Nanoparticle-Patterned Multicompartmental Chitosan Capsules for

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Controlled Release and Delivery Systems

Nanoparticle-patterned Multi-compartmental Chitosan Capsules for Oral Delivery of Oligonucleotides Taehyung Kim, Jeong Un Kim, Kyungjik Yang, Keonwook Nam, Deokyeong Choe, Eugene Kim, Il-Hwa Hong, Minjung song, Hyunah Lee, Jiyong Park, and Young Hoon Roh ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00806 • Publication Date (Web): 14 Nov 2018 Downloaded from http://pubs.acs.org on November 15, 2018

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ACS Biomaterials Science & Engineering

Nanoparticle-patterned Multi-compartmental Chitosan Capsules for Oral Delivery of Oligonucleotides Taehyung Kim†,‡, Jeong Un Kim†,‡, Kyungjik Yang‡, Keonwook Nam‡, Deokyeong Choe‡, Eugene Kim‡, Il-Hwa Hong§, Minjung Song∥, Hyunah Lee‡, Jiyong Park‡, Young Hoon Roh‡,*

‡Department

of Biotechnology, College of Life Science and Biotechnology,

Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea §Department

of Veterinary Pathology, College of Veterinary Medicine,

Gyeongsang National University, 501 Jinju-daero, Jinju, South Gyeongsang Province 52828, Republic of Korea ∥Department

of Food Biotechnology, Division of Bio Industry, Silla University,

140 Baegyang-daero, 700 beon-gil, Sasang-gu, Busan 46958, Republic of Korea

Corresponding Author

1 ACS Paragon Plus Environment

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*Email: [email protected]

ABSTRACT

Orally administered antisense therapy has been introduced as an effective approach for treating cancer in the gastrointestinal tract. However, its practical application has been limited by the instability of oligonucleotides and their inefficient delivery. To overcome these problems, we synthesized size-dependent, oligonucleotide nanoparticle-patterned chitosan/phytic acid (ODN/CS/PA) capsules with protective shields via a three-step process of self-assembly, nanoparticle encapsulation, and shell formation. The multi-compartmental capsule size and oligonucleotide nanoparticle-loading pattern were controlled by applying different potentials during the electrostatic extrusion process used for nanoparticle encapsulation. Over 95% of encapsulated oligonucleotides were protected from nuclease digestion (DNase I) and, depending on their size, showed 40–75% protection against simulated gastric fluid. Their controlled release


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from capsules correlated with the cellular delivery of released nanoparticles and the inhibition of protein expression in cancer cells. Specifically, large capsules showed approximately 32-fold greater delivery to cancer cells than non-encapsulated nanoparticles. We also confirmed delivery of oligonucleotide nanoparticles to the small intestine and colon of rats following oral administration. These findings demonstrate that the multi-compartmental ODN/CS/PA capsules can facilitate efficient oral delivery of oligonucleotides for cancer treatment.

Keywords: Multi-compartmental delivery system; Oral delivery; Oligonucleotide; Chitosan; Phytic acid; Cancer



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INTRODUCTION Antisense oligonucleotides have been proposed for treating various cancers because they can specifically regulate the expression of undruggable proteins associated with cancer development.1-4 Orally delivered oligonucleotides (ODNs) are expected to increase patient compliance and provide efficient delivery to cancer sites in the intestine. However, anatomical and physiological barriers including gastric juice, digestive enzymes, and mucus layers undermine the stability of ODNs after they are delivered.5-8 Moreover, the negative charge of ODNs is an inherent barrier against their intracellular delivery to intestinal cells. Formulating ODN nanoparticles with lipids and polymers is a frequently used strategy to overcome these barriers. Chitosan and its derivatives are commonly utilized because of their mucoadhesive properties that enable intestinal delivery and stable complex formation with polyanions for efficient oral delivery, and showed good efficacy in vivo.9-11 However, some problems remain to be solved, such as the lack of ODN nanoparticle stability under harsh digestive conditions


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and the inability to control the release of ODNs to specific regions of the gastrointestinal tract. Multi-compartmental

delivery

systems,

which

consist

of

one

or

more

compartment and protective outer shields, have been applied for drug delivery due to their ability to provide sustained and controlled release in diverse applications.12,

13

Particularly, both hydrophilic and hydrophobic therapeutic

molecules can be incorporated into these systems through sequential emulsion or encapsulation with stimuli-responsive polymers. For these reasons, various multi-compartmental systems have been developed for the oral delivery of nucleic acid therapeutics. For example, Peppas’s group recently developed microencapsulated nanogels for the oral delivery of small interfering RNA (siRNA).14 Nanogels that are synthesized with pH-responsive polycationic 2(diethylamino)ethyl

methacrylate

facilitated

cellular

uptake

and

endosomal

escape. These nanogels were loaded into microgels by crosslinking with pHresponsive

poly(methacrylic

acid-co-N-vinyl-2-pyrrolidone)

and

a

trypsin-

degradable peptide, allowing controlled release of nanogels in the intestine. 5 ACS Paragon Plus Environment


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This platform delivered siRNA to macrophages and showed significant inhibition of target protein expression. In addition, the nanoparticles-in-microsphere oral system (NiMOS) was reported by Amiji’s group.15,

16

This system encapsulates

gelatin nanoparticles that contain a plasmid vaccine or siRNA with poly-εcaprolactone, which is degraded only by an intestinal lipase, via a double emulsion-like

process.

Furthermore,

the

NiMOS

system

showed

improved

intestinal bioavailability and therapeutic efficacy in a murine inflammatory bowel disease

model.

In

addition,

several

multi-compartmental

delivery

systems

exhibited controlled release of plasmid DNA after applying an enteric coating with pectin (which is degraded by pectinase in the colon) and Eudragit (which confers gastro resistance and enables release in the intestine).17 These multicompartmental delivery system studies showed enhanced protective functions and payload-delivery efficiencies by increasing the exposure time of the therapeutic molecules to target cells, compared to nanoparticles. Despite these advancements in multi-compartmental delivery systems for delivering plasmids


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and siRNAs, more improvements such as controllability of the oral delivery region and stable formulation of complexes for ODN delivery are still needed. Chitosan (CS) is an abundant natural biopolymer derived from N-deacetylation of chitin that has been widely used as an oral delivery vehicle owing to its biocompatibility, biodegradability, mucoadhesiveness, and low toxicity.18-20 The positively charged amino groups of CS in acidic medium enable the formation of CS hydrogels via ionotropic crosslinking with negatively charged molecules.21, 22

This physical crosslinking reduces reagent toxicity and other side effects

associated with chemical crosslinking.23 The non-toxic, multivalent counterion tripolyphosphate (TPP) is often used as a physical crosslinker in CS hydrogels. However, the low mechanical strength of these hydrogels limits their use in biomedical

applications.24,

25

Phytic

acid

(PA),

also

known

as

inositol

hexakisphosphate, is a naturally occurring substance derived from grains and legumes. PA possesses moderate antioxidant properties and exerts therapeutic effects against cancer, diabetes mellitus, atherosclerosis, and coronary heart disease.26,

27

It has a mineral-chelating ability due to its unique structure with 7 ACS Paragon Plus Environment


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six negatively charged phosphate groups bound to an inositol, which attracts positively

charged

molecules.26

Recently,

PA

has

been

introduced

as

a

crosslinking agent to enhance the mechanical strength through an increased number of physical crosslinks with CS,28 which is attributable to three more negatively charged phosphates per molecule than TPP. By exploiting the strong physical interaction between CS and PA, we have successfully loaded protein drugs and probiotics into CS/PA capsules for stable oral delivery to the intestine, with minimal degradation by acidic gastric fluid.28,

29

Size-controlled

CS/PA capsules were also developed by combining ionic gelation with electrohydrodynamic atomization, which showed different release profiles of protein drugs, depending on the capsule size.30 In this context, applying stable and size-tunable CS/PA capsules to ODNs should greatly enhance their stability against digestive environments and enable release controllability. In this study, we developed a multi-compartmental ODN nanoparticle-patterned CS/PA (ODN/CS/PA) capsule, which overcomes the difficulties of delivering ODNs, such as the instability of ODNs in harsh digestive environments, the 8 ACS Paragon Plus Environment

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lack of controlled delivery to specific region of the gastrointestinal tract, and inefficient

intracellular

delivery.

In

more

detail,

our

platform

incorporates

nanoparticles into capsules for enhanced intracellular delivery and endosomal escape of ODNs. Moreover, CS–PA complexes significantly enhance the stability of ODNs by forming increased number of physical crosslinks. Furthermore, tuning the size of the ODN/CS/PA capsule, which induces different nanoparticle localization, enables control of the delivery region of ODN nanoparticles in the intestine. The synthesized multi-compartmental capsules were characterized in terms of morphology, structure, and the ODN nanoparticle-loading pattern. Moreover, the stability and release profiles of encapsulated ODNs were examined using simulated gastric fluid and DNase I solution. To investigate the potential therapeutic application of ODN/CS/PA capsules for colon cancer, we used HT-29 colon cancer cells to evaluate their cellular uptake, inhibition of protein expression, and cytotoxicity. In addition, we studied the intestinal payload delivery of ODN/CS/PA capsules following oral administration in rats.



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MATERIALS AND METHODS Materials. CS (degree of deacetylation = 90%, molecular weight = 44 kDa) was purchased from Biotech Co. (Mokpo, Korea). PA (50% w/w solution in H2O) was from Mitsui Fine Chemicals (Tokyo, Japan). All ODN sequences were referenced from a previous study,31 and synthesized by Integrated DNA Technologies

(Coralville,

IA,

USA).

An

ODN

with

a

sequence

of

5'-

GAGCTGCACGCTGCCGTC-3' was used to regulate green fluorescent protein (GFP) expression, and an ODN labeled with cyanine (Cy) 5 at the 5' end (Cy5ODN) was used to track the ODN. Additionally, the scrambled ODN sequence of 5'-GAGCATCCCCACCTCCA-3' was used as a negative control for inhibiting protein expression. Materials for cell culture including Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal

bovine

serum

(FBS),

penicillin–streptomycin

solution,

and

phosphate-

buffered saline (PBS) were purchased from Corning, Inc. (Corning, NY, USA). pLenti CMV GFP Blast (Plasmid #17445), psPAX2 (Plasmid #12260), and pMD2.G (Plasmid #12259) were purchased from Addgene (Cambridge, MA, 10 ACS Paragon Plus Environment

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USA). pLenti CMV GFP Puro was a gift from Eric Campeau (Addgene Plasmid #17448). All other reagents were from Sigma–Aldrich (St. Louis, MO, USA). Preparation

of

Multi-compartmental

ODN/CS/PA

Capsules.

Multi-

compartmental ODN/CS/PA capsules were prepared in a three-step process: self-assembly, nanoparticle encapsulation, and shell formation. First, 0.1% (w/v) CS prepared in 1.0% (v/v) acetic acid was mixed with ODN at an N/P ratio (i.e., the molar ratio of the amine groups in CS to the phosphate groups in ODN) of 4, under vigorous vortexing. In this step, ODN nanoparticles were formed by self-assembly of ODNs and CS through electrostatic attraction. Second, to induce nanoparticle encapsulation, 3.0% (w/v) CS was added to the ODN nanoparticle solution to a final CS concentration of 1.5% (w/v). A 0.75% (w/w) PA solution (pH 6.0) was prepared by diluting 50% (w/w) PA solution with distilled water and adjusting the pH with 5 N NaOH. The CS solution containing ODN nanoparticles was added dropwise to the PA solution through a 24-G needle using an encapsulator (VAR V1; Nisco Engineering AG, Zurich, Switzerland), leading to the formation of ODN/CS/PA hydrogels. The hydrogel 11 ACS Paragon Plus Environment


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size was controlled by adjusting an applied voltage, which determined the magnitude of the electrostatic force. Voltages of 0, 5, and 9 kV were selected to prepare hydrogels in three different size ranges. Lastly, the hydrogels were cured for 1 h in PA solution with stirring at 200 rpm, resulting in the formation of shells around the hydrogels (ODN/CS/PA capsules). Characterization of Multi-compartmental ODN/CS/PA Capsules. The dynamic light scattering (DLS) and zeta potential of the ODN nanoparticles were measured using a particle size & zeta potential analyzer (ELS-2000ZS; Otsuka Electronics Co., Osaka, Japan). The size of the ODN/CS/PA capsules was determined using a digital single-lens reflex (DSLR) camera (D5300, Nikon, Tokyo, Japan) and ImageJ software (v1.8, National Institutes of Health, Bethesda, MD, USA). The size distributions were analyzed using Minitab software (Minitab, State College, PA USA). The morphology of the capsules was examined with a fluorescence microscope (IX71; Olympus, Tokyo, Japan) and a field emission-scanning electron microscope (SU-8220; Hitachi, Tokyo,


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Japan). The capsules were mounted on stubs and coated with osmium for 8 s using an osmium coater (HPC-1SW; Vacuum Device, Tokyo, Japan). Loading Efficiency of ODN. ODN loading efficiency into the capsule was evaluated by measuring the amount of ODN released into the PA supernatant that leaked from ODN/CS/PA capsules during the preparation process. The collected supernatant was filtered through a 0.45-µm membrane for further analysis. The ODN concentration was determined with a high-performance liquid chromatography (HPLC) system equipped with a P680 HPLC pump, an ASI-100 autosampler, a TCC-100 thermostat column compartment, and a UVD340U diode array detector (Dionex, Sunnyvale, CA, USA). A 10-µL volume of each sample

was

injected

into

an

Inspire

5-µm

hydrophilic

interaction-liquid

chromatography column (250 × 4.6 mm; Dikma Technologies, Lake Forest, CA, USA) with a mobile phase of 0.1 M triethyl ammonium acetate/acetonitrile (95/5, v/v) at a flow rate of 1 mL/min. ODNs were detected at a wavelength of 260 nm. The ODN loading efficiency of each type of ODN/CS/PA capsule was calculated with eq 1. 13 ACS Paragon Plus Environment


% ODN loading efficiency = ([Wtotal − Wsup]/Wtotal) × 100

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(1)

where Wtotal is the total weight of ODNs used for multi-compartmental ODN/CS/PA capsule synthesis and Wsup is the measured weight of ODNs in the PA supernatant. ODN Loading Amount. The amount of ODN loaded in ODN/CS/PA capsules was also measured by gel electrophoresis. Capsules and naked ODNs (150, 300, and 450 ng) were loaded on a 2% agarose gel pre-stained with GelRed (Biotium, Hayward, CA, USA); naked ODNs were used as a reference. Electrophoresis was performed at a voltage of 10 V/cm for 30 min, and the gel was visualized with a gel documentation system (Gel Doc XR+; Bio-Rad Laboratories, Hercules, CA, USA). Images were analyzed with ImageJ software to quantify the amount of ODNs in each type of capsule. Localization of ODN Nanoparticles. To analyze the localization of ODN and CS in ODN/CS/PA capsules, fluorescein isothiocyanate (FITC)-labeled CS was synthesized by chemical reaction between the isothiocyanate group of FITC and the primary amino group of CS. Briefly, 20 mL of 1.0% (w/v) CS dissolved in 14 ACS Paragon Plus Environment

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1.0% (v/v) acetic acid solution was mixed with 20 mL of 0.1% (w/v) FITC dissolved in dehydrated methanol. After incubation for 3 h, FITC-CS was precipitated by increasing the pH to 10. Unreacted FITC and CS were removed by replacing the supernatant with distilled water. The purified FITC-CS was redissolved in 20 mL of 1.0% (v/v) acetic acid and dialyzed against 2.0 L of distilled water for 3 days in the dark.32 Cy5-ODN and FITC-CS were used to synthesize Cy5-ODN/FITC-CS/PA capsules to observe ODN and CS localization in the capsules, which were fixed with 10.0% (v/v) neutral buffered formalin solution (Sigma–Aldrich), embedded in paraffin, and sectioned at a thickness of 4

µm

with

a

microtome

(Leica

Microsystems,

Wetzlar,

Germany).

The

localization of Cy5-ODN and FITC-CS in each type of capsule was visualized by

confocal

laser

scanning

microscopy

(CLSM)

(LSM

700;

Carl

Zeiss,

Thornwood, NY, USA), and analyzed with Zeiss Zen software (Carl Zeiss, Thornwood, NY, USA). Stability of ODNs. The stability of multi-compartmental ODN/CS/PA capsules was

evaluated

under

enzymatic

and

acidic

conditions.

To

assess

the 15

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Page 16 of 79

resistance to enzymatic digestion, 3.0 µg of naked ODNs and ODNs in capsules were incubated at 37°C with 0.03 or 0.3 U/µL of DNase I (Sigma– Aldrich) for 10 min or 4 h. The ability of the capsule to protect the ODN was determined by measuring the amount of ODN remaining after DNase I digestion using gel electrophoresis and ImageJ analysis. To evaluate the stability in an acidic

environment,

naked

ODNs

and

each

type

of

multi-compartmental

ODN/CS/PA capsule were incubated in simulated gastric fluid (SGF, pH 1.2) at 37°C for 1 or 2 h, and the amount of ODN remaining in each type of capsule was quantified by gel electrophoresis and ImageJ analysis. ODN stability was calculated using eq 2. Intensity of treated DNA band

% ODN stability = Intensity of untreated DNA band × 100

(2)

The average ODN stability for each group was expressed as the mean ± SD with three different samples.

In Vitro Release of ODNs. In vitro ODN-release profiles of ODN/CS/PA capsules were determined by incubating each type of capsule in SGF and simulated intestinal fluid (SIF) prepared without enzymes. SGF (pH 1.2) was 16 ACS Paragon Plus Environment

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prepared with 0.2% (w/v) sodium chloride and 0.7% (v/v) hydrochloric acid, whereas SIF (pH 6.8) was prepared with 6.8% (w/v) monobasic potassium phosphate

and

0.9%

(w/v)

sodium

hydroxide.

Digestive

conditions

were

mimicked by sequentially incubating each type of ODN/CS/PA capsule in SGF for 2 h and in SIF for up to 70 h at 37°C. Throughout the incubation period, the capsules were agitated at 90 rpm in a shaking incubator (DS-310F; Dasol Science Co., Hwaseng, Korea). The remaining amount of ODN was determined by gel electrophoresis and ImageJ analysis after sequential incubation in SGF for 0.5, 1, or 2 h and in SIF for 24, 48, or 72 h. Preparation of GFP-Expressing HT-29 cells. GFP-expressing HT-29 cells were prepared by lentiviral infection to investigate the protein-regulatory function of ODNs released from ODN/CS/PA capsules. Briefly, human embryonic kidney 293T cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2. Lentivirus was prepared by transfecting the cells with the aforementioned plasmids using the Lipofectamine 17 ACS Paragon Plus Environment


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reagent (Invitrogen, Carlsbad, CA, USA).33 HT-29 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI 1640 medium supplemented with 10% FBS and 5% penicillin/streptomycin at 37°C in 5% CO2. The cells were transduced with the prepared lentivirus and cultured for 12–14 days in the presence of 10 µg/mL blasticidin to select GFP-expressing cells. Drug-resistant cells were expanded to generate a stably GFP-expressing HT-29 cell line. Cellular capsules

Uptake. to

Fluorescently

investigate

the

labeled

uptake

of

ODNs ODN

were

used

nanoparticles

to

synthesize

released

from

ODN/CS/PA capsules to colon cancer cells. The localization of Cy5-labeled ODN nanoparticles in cells was investigated using CLSM. HT-29 cells were seeded on a glass coverslip that was placed in a 24-well plate; the cells were seeded at a concentration of 1 × 105 cells/well. After incubation for 24 h, the cells were treated with each type of capsule for 2, 4, or 36 h. Cell nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA). The coverslips were mounted onto a slide glass to visualize, and analyzed the subcellular localization of the nanoparticles by CLSM equipped with Zeiss Zen 18 ACS Paragon Plus Environment

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software. The cellular uptake of ODN nanoparticles released from each type of capsule was quantified by flow cytometry. HT-29 cells were seeded in a 24-well plate at 5 × 104 cells/well and cultured in RPMI 1640 containing 10% FBS and 5% penicillin/streptomycin at 37°C. Each type of Cy5-ODN/CS/PA capsule was loaded into a 24-well plate, followed by incubation for 2 or 4 h. The cells were detached with 0.25% (w/v) trypsin/EDTA (Corning, Inc.) and centrifuged for 5 min at 300 × g and 4°C. After removal of the supernatant, the cells were washed with PBS and resuspended in 400 µL of PBS supplemented with 5% FBS and analyzed by flow cytometry (BD Accuri C6; BD Biosciences, San Jose, CA, USA). The cellular-uptake rate of each sample was determined by comparing the fluorescence intensity of Cy5-ODN/CS/PA capsule-treated cells to that of untreated cells.

In Vivo Uptake. Male Sprague–Dawley rats (5 weeks old) were purchased from Samtako (Osan, Korea) and housed at 22°C ± 2°C and 50% ± 10% humidity on a 12:12-h light/dark cycle, with free access to standard chow and water. All procedures involving animals were approved by the Animal Ethics 19 ACS Paragon Plus Environment


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Committee of Gyeongsang National University (approval no. GNU-180329R0015) and complied with institutional guidelines for the care and use of laboratory animals. After 1 week of acclimation, rats (180–200 g) were divided into control, small, and large capsule groups (n = 3 rats per group). The former group was treated with saline and the latter two groups were treated with small or large capsules containing Cy5-labeled ODN nanoparticles, and intestinal delivery of ODN nanoparticles was examined. Cy5-ODN in each type of capsule was orally administered to rats at an equivalent dose of 0.5 mg/kg. After 8 h, the rats were sacrificed and the small intestine and colon were excised and washed with PBS. The tissue was frozen in OCT compound (Sakura Finetek Co., Tokyo, Japan) and sectioned at a thickness of 10 µm on a cryotome (Leica Microsystems). Sections were stained with Hoechst 33342 to visualize nuclei. Images of small intestine and colon tissue sections were obtained by CLSM. Inhibition of Protein Expression. The inhibition of protein expression by ODNs released from each ODN/CS/PA capsule type was investigated in HT-29 20 ACS Paragon Plus Environment

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human colon cancer cells. After treating GFP-expressing HT-29 cells with the capsules, GFP expression was quantified by flow cytometry. Briefly, the cells were seeded in a 24-well plate at 5 × 104 cells/well. Cells in the 24-well plate were treated with each type of ODN/CS/PA capsule at an ODN concentration of 250 or 500 nM. After incubation for 72 h, the cells were detached and GFP expression levels of capsule-treated cells were analyzed by flow cytometry. The inhibition

efficacy

for

each

sample

was

determined

by

comparing

GFP

expression in cells treated with ODN/CS/PA capsules to that in cells treated with capsules containing ODNs with a scrambled sequence. Cytotoxicity Assay. The cytotoxicity of ODN/CS/PA capsules was evaluated with the Cell Counting Kit (CCK)-8 (Dojindo Molecular Technologies, Kumamoto, Japan). HT-29 cells were seeded in a 24-well plate at a concentration of 5 × 104 cells/well and treated with ODN/CS/PA capsules containing 250 or 500 nM ODN. After 24 h, CCK-8 reagent was added to the medium as recommended by the manufacturer, followed by incubation at 37°C for 1 h. The amount of water-soluble formazan in the medium was detected by measuring the 21 ACS Paragon Plus Environment


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absorbance of 450 nm with a multi-label microplate reader (Victor X5; Perkin Elmer, Waltham, MA, USA). Cell viability in each sample was determined by comparing the absorbance of ODN/CS/PA capsule-treated cells to that of untreated cells. Statistical analysis. Experimental data were expressed as the mean ± standard deviation (SD) for three or four sample per each group. Differences between groups were analyzed using the Scheffe and Dunnett T3 test function of the SPSS software package, version 24.0.

RESULTS AND DISCUSSION Preparation

of

Multi-compartmental

ODN/CS/PA

Capsules.

Multi-

compartmental ODN/CS/PA capsules with a triple shield were prepared through a three-step process of self-assembly, nanoparticle encapsulation, and shell formation (Figure 1). Prior to synthesizing the ODN/CS/PA capsules, ODN nanoparticles were prepared by inducing electrostatic interactions between the cationic amine groups of CS and the anionic phosphate backbone of ODNs 22 ACS Paragon Plus Environment

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(self-assembly step). Considering the effect of the N/P ratio on the binding affinity of CS for ODN loading and protection, the surface charge, and the size of ODN nanoparticles required for efficient intracellular uptake and therapeutic function,34 an ODN nanoparticle N/P ratio of 4 was selected based on our preliminary study, which showed efficient loading capacity and stable formulation of nanoparticles (data not shown). Stable complexation with CS protects ODNs from enzymatic digestion,35 representing the first shield for the capsules. In the next step, a highly concentrated CS solution was added to the synthesized ODN nanoparticles, providing a sufficient number of CS molecules for strong physical crosslinking with PA and nanoparticle encapsulation. CS and PA solutions with optimized concentrations and pH values were used to maximize the degree of crosslinking of the final products. The CS solution containing ODN nanoparticles was added dropwise from a needle to a PA solution by electrostatic extrusion, using an encapsulator. Voltages of 9, 5, and 0 kV were applied to the needle and PA solution, based on the optimal conditions determined in our previous study,30 creating differences in electrostatic potential 23 ACS Paragon Plus Environment


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that generated an appropriate droplet-size distribution during the nanoparticleencapsulation process. Specifically, a higher voltage caused premature release of the droplets, yielding smaller-sized droplets. As the droplets merged into the PA solution, a hydrogel began to form that functioned as the second shield (nanoparticle-encapsulation step). The third shield consisted of an outer shell gradually

formed

by

curing

the

hydrogels

in

PA

solution,

yielding

the

ODN/CS/PA capsule (shell-formation step). In this manner, we fabricated tripleshielded multi-compartmental ODN/CS/PA capsules of three different sizes, i.e., small, medium, and large.


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Figure 1. Schematic illustration of the process used to synthesize multicompartmental ODN/CS/PA capsules of three different sizes. (A) Illustration of an encapsulator used to apply different voltages for electrostatic extrusion during nanoparticle encapsulation. (B) Optical image of large ODN/CS/PA



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capsules after the curing process. Scale bar, 10 mm. (C) Representation of the molecular structures and ionotropic crosslinking of CS and PA.

Characterization of ODN/CS/PA Capsules. Each type of ODN/CS/PA capsule was characterized in terms of its morphology, size distribution, and ODN-loading efficiency. The morphology and structure of the capsules were visualized by light

microscopy

(LM).

The

micrographs

revealed

that

all

of

the

multi-

compartmental capsules were spherical and composed of a hydrogel core and separate film-like shell (Figure 2). The surface and cross-section of each type of capsule were analyzed by scanning electron microscopy (SEM). Each type of capsule had a similar spherical shape, in accordance with the LM observations. The shells were smooth and had a non-porous film-like structure, whereas the core had a mesh-like structure. Separation of the shell from the core was monitored by LM during the shell-formation step. The separation was clearly observed within 1 h, when the diameter of ODN/CS/PA capsules decreased by 26 ACS Paragon Plus Environment

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approximately 10% (Figure 3A, B). Meanwhile, 30% of PA concentration in the solution decreased within the first 30 min of incubation and did not decrease further after 30 min, showing a plateau curve. These results imply that PA molecules in the solution diffused into the hydrogel until the shell was formed. Therefore, it was verified that the PA influx led to shell formation and a more compact hydrogel during the curing process. Importantly, we observed the ODN nanoparticles encapsulated within the hydrogel core at a higher magnification. From the SEM images, we determined the average nanoparticle size as 88.2 ± 11.7 nm for all capsule types. This was comparable to the size of ODN nanoparticles measured by DLS analysis (Figure 2 and Table 1) prior to the nanoparticle-encapsulation step. The similar size distributions indicate that the nanoparticles maintained their original structure during the self-assembly step until they formed capsules.



Page 28 of 79

Figure 2. LM and SEM images of multi-compartmental ODN/CS/PA capsules of three different sizes. Shell and nanoparticle images were obtained from crosssectioned capsules. Scale bars, 500 µm (overall and surface), 100 µm (shell), and 500 nm (nanoparticle).


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Figure 3. Shell formation and size reduction of multi-compartmental ODN/CS/PA capsules, and reduction of the PA concentration during the curing process. (A) LM images of shell formation in an ODN/CS/PA capsule at different curing times.

Scale

bars,

500

µm.

(B)

Measurements

of

ODN/CS/PA

capsule



Page 30 of 79

diameters and absorbances, and PA concentrations taken after different curing times.


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Table 1 Particle sizes and zeta potentials of ODN nanoparticles with different N/P ratios N/P Ratio

Particle Size (nm)

Polydispersity Index

Zeta Potential (mV)

2

232.8 ± 66.3

0.322

30.4 ± 0.2

4

192.9 ± 55.4

0.261

30.9 ± 0.6

8

134.1 ± 39.9

0.286

30.4 ± 1.3

16

99.4 ± 29.7

0.288

30.5 ± 0.2

In addition, we compared the sizes of capsules synthesized at voltages of 9, 5, and 0 kV based on DSLR images, using ImageJ software. The capsule sizes were determined to be 961.9 ± 87.8, 1744.0 ± 121.6, and 2596.0 ± 99.7 µm, respectively, at the abovementioned voltages (Table 2). These results indicate that the capsule size was easily tuned by varying the applied voltage, with smaller capsules formed by applying a higher voltage. The ODN loading efficiency was evaluated by HPLC (Table 2). The loading efficiency was calculated as nearly 100% for all capsule types, as no ODN was present in the supernatant of PA solutions after ODN/CS/PA capsule preparation. We also measured the amount of ODN loaded into each capsule type by quantifying the



Page 32 of 79

gel electrophoresis results using ImageJ software. The amount of loaded ODNs increased with the capsule size (81.0, 99.6, and 159.6 ng/mg for small, medium, and large capsules, respectively). Taken together, these results demonstrate that

multi-compartmental

ODN/CS/PA

capsules

with

a

triple

shield

were

successfully synthesized at three different sizes with a high ODN-loading efficiency. Importantly, the ODN nanoparticles remained intact in the core of the capsule throughout capsule preparation.


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Table 2 Characteristics of ODN/CS/PA capsules of three different sizes. ODN/CS/PA Capsule Size

Applied Voltage (kV)

Capsule Size (μm)

ODN Loading Efficacy (%)

ODN Loading Amount (ng/mg)

Small

9

961.9 ± 87.8

100

81.1 ± 10.4

Medium

5

1744.0 ± 121.6

100

99.6 ± 11.1

Large

0

2596.0 ± 99.7

100

159.6 ± 5.8

Localization of ODN Nanoparticles in Multi-compartmental ODN/CS/PA Capsules. Fluorescence-labeled multi-compartmental ODN/CS/PA capsules were synthesized using FITC-labeled CS (FITC-CS) and Cy5-labeled ODN (Cy5-ODN) to determine the localization of ODN nanoparticles in sectioned capsules. The green fluorescence of FITC-CS and red fluorescence of Cy5-ODN were tracked by CLSM. CS was evenly distributed throughout each type of capsule. Importantly, the yellow fluorescent signal, representing localization of the ODN nanoparticle, varied according to the capsule size (Figure 4); ODN nanoparticles were dispersed throughout the capsule core in small capsules, but were mostly present at the core center in medium capsules. Images of a large capsule



Page 34 of 79

revealed that the capsule core had a three-layer structure comprising CS as the inner and outer layers with ODN nanoparticles in between. In agreement with a previous study, we speculate that the nanoparticles migrated as PA molecules

diffused

into

the

capsule,

crosslinking

with

CS

through

ionic

interactions that induce nanoparticle movement along the same direction of PA influx during the synthesis of multi-compartmental ODN/CS/PA capsules.36 According to a well-known ionic gelation model, i.e., the Mikkelsen–Elgsaeter model, the gelation rate of a spherical hydrogel is influenced by parameters including the particle radius, the ion and polymer concentrations, and the iondiffusion rate.37 Therefore, the localization of ODN nanoparticles in this platform are expected to be mainly related with capsule size because the other factors were fixed. In this context, the patterning of ODN nanoparticles in each type of capsule was easily controlled by tuning the capsule size. Furthermore, changing the pattern depends on the physicochemical properties of nanoparticles (such as the surface charge, size, and density of nanoparticles) should be undertaken


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in future work to investigate the exact mechanism of nanoparticle patterning in the capsules.



Page 36 of 79


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Figure 4. Localization of ODN nanoparticles in multi-compartmental ODN/CS/PA capsules. (A) CLSM images of multi-compartmental ODN/CS/PA capsules with different localization of ODN nanoparticles, depending on the capsule size. CS was labeled with FITC (green) and ODN was labeled with Cy5 (red). Scale bars, 500 µm. (B) Schematic illustration of the unidirectional movement of ODN nanoparticles during the crosslinking reaction.

Stability of ODN in a Digestive Environment. The stability of ODNs encapsulated

in

multi-compartmental

ODN/CS/PA

capsules

in

a

digestive

environment was evaluated by incubating the capsules with an enzyme in an acidic solution. The resistance of ODNs to enzymatic digestion was determined by measuring the amount of ODN remaining after DNase I treatment. After treatment with DNase I at a concentration of 0.03 U/µL for up to 4 h, the gel images showed that >95% of the ODNs were protected in the small, medium, and large capsules (Figure 5A, Figure S1, S2 in the Supporting Information). In



Page 38 of 79

contrast, naked ODNs were completely degraded, and only 37.2% of ODNs in non-encapsulated ODN nanoparticles remained after 10 min of treatment, which were completely degraded after 4 h of treatment. Furthermore, when ODNs loaded in small, medium, and large capsules were treated with DNase I at a higher concentration of 0.3 U/µL for 4 h, the capsules showed size-dependent stability, with 49.9%, 62.3%, and 74.5% of the ODNs remaining after incubation, respectively (Figure S3 in the Supporting Information). With each type of capsule, triple shielding showed superior protection of ODNs against DNase I, as compared to naked ODNs and non-encapsulated ODN nanoparticles. Given that ODNs are easily degraded in the acidic environment of the gastrointestinal tract by depurination, we investigated their stability in the capsules by measuring the amount of ODN remaining after incubation in SGF (pH 1.2). Because hydrogen ions are generally known to dissociate CS complexes, in acidic SGF they can cause either release or degradation of encapsulated ODN nanoparticles. After 2 h of incubation, 40.6%, 61.2%, and 75.5% of the initial ODNs remained in the small, medium, and large capsules, 38 ACS Paragon Plus Environment

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respectively, as compared to 11.0% of naked ODNs and 36.4% of ODNs in non-encapsulated

nanoparticles

(Figure

5B).

The

larger

capsules

were

dissociated to a lesser degree in the acidic environment due to their smaller surface area-to-volume ratio, providing greater protection to ODNs. In addition, the localization of ODN nanoparticles in the core of medium and large capsules protected the ODN by reducing their probability of contacting hydrogen ions. These results indicate that localization of the encapsulated ODN nanoparticles in the capsule core through induction of a strong physical interaction between CS and PA markedly enhances ODN protection against digestive environments.



Page 40 of 79

Figure 5. Stability of ODNs loaded in multi-compartmental ODN/CS/PA capsules against digestive environments. (A) Stability of ODNs in each type of capsule in the presence of digestive enzyme. Naked ODNs, non-capsulated nanoparticles (NPs), and capsules were treated with DNase I (0.03 U/µL) for 10 min or 4 h at 37°C. (B) Stability of ODNs in each type of capsule in SGF. Naked ODNs, NPs, and capsules were incubated for 1 or 2 h in SGF (pH 1.2) at 37°C. Pvalues were calculated using one-way analysis of variance (ANOVA) with a


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Scheffe and Dunnett T3 test (*p < 0.05, **p < 0.01, ***p < 0.005). Results were presented as mean ± SD (n = 3).

In

vitro

Release

of

ODNs.

The

release

rate

of

ODNs

from

multi-

compartmental ODN/CS/PA capsules was evaluated under gastrointestinal pH conditions. Specifically, the amount of ODNs released from the three types of capsules was calculated by quantifying the amount of ODNs remaining in the capsules after sequential incubation in SGF (pH 1.2) for 0.5, 1, or 2 h and in SIF (pH 6.8) for 24, 48, or 72 h. We found that 50.8%, 38.8%, and 24.5% of the ODNs were released from the small, medium, and large capsules, respectively, after 2 h in SGF, whereas 87.0%, 81.2%, and 32.2%, respectively, was released after 72 h of sequential SGF and SIF treatment (Figure 6A). All ODN/CS/PA capsule types showed an initial burst release of ODNs in SGF, followed by sustained release in SIF. The mechanisms of the drug release from chitosan (CS)-based particulate systems include release from the particle



Page 42 of 79

surface, diffusion through the swollen matrix, and release due to polymer erosion.38 The shift in the release profile between SGF and SIF was possibly due to a burst release of ODNs emanating from the shell of ODN/CS/PA capsules during SGF treatment and decreased swelling of CS-based capsules in response to the increase in pH under SIF conditions. In a strongly acidic solution, CS molecules are more protonated, leading to greater repulsion between each molecule.39 At neutral pH, the molecules are deprotonated, leading to stabilization of the capsule structure through the formation of more hydrogen bonds. A similar pH dependence was observed as capsules became swollen during SGF treatment (acidic pH) and then returned to a size similar to that of untreated capsules in SIF (neutral pH) (Figure 6B). Furthermore, the central localization of ODN nanoparticles in the core of medium and large capsules slowed the release rate of nanoparticles after capsule erosion and degradation.38

These

results

suggest

that

the

ODN/CS/PA

capsules

can

withstand the harsh digestive environment, allowing prolonged release of ODNs at specific sites in the intestine. 42 ACS Paragon Plus Environment

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Figure

6.

Release

profiles

of

ODNs

in

multi-compartmental

ODN/CS/PA

capsules. (A) In vitro release profiles of ODNs from each type of capsule in SGF and SIF. ODN/CS/PA capsules were incubated for 2 h in SGF (pH 1.2) and for up to 70 h in SIF (pH 6.8) at 37°C. (B) LM images of capsules of three different sizes in SGF and SIF.



Page 44 of 79

Cellular Delivery of ODN Nanoparticles. To achieve efficient cellular delivery of ODNs, we synthesized ODN nanoparticles with physicochemical properties (i.e., size and surface charge) that would promote cellular uptake.40,

41

As a

proof of concept, we evaluated the efficiency of ODN nanoparticle delivery from each multi-compartmental ODN/CS/PA capsule type to HT-29 colon cancer cells. The cellular uptake rate and subcellular localization of Cy5-labeled ODN nanoparticles were evaluated by CLSM after incubation for 2, 4, or 36 h. We found that nanoparticles released from each type of capsule were delivered to the cell nuclei over time. Images acquired after 2 h of treatment revealed that the red fluorescence signal corresponding to delivered ODN nanoparticles was localized to the cell periphery for each type of capsule. Nanoparticles released from the small capsules showed the highest rate of uptake. After treatment for 4 h, ODN nanoparticles were localized to the nucleus and cytosol, implying that they were successfully delivered to the target location to inhibit expression of the target protein. At 36 h, the large capsules showed the highest rate of nanoparticle uptake (Figure 7) and thus exhibited the most sustained release. 44 ACS Paragon Plus Environment

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Therefore,

nanoparticles

released

from

each

type

of

capsule

were

well

protected and were clearly delivered to colon cancer cells at a rate that depended on the capsule size. Moreover, the rate of nanoparticle uptake was dependent on both the release rate and capsule size, consistent with the obtained release profiles. Flow cytometric analysis quantitatively confirmed the size dependence of the ODN nanoparticle-uptake (Figure 7B); nanoparticles released from large capsules were taken up with approximately 30-fold lower fluorescence intensity than those released from small capsules, or with 32-fold lower fluorescence intensity than non-encapsulated nanoparticles after 2 h of treatment. Thus, sustained ODN delivery to colon cancer cells can be achieved by altering the capsule size. Furthermore, intracellular localization of delivered ODN nanoparticles confirmed the successful endosomal escape of ODNs and their potential to inhibit target protein expression, which can occur via several mechanisms including RNase H-mediated mRNA cleavage and steric hindrance of translational machinery.42-44



Figure

7.

Cellular

uptake

of

ODN

nanoparticles

Page 46 of 79

released

from

multi-

compartmental ODN/CS/PA capsules in colon cancer cells. (A) Size-dependent cellular uptake of ODN nanoparticles released from capsules of three different


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sizes analyzed by CLSM. HT-29 cells were exposed to each type of capsule for different times, up to 36 h. ODN nanoparticles were labeled with Cy5 (red), and nuclei were stained with Hoechst 33342 (blue). Scale bars, 10 µm. (B) Flow cytometric quantification of the cellular uptake of ODN nanoparticles. HT29 cells were exposed to non-capsulated nanoparticles (NP) and each type of the capsule for 2 h. P-values were calculated using one-way ANOVA with a Dunnett T3 test (***p < 0.005). Results were presented as mean ± SD (n = 3).

In Vivo Delivery of ODN Nanoparticles. For effective cancer treatment, it is critical that the therapeutic agent is preserved until delivery to the disease site. In the case of multi-compartmental ODN/CS/PA capsules, a triple shield protected the ODNs until their oral delivery to the cancer sites. We confirmed the stable delivery of ODNs in an in vivo study by evaluating ODN nanoparticle delivery in the digestive tract of rats. Small and large multi-compartmental Cy5ODN/FITC-CS/PA

capsules

were

synthesized,

and

the

uptake

of

ODN



Page 48 of 79

nanoparticles in the small intestine and colon was assessed by CLSM 8 h after oral administration by tracking the green fluorescence of FITC-CS and red fluorescence of Cy5-ODN (Figure 8, Figure S3). In the obtained fluorescence images, villi of the small intestine and crypts of the colon in rats treated with the capsules showed both red and green fluorescence, in contrast to those of rats that were treated with vehicle, indicating the successful protection and oral delivery of ODN nanoparticles. The small capsule-treated cells exhibited red fluorescence mostly in the small intestine, whereas the large capsule treated cells showed red fluorescence in both the small intestine and the colon. Moreover, higher red fluorescence was consistently observed in cells treated with large capsules compared to cells treated with small capsules, in both the small intestine and colon. In addition, green fluorescence was observed in intestinal epithelial cells treated with either small or large capsules. In more detail, the small capsule-treated colon cells showed lower fluorescence intensity than large capsule-treated cells, whereas the fluorescence intensities of small intestine cells treated with small or large capsules were similar. Collectively, 48 ACS Paragon Plus Environment

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these results imply that the ODN-delivery site could be controlled by varying the capsule size, which affected the degree of nanoparticle protection. These results confirmed that ODN nanoparticles were protected against the harsh conditions of the digestive tract to a greater extent in capsules, which thereby increased the number of nanoparticles delivered to the small intestine and colon for local payload delivery. Compared to a similar plasmid DNA-delivery system (the NiMOS from Amiji’s group), these results showed more sustained delivery especially in the small intestine.15 Furthermore, these results indicated that the capsule size should be tuned depending on the delivery site in the intestine. In the digestive tract of rats, small capsules showed appropriate delivery to small intestine, and large capsules exhibited efficient colon-targeted delivery. In addition, we expect that the size dependence of the uptake rate is attributable to the mucoadhesive properties of the CS and surface area of the capsule.45,

46



Figure

8.

In

vivo

delivery

of

ODN

nanoparticles

Page 50 of 79

released

from

multi-

compartmental ODN/CS/PA capsules to the small intestine and colon of rats. Each type of the capsule was orally administered to male Sprague–Dawley rats at an equivalent dose of 0.5 mg/kg (n = 3 rats per group). Images were obtained by CLSM. Scale bars, 100 µm.


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Inhibition of Protein Expression by ODNs. The functionality of the delivered ODN nanoparticles was investigated by examining the inhibition of target protein expression. GFP-expressing HT-29 colon cancer cells were treated with the three

types

of

multi-compartmental

ODN/CS/PA

capsules

at

various

concentrations for 72 h, and GFP fluorescence in the cells was detected by flow cytometry. GFP expression was lower in cells treated with ODN/CS/PA capsules containing ODNs complementary to the GFP as compared to cells treated

with

capsules

containing

ODNs

with

a

scrambled

sequence.

Consequently, GFP expression was inhibited in a concentration-dependent manner by all capsule types with the small capsule showing the highest efficiency of inhibition. The inhibition efficiency correlated with the amount of ODN

nanoparticles

delivered

to

cells

and

the

capsule

release

profiles,

demonstrating that ODNs released from each type of capsule were safely delivered to cells and suppressed target protein expression (Figure 9A). We evaluated

the

cytotoxicity

of

each

ODN/CS/PA

capsule

type

at

the

concentrations used to inhibit protein expression and found that cytotoxicity 51 ACS Paragon Plus Environment


Page 52 of 79

was negligible for all types of capsules (Figure 9B). Our results demonstrate that ODN nanoparticles released from the capsules and delivered to cells of interest effectively inhibited target protein expression in colon cancer cells in a capsule size-dependent manner with minimal cytotoxicity.


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Figure 9. Cellular efficacy and toxicity of ODN nanoparticles released from multi-compartmental ODN/CS/PA capsules in colon cancer cells. (A) Inhibition of GFP expression in HT-29 cells by ODN released from capsules of three



different

sizes.

The

mean

fluorescence

intensity

Page 54 of 79

was

analyzed

by

flow

cytometry. Cells were treated with ODNs at concentrations of 250 and 500 nM. P-values were calculated using one-way ANOVA with a Scheffe test (*p < 0.05, **p < 0.01, ***p < 0.005). Results are presented as mean ± SD (n = 3). (B) Viability of HT-29 cells treated with capsules containing 250 and 500 nM ODNs. Results are presented as mean ± SD (n = 3).

CONCLUSIONS In summary, multi-compartmental ODN/CS/PA capsules with a triple shield fulfill the criteria of an ideal nucleic acid delivery system compared to previously developed platforms, as the ODNs are protected from nuclease digestion and acidic environments by encapsulation within a highly crosslinked CS/PA hydrogel, they enable controlled ODN release to desired regions in gastrointestinal tract by tuning the capsule size, and offer improved ability for sustained release to cells or tissues. This platform was developed through


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sequential

steps

of

self-assembly,

nanoparticle

encapsulation,

and

shell

formation, resulting in orally delivered ODNs with remarkable stability. These capsules were synthesized in three different sizes to induce differing localization of

ODN

nanoparticles,

showing

size-dependent

stability

in

the

digestive

environment, differing release profiles from capsules, and a controlled cellular uptake rate in colon cancer cells. The delivered ODN nanoparticles clearly showed intracellular localization in the cytosol and nucleus, which indicates successful endosomal escape of the delivered ODNs and subsequent inhibition of protein expression in target cancer cells. In addition, the in vitro efficacy of delivered ODN nanoparticles was confirmed in size-dependent and dosedependent manners after 72 h of incubation. Results from in vivo experiments showed that the orally administered capsules selectively protected the ODNs until the nanoparticles were delivered to the small intestine or colon. This platform

can

be

applied

broadly

for

the

oral

delivery

of

nanoparticles

incorporating other nucleic acid therapeutics such as siRNAs, microRNAs, and plasmids to specific gastrointestinal sites by controlling the capsule size and 55 ACS Paragon Plus Environment


Page 56 of 79

nanoparticle formulation, representing a new promising treatment strategy for cancer.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI:

Additional gel images of DNase I stability experiments; Enzymatic stability of ODN Nanoparticles in Multi-compartmental ODN/CS/PA capsules at high concentration; CLSM images of small intestine and colon of vehicle treated rats. (PDF)

AUTHOR INFORMATION

Corresponding Author *Email: [email protected]

ORCID


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Young Hoon Roh: 0000-0002-7396-4156

Author Contribution †These authors contributed equally. Y.H.R. and J.P. conceived the idea and designed experiments. T.K., J.U.K., K.Y., E.K., I-H.H. and M.S. performed all the experiments and T.K., J.U.K., D.C., H.L and Y.H.R. analyzed data. T.K., J.U.K., K.Y., K.N. and Y.H.R. wrote the manuscript. All authors contributed to the general discussion and reviewed the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the Young Researcher Program (No. 2015R1C1A1A02037770), Basic Science Research Program (No. 2014M3A7B4051898) through the National Research Foundation of Korea (NRF) funded by the Korean Government. This work also supported by the BK21 plus program. Kyungjik Yang was supported by the NRF-2017-Global Ph. D. 57 ACS Paragon Plus Environment


Page 58 of 79

Fellowship Program. We thank Kyungsene Lee of Yonsei University in Korea for his technical assistant.

Conflicts of interest The authors confirm that there are no conflicts of interest.

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(11) Tahara, K.; Samura, S.; Tsuji, K.; Yamamoto, H.; Tsukada, Y.; Bando, Y.; Tsujimoto, H.; Morishita, R.; Kawashima, Y. Oral nuclear factor-κB decoy oligonucleotides delivery system with chitosan modified poly(d,llactide-co-glycolide) nanospheres for inflammatory bowel disease.

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For Table of Contents Use Only Nanoparticle-patterned Multi-compartmental Chitosan Capsules for Oral Delivery of Oligonucleotides Taehyung Kim†,‡, Jeong Un Kim†,‡, Kyungjik Yang‡, Keonwook Nam‡, Deokyeong Choe‡, Eugene Kim‡, Il-Hwa Hong§, Minjung Song∥, Hyunah Lee‡, Jiyong Park‡, Young Hoon Roh‡,*


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Figure 1. Schematic illustration of the process used to synthesize multi-compartmental ODN/CS/PA capsules of three different sizes. (A) Illustration of an encapsulator used to apply different voltages for electrostatic extrusion during nanoparticle encapsulation. (B) Optical image of large ODN/CS/PA capsules after the curing process. Scale bar, 10 mm. (C) Representation of the molecular structures and ionotropic crosslinking of CS and PA.



Figure 2. LM and SEM images of multi-compartmental ODN/CS/PA capsules of three different sizes. Shell and nanoparticle images were obtained from cross-sectioned capsules. Scale bars, 500 μm (overall and surface), 100 μm (shell), and 500 nm (nanoparticle).


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Figure 3. Shell formation and size reduction of multi-compartmental ODN/CS/PA capsules, and reduction of the PA concentration during the curing process. (A) LM images of shell formation in an ODN/CS/PA capsule at different curing times. Scale bars, 500 μm. (B) Measurements of ODN/CS/PA capsule diameters and absorbances, and PA concentrations taken after different curing times



Figure 4. Localization of ODN nanoparticles in multi-compartmental ODN/CS/PA capsules. (A) CLSM images of multi-compartmental ODN/CS/PA capsules with different localization of ODN nanoparticles, depending on the capsule size. CS was labeled with FITC (green) and ODN was labeled with Cy5 (red). Scale bars, 500 μm. (B) Schematic illustration of the unidirectional movement of ODN nanoparticles during the crosslinking reaction.


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Figure 5. Stability of ODNs loaded in multi-compartmental ODN/CS/PA capsules against digestive environments. (A) Stability of ODNs in each type of capsule in the presence of digestive enzyme. Naked ODNs, non-capsulated nanoparticles (NPs), and capsules were treated with DNase I (0.03 U/μL) for 10 min or 4 h at 37°C. (B) Stability of ODNs in each type of capsule in SGF. Naked ODNs, NPs, and capsules were incubated for 1 or 2 h in SGF (pH 1.2) at 37°C. P-values were calculated using one-way analysis of variance (ANOVA) with a Scheffe and Dunnett T3 test (*p < 0.05, **p < 0.01, ***p < 0.005). Results were presented as mean ± SD (n = 3).



Figure 6. Release profiles of ODNs in multi-compartmental ODN/CS/PA capsules. (A) In vitro release profiles of ODNs from each type of capsule in SGF and SIF. ODN/CS/PA capsules were incubated for 2 h in SGF (pH 1.2) and for up to 70 h in SIF (pH 6.8) at 37°C. (B) LM images of capsules of three different sizes in SGF and SIF.


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Figure 7. Cellular uptake of ODN nanoparticles released from multi-compartmental ODN/CS/PA capsules in colon cancer cells. (A) Size-dependent cellular uptake of ODN nanoparticles released from capsules of three different sizes analyzed by CLSM. HT-29 cells were exposed to each type of capsule for different times, up to 36 h. ODN nanoparticles were labeled with Cy5 (red), and nuclei were stained with Hoechst 33342 (blue). Scale bars, 10 μm. (B) Flow cytometric quantification of the cellular uptake of ODN nanoparticles. HT-29 cells were exposed to non-capsulated nanoparticles (NP) and each type of the capsule for 2 h. P-values were calculated using one-way ANOVA with a Dunnett T3 test (***p < 0.005). Results were presented as mean ± SD (n = 3).



Figure 8. In vivo delivery of ODN nanoparticles released from multi-compartmental ODN/CS/PA capsules to the small intestine and colon of rats. Each type of the capsule was orally administered to male Sprague– Dawley rats at an equivalent dose of 0.5 mg/kg (n = 3 rats per group). Images were obtained by CLSM. Scale bars, 100 μm.


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Figure 9. Cellular efficacy and toxicity of ODN nanoparticles released from multi-compartmental ODN/CS/PA capsules in colon cancer cells. (A) Inhibition of GFP expression in HT-29 cells by ODN released from capsules of three different sizes. The mean fluorescence intensity was analyzed by flow cytometry. Cells were treated with ODNs at concentrations of 250 and 500 nM. P-values were calculated using one-way ANOVA with a Scheffe test (*p < 0.05, **p < 0.01, ***p < 0.005). Results are presented as mean ± SD (n = 3). (B) Viability of HT-29 cells treated with capsules containing 250 and 500 nM ODNs. Results are presented as mean ± SD (n = 3).


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