Inhibition of brain tumor growth by intravenous poly
(β-L-malic acid) nanobioconjugate with pH-dependent
Hui Dinga, Satoshi Inouea, Alexander V. Ljubimovb,c, Rameshwar Patila, Jose Portilla-Ariasa, Jinwei Hua, Bindu Kondaa,
Kolja A. Wawrowskyd, Manabu Fujitab, Natalya Karabalina, Takako Sasakie, Keith L. Blacka, Eggehard Hollera,f,1,
and Julia Y. Ljubimovaa,c,1
Departments ofaNeurosurgery,bSurgery,cBiomedical Sciences, anddAcademic Affairs, Cedars-Sinai Medical Center, Los Angeles, CA 90048;eDepartment of
Experimental Medicine I, Nikolaus-Fiebiger Center of Molecular Medicine, University of Erlangen-Nürnberg, D-91954 Erlangen, Germany; andfInstitut für
Biophysik und Physikalische Biochemie der Universität Regensburg, D-93053 Regensburg, Germany
Edited by Alexander M. Klibanov, Massachusetts Institute of Technology, Cambridge, MA, and approved August 27, 2010 (received for review March 25, 2010)
Effective treatment of brain neurological disorders such as Alz-
heimer’s disease, multiple sclerosis, or tumors should be possible
with drug delivery through blood–brain barrier (BBB) or blood–brain
tumor barrier (BTB) and targeting specific types of brain cells with
drug release into the cell cytoplasm. A polymeric nanobioconjugate
drug based on biodegradable, nontoxic, and nonimmunogenic poly-
malic acid as a universal delivery nanoplatform was used for design
and synthesis of nanomedicine drug for i.v. treatment of brain
tumors. The polymeric drug passes through the BTB and tumor cell
membrane using tandem monoclonal antibodies targeting the BTB
and tumor cells. The next step for polymeric drug action was inhibi-
tion of tumor angiogenesis by specifically blocking the synthesis of
a tumor neovascular trimer protein, laminin-411, by attached anti-
sense oligonucleotides (AONs). The AONs were released into the tar-
get cell cytoplasm via pH-activated trileucine, an endosomal escape
moiety. Drug delivery to the brain tumor and the release mechanism
were both studied for this nanobiopolymer. Introduction of a trileu-
cine endosome escape unit resulted in significantly increased AON
in vivo, specific accumulation in brain tumors, and suppression of
intracranial glioma growth compared with pH-independent leucine
system that passes through the BTB, targets tumor cells, and inhibits
glioma growth gives hope for a successful strategy of glioma treat-
ment. This delivery system with drug release into the brain-specific
cell type could be useful for treatment of various brain pathologies.
glioma treatment|polymalic acid|laminin-411|endosomal escape|
At present, efficient drugs for treatment of gliomas are very
limited (1). A hallmark of current cancer treatment is inhibition of
tumor angiogenesis (2, 3). Antiangiogenic inhibitors of VEGF
receptors and VEGF-independent inhibitors combined with che-
motherapy have shown some promise, but the patients’ survival in
clinical trials was not significantly changed (4). We documented
overexpression of tumor-specific vascular basement membrane
protein laminin-411 in glioblastoma and its association with tumor
recurrence and decreased patients’ survival time (5, 6). Laminin-
411 consists of three different polypeptide chains, and it was
hitherto impossible to efficiently block its synthesis in vivo by
existing technologies. In our nanoconjugate, two antisense oligo-
nucleotides (AONs) against laminin α4 and β1 chains were co-
valently attached and delivered through the blood–brain tumor
barrier (BTB). Although the microvascular BTB is more permis-
sible than the blood–brain barrier (BBB), it still retains BBB
characteristics (7, 8), hampering drug delivery to brain tumors.
Antibodies to certain cell surface proteins, including transferrin
receptor (TfR), can pass the BBB and BTB by endothelial trans-
cytosis and then direct carrier systems with attached drugs into
tumor cells by receptor-mediated endocytosis (9–13).
delivery to the tumor with minimum side effects (3, 14, 15). The
endocytosed carriers, including polymers, are routed to the
endosomal pathway, and their design must ensure the drug escape
from the endosomes into the cytoplasm to avoid lysosomal deg-
radation. Cytoplasmic delivery using cell penetrating peptides
(CPPs) acting directly on cell membranes is a possible mechanism
for drug trafficking across the plasma membrane (16), but CPPs
are not specific to the cell type. Receptor-targeted cytoplasmic
delivery through endosome disruption is a safe and efficient ap-
proach avoiding potentially cytotoxic permeation through the
specific, membrane-disrupting moiety that takes advantage of the
low pH (5.0–6.5) in the endosome/lysosome compartments.
Several pH-dependent escape devices were designed, such as
polyethyleneimine (PEI), which is protonated during endosome
acidification (17).These polymers,however,areoflimitedusedue
as Glu-Ala-Leu-Ala (GALA) (19, 20) derived from certain viruses
or bacteria, are used for endosome escape of micelles and lip-
osomes (21) or are covalently bound to nanoconjugates to deliver
water-soluble proteins, antibodies, and nucleic acids (9, 22).
The nanoconjugate based on poly(β-L-malic acid) (PMLA) that
is introduced here meets all criteria for nanomedicine drugs (23)
and is a promising candidate for future clinical use, alone or in
combination with other treatments for brain cancer. This nano-
polymeric drug is designed for i.v. brain tumor treatment using
system might be used to treat other brain pathological conditions.
Synthesis of Polymalic Acid-Based Nanoconjugates. Nanoconjugates
of varying composition were synthesized. A schematic of a conju-
gate with allfunctional groupsisshown in Fig. 1A, anda simplified
chemical formula in the Fig. S1. NHS-activated carboxylates of
polymalic acid were first conjugated with H2N-Leu-Leu-Leu-OH
(LLL), H2N-Leu-Leu-Leu-NH2, H2N-Leu-ethylester (LOEt), 2-
Author contributions: H.D., A.V.L., M.F., K.L.B., E.H., and J.Y.L. designed research; H.D.,
S.I., R.P., J.P.-A., J.H., B.K., K.A.W., M.F., N.K., and J.Y.L. performed research; T.S. contrib-
uted new reagents/analytic tools; H.D., S.I., A.V.L., J.P.-A., T.S., K.L.B., E.H., and J.Y.L.
analyzed data; and H.D., A.V.L., E.H., and J.Y.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or ljubimovaj@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 19, 2010
| vol. 107
| no. 42
mercapto-1-ethylamine. The newly introduced sulfhydryl groups
were converted to disulfides with thiolated morpholino AON and
to thioethers with maleimidated mAbs and optionally with mal-
eimidated fluorophore Alexa Fluor 680. The composition of syn-
thesizednanoconjugate and pendant group functions areshown in
Fig. 1B. The composition was confirmed by analytical tests to
completely correspond to the synthetic strategy. The calculated
molecular weight values compared within 15% with experimental
absolute molecular weights by light scattering, which also con-
firmed the absence of particle aggregation. Sizes by dynamic light
scattering ranged from 6.6 nm for PMLA to 18 ± 2 nm for P/LLL/
AON/Hu/Ms and 22 ± 2 nm for P/LOEt/AON/Hu/Ms. The ζ
potentials (pH 7.0) ranged from −27 ± 1 mV for polymalic acid,
−9.4 ± 0.7 mV for P/LLL/AON/Hu/Ms to −5.2 ± 0.4 mV for P/
LOEt/AON/Hu/Ms; the potentials of LLL containing conjugates
was pH-dependent (Fig. 1C). The half-life of the lead nano-
conjugate P/LLL/AON/Hu/Ms was 24 h at 37 °C in PBS and
similarly in human plasma.
pH-Dependent Membrane Disruption for Nanoconjugate Escape from
membrane disruption. Two versionsofmembranedisruption units
were used: pH-dependent P/LLL (PMLA conjugated with 40%
LLL) and pH-independent P/LOEt (PMLA conjugated with 40%
LOEt) (24, 25). At pH 5 both nanoconjugates were membrane
AONs and mAbs were added to P/LLL/AON/IgG and P/LOEt/
AON/IgG nanoconjugates, the leakage remained high (80–90%)
(Fig. 2B), indicating that membrane disruption activity survived
conjugation of AONs and mAb. At physiological pH 7.4, P/LLL
conjugates remained (Fig. 2C). P/LOEt activity was high over the
and abruptly dropped at pH 6 (Fig. 2D). The pH dependence
mirrored the range of acidification in late endosomes/lysosomes.
This justified using P/LLL conjugates for endosome-specific es-
cape. In contrast to P/LOEt conjugates, the specific acidification
requirement forP/LLL conjugates wouldnot allowcell membrane
disruption at neutral pH.
Leucine and its derivatives may form strong hydrophobic inter-
directly into lipid membrane bilayers and provoke their disruption
(26, 27). This may be the mechanism of membrane interaction with
studied nanopolymers. However, LLL carries a charged terminal
carboxylate that cannot be accommodated into a lipohilic environ-
ment unless carboxylate isneutralized by protonation.By acid–base
P/LLL titration, this protonation followed a pKa5.5, and this pH
dependence paralleled the leakage dependence in Fig. 2D. P/LOEt
did not contain this ionizable group and did not show pH de-
pendence. When LLL was replaced by LLL–NH2with a non-
ionizable terminal amide group instead of carboxylate, pH
dependence and liposome leakage became similar to LOEt.
The simplest reaction mechanism implies that polymer and li-
posome bind before the lysis occurs. The concentration de-
pendence in Fig. 2 A–C may indicate similar binding affinities for
P/LLL and P/LOEt to the liposome membrane at pH 5.0 and
a very low affinity for P/LLL at pH 7.4 due to the carboxylate
charge. P/LOEt and P/LLL liposome membrane affinities dra-
matically diverged as indicated by confocal microscopy (Fig. 2E)
and ζ potential (Fig. 1C). P/LOEt readily bound to the liposome
membrane at neutral pH 7.4, but P/LLL amounts were too low to
bedetected(Fig.2E).The ζ potential ofP/LOEt didnotchange as
a function of pH either in the presence or absence of liposome
(Fig. 1C). The liposome ζ potential is near zero, and its contri-
bution is negligible. In the presence of liposome, ζ potential of P/
LOEt increased from −13 to −5. This change accounts for various
parameters that affect the electrophoresis underlying ζ potential
measurement and may indicate that the majority of P/LOEt was
sticking to the liposome after membrane disruption and leakage.
Minor amounts of free P/LOEt would not be detectable by this
technique. In contrast, P/LLL ζ potential is pH-dependent (Fig.
1C), the pH sensitivity correlating well with pH-dependent lipo-
some leakage activity. The inefficiency of P/LLL in staining lip-
osomes could indicate that small amounts of P/LLL bound to the
liposome were below the Zetasizer detection limit.
Overall, nanoconjugates with LOEt readily bound to lipid
membranes and in addition to their disruption gave rise to side
This “stickiness” at physiological pH could induce cell toxicity and
also systemic depletion of the circulating nanoconjugate after i.v.
injection, reducing its availability at the tumor site.
Cell Viability and Increased Suppression of Target Laminin-411
Synthesis with pH-Dependent Endosome Escape Unit. Using P/LLL-
and P/LOEt-containing nanoconjugates, cell viability was tested
on the human glioma cell line U87MG. P/LLL was nontoxic at all
concentrations (≤2 mg/mL), but P/LOEtdecreased cell viabilityat
(LLL or LOEt)
*Molecular weight calculated on the basis of Mn 100,000 for polymalic acid
**(%) denotes the percent fraction of polymer carboxylates conjugated
***mAb was substituted by human nontargeting IgG
escape (%) Anti-TfR mAb (%)
P/LLL + liposome
Zeta potential (mV)
acid platform and their ζ potentials. (A)
Cartoon of PMLA-based nanoconjugate.
(Left to Right) endosome escape unit
(LLL or LOEt), AONs to laminin-411 α4
and β1 chains, disulfide linkages cleaved
by cytoplasmic glutathione, capped un-
used sulfhydryl, mAb (Ms) targeting BTB
endothelium (mouse TfR), mAb (Hu) tar-
geting tumor cells (human TfR), tracking
dye Alexa Fluor 680, pendant carbox-
ylates for water solubility. (B) Table list-
ing nanoconjugates and their molecular
weight and composition. (C) ζ potential
for P/LLL and P/LOEt at variable pH. ζ
potential in the absence or presence
of liposomes was measured under con-
ditions when membrane disruption and
leakage had been completed within 5–
10 min. P/LOEt potential was signifi-
cantly shifted in the presence of lipo-
somes but only a marginal shift was seen
for P/LLL. This indicated that most P/LOEt was on the liposomes, but most P/LLL was in the free solute state. Measurements were performed with 200
μg/mL P/LLL or P/LOEt and in the presence of 160 μM lipid (liposomes).
Nanoconjugates with polymalic
| www.pnas.org/cgi/doi/10.1073/pnas.1003919107Ding et al.
as low as 0.15 mg/mL (Fig. 3A, Left). At 0.5 mg/mL P/LOEt, cells
shrunk and rounded up within 2 h, whereas no such changes were
human T98G and rat RG62 gliomas and human breast cancer
MDA MB-231 cells. By FACS analysis, the shrunk and rounded
cells were mainly in early apoptosis phase (annexin V-positive)
(Fig. 3A, Right), although some increase in necrosis [propidium
iodide (PI)-positive] (Fig. 3A, Right) was also evident.
Western blot analysis of conditioned media confirmed that
AONs were efficiently delivered by P/LLL/AON/Hu and P/LOEt/
AON/Hu into the cytoplasm of U87MG and T98G glioma cells
and that they inhibited the synthesis of laminin-411 α4 and β1
chains (Fig. 3B). In the absence of endosomal escape (in the
nanoconjugate P/AON/Hu), the synthesis was markedly less af-
fected. Free AONs that could not penetrate cell membranes
functional activities, including TfR binding and internalization,
endosomal uptake and escape, and inhibition of laminin-411 syn-
thesis. Nanoconjugates with LLL or LOEt were both active;
however, P/LLL/AON/Hu with pH-dependent endosomal escape
unit was more potent (Fig. 3B).
Colocalization of the Polymer Platform and Antisense Oligonucleotides
in Cytoplasm. To confirm that the polymer platform and AONs
entered the endosomal pathway together in U87MG and T98G
glioma cells, the nanoconjugate P/LLL/AON/Hu was double-
labeled with Lissamine at AON and Alexa Fluor 680 at PMLA. By
confocal microscopy (Fig. S2), the PMLA platform (green) and
AONs (red) colocalized (yellow) in intracellular entities, which
coincided (white) with stained endosomal membranes (blue) (Fig.
S2A). The number of green and red vesicles decreased over time
during a 3-h incubation period (Fig. S2B). The decrease coincided
with diffuse staining around these entities, in agreement with in-
duced leakage by membrane disruption. Statistical analysis of
colocalization expressed as Pearson’s correlation coefficients (28)
(Fig. S2C) indicated significant colocalization of PMLA and AON
and of each of them with endosomes at 0 h. However, at 3 h,
endosomal marker and nanoconjugate colocalization decreased.
Imaging Analysis of Drug Distribution and Accumulation in Tumor and
Normal Brain Tissues in Vivo.Fortheseexperiments,twomAbswere
attached to the PMLA platform. Anti-mouse TfR mAb was used
for transporting drugs through the mouse endothelial host system,
and anti-human TfR mAb served for targeting implanted human
tumor cells. Imaging analysis 24 h after i.v. injection showed that
nanoconjugate with anti-mouse TfR mAb delivered only a low
amount of drug into the intracranial human U87MG tumor (Fig.
4A, Upper Left). With anti-human TfR mAb attached to nano-
conjugate, drug accumulation in the tumor increased (Fig. 4A,
Upper Center). These data are consistent with the facilitation of
polymer passage through BTB by the enhanced permeability and
retention (EPR) effect. In the presence of both endothelium- and
tumor-targeting mAbs (anti-mouse and anti-human TfR, re-
spectively), P/LLL/AON/Hu/Ms drug predictably showed the
highest accumulation of all variants (Fig. 4A, Upper Right).
We next compared brain tumor-specific accumulation of poly-
meric drugs containing LLL or LOEt endosomal escape units. In
bothcases, the drug accumulation 24 h afteri.v. injectionpersisted
mainly in brain tumor and to some extent in drug-clearing organs,
livers and kidneys. The drug was no longer detected after 72 h. In
conjugates containing pH-dependent or pH-independent endosome escape
units. (A) Effects of LLL and LOEt endosomal escape units on U87MG cell via-
bility. Arrows relate to microscopic views (20× magnification) after 24 h
treatment with 0.5 mg of either nanoconjugate. The cells treated with P/LOEt
unlike P/LLL had low viability at high concentrations (Left) and were in early
apoptosis (Center). (Right) Representative FACS analysis of cell death after
double staining of cells with propidium iodide and FITC Annexin V. Note
markedly increased fraction of apoptotic cells after treatment of cells with P/
LOEt as compared with P/LLL. (B) Inhibition of laminin-411 α4 and β1 chain
synthesis in human U87MG and T98G glioma cells after treatment with PBS,
AON, P/AON/Hu (lacking endosomal escape unit), P/LOEt/AON/Hu, and P/LLL/
supernatant at the end of the treatment and subjected to Western blot
analysis. P/LLL/AON/Hu was the most effective in inhibiting the synthesis of
both laminin-411α4andβ1 chains. Secreted fibronectin wasused tonormalize
Human U87MG and T98G glioma cell treatment in vitro with nano-
to liposomes. (A) Concentration dependence of P/LLL and P/LOEt membrane
disruption activity at pH 5.0 measured by the liposome leakage assay. The
degree of leakage refers to complete leakage in the presence of 0.25% (vol/
vol) Triton-X 100. (B) Membrane disruption activity for nanoconjugates P/
LLL/AON/IgG and P/LOEt/AON/IgG at pH 5. It is not abolished over the range
of concentrations by the conjugation of AON and antibody. (C) Retention of
membrane-disrupting activity at pH 7.4 by P/LOEt and its loss by P/LLL. (D)
Membrane disruption for P/LLL and P/LOEt (each 50 μg/mL) as a function of
pH. Only P/LLL membrane disruption activity is pH-dependent following an
apparent pKa5.5. (E) Binding of P/LOEt and P/LLL to liposomes at neutral pH.
Confocal microscopy showing colocalization of P/LOEt and giant artificial
liposomes. P/LLL and P/LOEt were conjugated with rhodamine (red). Giant
liposomes were labeled with NBD [N(1)-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
yl)propane-1,3-diamine] (green). (Upper) Large amounts of P/LOEt stuck to
the vesicle membrane; liposomes and P/LOEt colocalized at pH 7.4 (yellow).
(Lower) Binding of rhodamine-labeled P/LLL to liposomes could not be de-
tected. Concentrations of conjugates were 20 μg/mL. The data correlate well
with ζ potential measurement (Fig. 1C).
Membrane disruption activity of P/LLL and P/LOEt and their binding
Ding et al.PNAS
| October 19, 2010
| vol. 107
| no. 42
24 h, drug-tumor accumulation of P/LLL/AON/Hu/Ms (Fig. 4A,
Upper Right) was 1.5 times higher (P < 0.03) than that of P/LOEt/
AON/Hu/Ms variant (Figs. 4A, Lower Center, and B). Because
both nanoconjugates use exactly the same targeting strategy, their
in membrane disrupting units LLL or LOEt, which corresponds
well to data shown on Fig. 2. Control P/LLL carrying a non-
targeting IgG (P/LLL/AON/IgG) accumulated in the tumor 90%
less than P/LLL/AON/Hu/Ms with two targeting antibodies (P =
0.0015) (Fig. 4A, Lower Left, and B).
injection of P/LLL/AON/Hu/Ms with labeled AON and PMLA
were analyzed by confocal microscopy. As shown in Fig. 4C, pos-
(AON label) were seen mainly in tumor cells and also in tumor
vessels positive for von Willebrand factor (vWF) staining. Both
moieties showed noticeable codistribution in the cytoplasm of
tumor cells (Fig. 4C, Lower). Normal brain regions of the same
animals showed little drug accumulation (Fig. 4C, Upper), in
agreement with whole brain imaging (Fig. 4A). The data suggest
that nanoconjugate is efficiently passing through BTB and in-
ternalizing into the tumor cells.
Significant Suppression of Tumor Growth and Vascularity by P/LLL/
AON/Hu/Ms Nanoconjugate. Systemic multiple treatments of mice
bearing intracranial human glioma U87MG with nanoconjugate
P/LLL/AON/Hu/Ms blocking laminin-411 synthesis significantly
suppressed tumor growth (Fig. 5A). The mean tumor volume was
4 mm3(P < 0.001 vs. PBS), compared with 18 mm3(P < 0.01 vs.
PBS) after P/LOEt/AON/Hu/Ms treatment and with 47 mm3in
PBS-treated controls. Tumor size reduction by P/LLL/AON/Hu/
Ms with pH-dependent LLL escape unit was highly significant,
resulting in 90% smaller tumors compared with PBS-treated ani-
mals. LLL-containing nanoconjugate was also more than 2-fold
more efficient in inhibiting tumor growth than the variant with
LOEt. This result fully corroborated in vitro data on laminin-411
inhibition (Fig. 3B). On brain sections of PBS-treated animals,
large invasively growing tumors were seen, whereas after P/LLL/
AON/Hu/Ms treatment, tumor remnants with significant necrosis
were typically found (Fig. 5B). Two conjugates without AON or
anti-TfR mAbs were used as controls with no effect on tumor
treatment compared with PBS, confirming that AONs against two
tumor growth. Morphometric analysis of vascularity on tumor
sections revealed largely similar vessel numbers in all treatment
groups. However, P/LOEt/AON/Hu/Ms and P/LLL/AON/Hu/Ms
treatments usually resulted in smaller vessels than in the PBS
for both nanoconjugates). The vessel area decrease was more
pronounced for P/LLL/AON/Hu/Ms (over 50% less than for PBS
group) than for P/LOEt/AON/Hu/Ms (P < 0.05) (Fig. 5C). On
typically strong in the tumor blood vessel basement membranes in
the PBS group, and many large vessels with irregular shape were
seen (Fig. 5D). After P/LOEt/AON/Hu/Ms, and especially P/LLL/
AON/Hu/Ms treatment, staining intensity was noticeably di-
minished for both laminin chains (Fig. 5D), and vessels became
more similar in size to those in normal brains.
A number of approaches for targeted drug delivery have emerged,
including encapsulation of cargo into micelles, nanospheres,
nanocapsules, and nanotubes (29). Nanoconjugates as covalent
delivery devices received wide interest after the introduction of
natural and synthetic polymers and dendrimers (2, 17, 22, 30–34).
The problems still shared by most delivery platforms include tox-
icity, immunogenicity, the absence of real biodegradability (i.e.,
lack of degradation to CO2and H2O), and the target cell’s cyto-
plasm drug delivery. Current drug design strategies are mostly
focused on bioavailability and tissue targeting but rarely address
drug delivery to specific intracellular compartments (35).
By introducing nanoconjugates, which use PMLA of the slime
mold Physarum polycephalum as a platform, we have overcome
most of the major drawbacks (9, 24, 30, 35–37). PMLA-based
P/LOEt/AON/Hu/Ms P/LLL/AON/Hu/Ms P/LLL/AON/IgG
PMLA AONMerge + DAPI
Relative fluorescence intensity
Signal - background
implanted in mice 24 h after i.v. injection of nano-
conjugate variants. Tumor cells (105) were implanted
intracranially and animals were treated with nano-
conjugates after 21 d. Brains were isolated and PBS per-
fused. The nanoconjugates were P/LLL/AON/Ms (Ms =
anti-mouse TfR), P/LLL/AON/Hu (Hu = anti-human TfR), P/
LLL/AON/Hu/Ms, P/LOEt/AON/Hu/Ms, and P/LLL/AON/IgG
(unrelated IgG). Representative results are shown. (A,
Upper) Alexa Fluor 680-labeled nanoconjugates with Ms,
Hu, and Ms/Hu were injected i.v. Different experiments
are shown in Upper and Lower. (Upper) the nano-
conjugate with both Ms and Hu mAbs showed the high-
est tumor accumulation. P/LLL/AON/Hu accumulated
markedly better than P/LLL/AON/Ms, although Hu in
contrast to Ms would not support transcytosis through
mouse endothelium. This effect was ascribed to efficient
drug withdrawal into the tumor cells after low-level EPR-
mediated delivery into the tumor interstitium. The with-
drawal effect could account for high accumulation.
(Lower) LLL presence ensured higher drug tumor accu-
mulation than LOEt, whereas a control nanoconjugate
with unrelated IgG showed low accumulation (Xenogen
IVIS 200 imaging). (B) Drug accumulation in A, Lower,
representing the signal with subtracted background was
quantitated by (F − Fo)/Fo. Averaged intensities F and Fo
refer to equally sized areas of the tumor and the refer-
ence nontumor area, respectively. Means ± SD of three independent measurements are shown. There is significantly higher accumulation of P/LLL/AON/Hu/Ms
than of P/LOEt/AON/Hu/Ms in the tumor tissue (P < 0.03). (C) Confocal microscopy of brain cryostat sections after i.v. injection of double-labeled P/LLL/AON/
Hu/Ms nanoconjugate in vivo. The PMLA platform was labeled with Alexa Fluor 680 (magenta) and the AONs with Lissamine (red). Vessels were revealed by
immunostaining for vWF (green). (Upper) There is little signal of PMLA and AON in the normal brain tissue contralateral to the tumor. (Lower) Both PMLA and
AON show distinct accumulation in the tumor tissue. They display significant colocalization in the tumor cell cytoplasm (purple, Right).
Imaging analysis of U87MG human brain tumor
| www.pnas.org/cgi/doi/10.1073/pnas.1003919107Ding et al.
nanoconjugates can deliver AON drugs into cells in vitro (38) or
upon injection into the tumor mass (30), but their systemic ad-
ministration with tumor cell-cytoplasm delivery (35) was not ex-
plored previously. Gliomas are highly invasive tumors, and only
systemic treatment could be really beneficial to treat this very ag-
gressive type of brain cancer. The problems now appear to be
largely solved by introducing a pH-dependent endosomal escape
unit, the tripeptide LLL that facilitates specific cytoplasm delivery
from late endosomes (Fig. 1A and Fig. S1).
a considerable pH-dependent membrane leakage both in lip-
osomes and cells. Out of many hydrophobically modified PMLA
topHinliposome leakage assay.Its pH-dependentmembranolytic
activity with an operational pKa5.5 matched acidification during
maturation from early to late endosomes. In contrast to the pre-
viously used membranolytic unit LOEt, the LLL unit was nontoxic
at all concentrations tested (Fig. 3). We explain the LOEt cyto-
toxicity by its lipophilicity at physiological pH ∼7 that renders it
sticky and destructive to cell membranes. In contrast, LLL is not
sticky at this pH due to its terminal negative charge. Importantly,
the absenceofstickinessto membranesmay alsoprevent lipophilic
hydrophobic sites of proteins such as opsonins of the re-
ticuloendothelial system (39), thus contributing to reduction of
nonspecific effects on nontarget cells.
The pH-restricted membranolysis is important for an optimal
as an increased inhibition of target laminin-411 production in vitro
and in vivo (Figs. 3B and 5D), higher tumor accumulation (Fig. 4),
and significantly increased antitumor efficacy possibly related to re-
(Fig. 5 C and D). The dual effect of reducing cytotoxicity and in-
creasing bioavailability to the target cells as opposed to nontarget
LLL drug delivery system. To our knowledge, the application of
brain cancer treatment has not been reported thus far.
A major problem in glioma treatment is the inefficiency of sys-
temic water-soluble delivery systems due to BTB, which was
bypassed in our nanoconjugate by tandem conjugated anti-mouse
(11, 40, 41). BTB permeation was additionally facilitated by EPR
effect (42). However, alone, transcytosis- and EPR-mediated
permeation were not efficient for nanoconjugate accumulation
in the tumor (Fig. 4A, Upper Left and Center). The second (anti-
human) mAb allowed the polymer to specifically bind to and get
nanoconjugate from interstitial space into the tumor cell cyto-
plasm mediated by anti-human TfR is important for successful
humans, two anti-species mAbs are unnecessary. However, the
technical possibility of attaching two mAbs to the polymer was
important in our study with xenogeneic tumors to prove the
receptor-mediated human tumor cell-specific targeting.
After endosome internalization, the LLL escape unit is acti-
vated during maturation to late endosomes concomitantly with
acidification. The delivery platform and AONs remain conjugated
until the disulfide linkage preserved during transcytosis (40) is
cleaved by reduction with cytoplasmic glutathione. After their
cytoplasmic release, free AONs can block synthesis of α4 and β1
in tumor neovasculature (5, 6, 30, 38). This systemic anti-
angiogenic approach resulted in a specific and significant in-
hibition of brain tumor vascularity and growth (Fig. 5).
Safety and efficiency are two primary concerns for designing
systems for AON delivery. Our PMLA-based nanoconjugates
meet both criteria and offer promising strategies in systemic drug
delivery to treat brain tumors and other brain degenerative con-
of their biodegradability, and lack of toxicity and immunogenicity,
variants of PMLA-based delivery systems can be designed for
a wide array of safe and efficient applications.
Materials and Methods
Cell Viability and Cell Death Assays. Cellviability wasquantifiedusingCellTiter
96 AQueous One Solution Cell Proliferation Assay kit (Promega) and a Spec-
P < 0.01
P < 0.001
Tumor size, mm3
Vessel area (%) per
x20 microscopic field
P < 0.001
P < 0.001
cape units in inhibiting brain tumor growth, vascularity, and target protein
expression. P/LLL/AON/Hu/Ms and P/LOEt/AON/Hu/Ms were injected i.v. at
5 mg/kg morpholino AONs to laminin-411 α4 and β1 chains. (A) Tumor size
quantitation after treatment with P/LLL/AON/Hu/Ms or P/LOEt/AON/Hu/Ms.
Both nanoconjugates significantly decreased tumor volume. However, vol-
ume decrease for P/LLL/AON/Hu/Ms (LLL) with pH-dependent unit was sig-
nificantly greater than for P/LOEt/AON/Hu/Ms (LOEt) with pH-independent
unit. (B)H&E-stained sections oftumorstreated byeither PBSorthelead drug
P/LLL/AON/Hu/Ms (LLL). Two different animals represent each group. In PBS-
injected mice (#35 and #37), invasively growing intact tumors are seen. In the
LLL-treated animals (#22 and #23), massive necrosis is visible with some tumor
remnants. (C) Morphometric analysis of microvessel area after various treat-
ments. Both P/LOEt/AON/Hu/Ms (LOEt) and P/LLL/AON/Hu/Ms (LLL) signifi-
cantly reduced vessel area (most pronounced in the latter group), compared
with PBS. In the LLL group, the reduction was significantly greater than in the
LOEt group (P < 0.05). Data are from 25 nonoverlapping fields of view per
group(fieldarea= 0.245μm2)using20× objective(fivepertumor,fivetumors
per group). Percentage of area occupied by vessels (revealed by laminin β1
chain immunostaining) to total field area is shown. (D) Immunostaining of
tumor sections for laminin α4 and β1 chains upon nanoconjugate treatment.
In the PBS group, vessels stained brightly for both chains and many large
vessels with irregular shapes were seen. Upon treatment with P/LOEt/AON/
Hu/Ms (LOEt) and especially P/LLL/AON/Hu/Ms (LLL), tumor staining intensity
for both chains was diminished and vessels became smaller, more similar to
normal brain vessels. Representative pictures for each group are shown. For
each antigen, exposure times were the same among groups.
Efficacy of different pH-dependent or -independent endosomal es-
Ding et al. PNAS
| October 19, 2010
| vol. 107
| no. 42
traMax Plus 384 ELISA reader (Molecular Devices) at A490for human gliomas Download full-text
U87MG and TG98 and breast cancer cell line MDA MB-231.
Apoptosis and/or necrosis induced by incubation of human glioblastoma
U87MG cells with 250 μg/mL of P/LLL and P/LOEt were assayed as follows.
Cells were seeded in 96-well plates (1 × 104cells per well) for 24 h and then
treated with P/LLL and P/LOEt in culture medium (Eagle’s MEM with 10%
FBS) for 24 h. Apoptosis was measured by flow cytometry using FITC Annexin
V apoptosis detection kit (BD Pharmingen). Cells stained with FITC Annexin V
represented apoptosis, and those positive for PI were presumed to be ne-
crotic (BD FACScan Flow Cytometer).
Tumor Treatment in Vivo. Animals were treated according to the approved
Cedars-Sinai Medical Center Institutional Animal Care and Use Committee
protocols. Athymic mice (CrTac:NCr-Foxn1nu homozygous) were from
Taconic. Human U87MG glioblastoma cells were stereotactically implanted
at 5 × 104into the right basal ganglia field of mice (n = 8 per group) as
reported (24). Nanoconjugate treatment began after day 8 by i.v. injecting
nanoconjugates at doses of 5 mg/kg AONs on every third day, amounting to
eight injections in total. Mice were killed on day 48 after cell inoculation,
and tumor volumes were measured using histological sections (43) stained
Imaging Analysis in Vivo. On days 20–30 after tumor implantation, 100 μL
solution of 3 μM Alexa Fluor 680-labeled nanoconjugates was injected i.v.
Mice were euthanized after 24 h. Following blood vessel clearance by
intraarterial PBS perfusion for 20 min, brains were removed for fluorescence
imaging by Xenogen 200 Living Image System 2.50 (Caliper Life Sciences).
Light intensities emitted from equally sized surface areas of tumor and
nontumor references were measured. In some cases, tumor/reference in-
tensity ratios were calculated. The reference area was in the brain but dis-
tant from the tumor. Nanoconjugates without fluorescent Alexa Fluor 680
(background) gave negative results. Injected free dye was rapidly cleared
and did not result in imaging signal (9).
Statistical Analysis. Statistical analysis was done using Prism4 statistical pro-
gram (GraphPad). Data in several groups were compared using ANOVA. Data
points represent mean ± SD of triplicates; in some cases in Fig. 2, SD bars
were too small to be shown. In some experiments, data are expressed as
mean ± SEM. P < 0.05 was considered significant.
Materials, Syntheses, Liposome Leakage Assay, Cell and Tissue Section
Immunostaining, Colocalization Studies, and Western Blot Analysis. Materials,
syntheses, liposome leakage assay, cell and tissue section immunostaining,
colocalization studies, and Western blot analysis are presented in detail in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. J. Young from the Department of
Comparative Medicine, Cedars-Sinai Medical Center, for help and advice
with animals. This work was supported by National Institutes of Health
Grants CA123495 (to J.Y.L.) and EY13431 (to A.V.L.), Winnick Family
Foundation, and M01 RR00425 (to A.V.L. and J.Y.L.), and a grant from the
Department of Neurosurgery, Cedars-Sinai Medical Center.
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| www.pnas.org/cgi/doi/10.1073/pnas.1003919107Ding et al.