Factors influencing in vivo disposition of polymeric micelles on multiple administrations

Article abstract of DOI:10.1021/ml500112u

Lactosome is a polymeric micelle composed of amphiphilic polydepsipeptide, poly(sarcosine)₆₄-block-poly(l-lactic acid)₃₀ (AB-type), which accumulates in solid tumors through the enhanced permeability and retention (EPR) effect. However, lactosome on multiple administrations changed its pharmacokinetics from accumulation in tumors to liver due to the production of antilactosome IgM, which was triggered by the first administration. This phenomenon is called the accelerated blood clearance (ABC). In order to reduce the production of antilactosome IgM, a novel nanoparticle composed of (poly(sarcosine)₂₃)₃-block-poly(l-lactic acid) ₃₀ (A₃B-type) was prepared. The A₃B-type lactosome at the second administration showed an in vivo disposition similar to that at the first administration due to suppression of antibody production. This study involving the AB- and A₃B-type lactosomes, with variation of conditions, revealed that the high local density of poly(sarcosine) chains of the A₃B-type lactosome should relate to the prevention of a polymeric micelle from interacting B-cell receptors.

Full text of DOI:10.1021/ml500112u

Letter
pubs.acs.org/acsmedchemlett
Factors Influencing in Vivo Disposition of Polymeric Micelles on
Multiple Administrations
Eri Hara,† Motoki Ueda,‡ Akira Makino,§ Isao Hara,∥ Eiichi Ozeki,∥ and Shunsaku Kimura*,⊥
†Department of Experimental Therapeutics, Institute for Advancement of Clinical and Translational Science, Kyoto University
Hospital, 53 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
‡Clinical Division of Diagnostic Radiology, Kyoto University Hospital, 53 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
§Division of Molecular Imaging, Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji-cho,
Yoshida-gun, Fukui 910-1193, Japan
∥Technology Research Laboratory, Shimadzu Corporation, 3-9-4 Hikari-dai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan
⊥Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto daigaku-katsura, Nishikyo-ku, Kyoto
615-8510, Japan
S
* Supporting Information
ABSTRACT: Lactosome is a polymeric micelle composed of amphiphilic polydepsipep-
tide, poly(sarcosine)64-block-poly(L-lactic acid)30 (AB-type), which accumulates in solid
tumors through the enhanced permeability and retention (EPR) effect. However, lactosome
on multiple administrations changed its pharmacokinetics from accumulation in tumors to
liver due to the production of antilactosome IgM, which was triggered by the first
administration. This phenomenon is called the accelerated blood clearance (ABC). In order
to reduce the production of antilactosome IgM, a novel nanoparticle composed of
(poly(sarcosine)23)3-block-poly(L-lactic acid)30 (A3B-type) was prepared. The A3B-type
lactosome at the second administration showed an in vivo disposition similar to that at the
first administration due to suppression of antibody production. This study involving the AB-
and A3B-type lactosomes, with variation of conditions, revealed that the high local density of
poly(sarcosine) chains of the A3B-type lactosome should relate to the prevention of a
polymeric micelle from interacting B-cell receptors.
KEYWORDS: Molecular imaging, nanoparticle, ABC phenomenon, IgM, pharmacokinetics, block polymer
However, the AB-type lactosome has a drawback of alteration
of in vivo disposition on multiple administrations.13 At the first
administration, the AB-type lactosome showed a long lifetime
in the bloodstream, but at the second administration, the AB-
type lactosome accumulated immediately in liver, resulting in
the rapid clearance from the bloodstream. The similar effect has
been already reported with PEGylated liposome and named as
the accelerated blood clearance (ABC) phenomenon.14−16 The
ABC phenomenon was reported with another polymeric
micelle using PEG as a hydrophilic block.17 It is therefore
suggested that the ABC phenomenon may be commonly
observed with nanoparticles. This phenomenon has been
clarified due to the production of antinanoparticle IgM upon
the first administration of nanoparticle. The hydrophilic
polymer chains surrounding the nanoparticles are found to be
T-independent antigens to induce the immune reaction.16 In
the case of lactosome, poly(sarcosine) covering the surface was
identified as an antigen by the competitive inhibition assay.13
anoparticles of polymeric micelles have high potential for
1,2
Ntumor imaging and antitumor therapy.
These micelles
accumulate in solid tumors through the enhanced permeability
and retention (EPR) effect of high permeability across blood
vessels and immature lymphatic drain systems.3,4 Indeed,
polymeric micelles loaded with antitumor drugs have been
widely studied for clinical applications.5
Lactosome is a polymeric micelle, which is composed of
amphiphilic polydepsipeptide with a hydrophilic poly-
(sarcosine) block and a hydrophobic poly(L-lactic acid) block
(AB-type).6 The amphiphilic polydepsipeptide is biodegradable
and biocompatible.7,8 Further, lactosome is hardly recognized
by the mononuclear phagocyte system (MPS) leading to a long
lifetime in the bloodstream. On the basis of the long in vivo
blood clearance time, the near-infrared fluorescent probe
(NIRF)-labeled lactosome was successful for imaging of various
solid tumors. Especially, the orthotopic liver tumor was clearly
imaged due to the low background of the healthy liver region
showing highly selective accumulation of lactosome in tumors.9
Lactosome has been studied as a nanocarrier of probes for
positron emission tomography (PET) imaging,10 tumor
targeting,11 and intraoperative imaging.12
Received: March 16, 2014
Accepted: June 18, 2014
© XXXX American Chemical Society
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In order to suppress the immune reaction against nano-
particles, there have been several reports that were recently
reviewed by Ishida et al.18 So far, factors of nanoparticle size,
different kinds of hydrophilic chains and core structure of the
core−shell micelle, and the administration regimen have been
discussed.19,20 We are here to discuss another factor, that of the
surface density of the hydrophilic polymer chains on the
polymeric micelle. This study is in the same line of the surface
modification with polymer brushes in the field of polymer
materials.21,22 When the surface modifying polymer chains
become a high-density polymer brush state corresponding to a
surface density of 0.3 chains/nm2 or over, the surface shows a
lubricating surface and also an antifouling property, etc. We
designed a novel amphiphilic polydepsipeptide having three
hydrophilic branch chains connected to one hydrophobic
poly(lactic acid) chain (A3B-type). The A3B-type polymeric
micelle size went down to ca. 22 nm from the AB-type of ca. 36
nm.23 However, the in vivo clearance time of the A3B-type
lactosome became shorter than that of the AB-type lactosome,
which factor was taken into consideration later (Table 1).
Figure S2. Accordingly, the surface density of poly(sarcosine)
chains on the A3B-type lactosome became 4 times higher than
that on the AB-type lactosome (Table 1). However, the half-
lifetime in the bloodstream of the A3B-type lactosome was
found to be 4.3 h, which was much shorter than the AB-type
lactosome of 17.2 h. The data suggest that the in vivo stability of
the A3B-type lactosome was impaired probably by accom-
modating so many hydrophilic chains into the shell layer of the
polymeric micelle. The critical association concentrations
(CAC) of the AB- and A3B-type lactosomes were found to
be as low as 1.3 × 10−8 and 1.9 × 10−9 M, respectively (on the
basis of the general evaluation method using a fluorescent
probe of pyrene (Supplemental Figure S3)). Although the in
vivo stability of the A3B-type lactosome was impaired, the self-
assembling propensity remains high.
Production of antilactosome IgM at 7 days after the AB- or
A3B-type lactosome administration was evaluated by ELISA
(Figure 2A). The production of antilactosome IgM was lower
Table 1. Characterizations of the AB- and A3B-Type
Lactotomes
hydrodynamic
poly(Sar) density
blood half-
c
a
b
type
diameter (nm)
PDI
(chain/nm2)
time (h)
AB
36.0 0.5
22.3 0.3
0.05
0.05
0.07
0.30
17.2
4.3
A3B
a
b
The data obtained by DLS measurement (n = 3). The details are
c
Figure 2. IgM productions upon the administrations of the AB- and
A3B-type lactosomes (A) (n = 3 per group). Antilactosome IgM
productions are normalized by taking the antilactosome IgM
production with the AB-type lactosome as a reference, 1.0.
Pharmacokinetic changes (NIRF images) upon multiple doses of the
AB- (B,D) and A3B-type (C,E) lactosomes. The images D and E were
taken at 7 days after the first administration of the AB- and A3B-type
lactosomes. The time schedule is shown in panel F.
shown in Supplemental Table S1. The decays of the lactosomes in the
bloodstream are shown in Supplemental Figure S1.
Further, we found that the hydrodynamic diameters of these
lactosomes can be precisely adjustable with loading benzylox-
ycarbonyl-poly(L-lactic acid)30 (Z-PLLA30). Therefore, we
could prepare the AB- and A3B-type lactosomes having the
same hydrodynamic diameter, which enabled us to evaluate
separately the two factors of the nanoparticle size and the
surface density of the poly(sarcosine) chains influencing the
ABC phenomenon.
The AB-type lactosome is composed of poly(sarcosine)64-
block-poly(L-lactic acid)30, and the hydrodynamic diameter was
ca. 36 nm (Figure 1A,C). However, the A3B-type lactosome
prepared from (poly(sarcosine)23)3-block-poly(L-lactic acid)30
shows a smaller hydrodynamic diameter of ca. 22 nm (Figure
1B,D). Transmission electron microscopy (TEM) images also
showed the spherical polymeric micelles of the AB- and A3B-
type polymers having diameters of ca. 34 and 21 nm,
respectively.23 The magnified views are shown in Supplemental
with the A3B-type lactosome than the AB-type lactosome,
indicating the lower immunogenicity of the A3B-type lactosome
than the AB-type lactosome.
In vivo dispositions of the lactosomes were analyzed by NIRF
imaging. The AB-type lactosome at the first administration
spread over the whole body and gradually accumulated in the
tumor region due to the EPR effect (Figure 2B). However, the
AB-type lactosome on the second administration at 7 days after
the first administration accumulated immediately in liver as
previously reported (Figure 2D).13 However, the A3B-type
lactosome spread over the whole body to accumulate in the
tumor region at either the first or the second administration
(Figure 2C,E).
Region-of-interest (ROI) analyses also support that the
accumulation amount of the A3B-type lactosome in the tumor
region (the right femoral region, Figure 2E) was 3−5 times
higher than that at the healthy left femoral region at the second
administration (Figure 3A). The accumulation ratios of liver
against background by ROI analyses also show that the liver
accumulation of the A3B-type lactosome was significantly low
just after the second administration followed by a moderate
increase, while the AB-type lactosome accumulated strongly in
liver even right after the second administration (Figure 3B).
The AB- and A3B-type lactosomes showed the different life
times in the bloodstream. In order to look into the effect of this
difference on the ABC phenomenon, we maintained the
Figure 1. Schematic presentation of the AB- and A3B-type polymeric
micelles (A,B). TEM images of the AB- and A3B-type micelles (C,D).
B
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nm (PDI = 0.038−0.070) (Figure 5A). Similarly, the AB-type
lactosome is variable in size from 38 to 52 nm (PDI = 0.052−
Figure 5. Productions of antilactosome IgM with lactosomes varying
the particle sizes. The A3B-type lactosomes increased the size from 22
to 39 nm with the addition of Z-PLLA30 from 0 mol % to 150 mol %
(A). The AB-type lactosomes increased the size from 38 to 52 nm with
the addition of Z-PLLA30 from 0 mol % to 100 mol % (B).
Antilactosome IgM productions are normalized by taking that with the
AB-type lactosome as 1.0. The p value: not significant (NS), *p < 0.05,
**p < 0.01.
0.123) with loading Z-PLLA30 (Figure 5B). Productions of
antilactosome IgM were evaluated by ELISA with using these
lactosomes of different nanosizes (Figure 5A,B). It is clearly
shown that the anti-A3B-type lactosome IgM productions were
lower than the anti-AB-type lactosome IgM productions
(Figure 5A). However, the size dependences of the
antilactosome IgM productions were not obvious within the
each group of the A3B- and AB-type lactosomes (Figure 5A,B).
However, a difference of antilactosome IgM productions
between two groups of the A3B- and AB-type lactosomes was
significant in the whole range of the micelle sizes examined here
(Figure 6). Notably, when the antilactosome IgM amounts
were compared between the A3B- and AB-type lactosomes of
the similar size of ca. 40 nm, the former became lower than the
Figure 3. Time profiles of the ratio of ROI at tumor against
background (tumor/background) (A). Time profiles of the ratio of
ROI at liver against background (liver/background) (B). The p value:
not significant (NS), *p < 0.05, **p < 0.01.
concentration of the A3B-type lactosome in blood by the
repeated administrations at 4 h intervals (0, 4, and 8 h). With
the repeated administrations, the concentration in blood was
kept high (Figure 4A), but the production of antilactosome
IgM was found to remain as low as the single administration of
the A3B-type lactosome (Figure 4B).
The nanoparticle size of the lactosomes can be increased by
loading Z-PLLA30 in the hydrophobic core of the polymeric
micelle.23 The A3B-type lactosome increased the hydrodynamic
diameter gradually with loading Z-PLLA30 up to 39 nm from 22
Figure 4. Time profiles of lactosome concentrations in blood using
ICG-labeled AB- and A3B-type lactosomes (A). Productions of the
antilactosome IgM with the single dose, the triplicate doses of the A3B-
type lactosome, and the single dose of the AB-type lactosome (B).
Antilactosome IgM productions are normalized by taking that with the
AB-type lactosome as 1.0. A3Bx3 represents the triplecate doses of the
A3B-type lactosome at 4 h intervals.
Figure 6. Size dependences of antilactosome IgM productions with
administrations of the AB- and A3B-type lactosomes. The graph is
reconstructed from data of Figure 5. Antilactosome IgM productions
are normalized by taking that with the AB-type lactosome as 1.0 (n = 3
per group).
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latter, suggesting that the size of the polymeric micelle should
not be critical for the antilactosome IgM production.
which is 4 times higher than 0.07 chain/nm2 for the AB-type
lactosome (Table 1). Even though the high surface density
should decrease with incorporation of Z-PLLA30 into the A3B-
type lactosomes, the three poly(sarcosine) chains are bundled
at the C-terminal resulting in locally a high density of
poly(sarcosine) chains (the molecular shape effect). However,
in the case of the AB-type micelle, a long poly(sarcosine) block
of 64-mer should generate a loose space at the peripheral
region of the shell layer of the polymeric micelle. The short
poly(sarcosine) blocks of 23-mer employed for the A3B-type
lactosome might be another reason for raising the local high
density of poly(sarcosine) chains because the shorter chains can
avoid the curvature effect.
Then, we currently have been trying to study the chain
length effect on the ABC phenomenon. Two other A3B-type
lactosomes having longer poly(sarcosine) chains of 33-mer and
50-mer were prepared. These A3B-type lactosomes also formed
polymeric micelles with a diameter less than 30 nm.
Interestingly, these lactosomes did not show the ABC
phenomenon either because the in vivo imaging showed that
the liver capture right after the second administration was not
significant (Supplemental Figure S4). Further, a long poly-
(sarcosine) block of 60-mer did not induce the ABC
phenomenon when the poly(sarcosine) block was expressed
densely at the surface of peptide-nanosheets prepared by self-
assembling of an amphiphilic polypeptide, poly(sarcosine)60-
block-(L-Leu-Aib)6.27 All these results support the interpretation
that the high local density of poly(sarcosine) chains rather than
the poly(sarcosine) chain length should be a major cause for
suppression of the ABC phenomenon. It is considered that the
locally packed poly(sarcosine) chains may exclude other
molecules from penetration into the surface region and prevent
the polymeric micelle from interacting with B-cell receptors.
One drawback of the A3B-type lactosome is, however, the
decrease of the tumor accumulation due to the shorter lifetime
in the bloodstream (Supplemental Figure S5). We are now
finding out the optimal chain length of poly(sarcosine) and
composition of lactosome to stabilize the polymeric micelle
structure to improve the accumulation in tumor with the
attenuated ABC phenomenon.
In regard to the ABC phenomenon of the AB-type
lactosome, the level of antilactosome IgM started to increase
from 3 days after the first administration up to a saturation level
at 5 days and later.13 The AB-type lactosome on the following
administration at later than 5 days from the first administration
accumulated immediately in liver due to the recognition by
antilactosome IgM, which was confirmed by NIRF imaging
using the NIRF-labeled lactosome. In the present study, we
have evaluated the A3B-type lactosome on the ABC
phenomenon. The A3B-type lactosome is composed of the
same hydrophilic and hydrophobic blocks as the AB-type
lactosome, but the hydrophilic block number in one molecule
of the A3B-type lactosome is three times higher than the AB-
type lactosome, which raises the local density of poly-
(sarcosine) chains on the surface. Notably, the A3B-type
lactosome attenuated the ABC phenomenon compared with
the AB-type lactosome as shown by NIRF imaging and the
antilactosome IgM production.
As far as the attenuation of the ABC phenomenon is
concerned, there have been already several reports; (1) the
PEGylated polymeric micelles decreasing the particle size to
less than 30 nm,24 (2) coating of nanoparticles with poly(N-
vinyl-2-pyrrolidone),25 (3) modification of liposome surface
with a polyglycerol-derived lipid,18 (4) a PEG-shell-possessing
polymeric micelle with a hydrophilic inner core (PEG-P(Lys-
DOTA-Gd),26 and (5) alteration of the administration regimen
of using high doses and the prolongation of a time interval
between injections.18 Actually, the A3B-type lactosome with a
22 nm diameter attenuated the ABC phenomenon, which may
be categorized into group (1) described above. However, there
are some controversies here against the interpretation that the
particle size is crucial for the ABC phenomenon. The A3B-type
lactosome with loading Z-PLLA30 to increase the particle size
up to 40 nm still showed the suppression effect on the
antilactosome IgM production as much as the A3B-type
lactosome of 22 nm (Figure 5A). Even though these lactosomes
have the similar particle size of 40 nm, the A3B-type lactosome
significantly suppressed the antilactosome IgM production
compared with the AB-type lactosome (Figure 6). Small
particle size should be favorable for suppression of the
antilactosome IgM production, but the size is not simply all
the trigger for the ABC phenomenon.
Applications of nanoparticles for diagnosis and therapy are
currently appealing. However, the ABC phenomenon with
nanoparticles should be solved out before putting the
nanoparticles on clinical researches. So far, there have been
several ways reported to attenuate the ABC phenomenon, but
the detailed mechanism remains to be solved. Here, the local
density of poly(sarcosine) chains of lactosomes was shown to
influence the ABC phenomenon. We think that the higher local
density of the surrounding hydrophilic polymer chains should
be related with prevention of the interaction between micelles
and B cell receptors, but further studies are needed to make it
clear.
We can now raise two factors for the ABC phenomenon.
One is the blood clearance time and the other is the local
density defined by the micelle size, the chain length of the
hydrophilic blocks, and the molecular shape. The half-lifetime
in the bloodstream of the A3B-type lactosome was 4.3 h, which
is considerably shorter than the AB-type lactosome of 17.2 h
(Table 1). The shorter lifetime of nanoparticles should result in
less chance to trigger the immune system. Indeed, Ishihara et al.
reported that the ABC phenomenon was not induced upon the
repeated injections of the PVP-coated nanoparticles, which
have a short lifetime in the bloodstream.25 Here, the triplicate
doses of the A3B-type lactosome at 4 h intervals with keeping
the high concentration in blood did not increase the IgM
production from that with the single dose. The shorter half-
time is therefore not the major factor for the reduction of the
ABC phenomenon (Figure 4).
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and supporting results. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
The surface density of the hydrophilic blocks differs
significantly between the two kinds of lactosomes. The surface
density reaches to 0.30 chain/nm2 for the A3B-type lactosome,
*(S.K.) Phone: +81-75-383-2400. Fax: +81-75-383-2401. E-
mail: shun@scl.kyoto-u.ac.jp.
D
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ACS Medicinal Chemistry Letters
Funding
Letter
percutaneous ethanol injection therapy. J. Nanopart. Res. 2013, 15,
2131.
This study is a part of joint research, which is focusing on the
development of the basis of technology for establishing COE
for nanomedicine, carried out through Kyoto City Collabo-
ration of Regional Entities for Advancing Technology
Excellence (CREATE), Japan Science and Technology Agency,
a part of the project entitled “Development of PET Probe for
Imaging Solid Tumors Using Nanocarriers” under the contract
of Innovation plaza Kyoto, Japan Science and Technology
Agency, and a part of the Innovative Techno-Hub for
Integrated Medical Bio-Imaging of the Project for Developing
Innovation Systems, from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT).
(12) Funayama, T.; Sakane, M.; Abe, T.; Hara, I.; Ozeki, E.; Ochiai,
N. Intraoperative near-infrared fluorescence imaging with novel
indocyanine green-loaded nanocarrier for spinal metastasis: A
preliminary animal study. Open Biomed. Eng. J. 2012, 6, 80−84.
(13) Hara, E.; Makino, A.; Kurihara, K.; Yamamoto, F.; Ozeki, E.;
Kimura, S. Pharmacokinetic change of nanoparticulate formulation
“lactosome” on multiple administrations. Int. Immunopharmacol. 2012,
14, 261−266.
(14) Dams, E. T. M.; Laverman, P.; Oyen, W. J. G.; Storm, G.;
Scherphof, G. L.; Van der Meer, J. W. M.; Corstens, F. H. M.;
Boerman, O. C. Accelerated blood clearance and altered biodis-
tribution of repeated injections of sterically stabilized liposomes. J.
Pharmacol. Exp. Ther. 2000, 292, 1071−1079.
(15) Ishida, T.; Maeda, R.; Ichihara, M.; Irimura, K.; Kiwada, H.
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Notes
The authors declare no competing financial interest.
(16) Koide, H.; Asai, T.; Hatanaka, K.; Akai, S.; Ishii, T.; Kenjo, E.;
Ishida, T.; Kiwada, H.; Tsukada, H.; Oku, N. T cell-independent b cell
response is responsible for ABC phenomenon induced by repeated
injection of PEGylated liposomes. Int. J. Pharm. 2010, 392, 218−223.
(17) Koide, H.; Asai, T.; Hatanaka, K.; Urakami, T.; Ishii, T.; Kenjo,
E.; Nishihara, M.; Yokoyama, M.; Ishida, T.; Kiwada, H.; Oku, N.
Particle size-dependent triggering of accelerated blood clearance
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ACKNOWLEDGMENTS
■
We thank Dr. M. Sugai (Department of Experimental
Therapeutics, Institute for Advancement of Clinical and
Translational Science, Kyoto University Hospital) for dis-
cussion.
(18) Abu Lila, A. S.; Kiwada, H.; Ishida, T. The accelerated blood
clearance (ABC) phenomenon: clinical challenge and approaches to
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