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reagents. Deacetylation of chitosan increases the summary charge
by this mean increasing the ability to form electrostatic complexes
with proteins. On the other hand it is known that DD decreases
biodegradability of chitosan [26]. Hypothetically, endosomal
escape of chitosan-based complexes occurs less readily with high
DD chitosans [27]. This can lead to a better antigen presentation of
proteins.
4. Experimental protocols
4.1. Antigens and chemicals
D
-galactose, D-mannose, D-glucosamine D-glucose, lactobionic
acid, N-hydroxysuccinimide, mono-chloroacetic acid, and hydrox-
ylamine were obtained from Fluka, Switzerland; EDC, NHS, MES-(2-
[N-morpholino]ethanesulfonic acid), sodium-acetate, sodium
hydroxide, 2-propanol, mono-chloroacetic acid, ovalbumin, and
bovine albumin were purchased from Sigma Co, USA; celloviridine
G20x and crab chitosan were obtained from Berdsk and ‘‘BioPro-
gress’’, accordingly (Russia). Recombinant Asp f 2 from A. fumigatus
fungi was a gift of Dr.V.P. Kurup, (USA).
High molecular weight chitosan obtained by traditional method
is poorly soluble in water and thus is of a limited usage in practice.
Chemical modifications – N-trimethylchitosan chloride, mono-N-
carboxymethylchitosan and others are used to overcome this
disadvantage [7]. In this work we obtained low-molecular weight
chitosan by proteolitic hydrolysis. This LMW-Chi possessed all the
advantages of chitin such as biodegradability, nontoxicity, positive
charge of the natural polysaccharide. At the same time it became
water soluble when dissolved in 50 mM HCl and then neutralized
by sodium hydroxide to reach pH 7.2–7.4. Succinylation increased
solubility and made LMW-Chi soluble in water without previous
acidification. However, succinyl derivatives unexpectedly demon-
strated decreased adjuvant properties of lipophilic LMW-Chi.
LMW-Chi effectively penetrates into the cells of different
origins. Earlier it was shown that chitosan interacts with murine
macrophage cell line RAW264 via mannose-like receptor [10,11].
So, we compared the effects of LMW-Chi on RAW264 cells, which
express mannose receptor, and on murine T-cell lymphoma BYRB,
and human epithelial cell lines HaCaT and HeLa which do not
express this receptor. Non-phagocytic cells were also stained with
Chi–FITC, however, we found two different subpopulations in HeLa
and BYRB cells showing that some subpopulations were responsive
to chitosan, while others did not bind it. It could be hypothesized
that FITC positive cells also express mannose receptor. Normally
these receptors behave as antigen uptake/processing receptors and
are highly expressed on professional antigen presenting cells such
as dendritic cells and macrophages, however, they are also found at
moderate levels on B-cells, at low levels on T- and NK cells [28], as
well as on epidermal cells [29]. On the other hand it can be
hypothesized that other mechanisms different from mannose
receptor binding could be involved in LMW-Chi penetration into
mammalian cells.
4.2. Chitosan oligosaccharides
LMW water-soluble chitosan was obtained from HMW crab
chitosan by hydrolysis using Celloviridine G20x as described [31].
The reaction was performed for 0.5–2 h in sodium-acetate buffer
ꢂ
(pH 5.2) at 55 C and 1:400 enzyme/substrate ratio, and termi-
nated by 1 M sodium hydroxide. The precipitate was isolated by
centrifugation at 5000 g for 15 min, resuspended in water and
extensively dialyzed using Spectra/Por membrane (Cole-Parmer,
USA) against distilled water. This protocol was used throughout
all final purifications. The yield of lyophilized LMW-Chi was 80%.
MW of LMW-Chi depended on the time of digestion. Chitosan
with various degrees of acetylation was produced by reacetylation
of the original chitosan (methanol/2% acetic acid at 54:51 v/v
ratio). The amount of acetic anhydride was in the range 0.1–
2.0 mmol per 1 g of chitosan [32]. The degree of chitosan deace-
tylation (DD) was estimated by conductometric titration [33]. A
panel of LMW-Chi with different MWs and DD (MW in kDa/DD)
was prepared: 150/0.85; 30/0.85; 22/0.85; 17/0.85; 7/0.85; 5/0.85;
22/0.77; 22/0.67; 22/0.42. Intrinsic viscosity was determined at
ꢂ
25.0 ꢁ 0.5 C in Ubbelohde viscometer using 0.2 m sodium-
acetate and 2% acetic acid (at ratio 1:1 v/v) as a solvent. The
viscosity-average MW was calculated according to the Mark-
a
Houwink equation:
h
¼ k ꢀ M . Molecular weights of chitosan
´
LMW-Chi is an ideal adjuvant because it can be easily modified
to prepare conjugates with desired properties. Earlier it was shown
that oligosaccharides target chitosan conjugates to dendritic cells,
which are the major antigen presenting cells in the body. Chitosan
modified by fat acids on the other hand sorbs better to mucosal
tissues and by this mean enhances hydrophobic molecules delivery
via mucosal layers [7,30]. Technically it is possible to conjugate
chitosan with any anchor residues such as oligosaccharides and/or
lipophilic compounds and with antigenic proteins of interest. In
this work we obtained LMW-Chi conjugated with various carbo-
hydrates and fat acids. As the standard procedures were not
applicable for incorporation of oleic acid in chitosan for this
samples were determined by high-performance liquid chroma-
tography. The weight-average MW (M ), the number-average
MW (M ) and polydispersion M /M for chitosan were deter-
w
n
w
n
mined using Ultrahydrogel 500 column 7.8 ꢀ 300 mm (Waters,
USA) in 0.15 M ammonium acetate, 0.05 M acetic acid, pH 5.2 and
the elution rate 0.5 ml/min.
4.2.1. Chitosan-mannose, chitosan-galactose, chitosan-glucose and
chitosan-glucosamine
Chitosan-mannose (Chi-Man), chitosan-galactose (Chi-Gal),
chitosan-glucose (Chi-Glc) and chitosan-glucosamine (Chi-GlcN)
were synthesized as described in Ref. [34] with minor modifica-
tions [35]. LMW-Chi (MW 15–24 kDa, DD ¼ 0.85) 0.5 g was dis-
solved in 0.02 M aqueous acetic acid at 1% (w/v). After that,
monosaccharide (mannose, glucose, galactose or glucosamine) was
dissolved in chitosan solution to a final monosaccharide concen-
tration of 1% (w/v). Every 24 h aliquots were collected for the
purpose we used
b-alanine as a special linker. First, we prepared
oleyl- -alanine, which was coupled with chitosan by HBTU-
b
method and the conjugate received was further modified with
succinic anhydride.
However, all the LMW-Chi-conjugates studied in this work were
not more active than unmodified LMW-Chi. We hypothesized that
unmodified glucosamine units of LMW-Chi are responsible for the
adjuvant effect on humoral immune response induced in mice.
Adjuvant effect of LMW-Chi was studied in vivo using two
strains of mice, two antigens and two routes of immunization to
diversify the results. In all cases the enhancement of IgG response
by chitosan derivatives was comparable to or lower than
unmodified LMW-Chi. Thus, water-soluble, unmodified, and thus
cheap LMW-Chi can be used as adjuvant in parental and mucosal
vaccines.
ꢂ
absorbance analysis at 420 nm. Reaction was conducted at 65 C for
5 days. After that the solution was centrifuged, dialyzed and then
lyophilized as described above. The yield of water-soluble chitosan-
saccharide derivatives was 55–60%. Substitution degree was
determined using N-alkylated monosaccharide chitosan deriva-
tives. To 5 ml aliquots of 10 mg/ml chitosan derivatives in 0.02 M
3 3
CH COOH, (pH 5.3–5.9) 5 mg of NaCNBH was added. Reaction was
conducted for 18–24 h. The yield of lyophilized N-alkylated
monosaccharide chitosan derivatives was 80–85%. Substitution
degree varied from 5 to 8%.