Synthesis and characterization of ramose tetralactosyllysyl-chitosan-5- fluorouracil and its in vitro release

Article abstract of DOI:10.1007/s11164-011-0473-x

In order to improve drug loading and achieve a good release effect, this paper adopts the ramose method, choosing chitosan as the carrier and 5-fluorouracil (5-Fu) as a model drug. Ramose chitosan-lysyl-5-Fu(3) and ramose tetralactosyllysyl- chitosan-5-Fu(6) were synthesized successfully, then the in vitro release of (6) was researched. The results show that the drug loading of (3) and (6) are 9.17 and 1.63% (w/w), respectively. The in vitro release behavior of (6) in pH 7.4 phosphate buffer solution and pH 1.2 HCl-KCl solution were studied. The zero order release time that (6) maintains in alkaline and acidic media are 64 and 24 h, and the total release by 184 h are 71.97 and 82.34%, respectively. The performance is smooth throughout the whole stage of release, and the concentration of cumulative release is lower in the alkaline environment than in the acidic environment over the same time. Springer Science+Business Media B.V. 2012.

Full text of DOI:10.1007/s11164-011-0473-x

Res Chem Intermed (2012) 38:1421–1429
DOI 10.1007/s11164-011-0473-x
Synthesis and characterization of ramose tetralactosyl-
lysyl-chitosan-5-fluorouracil and its in vitro release
He-ping Li
•
Long Qin
•
Zhou-dong Wang Shan Li
•
Received: 22 September 2011 / Accepted: 17 December 2011 / Published online: 4 January 2012
Ó Springer Science+Business Media B.V. 2012
Abstract In order to improve drug loading and achieve a good release effect, this
paper adopts the ramose method, choosing chitosan as the carrier and 5-fluorouracil
(
5-Fu) as a model drug. Ramose chitosan-lysyl-5-Fu(3) and ramose tetralactosyl-
lysyl-chitosan-5-Fu(6) were synthesized successfully, then the in vitro release of (6)
was researched. The results show that the drug loading of (3) and (6) are 9.17 and
1
.63% (w/w), respectively. The in vitro release behavior of (6) in pH 7.4 phosphate
buffer solution and pH 1.2 HCl–KCl solution were studied. The zero order release
time that (6) maintains in alkaline and acidic media are 64 and 24 h, and the total
release by 184 h are 71.97 and 82.34%, respectively. The performance is smooth
throughout the whole stage of release, and the concentration of cumulative release is
lower in the alkaline environment than in the acidic environment over the same
time.
Keywords Chitosan ꢀ Lysine ꢀ Lactose acid ꢀ Ramose ꢀ 5-Fluorouracil
Introduction
To date, polysaccharides have been widely used in pharmaceutical technology as
first choice excipients for production of traditional formulations. Their peculiar
physicochemical and biological properties have been exploited to develop
macromolecular prodrugs which can exhibit favorable biopharmaceutical properties
and enforce the therapeutic performance of the parent drugs. These polymers
display, in fact, high biocompatibility and biodegradability, multiple insertion
H. Li (&) ꢀ L. Qin ꢀ Z. Wang ꢀ S. Li
College of Chemical and Biologic Engineering, Changsha University of Science and Technology,
Changsha 410004, China
e-mail: lihp@csust.edu.cn
123

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H. Li et al.
points, and biological properties that can be advantageously exploited in drug
delivery [1]. Compared with most polysaccharides, chitosan is a better drug carrier.
Currently, chitosan, or chitosan derivatives, as a carrier to carry 5-Fu mostly uses
an encapsulated form. It has recently been prepared as nanoparticles or
microspheres, which can improve the targeting performance, but the greatest
disadvantage is instability release. Generally speaking, the reason for a burst release
phenomenon is that the mechanical strength of in the encapsulated microcapsule or
drug-containing gel is not strong enough, causing rapid leakage of in the drug, so we
have begun to research chemical-bonded drugs [2]. Microspheres or nanoparticles
can only achieve passive targeting. Refined targeting selectivity and high delivery
efficiency are two equally important goals in the development of drug delivery
systems [3]. So it is a good idea for us to choose a drug carrier with an active
targeting group. The galactose group can be recognised by liver cell surface
asialoglycoprotein (ASGP) receptors. ASGP receptors participate in the hepatic
metabolism of serum proteins and can recognize a glycoprotein with galactose
residues on the terminal position of the saccharide chain [4], so the compounds
based on in the galactose group generate a target property. Glycobiology studies
have shown that there are many target chambers on the surface of asialoglycoprotein
receptors which can identify galactosyl. In the recognition process, a number of
galactosyls combining with receptor proteins can generate synergy, which enhances
the targeting and binding force. The more ligand groups, the stronger the targeting
and binding effect, which is known as the ‘‘aggregation effect’’ or ‘‘multivalent
effect’’ [5, 6].
The successful synthesis of a water-soluble cationic graft copolymer, chitosan-
graft-poly (L-lysine), has been reported, and this novel material would be a good
candidate as a scaffold for cell culture and tissue engineering [7]. Liu et al. [8]
prepared polyion complex micelles (PIC micelles) based on methoxy-poly-
(ethylene glycol) (PEG)-graft-chitosan and lactose-conjugated PEG-graft-chitosan
for liver-targeted delivery of diammonium glycyrrhizinate (DG).
This paper chose 5-Fu as a model drug, galactose as a targeting group, and we
adopted the coupling method, hoping to synthesize the target compound to have
both good targeting property and superior release performance.
Experimental
Apparatus
FTIR spectra were recorded with a Nicolet Avatar FT/IR-360 spectrophotometer
(
USA). UV–Vis absorption spectra were recorded with a TU-1901 dual beam UV–
Vis spectrophotometer (Purkinje General Instrument, Beijing, China) between 200
and 800 nm. We also used HP6890/5973 Agilent 1,100 Liquid mass spectrometer,
Varian Mercury-VX 400-type (400 MHz) NMR, FD-1A-type freezer Dryer, WRS-
1-type melting point apparatus, dialysis bag, MWCO: 8,000–14,000; Beijing
Dingguo Biotechnology.
1
23

Synthesis and characterization of ramose
1423
Reagents
Chitosan (DD = 98.87%, Mg = 411KDa), self-restraint. Bromoacetic acid (AR);
Aladdin Reagent. L-lysine (BR); National Pharmaceutical Group Chemical Reagent.
5-Fu: 99%, J & K Chemical Reagent. Lactose acid (LA): 97%; J & K Chemical Reagent.
1
-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCꢀHCl): 98.5%;
National Pharmaceutical Group Chemical Reagent. 1-Hydroxy-2,5-pyrrolidinedione
(NHS): 98%; National Pharmaceutical Group Chemical Reagent. N,N-Dimethylpyridin-
4-amine (DMAP): 99%, Aladdin Reagent. All other chemicals were of analytical grade
and used without further purification.
Synthesis
Synthesis of N-carboxymethyl-5-Fu (1)
Synthesized according to Ref. [9]. The procedure is as follows: the KOH solid
1
.010 g (18 mmol) was added to a stirred 5-Fu 0.52 g (4 mmol) in distilled water
(
BrCH COOH dissolved in distilled water (2 mL) was added dropwise to the
2 mL), 50 °C constant magnetic stirring 0.5 h. Then, 0.8341 g (6 mmol)
2
mixture and the mixture was stirred for 3 h at reflux. In the whole process, a
moderate amount of KOH solid was added portionwise to maintain the pH value at
1
3. The resultant solution was cooled to r.t. and then the pH was adjusted with
concentrated HCl to 5.5. On being kept in the refrigerator for 4 h, if a white
precipitate appeared, then it was filtrated and the filtrate acidified with concentrated
HCl to pH 1, whereupon a white needle-like crystal appeared. It was again kept in
the refrigerator, for 12 h, filtrated, and the filter cake washed with 0 °C water three
times, and dried under vacuum for 48 h to yield a white needle-like crystal. yield:
-
0
.5595 g (73%); mp: 274–276 °C; MS: 186.8 [M-1] .
Synthesis of lysyl-5-Fu (2)
In a 100-mL round-bottom flask, 0.094 g (0.5 mmol) carboxymethyl-5-Fu was
dissolved in 4 mL distilled water, then EDCꢀHCl 0.1438 g (0.75 mmol), NHS
0
.0863 g (0.75 mmol) and DMAP 0.0122 g (0.1 mmol) dissolved in 5 mL distilled
water were added to it and the mixture was stirred for 4 h at room temperature.
After that, 0.0548 g (0.375 mmol) lysine was added to the mixture and stirred until
dissolved, then the pH value was adjusted by NaHCO solid powder to 7.5–8.0, and
3
?
the mixture was stirred for 24 h at 25 °C [10]; MS: 487.2 [M ? 1] .
Synthesis of ramose chitosan-lysyl-5-Fu (3)
EDCꢀHCl 0.1100 g (0.57 mmol), NHS 0.0656 g (0.57 mmol), DMAP 0.0122 g
(0.1 mmol) dissolved in 2 mL distilled water were added to (2), and the mixture was
stirred for 24 h at 25 °C. Chitosan 0.2000 g (1.25 mmol free NH ) dissolved in 8 mL
2
0.1 M HCl solution was added to activation solution (2), the reaction was stirred for 48 h
at 25 °C, dialyzed against deionized water over 3 days, and the product freeze-dried.
123

1424
H. Li et al.
-1
Compound (3) is yellowish sponge-like solid. IR (KBr): 3,376 cm (–OH and –NH
-1
stretching vibration mixed); 1,697 cm (amide I with C=O stretching vibration);
-1
-1
,616 cm (amide II with N–H deformation vibration); 1,308 cm (amide III with C–
1
N stretching vibration); 1,245 cm (C–F stretching vibration); 1,152 cm (C–O–C
-1
-1
-1
asymmetric stretching vibration); 1,082 cm (C–O stretching vibration of chitosan C3).
UV–Vis spectra showed a strong absorption peak at 269 nm.
Synthesis of branched trimeric lysine (4)
Lysine amino protection [11]: the Lysine 1.7520 g (12 mmol) was added to a stirred
mixture of 3 mL dioxane and 3 mL distilled water at reflux, and then 6 mL 2 M
NaOH solution was added to it. Under the ice bath, 26.2000 g (120 mmol) di-tert-
butyl dicarbonate((Boc) O) was added dropwise to the mixture in 1.5 h, and the
2
mixture was stirred for 1.5 h at room temperature. In the whole process, it is
necessary to control the pH value at 9 with 2 M NaOH solution. The resulting
solution was concentrated by a rotary evaporator at 75 °C, cooled to r.t. and then the
pH was adjusted with concentrated HCl to 2. This residue was extracted with ethyl
acetate (3 9 35 mL) and the extract was washed with saturated Na SO solution
2
4
(3 9 5 mL), and dried (anhydrous Na SO ), evaporation of ethyl acetate under
2 4
reduced pressure gave a light yellow viscous oil.
Synthesis of ramose trimeric lysine was according to Ref. [12]: the amino-
protected lysine dissolved in distilled water (2 mL) was added to the mixture of
EDCꢀHCl 0.8400 g (4.38 mmol), NHS 0.5050 g (4.38 mmol), and DMAP 0.0535 g
(
0.438 mmol) dissolved in distilled water (3 mL), and then the mixture was
activated for 4 h at room temperature. After that, 0.3200 g (2.19 mmol) lysine
dissolved in distilled water (2 mL) was added, then the pH value was adjusted by
NaHCO solid powder to 8, and the mixture was stirred for 24 h at 25 °C. without
3
separation, Evaporation was under reduced pressure with drying in the vacuum. The
dried material dispersed in 2 mL trifluoroaceticacid (TFA) was stirred for 2 h at
room temperature. Similarly, without separation, evaporation was under reduced
?
pressure and drying in the vacuum: MS: 403.2 [M ? 1] .
Synthesis of tetralactosyl-trimeric-lysine (5)
Synthesis of (5) was according to Ref. [12]. Lactose acid 0.2150 g (0.6 mmol)
dissolved in 2 mL distilled water was added to the mixture of EDCꢀHCl 0.1380 g
(
0.72 mmol), NHS 0.0829 g (0.72 mmol), and DMAP 0.0088 g (0.072 mmol). The
mixture was activated for 4 h at room temperature. After that, 0.1200 g (0.3 mmol)
4) was added then the pH value was adjusted by NaHCO solid powder to 7.5–8.0.
(
3
The mixture was stirred for 48 h at room temperature, and used directly for the next
reaction without separation.
Synthesis of ramose tetralactosyl-lysyl-chitosan-5-Fu (6)
The mixture of EDCꢀHCl 0.0900 g (0.45 mmol), NHS 0.0520 g (0.45 mmol), and
DMAP 0.0100 g (0.090 mmol) was added to (5), and activated for 4 h at room
1
23

Synthesis and characterization of ramose
1425
temperature. After that, 0.1000 g (3) dissolved in 0.1 M HCl solution was added,
then the pH value was adjusted with TEA to 7.5–8.0, and the mixture was stirred for
48 h at room temperature. After being dialyzed against deionized water over 3 days,
1
and freeze-dried, the product, compound (6) is a yellowish spongy solid. H NMR
(
C H); 2.735 (lactosyl C2OH); 3.033 (lysyl C6H); 3.246 (C6H of trimeric lysine
F COOD-DMSO-d , 20 °C): 1.075 (lysyl C4H); 1.536 (lysyl C5H); 1.751 (lysyl
3
6
3
which combine with lactosyl); 4.049 (5-Fu-CH (connected to C2 of lysyl)); 4.558
2
1
5-FuCH (connected to C6 of lysyl); 4.735 (lactosyl pyran ring C H); 7.623 (lysyl
(
C6NHCO); 8.058 (5-Fu ring C=C–H); 8.159 (NHCO in chitosan); 8.276 (C2NHCO
2
-
of lysyl) ppm. IR(KBr disk): 3,366 cm (O–H and N–H stretching vibration);
1
-
,978, 2,934, 2,869 cm (C–H stretching vibration of lysyl, lactosyl and chitosan);
1
2
1
-
1
-1
715 cm
(C4=O stretching vibration of pyrimidine ring); 1,699 cm
-
(C=O
1
stretching vibration of amide I); 1,650 cm (N–H deformation vibration of amide
-
II); 1,558, 1,539 cm (N–H deformation vibration of amino involving in lysyl and
1
-
1
chitosan); 1,367 cm (C–N stretching vibration of amide III); 1,251 cm (C–F
-1
-
1
stretching vibration); 1,155 cm
-
,081 cm (C3=O stretching vibration of chitosan ring) cm . UV–Vis spectra
(C–O–C asymmetric stretching vibration);
1
-1
1
show a strong absorption peak at 269 nm.
Determination of drug loading
Using a certain amount of (2) and (6) dissolved in 10 mL distilled water, UV–Vis
absorption spectra were scanned from 200 to 800 nm, and a strong absorption peak
showed at 269 nm.
-
1
Configuring a series of standard solutions with carboxymethyl 5-Fu: 0 mg L
-1
,
-
1
-1
-1
5
mg L , 10 mg L , 15 mg L , and 20 mg L , which were measured at
69 nm. according to this step, we got the standard curve and linear regression
2
equation as follows:
Abs ¼ 0:03984C=mg Lꢁ þ 0:00861ðR ¼ 0:9995Þ
1
ð1Þ
Then, 15 mg (3) and (6) were dissolved in 10 mL water, respectively, and we
filtered the suspended solid, measured absorbance at 269 nm, and calculated the
drug loading by the equation as follows:
the content of 5 ꢁ Fu in sample
Drug Loading ðDLÞ ¼
ꢂ 100%
ð2Þ
the amount of sample
The in vitro release study
The in vitro release was carried out according to Ref. [13]. An amount of 25 mg (6)
was suspended in PBS (5 mL, pH 7.4) and artificial gastric juice HCl-KCl (5 mL,
pH 1.2) buffer solution in two dialysis bags, respectively. The dialysis bag was
sealed and then slowly shaken (50 rpm) in 100 mL of PBS and HCl-KCl at 37 °C in
a 250-mL Erlenmeyer flask. Aliquots of the solution outside the dialysis membrane
(5 mL) were replaced with 5 mL of dialysis medium at various time intervals and
tested at 269 nm by UV–Vis spectrophotometer.
123

1426
H. Li et al.
Results and discussion
The synthetic route of compound (6) is shown in Scheme 1.
In the synthesis of compound (1), pH is a key factor, the yield was greatly
affected by the base, and the use of KOH increased the yield by up to 73%.
Coupling of compound (1) and lysine was performed using EDCꢀHCl/NHS/DMAP
in water at ambient temperature. The use of water as reaction medium has received
considerable attention in the context of green chemistry for several reasons: (1) it is
cheap, safe, and environmentally benign, (2) reactions in aqueous medium eliminate
the additional efforts in making the substrates and reagents dry before use and thus
reduce/eliminate the consumption of drying agents, energy, and time, and (3) the
unique physical and chemical properties of water can be utilized to realize reactivity
or selectivity that cannot be attained in organic solvents [14]. In pursuit of our recent
efforts to develop environmentally friendly synthetic methodologies by carrying out
reactions in water, very gratifyingly, the desired product was obtained in a easy
manner.
In the synthetic process of target compound (6), protection of lysine was with
Boc) O, because it is easy to crystallize, and there is a certain stability. More
2
(
importantly, it is easy to remove the Boc group. Generally speaking, removal of the
Boc group is by using TFA at room temperature. We still use the coupling agent
EDC for the acylation reaction, because its urea derivatives have a good water-
soluble property, which is conducive to the final step of dialyzing.
Characterization
It can be seen from the IR of compound (3) that the absorption peaks of C4=O,
C=C, and C–F are made obvious by introducing 5-Fu. Comparrf with chitosan, the
obvious change is that the absorption peak of the amide group appeared in
compound (3). There are three kinds of amino absorption peaks corresponding to
the amino group in the structure. This can be very good at proving the existence of
the ramose structure. Simultaneously, it also showed that the synthesis was
successful.
Compared with IR of compound (6) and compound (3), the absorption peak of
amide group and C4=O, C=C, and C–F in 5-Fu is obvious as shown before. But a
-
1
strong absorption peak at 1,155 cm appeared, which was attributed to C–O–C
asymmetric stretching vibration in the lactosyl group. We could judge from the IR
of compound (6) that the lactosyl group was successfully introduced.
The UV–Vis spectra of compounds (3) and (6) showed that characteristic
absorption peaks of 5-Fu ring all appeared, which showed that 5-Fu was
successfully connected to chitosan. The drug loadings of compounds (3) and (6)
are 9.17 and 1.63%, respectively.
1
H NMR of compound (6) showed that 3.033 ppm is the chemical shift of C6-H
after the acylation reaction between carboxymethyl 5-Fu and C6-NH of lysine;
2
4
carboxymethyl 5-Fu and C2 -NH of lysine; 4.558 ppm is the chemical shift of
.049 ppm is the chemical shift of 5-Fu-CH after the acylation reaction between
2
2
5
-Fu-CH after the acylation reaction between carboxymethyl 5-Fu and C6-NH of
2
2
1
23

Synthesis and characterization of ramose
1427
O
O
N
OH
O
Br
O
N
HO
HN
HO
HN
KOH
O
H
2
NH
2
NH
+
O
i)
OH
NH
O
F
O
F
O
EDC/NHS/DMAP
HN
O
O
N
O
N
O
(
1)
F
OH
O
HN
HN
F
O
(
2)
O
HO
OH
NH
O
x
O
x<n
O
HO
HN
NH
NH
2
n
O
F
O
EDC/NHS/DMAP
O
N
O
N
O
HN
HN
F
O
(3)
OH
O
H
2
N
N
BocHN
COOH
COOH
Lys
(
Boc) O
EDC/NHS/DMAP
HN
NH
2
ii)
Lys
O
O
Lys
Lys
H
2
BocHN
O
NH NH
Boc Boc
HN
NH
Boc Boc
OH
OH
O
Lys
Lys
r.t
TFA
LA-OH
HN
NH
HN
NH
EDC/NHS/DMAP
O
O
O
O
Lys
Lys
Lys
Lys
NH NH
LA LA
HN
NH
NH
2
NH2
H N
NH
2
2
LA LA
(4)
(5)
OH
O
OH
O
O
O
HO
HO
O
x
O
NH
y
x+y < n
NH
F
O
O
N
H
N
H
N
H
N
H
NH
O
N
O
O
O
HN
O
(
3)
HN
HO
OH HO
OH
OH O
NH
HO HO
OH OH
EDC/NHS/DMAP
O HO
iii)
+
(
HN
HO
O
OH HO
O
OH HO
OH
O
5)
O
O
O
N
O
OH
OH HO
OH HO
HO
O
O
O
O
HN
F
OH
HO
HO
OH
HO
OH
OH
OH
OH OH
(6)
O
OH
HO
OH
Lys =
LA =
OH
O
HO
HO
O
OH
OH
Scheme 1 The synthetic route of (6)
123

1428
H. Li et al.
lysine. This is good at proving the successful synthesis of (2). The chemical shift of
C6-H of lysine is 3.246 ppm after the acylation reaction between lactose acid and
C6-NH2 of lysine in trimeric lysine. However, the chemical shift of C6-H
connected with chitosan will not appear at this place, which proved the existence
of ramose structure. The chemical shift of 7.623, 8.159, and 8.159 ppm
corresponded to three kinds of amide bonds respectively, one is two amide bonds
of lysyl, the other is the formation between chitosan and lysine. In a word, the
synthesis is so successful.
The in vitro release behavior of compound (6)
As we all know, the pH of blood in human body is about 7.4, and pH of gastric juice
is about 1.2, so we simulated the environment of these two fluids. The in vitro
release property was tested by the standard method, and the result is shown in
Fig. 1.
It can be seen from Fig. 1 that compound (6) shows good slow-release
characteristics in alkaline environment. The time that maintains zero-order release is
6
6
4 h, and in this period, the accumulative release quantity reaches 54.65%; after
4 h, the release becomes slow, but still increases markedly, the accumulative
release quantity reached 71.97% in 184 h. In the acidic medium, the accumulative
release quantity is 82.34% in 184 h; the zero-order release time was maintained at
2
4 h. After that, the delivery rate become slow, but obviously also increases, and the
rate of increase is much higher than that in the alkaline medium. The common
feature is that the delivery rate become slow after the first fast release, which may be
caused by hydrolysis balance. First of all, the hydrolysis rate is much faster than the
delivery rate at pH 1.2. Secondly, in the acidic medium, the chitosan chain was
hydrolyzed, which accelerated the diffusion of the drug. That is because the chitosan
structure was considerably damaged after the modification with two large volumes
of branching moleculars.
100
pH=7.4
pH=1.2
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
50
100
150
release time/ h
Fig. 1 The in vitro release of (6)
1
23

Synthesis and characterization of ramose
1429
Conclusion
1
IR, H NMR, UV–Vis and LC/MS indicate successful synthesis of lysyl-5-Fu
1
)
(2), ramose chitosan-lysyl-5-Fu(3), and ramose tetralactosyl-lysyl- chitosan-5-
Fu(6).
2
)
The drug loading of ramose chitosan-lysyl-5-Fu(3) and ramose tetralactosyl-
lysyl-chitosan-5-Fu(6) were 9.17 and 1.63% (w/w), respectively, which is
determined by UV–Vis. On the other hand, we tested the behavior of the in
vitro release in pH 7.4 PBS buffer solution and pH 1.2 HCl-KCl solution. The
zero-order release time that compound (6) maintains in alkaline and acidic
media were 64 and 24 h, and the total release by 184 h were 71.97 and 82.34%,
respectively. The performance is smooth throughout the whole stage of release,
and the concentration of cumulative release is lower in the alkaline
environment than the acidic environment at the same time.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.
0775010), the Natural Science Foundation of Hunan Province of China (06JJ2038) and the Graduate
Innovation Fund of Hunan Province of China.
2
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