Accepted Manuscript

Title: Co-delivery of antineoplastic and protein drugs bychitosan nanocapsules for a collaborative tumor treatment

Author: Dong-Yan Wu Yu Ma Xiao-Shuang Hou Wen-JieZhang Pei Wang Huan Chen Bo Li Can Zhang Ya Ding

PII: S0144-8617(16)31292-9DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2016.11.027Reference: CARP 11739

To appear in:

Received date: 27-7-2016Revised date: 9-11-2016Accepted date: 9-11-2016

Please cite this article as: Wu, Dong-Yan., Ma, Yu., Hou, Xiao-Shuang., Zhang, Wen-Jie., Wang, Pei., Chen, Huan., Li, Bo., Zhang, Can., & Ding, Ya., Co-delivery ofantineoplastic and protein drugs by chitosan nanocapsules for a collaborative tumortreatment.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.11.027

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Co-delivery of antineoplastic and protein drugs by chitosan

nanocapsules for a collaborative tumor treatment

Dong-Yan Wu a,†, Yu Ma a,†, Xiao-Shuang Hou a, Wen-Jie Zhang a,b, Pei Wang a,b, Huan Chen c, Bo

Li a, Can Zhang b, Ya Ding a,*

a State Key Laboratory of Natural Medicines and Department of Pharmaceutical Analysis, China

Pharmaceutical University, Nanjing 210009, China.

b Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery,

China Pharmaceutical University, Nanjing 210009, China.

c Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical

University, Nanjing 210009, China

† The first two authors contributed equally to this work.

* Corresponding author. E-mail: ayanju@163.com (Y. Ding).

Highlights:

Nanocapsules were prepared by amino acid derivatives of chitosan.

A simple and quick emulsification method to prepare nanocapsules.

A general co-delivery system for chemical drug and protein.

Enhanced tumor cell inhibition and apoptosis in HeLa cells.

Abstract

Although combination delivery (co-delivery) shows much superiority in the defect

compensation of single-agent therapy, the construction and application of co-delivery systems are

still challenging, especially for protein-based joint systems. In this work, a series of chitosan

(CS)-amino acid derivatives (Arg-CS, Lys-CS, and Phe-CS) with different degrees of substitution

(DS) were synthesized to prepare CS nanocapsules (CNCs) using a simple emulsification method in

the presence of linoleic acid (LA). The hydrophobic drug can be loaded in LA droplets, and a

positively charged protein stabilized the optimized Arg-CS nanocapsules (Arg-CNCs) on their

negative surfaces. The in vitro antitumor efficacy of Arg-CNCs co-delivering paclitaxel and

recombinant human caspase-3 was evaluated in HeLa cells. The co-delivery system displayed much

lower IC50 values and a higher percentage of apoptotic cells compared with the control groups. This

system provides a promising and universal strategy for co-delivery, leading to collaborative tumor

treatment.

Keywords: chitosan, amino acid derivatives, nanocapsules, co-delivery, caspase-3

1. Introduction

Combination delivery (co-delivery) is a delivery strategy to simultaneously deliver more than

one type of therapeutic molecule. This strategy has attracted much attention due to its superiority in

the defect compensation of single-agent therapy, synergetic effects via different mechanisms, and

anti-multidrug resistance especially in tumor therapy (Khan, Ong, Wiradharma, Attia, & Yang, 2012;

Creixell & Peppas, 2012; Chen, Chen, & Shi, 2014). Combination systems delivering multiple

chemotherapeutics (Kolishetti et al., 2010; Duong, Marquis, Whittaker, Davis, & Boyer, 2011;

Wang et al., 2013) or nucleic acids (Tan, Zhang, & Huang, 2002) and chemical therapy-nucleic

acid-based joint systems (Wang, Gao, Ye, Yoon, & Yang, 2006; Saad, Garbuzenko, & Minko, 2008;

Kaneshiro & Lu, 2009; Zhu et al., 2010; Xiong & Lavasanifar, 2011; Zhu, Meng, Gao, & Hanagata,

2011) have been constructed and display pronounced advantages compared with monotherapy.

It is worth noting that reports of co-delivery systems containing therapeutic proteins are rare

(Martin-Ortigosa, Valenstein, Lin, Trewyn, & Wang, 2012; Borges et al., 2008; Yeste, Nadeau,

Burns, Weiner, & Quintana, 2012), although protein therapy is believed to be the most direct and

safe approach for treating diseases by delivering proteins into the cell to replace the dysfunctional

protein. This is partly due to the challenge of protein delivery arising from the poor stability and

low cellular uptake of proteins (Yan et al., 2010; Torchilin & Lukyanov, 2003; Gu, Biswas, Zhao, &

Tang, 2011). Therefore, a combination carrier possessing the capability of loading both chemical

and protein agents is expected to improve the treatment efficacy of diseases, especially cancer.

Chitosan (CS) is a mucoadhesive polysaccharide capable of opening the tight junctions

between epithelial cells (Rinaudo, 2006). CS-based delivery systems have been extensively and

intensively studied to enhance the absorption of small drug molecules (Kumar, Muzzarelli,

Muzzarelli, Sashiwa, & Domb, 2004), nucleic acids (Mao, 2001), and proteins (Amidi,

Mastrobattista, Jiskoot, & Hennink, 2010). To improve the biocompatibility of CS derivatives, a

functional moiety chosen from endogenous substances, such as amino acids, is desirable (Liu,

Zhang, Cao, Xu, & Yao, 2004). These amino-rich CS derivatives demonstrating good

anticoagulation property (Rinaudo, 2006; Song et al., 2010), desirable cell penetrating function

(Zhang et al., 2011; Lv et al., 2011), and lysosomal escape ability based on the proton sponge effect

(Behr, 1997; Yezhelyev, Qi, O’Regan, Nie, & Gao, 2008), can be promising materials to fabricate a

co-delivery system for joint chemical and protein therapy.

In this work, a series of CS-amino acid derivatives with different amino acids (Arg, Lys, and

Phe) and degrees of substitution (DS) are synthesized (Fig. 1A) to prepare CS nanocapsules (CNCs,

Fig. 1B). The preparation method involves a simple and quick emulsification process of an aqueous

solution containing linoleic acid (LA) using an amalgamator (Yang et al., 2011; Duncan et al.,

2015). Different from previous reports, CS-amino acid derivatives are employed instead of

Arg-modified gold nanoparticles (Arg-GNPs) to increase the biocompatibility and biodegradability

of the nanocapsule system. The nanocapsule is composed of a nano droplet of LA coated by the

above amino acid-modified CS derivatives via electrostatic interaction. The polymeric long chain

twining on the surface of the LA nanodroplet is expected to demonstrate higher stability compared

to uncrosslinked Arg-GNPs. LA oil droplets provide a hydrophobic environment to load insoluble

molecules. The positively charged protein will further adsorb and stabilize capsules with negative

zeta potentials. After the preparation process optimization of CNCs, Arg-CS nanocapsules

(Arg-CNCs, with DS of Arg-CS equal to 20.4%) were chosen as the test system to characterize and

investigate in vitro stability and drug release. We hypothesize that the prepared Arg-CNCs could be

a promising co-delivery system to improve the treatment efficacy of chemical and protein agents,

especially for cancer.

2. Materials and methods

2.1. Materials and characterizations

CS (molecular weight of 25.0 kDa, degree of deacetylation of 88.9%) was supplied by

Xincheng Biochemical Products Co., Ltd (Nantong, China). L-Arginine (Arg, 98%), L-Lysine (Lys,

98%), L-Phenylalanine (Phe, 99%), N-hydroxysuccinimide (NHS, 98%), and

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 98.5%) were purchased

from Aladdin Reagent Co., Ltd (Shanghai, China). Linoleic acid (LA, ≥99%), human transferrin

(Trf, ≥98%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were

purchased from Sigma-Aldrich (Shanghai, China). Nile Red (NR, 98%) was supplied by Nanjing

Duly Biotech Co., Ltd (Nanjing, China), and green fluorescent protein (GFP, ≥95.0%) was

obtained from ProSpec-Tany Technogene Ltd. (Israel). Recombinant Human Caspase-3

(Casp-3, >5,000 pmols/min/μg) was obtained from Sino Biological Inc. (Beijing, China). Unless

otherwise stated, all starting materials were obtained from commercial suppliers and used without

further purification. Solvents were dried using standard procedures. All aqueous solutions were

prepared using deionized water (>18 ΩU, Purelab Classic Corp., USA).

The chemical structures of CS derivatives in KBr discs were determined using a Tensor-27

Fourier transform infrared spectrometer (FT-IR, Bruker, USA) equipped with a liquid nitrogen

cooled mercury cadmium telluride (MCT) detector. 1H and 13C nuclear magnetic resonance

spectroscopy (1H and 13C NMR) spectra were recorded on a Bruker AV-500 NMR Spectrometer

(Bruker, Germany). Elemental analysis (EA) was performed using an Element Vario EL III analyzer

(Hanau, Germany). The hydrodynamic diameter and zeta potential of nanocapsules were measured

by a Zetasizer 3000HS instrument (Malvern, UK). For the morphological characterization, all

samples were imaged using a JEM-200CX transmission electron microscope (TEM, JEOL, Japan)

with an accelerating voltage of 200 kV. Analytical reverse-phase high performance liquid

chromatography (RP-HPLC) was performed using a Shimadzu-20AT series (Shimadzu, Japan) with

a Thermo BDS HYPERSIL C18 reversed-phase chromatography column (150 × 4.6 mm) and a UV

detector set at 227 nm. A mobile phase of methanol/water (70/30, v/v) was used with the flow rate

set at 1 mL/min (30 °C).

2.2. Synthesis of amino acid-modified CS derivatives

CS powder was dissolved in an aqueous solution of acetic acid (0.1%, v/v) in a three-neck flask.

Amino acids (Arg, Lys, or Phe), NHS, and EDC·HCl with a molar ratio of 1:1:3 were activated

previously in an aqueous solution of acetic acid (0.1%, v/v) for 2 h. The activated solution was

added into the CS solution and stirred at room temperature at a pH value of 6 for 48 h. The molar

ratios of amino acid/CS sugar units were set at 2.5:1, 5:1, 10:1, and 20:1. After the reaction, the

mixture solution was filtered. The filtrate was dialyzed against frequently changed distilled water

for 2 days. After the insoluble compound in the dialysate was filtrated, a light yellow powder of the

final product was obtained from the filtrate by the freeze-drying method (denoted as Arg-CS,

Lys-CS, and Phe-CS).

Arg-CS: 1H NMR (300 MHz, D2O): 4.79 (H1+DOH), 4.04~3.43 (H3~H6), 3.21 (H8), 2.89

(H2), 2.51~2.43 (H11), 1.84 (H9), 1.64 (H10). 13C NMR (300 MHz, D2O): 176.5 (C7), 162.0

(-COCH3), 159.4 (C12), 103.8 (C1), 80.1 (C4), 77.3 (C5), 74.5 (C3), 62.5 (C6), 57.6 (C2), 45.3

(C8), 43.2~30.8 (C9~C11), 24.4 (-COCH3).

Lys-CS: 1H NMR (300 MHz, D2O): 4.79 (H1+DOH), 4.04~3.43 (H3~H6), 3.30 (H8), 2.90

(H2), 2.43 (H12), 1.72 (H9), 1.51 (H11), 1.36 (H10). 13C NMR (300 MHz, D2O): 175.7 (C7),

161.7 (-COCH3), 103.8 (C1), 89.9 (C4), 77.4 (C5), 75.0 (C3), 62.7 (C6), 57.8 (C2), 45.3 (C8),

43.9~30.0 (C9~C11), 24.6 (-COCH3).

Phe-CS: 1H NMR (300 MHz, D2O): 7.33~7.24 (H10~12), 4.79 (H1+DOH), 3.88~3.74

(H3~H6), 3.36 (H9), 3.18 (H8), 2.88 (H2). 13C NMR (300 MHz, D2O): 176.7 (C7), 132.0 (C10),

131.3 (C11), 129.8 (C12), 101.1 (C1), 79.3 (C4), 77.4 (C5), 73.4 (C3), 62.7 (C6), 57.9 (C2), 45.3

(C8), 40.3~37.4 (C9~C11), 24.3 (-COCH3).

2.3. Preparation of CS derivative nanocapsules

Blank CNCs: The preparation method refers to the first step of a previous procedure for

GNP-stabilized nanocapsules (Yang et al., 2011; Duncan et al., 2015). Briefly, 1.0 μL of LA was

added in 499 μL of phosphate buffer (PB, 5.0 mM, pH 7.4) containing the CS derivative (1~100

μM). The mixture was emulsified using an amalgamator at a speed of 5,000 rpm for 99 s. The

resultant emulsified solutions were allowed to stand for 10 min to obtain CNCs (denoted as

Arg-CNCs, Lys-CNCs, and Phe-CNCs).

PTX-loaded Arg-CNCs (Arg-CNCs-PTX): 1.0 μL of LA suspension containing 80 mg/mL PTX

were emulsified in 499 μL of Arg-CS solution (30 μM in 5.0 mM PB, pH 7.4) using an

amalgamator (speed of 5,000 rpm for 99 s). The resulting emulsified solutions were allowed to

stand for 10 min to obtain Arg-CNCs-PTX.

Drug-and-protein-loaded Arg-CNCs: The preparation of Arg-CNCs loaded with both NR and

GFP (Arg-CNCs-NR/GFP) is described here as an example. NR was dispersed in LA at a

concentration of 5 mg/mL. Next, 1.0 μL of LA containing NR was emulsified in 499 μL of Arg-CS

solution (30 μM in 5.0 mM PB, pH 7.4) using an amalgamator (speed 5,000 rpm for 99 s). After 10

min of standing, 10 μL of GFP (2 μM) was mixed and incubated with 50 μL of NR-loaded

Arg-CNCs solution for 10 min to obtain Arg-CNCs-NR/GFP.

The preparation of Arg-CNCs-PTX/Trf and Arg-CNCs-PTX/Casp-3 was similar to the above

procedure, except for using a LA suspension containing 80 mg/mL of PTX to prepare

Arg-CNCs-PTX and then adding 50 μM Trf or 6.67 μM Casp-3 into the solution to finally obtain

Arg-CNCs-PTX/Trf or Arg-CNCs-PTX/Casp-3, respectively.

2.4. Stability

Arg-CNCs-PTX and Arg-CNCs-PTX/Trf were incubated at 37 °C under the following

conditions: 0.03 M PBS with different pH values (at pH 7.4 and pH 5.5), high ionic strength of 0.2

M PBS at pH 7.4, and 0.03 M PBS solution at pH 7.4 with 2% serum. Size was monitored at 0, 2, 4,

8, 12, 24, and 48 h after the prepared CNCs were added in the above media.

2.5. In vitro drug release

The release of PTX from Arg-CNCs-PTX and Arg-CNCs-PTX/Trf was investigated by a

dialysis method. The test samples containing 320 μg of PTX were dissolved in 1 mL of 0.03 M PBS

at pH 7.4 and pH 5.5, and placed in a dialysis bag (MWCO=3,500 Da). The above PBS solution

with different pH values containing Tween 80 (0.1%, v/v) was employed as the release medium.

The release system was stirred at 150 rpm at 37 °C. At predetermined time intervals, 200 μL

aliquots of the release medium were withdrawn, and the same volume of fresh medium was added.

The in vitro release behaviors of Arg-CNCs-PTX and Arg-CNCs-PTX/Trf were measured by

RP-HPLC analysis. Data are presented as the mean ± SD (n=3).

2.6. Cell culture

Human cervical cancer HeLa cells purchased from the China Center for Type Culture

Collection (Shanghai, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)

containing 10% (v/v) fetal bovine serum (FBS) and incubated at 37 °C in humidified atmosphere

with 5% CO2 for 24 h until they reached 80% confluence.

2.7. Cellular uptake of Arg-CNCs-NR/GFP

Fluorescent dyes NR and GFP, used as model hydrophobic and protein molecules, were loaded

in and on Arg-CNCs to investigate the cellular uptake of Arg-CNCs-NR/GFP. HeLa cells seeded in

cell plates were incubated overnight to reach 80% confluence and then treated with

Arg-CNCs-NR/GFP at a dose of 0.5 μM Arg-CS for 4 h and 8 h. The cells were observed by a laser

scanning confocal microscope (LSCM, Ti-C2, Nikon, Japan) after the nucleus was stained with

Hoechst 33342. The fluorescence intensities of intracellular NR and GFP were further determined

by flow cytometry (BD AccuriTM C6, USA).

2.7. In vitro antitumor efficacy

Cytotoxicity: The in vitro cytotoxicity of different Arg-CNC formulations was determined by a

MTT assay. Briefly, HeLa cells were seeded in a 96-well plate (1×104/well) using DMEM with 10%

FBS and then incubated for 24 h. The cells were then treated with a growth medium containing 5%

FBS and different concentrations of Taxol®, Arg-CNCs-PTX, Arg-CNCs-Casp-3, and

Arg-CNCs-PTX/Casp-3. After 24 h, the cells were incubated in a growth medium containing 0.5

mg/mL MTT for additional 4 h. The MTT-formazan generated by living cells was dissolved in

DMSO, and the absorbance at 490 nm was measured in each well using a microplate reader

(Bio-rad, USA). The cell viability (%) was determined by comparing the absorbance at 490 nm with

control wells containing only the cell culture medium. Data are presented as the means ± SD (n=5).

The concentration of PTX or Casp-3 that inhibited 50% cell growth compared with untreated cells

(IC50) was defined by curve fitting (LOGIT method) using the SPSS 18.0 software.

Apoptosis: HeLa cell apoptosis induced by different Arg-CNC formulations was detected by

flow cytometry (BD AccuriTM C6, USA). HeLa cells were seeded in 6-well plates at a density of

8×105 cells per well and incubated overnight to reach 80% confluence. The cells were treated with

Taxol®, Arg-CNCs-PTX, Arg-CNCs-Casp-3, and Arg-CNCs-PTX/Casp-3 with a PTX dose at 1.0

μM or a Casp-3 dose at 0.63 nM for 24 h and then stained by propidium iodide (PI) and Annexin

V-FITC Apoptosis Detection Kit according to the manufacturer's protocol. The percentage of cells

in each quadrant was evaluated using the BD Accuri C6 software.

3. Results and discussion

3.1. Synthesis and characterization of CS derivatives

The synthesis procedure of amino acid-modified CS is illustrated in Fig. 1A. All of the amino

acid-functionalized CS derivatives can be well dissolved in water. The chemical structures of the

final products were characterized using FT-IR and 1H/13C NMR (Figures 2-4). The DS of CS

derivative was determined by the elemental analysis method (Table 1).

FT-IR spectra of CS, Arg-CS, Lys-CS, and Phe-CS are shown in Fig. 2 from curve a to curve d,

respectively. Curve a shows the characteristic IR features of CS. The absorption bands located at

1380 and 1662 cm−1 are due to the adsorption of the amide III and amide I groups, respectively, and

the band at 1598 cm−1 arises from the bending vibration of –NH2. The absorption band at 1156 cm−1

can be assigned to the asymmetric stretching of the C–O–C bridge. The bands at 1075 and 1033

cm−1 are due to skeletal vibrations of the saccharine structure involving C–O stretching (Ding, Xia,

& Zhang, 2006; Ding, Xia, & Zhai, 2007). In the spectrum of Arg-CS (curve b), the characteristic

peak of the amide C=O vibration at 1659 cm−1 increases, which indicates successful substitution of

carbonyl groups at C2–NH2 in CS. At the same time, no significant decrease is observed at 1568

cm−1 corresponding to the amino N–H deformation vibration, again indicating the successful

grafting of guanidine group on the CS long chain. Similarly, the IR spectrum of Lys-CS (curve c)

also displays an increase in the vibration intensity of both amide and amino groups, demonstrating

the modification of CS by Lys. Moreover, in the IR spectrum of Phe-CS (curve d), in addition to the

above increases at approximately 1689 and 1571 cm-1, benzene infrared absorption is also present,

indicating Phe substitution on the CS molecule.

To further confirm the chemical structure of CS derivatives, the 1H and 13C NMR spectra of CS,

Arg-CS, Lys-CS, and Phe-CS were obtained and are shown in Fig. 3 and 4. The 1H NMR

assignments of CS (Fig. 3, curve a) are as follows: 1H NMR (D2O/F3COOH) δ= 4.76 (H1), δ= 3.09

(H2), δ= 3.81–3.43 (H3, H4, H5, H6), δ=1.96 (NHCOCH3) ppm (Ding, Xia, & Zhang, 2006; Ding,

Xia, & Zhai, 2007). Compared with the 1H NMR (D2O) spectrum of CS, the 1H NMR (D2O) spectra

of Arg-CS and Lys-CS (Fig. 3, curves b and c) show new peaks at around δ= 3.20 ppm. This peak is

assigned to the C8-H atom, i.e., the proton on the carbon atom connected by amide and amino

groups at the same time (Fig. 1A). Other signals attributed to protons in the amino acid moiety

appear and are in the chemical shift range below δ 2.75 ppm (Fig. 3, curves b and c), except in the

case of Phe-CS for the new proton signals on C8 and C9 located at 3.11 and 3.35 ppm, respectively.

The benzene ring protons appear at around δ 7.33 ppm (Fig. 3, curve d).

Moreover, the 13C NMR spectra of CS and its derivatives are shown in Fig. 4. The 13C NMR

assignments of CS (Fig. 4, curve a) are as follows: 13C NMR (D2O/F3COOH) δ= 97.5 (C1), δ= 76.5

(C4), δ= 75 (C5), δ= 70 (C3), δ= 60 (C6), δ =55.6 (C2) ppm (Ding, Xia, & Zhang, 2006; Ding, Xia,

& Zhai, 2007; Ding, Gu, & Xia, 2009). Similar to the 1H NMR spectra, the detailed attribution of

carbon atoms in Arg-CS, Lys-CS, and Phe-CS is shown in Fig. 4 (curves c-d), respectively.

The DS of amino acid-modified CS derivatives were detected and calculated using the

elemental analysis method (Shendage, Fro1hlich, & Haufe, 2004). From the results listed in Table 1,

it can be found that with the increase of the molar ratio of amino acid to saccharide units, the DS of

CS derivative increase accordingly. In addition, the DS values are different according to the amino

acid species. For example, at the same molar ratio of amino acid to saccharide units, the descending

order of DS (%) is Lys-CS > Arg-CS > Phe-CS. This phenomenon is due to the steric hindrance of

the chemical structure of amino acids.

3.2. Preparation and optimization of CNCs

To optimize the preparation process of CNCs, the different derivatives, DS, and concentrations

in the preparation solution were adjusted to investigate the hydrodynamic diameter, surface

potential, and storage stability of the as-prepared CNCs. The plots of hydrodynamic diameter and

zeta potential are demonstrated in Fig. 5A-C. The physical parameters of CNCs under the optimized

preparation process are presented in Fig. 5D. Among them, Arg-CNCs with DS of 20.4% show the

smallest size, desirable zeta potential value (Everett, 1998), and highest storage stability. The

aqueous solution of Arg-CNCs remained clear for more than 1 week, whereas visible precipitants of

Lys-CS and Phe-CS nanocapsules were found in the solution within 1-2 days. Therefore, Arg-CNCs

prepared with Arg-CS (DS 20.4%, 30 μmol/L-1) were used for the following studies.

3.3. Characterization, stability, and in vitro drug release of Arg-CNCs-PTX/Trf

To evaluate the general protein loading property of nanocapsules, three different proteins, i.e.,

Trf, GFP, and Casp-3, were used in this work. Trf was used in the preparation and characterization

and for investigating the stability and protection effect of protein on the nanocapsules. GFP with

green fluorescence was employed for the studies of cellular uptake. In vitro antitumor efficacy

involving Casp-3 would demonstrate the efficacy of therapeutic protein on tumor cells. First, the

pharmaceutical properties of the co-delivery system were evaluated. PTX and Trf were loaded in

and on Arg-CNCs as the model chemotherapeutic agent and protein molecule, respectively. The

morphologies of blank Arg-CNCs, Arg-CNCs-PTX, and Arg-CNCs-PTX/Trf are shown in Fig. 6A,

and their hydrodynamic diameters and zeta potentials are shown in Fig. 6B. Compared with blank

Arg-CNCs (Fig. 6A, image a), Arg-CNCs dyed with heavy metal salt (phosphomolybdate here, Fig.

6A, image b) show a clear spherical morphology and many dark dots. This is due to the binding of

phosphomolybdate with positively charged Arg-CS on the surface of nanocapsules. Comparing the

hydrodynamic diameters of Arg-CNCs (192.0 ± 4.2 nm) and Arg-CNCs-PTX (220.1 ± 6.4 nm), the

loading of PTX in the inner core of nanocapsules increases their sizes and leads to a dense core that

can be observed, even without the dyeing of phosphomolybdate (Fig. 6A, image c). The potentials

show no obvious change before (-32.9 ± 1.8 mV) or after (-31.4 ± 0.8 mV) the drug loading. It is

interesting that although the potential is enhanced (-9.2 ± 1.6 mV) due to the absorption of Trf, the

capsule size of Arg-CNCs-PTX/Trf does not increase much (223.5 ± 3.2 nm) compared with the

hydrodynamic diameter of Arg-CNCs-PTX (220.1 ± 6.4 nm). The small change of capsule size

shows the stabilization and contraction effect induced by the protein molecules. The TEM image of

Arg-CNCs-PTX/Trf is shown in Fig. 6A, image d.

The stability results of Arg-CNCs-PTX with and without Trf modification in different media

are shown in Fig. 7A. Under physiological conditions, such as 0.03 M PBS at pH 7.4, both

nanocapsules exhibit almost unchanged particle sizes (Fig. 7A, curves a), which is consistent with

the stability investigation in capsule optimization (section 3.2). A low pH environment, such as pH

5.5, causes a rapid increase in the capsule size (Fig. 7A, curve b). This phenomenon implies the

expansion and decomposition of the capsule structure and its subsequent drug release in the

microenvironment of tumors. It is worth noting that a high ionic strength leads to a large difference

in the stability of Arg-CNCs-PTX versus Arg-CNCs-PTX/Trf. After the media addition of 0.2 M

PBS at pH 7.4, the hydrodynamic size of Arg-CNCs-PTX increases rapidly from approximately 200

nm to 371.5 ± 8.4 nm and then to over 1,000 nm after 4 h (Fig. 7A, curve c in left). In contrast, the

particle size of Arg-CNCs-PTX/Trf changes from approximately 200 nm to ca. 320 nm, and then

this size remains unchanged for over 20 h (Fig. 7A, curve c in right). This indicates the stabilization

and protection effect of protein on the capsule surface, consistent with the contraction phenomenon

observed in the characterization studies above. Moreover, in the presence of 2% serum, both

formulations show good stability in 20 h, indicating again the stability effect induced by proteins

from the serum. Therefore, the modification of Trf on the surface of Arg-CNCs-PTX improves its

stability in all test conditions, especially under the high ionic strength condition. The results show

that the drug-loaded nanocapsules can maintain their stable structure in circulation and achieve drug

release under lower pH values (e.g., pH 5.5), which is confirmed by in vitro drug release

investigation (Fig. 7B). For Arg-CNCs-PTX/Trf, more sustained release than Arg-CNCs-PTX is

found; at the same time, the lower pH condition also shows more rapid drug release, indicating that

the modification of Trf does not change the pH-sensitive structure and subsequent drug release of

nanocapsules.

3.4. Cellular uptake of Arg-CNCs-NR/GFP

To assess the intracellular drug uptake delivered by Arg-CNCs, the hydrophobic dye NR with

bright red fluorescence imitated the insoluble drug, and GFP with green fluorescence was used as a

visible protein molecule. Arg-CNCs-NR/GFP was prepared and incubated with HeLa cells, and the

fluorescence of NR and GFP inside cells was monitored after 4 h and 8 h (Fig. 8). The results of

Laser Scanning Confocal Microscope (LSCM) (Fig. 8A) show that the red fluorescence of NR and

the green emission of GFP were simultaneously observed in the cytoplasm of Hela cells after 4 h

and 8 h of incubation, indicating the effective drug loading efficiency of Arg-CNCs and the cellular

uptake of Arg-CNCs-NR/GFP in HeLa cells. In addition, the fluorescence intensity of NR and GFP

inside the cells was quantified by flow cytometry. As shown in Fig. 8 B and C, HeLa cells display

strong red fluorescence intensity for NR and relatively weak green fluorescence intensity for GFP.

Since Arg-CNCs are negatively charged (-32.9 ± 1.8 mV) and GFP is positively charged (+3.7 ±

0.25 mV), they can interact with each other via electrostatic adsorption. However, due to the

relative large size of GFP (26.9 kDa) and small diameter of Arg-CNCs, relatively few protein

molecules are absorbed on the restricted capsule surface, and subsequently, the fluorescence

intensity of GFP inside the cells is weaker than that of NR.

3.5. In vitro antitumor efficacy of Arg-CNCs-PTX/Casp-3

In vitro cytotoxicity of Arg-CNCs-PTX/Casp-3 was evaluated on HeLa cells using a MTT assay.

As shown in Fig. 9A and B, cell viability decreases with the increase of PTX or Casp-3 amount in

all test samples, including Taxol®, Arg-CNCs-PTX, Arg-CNCs-Casp-3, and Arg-CNCs-PTX/Casp-3.

Arg-CNCs loaded with both PTX and Casp-3 show the lowest cell viability in the entire

concentration range, indicating that the Arg-CNC carrier can deliver PTX and Casp-3 into tumor

cells simultaneously. This conclusion is consistent with the results in the cellular uptake

experiments. The IC50 data of all test samples are shown in Fig. 9C. Arg-CNCs-PTX displays a

greater cytotoxicity towards HeLa cells, which has 24 h IC50 values of 3.51 ± 0.25 μM, i.e., nearly

two times lower than that of Taxol® (6.89 ± 0.08 μM). This result demonstrates that the Arg-CNC

carrier increases the tumor killing ability of PTX when compared with Taxol®. Compared to

mono-therapeutical formulations (Arg-CNCs-PTX and Arg-CNCs-Casp-3), Arg-CNCs-PTX/Casp-3

significantly and synergistically increases the cytotoxicity of PTX and Casp-3, giving the lowest

IC50 values. When 1.27 ± 0.04 μM PTX plus 0.79 ± 0.03 nM Casp-3 is loaded in Arg-CNCs, cell

growth is inhibited by 50%. Under the same conditions, Arg-CNCs loaded with either PTX or

Casp-3 reach their IC50 values at drug concentrations of 3.51 ± 0.25 μM or 2.91 ± 0.08 nM,

respectively. These values are far above (2.8- and 3.7-fold) the drug concentrations in a co-delivery

system. This phenomenon demonstrates that the present combination system could have synergetic

effects via different treatment mechanisms and would achieve improved performance for cancer

therapy.

To further evaluate the enhanced HeLa cell apoptosis induced by Arg-CNCs-PTX/Casp-3, flow

cytometry was performed after Annexin V-FITC/PI staining (Fig. 9D). The results show that the

Arg-CNCs-PTX/Casp-3 treated group has the highest late apoptosis rate among all samples. The

percentage of apoptotic cells in the Arg-CNCs-PTX/Casp-3 treated group is 53.2%, which is nearly

1.3-, 1.2-, and 2.7-fold higher than Taxol® (41.4%), Arg-CNCs-PTX (43.7%), and

Arg-CNCs-Casp-3 (20.0%) treated groups, respectively. This suggests that simultaneous PTX and

Caspase-3 encapsulation in Arg-CNCs was also effective for improving the apoptosis-inducing

activity of the combination system.

4. Conclusions

To achieve the simultaneous delivery of antineoplastic and protein drugs in a single carrier, a

series of CS derivatives were synthesized, and Arg-CS (with DS of 20.4%) was chosen in this work

to construct Arg-CNCs co-loaded with PTX and Casp-3. Here, based on a simple molecular

self-assembly strategy, the hydrophobic drug and protein can be effectively delivered

simultaneously to tumor cells by this Arg-CNC carrier. Much lower IC50 values and a higher

percentage of apoptotic cells treated by Arg-CNCs loaded with both PTX and Casp-3 displayed the

superiority of the co-delivery system and its collaborative treatment efficacy compared with all

control groups of monotherapy and commercial Taxol®. This confirms the high antitumor efficiency

through different treatment mechanisms. The nanocapsule system developed here can be a

promising tool for improving the performance of tumor therapy and provided the future prospects

of “cocktail therapy” application using a single carrier.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (31470916,

31500769), the Fundamental Research Funds for the Central Universities (2015PT036, 2016PT014),

A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education

Institutions, and the Open Project Program of MOE Key Laboratory of Drug Quality Control and

Pharmacovigilance (DQCP2015MS01).

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Fig. 1 (A) The synthesis procedure of Arg-, Lys-, and Phe-modified CS and (B) an illustration of

CNCs prepared by amino acid-modified CS derivatives using a one-step amalgamation method and

their co-loading process for the chemical drug and protein molecule.

4000 3000 2000 1000

Wavenumber / cm-1

1568

1662 1598

1659

16831641

1568

1571

16471689

29412891

3427

a

b

c

d

Fig. 2 FT-IR spectra of (a) CS, (b) Arg-CS, (c) Lys-CS, and (d) Phe-CS.

8 7 6 5 4 3 2 1 0

H9

H2H8

H8

H10

H2

H3~H6H8

/ ppm

a

b

c

d

H3~H6

H2H1

H7

H11H9

H2

H3~H6

H3~H6

H10

H11

H9H12

H10~12

H1+DOH

Fig. 3 1H NMR spectra of (a) CS, (b) Arg-CS, (c) Lys-CS, and (d) Phe-CS.

200 160 120 80 40 0

-COCH3

-COCH3

C9

-COCH3

-COCH3

C4C5 C8

C3

C7C12

C7

C10,C11

C9~C11

C6

C6

C6

C2

C2

C2

C1

C1

C12

C1

C7

C2C6

C3C5C4

/ ppm

a

b

c

d

C1

C3C5

C4

C3C5

C4

C8

C8

Fig. 4 13C NMR spectra of (a) CS, (b) Arg-CS, (c) Lys-CS, and (d) Phe-CS.

Fig. 5 The DS and concentration effects on the (a) hydrodynamic diameter and (b) zeta potential of

(A) Arg-CNCs, (B) Lys-CNCs, and (C) Phe-CNCs, and (D) the physical parameters of CNCs

prepared under the optimized preparation process.

Fig. 6 (A) TEM images of (a) blank Arg-CNCs, (b) blank Arg-CNCs dyed with phosphomolybdate,

(c) Arg-CNCs-PTX, and (d) Arg-CNCs-PTX/Trf. (B) The hydrodynamic diameters and zeta

potentials of Arg-CNCs, Arg-CNCs-PTX, and Arg-CNCs-PTX/Trf.

Fig. 7 (A) The stability studies of PTX-loaded Arg-CNCs with or without the surface modification

of Trf under different conditions: (a) 0.03 M PBS at pH 7.4, (b) 0.03 M PBS at pH 5.5, (c) 0.2 M

PBS at pH 7.4, and (d) 0.03 M PBS at pH 7.4 + 2% serum. (B) The accumulative drug release of

PTX-loaded Arg-CNCs with or without the surface modification of Trf in 0.03 M PBS media at pH

values of 7.4 and 5.5.

Fig. 8 Cellular uptake of Arg-CNCs using the fluorescent dye NR as the hydrophobic drug and GFP

as the protein molecule towards HeLa cells: (A) CLSM images (the nuclei were stained by Hoechst

33258), (B) flow cytometry analysis at 4 h and 8 h, and (C) The column graph of flow cytometry

results of (B): (a) NR and (b) GFP samples, showing the mean ± SD.

Fig. 9 (A) and (B) Cell viability of (a) Taxol®, (b) Arg-CNCs-PTX, (c) Arg-CNCs-PTX/Casp-3, and

(d) Arg-CNCs-Casp-3 with the same dose of PTX and Casp-3 for 24 h in HeLa cells. (C) IC50

values of various Arg-CNCs. Taxol® was used as a control. (D) Flow cytometric analysis of HeLa

cell apoptosis induced by (a) Taxol®, (b) Arg-CNCs-PTX, (c) Arg-CNCs-Casp-3, and (d)

Arg-CNCs-PTX/Casp-3 with the same dose of PTX and Casp-3 for 24 h using Annexin V-FITC/PI

staining. In each panel, the lower-left (Annexin V-FITC-, PI-), lower-right (Annexin V-FITC+, PI-)

and upper-right (Annexin V-FITC+, PI+) quadrants represent the populations of live cells, early

apoptotic cells, and late apoptotic cells, respectively. The average population (%) in each quadrant

is indicated by the numbers at the corner of the panels.

Table 1. Effects of the molar ratio of amino acid to saccharide units on the substitution degrees of

amino acid-modified CS derivatives.

Amino acid: saccharide unit Degree of substitution

Arg-CS Lys-CS Phe-CS

2.5: 1 0.20 0.33 0.09

5: 1 0.29 0.35 0.34

10: 1 0.34 0.78 0.47

20: 1 0.54 0.78 0.51