Finally, the coverslip was sealed with the acrylic glue in glass slide before examining in the confocal

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microscopy. The quantitative analysis of confocal micrograph was performed by using IN Cell Analyzer 1000

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software.

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2.6. Transfection efficiency and cellular uptake analysis in presence of endocytic inhibitor

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The transfection and cellular uptake efficiency of BSA-OC/pDNA polyplex were examined in CHO-K1 and

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HEK 293T cells in the presence of different endocytic inhibitors. The cells were seeded in 48 well plates at a

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density of 5×104 and after 90% confluence, the cells treated with polyplex and endocytic inhibitor for 3 hours.

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Subsequently, the transfection medium was replaced with fresh 10% DMEM medium and further incubated for

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another 24 hours. Finally, the cells were washed with PBS and nuclei of the cells were stained with Hoechst.

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The cellular uptake and transfection efficiency were calculated by using IN Cell Analyzer 1000 software.

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Transfection efficiency(%) =

𝐵𝑙𝑢𝑒 𝑐𝑜𝑢𝑛𝑡 𝑤𝑖𝑡ℎ 𝑔𝑟𝑒𝑒𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 ∗ 100 𝐵𝑙𝑢𝑒 𝑐𝑜𝑢𝑛𝑡

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2.7. Intracellular trafficking of the polyplexes

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The cells were seeded onto the glass coverslip in the 48-well culture plate as described above. For intracellular

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distribution of BSA-OC, the polyplex was prepared by using FITC labeled BSA-OC vector at a weight ratio of 5

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60:1 and incubated for 3 hours. After completion of the incubation period, the cells were treated with diluted

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LysoTracker Red DND (50 nM) in DMEM medium for 30 min. Following LysoTracker incubation, the cells

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were treated as stated previously for confocal imaging. The image was captured at excitation and emission

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wavelength of 488 nm and 560 nm for LysoTracker Red and further, the percentage of lysosome escape ability

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was calculated by using IN Cell Investigator software. Complex colocalization with LysoTracker Red is defined

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as:

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Complex colocalization (%) =

𝐴𝑟𝑒𝑎 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑔 𝑔𝑟𝑒𝑒𝑛 𝑎𝑛𝑑 𝑟𝑒𝑑 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 ∗ 100 𝐴𝑟𝑒𝑎 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑔 𝑔𝑟𝑒𝑒𝑛 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒

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2.8. Endo-lysosome membrane integrity

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Lysosome membrane integrity of treated cells with BSA-OC was investigated by acridine orange staining. The

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cells were seeded onto 48-well culture plate containing glass coverslip as described above. The cells were

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incubated with BSA-OC/pDNA polyplex for 3 hours and later treated with 10 µM acridine for 15 minutes.

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Finally, cells were washed with PBS and nuclei were stained with Hoechst. The cells were visualized by the

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confocal microscopy and the cell images were analyzed with IN Cell Analyzer 1000.

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2.9. Study of transfection efficiency in suspended cell lines

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For suspended cells, 1×106 cells were transfected in duplicates and the cell viability was maintained above 95%

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before transfection. The prepared polyplexes at a different weight ratio (10:1 to 60:1) were mixed to cells and

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incubated for 3 hours. After the incubation time, the medium containing polyplex was removed by

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centrifugation at 12000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and fresh

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medium was added to each well and incubated for another 24 hours. At the end of incubation time, the cells

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were washed with PBS and fixed with ethanol/acetic acid solution and the nucleus was stained with Hoechst

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33342. Following washing with PBS, the cells were mixed with Dako. A coverslip was covered and sealed by

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acrylic glue at the edge of the coverslip. Finally, the GFP expression and cell morphology were analyzed by IN

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Cell Analyzer 1000 microscope according to the above-stated protocol. The cell viability was also calculated by

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the automated investigation of the nuclear count (Hoechst filter; blue count):

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Cell viability(%) =

𝐵𝑙𝑢𝑒 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 ∗ 100 𝐵𝑙𝑢𝑒 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑐𝑒𝑙𝑙𝑠

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2.10. Blood cell hemolysis

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Freshly isolated mice blood cells were diluted with PBS (1:100 times) and treated with BSA-OC/pDNA

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polyplex at different weight ratios ranged from 10:1 to 300:1. Each weight ratio of prepared polyplex (100 μL) 6

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was then mixed with 100 μL of diluted blood separately, and incubated at 37°C for 2 hours. After the incubation

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period, the tubes were centrifuge at 1000 rpm for 3 min. Finally, 100 µl of supernatant was added to each well

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of the 96-well plate. Reading was taken at 576 nm by using a BioTek microplate reader. The blood cells mixed

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with sodium phosphate buffer was considered as a negative control (0% hemolysis), while cells treated with

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0.1% v/v Triton X-100 was considered as a positive control (100% hemolysis). Percentage of hemolysis was

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calculated as follows:

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Hemolysis (%) =

(𝑂𝐷𝑝𝑜𝑙𝑦𝑝𝑙𝑒𝑥 ― 𝑂𝐷𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) (𝑂𝐷𝑝𝑜𝑠𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝑂𝐷𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙)

∗ 100

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2.11. Biocompatibility of polyplexes in the in vivo condition

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Four-week-old CD1 (ICR) male mice were purchased from Biolasco Biotechnology Co., Ltd. (Taipei, Taiwan).

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All experiments involving the use of mice were performed in accordance with protocols approved by the

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“Animal Care and Use Committee” of Chang Gung Memorial Hospital (Approval Numbers: CGU107-125).

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Control group of mice were intravenously injected with 200 μL of PBS, 200 μg of vector, and 90 μg of DNA,

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separately. Whereas the experimental group of mice was intravenously injected with 200 μL of prepared

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polyplexes. Two different concentration of vector (60 μg and 90 μg) mixed with 60 μg of DNA, separately, to

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prepare the desired weight ratio of vector/DNA, i.e., 1:1 and 1.5:1. The ventral position of mice was observed at

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different duration after injection (2 days, 3 days, and 7 days) by using the IVIS Lumina II imaging system. The

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exposure time was fixed to 0.5 second.

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Results and discussion

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3.1. BSA-OC characterization and DNA condensation assay

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Particle size of the non-viral vectors plays a significant role in the cellular uptake and intracellular trafficking of

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polyplex. Generally, proteins are very sensitive to pH solution which affecting their conformation, surface

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charge, size, and stability [22, 23]. Fig. S1 represents the particle size and polydispersity index (PDI) of BSA-

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OC vector, which showed that the particle size decreased by increasing the pH value from acidic to a basic

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condition. The result that particle size increased slightly at pH 9 may be due to the aggregation of the vector

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near its isoelectric point (i.e. 8.34). When the zeta potential reaches zero around the isoelectric point, the 7

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interaction between molecules increases and prone to aggregation [24]. Furthermore, the increase in particle

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size at lower pH value indicates the protonated form of amine group which further limit the physical contact due

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to charge repulsion and thus form the larger nanoparticles. The deprotonation of amine groups has occurred at

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higher pH condition and thus decreased the particle size and PDI value [25]. The larger particle size at lower pH

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value also indicates the high buffering capacity caused from the accumulation of H+ ions. The nanoparticles

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absorb more protons triggering an inflow of chlorine ion. The flow of ions brings more fluid inside the

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endosomal vesicles leading to its breakdown and release of DNA into the cytoplasm. Simultaneously, the

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increase in particle size may also indicate the reduced interaction of vector with the DNA molecules and thus

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facilitate the DNA release from vector after the endosomal escape [26]. They reported that the smallest particle

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size and PDI value at pH 7 was the most suitable for the formation of polyplex and for gene delivery

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application. The smaller particle size could easily interact with the biological membranes due to large amount of

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surface to volume ratio [27]. Many studies revealed that the size and uniform dispersity of nanoparticle governs

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the cellular uptake efficiency and low PDI values indicate the uniformity of the nanoparticle [28, 29]. Nano-

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carriers around 200nm tend to form complexes that can enter the cells without damaging the cell membrane

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surface [30, 31].

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The cellular uptake mechanism of polyplex can influence the efficiency of gene expression by affecting the

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intracellular fate of the internalized nanoparticles [32]. The OC modified proteins can provide good gene

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transfection efficiency with low cytotoxicity. The transfection efficiency of these hybrid vectors depends on the

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weight ratio, amount of DNA, temperature effect, incubation time, and the presence of serum. A detailed

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investigation is requiring to understand the reasons of the high transfection efficiency using the cationic

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proteins. Therefore, we compared the DNA condensation and releasing ability of three vectors (i.e., BSA-OC,

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zein-OC, and OVA-OC) and then systematically investigated the internalization mechanism. The DNA binding

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affinity of the vector was analyzed through an EtBr retardation assay. The decrease in the relative fluorescence

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intensity of an EtBr-DNA complex following the addition of vector indicates the excellent formation of vector-

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pDNA polyplex. The interaction of EtBr-DNA complex with vector displaces the EtBr molecule from a double

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strand of DNA and further DNA associates with the vector, which decreases the fluorescence intensity of the

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EtBr-DNA complex. The higher reduction in fluorescence intensity indicates more condensation of DNA by the

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vector. In general, it has been reported that the increasing amount of polycations or amine contents reduces the

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fluorescence intensity of EtBr-DNA complex, which indicates the binding strength of polycation-DNA complex

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[33]. Our EtBr data also indicated that by increasing the concentration of the vector from 10 μg to 60 μg, the

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relative intensity of the EtBr-DNA complex decreases significantly, indicating that the positive charge of the

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vector interacts with a negative charge of DNA (Table S1). Moreover, zein-OC and BSA-OC showed higher 8

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fluorescence quench than that of OVA-OC at the 300:1 ratio. This may be due to the larger zeta potentials of

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zein-OC (21.2 ± 0.2mV) and BSA-OC (6.6 ± 2 mV) than OVA-OC (4.6 ± 4.8 mV). Similarly, Sun et al.

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indicated that the binding and release of DNA from the vector regulates the gene transfection efficiency [34].

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The positively charged carriers condense DNA tightly and impede the release of DNA in the intracellular

246

environment, which in turn reduces the gene transfection efficiency. Therefore, the DNA-releasing ability from

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the polyplex was analyzed in the presence of heparin using agarose gel electrophoresis. Heparin is a stronger

248

anionic macromolecule that competes with the DNA binding site and releases the weaker anionic molecules,

249

such as nucleic acid from the polyplex. The release of pDNA from the unpacking polyplex helps to correlate

250

with the gene transfection efficiency [35]. In addition, the defective release of DNA after condensation also

251

lowers the transfection efficiency of polyplex. As shown in Fig. S2, polyplex formed at a weight ratio of 60:1

252

showed the complete condensation of DNA by all three synthesized vectors, as no free DNA band was present.

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Moreover, the treatment with heparin manifests the release of DNA from all three polyplexes. Interestingly, the

254

DNA disassociated from the vector was placed in the same position as naked DNA, which indicates the good

255

condition of DNA. However, the released DNA from OVA-OC showed a faint band. In the case of zein-OC,

256

some amount of DNA was still entrapped with the vector and has not been completely released. This may be

257

due to the high surface charge of zein-OC tightly condensed the DNA and impedes the complete release of

258

DNA. Compared with zein-OC and OVA-OC, BSA-OC was proven as an ideal vector that exhibited a good

259

balance between DNA binding and release. Therefore, we chose the BSA-OC as the candidate for further

260

mechanistic investigation. Besides, the appropriate binding strength between the vector and DNA complex may

261

prompt the DNA-releasing ability and help to estimate the gene transfection efficiency in intracellular

262

conditions. Prior to exploring the mechanism of the BSA-OC vector, the pH sensitivity of the vector was

263

studied to examine the intracellular degradation of the nanoparticles over a pH ranging from basic to acidic.

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Further, we investigated the mechanism of the BSA-OC vector for intracellular delivery and identified the key

265

pathway driving the transfection.

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3.2. Quantitative measurement for the uptake of polyplexes

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To explore the cellular uptake of 150 nm of BSA-OC/pDNA polyplex with a 25 mV surface potential, the

268

vector was labeled with rhodamine. The cellular uptake mechanism and intracellular trafficking of polyplex is a

269

key step in the successful transfection. This process is also influenced by constituents of the designed vector,

270

type of modification, cell type, and culture medium [26]. The uptake analysis of BSA-OC was performed in two

271

different epithelial cell lines, i.e., CHO-K1 and HEK 293T. The cells were incubated with rhodamine-labeled

272

BSA-OC/pDNA polyplex for increasing time intervals, and Hoechst was used as a fluorescent stain for the cell

273

nuclei to determine the intracellular fate of polyplex using a confocal microscope. At each time point, HEK 9

274

293T exhibited more intense red fluorescence than CHO-K1, indicating that the internalization rates of polyplex

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was faster in HEK 293T cells (Fig. 1). Previously, we demonstrated that HEK 293T showed high gene

276

transfection efficiency as compared to CHO-K1 and HaCat cell lines at a weight ratio of 60:1. This may be due

277

to the presence of mannose receptor on the surface of HEK 293T cells, which ease the interaction of OC

278

molecules with the cell membrane [19]. Confocal microscopy image indicated that the polyplex was distributed

279

into the cytosol and minimum level of cellular uptake was observed after 0.5 hours of the incubation period.

280

The uptake of polyplex was increased up to 2-fold at 2 hours as compared to 1 hour and then reached a plateau

281

in Fig. 1(a). The distribution of rhodamine-labeled vector in cytoplasm suggests that the BSA-OC was able to

282

cross the cell membrane barrier and to enter into the nucleus successfully. The uptake of polyplex has reached a

283

plateau in both cell lines after 2 hours of incubation time indicating that the sufficient nanoparticles are

284

internalized and saturation of binding sites at the cell surface. Perumal et al. demonstrated that the key step for

285

the internalization process of cationic dendrimers is the ionic interaction of proteoglycans present on the cell

286

membrane with cationic dendrimers. The uptake of cationic dendrimers affects the adsorptive endocytosis

287

process and this process saturates after one hour due to the limitation of membrane binding sites [36].

288

Therefore, it is important to maintain an adequate incubation time in which a maximal rate of cellular uptake is

289

achieved.

290

The cell nuclei were labeled with Hoechst and the blue fluorescence intensity was used to analyze the toxicity

291

of the vector. Hoechst 33342, a non-intercalating dye that binds to dsDNA of the nuclei, emits blue fluorescence

292

at 350 nm of the excitation wavelength. The blue fluorescence intensity of the Hoechst is directly proportionate

293

to the chromatin state in cells that provide an understanding of cellular conditions and the physiological

294

characteristics of cells that reflect cellular viability [37]. As shown in Fig. 1(b), no significant difference was

295

observed in the blue intensity with increasing time duration for HEK 293T cells, but for CHO-K1 the intensity

296

was slightly reduced after 2 hours of treatment. The data suggested that BSA-OC was a safe carrier to transfer

297

genetic material into the mammalian cell lines. (a)

(b)

10

300

500

250

400

200

Blue intensity

Red fluroscence intensity

CHO-K1 HEK 293T

CHO-K1 HEK 293T

150

* 300

200

100 100

50

0

0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.0

Incubation time (hours)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Incubation time (hours)

(c)

HEK 293T

CHO-K1

Fig. 1. Effect of incubation time on cellular uptake efficiency of Rh-labeled BSA-OC/pDNA polyplex. (a) red fluorescence intensity, (b) blue fluorescence intensity, (c) confocal microscopy image of HEK 293T and CHO-K1 cells incubated with polyplex for 3 hours. The red and blue fluorescence intensity was quantified from individual cells by IN Cell Investigator software. Measurements were effectuated in duplicate with error bars representing the standard deviation. Stars (*) indicate the significant difference as compared with 0.5 hours of incubation time. The scale bar is 10μm in the photos. 298 299

3.3. Cellular uptake and internalization mechanism using endocytic inhibitors

300

Macromolecules are internalized into cells by a variety of mechanisms and their intracellular fates are usually

301

relevant with the uptake pathways. Generally, cationic gene delivery carriers are internalized into cells via the

302

endocytosis pathway as indicated in Table 1. To understand the internalization route of BSA-OC/pDNA

303

polyplex, two cell lines (CHO and HEK) were treated with a defined endocytic inhibitor to block the specific

304

endocytic pathways and analyzed by IN Cell Analyzer 1000. The cell viability of endocytic inhibitor in the

305

presence of polyplex was tested using the WST-1 assay according to a custom-developed protocol. As shown in

306

Fig. 2(c), the cells treated with polyplex were considered controls, showing no significant difference from those 11

307

treated with both endocytic inhibitor and polyplex. The data indicated that there were non-cytotoxic effects of

308

endocytic inhibitors. Later, the BSA-OC/pDNA polyplex uptake and transfection efficiency were studied in the

309

presence of endocytic inhibitor. Prior to endocytic study, treatment with sodium azide was performed to

310

understand the role of energy (ATP)-dependent pathways. The presence of sodium azide significantly reduced

311

the transfection efficiency and cellular uptake efficiency for both cell lines, which indicate that the endocytosis

312

is the dominant pathway for the internalization of BSA-OC/pDNA polyplex. The treatment with CPZ increased

313

the cellular uptake and transfection efficiency of the polyplex, which suggest that the CPZ increased the

314

compensatory pathways. However, the treatment with genistein significantly decreased the cellular uptake and

315

transfection efficiency for both CHO and HEK cell lines. Inhibition data in the presence of CPZ and genistein

316

indicate that the CvME plays a major role in the internalization of polyplex in both cell lines. The reduction of

317

cellular uptake and transfection efficiency for both cell lines by methyl-β-cyclodextrin (60%) confirmed the role

318

of cholesterol (Fig. 2 (a) and (b)). This result also suggests that the transportation of polyplex is not dependent

319

on the endosome-lysosome system and internalized via caveolae-mediated pathway by avoiding the chance of

320

pDNA degradation in lysosomes. The treatment with an endosome-lysosome acidification inhibitor can increase

321

the transfection efficiency of lipolyplexes by protecting the degradation of plasmid DNA in lysosomes [38]. The

322

impacts of six endocytic inhibitors on cellular internalization are summarized in Table 2. Fig. 2(d), (e)

323

illustrates the cell images of gene expression and the rhodamine labeled polyplex uptake by CHO-K1 and HEK

324

293T cells in presence of endocytic inhibitors. The inhibitor of the CvME pathway, genistein, could specifically

325

hinder the GFP expression and polyplex uptake using ATP as the energy. We now believe that the high gene

326

transfection efficiency of BSA-OC/pDNA polyplex is due to the involvement of the CvME pathway.

327

12

(a)

(b) 250

250 CHO-K1 HEK 293T

*

CHO-K1 HEK 293T

200

Transfection efficiency (%)

Cellular uptake (%)

* 150

100

* *

50

* *

* *

0 Control

CPZ

Gen

SA

MCD

Mon

200

*

* *

150

100

* * *

50

* *

0

PVP

Control

CPZ

Gen

SA

MCD

Mon

PVP

(c) 120 CHO-K1 HEK 293T

100

Viability (%)

80

60

40

20

0 Control

(d) Control

CPZ

genistein

CPZ

Gen

SA

Sodium azide

MCD

Mon

methyl-βcyclodextrin

PVP

Monensin

PVP

CHO-K1

HEK293 T (e) CHO-K1

HEK293 T

Fig. 2. Effect of the endocytic inhibitor on (a) cellular uptake efficiency, (b) transfection efficiency, (c) cell viability, (d) images of cells transfected with rhodamine labeled BSA-OC/pDNA polyplex, and (e) images of cells transfected by pEGFP-C3 plasmid. Measurements were in duplicate with error bars representing the 13

standard deviation. Stars (*) indicate the significant difference as compared with control. 328 329

3.4. Intracellular trafficking of the polyplexes

330

The caveolae-mediated pathway can escape the endo-lysosome compartments and thus deliver the DNA　cargo

331

without degradation [39]. But, it is completely not proven that polyplex internalized via caveolar vesicle could

332

always escape the endo-lysosome degradation. Because a few studies demonstrate that in some cases

333

cavesosome vesicles can fuse with acidic compartments and polyplex faces the low pH condition [40]. Hill et

334

al. have stated that the decrease level of caveolar coat protein (i.e., cavin) expression in caveolin leads to diffuse

335

of caveolin in the plasma membrane and become internalized into the endo-lysosome compartment [41].

336

However, the internalization route and endosome escape ability of polyplex play a significant role in enhancing

337

transfection efficiency [42]. Therefore, the intracellular distribution of BSA-OC/pDNA polyplex in both cell

338

lines was studied by confocal microscopy. To understand the intracellular distribution of polyplex, the BSA-OC

339

vector, cell nuclei, and lysosome were labeled with FITC, Hoechst, and LysoTracker Red. The FITC-labeled

340

BSA-OC was used to prepare polyplex with pDNA. Fig. 3 (a) and (b) represented the merged image of FITC

341

labeled BSA-OC/pDNA polyplex with acidic organelles dye (LysoTracker Red). The quantitative analysis of

342

confocal images suggested that more than 70% of polyplex escaped the lysosome in both cell lines in Fig. 3 (c).

343

Thus, most of the polyplexes escape the endosome and prevent polyplex degradation by lysosome enzymes. In

344

addition, the results of acidification inhibitors on transfection efficiency and uptake pathways demonstrate the

345

importance of the endosome-lysosome system as a gene carrier. The treatment with acidification inhibitors,

346

such as monensin and PVP, shows no inhibition effect on transfection efficiency, suggesting that acid

347

organelles did not play a role in the transport process of polyplex. The acidic lysosomal compartment was not

348

involved in the internationalization of polyplex and was thus internalized via a neutral pH condition through the

349

caveolar pathway in both cell lines.

350

14

351

(b)

(c) Percentage (%) colocalized/noncolocalized polyplex

(a)

120 Colocalized with LysoTracker Non-colocalized with LysoTracker 100

80

60

40

20

0 CHO-K1

HEK293T

Biopolymer/pDNA polyplex

Fig. 3. Confocal microscopy image of the intracellular distribution of BSA-OC/pDNA polyplexes with LysoTracker in (a) CHO-K1, (b) HEK 293T cells, (c) Confocal images of both cell lines were quantified by IN Cell Investigator software. The scale bar is 10 μm. The white box indicated the zoomed region of the cells showing colocalization. Blue fluorescence indicated the nuclei stained with Hoechst 33342. Green fluorescence indicated the FITC-labeled OC-modified proteins, and red fluorescence indicated the lysosome stained with LysoTracker Red. 352 353

Table 1 The internalization pathways followed by modified nanoparticles in literature. Nanoparticle composition Particle Cell line Pathway transfection size efficiency i. Self-branched and 76 nm HeLa cells CvME 30% trisaccharide-substitutes chitosan oligomer (SBTCO), 69 nm CME and CvME 5% ii. Linear chitosan (LCO) i. Hydrophobically modified 359 nm HeLa cells CvME, CME, and uptake of HGC glycol chitosan (HGC) macropinocytosis. nanoparticle was ii. Glycol chitosan (GC) 23% higher than 310 nm Not mentioned. GC Alginate-chitosan nanoparticles 157 nm 293T, CME and CvME. 293T- 15-20% COS7, and COS7- 1-3% CHO- negligible CHO cells Human serum albumin-DOTAP HeLa cells CME and endo- 1.2 *104 DOPE/DNA (HSA-lipoplexes) lysosome system. luciferase (ng)/protein (mg) i. Zein-OC 382 nm CHO-K1 CvME and 84% ii. OVA-OC 110 nm and HEK Lipid-mediated 70% pathways. 293T cells BSA-OC 120 nm CHO-K1 CvME pathway. 90% and HEK 293T cells 15

Ref [43]

[44]

[45] [46] [20] This study

354 355

Table 2 Summary of endocytic inhibitor on cellular uptake using BSA-OC polyplex Inhibitor

Function

Concentration

Cellular uptake CHO

HEK

Chlorpromazine

Clathrin-mediated endocytosis inhibitor

30 µM

+

+

Genistein

Caveolae-mediated endocytosis inhibitor

200 µM

-

-

Sodium azide

Active transport inhibitor

1%

-

-

Methyl-β-cyclodextrin

Cholesterol dependence endocytosis inhibitor

10 mM

-

-

Monensin

Block transport from Golgi complex to plasma membrane.

3 µM

±

±

Polyvinylpyrrolidone

Intracellular vesicular swelling

1mg/ml

±

±

356 357 358

Symbol (+) sign indicates the cellular uptake increased after treatment with a particular endocytic inhibitor as compared with control. Symbol (-) sign indicates the transfection efficiency decreased. Symbol (±) indicates no significant difference as compared with control.

359

3.5. Evaluation of endo-lysosome membrane integrity

360

To investigate the endo-lysosomal membrane integrity of CHO-K1 and HEK 293T cells after treatment with the

361

BSA-OC vector, the acridine orange (AO) staining technique was used. AO is a lysosomotropic weak base and

362

cell-permeable dye that shows diffuse green fluorescence in an uncharged state. The protonated form of AO

363

shows red fluorescence when it accumulates in acidic compartments. The distribution of red and green

364

fluorescence intensity in the organelles helps to understand the lysosome membrane integrity [47]. As

365

previously reported, cells treated with AO exhibit red fluorescence in the endosome and diffuse green

366

fluorescence in the cytoplasm in low pH conditions. An increase in orange-red fluorescence indicates cell

367

apoptosis [48]. Klimazewska-Wisniewska et al. performed AO staining to study cell autophagy. Upon treatment

368

with drugs, such as fisetin and paclitaxel, the A549 cells undergo autophagy and show high red fluorescence

369

intensity compared to positive control cells, representing an increase in acidic vesicular organelles (AVOs) [49].

370

As shown in Fig. 4 (a) and (b), cells transfected with BSA-OC/pDNA polyplex slightly increased the red

371

fluorescence intensity and had no significant difference from the controls. The cells transfected with BSA-

372

OC/pDNA polyplex did not induce the formation of AVOs. The cells transfected with Fe3O4@SiO2-SS-

373

PEI/siRNA show high colocalization of red and green fluorescence, confirming efficient endosomal disruption 16

374

and the efficient release of siRNA [50]. In the present study, HEK 293T cells show high entrapment of polyplex

375

with lysosomes (30%) compared to CHO-K1 (20%), which may disrupt lysosomes from releasing the entrapped

376

polyplex and further increase the red fluorescence intensity of HEK 293T after staining with AO. The data

377

suggest that BSA-OC is safe to transfer genetic material into mammalian cells.

378 379 (b)

(a) CHO-K1 cells (Control)

160

HEK 293T cells (Control)

140 Red fluroscence intensity

CHO-K1 cells (treated with BSA-OC)

Control Cells treated with BSA-OC vector

120 100 80 60 40 20

HEK 293T cells (treated with BSAOC)

0 CHO-K1

HEK 293T

Fig. 4. Endosome-lysosomes membrane disruption study by using acridine orange. (a) Confocal image of cells treated with acridine orange in presence/absence of BSA-OC vector. (b) Confocal images of both cell lines were quantified by IN Cell Investigator software. The scale bar is 10 μm. 380 381

3.6. Transfection in suspended cell lines

382

Transfection in a suspended cell line remains a major challenge. The most common reason for low transfection

383

efficiency in suspended cells is the low attachment ability of polyplex to the cell membrane[51]. To date, many

384

different approaches have been designed to deliver genetic material into a suspended cell line [52]. We found

385

that BSA-OC/pDNA polyplex showed a high endosome escape ability and gene transfer efficiency in the

386

adherent cell line. Therefore, we decided to test the transfection efficiency in a suspended cell line using BSA-

387

OC/pDNA polyplex. Hattori et al. showed that the reverse transfection procedure shows high transfection

388

efficiency and cell viability compared to the forwarding transfection procedure. In this procedure, the cells and

389

transfection complex mixed together at the time of seeding cells in the plate [53]. We chose the reverse 17

390

transfection to transfect a lymphoma cell line (BCRC 60252) and hybridoma cells (BCRC 60427 and CRL-

391

1754). Fig. 5 shows the transfection efficiency of BSA-OC vector/pDNA polyplex at various weight ratios from

392

10:1 to 60:1. The weight ratio was defined as different concentrations of the vector (10 μg to 60 μg) with 1 μg

393

DNA per well. By increasing the weight ratio, the transfection efficiency was increased and reached the

394

maximal 80%. BCRC 60252 cells showed high gene transfer efficiency in 40:1 and 60:1 compared to

395

hybridoma cells. Many authors have designed different techniques to transfect the suspended cells, such as

396

droplet electroporation and laser irradiation [51, 52]. But the obtained transfection efficiency and cell viability

397

remain unsatisfactory (the transfection efficiency was in the range of 60–72% and cell viability was 70–75%).

398

Furthermore, BSA-OC/pDNA polyplex could transfect three suspended cell lines with >85% cell viability (Fig.

399

5). The benefits of using the BSA-OC vector as a non-viral gene delivery vector are its simplicity, easy

400

conjugation, high transfection efficiency, and high gene expression. (a)

(b) 120

120

* *

80

*

*

80

* 60

252 cells 427 cells CRL cells

100

* * *

*

Viability (%)

Transfection efficiency (%)

100

252 cells 427 cells CRL cells

*

60

40

40

20

20

0

0 10:1

20:1

40:1

Control

60:1

10:1

20:1

40:1

60:1

Weight ratio (biopolymer: DNA)

Weight ratio (vector: DNA)

(c)

BCRC 60252 cells

BCRC 60427 cells

CRL-1754 cells

Fig. 5. GFP expression in suspended cells transfected by BSA-OC/pDNA polyplexes at various weight ratios (a) percentage of GFP-expressing cells, (b) cell viability after transfection, and (c) florescence images of BCRC60252, BCRC 60427, and CRL-1754 cells transfected at a weight ratio of 60:1. The images were obtained at a magnification of 10×. Measurements were effectuated in duplicate with error bars representing the 18

standard deviation. Stars (*) indicate the significant difference as compared with the weight ratio of 10:1. 401 402

3.7. Blood cell hemolysis

403

One of the major drawbacks of using the cationic polymer as a gene delivery vector for clinical purposes is the

404

non-specific interaction of the polymer with red blood cells (RBCs) that causes the aggregation of RBCs and

405

the risk of ischemic stroke [54]. The interaction of negatively charged blood components with positively

406

charged macromolecules causes cell lysis and releases the hemoglobin from RBCs [55]. Therefore, it is

407

essential to test the suitability of the cationic macromolecule when it interacts with the cell membrane with no

408

damaging effect. We tested the hemolytic activity of BSA-OC/pDNA polyplex at different weight ratios (10:1–

409

300:1) using mice blood. Triton X-100 (1%) and PBS (pH 7.4) were used as a positive control and a negative

410

control, respectively. The treatment of blood with Triton X-100 is considered 100% hemolysis, while treatment

411

with PBS is considered 0% hemolysis. As shown in Fig. 6, by increasing the weight ratio from 60:1 to 300:1,

412

the percentage of hemolysis increased from 2% to 34%, and no significant hemolytic effects were observed up

413

to 100:1 (500 μg/mL of BSA-OC), indicating no detectable membrane disruption of the red blood cell. The

414

BSA-OC vector showed high bio-compatibility toward blood cells and could be used for gene delivery using

415

parenteral administration.

40

*

Hemolysis %

30

*

20

10

0 0

10:1

20:1

40:1

60:1

80:1

90:1

100:1 200:1 300:1

Weight ratio (vector: DNA)

Fig. 6. Hemolytic activity of BSA-OC vector/pDNA polyplex at different weight ratio against RBCs. The polyplexes were prepared by mixing increasing concentration of vector from 10 µg to 300 µg with 1 µg of pDNA. Stars (*) indicate the significant difference (p<0.01) as compared with the weight ratio of 10:1. 416 417

3.8. Biocompatibility of polyplexes in the in vivo condition

418

High transfection efficiency and endosome escape ability of the BSA-OC vector were obtained in the in vitro

419

condition. The gene transfer ability was impeded in the in vivo condition due to the presence of inhibitory 19

420

biological fluid and the extracellular barrier [56]. The efficiency of BSA-OC vector for gene delivery in an in

421

vivo environment was examined using fluorescent tdTomato as a reporter gene. Two different vector/DNA ratios,

422

i.e., 1:1 and 1.5:1, were injected intravenously into CD1 (ICR) mice via a tail vein injection. The size and zeta

423

potential of two polyplexes was around 250±23nm and 28±3mV, respectively. Additionally, a mouse injected

424

with a high dosage of the vector (200 μg) was used to study the biocompatibility of the vector. Mice

425

individually injected with DNA (90 μg) and PBS were used as the controls. As shown in Fig. 7 (a) and (b), a

426

high level of tdTomato expression was observed in the 1:1 polyplex in the liver containing the reticulo-

427

endothelial system compared to that of 1.5:1 polyplex. The high tdTomato expression in RES organ is might

428

be due to the interaction of OC molecules with the mannose receptors of liver non-parenchymal cells. The

429

mixing ratio plays a significant role in successful transfection efficiency. Song et al. mentioned that a slight

430

excess of positive charge is suitable for gene transfection [57]. However, the large positive charge retards the

431

release of DNA from the complex and showed lower gene expression. By increasing the injection time period,

432

the fluorescence expression was decreased significantly. The fluorescence of tdTomato expression in control

433

mice was not detected; this indicated that even a high dose of DNA alone cannot show fluorescent protein

434

expression and will be degraded in an in vivo condition. One week after polyplex injection, the mouse did not

435

show any sign of apparent weakness and spontaneous animal death, suggesting the biocompatibility of the

436

vector in vivo environment. In conclusion, the data demonstrate that BSA-OC is a biocompatible vector for gene

437

delivery. (a)

DAY 2 A

Polyplexes

DAY 3 B

Control (b)

DAY 7

A

B

A

B

Polyplexes

Control

Polyplexes

Control

20

3.5e+5 Mice 1 Mice 2

Fluroscence intensity

3.0e+5 2.5e+5 2.0e+5 1.5e+5 1.0e+5 5.0e+4 0.0 2

3

7

Days after innjection

Fig. 7. In vivo bioluminescence imaging of CD1 mice with intravenous injection of polyplex sample. (a) Panel A is mice injected with prepared polyplex at a weight ratio of BSA-OC/pDNA polyplex (1) 60 µg vector/60 µg pDNA, and (2) 90 µg vector/60 µg pDNA; using tdTomato as a reporter gene. Panel B is the control mice injected with (3) vector only (200 µg), (4) DNA only (90 µg), and (5) PBS only. The mice were imaged using the IVIS spectrum after post-injection at a different time point (Day 2, 3, and 7), (b) quantitative estimation of tdTomato expression by using Image J software (mice 1: 60 µg vector/60 µg pDNA; mice 2: 90 µg vector/60 µg pDNA). 438 439

Conclusion

440

EtBr and agarose gel assay showed that BSA-OC had a good DNA condensation ability and a high DNA-

441

releasing capacity compared to zein-OC and OVA-OC. The data confirm the hypothesis that the type of protein

442

moiety influenced the surface charge of the vector and in thus balancing the good correlation between the DNA

443

binding and release capacity. Further, the internalization data indicated that the caveolae/lipid-mediated

444

pathway was the dominant pathway for the uptake of BSA-OC/pDNA polyplex in both cell lines CHO-K1 and

445

HEK 293T. The colocalization of polyplex with LysoTracker illustrates that BSA-OC/pDNA polyplex showed

446

70% endosome escape ability in the tested cells. By studying the endo-lysosome membrane integrity of BSA-

447

OC, this vector delivered the gene without inducing cell death or apoptosis in all tested cell lines. The

448

developed transfection process for suspended cell lines showed high gene expression with good cell viability

449

using BSA-OC/pDNA polyplex. Furthermore, the hemolysis percentage was less than 2% at the 500 μg/mL of

450

BSA-OC and showed good gene expression in the in vivo condition. Our data demonstrate the synthesized

451

BSA-OC vector is a promising candidate for plasmid delivery application.

452 453

Conflict of interest The authors declare that they have no conflict of interest. 21

454

Acknowledgements

455

We express gratitude to Ministry of Science and Technology (MOST 106-2221-E-182-050, 108-2221-E-182-

456

039), Chang Gung University (BMRP 758) and Chang Gung Memorial Hospital (2J0161, 2H0071, 2H0072) for

457

funding and supporting this research.

458

References

459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498

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594 595

24

Supporting Information

596 597 598

Table S1. Fluorescence quench of EtBr-DNA complex after the addition of vectors at various weight ratios. Weight ratio (vector/pDNA)

Fluorescence quench

599

Zein-OC BSA-OC OVA-OC 0:1 100 ± 0 100 ± 0 100 ± 0 50:1 70 ± 0.4* 78 ± 0.1* 84 ± 0.5 100:1 63 ± 0.7* 73 ± 0.3* 71 ± 0.3* 200:1 51 ± 0.2* 60 ± 0.2* 67 ± 0.1* 300:1 18 ± 0.3* 20 ± 0.1* 31 ± 0.7* Fluorescence quench was effectuated in duplicate and stars (*) indicate the significant difference as compared

600

with weight ratio of 0:1.

601

1.0

400 Particle size (nm) PDI

0.8

0.6

PDI

Particle size (nm)

300

200 0.4

100 0.2

0

0.0 pH 3

pH 5

pH 7

pH 9

pH 11

Fig. S1. Effect of different pH conditions on the particle size and poly-dispersity index of the BSA-OC vector. 602

25

Fig. S2. Agarose gel electrophoresis of vector/pDNA polyplex at a weight ratio of 60:1. Treatment with heparin (8 μg/μl) was used to analyze the DNA releasing efficiency from polyplexes. Naked DNA was used as a control. 603 604

26