Synthesis, nanosizing and in vitro drug release of a novel anti-HIV polymeric prodrug: Chitosan-O-isopropyl-5′-O-d4T monophosphate conjugate

Article abstract of DOI:10.1016/j.bmc.2009.11.013

A novel approach to improve the antiviral efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) and reduce their side effects was developed by constructing a nanosized NRTI monophosphate-polymer conjugate using d4T as a model NRTI. Firstly, a novel chitosan-O-isopropyl-5′-O-d4T monophosphate conjugate with a phosphoramidate linkage was efficiently synthesized through Atherton-Todd reaction under mild conditions. The anti-HIV activity and cytotoxicity of the polymeric conjugate were evaluated in MT4 cell line. Then the conjugate nanoparticles were prepared by the process of ionotropic gelation between TPP and chitosan-d4T conjugate to improve their delivery to viral reservoirs, and their physicochemical properties were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) techniques and X-ray diffraction (XRD). In vitro drug release studies in pH 1.1 and pH 7.4 suggested that both chitosan-d4T conjugate and its nanoparticles prefer to release d4T 5′-(O-isopropyl) monophosphate than free d4T for prolonged periods, which resulted in the enhancement of anti-HIV selectivity of the polymeric conjugate relative to free d4T due to bypassing the metabolic bottleneck of monophosphorylation. Additionally, the crosslinked conjugate nanoparticles can prevent the coupled drug from leaking out of the nanoparticles before entering the target viral reservoirs and provide a mild sustained release of d4T 5′-(O-isopropyl) monophosphate without the burst release. The results suggested that this kind of chitosan-O-isopropyl-5′-O-d4T monophosphate conjugate nano-prodrugs may be used as a targeting and sustained polymeric prodrugs for improving therapy efficacy and reducing side effects in antiretroviral treatment.

Full text of DOI:10.1016/j.bmc.2009.11.013

Bioorganic & Medicinal Chemistry 18 (2010) 117–123
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, nanosizing and in vitro drug release of a novel anti-HIV
polymeric prodrug: Chitosan-O-isopropyl-50-O-d4T monophosphate conjugate
a
a,
c
c
Lin Yang a, Liqiang Chen a, Rong Zeng b, , Chao Li , Renzhong Qiao , Liming Hu , Zelin Li
*
*
a State Key Laboratory of Chemical Resource Engineering, Department of Pharmaceutical Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China
b Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, PR China
c College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel approach to improve the antiviral efficacy of nucleoside reverse transcriptase inhibitors (NRTIs)
and reduce their side effects was developed by constructing a nanosized NRTI monophosphate-polymer
conjugate using d4T as a model NRTI. Firstly, a novel chitosan-O-isopropyl-50-O-d4T monophosphate con-
jugate with a phosphoramidate linkage was efficiently synthesized through Atherton–Todd reaction
under mild conditions. The anti-HIV activity and cytotoxicity of the polymeric conjugate were evaluated
in MT4 cell line. Then the conjugate nanoparticles were prepared by the process of ionotropic gelation
between TPP and chitosan-d4T conjugate to improve their delivery to viral reservoirs, and their physico-
chemical properties were characterized by transmission electron microscopy (TEM), dynamic light scat-
tering (DLS) techniques and X-ray diffraction (XRD). In vitro drug release studies in pH 1.1 and pH 7.4
suggested that both chitosan-d4T conjugate and its nanoparticles prefer to release d4T 50-(O-isopropyl)
monophosphate than free d4T for prolonged periods, which resulted in the enhancement of anti-HIV
selectivity of the polymeric conjugate relative to free d4T due to bypassing the metabolic bottleneck of
monophosphorylation. Additionally, the crosslinked conjugate nanoparticles can prevent the coupled
drug from leaking out of the nanoparticles before entering the target viral reservoirs and provide a mild
sustained release of d4T 50-(O-isopropyl) monophosphate without the burst release. The results sug-
gested that this kind of chitosan-O-isopropyl-50-O-d4T monophosphate conjugate nano-prodrugs may
be used as a targeting and sustained polymeric prodrugs for improving therapy efficacy and reducing side
effects in antiretroviral treatment.
Received 18 September 2009
Revised 4 November 2009
Accepted 5 November 2009
Available online 10 November 2009
Keywords:
Drug delivery
Chitosan-nucleoside monophosphate
conjugate
Anti-HIV activity
Polymeric prodrug
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the frequent administration of NRTIs in relatively high doses also
results in HIV-patient incompliance.6
Nucleoside reverse transcriptase inhibitors (NRTIs) such as
zidovudine (AZT), didanosine (ddI), lamivudine (3TC) and stavu-
dine (d4T) have been widely used in the treatment of human
immunodeficiency virus (HIV) infection. To exert their antiviral
activity, NRTIs are activated by cellular kinases to finally form
the corresponding 50-triphosphate derivatives, which can effi-
ciently terminate intracellular synthesis of the viral DNA from its
RNA by competing with natural nucleotides as a substrate for re-
verse transcriptase in infected cells.1–3 Although NRTIs have suc-
cessfully extended the lifespan of millions of HIV-infected
patients throughout the world in the past decades, these NRTIs
are still far from ideal for clinic use at the present time, since some
undesirable adverse effects such as neutropenia, peripheral neu-
ropathy and drug resistance are associated with them.4,5 Besides,
A number of methods have been developed to improve the anti-
viral efficacy of NRTIs and reduce their side effects based on the
consideration of bypassing the metabolic bottleneck, improving
cellular uptake efficacy, and targeting viral reservoirs, etc.6–8 For
instance, it was reported that the rate-limiting step for the intra-
cellular generation of the bioactive metabolite of d4T, d4T-triphos-
phate, seems to be the conversion of d4T to its monophosphate
derivative by nucleoside kinases. Accordingly, aryl phosphorami-
date derivatives of d4T exhibited enhanced antiviral activity and
selectivity since they can bypass the rate-limiting step of mono-
phosphorylation, and have better membrane permeability result-
ing from the presence of the lipophilic group.9,10
Except developing novel nucleoside analogue inhibitors of the re-
verse transcriptase by chemical modification, the design of drug
delivery systems (DDS) for NRTIs has been proven to be an efficient
approach to overcome the drawbacks of NRTIs, such as limited
stability, first pass metabolism and systemic toxicity.6 For instance,
drug-polymer conjugates as a special type of DDS have attracted
considerable attention due to their particular therapeutic
* Corresponding authors. Tel./fax: +86 20 85223271 (R.Z.); tel./fax: +86 10
82728926 (R.Q.).
E-mail addresses: tzengronga@jnu.edu.cn (R. Zeng), qiaorz@mail.buct.edu.cn
(R. Qiao).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.013

118
L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
properties, such as prolonged half-life, enhanced bioavailability, and
often targeting to specific cells, tissues or organs by attaching a
homing device.11 G. Giammona et al. reported the polymer-drug
conjugate of PHEA-50-O-succinylzidovudine with a prolonged dura-
tion of activity,12 and K. C. Chimalakonda et al. synthesized the mac-
romolecular prodrug of 3TC-dextran for selective antiviral delivery
to the liver.13 On the other hand, nanosized DDS is a very promising
way of improving drug bioavailability.14 Enhanced delivery and pro-
longed residence of NRTIs in monocytes/macrophages, central ner-
vous system (CNS) and the gastrointestinal tract could be observed
for nanoparticles or nanocapsules containing NRTIs.6,15–17
In this paper, we describe a combined strategy to improve the
antiviral efficacy of NRTIs and reduce their side effects by con-
structing a nanosized NRTI monophosphate-polymer conjugate
using d4T as a model drug. Chitosan, a well-known abundant
natural polysaccharide mainly composed of 2-amino-2-deoxy-b-
2. Results and discussion
2.1. Synthesis and characterization of chitosan-O-isopropyl-50-
O-d4T monophosphate conjugate
A
novel polymeric prodrug: chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate was efficiently synthesized through
Atherton–Todd reaction under mild conditions (Scheme 1). The
13
1H NMR and C NMR spectra of chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate have been shown in our previous
work.21 The degree of substitution (DS) of d4T monophosphate
moiety was estimated by 1H NMR spectra and confirmed by phos-
phorus content determination based on the ICP-AES method, was
equal to 17.0% (corresponding to a content of d4T of 17.5% w/w).21
The results suggested that Antherton–Todd reaction can pro-
vide an alternative approach for efficiently coupling nucleoside
drugs to chitosan by forming a phosphoramidate linkage, although
there exists the limitation of reactivity of H-phosphonate of d4T to
Cs-Tr resulted from steric hindrance of both chitosan and d4T
group. It’s also worth notice that various polymer phosphorami-
date bioconjugates with drugs, peptides, proteins or other biologi-
cally active components could be also successfully constructed
through Antherton–Todd reaction.
D-glucopyranose (D-glucosamine) residues was chosen as the poly-
meric vehicle since it is biodegradable, biocompatible, nontoxic,
and easy to form nanoparticles.18,19 Firstly, a novel chitosan-O-iso-
propyl-50-O-d4T monophosphate conjugate prodrug with a phos-
phoramide linkage between glucosamine and nucleoside
monophosphate was synthesized through Atherton–Todd reaction
under the mild conditions based on our previous work.20,21 The
anti-HIV activity and cytotoxicity of the polymeric conjugate were
evaluated in MT4 cell line. Further, chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate nanoparticles prepared by ionically
crosslinking with tripolyphosphate (TPP),22 which could be used
as a sustained polymeric nano-drug targeting to viral reservoirs.
The drug release characteristics of chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate and nanoparticles have also been inves-
tigated in vitro.
2.2. Anti-HIV activity and cytotoxicity of chitosan-O-isopropyl-
50-O-d4T monophosphate conjugate
The antiviral activity of chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate and the parent nucleoside d4T was
evaluated against HIV-1 in MT4 cells and summarized in Table 1.
The polymeric conjugate inhibited virus infection with an IC50
HO
OH
T
O
*
O
*
O
HO
NH2
3
n
1
a
PCl3
i-Pr-OH
Et3N
b
OTr
*
O
*
O
O
HO
i-Pr
NH2
O
P
O
n
H
OH
OH
2
T
*
O
O
*
O
O
O
HO
c
4
HO
NH
P
NH2
n-x
x
O
N
O
CH3
O
O
HN
T=thymine or
5
T
i-Pr
O
O
O
CH3
HN
O
HO
d4T=
O
N
Scheme 1. Synthetic route of chitosan-O-isopropyl-50-O-d4T monophosphate conjugate. Reagents and conditions: (a) (i) Phthalic anhydride, DMF, 130 °C, N2, 8 h, 83%; (ii)
TrCl, pyridine, 90 °C, N2, 24 h, 86%; (iii) NH2–NH2ꢀH2O, 80 °C, N2, 16 h, 81%; (b) (i) CH2Cl2, PCl3, ꢁ30 °C, 2 h; (ii) rt, 6 h; (iii) i-Pr-OH, 0 °C, 2 h; (c) (i) 2, Et3N, CCl4, DMA, 0 °C,
24 h; (ii) HCOOH, rt, 0.5 h.

L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
119
(concentration required to reduce virus replication by 50% in cul-
ture assays) of 0.027 g/ml, which was approximately 13-fold less
potent than d4T (IC50 value of 0.002 g/ml) against HIV-1 in cul-
ture assays. However, as the conjugate was much less toxic than
d4T (CC50 (concentration required to inhibit cell growth by 50%)
conditions had similar spherical structure but particle size smaller
than 90 nm.
l
l
Table 2 exhibited particle size, zeta potential and drug loading
capacity of chitosan-d4T conjugate nanoparticles and d4T-loaded
chitosan nanoparticles. The results demonstrated that chitosan-
d4T conjugate nanoparticles had a higher LC value than d4T-loaded
chitosan nanoparticles under the conditions used. It is also note-
worthy that hydrodynamic diameter of the nanoparticles mea-
sured by DLS was larger than the size estimated from TEM, since
chitosan-based nanoparticles dispersed in an aqueous phase for
the DLS experiments had high swelling capacity, while the TEM
experiments were performed in dry samples. Though chitosan-O-
isopropyl-50-O-d4T monophosphate conjugate nanoparticles and
d4T-loaded chitosan nanoparticles were formed by the same com-
plexation mechanism mainly based on the interaction between –
NH3+ of chitosan and TPP, chemical modification of chitosan would
greatly affected the size and surface properties of the obtained
nanoparticles. It could be seen that chitosan-d4T conjugate nano-
particles with larger diameter had a positive zeta potential equal
to ꢂ17 mV, while d4T-loaded chitosan nanoparticles with smaller
size had about 47 mV zeta potential. In comparison with the zeta
potential of the blank chitosan/TPP nanoparticles reported about
30–50 mV,26 the formation of phosphoramidate linkage of the
chitosan-d4T conjugate resulted in decrease of the zeta potential
value of nanoparticles.
Moreover, in order to identify the physical state and crystallin-
ity of d4T in polymeric conjugate or crosslinked nanoparticles, the
XRD spectra of chitosan, chitosan-O-isopropyl-50-O-d4T mono-
phosphate conjugate (DS = 17.0%), chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate nanoparticles and d4T-loaded chitosan
nanoparticles are presented in Figure 2, and the XRD pattern of
pure d4T is shown in Figure 3. As can be seen, the original chitosan
powder showed two major broad crystalline peaks at 2h of around
10.0° and 19.8°, respectively. While the diffraction peaks of chito-
san-O-isopropyl-50-O-d4T monophosphate conjugate were not re-
corded at the same position. The peak at 2h of around 10.0°
disappeared and instead new peaks at 2h of 23.6°, 26.3° and
35.6° with low intensity could be observed. These new peaks were
values of 658.6 and 30 lg/ml for the conjugate and d4T, respec-
tively), the overall selectivity index (SI) for the conjugate
(CC50/IC50) is superior to that of d4T. It is indicated that the synthe-
sized chitosan-d4T conjugate prodrug with a phosphoramide link-
age has remarkable anti-HIV activity with rather low cytotoxicity.
The results might be ascribed to the sustained release of d4T 50-(O-
isopropyl) monophosphate from the conjugate, which can bypass
the rate-limiting step of nucleoside phosphorylation and have bet-
ter membrane permeability resulting from the presence of the
lipophilic group.21
2.3. Preparation and characterization of chitosan-O-isopropyl-
50-O-d4T monophosphate conjugate nanoparticles
Although the synthesized water-soluble chitosan-d4T conjugate
can achieve a good approach to control drug concentration in
plasma and to improve the retention of the drug in the body by
sustained-releasing d4T 50-(O-isopropyl) monophosphate, it has
restricted access to the viral reservoirs, such as monocytes/macro-
phages, the brain and the gastrointestinal tract. Thus, chitosan-O-
isopropyl-50-O-d4T monophosphate conjugate nanoparticles were
prepared by ionically crosslinking with TPP to enhance their deliv-
ery to the viral reservoirs. Additionally, d4T-loaded chitosan nano-
particles were also prepared by the same method for comparison.
The formation of chitosan-O-isopropyl-50-O-d4T monophos-
phate conjugate nanoparticles occurs spontaneously upon incorpo-
ration negatively charged TPP into chitosan derivative (DS = 17.0%)
solution due to the complexation between oppositely charged spe-
cies.19,22 The formation process of complexes is very simple and
mild, avoiding the possible toxicity of chemical reagents and other
undesirable effects. As shown in Figure 1(a), the conjugate nano-
particles appeared spherical shape with the diameter ranged
between 100 and 200 nm, while Figure 1(b) illustrated that
d4T-loaded chitosan nanoparticles formed under the same
Table 2
Physicochemical characteristics of chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate nanoparticles and d4T-loaded chitosan nanoparticles
Table 1
Anti-HIV activity and cytotoxicity of chitosan-O-isopropyl-50-O-d4T monophosphate
Nanoparticles
Particle Polydispersity Zeta
LC
conjugate
size*
(nm)
index*
0.273
potential* (%)
(mV)
Compound
IC50
g/ml)
CC50
g/ml)
SI
(
l
(l
Chitosan-O-isopropyl-50-O-
d4T monophosphate
conjugate nanoparticles
d4T-Loaded chitosan
nanoparticles
228.9
+17.23
13.1
Chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate (DS = 17.0%)
d4T
0.027
658.6
24,393
0.002
30
15,000
166.8
0.297
+46.75
4.63
d4T 50-(O-Isopropyl) monophosphate
0.0008
104
130,000
*
Data measured by DLS.
Figure 1. Transmission electron microscopy (TEM) micrographs of nanoparticles: (a) chitosan-O-isopropyl-50-O-d4T monophosphate conjugate nanoparticles, and (b) d4T-
loaded chitosan nanoparticles.

120
L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
Figure 2. The XRD spectra of (a) chitosan, (b) chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate, (c) chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate nanoparticles, and (d) d4T-loaded chitosan nanoparticles.
Figure 4. Release of d4T 50-(O-isopropyl) monophosphate (j), d4T
( ) from
chitosan-O-isopropyl-50-O-d4T monophosphate conjugate and d4T 50-(O-isopropyl)
monophosphate ( ), d4T ( ) from chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate nanoparticles at pH 1.1 buffer solution at 37.0 0.1 °C.
also obvious in the chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate nanoparticles except the peak at 2h = 35.6° shifted to
38.1°. These new peaks may be attributed to a polymorph structure
transformation when the initial crystal form in not thermodynam-
ically stable and transform to the most stable form due to the
attachment of d4T 50-(O-isopropyl) monophosphate to chitosan
through a phosphoramidate linkage. In addition, the relative inten-
sity and position of XRD peaks of d4T-loaded chitosan nanoparti-
cles were quite similar with those of the conjugate nanoparticles,
but different from the simple addition of those of chitosan and
d4T, which indicated that the crystal form of loaded d4T was also
affected by the chitosan nanoparticles matrix.
from d4T-loaded nanoparticles was also investigated for compari-
son (shown in Fig. 6).
It could be seen that both d4T monophosphate derivative and
free d4T were released from chitosan-O-isopropyl-50-O-d4T mono-
phosphate conjugate under used experimental conditions for a
prolonged manner, and the amount of d4T 50-(O-isopropyl) mono-
phosphate was greater than that of free d4T. From the hydrolysis
profile at pH 1.1, the starting hydrolysis rate of d4T 50-(O-isopro-
pyl) monophosphate and d4T from the polymeric conjugate were
calculated to be 2.14%/h and 0.34%/h, respectively, and after 6 h
about 6.84% of d4T 50-(O-isopropyl) monophosphate and only
1.24% of free d4T were released. As for the hydrolysis at pH 7.4,
approximately 7.27% of d4T 50-(O-isopropyl) monophosphate was
released from the polymeric conjugate within 24 h with a starting
rate of 1.09%/h, whereas about 1.36% of free d4T was detected with
a starting rate of 0.29%/h. These results confirmed that the phos-
phoramidate bond between chitosan and d4T 50-(O-isopropyl)
monophosphate has higher hydrolytic activity than d4T 50-(O-iso-
propyl) monophosphate, and the chitosan-O-isopropyl-50-O-d4T
2.4. In vitro release studies
In order to obtain some preliminary information about the
potential use of chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate (DS = 17.0%) and its nanoparticles for antiretroviral
treatment, the polymeric conjugate and its nanoparticles were
subjected to in vitro hydrolysis studies in buffer solutions at pH
1.1 (simulated gastric juice) and pH 7.4 (extracellular fluids). The
results of drug release were determined by HPLC and shown in
Figures 4 and 5, respectively. In addition, the study of d4T releasing
Figure 5. Release of d4T 50-(O-isopropyl) monophosphate (j), d4T
( ) from
chitosan-O-isopropyl-50-O-d4T monophosphate conjugate and d4T 50-(O-isopropyl)
monophosphate ( ) from chitosan-O-isopropyl-50-O-d4T monophosphate conju-
gate nanoparticles at pH 7.4 buffer solution at 37.0 0.1 °C.
Figure 3. The XRD spectra of d4T.

L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
121
conjugate nanoparticles, and confirmed the capacity of the conju-
gate nanoparticles to mainly release d4T 50-(O-isopropyl) mono-
phosphate, which was prevented from leaking out of the
nanoparticles before entering the viral reservoirs.
By comparison, the d4T-loaded nanoparticles exhibited
a
releasing profile with two stages of an initial burst release period
followed by a period of slower release, as shown in Figure 6.
Approximately 65% of the drug content released in the initial per-
iod of burst release for 2 h both at pH 1.1 and at pH 7.4, which
associated with the dissolution of drug adsorbed or located close
to the surface of nanoparticles. Later the rate of release fell as the
dominant mechanism changed to diffusion through the swollen
rubbery chitosan matrix. Comparing data of the d4T-loaded nano-
particles, the crosslinked conjugate nanoparticles could avoid the
burst release effect to provide a quite mild release rate. These re-
sults suggested that the drug release from the conjugate nanopar-
ticles may involve three steps. First, water penetrates into the
particulate system, which causes swelling of the matrix; second,
a major amount of d4T monophosphate derivative with a little
amount of free d4T are released from polymer backbone due to
cleavage of chemical bond; the third step is the diffusion of drug
from the swollen rubbery matrix.19 It should be noticed that the
bond cleavage step is the rate-limiting step in this controlled re-
lease process. Therefore, the conjugate nanoparticles with the
capacity of targeting the viral reservoirs can provide a mild and
gentle sustained release of d4T 50-(O-isopropyl) monophosphate
without the burst release.
Figure 6. Release of d4T from d4T-loaded chitosan nanoparticles at pH 1.1 ( ) and
pH 7.4 (d) buffer solutions at 37.0 0.1 °C.
monophosphate conjugate prefers to release d4T 50-(O-isopropyl)
monophosphate than free d4T. Thus, d4T 50-(O-isopropyl) mono-
phosphate could be considered as the major product released from
the polymeric conjugate under both acidic and neutral conditions
compared with little amount of free d4T (as shown in Fig. 7). Since
the antiretroviral activity of nucleoside analogues may be more
critically dependent upon the rate-limiting step of their conversion
to the 50-O-monophosphate by nucleoside kinases in the intracel-
lular metabolism, a delayed release of the d4T 50-(O-isopropyl)
monophosphate from the conjugate was able to bypass the rate-
limiting step of monophosphorylation to improve therapy efficacy
and reduce side effects.
As for the conjugate nanoparticles, it could be seen that at pH
1.1 both d4T 50-(O-isopropyl) monophosphate and d4T were re-
leased with quite mild releasing rate compared with non-cross-
linked chitosan-O-isopropyl-50-O-d4T monophosphate conjugate.
The total released amount of d4T 50-(O-isopropyl) monophosphate
or d4T from the conjugate nanoparticles was less than half of re-
leased amount from the non-crosslinked conjugate within 6 h.
However, approximately 2.22% of d4T 50-(O-isopropyl) monophos-
phate was released from the conjugate nanoparticles within 24 h,
whereas free d4T was hardly detected at pH 7.4. The results sug-
gested that chitosan-O-isopropyl-50-O-d4T monophosphate conju-
gate can be protected from hydrolysis by forming the crosslinked
3. Conclusion
A novel water-soluble chitosan-O-isopropyl-50-O-d4T mono-
phosphate conjugate with DS value up to 17.0% was efficiently syn-
thesized through Atherton–Todd reaction under mild conditions.
The anti-HIV activity and cytotoxicity of the polymeric conjugate
evaluated in MT4 cell line exhibited that the resulting polymeric
conjugate has remarkable anti-HIV effect and rather low cytotoxic-
ity. In vitro drug release studies at pH 1.1 and pH 7.4 indicated the
polymeric conjugate prodrug prefers to release the d4T 50-(O-iso-
propyl) monophosphate than free d4T for a prolonged period. This
may lead to the enhancement of anti-HIV selectivity of the poly-
meric conjugate relative to the parent nucleoside analogue owing
to the effect of bypassing the rate-limiting step of monophosph-
orylation mediated by thymidine kinase.
Aiming to enhance the delivery to viral reservoirs of HIV, chito-
san-O-isopropyl-50-O-d4T monophosphate conjugate nanoparti-
Figure 7. Schematic representation of drug release from polymeric conjugate.

122
L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
cles were prepared by the process of ionotropic gelation using TPP.
Comparing with the data of chitosan-O-isopropyl-50-O-d4T mono-
phosphate conjugate and d4T-loaded nanoparticles, in vitro drug
release studies revealed that the crosslinked conjugate nanoparti-
cles can prevent the coupled drug from leaking out of the nanopar-
ticles in blood circulation and provide a mild sustained release of
d4T 50-(O-isopropyl) monophosphate without the burst release.
Further research on in vitro and in vivo biological activities of this
kind of chitosan-O-isopropyl-50-O-d4T monophosphate conjugate
nano-prodrugs could lead to their applications in antiretroviral
treatment.
(C4), 85.2 (C10), 90.2 (C13), 97.2, 101.2 (C1), 111 (C16), 125.5 (C12),
133.8 (C11), 137.8, 138.9 (C14), 152 (C17), 165.5 (C18), 174.6
(CH3–CO). Mw = 26.8 k, Mw/Mn = 1.63.
4.2. In vitro assays of anti-HIV activity and cytotoxicity of
chitosan-O-isopropyl-50-O-d4T monophosphate conjugate
MT4 cells, a human T4-positive cell line were infected with
HIV-1 at the multiplicity (MOI) of 0.1, and HIV-infected MT4 cells
were incubated for 1.5 h at 37 °C in a 5% CO2 incubator. Subse-
quently, cells were cultured in 96-well microtiter plates in the
presence of various concentrations of conjugate and aliquots of
culture supernatants were removed from the wells on the fifth
day after infection for p24 antigen assays. The applied p24 enzyme
immunoassay (EIA) was the unmodified kinetic which utilizes a
murine mAb to HIV core protein coated onto microwell strips to
which the antigen present in the test culture supernatant samples
binds. Percent viral inhibition was calculated by comparing the
p24 values from untreated infected cells. The 50% inhibition con-
centration (IC50) was determined from the concentration–response
curve using the median effect method.
Cytotoxicity of the conjugate was evaluated in parallel with
antiviral activity using the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) method. It was based on the viabil-
ity of mock-infected cells, as monitored by the MTT method. The
50% cytotoxic concentration (CC50) was determined from the con-
centration–response curve using the median effect method.
4. Experimental
Chitosan (low molecular weight) was purchased from Sigma–
Aldrich Chemicals Co, USA. Deacetylation degree of 88% was deter-
mined by 1H NMR. 6-O-trity1 chitosan was firstly synthesized from
chitosan in three steps, N-phthaloylation, 6-O-trityl protection and
de-phthaloylation, according to the known procedure reported by
Nishimura et al.23 D4T was purchased from Yingjie Biotechnology
Company (China), and dried at elevated temperature in vacuum.
Triethylamine, tetrachloromethane and sodium tripolyphosphate
(TPP) were purchased from Beijing Chemical Reagents Company
(China). All other solvents were commercially available reagents
of analytical grade, dried and purified by distillation before using.
The chemical structures of the synthesized chitosan-O-isopropyl-
50-O-d4T monophosphate conjugate were analyzed by 31P, 1H
NMR in 2% (v/v) CD3COOD/D2O and 13C NMR in 5% (v/v)
CF3COOD/D2O at 600 MHz using a Bruker AV600 NMR instrument.
Besides, the phosphorus content determination of the conjugate
was performed by the ICP-AES (Atomic Emission Spectrometry,
JY ULTIMA, France) method, thus the corresponding content of
d4T was obtained. The Mw and Mw/Mn of the polymeric conjugate
were measured by GPC (Waters 515-410, Column: Ultrahydrogel
250, Solvent: 0.1 M NaCl, 40 °C, Flow rate: 1.0 mL/min, Standards:
PEO).
4.3. Preparation of chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate nanoparticles
Chitosan-O-isopropyl-50-O-d4T monophosphate conjugate nano-
particleswerepreparedaccordingtotheprocedurereportedbyCalvo
et al. based on the ionic gelation of chitosan with TPP polyanions.25,26
Briefly, 30 mg of the synthesized polymer-d4T conjugate was dis-
solved in2%aceticaqueoussolution at 1 mg/ml. Undermagneticstir-
ring at room temperature, 10 mg TPP dissolved in 13 ml aqueous
solution was added dropwise into the conjugate solution, and stirred
continuously for 1 h to stabilize the nanoparticles through the elec-
trostatic interaction with TPP. Then the suspension of nanoparticles
dialyzed against deionized water and passed through a syringe filter
4.1. Synthesis of chitosan-O-isopropyl-50-O-d4T
monophosphate conjugate
The chitosan-O-isopropyl-50-O-d4T monophosphate conjugate
was prepared as described in a previous study.21 All experiments
involving water-sensitive compounds were conducted under dry
conditions. Briefly, O-isopropyl-50-H-phosphonate of d4T was syn-
thesized by using phosphorus trichloride as phosphorylation re-
agent according to the method reported by X. B. Sun et al.24
1.32 g (4 mmol) of O-isopropyl-50-H-phosphonate of d4T was dis-
solved in 10 mL dimethylacetamide (DMA), the solution was added
dropwise to 6-O-trityl chitosan (160 mg, ꢂ0.40 mmol of free NH2)
in a mixed solution of DMA (10 ml), triethylamine (1.76 ml) and
tetrachloromethane (2 ml) in ice-water bath. After a 24 h stirring,
the solution was filtered. The filtrate was added to EtOH
(200 mL) and the precipitate formed was collected by centrifuga-
tion. Then the precipitate was dissolved in 10 mL of formic acid
and was stirred for 0.5 h at room temperature. After removing for-
mic acid by rotary evaporation, the residue was dissolved in 2%
AcOH (20 mL), and then filtered. The filtrate was dialyzed with dis-
tilled water for 3 days and lyophilized to provide the water-soluble
target product, chitosan-O-isopropyl-50-O-d4T monophosphate
conjugate (69 mg). 31P NMR (263 K, 2% CD3COOD/D2O): d = 8.09,
8.27 (–NH–P–); 1H NMR (293 K, 2% CD3COOD/D2O): d = 1.15, 1.20
(d, H8), 1.79 (s, H15), 3.07, 3.12 (s, H2), 3.49–3.89 (m, H3, H4, H5,
H6, H7), 4.10 (br, H9), 4.77 (br, H1), 5.02 (s, H10), 5.91 (s, H11),
6.38 (s, H12), 6.80 (s, H13), 7.39 (s, H14), 8.15 (s, H16); 13C NMR
(293 K, 5% CF3COOD/D2O): d = 11.6 (C15), 22 (C8), 22.7 (CO–CH3),
55.8 (C2), 60.1 (C6), 66.8 (C9), 70 (C3), 73.5 (C7), 74.8 (C5), 76.8
(pore size 0.45 lm) prior to lyophilization. The drug loading capacity
(LC) of the conjugate nanoparticles was calculated from the content
of d4T coupled to chitosan.
For comparison, d4T-loaded chitosan nanoparticles were also
prepared by premixing d4T with chitosan acetic aqueous solution
at weight content of 18% and then following the above procedure
at the same chitosan to TPP weight ratio of 3:1. The drug loading
capacity (LC) of d4T-loaded chitosan nanoparticles was determined
by the separation of nanoparticles from the aqueous medium con-
taining non-associated d4T by centrifugation at 16,000 rpm for
30 min. The amount of free d4T in the supernatant was measured
by HPLC. Each experiment was performed in triplicate. The LC va-
lue was calculated from (Eq. 1), which indicated below:
LC ¼ ðA ꢁ BÞ=C ꢃ 100%
ð1Þ
A is the total amount of drug, B is the amount of free drug in super-
natant, and C is the weight of nanoparticles.
4.4. Physicochemical characterization of nanoparticles
For measurement of particle size, zeta potential and polydisper-
sity (size distribution) of freshly prepared chitosan-O-isopropyl-50-
O-d4T monophosphate conjugate nanoparticles and d4T-loaded
chitosan nanoparticles, Zetaplus (Brookhaven, USA) was used
which is based on the Dynamic Light Scattering (DLS) techniques.
All DLS measurements were done with a wavelength of 633 nm

L. Yang et al. / Bioorg. Med. Chem. 18 (2010) 117–123
123
at 25 °C with an angle detection of 90°. For zeta potential measure-
References and notes
ments, samples were diluted with 0.1 mM KCl and measured in the
automatic mode.
1. Furman, P. A.; Fyfe, J. A.; St Clair, M. H.; Weinhold, K.; Rideout, J. L.; Freeman, G.
A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya, H. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 8333.
2. Riddler, S. A.; Anderson, R. E.; Mellors, J. W. Antiviral Res. 1995, 27, 189.
3. Balzarini, J.; Herdewijn, P.; De Clercq, E. J. Biol. Chem. 1989, 264, 6127.
4. Tan, X. L.; Chu, C. K.; Boudinot, F. D. Adv. Drug Delivery Rev. 1999, 39,
117.
5. Rando, R. F.; Nguyen-Ba, N. Drug Discovery Today 2000, 5, 465.
6. Sosnik, A.; Chiappetta, D. A.; Carcaboso, Á. M. J. Controlled Release 2009.
doi:10.1016/j.jconrel.2009.05.007.
Morphological characteristics of the nanoparticles were ob-
served by Transmission Electron Microscope (TEM, Hitachi,
H-800, Japan). One drop of freshly made nanoparticles solution
was placed on 300 mesh copper grids coated with carbon film,
allowing to sitting until dried. The physical form (crystalline or
amorphous) of the lyophilized samples was determined by X-ray
diffraction (XRD) over the range 2h from 5° to 40°, a Rigaku D/
max 2500 diffractometer (Japan) with Bragg–Brentano geometry
(h, 2h) and Ni-filtered Cu Ka radiation.
7. De Clercq, E. J. Med. Chem. 1995, 38, 2491.
8. Wiebe, L. I.; Knaus, E. E. Adv. Drug Delivery Rev. 1999, 39, 63.
9. McGuigan, C.; Cahard, D.; Sheeka, H. M.; De Clercq, E.; Balzarini, J. J. Med. Chem.
1996, 39, 1748.
10. Venkatachalam, T. K.; Qazi, S.; Uckun, F. M. Bioorg. Med. Chem. 2006, 14,
5161.
4.5. In vitro drug release studies
11. Pasut, G.; Veronese, F. M. Prog. Polym. Sci. 2007, 32, 933.
12. Giammona, G.; Cavallaro, G.; Fontana, G.; Pitarresi, G.; Carlisi, B. J. Controlled
Release 1998, 54, 321.
13. Chimalakonda, K. C.; Agarwal, H. K.; Kumar, A.; Parang, K.; Mehvar, R.
Bioconjugate Chem. 2007, 18, 2097.
14. Vyas, T. K.; Shahiwala, A.; Amiji, M. M. Int. J. Pharm. 2008, 347, 93.
15. Chellat, F.; Merhi, Y.; Moreauc, A.; Yahia, L. Biomaterials 2005, 26, 7260.
16. Vinogradov, S. V.; Zeman, A. D.; Batrakova, E. V.; Kabanov, A. V. J. Controlled
Release 2005, 107, 143.
17. Hillaireau, H.; Le Doan, T.; Appel, M.; Couvreur, P. J. Controlled Release 2006,
116, 346.
18. Ravi Kumar, M. N. V.; Muzzarelli, R. A. A.; Muzzarelli, C.; Sashiwa, H.; Domb, A.
J. Chem. Rev. 2004, 104, 6017.
19. Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. J. Controlled Release
2004, 100, 5.
20. Zeng, R.; Fu, H.; Zhao, Y. F. Macromol. Rapid Commun. 2006, 27, 548.
21. Yang, L.; Zeng, R.; Li, C.; Li, G.; Qiao, R. Z.; Hu, L. M.; Li, Z. L. Bioorg. Med. Chem.
Lett. 2009, 19, 2566.
In vitro release studies of chitosan-O-isopropyl-50-O-d4T mono-
phosphate conjugate, conjugate nanoparticles and d4T-loaded
chitosan nanoparticles were carried out in buffer solutions at pH
1.1 (HCl, NaCl and glycine) and pH 7.4 (Na2HPO4, KH2PO4, NaCl).
Equal known aliquots of samples were suspended in equal volumes
of preheated buffer solutions at each different value, shaken in a
shaking water bath at 37.0 0.1 °C and sampled at predetermined
intervals. Each sample was directly analyzed by HPLC (Detection
wavelength: 263 nm, Analytical RP-18 Lichrospher column, Eluent:
CH3OH: 0.1% H3PO4 (20:80 v/v), flow rate: 1.0 mL/min) monitoring
the released amount of product, and confirmed the compounds by
ESI-MS.12 Each experiment was repeated in triplicate.
22. Janes, K. A.; Fresneau, M. P.; Marazuela, A.; Fabra, A.; Alonso, M. J. J. Controlled
Release 2001, 73, 255.
Acknowledgments
23. Nishimura, S.-I.; Kohgo, O.; Kurita, K.; Kuzuhara, H. Macromolecules 1991, 24,
4745.
24. Sun, X. B.; Kang, J. X.; Zhao, Y. F. Chem. Commun. 2002, 20, 2414.
25. Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J. L.; Alonso, M. J. J. Appl. Polym. Sci.
1997, 63, 125.
The authors would like to acknowledge financial support from
the National Natural Science Foundation of China (Grant Nos.
20872010, 20732004 and 20972014) and the National Basic
Research Program of China (No. 2009CB930203).
26. Gan, Q.; Wang, T.; McCarron, P. Colloids Surf., B 2005, 44, 65.