Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
One of the major objectives of advanced drug delivery
today is drug targeting with colloidal drug-delivery systems,
such as nanoparticles and liposomes. However, these
conventional carriers are rapidly cleaned from the systemic
circulation, and end almost exclusively in the mononuclear
phagocyte system (MPS) after iv injection, mainly in the
macrophages in the liver and spleen[1]. Of course, this
extensive uptake is advantageous for treating illness of the
reticuloendothelial system (RES) because it provides high local
concentrations of therapeutic agents. Unfortunately,
delivering drugs to sites other than RES is often desired. To meet
this requirement, stealth or long-circulating nanoparticles
have been investigated.
In the past two decades, there has been an increasing
interest in the development of stealth nanoparticles as drug
carrier systems[2,3]. One of the main methods for preparation
of stealth nanoparticles is to modify their surface with a
hydrophilic and non-ionic polymer, eg, poly
methoxy-ethyleneglycol (PEG)[4] or poloxamer derivatives
[5,6]. Previous studies demonstrate that the hepatic uptake of
nanoparticles can be reduced by coating these particles with
certain modification. Several mechanisms are discussed for
this modification of the body
distribution[6,8]: a dependence on the nanoparticle surface charge, a reduction in the
surface hydrophobicity reducing phagocytic uptake, a steric
hindrance preventing the contact between particles and blood
cells (theory of steric stabilization), and a change in the
adsorption of blood components determined by the surface
properties of the particles. The adsorption patterns differ
depending on the physicochemical properties of the
particles.
According to Peracchia et al, PEGylated
polycyanoacry-late nanoparticles are suitable for giving iv and capable of
circumventing capture by hepatic Kupffer
cells[9]. The increment of splenic nanoparticles led the authors to conclude
a splenic targeting mechanism. However, the investigation
was taken for a duration of only 24 h, and detailed
description of body distribution as a function of time in organs
other than liver and spleen was absent. Other authors have
studied the same vehicle by incorporating a protein drug,
recombinant human tumor necrosis factor
a[10]. In contrast to splenic accumulation, diversion of nanoparticles to
tissues other than the liver and spleen, that is tumors, was
observed.
Distribution of stealth nanoparticles to non-hepatic and
non-splenic organs or tissues make them prospective
vehicles for the delivery of active ingredients to these sites. In
the present study, biodistribution of
[14C]-labeled poly methoxyethyleneglycol
cyanoacrylate-co-n-hexadecyl cy anoacrylate (PEG-PHDCA) nanoparticles was evaluated for
the prolonged duration of 48 h. Distribution to organs other
than the liver and the spleen was studied, which is also of
interest for target stealth diversion. An in
situ phagocytic evading was also investigated in mouse peritoneum using
chemically induced macrophages and flow cytometry.
Materials and methods
Materials Methoxypolyethylene glycol (5000 Da of
molecular weight, MePEG 5000) was obtained from Sigma
Chemical Co. Cyanoacetic acid was obtained from Fluka Chemical
Co and [3-14C]-cyanoacetic acid was obatined from Moravek
Biochemicals. N, N¡¯-dicyclo-hexylcarbodimide and
4-dimethylaminopyridine were obtained from Shanghai
Chemical Reagent Co. Formaline (37%) was obtained from
Shanghai Jianxin Chemical Co. Aqueous dimethylamine (34%) was
provided by Shanghai Linfeng Chemical Co. Other reagents
were of analytical purity.
Synthesis of [14C]-labeled PEGylated and
non-PEGylated PHDCA polymers Synthesis of poly PEG-PHDCA
copolymers has been studied previously
[11]. The PEG-PHDCA 1:3 copolymer was synthesized by condensation of MePEG 5000
cyanoacetate with [14C]-hexadecylcyanoace-tate in ethanol,
in the presence of formaline and dimethylamine.
[3-14C] Cyanoacetic acid (490.4 mg, 552 μCi=20.4 MBq)
was dissolved in ethyl acetate (5 mL), and a solution of
hexadecanol (697.6 mg, 1.75 mmol) and
4-dime-thylamino-pyridine (1.1906 g, 1.75 mmol) in dichloromethane (9 mL) was
added. Then, N, N-dicyclo-hexylcarbodimide (129.38 mg,
0.63 mmol) was added. After stirring at room temperature for
16 h, the resultant mixture was filtered. Filtrate was
concentrated under vacuum, and [14C]hexadecylcyanoacetate was
collected as a waxy solid (1.0386 g, 87.4%, 400 μCi=14.8
MBq). [14C] Hexadecylcyano-acetate (519.3 mg, 1.68 mmol,
200 μCi=7.4 MBq) and MePEG 5000 cyanoacetate (2.9235 g,
0.56 mmol) was added to a 1:1 mixture of ethanol and
dichloro-methane (10 mL) , sequentially, 37% formaline (305 μL,
3.76 mmol) and 34% aqueous dimethylamine (515 μL,
388 mmol) was added. After stirring at room temperature for
16 h, the reaction mixture was concentrated under reduced
pressure. The residue was dissolved by dichloromethane
(100 mL) and washed with 10 mL of water. The organic phase
was dried over magnesium sulfate, and concentrated under
vacuum. Non-PEGylated [14C]-polymer PHDCA was also
synthesized by condensing [14C] hexadecylcyanoacetate with
cyanoacetate.
Preparation of [14C]-labeled and fluorescent
nanopar-ticles [14C]-labeled nanoparticles were prepared by a
nanoprecipitation/solvent diffusion
method[11]. The
[14C]labeled PEG-PHDCA (1:3) copolymer (100 mg) or the
[14C]PHDCA polymer was dissolved in 4 mLof
tetrahydro-furan, and the polymer solution was added, under magnetic
stirring, to 100 mL of demineralized water. Particle formation
occurred immediately. After solvent evaporation using a
rotatary evaporator, an aqueous suspension of nanoparticles
(1 mg/mL) was obtained. The nanoparticles were washed
twice with demineralized water after ultracentrifugation, and
then filtered (Millex AP, Millopore, 1.2 μm). The
radioactivity of the nanoparticle solution was determined just before
injection.
Flurescent nanoparticles were prepared following similar
procedures by incorporating 0.2% rhodamine B.
Characterization of nanoparticles The morphological
examination of nanoparticles was performed using
transmission electron microscopy (TEM) (HITACHI H-600)
following negative staining with sodium phosphotungstate
solution. The particle size and zeta potential of nanoparticles
were determined, respectively, by photon correlation
spectroscopy and laser Doppler anemometry (NICOMP 380/ZLS).
Influence of organic solvent, polymer concentration in
organic phase and preparation procedures on nanoparticle
structure were previously reported[11].
In situ evading of phagocytic uptake by macrophages
The phagocytic evading effect of PEG-PHDCA was observed
on chemically induced mouse peritoneal
macrophages[12]. The mice were injected with 1 mL of 3% sodium thioglycolate
solution ip as a stimulant for macrophages. After 3 d of
induction, fluorescent nanoparticles were injected ip and
incubated together with the macrophages for 2 h. Then, the
mice were killed by cervical dislocation, and each was
peritoneally injected with 5 mL of saline. The peritoneum of the
mouse was massaged for 1 min and the solution inside the
abdominal cavity was drawn out. Macrophages were washed
3 times with 1 mL of phosphate buffer. Cellular uptake of
PEG-PHDCA nanoparticles was examined with a FACScan
flow cytometer, operated under activation wavelength of
585 nm. Each assay counted 20 000 macrophages.
Non-stealth PHDCA nanoparticles were also studied as a contrast,
and saline as a control.
In vivo tissue distribution of
[14C]-labeled PEG-PHDCA
nanoparticles To evaluate the body distribution of
PEG-PHDCA and PHDCA nanoparticles, 2 groups of mice
(6 each) at 1 time interval were treated with
[14C]-PEG-PHDCA and
[14C]-PHDCA nanoparticles, respectively. A total of
200 μL nanoparticle suspension at the dose of 150 mg/kg
were injected into male mice (approximately 20 g in weight)
through the tail vein. The amount of given radioactivity was
1.7×105 Bq/kg. Animals were killed after 0.05, 0.5, 3, 6, 15, 24,
36, and 48 h of injection. Meantime, blood samples were
withdrawn, and hearts, livers, spleens, lungs, kidneys, brains,
and lymph nodes were carefully collected. The organs were
weighed immediately after removal. Alkaline tissue solubilizer
(2 mol/L NaOH) was added to organ samples, and heated at
12°C for 30 min, then cooled to an ambient temperature, and
30% H2O2 was added with occasional swirling until the color
subsided. After addition of 200 μL of acetic acid and 3 mL of
scintillation cocktail (PPO/POPOP), the prepared samples
were stored for approximately 2 d and then counted in the
scintillation counter (LKB 1210). For determining
radioactivity in blood, plasma was separated to exclude interference
of blood cell lysates. Recovery of radioactivity was found
to be between 95% and 103% for all tissues.
For the calculation of the radioactivity of the total dose
(percentage dose) and of 1 milligram of tissue (Bq/mg), the
injected radioactivity was taken as the total dose.
To evaluate the effect of specific accumulation of
nanoparticles in organs, areas under the radioactivity-time
curve (AUC0-48 h) were calculated and compared.
Statistical analysis In vivo radioactivity values were
calculated as a mean of the results of 6 mice determined
separately, and presented as mean±SD. Statistical
comparisons of the means were performed using multivariate
analysis of variance with SARS software.
Results
Synthesis of the [14C] labeled PEGylated and
non-PEGylated polymer According to our previous
findings[11], PEG-polycyanoacrylate copolymers were prepared by
condensation of a mixture of MePEG-cyanoacetate and
n-hexadecyl-cyanoacetate with high yields. After standing at
room temperature for 16 h and drying under vacuum, the
desired copolymers were obtained as pale yellow amorphous
solids. The structure and ratio between hexadecyl chains
and PEG chains were confirmed by integration of the
respective signals in 1H-nuclear magnetic resonance
(1H-NMR) spectroscopy. The structure of the material was also
confirmed by Fourier transform infrared spectrometer (FTIR) and
the molecular weight was determined by gel permeation
chromatography.
The fact that radiolabeling was integrated in the cyano
group, assured a great stability of the radiolabeled copolymer.
PEG-PHDCA and PHDCA were synthesized using similar
techniques, only substituting MePEG-cyanoacetate for
cyanoacetate.
Characterization of [14C]-labeled
nanoparticles Nano-particles prepared with PEG-PHDCA or PHDCA were
spherical in shape and uniform in size of approximately 200 nm
(Figure 1). PEGylated nanoparticles contrasted greatly with
non-PEGylated nanoparticles, with corolla-like stains.
The zeta potential of PEG-PHDCA and PHDCA
nanopar-ticles was -10.77 mV and -20.57 mV, respectively. A marked
decrease in the surface charge for PEG-PHDCA nanoparticles
was observed. Diameter of nanoparticles determined by
photo correlation spectrometry was within the range of
188.7 nm±67.2 nm, in accordance with TEM observation.
In situ evading of phagotytic uptake of PEG-PHDCA
nanoparticles Incorporation of fluorescent rohodamine B in
nanoparticles was verified under a research inverted system
microscope (IX-71, Olympus). Loading of rhodamine B was
approximately 0.2%, determined by spectrofluorometer
(LS-55, PerkinElmer), upon which the assumption that
toxicity of fluorescent marker to macrophages was negligible was
drawn. Flow cytometry diagrams are shown in Figure 2.
The value of relative fluorescence intensity of the saline,
PEG-PHDCA nanoparticles and PHDCA nanoparticles samples were 54.9, 156.5, and 1726.7, respectively, each
representing the stained cells. The higher the value was, the
more that the nanoparticles were captured by macrophages.
Fluorescence intensity for PEG-PHDCA nanoparticles was
approximately 3 times as much as that of saline control,
indicating limited phagocytosis by macrophages. For non-stealth
PHDCA nanoparticles, fluorescence intensity was almost 11
times as much as that of stealth PEG-PHDCA nanoparticles,
indicating profound phagocytosis by macrophages.
Evading of phagocytosis by macrophages was achieved through
PEGylation.
Long circulating in bloodstream PEG-PHDCA
nanopar-ticles had a remarkably higher accumulation in the
bloodstream than PHDCA (P<0.01). PEG-PHDCA nanoparticles
exhibited long-circulating characteristics, with sustained high
levels of blood-associated radioactivity, and 31% of the
radioactivity was still found in the bloodstream 48 h after
injection, assuming that radioactivity was still associated
with intact nanoparticles. In contrast, PHDCA nanoparticles
were cleaned up quickly from the bloodstream, and after
30 min, dropped abruptly to a much lower level, as expected.
At the end of 48 h, only approximately 4% of the
radioactivity was recovered from blood (Figure 3). The PEG-PHDCA
nanoparticles exhibited a `brush¡¯ PEG configuration at the
particle surface, and therefore performed the steric repulsion
efficiently[13].
Organ accumulation The distribution of stealth PEG-PHDCA nanoparticles and non-stealth PHDCA nanoparticals
in mice was poor in lung, kidney, and brain, and a little higher
in heart (Table 1, Figure 4).
There was a relatively higher accumulation in the spleen
and liver for both stealth PEG-PHDCA nanoparticles and
non-stealth PHDCA nanoparticals compared with heart, lung,
kidney, and brain (Table 1, Figure 4).
For stealth PEG-PHDCA nanoparticles, the accumulation
in the spleen was higher than that in the liver
(P<0.05). For non-stealth PHDCA nanoparticles, the accumulation in the
spleen was lower than that in the liver (P<0.05). The
accumulation of stealth PEG-PHDCA nanoparticles in the spleen
was 1.7 times as much as that of non-stealth PHDCA
(P<0.01, Table 1, Figure 4). But the accumulation of stealth
PEG-PHDCA nanoparticles in the liver was 0.8 times as much as
that of non-stealth PHDCA (P<0.05, Table 1, Figure 4).
The amount of radioactivity of stealth PEG-PHDCA
recovered from the spleen was much higher than that from
liver (P<0.01, Figure 5). Such a high spleen uptake was also
observed by other authors with PEGylated
nanoparticles[9].
Accumulation in lymph nodes was unusually higher for
both stealth PEG-PHDCA and non-stealth PHDCA nanoparticles compared with other organs. The
radioactivity was maintained at a high level for at least 36 h, typical of
lymphatic capture (Table 1). Lymphatic accumulation of
stealth PEG-PHDCA was lower than that of non-stealth
PHDCA nanoparticles (P<0.05, Figure 6).
The accumulation of stealth PEG-PHDCA nanoparticles
in the blood was 3.5 times as much as that of
non-stealth PHDCA (P<0.01, Table 1).
Discussion
In the present study, PEG-PHDCA was synthesized as a
diblock copolymer, where PEG chains brought about
hydrophilic modification of the nanoparticle surface, and hexadecyl
cyanoacrylate moieties guaranteed enough hydrophobicity
for the formation of nanoparticles. Structural importance
was testified by 1H-NMR and FTIR spectrum. Molecular
weight determination by gel permeation chromatography
showed a strong correlation between theoretical calculation
of total amount of MePEG and hexadecyl cyanoacrylate
present.
The nanoparticles were easily prepared by the
nanopre-cipitation/solvent diffusion method, with limited size
distribution as determined by NICOMP 380/ZLS and TEM. The
value of zeta potential of PEG-PHDCA nanoparticles was
much lower negative than that of PHDCA nanoparticles. It
should be mentioned that the PEG chains in nanoparticles
would be oriented towards the outer aqueous medium,
protecting the hydrophobic core. Change in surface zeta
potential was also indicative of modifications by PEG chains.
Particles with hydrophobic surfaces are removed readily
from the circulation through a mechanism of
opsonization[6], a process involving complement adsorption and following
recognition and phagocytosis by
macrophages[14]. Stealth nanoparticles with "water"-like hydrophilic chains poking
out interferred with adsorption of complements, and
subsequent capture by macrophages. It was simple and
informative to investigate interaction between vehicles and certain
cell lines before taking out in vivo study. Stealth properties
of nanoparticles have been studied in vitro in cultured
macrophage cell lines with a fluorescent
marker[15-17]. To rule out disturbing uncertainty associated with an
in vitro method, we developed a novel in
situ model to study the effect of stealth evading of phagocytic capture by macrophages.
Preliminary results were encouraging enough for us to take out
in vivo distribution studies.
Findings that the studied vehicles are long-circulating in
the bloodstream are similar to reports by Li
et al[10]. Sustained high levels of radioactivity of approximately one-third
of the original injection for 48 h indicated longer circulation
time of the vehicles, making it applicable carriers for
delivering active ingredients to non-RES targets or for sustained
release. Study on prolonged duration of long-circulating is
needed in the future. Biodegradation of PEG-PHDCA
nanoparticles has not been evaluated in the present study.
However, rapid degradation was not observed in the present
study, because radioactivity was embedded in the cyano
groups, and if biodegradation took place, it was only
associated with water-soluble small degradation remnants, which
was readily eliminated from the body. For at least 48 h,
nanoparticles did not undergo enormous degradation.
Splenotropic accumulation was also observed in the
present study, the accumulation of stealth PEG-PHDCA
nanoparticles in the spleen was 1.7 times as much as that of
non-stealth PHDCA. The propensity of the mouse spleen to
remove such particles from the blood was attributed to its
unique architecture (reticular meshwork of the red pulp and
interendothelial cell slits) and intrasplenic microcirculation
(anatomically open and physiologically closed
circulation)[19]. The mode of particle clearance from the blood by the spleen
appeared to be initially of mechanical
filtration[20]. Interestingly, large filtered sterically stabilized particles were
eventually phagocytosed by the splenic red pulp
macrophages[21]. Phagocytosis of such particles was, presumably,
derived either as a result of intrasplenic loss of the coating,
the steric barrier, or of certain intrasplenic opsonization processes. Predosing dramatically decreased the spleen
uptake of splenotropic spheres but had no effect on the
filtration of small sized particles (<150 nm). It is now evident
that opsonization of a `phagocyte-resistant¡¯ particle can even
occur, depending on the in vivo circumstances. Furthermore,
even stimulated macrophages can effectively recognize and
internalize `phagocyte-resistant¡¯ substrates independent
of opsonization processes. Spleen-targeted stealth
nanopar-ticles have the potential to be used to deliver nuclear
substances and active components for diagnostic purposes and
the therapeutic treatment of spleen-born abnormalities. In
contrast, because the spleen is the biggest resident site in
the human body for immune cells, such as T and B lymphocytes, delivery of vaccines into it might cause
profound immunoreactions.
In summary, PEGylation leads to long-circulation of
nanoparticles in the bloodstream, and splenotropic
accumulation which opens up the potential for further development
of spleen-targeted drug delivery.
References
1 Makino K, Yamamoto N, Higuchi K, Harada N, Ohshima H,
Terada H. Phagocytic uptake of polystyrene microspheres by
alveolar macrophages: effects of the size and surface properties
of the microspheres. Colloid Surf B 2003; 27: 33-9.
2 Moghimi SM, Szebeni J. Stealth liposomes and long circulating
nanoparticles: critical issues in pharmacokinetics, opsonization
and protein-binding properties. Prog Lipid Res 2003; 42: 463-78.
3 Moghimi SM, Hunter AC, Murray JC. Long-circulating and
target-specific nanoparticles: theory to practice. Pharmacol Rev
2001; 53: 283-318.
4 Neradovic D, Soga O, Nostrum CFV, Hennink WE. The effect of
the processing and formulation parameters on the size of
nanoparticles based on block copolymers of poly(ethylene glycol)
and poly(N-isopropylacrylamide) with and without
hydrolytically sensitive groups. Biomaterials 2004; 25: 2409-18.
5 Moghimi SM, Hunter AC. Poloxamers and poloxamines in
nanoparticle engineering and experimental medicine. Trends
Biotechnol 2000; 18: 412-20.
6 Moghimi SM. Mechanisms regulating body distribution of
nanospheres conditioned with pluronic and tetronic block
co-polymers. Adv Drug Del Rev 1995; 16: 183-93.
7 Vandorpe J, Schacht E, Dunn S, Hawley A, Stolnik S, Davis SS,
et al. Long circulating biodegradable poly(phosphazene)
nanoparticles surface modified with
poly(phosphazene)-poly(ethylene oxide) copolymer. Biomaterials 1997; 18: 1147-52.
8 Wilkins DJ, Myers PA. Studies on the relationship between the
electrophoretic properties of colloids and their blood clearance
and organ distribution in the rat. Br J Exp Pathol 1966; 47:
568-76.
9 Peracchia MT, Fattal E, Desmaele D, Besnard M, Noel JP, Gomis
MJ, et al. Stealth PEGylated polycyanoacrylate nanoparticles
for intravenous administration and splenic targeting. J Control
Release 1999; 60: 121-8.
10 Li YP, Pei YY, Zhang XY, Gu ZH, Zhou ZH, Yuan WF,
et al. PEGylated PLGA nanoparticles as protein carriers: synthesis,
preparation and biodistribution in rats. J Control Release 2001;
71: 203-11.
11 Huang M, Wu W. Synthesis of poly [poly (ethylene
glycol)-cyanoacrylate-co-hexadecyl cyanoacrylate] used for the
preparation of nanoparticles. Chin J Pharm 2005; 36: 152-5.
12 Nam YS, Kang HS, Park JY, Park TG, Han SH, Chang IS. New
micelle-like polymer aggregates made from PEI-PLGA diblock
copolymers: micellar characteristics and cellular uptake.
Biomaterials 2003; 24: 2053-9.
13 Gref R, Luck M, Quellec P, Marchand M, Dellacherie E, Harnisch
S, et al. `Stealth' corona-core nanoparticles surface modified by
polyethylene glycol (PEG): influences of the corona (PEG chain
length and surface density) and of the core composition on
phagocytic uptake and plasma protein adsorption. Colloids Surf B
Biointerfaces 2000; 18: 301-13.
14 Gaur U, Sahoo SK, De TK, Ghosh PC, Maitra A, Ghosh PK.
Biodistribution of fluoresceinated dextran using novel
nanoparticles evading reticuloendothelial system. Int J Pharm
2000; 202: 1-10.
15 Nguyen CA, Allémann E, Schwash G, Doelker E, Gurny R. Cell
interaction studies of PLA-MePEG nanoparticles. Int J Pharm
2003; 254: 69-72.
16 Bocca C, Caputo O, Cavalli R, Gabriel L, Miglietta A, Gasco MR.
Phagocytic uptake of fluorescent stealth and non-stealth solid
lipid nanoparticles. Int J Pharm 1998; 175: 185-93
17 Illum L, Hunneyball IM, Davis SS. The effect of hydrophilic
coatings on the uptake of colloidal particles by the liver and by
peritoneal macrophages. Int J Pharm 1986; 29: 53-65.
18 Moghimi SM. Mechanisms of splenic clearance of blood cells
and particles-towards development of splenotropic agents. Adv
Drug Del Rev 1995; 17: 103-15.
19 Moghimi SM, Porter CJH, Muir IS, Illum L, Davis SS.
Non-phagocytic uptake of intravenously injected microspheres in rat
spleen: influence of particle size and hydrophilic coating. Biochem
Biophys Res Commun 1991; 177: 861-6.
20 Moghimi SM, Hedeman H, Muir IS, Illum L, Davis SS. An
investigation of the filtration capacity and the fate of large filtered
sterically-stabilized microspheres in rat spleen. Biochem Biophys
Acta 1993; 1157: 233-40.
|
