Preparation, characterization, and in vitro efficacy of O-carboxymethyl chitosan conjugate of melphalan

Article abstract of DOI:10.1016/j.carbpol.2013.04.071

A series of melphalan-O-carboxymethyl chitosan (Mel-OCM-chitosan) conjugates with different spacers were prepared and structurally characterized. All conjugates showed satisfactory water-solubility (160- 217 times of Mel solubility). In vitro drug release behaviors by both chemical and enzymatic hydrolysis were investigated. The prodrugs released Mel rapidly within papain and lysosomal enzymes of about 40-75%, while released only about 4-5% in buffer and plasma, which suggested that the conjugates have good plasma stability and the hydrolysis in both papain and lysosomes occurs mostly via enzymolysis. It was found that the spacers have important effect on the drug content, water solubility, drug release properties and cytotoxicity of Mel-OCM-chitosan conjugates. Cytotoxicity studies by MTT assay demonstrated that these conjugates had 52-70% of cytotoxicity against RPMI8226 cells in vitro as compared with free Mel, indicating the conjugates did not lose anti-cancer activity of Mel. Overall these studies indicated Mel-OCM-chitosan conjugates as potential prodrugs for cancer treatment.

Full text of DOI:10.1016/j.carbpol.2013.04.071

Carbohydrate Polymers 98 (2013) 36–42
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation, characterization, and in vitro efficacy of
O-carboxymethyl chitosan conjugate of melphalan
∗
Bo Lu, Dan Huang, Hua Zheng , Zhijun Huang, Peihu Xu, Haixing Xu, Yihua Yin, Xia Liu,
Dan Li, Xueqiong Zhang
Department of Pharmaceutical Engineering, School of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of melphalan-O-carboxymethyl chitosan (Mel-OCM-chitosan) conjugates with different spacers
were prepared and structurally characterized. All conjugates showed satisfactory water-solubility (160-
Received 25 March 2013
Received in revised form 23 April 2013
Accepted 24 April 2013
Available online xxx
2
17 times of Mel solubility). In vitro drug release behaviors by both chemical and enzymatic hydrolysis
were investigated. The prodrugs released Mel rapidly within papain and lysosomal enzymes of about
0–75%, while released only about 4–5% in buffer and plasma, which suggested that the conjugates have
4
good plasma stability and the hydrolysis in both papain and lysosomes occurs mostly via enzymolysis.
It was found that the spacers have important effect on the drug content, water solubility, drug release
properties and cytotoxicity of Mel-OCM-chitosan conjugates. Cytotoxicity studies by MTT assay demon-
strated that these conjugates had 52–70% of cytotoxicity against RPMI8226 cells in vitro as compared
with free Mel, indicating the conjugates did not lose anti-cancer activity of Mel. Overall these studies
indicated Mel-OCM-chitosan conjugates as potential prodrugs for cancer treatment.
Keywords:
Melphalan
Carboxymethyl chitosan
Polymeric prodrug
©
2013 Elsevier Ltd. All rights reserved.
1
. Introduction
prodrug have several advantages including: (1) an increase in
water solubility of low soluble or insoluble drugs, and therefore,
enhance drug bioavailability; (2) protection of drug from deac-
tivation and preservation of its activity during circulation; (3)
an improvement in pharmacokinetics; (4) the ability of provide
passive targeting due to the enhanced permeability and reten-
tion (EPR) effect (Khandare & Minko, 2006; Mahato, Tai, & Cheng,
2011). Many polymeric prodrugs which contain alkylating agents
have been synthesized, including poly amino acid (poly-l-lysine
and poly-l-glutamic acid)-Mel (Yasunori et al., 1984), HPMA
copolymer-Mel (Duncan et al., 1991), albumin-chlorambucil (Kratz
et al., 1998), transferrin-chlorambucil (Beyer et al., 1998), PAMAM-
chlorambucil (Bielawski, Bielawska, Muszy n´ ska, Poplawska, &
Czarnomysy, 2011) and fluorodeoxyglucose-chlorambucil (Miot-
Noirault et al., 2011). These investigations proved that such
conjugates incorporating a covalent linkage for attachment of drugs
to carriers were suitable for alkylating agents delivery.
Melphalan is a cytotoxic drug that acts as a bifunctional alkyl-
ating agent on DNA and was introduced into clinic since the late
950s. Mel can be administered orally or systemically to treat
1
variety of cancers including myelomas, ovarian cancer and sarco-
mas. The main obstacles for Mel in clinical applications are due to
its extremely poor aqueous solubility, relatively instability, rapid
plasma clearance, poor bioavailability and biocompatibility (David,
Roger, & Valentino, 1999), which would cause severe side effects.
To solve these problems, many efforts have been made to syn-
thesize derivatives of Mel. The chemical structure of Mel invites
to modification of both the N- and C-termini as well as incorpora-
tion into peptides (Morris, Atassi, Guilbaud, & Cordi, 1997; Peyrode
et al., 2012; Sartania et al., 1999; Scutaru, Wenzel, & Gust, 2011;
Zhao, Meng, Yuan, & Lan, 2010). For instance, beta-alanyl-Mel (Tsay
&
Lloyd, 1987) and proline prodrug of Mel (Mittal et al., 2004) have
been prepared. These compounds showed much higher cytotoxic
activity and had the potential for enhanced tumor selectivity. Nev-
ertheless, these prodrugs were still water-insoluble and underwent
unavoidable rapid elimination of the drugs from circulation.
Conjugating drug molecules with biocompatible water-soluble
polymer is an alternative way to solve these problems. Polymeric
The appropriate polymeric carriers must be biocompatible and
water-soluble. OCM-chitosan is a water soluble chitosan derivative,
seems to be one of the most useful candidates for the drug car-
rier because of its non-toxicity, biodegradability (Fu et al., 2011),
biocompatibility and high water solubility (Zheng, Han, Yang, Liu,
2011; Zheng, Rao, et al., 2011). Besides, OCM-chitosan can be easily
derived from chitin, which is cheap and abundance in nature. OCM-
chitosan contains a large number of COOH and NH groups in the
2
∗ Corresponding author. Tel.: +86 27 87859019; fax: +86 27 87859019.
E-mail address: whutlvb@163.com (H. Zheng).
molecular that can be easily conjugated to drugs and proteins by
either direct attachment or through a linker. Chitosan derivatives
0
144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.04.071

B. Lu et al. / Carbohydrate Polymers 98 (2013) 36–42
37
based prodrugs have gained significant interest for drug delivery
Lee, Kim, Lee, & Jon, 2009; Sabaa, Mohamed, Mohamed, Khalil, &
Abd El Latif, 2010; Tokura, Miura, Johmen, Nishi, & Nishimura, 1994;
Wang et al., 2011) to increase the longevity of therapeutic agents
in the circulation and direct antitumor drugs to the tumor tissues
through passive accumulation in the tumor.
In polymeric prodrug design, polymer linkage plays a crucial
role in determining therapeutic potentials. Amino and small pep-
tide linkers are widely used (Greenwald, 2001; Mitsunori, Hiroyuki,
Toshiro, Takehiko, & Satoshi, 2000) due to their chemical versa-
tility for covalent conjugation and biodegradability, which can be
specific substrates for plasmin enzyme and proteinases whose con-
centration are much higher in various kinds of tumor mass (Ding
et al., 2012).
In this work, for improving water-solubility, systemic circula-
tion time, and pharmacokinetic profiles of Mel, Mel-OCM-chitosan
conjugates linked with different amino acid spacers (including l-
glycine (Gly), l-phenylalanine (Phe), l-leucine (Leu) and l-proline
2.2.2. Synthesis of Fmoc-Mel-amino acid
(
A stirred solution of Fmoc-melphalan (10 mmol, 5.43 g) and NHS
(1.05 equiv., 10.5 mmol, 1.21 g) in dry THF (100 mL) was treated at
◦
0 C with DCC (1.05 equiv., 10.5 mmol, 2.17 g). After 2 h, the mix-
ture was allowed to warm to room temperature and stirred for
22 h. The precipitate was removed by filtration and washed with
dry THF (2× 10 mL). The washes and filtrate containing Fmoc-
melphalan anhydride were combined. Amino acid (10 mmol, the
amino acid including Gly, Phe, Leu and Pro) was slowly added to
a mixture solution of NaHCO3 (1 equiv., 10 mmol, 0.84 g), water
(50 mL) and THF (30 mL). After stirring for 15 min, the THF solu-
tion of Fmoc-melphalan anhydride prepared above was added
to this suspension. The mixture was stirred at room tempera-
ture for 22 h, and then THF was removed as much as possible
◦
in vacuo at 30 C, then the mixture was partitioned between
ethyl acetate and water and adjusted to pH 2 with hydrochlo-
ric acid, the aqueous phase was extracted with ethyl acetate (3×
15 mL). The combined organic phases were washed with distilled
(
Pro)) were synthesized. The solubility, in vitro drug release, cell
water and brine and dried over MgSO . The resulting solution
4
cytotoxicity of Mel-OCM-chitosan conjugates were systematically
investigated. It was expected that the conjugates would be solu-
ble and stable during circulation, while in the targeted cells, the
conjugates would lead to a controlled release of Mel.
was concentrated and purified by column chromatography (hex-
ane:ethyl acetate = 1:1, v/v) to give the compound as off-white
solid.
Fmoc-Mel: 1H NMR (CD Cl, ppm) d: ı = 2.88–3.14 (m, 2H,
3
CH Ph), 3.48–3.78 (m, 8H, N(CH CH ) ), 4.2 (t, 1H, CH[Fmoc]),
2
2
2 2
4
.33–4.53 (m, 2H, CH2 [Fmoc]), 4.65 (m, 1H, CHCO), 5.4 (1H, m,
2
. Experimental
NH), 6.6 (d, 2H, arom meta), 7.0 (d, 2H, arom ortho), 7.28–7.83 (8H,
m, Ar[Fmoc]).
Fmoc-Mel-Gly: 1H NMR (CD Cl, ppm) d: ı = 2.95–3.12 (2H,
2.1. Materials
3
CH Ph), 3.45–3.72 (8H, N(CH CH ) ), 4.85–4.65 (6H, CH[Fmoc],
2
2
2 2
4
OCM-chitosan (molecular weight 8.6 × 10 , degree of deacety-
CH [Fmoc], CHCO, CH [Gly]), 5.25–5.4 (2H, NH CO), 6.5–7.0 (4H,
2 2
lation 91% and degree of substitution 85%) was purchased from
Qingdao Xunbo Biotechnology Co., Ltd. (China). Mel was pur-
chased from Suzhou Lide Chemistry Co., Ltd. (China). Amino
acids were purchased from Aladdin Reagent Co., Ltd. (China).
benzene ring), 7.25–7.8 (8H, Ar[Fmoc]).
Fmoc-Mel-Phe: 1H NMR (CD Cl, ppm) d: ı = 2.85–3.15 (4H,
3
CH Ph), 3.51–3.75 (8H, N(CH CH ) ), 4.15–4.8 (5H, CH[Fmoc],
2
2
2 2
CH [Fmoc], CHCO), 5.3–5.5 (2H, NH CO), 6.6–7.25 (8H, benzene
2
1
-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochlo-
ring), 7.28–7.85 (8H, Ar[Fmoc]).
1
ride (EDC·HCl), 1,3-dicyclohexyl carbodiimide (DCC) and
N-hydroxysuccinimide (NHS) were obtained from Sinopharm
Chemical Reagent Co., Ltd. (Shanghai, China). 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was
purchased from Sigma–Aldrich Chemical Co., Ltd. (St. Louis, MO).
Adult male Sprague-Dawley rats (200–225 g) were obtained from
Hubei Experimental Animal Center (Wuhan, China). All other
reagents were of analytical grade, and used as received without
further purification. RPMI8226 cells were purchased from ATCC
Fmoc-Mel-Leu: H NMR (CD Cl, ppm) d: ı = 0.91 (6H, CH(CH ) ),
3
3 2
1.5–1.75 (1H, CH(CH ) ), 2.88–3.14 (2H, CH Ph), 3.48–3.78 (8H,
3
2
2
N(CH CH ) ), 4.1–4.65 (7H, CH[Fmoc], CH [Fmoc]), CHCO, CH[Leu],
2
2
2
2
CH [Leu]), 5.68 (2H, NH CO), 6.6–7.1 (4H, benzene ring), 7.3–7.82
2
(8H, Ar[Fmoc]).
Fmoc-Mel-Pro: 1H NMR (CD Cl, ppm) d: ı = 1.51–2.25 (4H,
3
CH [Pro]), 2.85–3.25 (2H, CH Ph), 3.48–3.78 (10H, N(CH CH ) ,
2
2
2
2 2
CH [Pro]), 4.15–4.75 (5H, CH[Fmoc], CH [Fmoc], CH[Pro], CHCO),
2
2
5.35 (1H, NH CO), 6.5–7.2 (4H, benzene ring), 7.25–7.8 (8H,
Ar[Fmoc]).
(
USA) and grown in RPMI 1640 medium containing 12% fetal calf
serum, 2 mM l-glutamine, 100 U/mL penicillin and 100 ␮g/mL
streptomycin.
2.2.3. Synthesis of Mel-OCM-chitosan conjugates
Fmoc-Mel-amino acid (2 mmol) was mixed with EDC (383 mg,
2
.2. Synthesis of Mel-OCM-chitosan conjugates
2 mmol) in 20 mL N,N-dimethyl formamide (DMF) and reacted at
room temperature for 4 h. Then, NHS (230 mg, 2 mmol) was added
to the above mixture, and the whole mixture was stirred for 6 h at
room temperature to obtain NHS-activated Fmoc-Mel-amino acid.
OCM-chitosan was dissolved in 10 mL distilled water, and NHS-
activated Fmoc-melphalan-amino was added drop-wise to solution
of OCM-chitosan over 30 min. The mixture was reacted at room
temperature for 48 h, and the water of the solution was removed
2.2.1. Synthesis of Fmoc-Mel
The synthesis of Fmoc-Mel was referred to a previous report
(
(
Zhao et al., 2010). 9-Fluorenylmethyl N-succinimidyl carbonate
Fmoc-Osu, 3.71 g, 11 mmol) in dioxane (20 mL) was added to an
ice-cold solution of Mel (3.21 g, 10 mmol) in a mixture of diox-
ane (50 mL), distilled water (15 mL), and NaHCO (0.93 g, 11 mmol).
The mixture was stirred for 2 h at 0 C and then for 20 h at room
3
◦
◦
in vacuo at 50 C with adding suitable amount of toluene several
temperature. The reaction mixture was concentrated and parti-
tioned between ethyl acetate and distilled water. The mixture was
adjusted to pH 2 with hydrochloric acid, and then the aqueous
phase was extracted with ethyl acetate (3× 50 mL). The combined
organic phases were washed with distilled water and brine and
times. Then the N-Fmoc derivatives of Mel were deprotected with
piperidine.
Finally, the products were exhaustively dialyzed (MWCO
14,000) against DMF and deionized water, and then lyophilized
to obtain the white, cotton wool-like products. Mel-OCM-chitosan
conjugates without amino acid spacer was synthesized with
the same method. The products were synthesized and named
in Table 1.
dried over MgSO . The solid residue was purified by column chro-
4
matography (hexane:ethyl acetate = 3:1, v/v) to give the compound
as white solid powder.

3
8
B. Lu et al. / Carbohydrate Polymers 98 (2013) 36–42
Table 1
Synthesis of Mel-OCM-chitosan conjugates with different linkers and Mel contents.
Samples
Mel/OCM-chitosan (molar ratio)
Mel content (mg/100 mg)
Mel substitution degree (mol/mol)
Equivalent solubility (mg/mL)
Mel-OCM-chitosan
1:1
1:1
1:2
1:1
1:2
1:1
1:1
15.1
15.9
10.5
14.7
8.6
0.133
0.147
0.090
0.141
0.074
0.107
0.113
1.12
1.43
1.52
1.35
1.45
1.39
1.48
Mel-Gly-OCM-chitosan-1
Mel-Gly-OCM-chitosan-2
Mel-Phe-OCM-chitosan-1
Mel-Phe-OCM-chitosan-2
Mel-Leu-OCM-chitosan
Mel-Pro-OCM-chitosan
11.9
12.6
2
2.3. Instrumental analyses
50 ␮g/mL with R = 0.99636. The Mel content was calculated as
follows:
ꢀ
ꢁ
FT-IR spectrum analysis: Samples were prepared as KBr pel-
let and scanned against a blank KBr pellet background at range of
mMel
Mel% =
× 100
−1
mprodrug
6
00–4000 cm and characterized by FT-IR spectrometer (Bruker
Tesor 27, Germany).
1H NMR spectroscopy analyses: The 1H NMR spectrums were
determined on a Varian 600 spectrometer (Varian, USA) using
tetramethylsilane as internal standard at 25 C. The samples
2
.5. Solubility of Mel-OCM-chitosan conjugates
◦
To evaluate solubility of the conjugates and Mel, 20 mg Mel-
of Fmoc-Mel, Fmoc-Mel-Gly, Fmoc-Mel-Phe, Fmoc-Mel-Leu, and
OCM-chitosan conjugates or Mel was added into 1 mL deionized
water respectively, and the mixtures were vortexed for 5 min,
sonicated for 5 min, and centrifuged at 6000 rpm for 5 min. The
supernatants were collected and analyzed using UV spectropho-
tometer. The amounts of Mel were quantified by measuring UV
absorption at a wavelength of 261 nm.
Fmoc-Mel-Pro were dissolved in CDCl , while OCM-chitosan and
3
Mel-OCM-chitosan conjugates were dissolved in D O.
2
2.4. Drug content of Mel-OCM-chitosan conjugates
Content of incorporated melphalan was determined by mea-
surement of the UV absorption of the conjugates solution in
distilled water at 261 nm. The Mel content was determined
with the help of a calibration curve of Mel in mixture solu-
tion (methanol:water:acetic acid = 49.5:49.5:1) range from 2 to
2.6. In vitro drug release of Mel-OCM-chitosan conjugates
The chemical hydrolysis of the conjugates was carried out in
triplicates in phosphate-buffered saline (PBS) at pH 7.4 and 5.8
Fig. 1. (A) Synthesis of Fmoc-Mel-Gly, (B) synthesis of Mel-Gly-OCM-chitosan. Conjugates with other spacers (Phe, Leu, Pro) were synthesis with the same method.

B. Lu et al. / Carbohydrate Polymers 98 (2013) 36–42
39
Fig. 2. FT-IR spectra: (A) – (a) Mel, (b) Fmoc-Mel; (B) – (a) OCM-chitosan, (b) Mel-OCM-chitosan (c) Mel-Pro-OCM-chitosan.
◦
at 37 C, respectively. The conjugates were dissolved in PBS and
route of Mel-OCM-chitosan are illustrated in Fig. 1. The NH2 of
Mel were first protected with Fmoc-Osu. Then, the COOH was
stirred mildly. At the scheduled time, 20 ␮L of the solution was
removed and diluted with ice cold methanol (380 ␮L, containing
activated and chemically coupled with the NH of different amino
2
2
1
% acetic acid). The mixture was vortexed for 5 min, sonicated for
min, and centrifuged at 6000 rpm for 5 min. 20 ␮L of the super-
acid linkers by carbodiimide chemistry. In the following step, Fmoc-
Mel and Fmoc-Mel-amino acid were grafted onto the NH2 of
OCM-chitosan under the catalysis of EDC·HCl and NHS. The removal
of the Fmoc moiety was achieved with piperidine treatment.
The FT-IR spectra of Mel, Fmoc-Mel are shown in Fig. 2A. The
natants were injected and analyzed by HPLC method to determine
the release of Mel from conjugates.
The method for analysis of Mel release in plasma and enzyme
solution was similar to that of hydrolysis in PBS, except for the
pretreatment process. Rat blood and liver was obtained from
adult male Sprague-Dawley rats. Approximately 4 IU of hep-
arin was added to each mL of blood to prevent coagulation
−
1
basic characteristics of Mel (see curve a) are shown at: 3423 cm
( NH2 stretch), 2950 cm ( OH, CH , C H stretch), 1614 cm
−
1
−1
2
−
1
−1
−1
(C O stretch), 1519 and 1445 cm (C C stretch), 1350 cm (C N
stretch), 807.7 cm (C H bend of benzene ring), 732 cm (C Cl
stretch). In comparison with the Mel spectrum, the new absorp-
−
1
(Mehvar, Dann, & Hoganson, 2000). The blood was immedi-
◦
−1
−1
ately centrifuged at 6000 rpm, 4 C for 5 min to collect the pale
yellow supernatant as plasma. Rat liver lysosomal enzymes (tri-
tosomes) were prepared by differential centrifugation of the liver
homogenate solution according to the method of Bonifacino,
Dasso, Harford, Lippincott-Schwartz, and Yamada (2009, chaps.
tion band appearing at 3321 cm (amide N H stretch), 1708 cm
−
1
(amide I band C O stretch), 1047 cm (C O C stretch) and the
−
1
−1
(C H stretch), 1641 cm
increased intensities at 2950.2 cm
−
1
(amide II band C O stretch), 1520 cm (amide II band N H bend)
−
1
while the decreased intensity at 3423 cm (primary amines) in
the Fmoc-Mel spectrum (see curve b) indicated the formation
of Fmoc-Mel. Fig. 2B shows the FT-IR spectra of OCM-chitosan,
Mel-OCM-chitosan and Mel-Pro-OCM-chitosan. The character-
istics of OCM-chitosan are shown at: 3500–3200 cm
stretch overlapped with N H stretch), 1620 cm and 1417 cm
(COO antisymmetric and symmetric stretch), 1060 cm (C O C
stretching vibration in the glucopyranose ring). Compared with
OCM-chitosan, several new IR signals attributed to the Pro spacer
and Mel structure are shown in IR spectrum of Mel-Pro-OCM-
3
.0.1–3.0.7). Papain and tritosomes solution need to be activated
with 1 mM ethylenediaminetetra-acetic acid (EDTA) and 5 mM
reduced glutathione at 37 C for 15 min before used. In all cases,
a bounded melphalan concentration of 1 mg/mL was used.
◦
−
1
(O H
−
1
−1
−
−1
2.7. In vitro cytotoxicity
Evaluation of cytotoxicity of the conjugates was performed by
MTT method. RPMI8226 cells were seeded into 96-well microtiter
4
−1
plates at a density of 5 × 10 cells/well in RPMI 1640 medium and
chitosan. The new absorption band appearing at 738 cm (C Cl
−
1
−
1
incubation for 24 h. Mel or Mel-OCM-chitosan conjugates (corre-
sponding Mel concentrate of 20–100 ␮g/ml) were added to the
culture medium. After incubation 48 h, 10 ␮L MTT (5 mg/mL) solu-
tion in PBS (pH 7.4) was added to each well and further incubated
stretch), 873 cm (C H bend of benzene ring), 1250 cm (C H
−
1
stretch of benzene ring) and 1450 cm (C C stretch of benzene
−
1
ring) can be attributed to Mel. And new absorbance at 1519 cm
−
1
and 1625 cm
(amide II band N H bend) belong to the amide
◦
in 5% CO2 incubator at 37 C for 4 h. After removal of the MTT
bond between OCM-chitosan and Pro and Mel. In addition, a new
−
1
containing medium, 100 ␮L DMSO was added to dissolve the form-
azan crystals formed in live cells. Finally, the absorbance was
measured at 570 nm using a microplate reader (BIO-RAD, Model
absorption band appearing at 2860 cm and the increased inten-
−
1
sity at 2920 cm were corresponded to C H stretching mode of
the methylene in Mel and Pro.
5
50, USA). The relative cell inhibition ratio (%) was calculated by
ODcontrol − ODsample)/ODcontrol × 100. The data were all corrected
by the blank group with media in the absence of RPMI8226 cells.
1H NMR spectra of OCM-chitosan, Mel-OCM-chitosan and
Mel-Pro-OCM-chitosan are shown in Fig. 3F. The protons of OCM-
(
chitosan are assigned as follows (ppm): 1.98 ( COCH ), 2.78 (CH,
3
carbon 2 of glucosamine ring), 3.3–3.9 (CH, carbon 2, 3, 4, 5
and 6 of glucosamine ring), 4.47 (CH, carbon 1 of glucosamine
ring), 4.75 (O CH2 COOH). Comparing Mel-OCM-chitosan spec-
trum with OCM-chitosan spectrum, new peaks appears at 6.90 and
3
. Results and discussion
3.1. Synthesis and structural analysis of intermediates and
prodrugs
7
.05 ppm were assigned to the protons of benzene rings of Mel. By
In this study, we used different amino acid as spacers to syn-
thesize a serial of Mel-OCM-chitosan conjugates and the synthesis
comparison of Mel-Pro-OCM-chitosan conjugate with Mel-OCM-
chitosan, additional chemical shifts at 4.3–4.5 and 2.2 ppm can be

4
0
B. Lu et al. / Carbohydrate Polymers 98 (2013) 36–42
Fig. 3. 1H NMR spectra: (A) Fmoc-Mel; (B) Fmoc-Mel-Gly; (C) Fmoc-Mel-Phe; (D) Fmoc-Mel-Leu; (E) Fmoc-Mel-Pro; (F) – (a) OCM-chitosan, (b) Mel-OCM-chitosan, (c)
Mel-Pro-OCM-chitosan.
attributed to Pro spacer. In addition, the almost disappearance of
the signals at 7.28–7.83 (8H, m, Ar[Fmoc]) indicated that Fmoc was
completely de-protected by piperidine.
conjugates showed good water solubility mainly due to the good
aqueous solubility of OCM-chitosan and the possibility of micellar
formation self-assembled by Mel-OCM-chitosan conjugates in the
aqueous solution. It was reported that hydrophobic modified OCM-
chitosan can self-assemble into nanoparticles with a hydrophobic
core and a hydrophilic shell (Gong et al., 2012; Zheng, Han, et al.,
3.2. Drug content and water solubility
2
011; Zheng, Rao, et al., 2011). The hydrophilic shell could increase
It was tested that free Mel and Mel-OCM-chitosan conjugates
the water solubility of the prodrugs. In addition, the existence of
spacers also slightly enhanced the water solubility of the conjugates
although it showed no obvious regularity among different spacers.
have the same UV absorption characteristic and both showed an
absorption peak at around 261 nm. Table 1 shows the Mel con-
tent in all Mel-OCM-chitosan conjugates. Keeping OCM-chitosan
feed ratio unchanged, it was found that Mel content in Mel-OCM-
chitosan conjugates decreased from 15.9 to 11.9, Mel substitution
degree decreased from 0.147 to 0.107, as the spacer changed from
Gly to Leu. The result indicated that selection of amino acid spacer
played an important role in adjusting Mel content.
The solubility of Mel in water was measured to be approximately
–8 ␮g/mL. Compared with the extremely poor solubility of Mel,
all of the tested conjugates showed satisfactory water solubility,
which was 160-fold (Mel-OCM-chitosan, 1.12 mg/mL) at least and
17-fold (Mel-Gly-OCM-chitosan-2, 1.52 mg/mL) at most of Mel (as
3.3. In vitro drug release of Mel-OCM-chitosan conjugates
Chemical and enzymatic hydrolysis was carried out to evaluate
the drug release behavior of Mel-OCM-chitosan conjugates by using
HPLC method. The accumulative release rate of Mel from the differ-
ent conjugates and hydrolysis conditions are shown in Figs. 4 and 5.
The results displayed several important clues as follow.
6
Firstly, the drug release behavior toward chemical hydroly-
◦
2
sis was evaluated at pH 7.4 and 5.8 in PBS (37 C) to mimic the
shown in Table 1). The solubility of the conjugates with same amino
acid spacer decreased with Mel substitution degree increased. The
physiological and cell lysosomal pH environments, respectively.
The release of Mel from Mel-OCM-chitosan conjugates in both PBS

B. Lu et al. / Carbohydrate Polymers 98 (2013) 36–42
41
model of cysteine proteases. Comparing to the chemical hydroly-
sis, the obvious Mel release could be observed within an hour in
both papain and tritosomes. These observations stand in contrasts
with previous reports (Duncan et al., 1991) that HPMA-melphalan
conjugates with Gly spacer was not degradable by thiol-proteases.
So it is noteworthy that the nature of the polymeric backbone also
serves to influence the rate and specificity of enzymatic cleavage of
amino spacers. The rapid drug release in lysosome and papain while
slow drug release in plasma ensure the effective release of bioac-
tive molecules in tumor cells after the conjugates passive target the
tumor cells through EPR effect.
Thirdly, connecting Mel and OCM-chitosan molecules with
amide bond directly, displayed the highest stability toward both
chemical and enzymatic hydrolysis. In contrast, conjugates with
amino acid spacers showed relative fast and increased drug
release velocity, which means that these conjugates have enzyme-
responsive characteristic, and enzymatic release of melphalan from
Mel-OCM-chitosan conjugates was clearly dependent on the amino
acid used to link drug to polymer. In all cases, the rate of release was
as follows: Mel-Gly-OCM-chitosan > Mel-Pro-OCM-chitosan > Mel-
Phe-OCM-chitosan > Mel-Leu-OCM-chitosan. The results demon-
strated that increased steric hindrance provided by spacers with
large side chain could lead to a lower and slower free drug release
under the same environmental condition. This can be attributed
that the large side chain induced steric hindrance would become
an obstacle for the enzymatic hydrolysis.
Fig. 4. Release of Mel from Mel-Gly-OCM-chitosan in plasma and PBS.
indicated a slow Mel release and which shown no apparent differ-
ence between all the conjugates. After 48 h, only 4.1% of the Mel was
released from Mel-Gly-OCM-chitosan in PBS (pH 7.4) and 5.0% in
PBS (pH 5.8). We can notice that lower pH environment led to faster
releasing speed. The differences may be ascribed to that amido bond
is more stable in neutral environment than in acid environment.
The pH dependant release can be also attributed to the swelling
property of the polymer matrix. At lower pH, the residual amine
groups is protonated and creates a repulsive force between the
adjacent positive charge, and thereby more amount of conjugated
drug will be exposed to be hydrolyzed (Anitha et al., 2011). The
rate of Mel release from Mel-OCM-chitosan conjugates in plasma
showed no big different from those in buffer, which suggested that
the few and low concentrate of the enzyme exist in plasma had no
obvious effect on the drug stability in plasma.
3.4. In vitro cytotoxicity
The RPMI8226 cells were selected as a model for in vitro cyto-
toxicity study of the Mel-OCM-chitosan conjugates. It was found
in Fig. 6 that free Mel had the maximum inhibition ratio and
all conjugates exhibited obvious cytotoxicity against RPMI8226
cells, indicating polymeric prodrugs did not lose anti-cancer
activity of Mel. At the same time, we can notice a relatively
lower cellular cytotoxicity induced by the polymeric derivatiza-
tion of OCM-chitosan and amino acid, which probably due to the
self-assembled behaviors of the amphiphilic polymers to nanopar-
ticles. Mel was probably chemically embedded in the interior
core of the self-assembled nanoparticles of the conjugates, which
can protect Mel from interaction with the tumor cells. How-
ever, only slight activity loss was observed with the conjugated
form, suggesting that free Mel was released from the conjugates
through cleavage of the amino acid linker between Mel and OCM-
chitosan. It is worth noticed that the conjugates with the amino
acid linkers were more active than the conjugates without the
linkers, suggesting the cell cytotoxicity was mainly due to the
Secondly, drug release by enzymatic hydrolysis was tested in the
presence of a model enzyme (papain) and lysosome. It has reported
a higher level of a number of enzymes expressing in tumor extracel-
lular space for tumors to survive, grow and metastasize (Chau, Tan,
&
Langer, 2004). Among the many tumor-associated enzymes, we
have focus on selected cathepsins (including Cathepsin B, Cathep-
sin H, Cathepsin X and so on), which belong to cysteine proteases
and demonstrated over-expressing in malignant tumor (Repnik,
Stoka, Turk, & Turk, 2012). Cysteine proteases degrade polypep-
tides share a common catalytic mechanism. Papain was used as a
Fig. 5. Enzymatic degradation of Mel-OCM-chitosan conjugates. Degradation by (A) papain and (B) tritosomes is shown.

4
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FT-IR, 1H NMR. These Mel-OCM-chitosan conjugates showed sat-
isfactory water solubility comparing with free melphalan. In vitro
study showed that the conjugates are stable in plasma but rapidly
degraded in enzyme solution. The selection of amino acid spac-
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drug release behavior of polymeric prodrugs. The conjugates with
Gly spacer was considered to be the best compound, as it had
good water solubility and the highest antitumor activity compared
with other conjugates. These results demonstrated that the poly-
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of melphalan and have the potential to be used as new polymeric
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(
[
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This work was supported by National Natural Science Founda-
tion of China (51273156, 21204071), Chen Guang Project of Wuhan
2
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(
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&
I study of beta-alanyl-melphalan
a
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