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Yingyuan Zhao, Junli Liu, Lei Guan, Yaping Zhang, Ping Dong,
Jing Li, Xingguo Liang, Makoto Komiyama
PII: S0141-8130(18)30065-5
DOI: doi:10.1016/j.ijbiomac.2018.05.054
Reference: BIOMAC 9655
To appear in:
Received date: 5 January 2018
Revised date: 10 May 2018
Accepted date: 10 May 2018
Please cite this article as: Yingyuan Zhao, Junli Liu, Lei Guan, Yaping Zhang, Ping Dong,
Jing Li, Xingguo Liang, Makoto Komiyama , Fabrication of aqueous nanodispersion from
natural DNA and chitosan as eminent carriers for water-insoluble bioactives. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Biomac(2017), doi:10.1016/j.ijbiomac.2018.05.054
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a. College of food Science and Engineering, Ocean University of China, Qingdao 266003, PR China
b. Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science
and Technology, Qingdao 266235, PR China
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c. World Premier International (WPI) Research Centre for Materials Nanoarchitectonics (MANA),
National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan
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*Corresponding authors
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To whom correspondence should be addressed. Tel: 0086-532-82031086; Fax: 0086-532-82031086;
Present Address: College of Food Science and Engineering, Ocean University of China, Qingdao
266003, China
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Email: lijouc@ouc.edu.cn (J. Li); liangxg@ouc.edu.cn (X. Liang)
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Abstract
For high-valued application of natural DNA as raw materials, we prepared nanocarriers by using
salmon sperm DNA and chitosan to encapsulate water-insoluble bioactives. Here, water dispersible
astaxanthin/DNA/chitosan nano-aggregates (ADC-NAs) were prepared by co-assemble evaporation
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method. The key point for preparing well formed ADC-NAs was specifically discussed. The resultant
ADC-NAs were spherical with 100-300 nm diameter measured by dynamic light scattering (DLS)
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and transmission electron microscopy (TEM), and their homogeneous dispersions were sufficiently
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stable at room temperature. One important feature of these nanocarriers is enormously high loading
amount of cargo (about 40 wt.-%). According to the UV-vis spectra of the nanosuspension, we
deduced that astaxanthin is encapsulated as uniquely structured J-aggregates. Fourier transform
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infra-red (FTIR) spectroscopy proved fabrication was successfully and astaxanthin was embedding
in DNA/chitosan nanocarriers. Cytotoxicity was examined in vitro using cell culture in L929 cell
lines. When necessary, these nano-aggregates can be degraded by DNase I. Homogeneous
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dispersions of other non-charged guest molecules are also prepared by using DNA/chitosan
nanocarriers. These dispersions are cheaply and easily obtainable from naturally occurring DNA and
chitosan, and should be useful for versatile applications.
Key words: DNA, chitosan, polyion complex, nanoparticles, water dispersion, astaxanthin
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1. Introduction
Deoxyribonucleic acid (DNA) has many characteristic features and is one of the most ideal and
promising raw materials for medicine, biotechnology, food science, and others. First, it is cheaply
obtainable in large amounts from the nature (more than 100 tons of DNA is being extracted every
year from salmon sperm) [1-3]. Second, its unique properties (non-immunogenicity, enzymatic
degradability, nano-scale controllability, selective binding with other molecules, etc.) cannot be
otherwise fulfilled. Thirdly, its non-toxicity has been well documented. The importance of this
natural polymer has been further substantiated by recent remarkable progresses in DNA science and
technology [4]. In almost all previous studies on DNA, however, oligonucleotides produced by DNA
synthesizers were employed as the source [4-13], and naturally occurring DNA has been used for
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only limited purposes [14,15]. It has been long believed that the DNA from the nature has a simple
and rigid double-stranded structure and thus cannot be easily fabricated to complicated
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nanostructures. However, the DNA from synthesizers is too expensive and the amounts obtainable
are too small for practical applications (less than a few milligrams are usually produced per one
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synthesis). Thus, the methodology to control the aggregation features of naturally occurring DNA
and construct designated products is vitally important from the viewpoints of both fundamental
science and practical applications.
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Aqueous dispersions of water-insoluble molecules (e.g., medicines and food additives) are
important in many fields [16-21]. Very recently [22], we found that astaxanthin
(3,3’-dihydroxy-β,β’-carotene-4,4’-dione) can be dispersed in water by use of the combination of
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naturally occurring DNA and chitosan (the molecular structures of the components are presented in
Fig. 1). The cellular uptake and antioxidation capability were enormously improved. This
ketocarotenoid is eminent as antioxidant [23-25], nutritional agent, and food colorant [26,27]. Its
medicinal effectiveness has been also evidenced [28-30]. However, further applications of this
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chemical have been restricted by its poor water-solubility and chemical instability. Although
emulsification technology successfully increased the solubility [31-35], undesirable organic solvents
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and surfactants must be used there [36-38]. Thus, the DNA/chitosan systems developed by our group
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successfully prepared from naturally occurring DNA (from salmon sperm) and chitosan. First, the
method to prepare astaxanthin/DNA/chitosan dispersions is established, and their structure and
properties are characterized in detail. Then, the following features of DNA/chitosan nanocarriers,
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which are essential for versatile applications, are evidenced: (i) the encapsulating activity is
enormously high, (ii) astaxanthin is encapsulated as specifically structured nanocrystals
(J-aggregates) which are otherwise unstable in water, (iii) no measurable cytotoxicity is detectable,
(iv) when necessary, the DNA therein can be digested by nucleases, and (v) versatile non-charged
water-insoluble molecules can be efficiently encapsulated by the nanocarrier by the same method.
The formation mechanism of the nano-aggregates, as well as potential applications of the nanocarrier,
is discussed in terms of these results. Polyion complex formation of chitosan with plasmid DNA was
already used as a means to deliver this DNA to target site [39-42]. However, nanocarriers of
water-insoluble compounds, based on this polyion complex, have been for the first time envisioned
in this study.
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Astaxanthin was dissolved in ethanol at 25oC, and then the solution was centrifuged at 8,000 g for 5
min to remove solid impurities. Chitosan was dissolved in 0.1 M hydrochloric acid, diluted with
distilled water, and the pH was adjusted to 5.0 with small amount of sodium hydroxide solution.
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Fucoxanthin, curcumin, β-carotene and paclitaxel were dissolved in ethanol at 25oC for appropriate
concentration.
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2.2 Preparation of astaxanthin/DNA/chitosan nano-aggregates
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In Protocol I in Fig. 2, ethanol solution of astaxanthin and aqueous solution of chitosan (pH 5.0)
were first mixed, and stirred for 10 min at room temperature. Then DNA in water was gradually
added in 1-2 min with magnetic stirring (300 rpm). The mixture was further stirred for 10 min, and
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was subjected to a vacuum rotational evaporator (Buchi RE 121, Switzerland) to remove the ethanol
under reduced pressure (8 mbar) at 30oC on a water bath. Most (> 99%) of the ethanol used for the
preparation was removed by the evaporation step, as evidenced by gas chromatography. In Protocol
II, astaxanthin in ethanol was first mixed with DNA in water, and then chitosan in water (pH 5.0)
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was gradually added in 1-2 min. Otherwise, the procedure was the same as in Protocol I. The mole
ratio (A/P) of the amino groups in chitosan to the phosphate groups in DNA was calculated by
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(MCS×326.9)/(MDNA×165.3), where MCS and MDNA are the weights of chitosan and DNA,
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respectively.
Several batches were prepared for each experiment. Particle sizes, zeta potentials, and astaxanthin
contents were immediately measured, and then all the samples were stored in screw-capped amber
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acid staining solution for 3-5 min. Samples were subjected to TEM after natural drying.
2.3.3 UV spectroscopy
The UV-vis absorption spectra were recorded at normal incidence with a Shimadzu UV-1800
spectrophotometer. Samples were held in a quartz cuvette with 1 cm path length. The wavelength
was ranged from 200 nm to 800 nm with a slit of 1.0 nm and a high scan speed model. All the
experiments were carried out at 25 oC.
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4000-400cm-1 by using 32 scans at a resolution of 4 cm-1 by using a model Vertex70 spectrometer
( Bruker Corporation, Karlsruhe, Germany) The powdered sample (2-3 mg) was mixed with spectral
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purity KBr (200-300 mg) and created thin film with pressure (60 MPa).
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2.3.5 Determination of encapsulating capacity
The encapsulating activity of the particles was evaluated in terms of loadage of astaxanthin (the
weight percentage of the guest in the whole nanocarriers). The encapsulated astaxanthin was
extracted three times using a 1:1 dichloromethane/methanol solution and through a 0.22 μm
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membrane. According to the method previously [22], astaxanthin content analysis was performed in
a D-2000 type of HPLC system (Hitachi, Japan) with an YLT Sino Chrome C18 column (5 µm, 200
mm × 4.6 mm). 10 μL of samples was eluted at 25 oC with an isocratic mobile phase consisting of
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85% v/v methanol, 5% v/v dichloromethane, 5% v/v acetonitrile and 5% v/v water. The flow rate
was 1.0 mL/min.
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modified Eagle medium (DMEM), L929 cells were cultured at 37 oC in a CO2 (5%) incubator. These
cells were seeded in a 96-well plate at an initial density of 1×104 cells/well in 100 µL of complete
DMEM, and cultured for 24 h to reach a 60-70% confluence. These cells were then co-treated with a
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required amount of ADC-NAs, and incubated with those nanoparticles for 48 h. After specified
durations, 20 µL of MTT solution (5 g/L in PBS buffer) was added to the cultured supernatants and
incubated for 4 h. The medium in each well was removed, and 200 µL of DMSO was added to
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dissolve the internalized purple formazan crystals. Finally, the plate was gently agitated for 15 min
and the absorbance at 570 nm was recorded by a microplate reader (VARIOSKAN FLASH, Thermo
Scientific, Finland). The cell viability was calculated by (ODsample - ODblank)/(ODcontrol - ODblank).
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SGC: Stock solutions of pepsin, digestion buffer (pH 3.0) and H2O were mixed together at a ratio
of 5:5:8 (v/v/v). The mixture was reacted at 37 °C for 0~24 h.
SIF: Stock solutions of trypsin, digestion buffer (pH 8.5) and H2O were mixed together at a ratio
of 1:1:1 (v/v/v). The mixture was reacted at 37 °C for 0~8 h.
DNase I solution: Stock solutions of DNase I, ADC-NAs, DNase I 10 × reaction buffers (pH 7.6)
and H2O were mixed together at a ratio of 1:4:15 (v/v/v). The mixture was reacted at 37 °C for 0~10
min.
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mean ± standard deviation (S.D.). Experimental statistics were performed using a SPSS 17 software.
The analysis of variance (ANOVA) with Duncan’s multiple-comparison test was used. Differences
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were considered significant at p < 0.05.
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3. Results and discussion
3.1 Preparation and characterization of astaxanthin/DNA/chitosan nano-aggregates
3.1.1 Critical importance of addition order of reagents
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By combining macromolecular co-assembly with solvent evaporation, water dispersible
astaxanthin/DNA/chitosan nano-aggregates (ADC-NAs) were prepared. Two protocols involving
different addition orders of reagents were attempted (see Fig. 2A). In Protocol I, DNA was added
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after mixing chitosan with ethanol solution of astaxanthin, while chitosan was added after mixing
DNA with ethanol solution of astaxanthin in Protocol II. It was found that the order of addition of
reagents is one of the key factors to obtain homogeneous dispersions of astaxanthin. Note that all the
preparations were achieved under the conditions that the concentrations of DNA and chitosan largely
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exceeded the critical assemble concentration (1.0 x 10-3 g/L), which was independently determined
(Fig. S1).
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In Protocol I in Fig. 2, 4 mL of ethanol solution of astaxanthin (38 mg/L) was first mixed with 8
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mL of aqueous pH 5.0 solution of chitosan (200 mg/L). After mixing for 10 min, 4 ml of aqueous
solution of DNA (200 mg/L) was slowly added to this astaxanthin/chitosan mixture in 1-2 min under
continuous stirring. Finally, the ethanol was removed by evaporation. In this preparation, the
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ethanol/water ratio (prior to the evaporation) was 1:3, whereas the A/P ratio (the ratio of the amino
groups in chitosan to the phosphate groups in DNA) was 4:1. As the result, a homogeneous
pink-purple dispersion was successfully obtained (see the picture in Fig. 2B). About 47% of the
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astaxanthin used was encapsulated in the nanoparticles, and this cargo occupied 8.2 % of the total
weight of the nanoparticles. The dispersion was highly stable and no apparent change was perceived
when it was stored at room temperature for more than one month.
In Protocol II, astaxanthin in ethanol was first mixed with aqueous solution of DNA, and aqueous
solution of chitosan (pH 5.0) was added to this astaxanthin/DNA mixture. In contrast with the
successful preparation of homogeneous dispersions by Protocol I, purple linear-shaped flocculations
were formed in the mixture (see Fig. 2C). The flocculation started in the first step of the preparation
(mixing of the ethanol solution of astaxanthin with the aqueous solution of DNA), probably due to
some aggregations of astaxanthin molecules. Upon the removal of the ethanol by evaporation, the
flocculation became still more evident and precipitates were finally formed.
In the presence of chitosan in Protocol I, astaxanthin molecules are immediately bound to the
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chitosan. The cation- interactions of the ammonium ions of chitosan with -conjugated system of
astaxanthin [45, 46] and/or their cation-dipole interactions with the carbonyl groups are primarily
responsible for this binding. With Protocol II, however, DNA coexists with astaxanthin in the
ethanol/water mixtures. The interactions of the astaxanthin molecules with DNA are not so strong
that stable dispersions cannot be formed. The sole organic solvent used for the preparation of the
dispersions is ethanol, which is labeled by U.S. Food and Drug Administration as a Generally
Recognized as Safe (GRAS). Furthermore, this ethanol is removed by evaporation, rendering the
dispersions further friendly to human health. Thus, it can be concluded that stable and homogeneous
dispersion of astaxanthin/DNA/chitosan nano-aggregates can be prepared only by adding DNA (in
water) to astaxanthin/chitosan mixture in ethanol/water solutions, followed by the evaporation of the
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ethanol. Throughout the subsequent parts of this paper, all the specimens of
astaxanthin/DNA/chitosan composite were prepared by Protocol I, unless noted otherwise.
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3.1.2 Properties of nanoparticles of ADC-NAs
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Table 1 shows the results of preparation of dispersions by Protocol I with different A/P ratios. The
ethanol/water ratio (prior to the evaporation) was kept constant at 1:3. Homogeneous dispersions
were successfully obtained in a wide range of A/P ratio (from 14:1 to 3:1), where the amino groups
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of chitosan were in large excess with respect to the phosphate groups of DNA. According to DLS
analysis, the nanoparticles were mostly in the size of 100-300 nm in diameter (Fig. 2D). The zeta
potentials were in the range of +43 to +19 mV, which are primarily associated with excess amino
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groups of chitosan to the phosphate groups of DNA. Consistently, TEM showed highly spherical
nanoparticles of 100-300 nm size (Fig. 2E). For the purpose of comparison, homogeneous
dispersions of DNA/chitosan nanoparticles (without astaxanthin) were also prepared under
comparable conditions (see Fig. S2). These nanocarriers had particle sizes of 150-250 nm, and their
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zeta potentials were +20-40 mV. Both values were almost the same as the values for ADC-NAs,
showing that astaxanthin in ADC-NAs was encapsulated without causing much structural
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disturbance of the DNA/chitosan nanocarriers. It is strongly indicated that these guest molecules are
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located in the nano-scaled void space formed in the polyion complexes between DNA and chitosan.
When the A/P ratio was decreased to 2:1 or 1:1, however, flocculation notably occurred during the
preparation. Almost complete neutralization of positive charges of chitosan with negative charges of
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DNA diminishes the water solubility of polyion complexes and is unfavorable to form homogeneous
dispersions. This argument is supported by the fact that, at still smaller A/P ratio (e.g., 1:2, 1:3, and
1:4) in which DNA is in excess to chitosan, homogeneous dispersions were also successfully
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obtained (Table S1). Thus, negatively charged DNA/chitosan nanocarriers can be also prepared.
However, positively charged particles would be in general more favorable for practical in vivo
applications, due to feasible penetration through cell membranes, so that the following part of this
paper is restricted to the nanocarriers of A/P > 1.
Average size of
A/P ratio State of dispersionb Zeta potential (mV)c
particles (nm)c
14:1 H 133±3α +43±5δ
12:1 H 119±6α +31±2βγ
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“c”. Values with different letter “α, β, γ, δ” are statistically different in the same column.
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3.1.3 Enormously high loadage of astaxanthin in ADC-NAs
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One of the most important advantages of the present DNA/chitosan nanocarrier is enormously high
loadage of astaxanthin (the fraction of astaxanthin in the nano-aggregates). Under the typical
preparation conditions in Fig. 2, the encapsulated astaxanthin occupied 8.2% of the total weight of
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ADC-NAs. This value is satisfactorily high, but still further improvements were attempted through
optimization of the preparation conditions. In Table 2, the A/P ratio was kept at 4:1. It was found that
the loadage of astaxanthin notably increases with decreasing amount of DNA/chitosan nanocarrier
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used. On the basis of this finding, various factors were changed to maximize the loading amount. As
the result, the highest loadage of astaxanthin (40 wt.-%) was obtained, when 9 mL of ethanol
solution of astaxanthin (38 mg/L) was first mixed with 6 mL of aqueous solution of chitosan (20
mg/L), and then 3 mL of aqueous solution of DNA (20 mg/L) was added to the mixture (the 2nd line
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from the bottom in Table 2). In other words, 10 g of the DNA/chitosan nanocarrier thus prepared can
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rather than as single molecules, and thus such a remarkably high loadage is attainable. To our best
knowledge, this is one of the most efficient colloidal loading systems towards water-insoluble
organic compounds. Applications to drug delivery systems are promising (vide infra).
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monomeric form (dotted line). This remarkable red-shift shows that astaxanthin is encapsulated in
the DNA/chitosan nanocarrier as tiny crystals of J-aggregates, in which the molecules are mutually
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arranged in an ordered fashion. It is well known that dye molecules occasionally form either
H-aggregates or J‐ aggregates, which induce hypsochromic and bathochromic shifts, respectively
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[47, 48]. H-aggregates are composed of parallel plane-to-plane stacking of dye molecules, whereas
J-aggregates involve head-to-tail stacking (Fig. 3B). According to previous studies, the max of the
H-aggregate of astaxanthin is 388 nm in methanol/water solutions [49], whereas the maximum of the
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J-aggregate is located at around 562 nm in acetone/water mixtures [50]. As shown by the chain
dotted line in Fig. 3A, ADC-NAs in water almost exclusively involve the J-aggregates and the
contribution of the H-aggregates is negligible.
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solutions of different ethanol/water ratios (Fig. S3). Addition of either chitosan or DNA did not much
change the equilibrium. Consistently, J-aggregates of astaxanthin were previously prepared only in
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organic solvent/water mixtures of low water content (e.g., 1:1 methanol/water), and the H-aggregates
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prevailed at higher water contents [49, 50]. On the other hand, the dispersions of astaxanthin
prepared by emulsification method involved only monomeric species [31, 32, 51-53]. Thus, the
DNA/chitosan nanocarriers are highly unique in that the J-aggregates, which are otherwise
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thermodynamically unstable and hard to obtain in aqueous phase, are efficiently stabilized and stably
accommodated in water. Applications of J-aggregates of astaxanthin to physical and material science
have been proposed [54]. Furthermore, J-aggregates are advantageous especially for biological
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applications, since they absorb the light of longer wavelength which is characterized by both higher
tissue penetration and lower phototoxicity.
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Consistently, the infrared vibration bands of astaxanthin at 1552 cm-1 and 976 cm-1 (stretching of
C=C and C-H, respectively, in the conjugation system) were greatly weakened upon the
encapsulation. These results indicate that the aromatic ring of astaxanthin was encapsulated in the
DNA-chitosan nanocarriers. Similar changes were observed, when astaxanthin formed an inclusion
complex with hydroxypropyl--cyclodextrin and was accommodated in its apolar cavity [56].
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DC-NAs (gray bars), the cell viability was higher than 100%, confirming that both of them are
almost completely nontoxic and biocompatible to living cells, as expected.
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3.5 Enzymatic degradation of ADC-NAs
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For practical applications, ADC-NAs must be sufficiently stable to deliver the encapsulated
water-insoluble molecules and furthermore, when necessary, should be decomposed to release the
cargo at target sites. Thus, their stability against several enzymes was investigated (Fig. 6). It was
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found that ADC-NAs were easily digested by DNase I (lanes 2, 4, 6 and 8 in Fig. 6A). Only in a few
minutes, the DNA used for ADC-NAs (>10 kbp) was mostly converted to the fragments smaller than
1 kbp. However, these nano-aggregates were resistant against pepsin (Fig. 6B) and trypsin (Fig. 6C).
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Although pepsin digested naked DNA (lanes 1 and 3 in Fig. 6B) as we previously showed [44], the
DNA in ADC-NAs was never digested. Thus, among these three enzymes, DNase I can be a
promising candidate of a trigger to release the cargo from ADC-NAs.
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curcumin, -carotene, and paclitaxel) in water (see Fig. S4 and Table S2). Interestingly and
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importantly, in order to prepare homogeneous dispersions, the order of reagent addition must be
changed depending on the guest. For curcumin, Protocol I (used to obtain the astaxanthin
dispersions), was directly applicable. On the other hand, notable flocculation occurred, when
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-carotene, fucoxanthin and paclitaxel were the guests and Protocol I was used. In these cases,
however, homogeneous dispersions were produced with the use of Protocol II in which chitosan was
added to the guest/DNA mixture in ethanol/water solutions. These results indicate that complicated
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intermolecular interactions in the solutions are responsible for the preparation of homogeneous
dispersions (vide infra). In all the cases, homogeneous dispersions, prepared by either Protocol I or II,
were sufficiently stable, and no precipitations were observed even in longtime storages. Versatile
utility of the present methodology has been further confirmed.
4. Conclusion
It has been shown that the polyion complexes between two naturally occurring polymers, DNA and
chitosan, serve as eminent nanocarriers of hydrophobic molecules. Various non-charged molecules
can be successfully entrapped in these carriers, and dispersed in water. They are highly
biocompatible, easily available, and of low cost. As an example, stable dispersions of
astaxanthin/DNA/chitosan nano-aggregates (ADC-NAs) in water were prepared by using
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far longer wavelength (max = 580 nm) than the monomeric astaxanthin (max = 480 nm).
Applications of these nano-aggregates to phototherapy using long-wavelength light are promising,
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and these studies are currently underway in our laboratory.
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Conflicts of interest
There are no conflicts of interest to declare. NU
Acknowledgements
This work was supported by the National Natural Science Foundation of China [Grant No.
31301420, No. 31571937 and No. 31201327]; and China Postdoctoral Science Foundation Funded
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Figure Captions:
Fig. 1. Molecular structures of DNA (A), chitosan (B), and astaxanthin (C). In the chitosan used in this study,about 10%
of the amino groups are acetylated.
Fig. 2. Scheme for preparation and characterization of astaxanthin/DNA/chitosan nano-complex. (A) Two protocols to
prepare astaxanthin/DNA/chitosan nano-aggregates, (B) photo-image of ADC-NAs suspension formed by Protocol I, and
(C) photo-image of ADC-NAs formed by Protocol II (flocculation is evident). In (D) and (E), size distribution and TEM
image of nanoparticles, obtained by Protocol I, are presented. Note that homogeneous dispersions are obtainable only by
Protocol I. The specimens in (B) were prepared from 4mL of ethanol solution of astaxanthin (38 mg/L), 8 mL of aqueous
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solution of chitosan (200 mg/L), and 4 ml of aqueous solution of DNA (200 mg/L); see text for more details.
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Fig.3. (A) UV-Vis spectraof astaxanthinin ethanol (dotted line) anddispersion of ADC-NAs (chain dottedline). For the
purpose of comparison, the spectrumof aqueous solution of DNA(broken line)and chitosan (solid line)werealso
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shown.The specimens were prepared from 3mLof ethanol solution of astaxanthin(6mg/L), 6mLof aqueous solution of
chitosan (20mg/L), and 3mL of aqueous solution of DNA (20mg/L) according to Protocol I.(B) Typicalstructures of
J-aggregateand H-aggregate.
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Fig.4.FTIR spectra of astaxanthin (a), chitosan (b), DNA (c), simple mixture of astaxanthin, DNA, and chitosan (d),
and ADC-NAs (e).
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Fig.5. In vitrocytotoxicity of ADC-NAs toL929 cells for 48 h(black bars).Forthe purpose of comparison, the cytotoxicity
of DC-NAs, composedof only DNA and chitosan withoutastaxanthin, is also presented by gray bars.
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Fig. 6.Digestion of ADC-NAs at 37oC by (A) DNase I(pH 7.6),(B) pepsin(pH 3.0),and (C) trypsin(pH 8.5).Lanes1, 3, 5,
and 7, naked salmon sperm DNA treated with the enzyme. Lanes2, 4, 6 and8, ADC-NAs treated with theenzyme. M: 1
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kbpDNA ladder; C: salmon sperm DNA; S: ADC-NAs.In lanes 2 and 4 in (B) and (C), most of the specimens retained in
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the gel holes even after the enzymatic treatments, showing that the DNA in ADC-NAswas not digestedat all. The enzyme
concentrations of DNase I, pepsin, and trypsin were125 U/mL,5 mg/mL, and 2.5 mg/mL,respectively.
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Highlights
Novel nanocarriers prepared from natural DNA and chitosan for encapsulating and delivering
water-insoluble bioactive molecule were sufficiently validated.
Addition order of reagents is the key factor to obtain homogeneous bioactive-loaded
nanodispersions in the co-assemble evaporation method.
Uniquely structured tiny crystals (J-aggregates) of astaxanthin can be highly and stably loaded in
the DNA/chitosan nanocarriers.
Astaxanthin/DNA/chitosan nano-aggregates can be degraded by DNase I when necessary for
delivery.
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Graphics Abstract
Figure 1
Figure 2
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Figure 6