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Tumor targeting using magnetic nanoparticle Hsp70 conjugate
in a model of C6 glioma
Maxim A. Shevtsov, Ludmila Y. Yakovleva, Boris P. Nikolaev, Yaroslav Y. Marchenko, Anatolii V. Dobrodumov,
Kirill V. Onokhin, Yana S. Onokhina, Sergey A. Selkov, Anastasiia L. Mikhrina, Irina V. Guzhova, Marina G. Martynova,
Olga A. Bystrova, Alexander M. Ischenko, and Boris A. Margulis
Institute of Cytology of the Russian Academy of Sciences, St. Petersburg, Russia (M.A.S., K.V.O., I.V.G., M.G.M., O.A.B., B.A.M.); Research
Institute of Highly Pure Biopreparations, St. Petersburg, Russia (L.Y.Y., B.P.N., Y.Y.M., A.M.I.); Institute of Macromolecular Compounds of the
Russian Academy of Sciences, St. Petersburg, Russia (A.V.D.); D. O. Ott Institute of Obstetrics and Gynecology, Northwestern Division of the
Russian Academy of Medical Sciences, St. Petersburg, Russia (Y.S.O., S.A.S.); I. M. Sechenov Institute of Evolutionary Physiology and
Biochemistry of the Russian Academy of Sciences, St. Petersburg, Russia (A.L.M.)
Corresponding author: Maxim A. Shevtsov, MD, PhD, Laboratory of Cell Protection Mechanisms, Institute of Cytology of Russian Academy of Sciences,
Tikhoretsky Ave. 4, 194064, St. Petersburg, Russia (shevtsov-max@mail.ru).
Background. Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function
both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein
Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental
C6 glioma.
Methods. The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was
assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously admi-
nistered was analyzed in the experimental model of C6 glioma in the MRI scanner.
Results. Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance
signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by con-
focal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-
SPIONs in comparison to non-modified SPIONs. Histologicalanalysis of the brain sections confirmed the retention of the Hsp70-SPIONs in
the glioma tumor but not in the adjacent normal brain tissues.
Conclusion. The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the
tumors and enhanced the contrast of their MR images.
Keywords: glioma, Hsp70, magnetic nanoparticles, SPION, targeted delivery.
Superparamagnetic iron oxide nanoparticles (SPIONs) have
attracted attention in the past decades due to their possible appli-
cations in brain tumor therapy, imaging, or drug delivery.
1,2
Since
nanoparticles cross the blood–brain barrier (BBB), they could be
applied in the development of novel therapeutic modalities. One
of the most promising approaches is based on the application of
localized hyperthermia, when magnetic nanoparticles (MNPs)
absorb energy from alternating magnetic fields and transform
this energy into heat.
3,4
The efficacy of this method was demon-
strated in numerous preclinical and clinical studies.
5 –9
Recently,
Maier-Hauff et al,
10
in a single-arm phase II study of intratumoral
thermotherapy combined with external beam radiotherapy in
patients with recurrent glioblastoma, showed significant increase
in overall survival (up to 23.2 mo) in comparison with historical
control of 14.6 months reported by Stupp et al.
11
Currently, the delivery of MNPs is based on a direct intratumoral
injection, which limits the clinical application of this method.
10
Further improvement of tumor targeting by the SPIONs requires a
special surface coating, which can provide the specificity of
SPION delivery to the tumor cells in vivo.
12
Thus, conjugation to
the purified antibody that selectively binds to the epidermal
growth factor receptor deletion mutant (EGFRvIII) present on glio-
blastoma cells significantly elevated the efficacy of the accumula-
tion of MNPsin the tumor site.
13
In the elegant study by Basel et al,
7
the authors used cytotherapy-directed hyperthermia when MNPs
were loaded into monocyte/macrophage-like cells, which have
Received 20 May 2013; accepted 29 July 2013
# The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com.
Neuro-Oncology
Neuro-Oncology 16(1), 38–49, 2014
doi:10.1093/neuonc/not141
Advance Access date 4 December 2013
38
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been shown to specifically migrate into the tumor. Further studies
demonstrated that the therapeutic efficacy of local hyperthermia
could be increased by combination with other methods, including
use of various anticancer drugs.
6,14
Thus, Fe
3
O
4
MNPs combined
with chemotherapy and hyperthermia could overcome the multi-
drug resistance in an in vivo model of leukemia.
6
Earlier we developed MNPs with a size ,100 nm that were con-
sidered to have low toxicity.
15
For elevating the efficacy of tumor
targeting, we conjugated MNPs with epidermal growth factor
(EGF), whichincreased the selectivity of the MNP-EGFaccumulation
in cancer cells in a melanoma mouse model.
15
The developed for-
mulation of the MNP-EGF conjugates was characterized by the
coefficients of magnetic relaxation efficacy (R
1
, R
2
, R
∗
2
), which
were close to the characteristics for the negative contrast agents
for MRI. This resulted in the generation of a strong hypointense
T
2
-weighted contrast on MRI.
15
In the present study, for brain
tumor targeting, we decided to use recombinant heat shock
protein (Hsp)70 covalently conjugated to the surface of the
SPIONs. Several reasons were taken into consideration for the ap-
plication of Hsp70. First was the possibility of selective brain
tumor targeting due to the overexpression by the glioma Hsp70
cell receptors. Previously, various receptors to Hsp70 have been
identified, including CD91, lectin-type oxidized low-densitylipopro-
tein receptor 1 (LOX-1), Toll-like receptors (TLR-2/TLR-4), and
CD40.
16 – 18
In several studies it was observed that on the cell
surface of glioma cells, CD40 was highly expressed in comparison
with the surrounding normal tissues.
19 – 21
The prevailing expres-
sion of CD40 in the glioma can cause the accumulation of the
SPIONs conjugated with Hsp70 in the tumor site. The second
reasonfor the application of Hsp70 was its immunomodulatory ac-
tivity, as Hsp70 is involved in the generation of adaptive and innate
antitumor immune responses.
22
This immunomodulatory func-
tion could be used for augmentation of the immune response
that is generated by the localized hyperthermia of the tumor. Pre-
viously, Ito et al
23
demonstrated in a model of melanoma the
therapeutic potential of local intratumoral delivery of Hsp70 com-
bined with magnetic nanoliposome-based hyperthermia.
In the current study, in the model of the intracranial C6 glioma,
we demonstrate the possibility of glioma targeting by
Hsp70-SPION conjugates that could be intravenously injected.
Materials and Methods
Preparation of Recombinant Hsp70
Recombinant human Hsp70 was prepared from Escherichia coli trans-
formed with a pMSHsp70 plasmid. Hsp70 was purified by anion exchange
chromatography using diethylaminoethanol-sepharose (GE Healthcare)
followed by ATP-affinity chromatography on ATP-agarose (Sigma). Endo-
toxin was depleted by polymixin B-sepharose endotoxin removing gel
(Sigma). Quantitationof endotoxinwas performed using the Limulusamoe-
bocyte lysate assay (QCL-1000, Cambrex Bio Science). The resulting endo-
toxin content was below 0.1 endotoxin unit per milligram. For the
analysis of Hsp70 uptake, in both in vitro and in vivo experiments the chap-
erone was conjugated with Alexa Fluor 555 (Invitrogen) according to the
manufacturer’s protocol.
Synthesis of Iron Oxide MNPs
SPIONs were prepared by coprecipitation in alkaline media at 808C.
24
Iron
salts FeSO
4
and FeCl
3
at Fe2+/Fe3+ ratio 1:2 were dissolved in distilled
water with addition of some salt. The precipitation was performed by the
dropwise addition of NH
4
OH to the iron salt solution under nitrogen
gaseous atmosphere with vigorous stirring. The magnetite formation
follows the reaction
2Fe(+3)+Fe(+2)+8OH
−
Fe
3
O
4
+ 4H
2
O.
Magnetic crystal formation was completed by stirring the stock solution
after 5 min. To stabilize suspension for storage, the particles were coated
by low molecular dextran (molecular weight, 10 kD; Sigma). For best
surface modification, ultrasound sonication at 22 kHz was applied. The
stock solution of MNPs was washed and size-fractionated to 4 fractions
by centrifugation and ultrafiltration using fiber membranes (0.2 mm;
Millipore). The finest fraction of nanoparticles was stored in water at 48C
for analysis and further conjugation.
Magnetic Hsp70 Conjugate Synthesis
The synthesis of magnetic conjugates of Hsp70 withSPIONs was carried out
in accordance with the scheme shown in Figure 1A. Dextran-coated MNPs
were crosslinked with epichlorohydrin and aminated. MNPs were sus-
pended in phosphate buffered saline (PBS) solution with 60 mg of Hsp70,
and conjugation was carried out for 1 h at 208C in a shaker. Activated by
water-soluble carbodiimide, dextran was coupled to carboxyl groups of
Hsp70 protein, producing magnetic conjugate. The size of SPIONs or
Hsp70-SPIONs was assessed using atomic force microscopy (AFM).
Briefly, nanoparticles in 10 mLof the sample were placed on the microscope
slide and were allowed to air dry. Samples were then processed with AFM on
an NTEGRA Prima scanning probe microscope (NT-MDT). Closed-loop feed-
back semicontact modewas used at a rateof 0.6 Hz. Scanning started from
the 50-mm area, going down to 5 mm. The images obtained were analyzed
with NT-MDT image analysis software v2.2. At least 25 individual particles
were measured from 3 different positions, and the average diameter was
reported. The surface morphology of the sample was observed in a
large-scale scanning area of 700 nm× 700 nm and 1000×1000 pixels.
The Hsp70 content in conjugate samples was measured by ELISA. The
biological activity of Hsp70 in the conjugate was assessed by the chaperone
ELISA and found not to be changed by the conjugation with SPIONs.
25
The
magneticHsp70 conjugatesampleswere analyzed forcontentof total Fe by
spectrophotometry of thiocyanate Fe(+3) complex obtained after HNO
3
dissolution.
Cells
The C6 rat glioma cell line was obtained from the Russian cell culture collec-
tion of the Institute of Cytology in St Petersburg. C6 cells were grown in Dul-
becco’s modified Eagle’smedium/F12supplementedwith 10% fetal bovine
serum, 2 mM
L-glutamine, and antibiotics (100 U/mL penicillin G and
0.1 mg/mL streptomycin). Cells were grown in a CO
2
incubator in an atmos-
pherewith 6% CO
2
and 90% humidity. For in vivo experiments, C6 cells were
also infected with recombinant lentivirus vectors (including LVTHM, which
expresses green fluorescent protein [GFP]), and C6-GFP+ cells were ana-
lyzed with the aid of flow cytometry (Cytomics FC500, Beckman Coulter).
Before experiments, cells were harvested in log phase of growth, and
their viability was determined by 0.4% trypan blue exclusion.
Model of Intracranial C6 Glioma in Rats
Before being mounted in a stereotactic frame (David Kopf Instruments),
male Wistar rats (250–300 g) were anesthetized with 10 mg tiletamine
hydrochloride and zolazepam (Zoletyl-100, Virbac) and 0.2 mL 2% xylazi-
num hydrochloride (Rometar, Bioveta) intraperitoneally. A burr hole was
made 1 mm posterior to the bregma and 3 mm to the right of the
midline. Rat C6 glioma (10
6
) cells in 10 mL PBS were injected 3 mm below
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
Neuro-Oncology 39
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the cortical surface using a Hamilton microsyringe. Stereotactic coordi-
nates corresponded to the nucleus caudatus dexter (per the stereotactic
atlas of Pellegrino). All animal experiments were approved by the local
ethical committee.
Magnetic Relaxometry and MRI Study
Nuclear magnetic resonance (NMR) spectra and magnetic relaxation times
T
1
, T
2
, T
2
*
were measured with the help of an NMR spectrometer (CXP-300,
Bruker) in a magnetic field of 7.1 T. The NMR spectra were recorded by
Fourier transform of one-pulse induction decay. T
2
*
was calculated from
line width at half height. To estimate magnetic relaxation times, inversion
recovery and Carr-Purcell-Meiboom-Gill pulse sequences were applied.
Proton relaxation times were studied dependent on the concentration of
magnetic Hsp70 conjugates in buffer solution in 5-mm tubes. The coeffi-
cients of relaxation efficiency R
1
, R
2
, R
2
*
(relaxivity) were determined from
the slopes of concentration plots of inverse times of magnetic relaxation.
The rat and gel phantom images were acquired with the help of a Bruker
Avance II NMR spectrometerequipped with a microtomographic accessory
at a magnetic field of 11 T. The measurements were carried out using
gradient echo fast imaging and multiscan-multiecho imaging. The T
1
, T
2
-
weighted images were obtained under the scanning regimes of rapid
acquisition with relaxation enhancement (RARE)–T
1
and Turbo-RARE-T
2
.
A series of transverse and coronal sections of tumor were acquired after
intravenous injection of Hsp70-SPION conjugates at different times and
under different regimes of acquisition.
Assessment of Distribution of Hsp70 in C6 Glioma
On the 20th day following intracranial implantation of the C6 glioma cells or
C6 cells labeled with GFP, the Hsp70–Alexa Fluor 555 conjugate was i.v.
injected into the tail vein (5 mg/kg in 200 mL of saline solution, for 3
animals in each group). Twenty-four hours after the protein injection,
animals were sacrificed, brains were extracted and fixed in 4% paraformal-
dehyde (PFA), and serial frozen sections were obtained. Nuclei were stained
with 4
′
,6
′
-diamidino-2-phenylindole(DAPI). Additionally, the brain sections
were stained with anti-CD40 fluorescein isothiocyanate (FITC)–conjugated
monoclonal antibodies (1/100; BD Biosciences). Immunofluorescence
images were captured with a Leica TCS SP5 confocal system.
Assessment of Hsp70-SPION Conjugate Uptake by C6 Cells
C6 glioma cells were allowed to settle on glass slides coated with
poly-
L-lysine. Cells were incubated with PBS, SPIONs (0.05 mg/mL), or
Fig. 1. The preparation and microscopic image of Hsp70-SPION conjugate. (A) Scheme of synthesis of Hsp70-SPION conjugates. (B) Transmission electron
microscopy image of Hsp70-SPION conjugates. Scale bar, 1 mm. (C) Atomic force microscopy of the SPIONs and Hsp70-SPION conjugates.
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
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Hsp70-SPION conjugates (0.05 mg/mL) in cell culture medium for 1, 6, 12,
and 24 h at 378C, 6% CO
2
. Following incubation with nanoparticles, cells
were excessively washed and fixed with 4% PFA. Nuclei were stained with
DAPI. Immunofluorescence images were captured with a Leica TCS SP5
confocal system at 488 nm (Ar/Kr) and 405 nm on a Leica DM IRBE micro-
scope. We also analyzed the role of CD40 receptor in the uptake of
Hsp70-SPION complexes by the C6 cells. Sells were incubated with blocking
anti-CD40 monoclonal antibodies (dilution 1:100; BD Biosciences) for 2 h.
Following incubation with antibodies, C6 cells were incubated with
Hsp70-SPION conjugates for 1, 6, 12, and 24 h at 378C, 6% CO
2
, then
washed and fixed with 4% PFA. The internalization of the conjugates was
assessed with confocal microscopy.
For evaluation of the precise intracellular localization of the nanoparti-
cles, electron microscopy was performed. Experiments were conducted
as follows: C6 cells were exposed to SPIONs or Hsp70-SPIONs at a concen-
tration of 0.05 mg/mL in cell culture medium for 24 h at 378C, 6% CO
2
. Fol-
lowing incubation with nanoparticles, probes were centrifuged and the
pellet was postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer
for 30 min, dehydrated in a graded series of ethanol dilutions, and embed-
ded in Durcupan (Fluka). After polymerization, serial ultrathin sections from
the pellet were cut on an Ultracut ultramicrotome (Reichert-Jung) and col-
lected on Nickel 200 mesh square grids. Sections were counterstained with
lead citrate and examined with a JEM1200EX electron microscope (Jeol).
Additionally we performed immunogold labeling of the C6 cells by mono-
clonal anti-CD40 antibodies (BD Biosciences). For immunocytochemical
examination, ultrathin sections mounted on nickel grids were first treated
with hydrogen peroxide for 20 min to loosen the resin. Three washes in
PBS for 2 min each were followedby incubation with anti-CD40 monoclonal
antibodies diluted 1:100 in 0.05 M Tris-HCl buffer, pH 7.4, containing 1%
bovine serum albumin and 0.1% cold water fish gelatin overnight, at 48C
in a moist chamber. Gold-conjugated (10 nm) goat anti-mouse immuno-
globulin G (Sigma) diluted 1:10 was used as secondary antibody, and sec-
tions were incubated for 1 h at room temperature. For the control, the
primary antibody was omitted or replaced by irrelevant antibodies.
Immunofluorescence Analysis of Nanoparticle
Uptake In vivo
Twenty-four hours following i.v. injection of Hsp70-SPIONs or SPIONs,
animals were sacrificed with CO
2
and transcardially perfused with PBS for
15 min (20 mL/min), followed by 4% PFA (in 0.1 M phosphate buffer, pH
7.4) fixation for 15 min (20 mL/min). Tumor tissue samples were harvested
into Tissue-Tek compound for sectioning and stored at 2808C. Serial 7-mm
frozen sec tions were stained with DAPI. Sections were analyzed on the
confocal microscope in reflected-light laser scanning with a Leica TCS SP5
confocal system.
Statistical Analysis
Continuous variables were compared using a paired Student’s test.
Statistical significance was determined at the P , .05 level. A 2-tailed
Mann–Whitney log-rank test was used to compare study and control
groups.
Results
Magnetic Relaxometry and Size Analysis of
Hsp70-SPION Conjugate
The prepared suspensions of SPIONs and their conjugates with dif-
ferent iron contents were investigated by magnetic relaxometry
and AFM. The size of the nanoparticles was determined by AFM
on solid surfaces after drying. The microscopy data indicated the
existence of single spherical particles with diameters ranging
from 15 to 25 nm (SPIONs) and mean particle size of 20 nm,
respectively (Fig. 1C). For Hsp70-SPION conjugates, the size
ranged from 20 to 40 nm, and the mean size was 35 nm (Fig. 1B
and C). Also, some clusters were observed with a mean size of
about 100 nm. Fresh samples of suspensions were transparent
brown solutions that were stable at 48C storage. Magnetic relaxo-
metry analysis showed the paramagnetic property of the suspen-
sion. The addition of SPIONs or conjugates into distilled water
caused strong decreases of relaxation time T
1
, T
2
of water
protons from 3 s to 100–300 ms. The decrease of relaxation time
correlated with the growth of SPION concentrations. The recipro-
cals of magnetic relaxation times R
1
, R
2
were estimated from a
linear fit of logarithmic echo amplitude versus spin echo time
(Fig. 2). The commercial MR contrast agent fluid MAG-DX (Chemi-
cell) was used for comparative control. The R
1
, R
2
, R
∗
2
relaxivity
values of synthesized SPION-Hsp70 conjugates were 0.37, 113,
and 230 mM
21
s
21
, respectively. The range of relaxivity was no
worse than for MAG-DXand corresponded to properties of negative
contrast agents. These results suggest the strong relaxation effi-
ciency of magnetic Hsp70 conjugates in aqueous dispersions.
The action of the strong magnetic field of 7.1 T on aggregation
of Hsp70-SPIONs was investigated by relaxationstudy in dynamics.
Proton magnetic relaxation rates r1, r2, r2* were recorded after
inserting the samples with conjugated and nonconjugated
SPIONs into a uniform magnetic field of the NMR spectrometer
for long periods of time. NMR acquisition was performed step by
step by applying impulse sequences at different time points.
A 2-phase behavior of the magnetic relaxation rates of water
protons r1, r2, r2* time in suspensions of MNPs was observed. The
firstphase of the fall of the rateofmagnetic relaxation occurredim-
mediately after inserting the sample into the magnetic field. This
stage of transition to equilibrium lasted for about 3 h. The second
phasewascharacterized byalmostconstant values of the magnet-
ic relaxation rates for periods from 3 h to 20 h. Further observation
did not reveal any changes in relaxation behavior. The largest drop
in relaxation rate (almost 2-fold) was observed when measuring
the value of r2* (Fig. 2).The similar plots for r1, r2 are not shown
for their resemblance in general form. After a long prehistory
(20 h) in the 7.1-T magnetic field, the rate of magnetic relaxation
of SPIONs and Hsp70-SPION conjugates remained constant in
time. The stability of the magnetic Hsp70 conjugate was shown
to be sufficient for further MRI studies of tumor-bearing rats.
The magnitude of magnetic relaxation rate of nanoparticle
conjugates matches well with their appreciable contrast mani-
festation in a phantom study made by MRI. Representative
images of phantom agar-agar samples loaded by magnetic
Hsp70 conjugates at various Fe concentrations are shown in
Fig. 2B. The signal intensity of images decreased with the
growth of iron oxide in gel. The highest contrast was achieved
in T
2
-weighted and gradient spin echo regimes in accordance
with the strong influence of T
2
–outer sphere mechanism relax-
ation. These relaxation measurements provide evidence that con-
jugation of nanoparticles with Hsp70 through a carbodiimide
linker does not interfere with the nanocrystal structure of the
magnetite core. The dextran surface prevents the magnetic
core of the nanoparticles from the chemical transformation into
a diamagnetic state. The ratio of the relaxation efficiency
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Neuro-Oncology 41
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coefficient R
2
/R
1
ranges from several tens to hundreds, which is
the basis for regarding the synthesized conjugates of Hsp70 as
powerful negative contrast agents.
Accumulation of Hsp70 in the CD40 + C6 Glioma Cells
Assessment of the intratumoral accumulation of Hsp70 was per-
formed in the case of the i.v. injection of the fluorochrome-labeled
protein. Twenty-four hours after the infusion of Hsp70–Alexa Fluor
555 (5 mg/kg in saline solution), the protein could be observed in
the tumor site (Fig. 3A). Hsp70 was distributed throughout the
gliomaaccumulatingwithin thecytoplasmandthenucleusenviron-
ment. Insome cells,Hsp70appearedto formaggregatesinthecyto-
plasm. For the confirmation of the incorporation of the Hsp70 inside
C6 glioma cells, the latter were labeled with GFP and inoculated into
the rat brain. The subsequent i.v. injection of Hsp70–Alexa Fluor 555
confirmed the accumulation of the protein inside the C6 cells
(Fig. 3B). The Hsp70 accumulated within the cytoplasm of the
GFP+ cells. Following the assessment of intratumoral distribution
of Hsp70, we analyzed the possible accumulation of the labeled
protein in the CD40+ glioma cells,as CD40 was describedasa recep-
tor for Hsp70.
16,26
Immunofluorescence images of the C6 tumor
show the presence of the CD40+ cells throughout the tumor
(Fig. 3C). When we stained the frozen tumor sections of the
animals being i.v. injected with Hsp70–Alexa Fluor 555 with
anti-CD40 antibodies, we were able to confirm the accumulation
of Hsp70 inside the CD40+ cells (Fig. 3D).
Confirmation of Hsp70–SPION Conjugates Binding
to C6 Glioma Cells
Rat C6 glioma cells were incubated with control (PBS), SPIONs
(0.05 mg/mL), and Hsp70-SPIONs (0.05 mg/mL) for 1, 6, 12, and
24 h. At first, the cell viability with 0.4% trypan blue was assessed
for all of the incubation times with nanoparticles. There was no sig-
nificant toxicity found within C6 cells after treatment with SPIONs
or Hsp70-SPIONs. Following viability assessment, the inclusion of
nanoparticles inside C6 cells was analyzed with the help of con-
focal microscopy (Fig. 4A–C). SPIONs or Hsp70-SPION conjugates
gradually deposited onto C6 glioma cells and passed through the
plasma membrane. Nanoparticles appeared to be present within
the cytoplasm and to surround the nucleus. The particles did not
seem to penetrate into the nucleus, instead forming coarse aggre-
gates within the cytoplasm; more aggregates were detected in
the cytoplasm at 24 h. Intriguingly, the quantity of nanoparticles
within the cells was higher in the case of Hsp70-SPIONs than in
nonconjugated SPIONs. The cytoplasm of C6 cells incubated
with Hsp70 conjugates was completely filled with magnetic
Fig. 2. The magnetic contrast study of Hsp70-SPION conjugate in vitro. (A) Relationship of magnetic relaxation rate r
∗
2
,r
2
and r
1
of water protons in
Hsp70-SPION conjugate dispersion vs Fe concentration at 208C. (B) MR transverse images of agar phantom loaded with Hsp70-SPION conjugates in
concentration (anticlockwise) 0.1, 0.2, 0.4, 0.8% obtained under gradient echo fast imaging (GEFI), rapid acquisition with relaxation enhancement
(RARE)–T
1
and Turbo-RARE-T
2
scanning regimes.
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nanoparticles (Fig. 4C). Electron microscopy images show the
nanoparticles containing vesicles within the cytoplasm of the C6
cells incubated with SPIONs or Hsp70-SPIONs (Fig. 4D and F). In a
few cases we observed the adsorbed particles on the cell surface,
which is a prerequisite for cell internalization. In the case of the
Hsp70-SPIONs, we observed the massive presence within the
cytoplasm of aggregates that were not surrounded by the mem-
brane (Fig. 4E).Visual examination of the different cell samples
demonstrated endosomes containing particles not included in
the endosomes in the case of the Hsp70 conjugates. Immunogold
labeling with anti-CD40 monoclonal antibodies demonstrated the
presence of the Hsp70-SPIONs inside the membrane structures
containing CD40 receptor (Fig. 4H). The role of CD40 receptor in
the internalization of the Hsp70-SPION conjugates was confirmed
with blocking anti-CD40 receptors. When the anti-CD40 antibodies
were applied, the significantly reduced uptake of the Hsp70-SPIONs
was observed (Fig. 4G).
In control cells containing no nanoparticles, there were few
endosomes, indicating that the C6 cells were not phagocytically
activated; no electron-positive granules were found inside the
endosomes in the control cells (data not shown).
Distribution of the Hsp70-SPION Conjugates
in a C6 Glioma Model
Animals on the 20th day after tumor inoculation were divided into 4
groups (3 animals each) according to the subsequent i.v. treatment
with nanoparticles: (i) a control group with PBS solution treatment,
(ii) SPIONs (0.15 mg/mL, 200 mL), (iii) Hsp70-SPIONs (0.15 mg/mL,
200 mL), and (iv) Hsp70-SPIONs (0.15 mg/mL, 200 mL) with the pre-
liminary injection of Hsp70 (5.0 mg/kg, 24 h before the infusion of
nanoparticles). MR scanning was performed 24 h after infusion to
determine localization and relaxivity times of the nanoparticles
administered (Fig. 5). Intravenous infusions of the Hsp70-SPIONs
or nonconjugated SPIONs were safe, as all animals survived the pro-
cedures and showed no signs of the toxicity. In the control group of
animals without particle treatment, thetumorcouldbe observed on
the MR scans with different patterns of growth, including cerebro-
spinal fluid dissemination into the ventricles. The glioma was char-
acterized by the hypointense signal in the T
1
-weighted imaging
and hyperintense in the T
2
-weightedregimen(Fig. 5A). After MNP in-
fusion, the most significant change in contrast was observed in the
tumor on T
2
-weighted images. Following i.v. infusion of the SPIONs,
Fig. 3. Accumulation of Hsp70 in C6 glioma cells in vivo. (A) Fluorochrome-labeled (Alexa Fluor 555) Hsp70 was infused intravenously (5 mg/kg) in the
tumor-bearing animal on the 20th day following tumor implantation. After 18 h the animal was dissected and assessed for the presence of the
labeled chaperone inside tumor cells. Sections were stained with 4
′
,6
′
-diamidino-2-phenylindole (DAPI; blue). The labeled Hsp70 (red) accumulated
inside the cytoplasm of tumor cells. Scale bar, 75 mm. (B) For confirmation of the localization of exogenous Hsp70 inside glioma cells, the latter were
infected with GFP (green). In the same set of experiments, we assessed the distribution of i.v. injected Hsp70–Alexa Fluor 555 protein inside the
glioma. The protein accumulated inside the GFP+ C6 cells as red aggregates within the cytoplasm. Scale bar, 25 mm. (C) Immunofluoresence image of
C6 glioma tissue section with CD40+ cells detected by FITC-conjugated monoclonal antibodies (green). Nucleus was stained with DAPI (blue). Scale
bar, 20 mm. (D) Immunofluorescence image of C6 glioma with CD40+ cells detected by FITC-conjugated monoclonal antibodies (green; shown by
white solid arrows). The fluorochrome labeled Hsp70–Alexa Fluor 555 is localized within the cell cytoplasm (red). Nuclei were stained with DAPI (blue).
Scale bar, 25 mm.
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
Neuro-Oncology 43
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after 24 h there was a decrease of the T
2
relaxivity time in compari-
son with the control animals (P , .05; Fig. 5B). As can be seen from
experiment, a 10% reduction of the transverse relaxation time T
2
was accompanied by the growth of image contrast due to the
darkening of sites of MNP accumulation. Application of the
Hsp70-SPIONs further reduced the relaxationtime in the T
2
-weighted
regimen, and numerous zones of conjugate aggregates could be
observed as areas of signal decrease (especially in the fast lo w
angle shot regimen; P , .001; Fig. 5C). On the subsequent post-
mortem fluorescence images of the brain sections in reflecting-laser
light scanning, we confirmed the accumulation of the Hsp70-SPIONs
in the glioma tumor. Conjuga tes appeared to localize within
the cytoplasm surrounding the nucleus (Fig. 5,rightcolumn).
Hsp70-SPIONs not only formed the aggregates within the cells but
also were pr esent in the extr acellular space. Visual examina tion dis-
cov er ed more nanoparticles in the case of the Hsp70 conjugate
Fig. 4. Fluorescent and transmission electron microscopy images of C6 glioma cells in vitro. (A) ControlC6 cells and (B) cells incubated in slide chambers for
24 h with SPIONs (50 mg/mL) or (C) cells incubated with Hsp70-SPION sonjugates (50 mg/mL) were fixed with 4% PFA, stained with
4
′
,6
′
-diamidino-2-phenylindole (DAPI). Images of cell nuclei (blue) and nanoparticles (green) were acquired sequentially. Nanoparticles appear as
green dots or larger coarse aggregates inside the cytoplasm. Scale bar, 25 mm. (D) Electron microscopy images of C6 cells incubated with SPIONs
(50 mg/mL) for 24 h. Nanoparticles within the cytoplasm of C6 cells as electron-positive inclusions in the membranous structures are shown by red
arrow. Scale bar, 1 mm. (E) Hsp70-SPION conjugates presented as electron-positive inclusions in nonmembranous structures and (F) endosomes within
the cytoplasm. Scale bar, 1 mm. (G) Fluorescent image of C6 glioma cells incubated with Hsp70-SPION conjugates (50 mg/mL) for 24 h following
treatment with blocking anti-CD40 antibodies. Scale bar, 25 mm. (H) Immunocytochemistry of C6 cell stained with anti-CD40 antibodies. The presence
of Hsp70-SPION conjugates (red arrows) inside the CD40+ (blue arrows) membrane structures could be shown. Scale bar, 500 nm.
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
44
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application than in the case of nonconjugated SPIONs. The Hsp70 re-
ceptor saturation by the i.v. injection of the purified Hsp70 dr ama tic-
ally increased the level of the Hsp70-SPIONs in the tumor (Fig. 5D).
C onjuga tes on the MR T
2
-weighted scans appeared as negative con-
tras t “dark” zones and could thus reve al that the tumor was ,1mm
in diameter. As observ ed on T
2
-weighted images, the “dark” core
inside the tumor could be attributed to the necrotic area. Fo r the dif-
ferential diagnosis between the specific accumulation of the nano-
particles inside the glioma and tumor necrosis, we applied contras t
solution (1.0 mL of 1.0 mmol/mL; Gadovist) for animal #2 from the
4th group. Before mounting the animal in the MR scanner, we i.v.
injected Gadovist into the tail vein. Prev iously it was rep orted that
on T
1
-weighted gadolinium (Gd)-enhanced images the tumor nec-
rotic core shows hyperintensities due to the disruption of the BBB.
27
In our study on the subsequent Gd-enhanced T
1
-weighted images,
we observed only a slight increase in contrast, although the tumor
zone remained hypointensive, which indicated the accumulation of
Hsp70-SPIONs inside the tumor (Fig. 6).
Discussion
The acquired results of the study of synthesized magnetic conju-
gates of Hsp70indicatethat conjugates appear as small biocompat-
ible nanoparticles with high magnetic moments. The measured
magnetic relaxation times T
1
, T
2
, T
∗
2
in aqueous Hsp70-SPION con-
jugate suspensions are in close relation to the values of initial iron
oxide nanoparticles (Figs 1 and 2). Acceleration of magnetic relax-
ation of water protons in suspensions follows from spatial distribu-
tion of magnetic field induced by SPION cores. All absolute values
of T
1
, T
2
, T
∗
2
and the ratio of T
2
/T
1
,T
2
/T
2
correspond to the magnetic
properties of commerciallyavailable negative contrast agents, such
as CombiDex and Feraheme, which lead to decreased MRI signals.
Therelaxivityof conjugate Hsp70-SPIONcalculated fromconcentra-
tion dependencies is comparable to previously reported data of iron
oxide nanoparticles conjugated with EGF, vascular endothelial
growth factor receptor–targeted antibodies, transferin, aptamers,
receptor active peptides, and other bioligands.
15,28
The negative contrast agents are known to reduce magnetic
relaxation times, which results in a hypointensive change of reson-
ance signal in MR scanning. The observed short times of magnetic
relaxation of water protons in the presence of magnetic Hsp70
conjugates are due to the intense relaxation of spins in an inhomo-
geneous magnetic field induced by the magnetic nuclei of
superparamagneticmagnetite nanoparticles in conjugate. The dif-
fusion of water molecules around the magnetic centers leads to
the partial averaging of local magnetic fields experienced by a
spin during the precession in the magnetic field of the spectrom-
eter. In the approximation of the total diffusion averaging, one
can expect a strong deviation of the ratio T
2
/T
1
of 1. The data dem-
onstrate the performance of relationship T
2
/T
1
. 1 and T
2
*. T
2
,
which is consistent with the mechanism of the inhomogeneous
line broadening due to the strong interference of the magnetic
field induced by MNPs. In accordance with the diffusion
Fig. 5. MRIs of (A) C6 glioma for control, (B) SPIONs i.v. injected, (C) Hsp70-SPION conjugates i.v. infused, and (D) the infusion of Hsp70-SPIONs following
preliminaryinjection of Hsp70.Images weretakenfollowing24 hafter nanoparticle infusion atdifferentacquisitionregimes: RARE-T1,Turbo-RARE-T2,fast
low angle shot MRI (FLASH), and multiscan-multiecho (MSME). Scale bar, 1 cm. Red solid arrows point to the zones of “darkening” inside the glioma that
correspond to the nanoparticle accumulation. Following assessment on MR scanner, the brains were extracted, dissected, stained by
4
′
,6
′
-diamidino-2-phenylindole (DAPI; blue), and analyzed for nanoparticle presence with the help of confocal microscopy. Nanoparticles appear as
green dots or larger aggregates (shown by white solid arrows) within the cytoplasm and in the extracellular space. Scale bar, 25 mm.
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
Neuro-Oncology 45
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mechanism of relaxation of the MNPs, the reduction of the relax-
ation efficiency must be related to the decreasing magnetic
moment of the particles and the narrowing of the distorted
region of the magnetic field near the core due to a decrease in
spatial access for water protons. According to AFM, the bound
Hsp70 layer leads to a 2-fold increase of the effectivehydrodynam-
ic diameter of MNP and, consequently, the magnetic shielding of
the magnetic core for the water protons. Shielding of MNPs
should cause a decrease in the relaxation rate r2m, as was
shown in a long-term study of proton magnetic relaxation in sus-
pensions of magnetic Hsp70 conjugates. Dextran-coated SPIONs
showed slightly higher T
2
relaxivity than Hsp70 conjugates;
however, this difference is not principal for contrast enhancement
in MRI. The formation of an Hsp70 shell around SPION does not
shield the magnetic core from water. Water protons perceive the
influence of the magnetic field generated by cores of SPIONs
coming close to the center due to intense diffusion and small
proton size.
MRI of agar phantoms saturated by Hsp70-SPION conjugates
also confirmed the high relaxivity of gel preparation. As a result
of growth of relaxation under the influence of SPION-Hsp70, the
phantom images have a view of dark spots with low NMR intensity.
The magnetic characteristics of Hsp70-SPIONs obtained in vitro
make it possible to use one as an effective contrast agent for MRI
of the rat brain model in the case of sufficient biocompatibility
and targeted accumulation in cancerous tissue. The observed
rate of relaxation times was proportional to the Fe content in sus-
pension. The intensity map of the MRI is known to depend on the
distribution of T
1
and T
2
times associated with voxel position;
therefore, the distribution of intensity NMR signal across the T
2-
-weighted images will evidence the potential accumulation of
magnetic conjugateHsp70-SPION in tumorcells. The measured re-
laxation characteristics of Hsp70-SPION conjugate in suspension
remained constant in fractions after centrifugation and separation
procedures on magnets. The stability and particle size of prepared
SPIONs (,100 nm) are sufficient for in vivo MRI diagnostic experi-
ments.
Biocompatibility of magnetic conjugate is another substantial
demand for targeted delivery of drugs into brain tumors. The toxi-
cology study shows that the Hsp70-SPION conjugates at diagnos-
tic concentrations exert no toxicity toward C6 glioma cells. In vivo
experiments also confirmed the safetyof i.v. injections of SPIONs or
Hsp70-SPION conjugates, as all of the animals survived the proce-
dures and showed no signs of toxicity. Acute toxicity was not found
for dose 0.01–1.6 mg/mouse SPION conjugates.
15
These data are
in accordance with those of Hadjipanayis et al,
13
who studied the
toxicity of the iron oxide nanoparticles on glioblastoma cells or
normal human astrocytes and found no significant toxicity 3
days after treatment with nanoparticles. Biocompatible SPIONs
were degraded and cleared from circulation by the endogenous
iron metabolic pathways; iron content in blood revealed no differ-
ence from control after 2 weeks.
Biocompatible conjugates Hsp70 are not inert extracellular par-
ticles; the results point to the notable intracellular absorption. Our
in vitro experiments demonstrated the incorporation of nanoparti-
cles within C6 cell cytoplasm; the elec tron-dense Hsp70-SPION
conjugates accumulated inside the endosome structures (Fig. 4).
The possible mechanism of conjugate uptakeis receptor-mediated
endocytosis. Previously, several receptors for Hsp’s were described
onvarious cells, including scavenger receptors SR-A, SR-F1 (scaven-
ger receptor class F–1/scavenger receptor expressed on endothe-
lial cells–1), stabilin-1, and LOX-1
29 – 32
; c-type lectins such as
Lectin-1, CD94, NKG2D,DC-SIGN
33 – 35
;receptor for a2macroglobu-
lin CD91
36
; and the Toll-like receptor (TLR-2, TLR-4) family.
37,38
In
our study we assessed the other receptor for Hsp70, a member
of the tumor necrosis factor receptor family, CD40, which is
known to be expressed not only on antigen-presenting cells but
also on glioma cells.
18 – 21,39
We observed a dramatic increase of
CD40+ cells inside the glioma tumor in vivo (Fig. 3). We supposed
that the microenvironment inside the tumor would drive the
growth of the CD40 expression. In vitro, we modulated the oxida-
tive stress and confirmed the hypothesis of the CD40 expression
growth on the gliomacells under the stress stimuli (Supplementary
material, Fig. S1). The redox status in the cell is determined by the
balance of reactive oxygen and nitrogen species, free radicals such
as superoxide (O
2
), a hydroxyl radical (HO), and nonradicals that
could generate free radicals (such as H
2
O
2
).
40
Previously it was
shown that oxidative stress activates various pathways, including
mitogen-activated protein kinase and response mediated by
nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-kB), which mediate intracellular signal transduction.
41 – 43
It
is believed that the activation of the NF-kB pathway in oxidative
stress conditions promotes the expression of CD40 on the glioma
cells
44
; CD40 was expressed in only the C6 glioma site but not in
other normal brain areas. Conceivably, this could explain the pref-
erential accumulation of fluorochrome-labeled Hsp70 inside the
C6 glioma cells but not in the normal brain tissues when being i.v.
administered to the tumor-bearing animal (Fig. 3). The presented
data are in compliance with our previously obtained results when
Fig. 6. MRIs of animal #2. Following i.v. infusion of Hsp70 (5 mg/kg), the
Hsp70-SPION conjugates (150 mg/kg) were i.v. injected. Before mounting
the animal in the head coil i.v., we injected contrast agent (Gadovist,
1.0 mmol/mL, 1 mL). The T
1
- and T
2
-weighted coronal and sagittal brain
sections are presented. Red solid arrows point to the location of the C6
tumor. The accumulation of nanoparticles appears as a “dark” core at the
T
2
-weighted regimen. On the Gd-enhanced T
1
-weighted images, only the
slight increase in contrast in the tumor zone is observed.
Shevtsov et al.: Nanoparticle Hsp70 conjugate for tumor targeting
46
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Hsp70-containing hydrogel was applied on the surface of a
7-day-old B16F10 melanoma tumor. According to the histochem-
istry, Hsp70 diffused through the skin layer inside the B16 tumor.
45
As well as receptor-mediated cellular uptake of Hsp70, there have
been other mechanisms described that could play a certain role in
the Hsp70-SPION uptake, including anchoring to the lipid rafts.
46–48
The cellular uptake of Hsp70 gives reason to assume that the
notable accumulation of SPIONs conjugated with Hsp70 can be
realized in glial tissue in vivo. Actually, the study demonstrated
that Hsp70-SPION conjugate i.v. injected into a C6 rat glioma
model accumulates in the tumors and enhances the contrast of
their MRI images. The magnetic Hsp70 conjugate after i.v. injection
was specifically delivered to malignant areas of implanted tumors
(Fig. 5). According to the confocal microscopy data, we could
observe the presence of nanoparticle aggregates within the cell
cytoplasm or the extracellular space in the tumor site but not in
the normal adjacent brain tissues (Fig. 5, right column). The pres-
ence of the SPIONs or Hsp70-SPIONs inside the glioma could be
attributed to the destruction of the BBB, which is the main
feature of malignant tumors.
49,50
Earlier it was shown that nano-
particles have the ability to cross the BBB, thus providing brain
tumor targeting.
5,51,52
The case when the level of Hsp70-SPION re-
tention in the glioma site was significantly higher compared with
the nonconjugated SPIONs could be explained by the specific
receptor-mediated endocytosis of the Hsp70-SPION conjugates
by the CD40+ glioma cells. In favor of this are the in vitro data,
where it was demonstrated (by means of confocal microscopy)
that the magnitude of Hsp70-SPION uptake by C6 cells was
higher than that of nonconjugated SPIONs (Fig. 4). The similar
pattern of nanoparticle internalization (with a higher uptake of
Hsp70-SPIONs in comparison with nonconjugated SPIONs) was
observed in the case of HeLa cells (Supplementary material,
Fig. S2). Hsp70-SPION conjugates show high relaxivity in the T
2-
-weighted regimen and can be used as negative contrast agents
for MRI diagnostics of malignant tissues with overexpressed recep-
tors for Hsp70. As a result of targeted delivery, the images become
more enhanced with regard to resolution. The degree of negative
contrast was comparable to that achieved by other contrast
agents, including the earlier report of the EGFRvIII antibody conju-
gated with iron oxide nanoparticles.
13
The magnitude of the tumor
accumulation of the Hsp70-SPIONs could be increased by the pre-
liminarysaturationofthe Hsp receptors with i.v. injection ofpurified
Hsp70 prior to the infusion of the Hsp70-SPION conjugates (Figs 5D
and 6). We proposed that various receptors to the Hsp’s, including
Hsp70, exposed on different cells and mostly on the reticulo-
endothelial system cells,
16
thus contribute to receptor-mediated
Hsp70-SPION internalization, affecting the accumulation inside
the tumor tissue. Consequently, receptor saturation by the i.v. infu-
sion of the purified Hsp70 could increase the retention of
Hsp70-SPIONs inside the glioma. Previously, Binder et al
26
studied the saturation concentrations (varying from 0 to 200 mg/
mL) for gp96, Hsp90, and Hsp70 with CD11b+ cells, and Hsp70
did not reach saturable binding at 200 mg/mL. According to our
data, the accumulation of Hsp70 –Alexa Fluor 555 conjugate
inside the C6 glioma starts to be detectable at concentrations
over 5 mg/kg (Fig. 3). Following i.v. treatment by purified Hsp70,
we injected Hsp70-SPIONs and showed the dramatic increase of
the hypointense zone in the tumor site on the T
2
-weighted
images compared with injection of conjugates without the prelim-
inary saturation of the Hsp70 receptors (Fig. 5D).
In summary, the presented work shows the feasibility of glioma
targeting by Hsp70-SPION conjugates via i.v. administration. The
level of intratumoral accumulation is comparable to that of local
infusion, which broadens the clinical application of these conju-
gates for targeted delivery to the metastatic or diffuse tumors
and tumors located in eloquent brain areas. A possible way to
further enhance the retention of a targeted Hsp70 drug at the ma-
lignant site is to focus Hsp70-SPIONs by a specially configured
magnetic field via a magnet implant. Further steps for develop-
ment of this targeted nanoconstruction include the improvement
of stability, a pharmacokinetic study, and optimization of medical
application.
Supplementary Material
Supplementary material is available at Neuro-Oncology Journal
online (http://neuro-oncology.oxfordjournals.org/).
Funding
This study was supported by the grant of the Program of Russian Academy
of Sciences (RAS) “Molecular and Cell Biology,” grants of Russian Fund for
Basic Research No 10-0401049 and No 11-08-00045, Governmental
grant (20.11.2012) No 14.N08.11.0001.
Acknowledgments
Authors thank G. I. Stein and M. L. Vorobiev for the technical assistance in
the confocal microscopy analysis. Authors are grateful to P. P. Klein for
the preparation of illustrations.
Conflict of interest statement. None declared.
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