Combination anti-HIV therapy with the self-assemblies of an asymmetric bolaamphiphilic zidovudine/didanosine prodrug

Article abstract of DOI:10.1021/mp100457d

Combination anti-HIV therapy is important for AIDS treatment. A bolaamphiphilic prodrug, zidovudine-phosphoryl-deoxycholyl didanosine (ZPDD), was synthesized, combining zidovudine (AZT) and didanosine (ddI) in one molecule. As one lipid derivative of nucleosides, ZPDD showed special solubility with free soluble in chloroform and tetrahydrofuran but was slightly soluble in cyclohexane. The amphiphilicity of ZPDD was shown according to the monolayers at the air-water interface. ZPDD self-assembled to the spherical vesicles in water with 174 nm in size and -31.3 mV of zeta potential. The stability of assemblies depended on pH because the phosphoryl zidovudine group could release hydrogen ions. ZPDD was rapidly degraded to AZT in the plasma and tissues of mice. ZPDD self-assemblies had high anti-HIV activity in vitro with the half effective concentration (EC₅₀) of 5 nM. ZPDD self-assemblies may be targeting macrophages since ZPDD was found in macrophage-rich tissues in vivo and rapidly released AZT in the targeted tissues after intravenous administration to mice. The bioavailability of ZPDD was 90.5% and 30.8% for the intraperitoneal and oral administrations compared with the venous route. The self-assemblies of bolaamphiphilic prodrugs could simultaneously deliver two types of drugs to targeted tissues and would become a promising nanomedicine.

Full text of DOI:10.1021/mp100457d

ARTICLE
pubs.acs.org/molecularpharmaceutics
Combination Anti-HIV Therapy with the Self-Assemblies of an
Asymmetric Bolaamphiphilic Zidovudine/Didanosine Prodrug
Yiguang Jin,*,†,‡ Rui Xin,†,‡ Li Tong,§ Lina Du,† and Miao Li†
†Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing 100850, China
‡Institute of Pharmacy, Pharmaceutical College of Henan University, Henan 475004, China
§College of Life Sciences, Beijing Normal University, Beijing 100875, China
S Supporting Information
b
ABSTRACT: Combination anti-HIV therapy is important for
AIDS treatment. A bolaamphiphilic prodrug, zidovudine-
phosphoryl-deoxycholyl didanosine (ZPDD), was synthesized,
combining zidovudine (AZT) and didanosine (ddI) in one
molecule. As one lipid derivative of nucleosides, ZPDD showed
special solubility with free soluble in chloroform and tetra-
hydrofuran but was slightly soluble in cyclohexane. The amphi-
philicity of ZPDD was shown according to the monolayers at
the airꢀwater interface. ZPDD self-assembled to the spherical
vesicles in water with 174 nm in size and ꢀ31.3 mV of zeta potential. The stability of assemblies depended on pH because the
phosphoryl zidovudine group could release hydrogen ions. ZPDD was rapidly degraded to AZT in the plasma and tissues of mice.
ZPDD self-assemblies had high anti-HIV activity in vitro with the half effective concentration (EC50) of 5 nM. ZPDD self-assemblies
may be targeting macrophages since ZPDD was found in macrophage-rich tissues in vivo and rapidly released AZT in the targeted
tissues after intravenous administration to mice. The bioavailability of ZPDD was 90.5% and 30.8% for the intraperitoneal and oral
administrations compared with the venous route. The self-assemblies of bolaamphiphilic prodrugs could simultaneously deliver two
types of drugs to targeted tissues and would become a promising nanomedicine.
KEYWORDS: anti-HIV, bolaamphiphile, didanosine, prodrug, self-assembly, zidovudine
’ INTRODUCTION
intravenous (iv) infusion and oral administration.13 Low bio-
availability and weak targeting could lead to frequent high-dose
administration of the drugs and further patient incompliance and
drug resistance.2 Macrophage is demonstrated as a reservoir of
HIV. But many anti-HIV agents distribute little in the cells.14,15
Nanoscale particles are preferred as the macrophage-targeted
system based on optimization in circulation and macrophage-
specific reorganization, so that the anti-HIV agent-loaded nano-
particulate systems are used for macrophage targeting.16ꢀ18
Bolaamphiphilic molecules or bolaamphiphiles are one special
type of amphiphilic molecules and defined as the molecules
containing a hydrophobic skeleton and two water-soluble groups
on both ends.19 The unique structure of bolaamphiphiles makes
them to own some special functions different from traditional
amphiphiles with one polar head. Bolaamphiphiles are inclined to
form monolayers but not bilayers unless the hydrophobic
skeleton is very soft and long, and the monolayer may be very
strong even under harsh environments. Archaebacteria, one type
of ancient bacteria with bolaamphiphilic monolayer membranes,
can survive in the volcanic environment, enduring 80 °C high
Combination pharmacotherapy is commonly applied to treat
AIDS, cardiovascular diseases, cancer, and so on.1ꢀ4 Usually, it is
difficult to simultaneously deliver all of the components of
combination drugs to targeted cells and maintain an appropriate
concentration. Two or more drugs are covalently combined to
form codrugs to address the above problem though the conjuga-
tion may be complicated.5ꢀ7 Carriers, such as liposomes and
nanoparticles, could entrap two types of drugs, although the drug
loading efficiency and the entrapment efficiency are generally
low.8,9 Furthermore, the carriers could be destroyed in circula-
tion followed by rapid release of the cargos before reaching
targets. It is necessary to develop a method to simultaneously
deliver combination drugs to targets and release them.
Highly active antiretroviral therapy (HAART), combining at
least three antiretroviral drugs, is a standard method to treat
AIDS. Anti-HIV codrugs are also studied.10 Dinucleoside com-
pounds combine two nucleoside analogues with either one type
or two types against viruses.11,12 30-Azido-30-deoxythymidilyl-
(50,50)-20,30-dideoxy-50-inosinic acid (AZT-P-ddI, IVX-E-59,
Scriptene) was prepared and showed enhanced anti-HIV activity
and selectivity in vitro compared with AZT and ddl alone.
However, the pharmacokinetic study in humans showed that
AZT-P-ddI was rapidly metabolized to β-glucuronide derivative
of zidovudine (AZT) and a metabolite of didanosine (ddI) after
Received: December 26, 2010
Accepted: May 9, 2011
Revised:
April 27, 2011
Published: May 09, 2011
r
2011 American Chemical Society
867
dx.doi.org/10.1021/mp100457d Mol. Pharmaceutics 2011, 8, 867–876
|

Molecular Pharmaceutics
ARTICLE
Figure 1. Synthesis route of ZPDD.
temperature and hot sulfuric acid.20 Synthetic bolaamphiphiles
may become the components of interesting structures, for
example, nanowells, ion pores, electron conductors, DNA-like
nanofibers, archaebacteria-mimic membranes, or nanotubes.19,21ꢀ24
Some bolaamphiphiles are also used to form carriers to deliver
genes or drugs.25ꢀ27
of reagent grade. Purified water was prepared with the Heal Force
Super NW Water System (Shanghai Canrex Analytic Instrument
Co. Ltd., Shanghai, China), and always used otherwise specially
indicated. UV spectra and 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded respectively on a Shimadzu
UV-2501PC spectrophotometer, a JNM-ECA-400 NMR spec-
trometer. IR spectra were recorded on a Bio-RAD FTS-65A IR
spectrophotometer. FAB-MS was recorded on a Micromass
ZabSpec high-resolution mass spectrometer.
MT4 cells and human immunodeficiency virus type-1
(HIV-1IIIB) virus were from the Center of AIDS, Beijing
Institute of Microbiology and Epidemiology. Plasma from
Kunming mice and SpragueꢀDawley rats was prepared in our
lab. Plasma from beagle dogs, monkey, and healthy humans
was donated by Prof. G. Dou of Beijing Institute of Transfu-
sion Medicine.
Kunming mice from the Laboratory Animal Center of Beijing
Institute of Radiation Medicine (BIRM) were used. Principles in
good laboratory animal care were followed, and animal experi-
mentation was in compliance with the Guidelines for the Care
and Use of Laboratory Animals in BIRM. Mice were sacrificed by
euthanasia to remove tissues. Mouse tissue homogenates used in
the experiments of chemical stability and tissue distribution were
prepared in tissue/water (1:1, w/w).
A method was developed to perform combination anti-HIV
therapy with dual drugs in this study. A bolaamphiphilic prodrug,
zidovudine-phosphoryl-deoxycholyl didanosine (ZPDD), was
prepared. Zidovudine (AZT) and didanosine (ddI) were com-
bined to treat AIDS.28,29 ZPDD is the conjugate of the two drugs
with deoxycholyl as the spacer. The prodrug can form the nano-
scale self-assemblies with high anti-HIV activity in vitro and
macrophage targeting in vivo. The assemblies could release
parent drugs or active intermediates in the targeted tissues.
ZPDD self-assemblies belong to the self-assembled drug delivery
system (SADDS). SADDS has been developed in our lab for
several years, defined as the self-assembled nanoparticulates of
amphiphilic prodrugs. Many amphiphilic prodrugs were pre-
pared and self-assembled to the assemblies with various mor-
phologies though most of them only had one polar head and a
long lipid chain.30ꢀ33 Compared with traditional drug delivery
systems, the unique advantages of SADDS include high stability,
high drug loads, and controlled release in targets.32
Synthesis of the Prodrug. The synthesis procedure of ZPDD
was divided into four steps (see Figure 1). Simply, deoxycholic
acid was transformed to dehydrodeoxycholic acid followed by
conjugation with ddI. The intermediate was reduced to deoxy-
cholate that was then phosphorylated with phosphorus oxy-
chloride. Finally, AZT was conjugated to obtain ZPDD. The
details are described as follows.
’ EXPERIMENTAL SECTION
Materials. AZT and ddI were from Zhang Jiang Desano
Science and Technology Co., Ltd., Shanghai, China. Deoxycholic
acid was from Anhui Kebao Bioengineering Co., Ltd. (Anhui, China).
Organic solvents were of analytical grade. Other chemicals were
868
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
Deoxycholic acid (20 mmol) was dissolved in acetone
(100 mL) followed by incubation in 4 °C bath and agitation
for 10 min. A Jones reagent (20 mmol, 10 mL) was added. The
reaction proceeded for 2 h. Most of solvents were removed under
vacuum. The residue liquid was transferred to a separatory funnel
and diluted with dichloromethane (DCM, 30 mL). The organic
solution was washed with the saturated NaHCO3 solution repea-
ted until the color disappeared. The DCM phase was separated
and dried with anhydrous Na2SO4. The solvent was removed to
obtain the white solid of dehydrodeoxycholic acid (C24H36O4)
with about 80% yield.
150.2 (2CO), 163.8 (CO); FAB-MS (þ): 941.60, (M þ H)þ
(17).
Solubility Measurement. A certain amount of ZPDD was
mixed with solvents to obtain the suspensions that were agitated
at 25 °C for 48 h to sufficiently dissolve. The saturated solutions
were filtrated through the 0.45 μm membranes, and ZPDD in the
filtrates was determined using high-performance liquid chroma-
tography (HPLC).
Langmuir Monolayers at the AirꢀWater Interface. Surface
pressureꢀarea (πꢀA) isotherms were obtained using a KSV
minitrough II film balance (KSV Instrument, Finland) equipped
with the dual barriers and a Pt Wilhelmy plate sensing device.
The area of the Teflon trough is 24 300 mm2. The subphases
were purified water, amine acetate buffered solution (pH 4.0,
20 mM), and Tris-HCl buffered solution (pH 9.0, 20 mM),
respectively, and the temperature of subphases were controlled
to 35 °C with the circulation water bath. ZPDD solution in
chloroform (25 μL) was deposited onto the subphase with a
Hamilton microsyringe precisely. Compression was initiated
after a delay of 15 min to allow evaporation of the spreading
solvent. The compression rate was 10 mm/min. πꢀA isotherms
were recorded with a computer.
Preparation of the Self-Assemblies. The preparation of
ZPDD self-assemblies was similar to the other self-assemblies
of nucleoside lipid derivatives.33,34 An injection method was used
to prepare ZPDD self-assemblies. The ZPDD solution (5 mg/
mL) in tetrahydrofuran (THF) was slowly injected into water
using a 100 μL microsyringe under vortex agitation to get a
homogeneous and slightly blue scattering transparent suspen-
sion of about 1 mg ZPDD/mL. After removing solvents and
partial water by heat under vacuum, a stable concentrated
suspension of ZPDD self-assemblies was obtained with the high
concentration of ∼10 mg ZPDD/mL.
Dehydrodeoxycholic acid (60 mmol), ddI (20 mmol), N,N0-
dimethyl aminopyridine (DMAP, 4 mmol), and N,N0-dicyclo-
hexyl carbodiimide (DCC, 24 mmol) were together dissolved in
N,N0-dimethylformamide (DMF, 100 mL) followed incubation
at 45 °C and agitation for 2 days. Most of solvents were removed
under vacuum. The residue liquid was transferred to a separatory
funnel and diluted with DCM (50 mL). The organic solution was
washed with the citric acid solution (0.5 mmol/L), the saturated
NaHCO3 solution, and water in turn. The DCM phase was sep-
arated and dried with anhydrous Na2SO4. The solvent was remo-
ved to obtain a slightly yellow solid. The solid was then purified
on a silica chromatographic column with eluent of DCM/ethanol
of the volume ratios from 50:1 to 30:1 to obtain the white powder
of didanosine dehydrodeoxycholate (C34H46N4O6) with about
70% yield.
Didanosine dehydrodeoxycholate (1 mmol) and sodium
borohydride (1 mmol) were dissolved in ethanol (20 mL) fol-
lowed by agitation at room temperature for 2 h. Most of solvents
were removed under vacuum. The residue liquid was mixed with
water (20 mL) and then extracted with ethyl acetate (20 mL).
The ethyl acetate phase was separated and dried with anhydrous
Na2SO4. The solvent was removed to obtain the white solid of
didanosine deoxycholate (C34H50N4O6) with more than 90%
yield.
Didanosine deoxycholate (1 mmol) and POCl3 (2.2 mL) were
dissolved in DCM (20 mL) and agitated in ice bath for 2 h.
Zidovudine (1 mmol) was dissolved in DCM (20 mL) and then
added with triethylamine (0.4 mL). The solution was dropped
into the didanosine deoxycholate solution and agitated at room
temperature overnight. The reaction solution was transferred to a
separatory funnel and washed with the citric acid solution
(0.5 mM), the saturated NaHCO3 solution and water in turn.
The organic phase was separated, dried with anhydrous Na2SO4.
The solvent was removed to obtain a semisolid. The solid was
purified on a silica chromatographic column with eluent of
DCM/ethanol of the volume ratios from 30:1 to 15:1 to obtain
the white powder of ZPDD (C44H62N9O12P) with 45% yield.
TLC: chloroform/methanol (90:10, v/v), Rf = 0.7; UV
(MeOH): λmax = 266 nm; IR νmax: 3409, 3194, 3065, 2930,
2818, 2106, 1690, 1551, 1466, 1371, 1319, 1269, 1181, 1036, 918,
and 557 cmꢀ1; δH (400 MHz, CDCl3): 0.6ꢀ2.2 (m, 33H),
2.2ꢀ2.5 (m, 9H), 3.57 (q, 1H, J = 3.4 Hz, OPOꢀCH,
deoxylcholyl), 3.76 (q, 1H, J = 2.5 Hz, CH2CH(OH)C,
deoxylcholyl), 3.90, 4.23 (m, 4H, OPO-H, OCOCH), 3.93 (s,
1H, OH), 4.12 (q, 1H, J = 7.3 Hz, OꢀCHCH2ꢀOPO), 6.20 (t,
1H, J = 6.4 Hz, CH-thymidine), 6.25 (t, 1H, J = 6.5 Hz, CH-
inosine), 7.29 (s, 1H), 7.45 (s, 1H), 7.54 (s, 1H), 9.39 (s, 1H),
9.41 (s, 1H); δC (100 MHz, CDCl3): 12.6ꢀ32.6 (C-deoxycholic
acid), 12.8 (CH3), 38.0, 38.3 (HCCH2CH2CH), 60.8 (CHCH),
66.6 (N3CC), 67.5 (2C, CCO), 83.6, 83.2 (3C, OCC), 104.0 (C,
CCCO), 104.7 (NCCCH3), 110.9 (NCCN), 135.4 (2C, NCN),
Characterization of the Self-Assemblies. The self-assem-
blies were observed using a Philips CM120 80 kV transmission
electron microscope (TEM). Five microliters of the suspensions
were dropped onto the carbon-coated copper nets and main-
tained for one minute. The redundant liquid was removed by
filter paper from the edge of nets. A solution containing 2%
sodium phosphotungstate (pH 6.5) was also dropped onto the
above nets and processed as the above. The resulted negative-
stained samples were air-dried at room temperature and moved
to the TEM for observation as soon as possible.
The dynamic lighting scattering method was used to measure
the particle size and size distribution of the assemblies on
Zetasizer Nano ZS (Malvern, UK). The suspension was diluted
with water until to about 0.1ꢀ0.6 mg/mL ZPDD, and then
measured at 25 °C. The zeta potential of self-assemblies was also
measured with the above instrument and the same process.
HPLC Measurement. HPLC experiments were performed on
a Hitachi L-2130 HPLC system (Japan), consisting of L-2130
pump, L-2400 UV detector, and Hitachi D-2000 chromato-
graphic workstation software. The Diamonsil C18-ODS columns
(5 μm, 250 mm ꢁ 4.6 mm) and the EasyGuard C18-ODS guard
columns (5 μm, 8 mm ꢁ 4 mm) were purchased from Dikma
Co., Ltd. (China). A manual injection valve and a 20 μL loop
(7725i, Rheodyne, USA) were used. The HPLC column tem-
perature was kept at 30 °C with a HT-230A heater and cooler
(Tianjin Hengao Technology Development Co., Ltd.). UV
wavelengths were 266 nm. ZPDD and the possible degradation
products (mainly AZT and ddI) in suspensions or in biological
samples were simultaneously determined on HPLC with the
869
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
gradient mobile phase of the mixture of methanol and water. The
mobile phase condition included: the volume ratios of methanol
in the mixture ranging from 20% to 95% within 0 to 15 min, and
the flow rate was 1.0 mL/min. The retention times (tR) of ddI,
AZT, and ZPDD were about 6.4, 9.5, and 13.4 min, respectively.
Stability Investigation of the Self-Assemblies. ZPDD self-
assemblies belong to the nanoparticulate systems so that the
physical stability should be considered. ZPDD is a prodrug. It is
necessary for ZPDD to degrade to the parent drugs or active
intermediates to perform treatment. Therefore, the chemical sta-
bility should be explored.
Heat could affect the physical stability of ZPDD self-assem-
blies. The self-assembly suspensions were sealed in ampules and
processed with autoclave (121 °C, 15 pounds, 30 min) or boiling
at 100 °C for 30 min. The sizes of ZPDD self-assemblies were
measured after being cooled. Gravity could induce aggregation of
the assemblies. The suspensions were centrifuged from 2000 to
10000 rpm for 5 min and resuspended for size measurement. The
effect of long-term storage was also investigated. The suspen-
sions of ZPDD self-assemblies were diluted with the buffered
solutions of different pH values (pH 7.4, 5.5, 5.0, 4.0, and 3.0)
and maintained for several hours. The sizes and zeta potentials
were measured.
The degradation of ZPDD in buffered solutions, plasma, and
tissue homogenates was investigated. The suspensions were
diluted with the 3-fold volume of buffered solutions, including
0.01 M hydrochloride acid solution (pH 2.0) and 20 mM
phosphate buffers (pH 5.0 and 7.4), and incubated at 37 °C.
At predetermined time intervals, one aliquot of 10 μL was
pipetted out and 10-fold diluted with methanol. The samples of
plasma and tissue homogenates needed centrifugation at 5000 g
for 10 min after methanol dilution. The dilutions or supernatants
were determined with HPLC.
ICp software using the LOGIT method (free software supplied
by Dr. Tang Tao). AZT solution was used as the control.
Pharmacokinetic Study. The in vivo fate of ZPDD were
studied after administration of ZPDD self-assemblies to mice
(body weight, 20ꢀ24 g) through three routes including iv,
intraperitoneal (ip), and per oral (po). The suspensions were
quantitatively determined by HPLC and sterilized with the 0.22 μm
filters. For the iv or ip route, the drug was injected through tail
vein or peritoneum with the dose of 100 mg ZPDD/kg mice.
The blood samples were collected from the opposite tail veins,
put into heparinized centrifuge tubes at the predetermined time
points, and centrifuged to isolate plasma at 3000 rpm for 10 min.
The drug in plasma was determined as the above stability
experiment. In the tissue distribution experiment, the animals
were ethically sacrificed at the predetermined time points, and
the tissues were removed, cleared, weighed, and disrupted to
homogenates followed by the same process as above. For the po
route, the same dose of ZPDD was orally administered after po
administration of 0.2 mL antiacid agent (NaH2PO4-citric acid,
pH 5.5, 50 mM) to each mouse in advance.
The bioavailability for the ip or po route can be calculated
according to the following equation.
AUC0ꢀ120minðpo=ipÞ=Dose
F ¼
ðpo=ipÞ ꢁ 100%
ð1Þ
AUC0ꢀ120minðivÞ=Dose
ðivÞ
where F is the bioavailability, and AUC is the area under the drug
concentrationꢀtime curve. Because the dose is the same for all of
the routes, the above equation can be abbreviated as the
following equation.
AUC
F ¼
0ꢀ120minðpo=ipÞ ꢁ 100%
ð2Þ
AUC0ꢀ120minðivÞ
Cell Experiments. The anti-HIV effect of ZPDD self-assem-
blies was performed with reference to our previous research.31,34
The HIV-1 infected MT4 cells were used as a model. The cultural
medium was the RPMI-1640 (Sigma) solution supplemented
with 10% calf serum and 1 ꢁ 105 units of penicillin and
streptomycin. The suspension of self-assemblies and AZT aqu-
eous solution were sterilized by the film filtration with the
0.22 μm filters. The medicines were 10-fold gradient-diluted
with the cultural medium. The concentration of ZPDD in cell
cultures was from 10 μM to 0.1 nM, in six levels with a 10-fold
decreasing gradient and in triplicates every level. AZT aqueous
solutions were as the controls with the same molar concentra-
tions. After 72 h of incubation at 37 °C, the cytopathic effect
(CPE) assay was performed with a light microscope, and the 50%
effective concentration (EC50) was deduced.
The toxicity of ZPDD self-assemblies was determined using
the MTT test by measuring mitochondrial dehydrogenase
activity on the above MT4 cell model. ZPDD was gradient-
diluted by the cultural medium to 590.5, 118.1, 11.81, and
1.181 μM, respectively. The diluted medicines were added to
the cells followed by incubation for 72 h. Every level was tested in
triplicate. After 72 h, the MTT solution (5 mg/mL, 20 μL,
Amresco) was added to each well. Four hours later, the cultural
media were removed. The formed formazan crystals were
dissolved in 150 μL dimethyl sulfoxide per well. The absorbance
of converted dye was measured at 490 nm using a microplate
reader (Multiskan MK3, Thermo Scientific). The cell viability
was equal to experimental absorbance/control absorbance ꢁ
100%. The half toxic concentration (TC50) was calculated by the
Pharmacokinetic parameters are calculated with the 3p87
pharmacokinetic software supplied by Prof. H. Song of BIRM.
Statistically significant differences for multiple groups were
deduced using a one-way ANOVA with a Dunnett t test. All
testing was done using SPSS 16.0 (SPSS Inc.).
’ RESULTS
Solubility of ZPDD. The solubility of ZPDD was different
from traditional lipophilic compounds. The solubility of ZPDD
in chloroform and THF was high (>125 mg/mL) but low in
cyclohexane (3.6 mg/mL). The nucleobase-containing chemi-
cals tend to form intermolecular hydrogen bonding between
themselves in noncompetitive solvents (the solvents having poor
hydrogen bonding donors and acceptors) such as chloroform
and THF.35 The hydrogen-bonding association of ZPDD in
chloroform and THF may lead to the high solubility. ZPDD was
soluble in octanol (15.8 mg/mL), slightly soluble in methanol
(2.64 mg/mL), and insoluble in water.
Monolayer Behavior of ZPDD at the AirꢀWater Interface.
The πꢀA isotherms of Langmuir monolayers can provide the
conformation information of amphiphilic molecules at the
interface.36 ZPDD showed monolayer behavior different from
normal amphiphiles at the airꢀwater interface. The πꢀA
isotherms of ZPDD monolayers had a much larger onset
molecular area than that of another amphiphilic prodrug, cho-
lesteryl-phosphoryl zidovudine (CPZ), containing the same
polar head as ZPDD. The difference should result from the
two polar heads of the bolaamphiphilic ZPDD molecules that
870
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
Figure 4. Degradation of ZPDD in the different plasma. The data are
the means of two replicates.
Figure 2. πꢀA isotherms of ZPDD monolayers at the different
interfaces and the illustration of monolayer behavior. The interfaces
are airꢀwater (Curve 1), airꢀamine acetate buffered solution (pH 4.0,
20 mM) (Curve 2), and Tris-HCl buffered solution (pH 9.0, 20 mM)
(Curve 3), respectively. The larger filled circles represent the phosphoryl
zidovudine groups, the smaller circles are the ddI groups, and the long
sticks are the deoxycholyl groups. The arrow point indicates the
inflections that all of the curves have.
suggesting that the molecules could basically stand at this time.
After the inflection, the surface pressure increased along with the
further compression, similar to the behavior of CPZ monolayer.
In the end of compression, the heads could be squeezed out over
the interface. Unfortunately, the compression did not proceed
after the molecular area got to about 0.5 nm2 because of the limit
of trough area. Interestingly, the πꢀA isotherms of ZPDD
monolayers were sensitive to the subphase pH values. Compar-
ing the isotherms with three types of subphases including pH 9.0
buffers, water, and pH 6.0 buffers, the first isotherm showed the
earliest lift-off point and the largest onset molecular area than the
others (Figure 2).
Characteristics of the Self-Assemblies. ZPDD self-assem-
blies were the spherical vesicles according to the TEM image
(Figure 3). The self-assembly suspension was homogeneous and
nearly transparent. ZPDD self-assemblies had a small mean size
of 174 nm and a narrow size distribution. The high zeta potential
(ꢀ31.3 mV) of assemblies was related to phosphoryl dissociation
and improved repulsion between particles. A concentrated
suspension (∼10 mg/mL ZPDD) was obtained without any
precipitant.
Interestingly, the sizes and zeta potentials of assemblies
depended on pH. The mean sizes were 143, 144, 270, 288, and
1107 nm at pH 7.4, 5.5, 5.0, 4.0, and 3.0, respectively. The zeta
potentials were ꢀ32.5, ꢀ27.8, ꢀ19.2, ꢀ8.4, and ꢀ2.3 mV in the
above turn, respectively. The monolayer experiment had shown
that acid inhibited phosphoryl association. Therefore, the low pH
environment resulted in the low surface charge of assemblies, and
then the little charged assemblies was prone to aggregation. The
phenomena demonstrated that the phosphoryl zidovudine
groups could dominate in the outer surface of self-assembled
vesicles, while the ddI groups could dominate in the inner surface.
Stability of ZPDD Self-Assemblies. The heat process did not
change the size of ZPDD self-assemblies when autoclave or
boiling was applied. The size of assemblies was only from 174 to
178 nm after heating. However, it was found that the degradation
of ZPDD happened in the heated samples. Therefore, heat
sterilization could not be accepted. The film filtration was used
for sterilization. To accelerate gravity effect on the stability of
ZPDD self-assemblies, the high centrifugation was applied. It was
found that ZPDD self-assemblies kept stable under high cen-
trifuge. Furthermore, the storage at room temperature for more
than one month did not affect the appearance and size of ZPDD
self-assemblies.
Figure 3. TEM image of ZPDD self-assemblies.
could lie on the interface with two heads contacting the subphase
(Figure 2). In contrast, the amphiphilic CPZ molecules could
only stand or tilt at the interface at the initial compression field.
The lying conformation should lead to the large onset molecular
area of ZPDD monolayer. In the whole compressing process, the
phosphoryl zidovudine groups were prone to insert into the
subphase due to its larger volume and higher hydrophilicity than
the other polar head ddI group. When compression continued,
the molecules could undergo a tilt-to-stand process, and the
highly compressed molecules could partially overlap. The follow-
ing slowly lifting curves of ZPDD monolayers could indicate the
molecules inserting and overlapping the adjacent ones. The
isotherms of ZPDD monolayers had the inflections at 20 mN/m,
871
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
Figure 5. Possible metabolic routes of ZPDD.
ZPDD needs to degrade to the active parent drugs or inter-
mediates. It was found that ZPDD degradation highly depended
on pH. pH 2.0, 5.5, and 7.4 represent the environment in the
stomach, intestine, and blood, respectively. ZPDD degraded in
acidic solutions but kept stable in neutral media. All ZPDD was
nearly degraded within 0.5 h at pH 2.0. The degradation half-life
(t1/2) of ZPDD was 61.3 h at pH 5.5 and more than 1000 h at
pH 7.4.
ZPDD degradation was different in the plasma of humans,
dog, monkey, and mouse (Figure 4), wherein the mouse plasma
had the strongest ability with the degradation t1/2 of 9 h. The
species differences of enzyme activity is the major reason.37 Fur-
thermore, the t1/2 in mouse liver homogenates was only 3 h. The
rapid degradation of ZPDD might produce the parent drugs or
active intermediates such as phosphoryl zidovudine. The possible
degradation mechanism of ZPDD is shown in Figure 5.
In Vitro Antiviral Activity of the Self-Assemblies. Anti-HIV
action and the toxicity of ZPDD self-assemblies were explored on
the HIV-1 infected MT4 cells with AZT solution as a control.
ZPDD had the high anti-HIV activity with the half effective
concentration (EC50) of 5 nM, equal to the EC50 of AZT. The
half toxic concentration (TC50) of ZPDD was 42.22 μM, so that
the selection index (SI) of ZPDD self-assemblies was 8444. In
contrast, the TC50 of AZT was 283.17 μM.
lymph node, thymus, liver, and lung (Figure 6b), and a great
number of phagocytes were present in the latter tissues.38 The
phagocyte targeting effect was related to the nanoscale nature
of assemblies. Interestingly, the molar concentration of AZT
was much higher than that of ZPDD in the targeted tissues
(Figure 6c). The rapid degradation of ZPDD in the targeted
tissues should be responsible for this. The in situ high con-
centration of AZT would favor AIDS treatment. However, the
concentration of ddI was comparable with that of ZPDD in the
targeted tissues (Figure 6d). Maybe ddI was produced more
slowly than AZT, and the metabolites of ddI were pro-
duced.
The detailed pharmacokinetic parameters are shown in
Table 1. The bioavailability of ZPDD was 90.5% and 30.8% after the
ip and po administration compared with the iv route. The high ip
bioavailability may be related to the lipophilic property of ZPDD.39
The low oral bioavailability may be related to the instable proper-
ties in stomach, the enzyme damage, and the insufficient absorp-
tion of ZPDD self-assemblies, although one antiacid agent was
administered in advance. Interestingly, in the case of the ip route,
the elimination rate constant (ke) was smaller than the other
routes, and a longer elimination t1/2 was shown. The absorption
of ZPDD could be slow from peritoneum, and there was a
continual absorption in the field of elimination based on the
pharmacokinetic profile (Figure 6a). For the po route, the
apparent distribution volume (Vd) was much more than the
other routes. This might be related to the lymph absorption of
ZPDD from small intestine due to the lipophilic and nanoparti-
culate properties.40,41
In Vivo Fate of ZPDD Self-Assemblies. ZPDD self-assem-
blies showed the different pharmacokinetic profiles and tissue
distribution after administration to mice through the iv, ip, and
po routes (Figure 6). Little ZPDD appeared in the heart and
brain after administration, but a lot occurred in the spleen,
872
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
Figure 6. Profiles of ZPDD concentration in circulation (a) after administration of ZPDD self-assemblies to mice through three routes, and the tissue
distribution of ZPDD (b), AZT (c), and ddI (d) after the iv administration of ZPDD self-assemblies to mice. N = 5.
The amphiphilic ZPDD had the special solubility in different
solvents, suggesting that ZPDD is lipophilic and tends to form
hydrogen bonds. The phenomenon is similar to the other lipid
derivatives of AZT and ddI.33 The highly concentrated solution
of ZPDD in THF is also a good option as the injection solution to
prepare self-assemblies.
Table 1. Pharmacokinetic Parameters of ZPDD after Ad-
ministration of ZPDD Self-Assemblies to Mice
valuesa
parameters
iv
ip
po
ke/minꢀ1
t1/2/min
Vd/mL
0.047 ( 0.023 0.026 ( 0.006 0.053 ( 0.018
Langmuir monolayer experiment is a common method to
characterize amphiphiles. ZPDD showed the unique Langmuir
monolayer behavior. ZPDD molecules could lie on the water
surface with two heads contacting water at the beginning of
compression. The special conformation is the typical behavior of
bolaamphiphiles at the interface.31,42 Significantly, the phospho-
ryl groups of ZPDD tended to dissociate hydrogen ions into the
subphases, and the dissociation should be inhibited by acid and
improved by alkaline. The stronger ZPDD dissociation was, the
higher the head charges, and then the stronger the intermolecular
repulsion. Therefore, ZPDD had a earlier lift-off point and larger
molecular area in the higher pH subphase than the lower pH
subphase. The pH sensitivity also demonstrated that the phos-
phoryl zidovudine groups should dominate at the interface.
The driving force of ZPDD self-assembly should be hydro-
phobic interaction between the deoxycholyl groups, like other
nucleoside lipid derivatives.33 The interaction could lead to the
formation of soft ZPDD monolayers further bending to the
closed vesicles. Which surface of the monolayer one of two heads
of ZPDD dominated is not known now. There is no suitable
method to give sufficient proofs to indicate which head domi-
nates in the inner or outer surface of vesicles. However, according
to the volume of heads, most of the larger phosphoryl zidovudine
17.8 ( 6.5
28.1 ( 7.8
15.7 ( 7.6
7.96 ( 7.40
0.30b ( 0.15
10.0 ( 4.8
9.77 ( 4.50
575 ( 630
1.42 ( 0.28
2.44 ( 1.30
Cl/mL minꢀ1
0.065 ( 0.026 0.063 ( 0.040
3
Cmax/μg mLꢀ1
—
41.4 ( 26.0
8.24 ( 2.80
1690 ( 438
3
tmax/min
—
AUC0ꢀ120 min
/
1868 ( 829
μg mLꢀ1 min
3
3
a The values are the means ( SD, n = 5. ke, the elimination rate constant.
t1/2, the elimination half-life time. Vd, the apparent distribution volume.
Cl, the clearance rate. Cmax, the maximum drug concentration. tmax, the
time reaching Cmax. AUC0ꢀ120 min, the area under the drug concentrationꢀ
time curve between 0 to 120 min. b P < 0.01, po vs iv or ip
’ DISCUSSION
The aim of this study is to obtain a stable self-assembled
system of bolaamphiphilic prodrugs to target to pathological
tissues and release the active drugs with an appropriate rate.
Therefore, the self-assembling property, stability, activity,
body deposition, and metabolism of ZPDD are focused on.
The design of ZPDD is also based on the above aim and the
related requests.
873
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
groups might distribute in the outer surface due to the larger area,
while most of the smaller ddI groups might exist in the inner
surface. The high negative zeta potential of ZPDD self-assem-
blies indicated the release of hydrogen ions of phosphoryl groups
to the surroundings, which was evidence of the phosphoryl
zidovudine groups existing in the outer surface. In addition, the
pH sensitivity of self-assemblies also indicated that phosphoryl
groups could dominate the outer surface of self-assemblies.
The degradation of ZPDD was highly related to its molecular
structure. ZPDD had three ester bonds, resulting in rapid
degradation in the high or low pH media, as in the other re-
ports.31,32,34 Therefore, antiacid agents should be orally adminis-
tered before po administration of the assemblies. Esterases were
widely present in body so that it was not surprising that ZPDD
was rapidly degraded in plasma or tissues. The rapid degradation
was favorable because the inhibition of virus needed enough
drugs. In our previous study, no antiviral activity was observed
because the degradation of the lipid derivative of ddI was very
slow.43
The high in vitro anti-HIV activity of ZPDD demonstrated
that the self-assemblies could enter into cells and rapidly degrade
to active drugs. The active drugs could include AZT, and the
intermediate phosphoryl zidovudine that had been demonstrated
as a potent anti-HIV intermediate.44 However, the release of ddI
could be slow because the bond between carboxyl and ddI might
be destroyed with difficulty.43 A little toxicity of ZPDD could be
related to its amphiphilicity, involving the molecular insertion of
cell membranes. Another amphiphilic nucleoside derivative also
showed the similar effect.32 However, the self-assemblies could
be quickly optimized by plasma proteins in circulation after iv
administration so that there was no opportunity to destroy the
cell membranes such as erythrocytes before reaching targeted
sites. Our previous research showed that another self-assembly
does not lead to hemolysis in the whole blood of rabbits
because of protein absorption in spite of the hemolysis of
erythrocytes.32
ZPDD self-assemblies are mainly distributed in the macro-
phage-sufficient tissues. Macrophages constitute one of the most
important viral reservoirs outside the bloodstream and are able to
transport HIV into the central nervous system. Most of current
anti-HIV drugs distribute in monocytes/macrophages not well
so that eradication of HIV is very hard.16,17 Therefore, ZPDD
self-assemblies could be a good alternative method against latent
HIV. ZPDD self-assemblies might be iv administered to elim-
inate the virus in macrophages after a long-term anti-HIV therapy
with traditional HAART. The good bioavailability of ip admin-
istration indicates that ZPDD self-assemblies might be adminis-
tered through intramuscular injection in the future clinical
application. Generally, nanoparticles could be instable in the
gastrointestinal tract. ZPDD could be absorbed in the molecular
state, and the absorption efficacy was determined by its intrinsic
property.
The asymmetric bolaamphiphilic prodrugs like ZPDD only
contain two drug heads, so that the drug ratio is limited to 1:1.
However, the 1:1 molar ratio may not be a good combination of
drugs for some diseases. Furthermore, the current combination
therapy is constructed based on free drugs, not bolaamphiphilic
prodrugs, though some codrugs have been prepared. Therefore,
the appropriate drugs for the preparation of bolaamphiphilic
prodrugs should be explored in detail. We also look forward to
find a novel prodrug technique combining the suitable drug
ratios rather than 1:1.
In this study, a novel bolaamphiphilic prodrug-ZPDD was
designed based on the SADDS theory and the need of anti-HIV
therapy. Unlike AZT-P-ddI that was rapidly metabolized before
reaching the target cells, the bolaamphiphilic ZPDD can simul-
taneously deliver two different drugs to the targeted cells, also the
HIV reservoir, macrophages. This is a new progress of antiviral
therapy and SADDS research. High stability, rapid release of at
least one of the two parent drugs, and potentially good macro-
phage targeting effect of ZPDD self-assemblies demonstrate that
the assemblies would become one promising anti-HIV nanome-
dicine. Combination therapy is widely applied in various diseases.
However, the components of combination could not be sure to
enter into the targeted cells simultaneously and maintain the
suitable concentration. One bolaamphiphilic prodrug of double
AZT moieties with the spacer of pentadecanedioyl was pre-
viously prepared in our lab. Its self-assembly and the in vivo
behavior were explored.31 The self-assemblies mainly distributed
in the liver, spleen, and testis after iv administration to rabbits
followed by rapid production of AZT. The merit of ZPDD self-
assemblies is to combine two different types of antiviral agents
into one molecule so that they could simultaneously enter the
targeted cells. ZPDD represents a novel combination technique,
involving prodrug, bolaamphiphilic molecules, nanotechnology,
biology, and medicine.
’ ASSOCIATED CONTENT
S
Supporting Information. Table S1. Solubility of ZPDD;
b
Figure S1. πꢀA isotherm of cholesteryl-phosphoryl zidovudine
(CPZ); Figure S2. Structures of CPZ and ZPDD; Figure S3.
Illustration of ZPDD monolayer behavior at the airꢀwater
interface with the surface pressure increasing; Figure S4. Photo-
graph of the suspension of ZPDD self-assemblies; Figure S5. The
relationship between pH and zeta potentials of ZPDD self-
assemblies; Figure S6. The relationship between pH and sizes
of ZPDD self-assemblies; Table S2. Effect of centrifugation on
the size of ZPDD self-assemblies. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Beijing Institute of Radiation Medicine, Department of Phar-
maceutical Sciences, 27 Taiping Road, Beijing 100850, China.
Tel.: þ86-10-8821-5159. Fax: þ86-10-6821-4653. E-mail: jinyg@
bmi.ac.cn (Y.J.).
’ ACKNOWLEDGMENT
This work was supported by the National Natural Science
Foundation of China (No. 30672542) and the National Key
Technologies R&D Program for New Drugs (No. 2009ZX-
09301-002). The authors also acknowledge Prof. Dr. Minghua
Liu of CAS for useful discussion.
’ REFERENCES
(1) Barreiro, P.; García-Benayas, T.; Rendꢀon, A.; Rodríguez-Novoa,
S.; Soriano, V. Combinations of nucleoside/nucleotide analogues for
HIV therapy. AIDS Rev. 2004, 6, 234–243.
ꢀ
(2) Sosnik, A.; Chiappetta, D. A.; Carcaboso, A. M. Drug delivery
systems in HIV pharmacotherapy: What has been done and the
challenges standing ahead. J. Controlled Release 2009, 138, 2–15.
874
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
(3) Falandry, C.; Canney, P. A.; Freyer, G.; Dirix, L. Y. Role of
combination therapy with aromatase and cyclooxygenase-2 inhibitors in
patients with metastatic breast cancer. Ann. Oncol. 2009, 20, 615–620.
(4) Sanz, G.; Fuster, V. Fixed-dose combination therapy and sec-
ondary cardiovascular prevention: rationale, selection of drugs and
target population. Nat. Clin. Pract. Cardiovasc. Med. 2009, 6, 101–110.
(5) Cappellacci, L.; Franchetti, P.; Vita, P.; Petrelli, R.; Grifantini, M.
Synthesis and antitumor activity of a heterodinucleotide of BVDU and
gemcitabine. Nucleosides, Nucleotides Nucleic Acids 2008, 460–468.
(6) Guenther, S.; Nair, V. Binding modes of two novel dinucleotide
inhibitors of HIV-1 integrase. Bioorg. Med. Chem. Lett. 2002, 12, 2233–
2236.
(23) Iwaura, R.; Yoshida, K.; Masuda, M.; Yase, K.; Shimizu, T.
Spontaneous fiber formation and hydrogelation of nucleotide bolaam-
phiphiles. Chem. Mater. 2002, 14, 3047–3053.
(24) Kim, J.-M.; Thompson, D. H. Tetraether bolaform amphiphiles
as models of archaebacterial membrane lipids: synthesis, differential
scanning calorimetry, and monolayer studies. Langmuir 1992, 8,
631–644.
(25) Weissig, V.; Torchilin, V. P. Cationic bolasomes with deloca-
lized charge centers as mitochondria-specific DNA delivery systems.
Adv. Drug Delivery Rev. 2001, 49, 127–149.
(26) Fabio, K.; Gaucheron, J.; Giorgio, C. D.; Vierling, P. Novel
galactosylated polyamine bolaamphiphiles for gene delivery. Bioconju-
gate Chem. 2003, 14, 358–367.
(7) Romeo, S.; Parapini, S.; Dell'Agli, M.; Vaiana, N.; Magrone, P.;
Galli, G.; Sparatore, A.; Taramelli, D.; Bosisio, E. Atovaquone-statine
“double-drugs” with high antiplasmodial activity. ChemMedChem 2008,
3, 418–420.
(27) Muzzalupo, R.; Trombino, S.; Iemma, F.; Puoci, F.; Mesa, C. L.;
Picci, N. Preparation and characterization of bolaform surfactant vesi-
cles. Colloids Surf., B 2005, 46, 78–83.
(8) Song, X. R.; Zheng, Y.; He, G.; Yang, L.; Luo, Y. F.; He, Z. Y.; Li,
S. Z.; Li, J. M.; Yu, S.; Luo, X.; Hou, S. X.; Wei, Y. Q. Development of
PLGA nanoparticles simultaneously loaded with vincristine and verapa-
mil for treatment of hepatocellular carcinoma. J. Pharm. Sci. 2010,
99, 4874–4879.
(9) Tardi, P. G.; Gallagher, R. C.; Johnstone, S.; Harasym, N.; Webb,
M.; Bally, M. B.; Mayer, L. D. Coencapsulation of irinotecan and
floxuridine into low cholesterol-containing liposomes that coordinate
drug release in vivo. Biochim. Biophys. Acta 2007, 1768, 678–687.
(10) Zhan, P.; Liu, X. Designed multiple ligands: an emerging anti-
HIV drug discovery paradigm. Curr. Pharm. Des. 2009, 15, 1893–1917.
(11) Busso, M.; Mian, A. M.; Hahn, E. F.; Resnick, L. Nucleotide
dimers suppress HIV expression in vitro. AIDS Res. Hum. Retroviruses
1988, 4, 449–455.
(28) Bussmann, H.; Wester, C. W.; Thomas, A.; Novitsky, V.;
Okezie, R.; Muzenda, T.; Gaolathe, T.; Ndwapi, N.; Mawoko, N.;
Widenfelt, E.; Moyo, S.; Musonda, R.; Mine, M.; Makhema, J.; Moffat,
H.; Essex, M.; Degruttola, V.; Marlink, R. G. Response to zidovudine/
didanosine-containing combination antiretroviral therapy among HIV-1
subtype C-infected adults in Botswana: two-year outcomes from a
randomized clinical trial. J. Acquir. Immune Defic. Syndr. 2009, 51, 37–46.
(29) Luzuriaga, K.; Bryson, Y.; Krogstad, P.; Robinson, J.; Stechenberg,
B.; Lamson, M.; Cort, S.; Sullivan, J. L. Combination treatment with
zidovudine, didanosine, and nevirapine in infants with human
immunodeficiency virus type 1 infection. N. Engl. J. Med. 1997, 8,
1343–1349.
(30) Jin, Y.; Chen, S.; Xin, R.; Zhou, Y. Monolayers of the lipid
derivatives of isoniazid at the air/water interface and the formation of
self-assembled nanostructures in water. Colloids Surf., B 2008, 64,
229–235.
(31) Jin, Y.; Qi, N.; Tong, L.; Chen, D. Self-assembled drug delivery
systems. Part 5: Self-assemblies of a bolaamphiphilic prodrug containing
dual zidovudine. Int. J. Pharm. 2010, 386, 268–274.
(12) Rossi, L.; Serafini, S.; Franchetti, P.; Cappellacci, L.; Fraternale,
A.; Casabianca, A.; Brandi, G.; Pierigꢀe, F.; Perno, C.-F.; Balestra, E.;
Benatti, U.; Millo, E.; Grifantini, M.; Magnani, M. Targeting nucleotide
dimers containing antiviral nucleosides. Curr. Med. Chem. Anti-Infect.
Agents 2005, 4, 37–54.
(13) Zhou, X. J.; Squires, K.; Pan-Zhou, X. R.; Bernhard, S.;
Agrofoglio, L.; Kirk, M.; Duchin, K. L.; Sommadossi, J. P. Phase I
dose-escalation pharmacokinetics of AZT-P-ddI (IVX-E-59) in patients
with human immunodeficiency virus. J. Clin. Pharmacol. 1997,
37, 201–213.
(14) Aquaro, S.; Caliꢁo, R.; Balzarini, J.; Bellocchi, M. C.; Garaci, E.;
Perno, C. F. Macrophages and HIV infection: therapeutical approaches
toward this strategic virus reservoir. Antiviral Res. 2002, 55, 209–225.
(15) Gavegnano, C.; Schinazi, R. F. Antiretroviral therapy in macro-
phages: implication for HIV eradication. Antiviral Chem. Chemother.
2009, 20, 63–78.
(32) Jin, Y.; Tong, L.; Ai, P.; Li, M.; Hou, X. Self-assembled drug
delivery systems. 1. Properties and in vitro/in vivo behavior of acyclovir
self-assembled nanoparticles (SAN). Int. J. Pharm. 2006, 309, 199–207.
(33) Jin, Y.; Xin, R.; Ai, P.; Chen, D. Self-assembled drug delivery
systems. 2. Cholesteryl derivatives of antiviral nucleoside analogues:
synthesis, properties and the vesicle formation. Int. J. Pharm. 2008,
350, 330–337.
(34) Jin, Y.; Xing, L.; Tian, Y.; Li, M.; Gao, C.; Du, L.; Dong, J.;
Chen, H. Self-assembled drug delivery systems. Part 4. In vitro/in vivo
studies of the self-assemblies of cholesteryl-phosphonyl zidovudine. Int.
J. Pharm. 2009, 381 (1), 40–48.
(16) Gupta, U.; Jain, N. K. Non-polymeric nano-carriers in HIV/AIDS
drug delivery and targeting. Adv. Drug Delivery Rev. 2010, 62, 478–490.
(17) Neves, J. d.; Amiji, M. M.; Bahia, M. F.; Sarmento, B. Nano-
technology-based systems for the treatment and prevention of HIV/
AIDS. Adv. Drug Delivery Rev. 2010, 62, 458–477.
(18) Mamo, T.; Moseman, E. A.; Kolishetti, N.; Salvador-Morales,
C.; Shi, J.; Kuritzkes, D. R.; Langer, R.; von Andrian, U.; Farokhzad,
O. C. Emerging nanotechnology approaches for HIV/AIDS treatment
and prevention. Nanomedicine 2010, 5, 269–285.
(35) Lutz, J.-F.; Pfeifer, S.; Chanana, M.; Thunemann, A. F.; Bienert,
R. H-bonding-directed self-assembly of synthetic copolymers containing
nucleobases: organization and colloidal fusion in a noncompetitive
solvent. Langmuir 2006, 22, 7411–7415.
(36) Jin, Y.; Chen, S.; Xin, R.; Zhou, Y. Monolayers of the lipid
derivatives of isoniazid at the air/water interface and the formation of
self-assembled nanostructures in water. Colloids Surf., B 2008, 64 (2),
229–235.
(37) Minagawa, T.; Kohno, Y.; Suwa, T.; Tsuji, A. Species differ-
ences in hydrolysis of isocarbacyclin methyl ester (TEI-9090) by blood
esterases. Biochem. Pharmacol. 1995, 49, 1361–1365.
(19) Fuhrhop, J.-H.; Wang, T. Bolaamphiphiles. Chem. Rev. 2004,
104, 2901–2937.
(20) Li, S.; Zheng, F.; Zhang, X.; Wang, W. Stability and rupture of
archaebacterial cell membrane: a model study. J. Phys. Chem. B 2009,
113, 1143–1152.
(38) Dou, H.; Destache, C. J.; Morehead, J. R.; Mosley, R. L.; Boska,
M. D.; Kingsley, J.; Gorantla, S. Development of a macrophage-based
nanoparticle platform for antiretroviral drug delivery. Blood 2006,
108, 2827–2835.
(39) Di Stefano, A.; Carafa, M.; Sozio, P.; Pinnen, F.; Braghiroli, D.;
Orlando, G.; Cannazza, G.; Ricciutelli, M.; Marianecci, C.; Santucci, E.
Evaluation of rat striatal L-dopa and DA concentration after intraper-
itoneal administration of L-dopa prodrugs in liposomal formulations.
J. Controlled Release 2004, 99, 293–300.
(21) Ambrosi, M.; Fratini, E.; Alfredsson, V.; Ninham, B. W.; Giorgi,
R.; Nostro, P. L.; Baglioni, P. Nanotubes from a vitamin C-based
bolaamphiphile. J. Am. Chem. Soc. 2006, 128, 7209–7214.
(22) Iwaura, R.; Yoshida, K.; Masuda, M.; Ohnishi-Kameyama, M.;
Yoshida, M.; Shimizu, T. Oligonucleotide-templated self-assembly of
nucleotide bolaamphiphiles: DNA-like nanofibers edged by a double-
helical arrangement of A-T base pairs. Angew. Chem., Int. Ed. 2003,
42, 1009–1012.
(40) Garzon-Aburbeh, A.; Poupaert, J. H.; Claesen, M.; Dumont, P.
A lymphotropic prodrug of L-dopa: synthesis, pharmacological
875
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876

Molecular Pharmaceutics
ARTICLE
properties, and pharmacokinetic behavior of 1,3-dihexadecanoyl-2-[(S)-
2-arnino-3-(3, 4-dihydroxyphenyl)propanoyl]propane-l,2,3-triol. J. Med.
Chem. 1986, 29, 687–691.
(41) Nishioka, Y.; Yoshino, H. Lymphatic targeting with nanoparti-
culate system. Adv. Drug Delivery Rev. 2001, 47, 55–64.
(42) Meister, A.; Weygand, M. J.; Brezesinski, G.; Kerth, A.;
Drescher, S.; Dobner, B.; Blume, A. Evidence for a reverse U-shaped
conformation of single-chain bolaamphiphiles at the air-water interface.
Langmuir 2007, 23, 6063–6069.
(43) Jin, Y.; Ai, P.; Xin, R.; Tian, Y.; Dong, J.; Chen, D.; Wang, W.
Self-assembled drug delivery systems. Part 3. In vitro/in vivo studies of
the self-assembled nanoparticulates of cholesteryl acyl didanosine. Int. J.
Pharm. 2009, 368 (1ꢀ2), 207–214.
(44) Mavromoustakos, T.; Calogeropoulou, T.; Koufaki, M.; Kolocouris,
A.; Daliani, I.; Demetzos, C.; Meng, Z.; Makriyannis, A.; Balzarini, J.; Clercq,
E. D. Ether phospholipid-AZT conjugates possessing anti-HIV and antitumor
cell activity. Synthesis, conformational analysis, and study of their thermal
effects on membrane bilayers. J. Med. Chem. 2001, 44, 1702–1709.
876
dx.doi.org/10.1021/mp100457d |Mol. Pharmaceutics 2011, 8, 867–876