A novel melphalan polymeric prodrug: Preparation and property study

Article abstract of DOI:10.1016/j.carbpol.2014.04.062

The clinical application of melphalan (Me), an anticancer drug for the treatment of hematologic malignancies, has been limited due to its poor water solubility, rapid elimination and lack of target specificity. To solve these problems, O,N-carboxymethyl chitosan-peptide-melphalan conjugates were synthesized and characterized. All polymeric prodrugs showed satisfactory water solubility. It was found that the molecular weight of O,N-carboxymethyl chitosan (O,N-CMCS) and the peptide spacer played a crucial role in controlling the drug content, diameter and drug release properties of O,N-carboxymethyl chitosan-peptide-melphalan conjugates. The studies of in vitro drug release and cell cytotoxicity by MTT assay revealed that, employing the polymeric conjugation strategy and using the peptides glycylglycine (Gly-Gly) as a spacer, the conjugates have good cathepsin X-sensitivity and lower toxicity and the drug release behavior improved remarkably. In conclusion, O,N-carboxymethyl chitosan-peptide-melphalan conjugates could be promising prodrugs for anticancer application.

Full text of DOI:10.1016/j.carbpol.2014.04.062

Carbohydrate Polymers 111 (2014) 928–935
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
A novel melphalan polymeric prodrug: Preparation and property
study
Dan Lia, Bo Lua,b,∗, Zhijun Huanga,b, Peihu Xua,b, Hua Zhenga,b, Yihua Yina, Haixing Xua,b
,
Xia Liua, Lingyun Chenc, Yiceng Loua, Xueqiong Zhanga, Fuliang Xionga,b
a Department of Pharmaceutical Engineering, School of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, PR China
b Engineering Technology Center of Hubei traditional Chinese Medicine Preparation, Wuhan University of Technology, Wuhan 430070, PR China
c Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5
a r t i c l e i n f o
a b s t r a c t
Article history:
The clinical application of melphalan (Me), an anticancer drug for the treatment of hematologic malig-
nancies, has been limited due to its poor water solubility, rapid elimination and lack of target specificity.
To solve these problems, O,N-carboxymethyl chitosan–peptide–melphalan conjugates were synthesized
and characterized. All polymeric prodrugs showed satisfactory water solubility. It was found that the
molecular weight of O,N-carboxymethyl chitosan (O,N-CMCS) and the peptide spacer played a cru-
cial role in controlling the drug content, diameter and drug release properties of O,N-carboxymethyl
chitosan–peptide–melphalan conjugates. The studies of in vitro drug release and cell cytotoxicity by MTT
assay revealed that, employing the polymeric conjugation strategy and using the peptides glycylglycine
(Gly-Gly) as a spacer, the conjugates have good cathepsin X-sensitivity and lower toxicity and the drug
release behavior improved remarkably. In conclusion, O,N-carboxymethyl chitosan–peptide–melphalan
conjugates could be promising prodrugs for anticancer application.
Received 26 October 2013
Received in revised form 15 April 2014
Accepted 19 April 2014
Available online 20 May 2014
Keywords:
Carboxymethyl chitosan
Cathepsin X
Melphalan
Nanoparticles
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
developed for treating leukemia. However, only a limited num-
ber of systems have been developed targeting multiple myeloma
Recent advances in prodrug have led to the development of
controlled drug delivery systems with desirable properties that
can overcome the limitations of traditional therapeutic agents
(Bildstein, Dubernet, & Couvreur, 2011; She et al., 2013; Chhikara,
Mandal, & Parang, 2010). Various water-soluble polymers and their
conjugates with peptides are used as carriers for improving the
utilization of anticancer agents. A successful drug delivery system
relies on an appropriate selection of the polymer carrier, targeting
residues and the linkage spacer (Jain, Kesharwani, Gupta, & Jain,
2012). The release rate of prodrugs can be controlled by site-specific
hydrolysis.
(Dinauer et al., 2005; Parekh, Weniger, & Wiestner, 2011; Zheng
et al., 2011). Multiple myeloma (MM) is a relatively common clonal
B-cell malignancy (Mincher et al., 2008). MM accounts for 13.4% of
all hematologic malignancies diagnosed, 19% of all deaths result-
ing from hematologic malignancies, and 2% of all cancer deaths
(Mahindra, Hideshima, & Anderson, 2010).
Melphalan (Me) is an anticancer drug for the treatment
of hematologic malignancies, especially multiple myeloma and
chronic lymphocytic leukemia (Hjertner, Börset, & Waage, 1996;
Machana, Weerapreeyakul, Barusrux, Thumanu, & Tanthanuch,
2012). Melphalan exhibits beneficial treatment effect, and the
major disadvantages are poor water solubility and lack of target
specificity. Most studies are concentrated on chemical modifica-
tions and formation of prodrugs or nanosystems to reduce the
toxicity of melphalan (Wickström et al., 2010; Rajpoot, Bali, &
Pathak, 2012). Although the water solubility and toxicity of mel-
phalan have been improved in these studies, only limited efforts
have been focused on its targeted delivery.
Prodrugs combining therapeutic agents and targeting residues
are an emerging technique to improve the therapeutic effect
of hematologic malignancies. Among hematologic malignancies,
there are over two-dozen drug delivery systems that have been
∗
Corresponding authors at: Department of Pharmaceutical Engineering, 122
Higher expression of unique enzymes in cancer cells than in
normal cells can be exploited for development of site-selective
delivery systems (Vig, Huttunen, Laine, & Rautio, in press). Many
Luoshi Road, Wuhan, Hubei, Wuhan 430070, PR China. Tel.: +86 27 87859019;
fax: +86 27 87859019.
E-mail addresses: whulb@163.com (B. Lu), whutlvb@163.com (H. Zheng).
http://dx.doi.org/10.1016/j.carbpol.2014.04.062
0144-8617/© 2014 Elsevier Ltd. All rights reserved.

D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
929
cathepsin-sensitive peptides were invoked as spacers and linked
with anticancer drugs such as paclitaxel, doxorubicin, methotrex-
ate, geldanamycin, solamargine (Burke et al., 2009; Liang et al.,
2012; Sun et al., 2010; Jeffrey et al., 2006; Ofner, Pica, Bowman, &
Chen, 2006). Ruth et al. (1991) have proved that P-Gly-Phe-Leu-Gly-
Me had a response toward cathepsin B (CB) in terms of melphalan
release. Moreover, proline prodrugs of melphalan (Chrzanowski,
Bielawska, & Pałka, 2003) and methotrexate (Wu, Shah, Patel, &
Yuan, 2010) were designed for their targeted delivery to tumor cells
where the spacers can be cleaved by specific enzymes (imidodipep-
tidase and prolidase, respectively) to release the conjugated drugs.
Cathepsin X belongs to the papain family of cysteine proteases
and is over-expressed in cancer cells (Nägler and Ménard, 1998;
Obermajer, Doljak, Jamnik, Fonovic´, & Kos, 2009; Kirschke, 2007).
More recent findings indicate that cathepsin X can release C-
terminal amino acids of polypeptides and proteins and has the
possibility of being used for development of cancer therapy (Paulick
& Bogyo, 2011; Nägler et al., 1999). For example, glycylglycine pep-
tides (Gly-Gly) were intended to be a highly specific substrate for
cathepsin X and the linkage between the polymer and the drug
(Devanathan et al., 2005).
The extraction was dissolved again, 12% (v/v) HCl was used for
acidization with the aqueous phase. The precipitate was collected,
cooled over night and then washed with acetic ester, deionized
water and saturated sodium chloride solution, and finally dried
under vacuum. Fmoc-Melphalan was synthesized by the same
method as Fmoc-(Gly-Gly)n.
2.3.2. Synthesis of (Gly-Gly)n-Me
Fmoc-Melphalan (1 mmol) was in addition to Wang resin (4-
(Hydroxymethyl) phenoxymethyl Polystyrene Resin, 2.0 g) after
5 min of preactivation in DMF using DMAP (150 mg) and DIC
(100 mg) as catalysts. After washing with DMF (4–6 times with
20 ml), piperidine solution was inserted twice for 5 + 10 min to
remove Fmoc- group and then washed in DMF. Fmoc-(Gly-Gly)n
(0.5 mmol), HBTU (3 mmol) and DIEA (0.5 ml) were added to react
for 40 min and followed by washing with DMF. The product was
cleaved from the resin with pyridine, neutralized with acetic anhy-
dride (volume ratio of 1:1) and purified by preparative RP-HPLC.
Removed from Wang resin, Fmoc-(Gly-Gly)n-Me was dissolved in
20 ml dichloromethane with 10 times amount of diethylamine as
a catalyst at room temperature under constant stirring for 24 h.
Subsequently, the solids were purified by crystallization in a sol-
vent of petroleum ether, thoroughly washed with deionized water,
and dried under vacuum to afford the desired product as a white
powder (yield = 34%).
In this work, O,N-CMCS-(Gly-Gly)n-Me prodrug with Gly-Gly
as a cathepsin X substrate was synthesized. Then nanoparticles
were formed by self-assembly of O,N-CMCS-(Gly-Gly)n-Me poly-
mers. Morphology and size of nanoparticles, in vitro drug release,
and cell cytotoxicity of the polymeric prodrugs were investigated
to demonstrate the importance of molecular weight of polymers
and the effect of spacer in design of polymeric prodrugs.
2.3.3. Synthesis of O-CMCS-(Gly-Gly)3-Me
O-CMCS (600 mg) was suspended in 15 ml of ultrapure deion-
ized water using EDC·HCl (260 mg, 1 mmol) and NHS (230 mg,
1.05 mmol) as couple reagents for activation of carboxyl group.
After 6 h, (Gly-Gly)3-Me (300 mg, 0.5 mmol) were dissolved in 5 ml
DMSO and was added to the reaction system dropwise. The reaction
mixture was stirred at room temperature for 24 h. After dialysis and
lyophilization, the products were obtained as a white fibrous (62%
yield).
2. Materials and methods
2.1. Materials
Carboxymethyl chitosan (CMCS) was acquired from Jinke
Zhejiang, China. Melphalan was provided by Suzhou Lide Inc.
China. N,N-dimethylformamide (DMF), 1,4-diethylene dioxide, 1-
ethyl-(3-dimethyllaminopropyl) carbodiie hydrochlide (EDC·HCl),
N-hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), eth-
ylene diamine tetraacetic acid (EDTA) and glutathione (GSH)
were recruited from National Medicines (Group) Co., Ltd,
and purified before use. Fmoc N-hydroxysuccinimide este
(Fmoc-OSu), N,Nꢀ-diisopropylcarbodiimide (DIC), O-benzotriazole-
N,N,N,N-tetramethyl-uronium-hexafluorophosphate (HBTU), 4-
dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine
(DIEA) were purchased from Shanghai Medpep (Group) Co., Ltd.
All the other chemicals were of analytical or reagent grade and
were obtained from commercial channels without any purifica-
tion. Ultrapure deionized water was used in all buffer solutions and
experiments.
2.3.4. Synthesis of O,N-CMCS-(Gly-Gly)3-Me
O-CMCS-(Gly-Gly)3-Me (2.5 g) dissolved in 30 ml isopropanol.
After 1 h, an aqueous NaOH solution (10 ml, 40% w/v) was added
dropwise into the reaction system at 30 ◦C. The emulsion was
stirred overnight for alkalization. Chloroacetic acid (12 g, 0.13 mol)
in a 20 ml isopropanol was slowly added to this solution at 60 ◦C.
The reaction mixture was stirred for 3 h until a uniform solution was
obtained. 15 ml 1 M NaOH was used to neutralize and the crude
product was precipitated by ethanol. The attained solid was dis-
solved in 500 ml ultrapure waters. Hydrochloric acid was used to
adjust pH to the isoelectric point. White precipitation appeared was
washed twice with ethanol. The white fibrous products in 77% yield
were achieved after vacuum drying at 50 ◦C.
2.2. Cell lines and culture conditions
2.4. Characterization of (Gly-Gly)3-Me and CMCS-(Gly-Gly)3-Me
The RPMI8226 human myeloma cell line was obtained from
American Type Culture Collection (ATCC, Rockville, MD, USA) and
maintained in RPMI1640 medium containing 10% fetal bovine
serum (FBS) (Gibco, Grand Island, NY, USA), 100 units/ml peni-
cillin, and 100 g/ml streptomycin at 37 ◦C with 5% CO2. The cells
for experiments were in their logarithmic growth phase.
2.4.1. Mass spectrometric analysis of (Gly-Gly)3-Me
LC–MS/MS was performed using 1100 LC/MSD Trap mass spec-
trometer (Agilent, USA) equipped with an Accela pump and a
deconvolution software. Electrospray-ionization was performed in
the negative ion mode. Nitrogen was used as nebulizer gas set at
1.5 l/min. The mobile phase was 50% H2O and 50% ACN with a flow
rate of 0.2 ml/min. The spray voltage was 3.5 kV, DL temperature
250 ◦C, block temperature 200 ◦C.
2.3. Synthesis of O,N-CMCS-(Gly-Gly)3-Me delivery system
2.3.1. Synthesis of Fmoc-(Gly-Gly)n and Fmoc-Melphalan
2.4.2. FTIR of CMCS-(Gly-Gly)3-Me
Fmoc-(Gly-Gly)n was synthesized by mixing Gly-Gly 1.0 mmol
and Fmoc-OSu 1.2 mmol in 1,4-diethylene dioxide 20 ml at room
temperature under constant stirring for 24 h. The reaction mixtures
were diluted with 150 ml deionized water and extracted with ether.
The IR spectra of (Gly-Gly)3-Me and CMCS-(Gly-Gly)3-Me con-
jugates were measured on a FT-IR spectrometer (Bruker TENSOR
27, German) using KBr pellets.

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D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
2.4.3. NMR of CMCS-(Gly-Gly)3-Me
or tritosomes was used as a control sample. The tritosomes were
extracted from the liver of Kunming strain mice (20 2 g) supplied
by Hubei Experimental Animal Center (Juan & Bonifacino, 2009).
The release studies were carried out for 24 h at an oscillation condi-
tion of 37 ◦C, 50 rpm. At specific time intervals, 10 ␮l samples were
withdrawn and analyzed with HPLC and concentrations of mel-
phalan were determined using the standard curve prepared under
the same conditions. Various tritosomes concentrations were used
to determine tritosomes dependent release kinetics of O,N-CMCS-
(Gly-Gly)3-Me.
Prodrug was also incubated at a concentration of 100 ␮M with
5 × 10−6 M papain suspension in 0.1 M PBS (pH = 5.5). EDTA (1 mM)
and GSH (1 mM) were added to activate papain. The mixture was
incubated at 37 ◦C. The papain dependent release was also deter-
mined by the same method as the tritosomes dependent release.
High performance liquid chromatography (Agilent 1100N, USA)
was used for analysis of melphalan release in medium with dif-
ferent enzymes (Pinguet et al., 1996). The eluent mixture was
comprised of methanol, purified water and acetic acid (49.5:49.5:1,
v/v) and deaerated by ultrasound prior to use. The mobile phase
was pumped onto the column at 1.0 ml/min, which corresponds to a
pressure of about 120 bar. The volume injected was 20 ␮l, the chro-
matography was performed at 35 ◦C and the analytes were detected
at 254 nm. The calibration curve was found to be linear in the range
of 0.1–50 ␮g/ml with the r2 value of 0.9966.
(Gly-Gly)3-Me and CMCS-(Gly-Gly)3-Me were lyophilized and
re-dissolved in deuterated DMSO (d6-DMSO) and D2O and charac-
terized by 1H NMR at 500 MHz and 13C NMR at 100 MHz (Mercury
Plus 400, Varian, USA).
2.4.4. Determination of drug loading capacity
Content of incorporated melphalan was determined by mea-
suring the UV absorption of the conjugate solution in distilled
water at 263 nm. The reference solution having the identical sol-
vent with the test solution was prepared. Me content was identified
with the help of a calibration curve of Me in a mixture solu-
tion (methanol:water:acetic acid = 49.5:49.5:1) range from 2 to
50 ␮g/ml with r2 = 0.99636. Me content was calculated as follows:
ꢀ
ꢁ
mMe
Me% =
× 100%
mprodrug
2.5. Preparation of O,N-CMCS-(Gly-Gly)3-Me nanoparticles
O,N-CMCS-(Gly-Gly)3-Me polymer dissolved in pH 7.4
phosphate-buffered saline (PBS) to the desired concentration
(10 mg/ml) with uninterrupted magnetic stirring. Then it was
sonicated twice at 100 W for 4 min, using a probe-type sonifier
(JY92-2D, Ningbo Scientz Biotechnology CO., Ltd, China). To protect
the solution from heat build-up, a pulse function was used (pulse
on, 2.0 s; pulse off, 2.0 s).
2.8. Cell cytotoxicity assays
Evaluation of the cytotoxicity of polymers was performed by
MTT method (Mao, Ma, Zhou, Gao, & Shen, 2005). The RPMI8226
human myeloma cells were seeded in the 96-well plate at a density
of 4 × 105 cells/well in 100 ␮l DMEM containing 10% fetal bovine
serum (FBS), 100 units × ml−1 penicillin, and 100 g × ml−1 strep-
tomycin. After incubation for 24 h, polymer solutions were added
to the culture medium. Cell viability was tested after the addi-
tion of polymer for 48 h. After incubation, 10 ␮l MTT (5 mg/ml)
solution in PBS (pH = 7.4) was added to each well and further incu-
bated in 5% CO2 incubator at 37 ◦C for 4 h. After removal of the
MTT containing medium, 100 ␮l DMSO was added to dissolve the
formazan crystals formed in living cells. Finally, the absorbance
was measured at 490 nm using a microplate reader (BIO-RAD,
Model 550, USA). The relative cell viability was calculated as: Cell
viability (%) = (ODsample)/(ODcontrol) × 100, where ODcontrol (control
optical density) was obtained in the absence of polymers and
ODsample (optical density of the sample) was obtained in the pres-
ence of polymers. Cells incubated in the absence of nanoparticles
were used as a control. Each value was averaged from 6 parallel
experiments.
2.6. Characterization of nanoparticles
2.6.1. Measurements of critical micelle concentration
The critical micelle concentration (CMC) of the copolymers was
established by fluorescence spectroscopy using pyrene as a flu-
orescence probe (Cammas et al., 1997; Jie, Venkatraman, Min,
Freddy, & Huat, 2005). Pyrene solution in methanol (0.22 g/l) was
put into vials. Methanol was evaporated by a nitrogen stream.
Nanoparticles solutions in water were added to the vials containing
pyrene ([py] = 6.0 × 10−7 M). The concentration of micelles ranged
from 10 mg/ml to 0.01 mg/ml. The mixture was stored overnight
for equilibrium. Emission spectra (ꢀem = 350–450 nm, ꢀex = 335 nm,
Ex(SBW) = 3 nm, Em(SBW) = 1.5 nm) were accounted for. Upon for-
mation of nanoparticles, pyrene would move into the inside of the
nanoparticles from the aqueous phase which is expected to result
in an alteration in the intensity ratio (I1/I3) of pyrene fluorescence
bands I (372 nm) and III (383 nm).
2.6.2. Particle size, morphology, and zeta-potential of
nanoparticles
The morphology of the self-aggregate sample was obtained by
transmission electron microscopy (TEM) analysis using JSM-6700F,
made by JEOL, Japan operated at 200 kV. To prepare the TEM sample,
a drop of solution (5 mg/ml) was deposited onto 200 mesh copper
grids and dried at room temperature.
The average particle size and size distribution were determined
by dynamic light scattering (DLS) analysis using a Malvern Zetasizer
Nano ZS (Malvern, UK) at a polymer concentration of 5 mg/ml. The
nanoparticles were sonicated before measurement. DLS measure-
ments were performed at least in triplicate for each sample.
2.9. In vitro cellular uptake
The cellular uptake of O,N-CMCS-(Gly-Gly)3-Me nanoparticles
by cancer cells were supported by a confocal laser scanning
microscope (FV1000 confocal laser scanning microscope (CLSM),
Olympus Corporation, Tokyo, Japan) (Anitha et al., 2011). The
RPMI8226 human myeloma cells were seeded in a 24-well tissue
culture plate at a density of 106 cells per well, and were allowed to
grow for 24 h. In order to characterize cellular uptake of O,N-CMCS-
(Gly-Gly)3-Me nanoparticles, the nanoparticles were fluorescently
labeled with Dylight-488 dye. After 24 h of growth, the nanoparti-
cles were added into the culture plate and incubated at 37 ◦C for 1 h.
After incubation, the cells were washed with PBS. The cells were
centrifuged, and the supernatant was discarded. The cells were
resuspended in PBS solution, and subsequently measured.
2.7. In vitro drug release
O,N-CMCS-(Gly-Gly)3-Me dissolved in 0.15 M PBS (pH = 5.5) was
put in the release media (1 ml) containing rat liver tritosomes to
initiate the release of melphalan. The medium without enzyme

D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
931
Scheme 1. Synthesis route of O,N-CMCS-Gly-Gly-Me.
Fig. 1. Spectra of (Gly-Gly)3-Me and O,N-CMCS-(Gly-Gly)3-Me. (A) MS spectra of (Gly-Gly)3-Me; (B) 1H NMR spectra of O,N-CMCS-(Gly-Gly)3-Me in D2O; (C) 13C NMR spectra
of O,N-CMCS-(Gly-Gly)3-Me in d6-DMSO.
3. Discussion and results
Fig. 2B and D shows the FT-IR spectra of CMCS and O,N-CMCS-
(Gly-Gly)3-Me prodrug, The band assignment of CMCS is as follows:
3450–3408 cm−1 (O H stretch overlapped with N H stretch),
2851 cm−1 (C H stretch), 1630 cm−1 and 1431 cm−1 (COO− anti-
symmetric and symmetric stretch), 1315 cm−1 (C N stretch),
3.1. Synthesis and characterization of (Gly-Gly)n-Me and
O,N-CMCS-(Gly-Gly)n-Me
Sample preparation procedures were illustrated in Scheme 1.
(Gly-Gly)n-Me was prepared by solid phase peptide syntheses.
To synthesize O,N-CMCS-(Gly-Gly)n-Me prodrug, the C6-carboxyl
group of O-CMCS was firstly activated by NHS and then chemically
coupled with the amino group of (Gly-Gly)n-Me by formation of an
amide bond using “zero length” crosslinker of NHS. Then making
n = 3 as an example for structure analysis.
As showed in Fig. 1A, the measured molecular weight as revealed
by MS data is consistent with the value of (Gly-Gly)3-Me. The
samples were further analyzed by FTIR and NMR spectroscopy.
In comparison to the melphalan spectrum (Fig. 2A), additional
absorption bands were observed for (Gly-Gly)3-Me (Fig. 2C) at
1617 cm−1(amide II band C O stretch), 1546 cm−1(amide II band
1240 cm−1 (bridge O stretch), 1082 cm−1 (C
O C stretching vibra-
tion in the glucopyranose ring). Compared to CMCS (Fig. 1B), the
bands of O,N-CMCS-(Gly-Gly)3-Me (Fig. 1D) at 3330–3320 cm−1
(O H stretch overlapped with N-H stretch) and 1650–1550 cm−1
(C O stretch overlapped with N H bend) obviously increase. New
absorption band of O,N-CMCS-(Gly-Gly)3-Me at 1240 cm−1 (C
H
stretch of the benzene ring), 1576 cm−1 (amide II band N H bend)
appears.
In the 1H NMR spectrum of O,N-CMCS-(Gly-Gly)3-Me (Fig. 1B),
the peaks at 7.37 ppm and 7.44 ppm in 1H-NMR correspond to the
protons in the benzene ring and the amide bond protons linking
glycylgcine with melphalan. The signal at 3.5–4.0 ppm corresponds
to the saccharide ring and carboxymethyl group protons of O,N-
CMCS, N(CH2CH2)2 groups and CH2 of glycine. In Fig. 1C, the
peaks at 60–80 ppm in 13C-NMR correspond to the carbons in
the saccharide ring of O,N-CMCS. The signal at 169.6 ppm corre-
sponds to the carbons of glycine. The peak at 111.9 ppm is from the
N(CH2CH2)2 groups of melphalan. These results indicate that the
N
H bend). Meanwhile, the characteristic absorptions of mel-
phalan remained at: 3418 cm−1
( ( OH,
NH2 stretch), 2961 cm−1
CH2, C H stretch), 1518 and 1443 cm−1 (C C stretch), 1348 cm−1
(C N stretch), 806 cm−1 (C H bend of benzene ring), and 745 cm−1
(C Cl stretch).

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D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
Fig. 3. The relationship of I372/I383 from fluorescence spectra of pyrene (6 × 10−7 M)
with lgC of O,N-CMCS-(Gly-Gly)3-Me.
Fig. 2. FTIR spectra: (A) Melphalan; (B) CMCS; (C) (Gly-Gly)3-Me; (D) O,N-CMCS-
(Gly-Gly)3-Me).
of the sample, whereas the size measured by laser light scattering
method is a hydrodynamic diameter (hydrated state). The nanopar-
ticles showed a larger hydrodynamic volume due to the hydration
effects (She et al., 2013). Compared with freshly prepared, the
nanoparticles have no obvious change when stored at 4 ◦C in a
PBS solution for one month (still 20–120 nm in diameter), which
indicates that the nanoparticles had excellent stability (shown in
Fig. 4).
In addition, characterization of nanoparticles with different
polymer molecular weight was measured, shown in Table 1. The
mean size of nanoparticles increased from 80 to 160 nm with the
increase of molecular weight from 6.0 × 104 to 3.4 × 108. These
results suggest that the mean size could be controlled by choosing
polymer molecular weight to match requirements. The nanoparti-
cles had a negative zeta potential (from −19.6 mV to −11.8 mV). It
may be attributed to carboxyl groups on the particle surface.
polymer prodrug O,N-CMCS-(Gly-Gly)3-Me was synthesized suc-
cessfully.
The weight percentage (wt%) of Me in the O,N-CMCS-(Gly-Gly)3-
Me conjugates was analyzed using a UV spectrophotometer by
measuring absorption at a wavelength of 263 nm. As showed in
Table 1, the drug loading content reduces from 12.22 to 6.17%
with increasing the molecular weight from 6.0 × 104 to 3.4 × 108.
This may be attributed by the level of chain extension during the
synthesis process. The results indicate that the Me content of O,N-
CMCS-(Gly-Gly)3-Me conjugates could be related to their molecular
weight.
3.2. Characterization of O,N-CMCS-(Gly-Gly)3-Me nanoparticles
It is noted that amphiphilic polymers with an appropriate
hydrophilic/hydrophobic balance can self-assemble into nanopar-
ticles in aqueous media. The amphiphilic O,N-CMCS-(Gly-Gly)3-Me
polymers, consisting of carboxyl and Me segments, were easily
self-assemble into nanoparticles in PBS solutions. During prepa-
ration, there was no drug released from the polymers in aqueous
media determined by HPLC. This demonstrates that the resulting
nanoparticles are stable. Since pyrene is hydrophobic and has a
very low solubility in water, it tends to solubilize in the hydrophobic
region of nanoparticles, and the fluorescence intensity will be influ-
enced by environmental change around pyrene. It is reported that
the intensity ratio (I372/I383) of first (372 nm) and third (383 nm)
peaks in the emission spectra is quite sensitive to the environ-
mental change of pyrene. Therefore, this parameter was analyzed
for determining critical micelle concentration (CMC) of O,N-CMCS-
(Gly-Gly)3-Me. As showed in Fig. 3, the intersection point of the two
straight lines represents the critical micelle concentration, and CMC
value was 0.1170 mg/ml.
A small enough particle size is an important prerequisite to
enhance the cellular uptake of nanoparticles. Particle size of the
nanoparticles was estimated by TEM. Fig. 4 displayed the mor-
phology and size of the nanoparticles of O,N-CMCS-(Gly-Gly)3-Me
in PBS solutions. The typical images show that the nanoparticles
in PBS solutions took near spherical shape and had no obvious
agglomeration. Moreover, the nanoparticles possess a particle size
of roughly 20–120 nm obtained by TEM images. However, the size
was smaller than that measured by DLS, shown in Table 1. This
might be mainly due to the process involved in the preparation of
the sample. TEM images depict the actual size at the dried state
3.3. In vitro drug release
Prodrugs were incubated with a model enzyme papain, cathep-
sin X and rat liver lysosomal enzymes (tritosomes) to study the drug
release profiles. The in vitro drug release profiles from nanoparti-
cles were shown in Fig. 5. Melphalan was continuously released up
to 20 h, and the drug release rate varies depending on the enzyme
used. Melphalan is being released more rapidly from nanoparti-
cles incubated with cathepsin X and tritosomes (50%) than with
papain (25%), suggesting that a cathepsin X-sensitive system has
been successfully prepared (Fig. 5A, D and F). In addition, Fig. 5E–G
displays an increasing drug release rate with longer spacer. This
may be attributed that the longer spacer arm prevented steric hin-
drance and accelerated the drug release. The self-assembly process
and drug release are displayed in Fig. 6. Amphiphilic copolymers
can self-assemble into core-shell nanoparticles in aqueous envi-
ronment. Thus the melphalan release from the polymeric prodrugs
mainly depends on the type of enzyme. Therefore, this cathepsin
X-sensitive system could be applied to enhance drug delivery.
In larger nanoparticles with the molecular weight of 2.5 × 107
and 3.4 × 108, 39% and 46% of melphalan had released from the
particles after 10 h, respectively (Fig. 5B and C). In the small size
groups with the molecular weight of 6.0 × 104, only 24% of drug
was released (Fig. 5A). At the end of 20 h, no significant difference
was observed in drug release levels. The drug release appears to
be controlled by the swelling behavior of the nanoparticles. The
larger nanoparticles swelled faster, resulting in the difference in
drug release before 10 h. The release is only due to enzymatic

D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
933
Table 1
Loading capacity and particle size of nanoparticles (n = 3).
Molecular weight
Drag content (%)
Restored time (d)
Diameter (mn)
PDI
Zeta potential (mV)
6.0 × 104
6.5 × 105
2.5 × 107
3.4 × 108
6.5 × 105
12.13
12.07
8.99
6.08
–
1
1
1
1
15
81
83
122
158
127
6
4
3
4
6
0.402
0.247
0.556
0.265
0.538
−19.6
−11.8
−13.0
−13.6
−13.7
Fig. 4. TEM morphology of nanoparticles of O,N-CMCS-(Gly-Gly)3-Me (5 mg/ml) in PBS (pH 7.4) solution. (A) Freshly prepared; (B) stored for one month.
degradation after swelling process, so the total amount of release
drug was similar across all groups after 20 h.
Sustainable release of melphalan for a 24-h period suggests that
O,N-CMCS-(Gly-Gly)3-Me can be developed as potential anticancer
agents.
3.4. Cytotoxicity of O,N-CMCS-(Gly-Gly)3-Me
Fig. 7 is in vitro cell viability of multiple myeloma cells RPMI8226
incubated with different concentrations of melphalan or nanopar-
ticles for 48 h. The cells incubated in culture media containing 30 ␮l
tritosomes were also measured.
As showed in Fig. 7, free melphalan exhibits the maximum
inhibition efficiencies while others have the lower values at each
Fig. 5. Enzymatic degradation of polymer bound melphalan ((A) Mw = 6.0 × 104
NPs with triple spacer incubated with tritosomes; (B) Mw = 2.5 × 107 nanoparti-
cles with triple spacer incubated with tritosomes; (C) Mw = 3.4 × 108 nanoparticles
with triple spacer incubated with tritosomes; (D) Mw = 6.0 × 104 nanoparticles with
triple spacer incubated with papain; (E) Mw = 6.0 × 104 nanoparticles with quintu-
ple spacer incubated with cathepsin X; (F) Mw = 6.0 × 104 nanoparticles with triple
spacer incubated with cathepsin X; (G) Mw = 6.0 × 104 nanoparticles with single
spacer incubated with cathepsin X).
Fig. 7. Inhibition of RPMI-8226 cells proliferation by the compounds and melphalan
after 48 h. All the experiments were performed in six times and average values were
reported.
Fig. 6. Schematic illustration of the formation of self-assembled nanoparticles and
the process of melphalan release.

934
D. Li et al. / Carbohydrate Polymers 111 (2014) 928–935
Fig. 8. Fluorescence images and intensity of O,N-CMCS-(Gly-Gly)3-Me nanoparticles.((A) Fluorescent microscopy in 1 h; (B) fluorescent microscopy in 4 h; (C) fluorescent
microscopy in 12 h; (D) fluorescent microscopy in 24 h; (E) fluorescence intensity).
concentration. The cytotoxicity was caused by drug-protein inter-
actions. O,N-CMCS-(Gly-Gly)3-Me are a kind of block copolymers
composed of hydrophilic carboxymethyl and hydrophobic mel-
phalan. Melphalan is chemically embedded in the interior core
of the self-assembled nanoparticles, thus avoiding burst release.
However, the nanostructures were destroyed by enzyme-mediated
process, and more melphalan was released with larger inhibition
ratios. These results indicate that the enzyme-sensitive polymer
would be a useful tool for targeted cancer therapeutics.
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