A virus-mimetic nanogel vehicle

Article abstract of DOI:10.1002/anie.200704121

Delivering the goods: A pH-sensitive nanogel consists of a hydrophobic copolymer core and two layers of hydrophilic shell (see picture). The core is loaded with a model anticancer drug, doxorubicin (DOX). The nanogel infects tumor cells in a receptor-dependent manner, kills the cells, and migrates to neighboring cells like a virus. BSA = bovine serum albumin, F = folate, PEG = polyethylene glycol. (Figure Presented).

Full text of DOI:10.1002/anie.200704121

Communications
DOI: 10.1002/anie.200704121
Drug Delivery
AVirus-Mimetic Nanogel Vehicle**
Eun Seong Lee, Dongin Kim, Yu Seok Youn, Kyung Taek Oh, and You Han Bae*
Viruses infect specific cells within host organisms, replicate,
destroy the cells, and spread from cell to cell in infectious
cycles, thus causing disease.[1] These viral properties have
inspired synthetic designs of various delivery vehicles,[2–6]
particularly for toxic anticancer agents that exhibit numerous
side effects. Drug-delivery vehicles often mimic viral aspects,
such as size and surface properties, to improve cell entry and
residence within the body before being cleared.[2–6] Recent
efforts in biomimetic drug-carrier design have aimed to
endow advanced functionality.[3] Herein, we describe a
synthetic nanosized polymer vehicle that mimics viral proper-
ties more significantly than any known delivery systems so far
reported. This virus-mimetic nanogel (VM-nanogel) should
prove valuable for treating several major disease classes, such
as tumors, with greater efficacy.
The VM-nanogel we have developed consists of a hydro-
phobic polymer core and two layers of hydrophilic shell
Figure 1. Preparation and structure of the VM-nanogel. See text for
(Figure 1). A particle core made of poly(l-histidine-co-
details; PBS=phosphate-buffered saline.
phenylalanine) (poly(His32-co-Phe6),[4] where the numerals
indicate the numbers of His and Phe units in the block) is
loaded with a model anticancer drug, doxorubicin (DOX).
Polyethylene glycol (PEG; number-average molecular weight
2000 Da) forms the inner shell. One PEG end is linked to the
core polymer and another to bovine serum albumin (BSA),
which forms a capsid-like outer shell.[5] The core and inner
shell were constructed by an oil-in-water emulsion method.[2,6]
A single BSA molecule could be linked to multiple PEG ends,
as depicted in Figure 1. The BSA and PEG components on
the surface may help to avoid potential immune responses.[7]
Extensive study of the biocompatibility and toxicity of
PEG-b-poly(His-co-Phe) is under way in our laboratory, and
no apparent cytotoxicity or systemic toxicity have been found
in mouse models for PEG-b-polyHis. The BSA shell is
subsequently conjugated with folate ligands for specific
recognition of the folate receptor (FR), which is overex-
pressed on many tumors.[8] Folate conjugation will contribute
to the enhancement of the antitumor activity of VM-nanogels
by providing an active entry mechanism.[9]
The sensitivity of VM-nanogel to the pH of the solution
was assessed by the optical transmittance of an aqueous VM-
nanogel solution (Figure 2a). The percentage transmittance
(%T) dropped sharply over the narrow pH range of 6.6–6.4
upon reduction of the pH from 7.4 to 5.5. When the pH was
increased again, %T recovered with a small hysteresis. The
observed change in %T is attributed to the dynamic dimen-
sional changes of the VM-nanogel with pH. The pKb of l-
histidine in the core poly(His32-co-Phe6) is near 6.4,[4] as
measured by an acid–base titration method. At high pH, the
VM-nanogel core is rigid. However, the core swells sponta-
neously when the polymer His residues are protonated at
lower pH values[4] (Figure 1).
[*] D. Kim, Dr. Y. S. Youn, Dr. K. T. Oh, Prof. Dr. Y. H. Bae
Department of Pharmaceutics and Pharmaceutical Chemistry
University of Utah
When the pH was cycled between 7.4 and 6.8 (the typical
cytosolic pH range[10]), the VM-nanogels reversibly swelled
and shrank. However, the magnitude of the reversible
changes of the VM-nanogel between pH 7.4 and 6.4 (early
endosomal pH[11]) was enormous (Figure 2b): 55 nm in
diameter at pH 7.4 and 355 nm at pH 6.4. Apparently, the
structure of the VM-nanogel is maintained during this size
change by virtue of the capsid-like[5] BSA shell that holds
multiple interior core block copolymers by PEG linkages,
which prevents them from dissociation even after ionization
of the core His component at lower pH values. Flexible
tethering of the BSA corona to the pH-sensitive core by the
PEG units accommodates a large amount of expansion
without particle disassembly. Hydrophobic interaction
421 Wakara Way, Suite 315, Salt Lake City, UT 84108 (USA)
Fax: (+1)801-585-3614
E-mail: you.bae@utah.edu
Homepage: http://www.pharmacy.utah.edu/pharmaceutics/
groups/bae/
Dr. E. S. Lee
Division of Biotechnology
The Catholic University of Korea
43-1 Yeokgok 2-dong, Bucheon-si 420-743 Republic of Korea
[**] We thank Dr. Grainger (Department of Pharmaceutics and Phar-
maceutical Chemistry, University of Utah) for his kind suggestions
and proofreading. This work was funded by NIH CA122356 and NIH
CA101850.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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as a result of surface-localized fluorescein isocyanate (FITC,
green fluorescence) on the BSA shell. The dimensions of the
VM-nanogels in both states coincide with the particle sizes
measured by light scattering (Figure 2b).
This substantial volumetric expansion of VM-nanogels
and the known proton buffering effect of polyHis or poly-
(His32-co-Phe6)[4] are considered able to physically disrupt
endosomal membranes (see the Supporting Information).
This allows the VM-nanogels to transfer from the endosomes
to the cytosol, where the VM-nanogels rapidly shrink back to
their original size. Furthermore, the pH-induced reversible
swelling/deswelling of the core is closely linked to the release
rate of incorporated DOX[9] (see the Supporting
Information). The VM-nanogels released
a significant
amount of DOX at endosomal pH (e.g., pH 6.4), while
reducing the DOX release rate at cytosolic or extracellular
pH (e.g., pH 6.8–7.4).
Figure 3. Migration of DOX-loaded VM-nanogels (equivalent DOX
10 mgmLꢀ1) from infected A2780/AD cells to untreated cells. A2780/
AD cells grown on a glass slide fragment (dimensions 1”1 cm; A)
were pretreated with DOX-loaded VM-nanogels for 4 h. After washing
the treated cell plate with PBSof pH 7.4, it was co-cultured with fresh
cells (B-0) seeded on another glass slide fragment for 20 h in fresh
culture medium. The two plates were 1 mm apart in a culture dish,
continuously sharing the culture medium. B-0 became B-1 after 20 h of
co-culture with A. The procedure was repeated to yield B-2 and B-3 in
subsequent cycles (that is, B-2 refers to the cells on a plate co-cultured
with B-1 for 20 h). Three control experiments were conducted: DOX-
loaded control NPs (equivalent DOX 10 mgmLꢀ1), free DOX
Figure 2. a) Relative transmittance of the VM-nanogel solution mea-
sured at selected pH values with respect to transmittance at pH 7.4.
*
The solution pH is gradually changed from 7.4 to 5.5 ( ) and from
*
pH 5.5 to 7.4 ( ). b) Size changes of VM-nanogel (determined by
dynamic light scattering) with pH value. The solution pH is succes-
sively adjusted in the order A (pH 7.4)!B (pH 6.8)!A!B (upper
row) and A (pH 7.4)!C (pH 6.4)!A!C (lower row), with equilibra-
tion for 1 h at each pH. The particle size distribution at each pH was
obtained from the particle scattering intensity. c) Confocal slice images
of the VM-nanogels in a living cell. Ovarian A2780 cells were incubated
with FITC-labeled VM-nanogels (10 mgmLꢀ1, pH 7.4) and confocal slice
images were obtained at 60, 80, and 90 min.
(10 mgmLꢀ1), and FITC-labeled blank VM-nanogels (no DOX, VM-
nanogel 50 mgmLꢀ1). All confocal images were sliced.
To verify VM-nanogel migration to neighboring cells
(Figure 3), an A2780/AD[12] cell cluster seeded on a glass plate
was pretreated with DOX-loaded VM-nanogels for primary
infection for 4 h (see the Supporting Information). A
neighboring glass plate with adherent untreated cells was
used as a potential site for secondary VM-nanogel infection.
The primary infected cell group (Figure 3, VM-nanogel, A)
can be seen by confocal microscopy, where cells appear red
because of the intrinsic DOX fluorescence within the VM-
nanogel. DOX spreads evenly within the cells, even in the
nucleus, while the untreated cell group appears dark
among Phe components in the core[4] is also thought to help
prevent dissociation.
VM-nanogels are designed to interact with the target cells
through folate–FR coupling[8,9] and subsequent localization
within early endosomes.[8] Figure 2c presents the coexistence
of unswollen and swollen VM-nanogels in live human ovarian
carcinoma A2780 cells, traced by a series of confocal slice
images taken for a single cell. The red and white arrows
indicate unswollen and swollen VM-nanogels, respectively.
Swollen VM-nanogels appear after 60 min as a hollow sphere,
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Communications
(Figure 3, VM-nanogel, B-0). After co-incubation of the
primary VM-nanogel “infected” cells and untreated cells for
20 h, the red DOX fluorescence appears in the untreated cell
group, even in the cell nucleus (Figure 3, VM-nanogel, B-1).
This may be because DOX action on the cells induces
apoptosis and the VM-nanogels are released from the dead
cells for subsequent infection in neighboring cells (see the
Supporting Information).
The infection procedure was repeated and another
infection cycle occurred (Figure 3, VM-nanogel, B-2, B-3).
In addition, the weakened “infection signal” (that is, fluores-
cence) over repeated cycles is most probably caused by the
loss of VM-nanogel by medium replacement, DOX depletion
from the VM-nanogel, and less release of DOX during each
cell cycle, which eventually falls below the drug concentration
required to induce cell death.
Three control experiments support the unique repeated
infection capabilities of the VM-nanogels (Figure 3). Control
nanoparticles (NPs; ꢁ 70 nm in diameter),[13] which comprise
a pH-insensitive central core (poly(l-lactic acid) and DOX)
and a hydrophilic shell (PEG–folate), have no capability for
endosomal escape, thus resulting in cytosolically trapped
DOX[13] and limited cell death. The control NPs were only
able to produce secondary infection very weakly in the first
cycle after 20 h.[13] Experiments with free DOX produced
limited fractions of DOX diffusing into cells in the primary
infection cycle because of the drug-resistant, active P-
Figure 4. Antitumor activity of DOX-loaded VM-nanogels (white),
DOX-loaded control NPs (gray), and free DOX (black). The DOX
equivalent content of each sample is 5 mgmLꢀ1. a) A2780 or resistant
A2780/AD tumor cells were treated with each sample for 48 h in
culture media of pH 7.4 or 6.8. b) A549 cells were treated with each
sample for 48 h in culture medium (pH 6.8). Each data point in (a)
and (b) represents an average standard deviation (n=9); ** indicates
p<0.05 for free DOX.
glycoprotein DOX efflux pumps in A2780/AD cells.[12]
A
third control utilized VM-nanogels without DOX (Figure 3,
blank VM-nanogels). After entry into the primary infected
cells, the blank VM-nanogel with FITC (green color)
distributed evenly within the cells.[13] No noticeable secondary
infection was detected because of a lack of cell death[13] and
lytic release of VM-nanogels.
Recently, Nagasaki et al. reported pH-sensitive PEGy-
lated nanogels with a sharp volume transition at pH values
around 7.0, as a result of the protonation of cross-linked
poly[2-(N,N-(diethylamino)ethyl methacrylate)] in the
core.[14,15] These nanogels facilitated triggered DOX release
at endo/lysosomal pH for effective chemotherapy.[15] How-
ever, these nanogels did not show cell specificity and recycled
infection.
Importantly, when a drug carrier completely releases its
payload upon cell internalization to achieve high drug
concentrations within the cells,[9,13] this often results in
excessive drug over the threshold of its cytotoxicity. The
excess drug could be ineffective against neighboring cells if
these cells are drug resistant.[12] However, the VM-nanogel
system may be able to maximize the effect of the drug by
pulsatile drug release modulated by pH and repeated entry
into cancerous cells. Once the VM-nanogel escapes the
endosomes, the DOX release rate is minimized. This indicates
that the amount of DOX released while the VM-nanogels are
entrapped in the endosomes for approximately 1–1.5 h
(Figure 2c) before endosomal disruption should be high
enough to provide a drug concentration sufficient to kill
cancer cells. The amount of DOX released for 1 h at pH 6.4 in
each cycle is expressed in the Supporting Information.
Figure 4a presents an impressive contrast for cell killing in
the various cases. The viability of both drug-sensitive A2780
cells and drug-resistant analogues was below 20% when
treated at a DOX equivalent concentration of 5 mgmLꢀ1
carried by the VM-nanogels. However, DOX carried by the
control NPs was not effective in cell killing. Free DOX at the
same concentration showed a viability of 30% for sensitive
cells but 80% for resistant cells. Similar results with free DOX
were obtained when the same experiments were conducted at
pH 6.8, which is close to the tumor extracellular pH.[4,9,13] The
results suggest that the VM-nanogels do not distinguish
between sensitive and resistant cells in tumor-cell killing
capability, by preserving a sufficient cytosolic dose of DOX.[9]
More significantly, the VM-nanogels do not infect (see the
Supporting Information) or kill A549 human nonsmall lung
carcinoma cells[16] which do not express the FR (Figure 4b).
In conclusion, the VM-nanogels infected cells in a
receptor-dependent manner, effectively killed the tumor
cells, and migrated to neighboring cells as a virus does.
When coupled with other specific targeting moieties or gene
vectors, this VM-nanogel nanotechnology could have great
potential for treating solid tumors, inflamed tissues, and other
diseases in an effective manner. To confirm and realize this
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Keywords: antitumor agents · drug delivery · gels ·
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