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Y. Xiao et al. / International Journal of Pharmaceutics 465 (2014) 143–158
The main problem affecting the efficacy CS is its poor intestinal
2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide
(DMSO) and 1,9-dimethyl- methylene blue (DMMB) were obtained
absorption, resulting fromitshigh molecular weight, charge density,
as well as hydrophilicity (Baici et al., 1992). Therefore, intestinal
absorption became one of critical factors for the successful
applicationofCSandCSderivativesinthetreatmentofosteoarthritis
and atherosclerosis. Some results reported in recent years have
demonstrated that oral delivery systems, including amphiphilic
polysaccharides, liposomes, enteric coatings, and the addition of
absorption enhancers, could be possible ways to improve the oral
absorption of polysaccharides (Kim et al., 2006; Mo et al., 2011; Qian
et al., 2013; Salartash et al., 2000). Drug carriers can be employed to
target transport carrier proteins or to open tight junctions between
the epithelial cells, so as to facilitate the absorption of polysacchar-
ides. Nano-sized polymeric micelles have been successfully applied
in oral drug delivery system. The core-shell structure of such self-
assembling systems presents advantages besides the improvement
of drug absorption, such as lower cytotoxicity, higher solubility, and
good stability(Yao et al., 2011; Zhang et al., 2010). What is more,
almost all of the nano-sized polymeric micelles of polysaccharides
are documented to be chitosan, heparin, pullulan (Duhem et al.,
2012; Kim et al., 2006; Mo et al., 2011; Salartash et al., 2000).
However, few works have focused on the use of self-assembling
micelles as the drug carrier systems for oral administration of CS.
Here, we want to figure out whether low molecular weight
amphiphilic polysaccharides-based self-assembling micelles would
enhance the oral absorption of CS. Esterification is considered as a
method to improve the hydrophobicity for many polysaccharides.
from Sigma–Aldrich (St. Louis, MO, USA).
a-Linolenic acid, 5-
aminofluorescein (5-AMF), 1-ethyl-(dimethylaminopropyl) carbo-
diimide (EDC), 4-Dimethylaminopyridine(DMAP), Hoechst 33342
and pyrene were purchased from Aladdin Chemistry Co., Ltd.
(Shanghai, China). Dialysis membranes (MWCO 1000), Tris
(hydroxymethyl) aminomethane (Tris), Triton X-100 and phenol
(saturated with water) were obtained from the Shanghai Medical
Chemical Reagent Co., Ltd. (Shanghai, China). Dulbecco’s Modified
Eagle Medium (DMEM), Fetal bovine serum (FBS), 0.25% trypsine
and Hanks balance salt solution (HBSS) were purchased from Gibco,
Invitrogen Corp (Ontario, USA). Confocal dishes, 25 cm2 plastic
culture flasks, 6-well and 96-well tissue culture plates were
obtained from Costar (Corning Incorporated, USA). Millicell 6-well
plate (12 mm, 3.0 mm pore size inserts) and actinase E were
purchased from Merck Millipore (Shanghai, China). Rhodamin-
phalloidin was purchased from Biotium, Inc. (Hayward, USA). 10-
dioctadecyl-3,3,30,30-tetramethylindocarbocyanine
(DiI) was purchased Beyotime Institute of Biotechnology (Jiangsu,
China). All the other chemicals and reagents used were of analytical
purity grade or higher, obtained commercially.
Clean grade Sprague–Dawley rats weighing 230 ꢀ 10 g were
obtained from Beijing HFK Bioscience Co., LTD (China, Document
No. SCXK 2009-0004). Rats were housed under 12 h light/dark cycle
conditions, with food and water freely available. Temperature and
relative humidity were maintained at 25 ꢀ 1 ꢁC and (55 ꢀ 5%),
respectively. All care and handling of animals were performed with
the approval of the Institutional Animal Care and Use Committee of
Shandong University. Guidelines of Institutional Animal Ethics
Committee were followed for in vivo experiments.
perchlorate
a
-Linolenic acid (a-LNA) is an essential fatty acid that cannot be
synthesized in humans body but only obtained from the diet.
a-LNA
can be converted into eicosapentaenoic acid (EPA) and docosahex-
aenoic acid (DHA) in the body, although conversion only occurs to a
limited extent(Brenna et al., 2009; Burdge and Calder, 2006). Apart
from potential indirect effects on cardiovascular diseases via
2.2. Synthesis of
sulfate polymers
a-linolenic acid-low molecular weight chondroitin
conversion into EPA and DHA,
a-LNA also has direct anti-
inflammatory (Stark et al., 2008), antiarrhythmic (Albert et al.,
2005), anti-thrombotic (Campos et al., 2008) and neuroprotective
effects (Nguemeni et al., 2010). Therefore, the modification of low
a
-LNA–LMCS was synthesized by the method as follows:
1 mmol of LMCS and 0.1 g of DMAP were dissolved in 10 mL of
molecular weight chondroitin sulfate (LMCS) with
a-LNA may
formamide by vigorous stirring at 55 ꢁC. To this solution, 0.1, 0.5, 1,
improve the hydrophobicity of LMCS, thereby leading to a positive
synergistic effect on anti-inflammation and anti-atherosclerosis.
or 2 mmol of a-linoleoyl chloride were added to change the
esterification degree. The mixture was stirred at 55 ꢁC for 1 h. The
product was dialyzed using dialysis membrane (MWCO 1000) in
deionized water for two days to remove excess reagents and
formamide, and further dialyzed against 95% ethanol for two days
In this work, we prepared LMCS micelles using
hydrophobic chain of the micelle-forming materials. The physico-
chemical characteristics of -LNA–LMCSs were investigated by
FTIR, 1HNMR, TGA/DSC, TEM, laser light scattering and zeta
potential. The oral bioavailability of -LNA–LMCS micelles in vivo
was evaluated by determining the -LNA–LMCS concentrations in
a-LNA as the
a
to remove unconjugated
a-linolenic acid. A yellowish precipitate
a
was thus obtained and purified by precipitation with 50 mL of 95%
ethanol. The solid material was vacuum-dried for 24 h, and a
yellowish powder was obtained. The resulting products were
designated as LNA–LMCS1, LNA–LMCS2, LNA–LMCS3 and
a
plasma levels following oral administration to rats in comparison
with CS, and Caco-2 cell monolayers representing in vitro model of
the intestinal epithelial barrier were used to determine the
LNA–LMCS4, respectively. The synthetic procedure of
a-LNALMCS
intestinal transport ability of
a-LNA–LMCS micelles. All these
polymers was shown in Fig. 1.
results indicated that the -LNA–LMCS micelles could enhance the
a
intestinal absorption of CS. Furthermore, we used confocal laser
scanning microscope (CLSM) to study the possible mechanisms,
and found that the enhancement of absorption was depending on
the paracellular pathway and endocytosis.
2.3. Preparation of a-LNA–LMCS micelles
a
-LNA–LMCS micelles were prepared via dialysis (Yang et al.,
2009) -LNA–LMCS(5 mg) was dissolved in 3 mL of formamide. To
a
form the nanomicelles, the prepared solution was dialyzed using
MWCO 1000 membrane in deionised water. After two days, the
dialysates were collected and diluted to 10 mL with water (final
2. Materials and methods
2.1. Materials and animals
concentration 0.5 mg/mL). The solution of a-LNA–LMCS micelles
wasstored at 4 ꢁC. The totalpreparationprocesswas shown in Fig. 2.
The average molecular weights of chondroitin sulfate (CS)
(Huamao Shuanghui Co., Ltd., Luohe, China) and Low molecular
weight chondroitin sulfate (LMCS, prepared in our lab) were
17.5 kDa and 4.1 kDa, respectively. The LMCS was obtained by
chemical depolymerization with hydrogen peroxide. Chondroitin
sulfate sodium salt from bovine cartilage, 3-(4,5-dimethylthiazol-
2.4. Physicochemical characterization of CS and its derivatives
2.4.1. Recording of Fourier transform infrared spectra
Fourier transform infrared (FTIR) spectra were recorded with a
NEXUS 470 spectrometer (Nicolet, USA) by KBr method.