NO-Responsive vesicles as a drug delivery system

Article abstract of DOI:10.1039/c7cc00918f

A rationally designed amphiphile containing a hydrophobic Hantzsch ester and a hydrophilic phosphate ester was able to form vesicles in aqueous solution, and resulted in the first example of a NO-responsive drug delivery system.

Full text of DOI:10.1039/c7cc00918f

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NO-Responsive Vesicles as Drug Delivery System
Zhi-Heng Li,a Zheng-Li Tan,a Ai-Xiang Ding,a Bing Gong, a,c Zhong-Lin Lu,a* and Lan He,b*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
rationally designed amphiphile containing a hydrophobic There are three isoforms of NOS: endothelial NOS (eNOS),
Hantzsch ester and a hydrophilic phosphate ester was able to neuronal NOS (nNOS) and inducible NOS (iNOS). eNOS and
form vesicles in aqueous solution, and resulted in the first nNOS are referred to as constitutive NOS (cNOS), and their
example of a NO-responsive drug delivery system.
activity is mainly dependent on the concentration of calcium
ions. The concentration of NO produced by cNOS generally
ranges from fM (10ꢀ15 M) to pM (10ꢀ12 M). Unlike cNOS, variꢀ
ous factors, including diseases, have a big impact on the activiꢀ
ty of iNOS, leading to a concentration of NO higher than nM
(10ꢀ9 M). As a result, NO relates to various diseases, including
cancer, Parkinson’s disease, hypertension, heart disease, etc.19ꢀ
21 Most of all, moderateꢀtoꢀhigh concentrations of NO produced
by iNOS are thought to cause DNA damage and promote neoꢀ
plastic transformations, and become an important factor which
results in tumorigenesis.19 For instance, genetic disruption of
iNOS produced an 80% reduction in urethaneꢀinduced lung
tumor formation.22 Therefore, a NOꢀresponsive DDS would
possess potential value for treating these diseases, especially
cancer.
Various probes for NO detection have also been studied.23ꢀ26
Recently, our group reported several fluorescence probes for
NO detection based on the 4ꢀsubstituted Hantzsch 1,4ꢀ
dihydropyridines (DHP).27ꢀ29 According to the former work,
DHP is not only able to quantitatively react with NO, but also
exhibits a good selectivity for NO against to other interfering
reactive oxidative species (ROSs). Interestingly, when the subꢀ
stituent in the 4ꢀposition is an isopropyl or benzyl group, it will
break away from the pyridine ring when the DHP is treated
with NO.26,30
Drug delivery systems (DDSs) are extremely valuable in the
treatments for various diseases. The main advantages of DDSs
are the protection of reactive drugs under physiological condiꢀ
tions, improvement of drug solubility, fewer side effects, and
better efficacy against resistant diseases. Generally, to design
an effective DDS, two basic aspects should be considered: the
type of the system, and the mechanism of the triggerꢀrelease.
Concerning the structure of DDSs, commonly reported examꢀ
ples include dendrimers, micelles, vesicles, gels, etc.1ꢀ5Among
them, vesicles have attracted increasing attention due to their
unique structures and outstanding abilities, such as encapsulaꢀ
tion and release of both hydrophilic and hydrophobic drugs in
the aqueous compartments and lipid membranes. The triggerꢀ
release strategies have been extensively studied during the past
years. For instance, pH, enzyme activity, redox conditions,
temperature, competitive binding, magnetic field effects, light,
etc.6ꢀ14 However, NOꢀresponsive DDSs have never been reportꢀ
ed.
Nitric oxide (NO) is one of the smallest gaseous molecules
found in nature. It is a highly reactive and multifunctional free
radical, and was primarily accepted as an environmental polluꢀ
tant in early years.15,16 Recently, researchers have discovered
that NO also serves as an indicator for many biological proꢀ
cesses in the human immune system, nervous system, epitheliꢀ
um system and cardiovascular system.17,18 NO is synthesized
from Lꢀarginine, NADPH and oxygen by NO synthase (NOS).
Taking the above information into consideration, we herein
report on a rationally designed amphiphile which combines a
hydrophobic Hantzsch ester and a hydrophilic phosphate ester
via a tyrosol linkage (Scheme 1, VNO), VNO successfully
proved to form NOꢀresponsive vesicles for the first time acꢀ
cording to our knowledge. 4ꢀBenzylmodified 1,4ꢀdihydroꢀ
pyridine is the key switch unit, its oxidation to pyridine by NO
would result in the breakage of the benzyl linkage in the amphiphile
and the rupture of the vesicles formed. The potential applicaꢀ
tions of the VNO vesicles in NOꢀresponsive drug delivery were
also investigated in vivo and in vitro
.
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pyrene as a polarity sensor. To a certain extent, the value sugꢀ
gested the resulting aggregates have a reasonable stability
which may be suitable for other practical applications.
Particle size is an important parameter to evaluate the potenꢀ
tial application in the encapsulation of drugs and cellular uptake.
The size of the aggregates derived from VNO was determined
by the results of DLS (Fig. S3). The measurements showed that
the averaged diameters of the aggregates were around 138 nm
with a narrow size distribution in aqueous solution, which were
in the desired aggregate size range for the encapsulation and
delivery of hydrophilic drugs.31
The appearance of VNO aggregates was further supported by
TEM (Fig. 2). The black outline, which represent the lipid biꢀ
layer, indicating the aggregates are vesicles. The averaged
Scheme 1. Structure of VNO and its decomposition in the
presence of NO.
The synthesis of VNO started from 4ꢀbromobutoxy(tertꢀbutyl)ꢀ
dimethylsilane by the reaction with tyrosol in the presence of K2CO3,
further oxidation with DessꢀMartin periodinane and reaction with
hexadecyl 3ꢀoxobutanoate in the presence of ammonia, resulting in
VNO-H. Removal of the TDMS protecting group in VNO-H, furꢀ
ther phosphoration with 2ꢀchloroꢀ1,3,2ꢀdioxaphospholaneꢀ2ꢀoxide,
and the final formation of the quaternary ammonium group afforded
the target compound VNO. The details of the synthesis and full
characterization of the new compounds can be seen in the supporting
information.
According to the former work,26ꢀ29 Hantzsch ester, a dihyꢀ
dropyridine derivative, is not only able to quantitatively react
with NO, but also exhibits a good selectivity for NO against
other interfering ROS, such as peroxynitrite (ONOOꢀ), superoxꢀ
ꢀ
ide (·O2 ), hydroxyl radicals (·OH), etc. To evaluate the reactivꢀ
ity and selectivity of VNO with NO, 1H NMR spectroscopy
was applied to monitor the reaction process. Due to the purifiꢀ
cation problem with VNO-a, the standard product of VNO, the
precursor VNO-H was used in the assay. The standard products
VNO-b and VNO-c were synthesized by the reaction of VNO-
H
with NO in high yields (Fig. 1). VNO-b was also confirmed
by the reaction of VNO with NO in dichloromethane (See the
supporting information).
Upon the addition of the NO stock solution (1.9 mM in deꢀ
ionized water) to the solution of VNO-H (100 ꢁM, DMSOꢀd6
containing 20% of H2O), the intensity of peaks of H1, H2 and
H3 gradually decreased, while the intensity of peaks of H1a,
H2a gradually increased (Fig. 1A). As the proportion of NO
increased to 3.5 equiv., the peaks of H1, H2 and H3 totally disꢀ
appeared. However, due to the insolubility of VNO-b in the
tested solvents, the peak of H4 was never been found. Furtherꢀ
more, the responses of VNO-H to NO and other ROSs were
also compared (Fig. 1B). Except for NO, no new peaks apꢀ
ꢀ
peared upon the addition of H2O2, ClOꢀ, NO3 or any other
ROSs, which exhibited a superb selectivity of VNO-H for NO.
We can infer that VNO also shares the same character.
The aggregation behavior of VNO in aqueous solution was
confirmed through the following experiments including dynamꢀ
ic light scattering (DLS), transmission electron microscopy
(TEM). Critical micelle formation (CMC) is a good indicator
that describes the aggregation behavior and the stability of the
aggregates formed by VNO, which was determined to be about
18.2 mg/ L (18 ꢁM) by the fluorescence probe technique with
Fig. 1 1H NMR spectra of the VNO-H (100 μM, DMSO-d6 con-
taining 20% of H2O) with (A) different concentrations of NO
and (B) different ROSs (1 equiv.).
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Fig. 2 TEM images of the VNO vesicles.
diameter of spherical vesicles is in accord with the data of DLS
measurements. However, some vesicles seem to be smaller in
size than the average level obtained from DLS measurements.
This is probably due to collapse of vesicles during solvent
evaporation in vacuum.
Fig. 3 (A) Fluorescent spectra of vesicle stock suspension be-
fore and after treated with 0.5 % of Triton X-100; (B) Time-
dependent fluorescent spectra of vesicles in the presence of
NO with different concentrations; (C) Fluorescent spectra of
vesicle stock suspension after treated with NO at different
concentrations for 30 min; (D) VNO vesicle solutions exposed
to laser irradiation in the presence (left) and absence (right) of
5.0 equiv. of NO.
To investigate the encapsulation and controlled release abilꢀ
ity of the vesicles, carboxyfluorescein (CF), a common fluoresꢀ
cent dye for biological assays as a model drug, was encapsulatꢀ
ed in vesicles at a selfꢀquenching concentration (200 mM).31
After treated with Triton Xꢀ100, a detergent which could deꢀ
struct vesicles, the dye was completely released and diluted; the
increase in the fluorescence intensity at 520 nm was measured
as the final release value of 100% (Fig. 3A). The release behavꢀ
ior proves the existence of the aqueous compartments, which is
also a strong evidence indicating the aggregates are vesicles.
Then the vesicle stock suspension was treated with the NO
stock solution (1.9 mM)32 at various concentrations to evaluate
the NOꢀtriggered release of CF from the vesicles (Fig. 3B, C).
Not surprisingly, as the concentration of NO increased from 0
to 50 ꢁM (5 equiv. to the concentration of VNO), the fluoresꢀ
cence intensity at 520 nm was enhanced therewith; implying
the release of CF was increasing, due to the particular reaction
of DHP with NO. That was the main phenomenon to exhibit the
NOꢀtriggered drug release in vitro. CF release tended to be
complete after treatment with 5.0 equiv. of NO for about 20
min at 37 °C, and over 80% fluorescent intensity was recovered.
As a supplement, the reaction mixture of the vesicles with NO
(1 equiv.) was characterized with MS (Fig. S2), and the domiꢀ
nant peak (m/z = 644.5286) given by the [M + H+] ion of the
major product VNO-b revealed that the triggerꢀrelease mechaꢀ
nism of the vesicles was indeed due to the cleavage of the benꢀ
A549, Hek293T and Raw264.7 by the MTT assay was applied at
different concentrations. Both compounds exhibited low cytotoxicity
to all the four kinds of cells (Fig. S3). These results suggest comꢀ
pound VNO and its reaction product VNO-b are harmless to various
cells, and vesicles prepared by VNO can act as drug carriers in bioꢀ
medical applications.
The mouse macrophage cell RAW264.7 was chosen to evaluate
the feasibility of NOꢀtriggered drug release from the vesicles in livꢀ
ing cells. Cells were first treated with different inducers, and then
vesicle stock suspensions (10 ꢁM for the concentration of VNO)
were added and kept for another 20 min. iNOS can be induced by
lipopolysaccharide (LPS) to produce large amount of intracellular
NO33. NGꢀmethylꢀLꢀarginine (LꢀNMA) is often used as an inhibitor
to block the production of intracellular NO34. It can be seen that
strong fluorescence in the green emission channel was observed as
there is intracellular NO in cells (Fig. 4B), or exogenous NO
zyl linkage in VNO
.
The vesicle stock suspension was irradiated by a laser to exꢀ
hibit Tyndall effect (Fig. 3D), indicating the presence of colloiꢀ
dal particles. These particles disappeared after treatment with
5.0 equiv. of NO for 30 min, and the optical path of laser light
was no longer visible. These observations also implied the disꢀ
ruption of VNO vesicles in the presence of NO stimulus.
Fig. 4 Fluorescence (top) and bright field (bottom) microscopic
images of RAW264.7 cells treated with (A) vesicles only; (B)
LPS (1 μg/ mL) for 4 h, then loaded with vesicles; (C) NOS inhib-
itor (L-NMA, 2 mM) 1.5 h and incubated with LPS (1 μg/ mL) 4 h,
then loaded with vesicles; (D) NO (30 μM) for 10 min, then loaded
with vesicles. Scale bars: 50 μm; vesicles stock solution: 10 μM.
To evaluate the cytotoxicity of VNO and its product VNO-b
,
the cell viability test on four kinds of cells, including Hela,
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In conclusion, a rational designed amphiphile VNO was successꢀ
fully synthesized through the combination of
a hydrophobic
Hantzsch ester and a hydrophilic phosphate ester via tyrosol linkage
and showed high reactivity and selectivity to NO. The vesicles
formed by VNO in aqueous solution were investigated by DLS,
TEM and fluorescent measurements. The disassembly of vesicles
and the release of CF were fluorescently evaluated in the presence of
NO. Moreover, the intracellular triggerꢀrelease of the encapsulated
simulate drug was observed directly using fluorescence microscopy.
This is the first example of NOꢀresponsive drug delivery system
with the potential applications in biomedicines. Further work to
improve the sensitivity and put into practical applications is in proꢀ
gress.
Acknowledgements: This article was supported by the financial
assistance from the Nature Science Foundation of China
(21372032 and 91227109), the Fundamental Research Funds
for the Central Universities, Beijing Municipal Commission of
Education, the Program for Changjiang Scholars and Innovative
Research Team in Universities.
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